7

Targeted Therapies for Localized Prostate Cancer: The Urologists Perspective

JAMES JOHANNES, LEONARD G. GOMELLA, AND EDOUARD J. TRABULSI
Department of Urology, Kimmel Cancer Center, Thomas Jefferson University, Philad
elphia, Pennsylvania, U.S.A.
INTRODUCTION There have been great advances in both detection and treatment of p
rostate cancer over the past 20 years. Largely, because of the application of ro
utine prostate-specific antigen (PSA) screening, cancer-specific mortality from
prostate cancer has decreased over this time period (1). With early detection an
d modern treatment modalities, prostate cancer five-year survival in the United
Stated is now greater than 99% for all patients (2). In addition to these improv
ements, routine PSA screening has led to a marked stage migration with the major
ity of newly diagnosed patients presenting with localized disease. These epidemi
ological changes are prompting a reexamination of prostate cancer risk stratific
ation and treatment algorithms. Patients with low-risk localized disease are eag
er to pursue minimally invasive, targeted therapies to avoid radical surgery or
radiation therapy, because of the side effects and complications commonly seen w
ith traditional treatment options. Treatment options for localized prostate canc
er are expanding. Historically, patients decided among radical surgery, radiothe
rapy, or active surveillance (watchful waiting) (3). Treatment direction should
be made after a personalized discussion, ideally, in a multidisciplinary fashion
(4). With the advent of laparoscopic and robotic radical prostatectomy, much of
the perioperative morbidity of the procedure is minimized while offering the
benefits of a curative procedure with improved convalescence and cosmesis. Despi
te the technical advantages of laparoscopy, patients still face similar postoper
ative functional and quality of life side effects as with standard open radical
prostatectomy. In a recent review of roboticassisted laparoscopic prostatectomy
(RALP), between 82% and 96% of patients required 0 to 1 pad daily for incontinen
ce and only between 38% and 66% were able to perform sexual intercourse six mont
hs after surgery (5). Three-dimensional conformal external beam radiotherapy (3D
CRT), intensity-modulated radiotherapy (IMRT), and prostate brachytherapy are ta
rgeted radiotherapeutic options for the treatment of localized prostate cancer.
Technological improvements in modern radiotherapy have improved the accurate tar
geting of prostate cancer. These advances have decreased the dose administered t
o the adjacent tissues such as the bladder and rectum, significantly reducing to
xicity, while concomitantly allowing a higher and potentially more efficacious d
ose administered to the target (prostate) tissue (6). However, there exists a si
gnificant number of patients who fail to get localized treatment from presumed u
nderstaging or radioresistant tumors (7). Finally, active surveillance in the co
rrect patient population avoids the morbidity of treatment and may not affect th
eir prostate cancerspecific mortality (8). However, many of these patients eventu
ally opt for treatment or progress to advanced disease.
71
72
Johannes et al.
Today is an exciting time for prostate cancer specialists, both in the refinemen
t of older technologies and development of new approaches for prostate cancer. C
linicians today have new minimally invasive treatment options including cryother
apy and high-intensity-focused ultrasound (HIFU) ablation. By implanting fiducia
l markers to improve radiation therapy, multimodality approaches to prostate can
cer are expanding into treatment. Although these modalities are not yet widely a
vailable, tomorrows urologist will likely see these technologies become mainstrea
m. CRYOTHERAPY History The first use of freezing for the treatment of cancer in

medical literature dates to 1850 (9). The first closed cryosurgical system was r
eported in 1938 when hollow bore instruments carried an ice mixture to treat met
astatic tumors (10). Following the development of liquid nitrogen closed system
technology, cryosurgery of the human prostate was described as a treatment for b
enign prostatic hyperplasia (BPH) and cancer (11,12). Innovations in cryotherapy
over the past several decades have marked milestones in the improvement of this
treatment modality (Table 1). First Generation Early cryosurgery of the prostat
e was used only on nonsurgical candidates. In the late 1960s and 1970s, a direct
transurethral or transperineal approach using liquid nitrogen was used (13,14).
In these early techniques, cystoscopic and digital rectal monitoring of the fre
eze sought to minimize injury or damage to periprostatic tissues. The morbidity
from these procedures was extreme by modern standards. Despite the high risks of
incontinence, severe urethral sloughing, and stricture, first generation cryosu
rgery compared favorably to the radiation and radical surgery morbidity of the e
ra (15). Second Generation Several technological advancements in the 1980s promp
ted reinvestigation in prostatic cryosurgery. Transrectal
Table 1 Evolution of Cryotherapy Generation 1st (1960s) 2nd (1990s) 3rd (current
) Cryogen Liquid nitrogen Liquid nitrogen Argon/helium Probe size 6.3 mm 3.4 mm
17 gauge
ultrasound proved to be an improved method of observing the extent of ice ball f
ormation (16). Constant flow urethral warming catheters marked a major breakthro
ugh in limiting cryotherapy morbidity. By keeping the urethral tissue from freez
ing, the rate of postsurgical urethral sloughing, stricture, and fistula improve
d greatly (17). The use of multiple cryoprobes improved the uniformity of freeze
and improved efficacy by simultaneously freezing the entire gland (18). These t
echnical advances were made in the setting of modern PSA testing. PSA screening
identified prostate cancer earlier and provided a valuable method of monitoring
treatment response. It proved that cryotherapy was efficacious in properly selec
ted patients with localized cancer (19). Third Generation Although cryotherapy i
s described in three generations, the modifications in technique and equipment w
ere added as they became available, ultimately evolving into what is commonly de
scribed as the third generation of cryosurgery. A major limitation of liquid nit
rogen is the requirement of relatively large bore needles. Utilizing the Joule-T
hompson effect of gas compression, a new cryogen using argon and helium gasses m
ade cryotherapy through small-bore needles possible. These thin needles now allo
w a direct percutaneous approach and multiple needle placements providing a more
exact freeze. Multiple small cryoprobes are inserted percutaneously in the peri
neum through holes in a brachytherapy-like template. Several coordinated systems
optimize the treatment while seeking to minimize injury to nontarget tissues. T
he edge of the advancing ice ball is monitored not only by transrectal ultrasoun
d but also by thermocouplers placed at the target margins. Urethral warmers prev
ent excessive urethral sloughing and its side effects. Finally, optimal treatmen
t plans are calculated using computer modeling. Together with better patient sel
ection, these advances culminating in todays cryosurgical technique have minimize
d the morbidity and optimized the efficacy of prostate cryosurgery. Tissue Destr
uction Physiology Refinements in cryoablative surgery have improved the ability
to efficiently kill cancer cells. Optimizing the
Ultrasound guidance No Yes Yes
Urethral warmer No Yes Yes
Temperature monitoring Manual Manual Thermocouplers
Targeted Therapies for Localized Prostate Cancer
73
freeze temperature, duration of freeze, speed of cooling, and the number of cycl

es maximizes tumor death. The lethality of extreme cold to prostate tissue is du

e to both direct cell effects and secondary vascular causes. A rapid and prolong
ed freeze with a slow thaw maximizes lethality by a combination of intracellular
ice crystal formation and disruption of normal osmotic gradients (20). Furtherm
ore, a double freeze-thaw cycle has been shown to have lower posttreatment posit
ive biopsy rates and fewer PSA recurrences (21). In addition to intracellular ef
fects, the freeze-thaw cycle destroys prostatic microvasculature that contribute
s to a delayed necrosis (22). Patient Selection Careful patient selection is cru
cial for all treatment modalities for prostate cancer and is particularly import
ant for cryotherapy. As the indications for cryotherapy of prostate cancer conti
nue to evolve, its applications are broadening. Most commonly, it is used as a p
rimary treatment in patients who are nonsurgical candidates and are eligible for
radiation therapy and decide against active surveillance or watchful waiting. A
lthough use as a salvage therapy is controversial, secondary cryotherapy may hav
e a promising future as a surgical option for local recurrence after initial rad
iotherapy treatment. Clinical risk stratification in patients with presumed orga
n-confined prostate cancer is a primary determinant of treatment options. With r
outine PSA screening, the majority of patients with localized cancer present wit
h clinical stage T1c disease (23). Risk stratification is estimated by pretreatm
ent PSA, Gleason score on TRUS biopsy, and clinical stage on digital rectal exam
. Several nomograms are used routinely by urologists and radiation oncologists t
o estimate pathological stage and recurrence rates on the basis of these variabl
es (2330). Primary cryotherapy for localized prostate cancer is an option, in gen
eral, for patients with clinical T1c to T3 disease. However, patients with periu
rethral disease are poor candidates as urethral warmers prevent an adequate kill
temperature from reaching this tissue zone. Furthermore, high impotency rates a
ssociated with cryotherapy makes patients interested in preserving potency poor
candidates. Prostate size is an important consideration in offering cryotherapy.
Patients with large pretreatment volumes (larger than 50 cm3) or those with a s
erum PSA level >10 ng/mL, may benefit from neoadjuvant hormone blockade (21,31).
After hormonally induced size reduction, the entirety of the gland is more reli
ably treated. Additionally, shrinking the prostate can help separate it from the
rectum, preventing rectal wall damage (32).
Despite these limitations, cryotherapy offers advantages over primary radiation
therapy or surgery. First, it is administered in a single day as compared to the
several week course of treatment for radiation therapy. Due to the minimal bloo
d loss, shorter operative time, and quick recovery, patients with a prohibitive
operative risk can typically undergo cryotherapy safely, even under spinal anest
hesia. In patients with high-risk disease, a pretreatment laparoscopic pelvic ly
mph node dissection can help determine the status of lymphatic metastasis (33).
Patients with evidence of extraprostatic involvement should not be offered cryot
herapy, but counseled on other treatment options. Finally, cryotherapy is safe t
o repeat, improving local control of locally recurrent disease. Follow-up After
Cryotherapy Patient follow-up after cryotherapy is based on PSA surveillance. In
itially, PSA is checked every three months for the first year and every six mont
hs thereafter for the first few years. Due to cell necrosis, there is an initial
bump in serum PSA that falls over time to a nadir (34). Unlike the postprostate
ctomy setting, in which the PSA should nadir to undetectable, and similar to rad
iation therapy, there is no consensus postcryotherapy nadir PSA indicative of bi
ochemical failure. Biochemical failure has been variably defined in a range from
0.3 up to 1.0 ng/mL (35,36). Others advocate the use of the accepted American S
ociety for Therapeutic Radiology and Oncology (ASTRO) criteria of three consecut
ive PSA rises following nadir (37). There have been several studies describing f
ive-year follow-up of patients treated with cryotherapy (Table 2). For low-risk
patients, the biochemical recurrencefree range from 60% to 80% (36,38,39). The la
rgest single institution experience reported cryotherapy outcomes in 590 consecu
tive patients with seven-year follow-up (36). Although the first 350 patients we
re treated with a second generation machine, the results are consistent with oth
er smaller more recent series (Table 2). Overall, 15.9%, 30.3%, and 53.7% were l

ow-, intermediate-, and highrisk patients, respectively. All patients were follo
wed with both PSA measurements and routine biopsies at 6, 12, 24, and 60 months
postoperatively. Using the ASTRO criteria, the seven-year actuarial biochemical
diseasefree survival (DFS) was approximately 90% for all risk groups. Overall, 13
% of patients had a positive posttreatment positive biopsy although the authors
did not stratify these patients according to preoperative risk. Together, these
results suggest there were some recurrences or treatment failures identified by
biopsies that were not identified by PSA recurrence. Although definitions of rec
urrence vary,
74 Table 2 Outcomes in Modern Prostate Cryotherapy Series Series Han et al. (45)
Donnelly et al. (39) Year 2003 2002 Number of patients 122 76 Generation 3rd 2n
d Length of follow-up 12 mo 5 yr PSA recurrence criteria (ng/mL) !0.4 >1.0 >0.3
>0.4 ASTRO
Johannes et al.
PSA recurrence-free rate % (risk) 76 73 75 89 76 60 77 48 84 92 92 89 87 79 71 6
1 68 61 76 71 61 60 61 36 (low) (all) (low) (intermediate) (high) (low) (interme
diate) (high) (low) (intermediate) (high) (low) (intermediate) (high) (low) (hig
h) (high) (low) (intermediate) (high) (low) (intermediate) (high)
Ellis (44) Bahn et al. (36)
2002 2002
75 590
3rd 2nd/3rd
3 mo 7 yr
>1.0 >0.3 Long et al. (38)a >1.0 >0.5
2001
975
2nd/3rd
5 yr
a
Pooled analysis of five series. Abbreviations: ASTRO, American Society for Thera
peutic Radiology and Oncology; PSA, prostate-specific antigen. Source: From Ref.
81.
patients generally undergo biopsy once recurrence is suspected. A smaller series
reporting results of 65 men with highrisk prostate cancer defined as a preopera
tive PSA >10 ng/mL or Gleason score !8 utilizing the ASTRO criteria for PSA recu
rrence shows that cryotherapy is a viable primary option for high-risk cancer (4
0). Eighty three percent of their patients were free of biochemical recurrence a
t a median of 35-month follow-up. Moreover, only one of the eight patients who u
nderwent biopsy had histological evidence of cancer. This highlights the difficu
lty in using both the ASTRO criteria to screen for recurrence and reliability of
the small sample prostate biopsy to confirm recurrence. Although these numbers
are promising, ten-year follow-up data with modern cryotherapy is awaited to det
ermine the long-term recurrence-free rates. Although cryotherapy is often a seco
ndary option for many patients who cannot be offered radical surgery and choose

against radiotherapy, the cryotherapy cancer control rates are comparable to rep
orted rates for radical prostatectomy and radiotherapy. A representative study o
f 743 patients receiving dose escalation conformational
radiation therapy were followed prospectively for PSA recurrence (41). These pat
ients were stratified into favorable risk (stage T1 or T2, Gleason score 6 or le
ss, PSA 10 or less), intermediate risk [one adverse feature or unfavorable (two
adverse features)]. The five-year PSA-free recurrences were 85%, 65%, and 35%, r
espectively. For patients treated with brachytherapy, similar results are seen.
One study of brachytherapy with a long mean follow-up period of 82 months showed
PSA-free recurrence rates according to ASTRO criteria in low-, intermediate-, a
nd high-risk patients of 91%, 80%, and 66%, respectively (42). Unfortunately, a
randomized trial comparing cryotherapy and radiotherapy options that could compa
re the oncological efficacy of these modalities has not been performed, and most
clinicians shy away from cryotherapy for higher-risk patients. Cryotherapy as a
n Option for Recurrent Disease Cryotherapy has an emerging role as salvage thera
py for local recurrence or progression following primary radiotherapy modalities
, based on PSA or biopsy data.
Targeted Therapies for Localized Prostate Cancer
75
Radiation therapy has long been a salvage therapy for patients with biochemical
failure and presumed local recurrence after prostatectomy. Similarly, cryotherap
y is a potential salvage therapy for local recurrence after radiation failure. O
nce residual cancer is confirmed by biopsy and evidence of systemic disease is e
xcluded, curative options for these patients are limited. Salvage cryotherapy of
fers an attractive alternative to salvage prostatectomy, which is technically di
fficult and carries significant risk and morbidity than primary radical prostate
ctomy. Salvage cryotherapy, in properly selected patients, may be an appropriate
choice for radiotherapy failures. In the largest cohort of patients failing pri
mary radiotherapy, salvage cryotherapy showed a five-year 79% diseasespecific su
rvival (DSS) and 40% DFS rate (43). Examining the relatively modest five-year DF
S, it is clear that patient selection is paramount, since the majority of patien
ts will fail despite additional local therapy, indicative of likely micrometasta
tic disease at the time of salvage treatment. Several factors predictive of a du
rable response to salvage cryotherapy were identified, including hormone-sensiti
ve prostate cancer, PSA <10 ng/mL, Gleason score 8 or less, and preradiation tre
atment clinical stage of T1 to T2. However, further studies are needed to compar
e salvage cryotherapy with other salvage treatment modalities. Complications of
Cryotherapy and Effect on Quality of Life Potential complications and quality of
life side effects weigh heavily in the decision process for treatment of locali
zed prostate cancer. Cryotherapy in the past was limited by its high complicatio
n rates. However, current third generation technical improvement has limited muc
h of the morbidity associated with treatment. Complication rates are summarized
in Table 3. Despite the improvements of third generation cryotherapy, erectile d
ysfunction remains the most common complication of cryotherapy. Several recent s
eries measured the rate of erectile dysfunction to be between 82% and 95% (36,44
,45). The high rate of postoperative impotence
Table 3 Complication Rates in Modern Prostate Cryotherapy Series Series Han et a
l. (45) Donnelly et al. (39) Ellis (44) Long et al. (38) Bahn et al. (36) Year 2
003 2002 2002 2001 2002 Number of patients 122 76 75 975 590
is attributed to extension of the ice ball to the neurovascular bundles. Althoug
h lower rates of impotence have been reported to be as low as 53% with the help
of erectile aids (46), patients wishing to preserve erectile function should con
sider other therapy. Urinary side effects are not eliminated by the current thir
d generation machinery. Cryotherapy can lead to stress or urge incontinence by a
number of putative mechanisms, including damage to the striated sphincter, uret

hral sloughing, nerve damage, and subsequent detrusor instability. Variable defi
nitions of incontinence make direct comparisons among published series difficult
. Recent series report incontinence rates between 4.3% and 7.5% (36,38). Althoug
h these relatively low rates reflect the advantages of third generation cryother
apy, patients requiring a postoperative transurethral resection of the prostate
(TURP) appear to have a significantly higher stress incontinence rate of almost
50% compared to 4% of those not requiring TURP (21). Several complications uniqu
e to cryotherapy are also seen less frequently today. Urethral sloughing from da
mage or urethral mucosa can cause intermittent obstruction, stricture, or calcif
ication necessitating intermittent catheterization, dilation, or transurethral r
esection. Representative modern series show a low rate of sloughing between 5.8%
and 6.7% (44,45). Although these low rates reflect refinement in technique, the
re should be a low index of suspicion to evaluate patients for these complicatio
ns both in perioperative and long-term follow-up period. Complaints of pelvic pa
in or perineal sensation alterations are common after cryotherapy. In a large mu
lticenter experience, pelvic pain was a relatively infrequent complication occur
ring in 6% of patients treated with cryotherapy (45). This same study reports pe
nile numbness occurring in 2% of patients. All of these patients experienced res
olution of their symptoms with conservative management. Fistula formation betwee
n the rectum and prostate, bladder, or urethra can be a catastrophic complicatio
n after cryotherapy. In older generation cryotherapy machines, this dreaded comp
lication was unfortunately much more common. Fortunately, it is a rare complicat
ion
Erectile dysfunction (%) 87 47 82.40 93 94.90
Fistula rate 0 0 0 0.50% <0.1
Incontinence (%) 4.30 1.3 5.50 7.50 4.30
Urethral sloughing and/or TURP (%) 5.80 3.9 6.70 13 5.50
Abbreviation: TURP, transurethral resection of the prostate.
76
Johannes et al.
today. Han et al. (45) reported no rectourethral fistulas in their experience of
106 patients, although a larger experience of 975 patients showed an overall in
cidence of 0.5% at a five-year follow-up (38). Although conservative treatment w
ith catheter drainage can be successful, often a fecal or urinary diversion is r
equired, with subsequent complex reconstructive procedures. Fistulas can be espe
cially problematic in previously irradiated fields when cryotherapy is used as l
ocal salvage therapy. Using the Functional Assessment of Cancer TreatmentProstat
e (FACT-P) and Sexuality Follow-up Questionnaire (SFQ), standardized metrics of
patients quality of life after treatment for prostate cancer have been measured.
In a single cohort, cryotherapy patients reported no significant changes in thei
r quality of life between one and three years postoperatively with the exception
of sexual function (46,47). Moreover, the FACT-P scores in cryotherapy patients
are very similar at one year after treatment to patients undergoing radical pro
statectomy, external beam radiation, or brachytherapy (48,49).
with localized prostate cancer were treated (52). In this study, 50% of the pati
ents had negative posttreatment biopsies and no major complications were observe
d. Since then broader trials performed on patients with cT1 to cT2 disease as bo
th primary or salvage therapy have confirmed efficacy and defined the future rol
e of HIFU treatment of prostate cancer.
Principles and Physiology of HIFU HIFU is in principle similar to diagnostic ult

rasound. The relatively low intensity and diffuse propagation of ultrasound wave
s in diagnostic ultrasound do not cause damage to target tissues. Conversely, HI
FU uses a highintensity ultrasound wave that delivers energy many orders of magn
itude higher than diagnostic ultrasound. This energy when focused to a small tar
get area causes necrosis of the target tissue. In prostatic HIFU, tissue destruc
tion results from two main mechanisms, thermal destruction and cavitation (53).
Coagulative necrosis has been observed in target tissues at a temperature of 568
C for one second (54). HIFU heats tissue well above that level. Due to the rapid
extreme heating, tissue outside the targeted site is spared destruction and has
a normal histological appearance (55). Cavitation occurs because of the creatio
n of microbubbles by ultrasound energy absorbed into subcellular organelles and
fluids. These bubbles cause mechanical stress and dispersion of energy in the mi
croenvironment, further contributing to direct thermal destruction of tissue. Th
ere are currently two main devices in use for prostatic HIFU, the Sonablate-500
(56) and Ablatherm (57). Although there are design differences, the principles e
mployed in both machines are the same. A rectal probe inserted into the rectum c
ontains the HIFU transducer, a normal diagnostic ultrasound transducer, and a co
oling mechanism to protect the rectal wall. Using a combination of computer mode
ling and real-time imaging, a series of ablation parameters determine the course
of treatment. In general, several overlapping ellipses of HIFU energy sequentia
lly ablate the entire prostate. The procedure is performed under spinal or gener
al anesthesia. Newer protocols include a routine TURP prior to therapy to reduce
gland volume and have significantly reduced postoperative urinary retention, wh
ich has been a common complication (58).
HIGH-INTENSITY-FOCUSED ULTRASOUND History Together with the newest generation of
cryotherapy, HIFU offers another minimally invasive option to prostate cancer p
atients. It is an older technology that has shown promise in a limited but expan
ding international experience. The first clinical applications of HIFU date back
to the middle of the twentieth century as it was first used to selectively abla
te brain lesions (50). Earlier techniques, however, were severely limited as the
re was no reliable way to image in three dimensions and monitor the ablation. Ad
vances in the past twenty years in computed tomography (CT) and transrectal ultr
asound have allowed a minimally invasive application of this technology to ablat
e prostatic tissue. The eagerness of both patients and practitioners to have min
imally invasive nonsurgical treatment of localized prostate cancer is advancing
HIFU into the arena of mainstream prostate cancer therapeutics. The first use of
HIFU specifically for prostate cancer was reported in 1995 (51). In this study,
29 patients with cT2 prostate cancer underwent treatment to study the in vivo e
ffect of varying focal length and intensity. All of these patients underwent imm
ediate radical prostatectomy under the same anesthesia. Pathological examination
confirmed coagulative necrosis in every specimen, although the extent of tissue
ablation was dependent of focal length and higher intensities. Shortly thereaft
er, HIFU was investigated in a feasibility study where a small cohort of inopera
ble patients
Patient Selection In general, HIFU has the same indications and patient eligibil
ity as cryotherapy. The current published trials
Targeted Therapies for Localized Prostate Cancer
77
were performed in patients with cT1 or cT2 Nx-0 M0 disease. It should only be co
nsidered a primary option for patients who are not suitable candidates for radic
al surgery. These patients generally cannot receive or refuse radiotherapy. Ther
e are specific contraindications to HIFU therapy. Prostatic size is a crucial fa
ctor to the success of the procedure. In general, gland volume should be less th
an 40 g. To downsize larger glands, urologists either perform a TURP or shrink t
he gland with hormone deprivation. Further considerations, such as rectal pathol

ogy and pelvic anatomy may be contraindications. Prostatic calcifications, which

are commonly seen on diagnostic prostate ultrasound, will cause shadowing and i
nterfere with transmission of HIFU waves, which impairs adequate treatment of pr
ostate tissue downstream of the calcification. Calcifications must be removed by
TURP prior to therapy, or the patient must be counseled against HIFU treatment.
HIFU Outcomes With the first American trials investigating HIFU only beginning
in 2006, the European experience comprises the published outcome data for HIFU t
reatment of prostate cancer. Table 4 shows a summary of selected trials. All tri
als included patients with cT1 to cT2 disease with no evidence of metastatic dis
ease. As in radiotherapy and cryotherapy, PSA nadir is an important marker of ou
tcome. HIFU theoretically ablates the entire prostate and should leave a low PSA
nadir. This is reflected in the mean nadir PSA of less than 0.5 ng/mL in most s
eries (Table 4). Since HIFU is a new technology, long-term follow-up data is not
yet available. Acknowledging limitations in extrapolating short-term data, PSA
recurrence and biopsy results offer insight into the efficacy of HIFU for prosta
te cancer treatment. Although there are no broadly accepted criteria for determi
ning biochemical recurrence after HIFU, most
Table 4 Outcomes in Current Prostate HIFU Series Number of patients 227 30 146 2
71 402
studies use the ASTRO criteria. The study with the longest follow-up to date is
of 227 patients treated with HIFU (59). Based on both ASTRO PSA criteria and pos
toperative biopsy, the authors report a five-year disease-free rate of 66%. This
is comparable to the other selected studies (Table 4). Interestingly, clinical
stage, Gleason score, or neoadjuvant hormone therapy were not significant predic
tors of DFS. However, improved DFS was noted for patients with lower pretreatmen
t PSA values, with DFS for preoperative PSA <4.0, 4.1 to 10, and >10 of 90%, 57%
, and 61% at five years, respectively (59). The authors postulated that these ob
servations were indicative that HIFU effectively destroyed all treated tissues,
with Gleason score or more aggressive tumors being equally susceptible to HIFU t
reatment. Additionally, the lack of effect of pretreatment with hormonal therapy
may be indicative of lack of synergy between hormonal ablation and HIFU, unlike
that seen with hormonal therapy and radiotherapy. In 2003, the results from the
European Multicentric Study of HIFU for prostate cancer were reported (60). Thi
s multiinstitutional nonrandomized clinical trial is one of the largest HIFU ser
ies published. The results reported were preliminary, with a median follow-up of
slightly less than one year. Additionally, the treatment protocols were heterog
eneous between centers, and retreatments were allowed, with a mean of 1.5 HIFU t
reatments per patient. This study showed an overall posttreatment negative biops
y rate of 87%. In low-, medium-, and high-risk patients, the negative biopsy rat
e was 92%, 86%, and 82%, respectively. In patients available for follow-up for a
period greater than six months, the mean nadir PSA was 1.8 ng/mL. This relative
ly high mean is skewed higher due to inclusion of nonresponders. To further emph
asize the importance of patient selection, patients with a gland size less than
40 cc had a median nadir PSA of 0.4 (60). Because of the short follow-up and het
erogeneous treatment plans used in this study, conclusions about efficacy
Series Poissonnier et al. (59) Vallancien et al. (58) Blana et al. (82) Chaussy
and Thuroff (62) Thuroff et al. (60)
Year 2007 2004 2004 2003 2003
Mean or median PSA follow-up (mo) 27 20 22.5 14.8 13.1
Median nadir PSA (ng/mL) 0.1 0.9 0.07 0 0.6
PSA recurrence criteria (ng/mL) Routine biopsy and ASTRO Routine biopsy and PSA
< 0.9 PSA < 1.0 ASTRO Routine biopsy
Cancer-free survival (%) (risk) 66.00 73.30 84.00 82.10 92 (low) 86 (medium) 82

(high) 75.0
Uchida et al. (83)
2003
63
23
1
ASTRO
Abbreviations: ASTRO, American Society for Therapeutic Radiology and Oncology; P
SA, prostate-specific antigen.
78
Johannes et al.
must be confirmed with future prospective studies with longer follow-up. A recen
t small retrospective study examined the nadir PSA after HIFU treatment (61). On
e hundred three patients with a median follow-up of 4.9 years after HIFU treatme
nt were stratified according to the PSA nadir achieved for each patient. Using P
SA nadir levels <0.2 ng/mL, 0.2 to 1.0 ng/mL, and >1 ng/mL, the actuarial five-y
ear DFS rates were 95%, 55%, and 0%, respectively. Therefore, in addition to the
ASTRO definition, absolute PSA nadir achieved after HIFU may be an accurate ass
essment of treatment efficacy and allows quicker implementation of salvage thera
py or HIFU retreatment to improve long-term oncological outcomes. It is importan
t to emphasize that very little long-term data is available for evaluating HIFU
for prostate cancer. This should be emphasized when counseling patients on treat
ment options for prostate cancer. Starting in 2006, a multicenter American equiv
alency trial was initiated to compare the outcomes of HIFU and cryotherapy. Sele
ct centers offering either HIFU or cryotherapy will enroll patients with localiz
ed disease who are not surgical candidates. This will be a landmark study direct
ly comparing the outcomes between these two modalities and largely determine the
future of HIFU beyond an experimental treatment in the United States.
HIFU Complications and Effects on Quality of Life Complication rates are well do
cumented in all HIFU trials to date. Table 5 summarizes the adverse events seen
in several HIFU trials. Urinary retention is a common complication and will be p
resent in almost all patients immediately following treatment. Urinary retention
can be caused by a number of treatment-specific mechanisms, including tissue sl
oughing and prostate edema. All patients require postoperative catheter drainage
with a Foley catheter or suprapubic tube. The duration of catheter drainage is
not easy to predict, because of the multiple
Table 5 Complication Rates in Current Prostate HIFU Series Series Poissonnier et
al. (59) Vallancien et al. (58) Blana et al. (82) Chaussy and Thuroff (62) Thur
off et al. (60) Uchida et al. (83)
a b
machine prototypes and treatment plans used in the various published studies. Co
ncomitant TURP with HIFU has made a significant improvement in urinary retention
rates. One study comparing two cohorts of HIFU and TURP with HIFU alone shows a
marked difference in the duration of urinary catheterization, with a median of
40 and 7 days, respectively (62). Patients receiving a TURP had a lower rate of
incontinence, 15.6% versus 6.9%, respectively, and postoperative urinary tract i
nfection, 47.9% and 11.4%, respectively. For these reasons, a TURP is considered
the current standard of care in Europe for HIFU unless contraindicated. One con

cern with the current American trial is that TURP is not allowed in the HIFU coh
ort, and the duration of urinary catheterization will be closely analyzed. Incon
tinence rates in HIFU are low in the later reported series and a result of impro
ved technique. Stress urinary incontinence is observed in 0.6% to 16% of patient
s (Table 5). Refinements in administration of HIFU, specifically leaving a 6-mm
safety margin at the prostate apex, have been credited with significantly reduci
ng stress incontinence (59). However, the long-term effect on oncological outcom
e from leaving this portion of viable prostatic tissue is worrisome. In the Euro
pean Multicentric Study, grade I or II incontinence was seen in 10.6% and 2.5%,
respectively and only six cases of grade 3 incontinence requiring invasive treat
ment (60). Impotency rates in HIFU are similar to the high rates observed in oth
er targeted therapies. Different definitions of potency show varying impotency r
ates in HIFU (Table 5). A nervesparing approach has been proposed that limits th
e lateral extent of the ablation (63). By sparing the contralateral neurovascula
r bundle in unilateral disease, these authors preserved potency in almost two-th
irds of patients but had a 15% higher recurrence rate. Patients should be carefu
lly counseled as the long-term effect on survival of this recurrence risk is unk
nown. The risk of bladder outlet obstruction from stricture has greatly improved
in the era of TURP and HIFU. Previously, in HIFU alone, stricture rates were as
high 22% (64),
Year 2007 2004 2004 2003 2003 2006
Number of patients 227 30 146 271 402 63
Impotence 39.0% 21.4% 49.8% 35.9% 8.7%b 20.0%
Incontinence 13.0% 3.0% 7.6% 15.4/6.9%a 14.6% 0.6%
Urinary retention Not reported 6.0% Not reported Not reported 8.6% 0.6%
Bladder outlet obstruction 12.0% 0.0% 19.7% Not reported 3.6% 22.0%
Fistula (%) 0.0 0.0 0.5 0.0 1.2 1.0
HIFU alone/HIFUTURP. Pretreatment data potency not recorded. Abbreviations: HIFU,
high-intensity-focused ultrasound; TURP, transurethral resection of the prostat
e.
Targeted Therapies for Localized Prostate Cancer
79
but the combination of TURP with HIFU has significantly reduced this risk (62).
Finally, the most serious complication of rectourethral fistula is rare. The uni
versal adoption of rectal cooling devices, better patient selection, and improve
d technique has driven this risk to a minimum (Table 5).
FIDUCIAL MARKERSTHE UROLOGISTS ROLE IN EXTERNAL BEAM RADIATION THERAPY External be
am radiation has advanced significantly in the past decade. The concept of dose
escalation and subsequent application in the form of conformational radiation th
erapy and IMRT has had profound effects on the treatment of localized prostate c
ancer. These technologies allow a higher dose of radiation to be focused on a sm
aller area. Application of this simple concept using modern technology has decre
ased the incidence of rectal and bladder complication related to radiation toxic
ity. External beam radiotherapy, unlike other targeted therapies such as cryothe
rapy and HIFU, is a repetitive, sequential treatment repeated multiple times ove
r a course of several weeks. The position of the prostate is not predictably con
sistent over the course of radiation treatment (65). CT and ultrasound have been
used prior to every treatment to better localize the prostate (66,67). The impo

rtance of prostate position is highlighted by the study by de Crevoisier et al.,

examining the effect of rectal distention noted on CT during 3DCRT (68). Patien
ts did not have daily imaging or repositioning during treatment, and those with
a distended rectum on planning CT had a significantly higher risk of biochemical
failure, indicating that rectal distention at the time of simulation altered th
e position of the prostate, and subsequent treatments without rectal distention
potentially missed the prostate. The use of a rectal balloon for each treatment to
immobilize the prostate and to allow accurate daily targeting has also been inv
estigated to improve the accuracy of IMRT. These manipulations appear to improve
both the accuracy of treatment and lowering the dose to the rectum, and thereby
lowering rectal toxicity (69). Within the past decade, attempts to allow easy i
dentification of the prostate on CT and plain film images during 3DCRT and IMRT
led to the development of implantable fiducial markers. This procedure can be pe
rformed in the outpatient setting under local anesthesia. Fiducial markers are r
adiopaque gold seeds that are directly implanted into the prostate by a techniqu
e similar to a routine transrectal ultrasound guided prostate biopsy. Typically,
three markers are used, at the right and left base laterally and the midline at
the apex of the prostate. Combining fiducial markers with an automated
realignment system has been shown to both be feasible and reduce treatment margi
ns (70). Concerns that fiducial markers adversely affect the prostate during tre
atment have been shown to be clinically insignificant. An initial experience wit
h 10 patients showed there was no significant deformation of the gland secondary
to implantation of fiducial markers (71). Moreover, the seeds have been observe
d to remain aligned and have no significant migration during a complete course o
f IMRT treatment in 56 patients (72). Overall, fiducial markers are considered s
afe with no major complications reported. In our institutional experience with o
utpatient placement of fiducial markers, no patients have experience of urinary
retention, gross hematuria, or infection. Major advances in radiation therapy in
cluding innovations in modalities are complimented by improving prostatic locali
zation. Implantation of fiducial markers will play an important role in future a
dvancements in radiation therapy for prostate cancer. TARGETED FOCAL THERAPY FOR
LOCALIZED PROSTATE CANCER Although refinements in targeted therapies for prosta
te cancer have improved much of the long-term morbidity, side effects especially
impotence are still common. Cryotherapy, brachytherapy, and HIFU were first dev
eloped to offer minimally invasive alternatives to radiation therapy in patients
who could not tolerate surgery. Since the development of nerve-sparing prostate
ctomy, many patients maintain erectile function after radical surgery. Similarly
, advocates of cryotherapy suggest that partial or focal ablation of the prostat
e sparing one neurovascular bundle may help prevent the almost definite impotenc
e seen in whole prostate cryotherapy. Similar to the progression from radical ma
stectomy in women with breast cancer to current standards of lumpectomy, patient
advocates have called for similar partial prostate treatments to alleviate compli
cations and quality of life side effects. Although published series are limited,
they have generated an important discussion on the possibility of focal therapy
for prostate cancer, the so-called male lumpectomy. There are some conceptual l
imitations to focal therapy for prostate cancer. The majority of prostate cancer
is multifocal. Prostate biopsy routinely misses areas of cancer, when compared
with the surgical pathology available from radical prostatectomy. Therefore, are
as that are untreated after focal therapy may contain an undiagnosed prostate ca
ncer. Moreover, it is thought that patients with prostate cancer in one part of
the gland are at a higher risk of developing a second lesion in the remaining no
rmal tissue. These satellite lesions, however, may be clinically insignificant, an
d it has been proposed that as much
80
Johannes et al.
as 80% of men could undergo focal ablation without leaving clinically significan

t cancer behind (73). However, there is no follow-up data in patients who have u
ndergone focal ablation to support this claim. The ability to accurately identif
y patients with true focal disease is limited using todays technology. Routinely,
patients undergo a standard 6 or 12 core TRUS biopsy. False negative rates for
TRUS biopsy are high but have been improved by an extended core and saturation b
iopsy techniques (74,75). In one study, Walz and colleagues found that saturatio
n biopsy found cancer in 41% of patients who previously had two negative prostat
e biopsies (75). New radiological technologies such as endorectal coil MRI (76),
dynamic contrast enhanced MRI (77), and MRI spectroscopy (78) show some promise
in identifying prostate cancer nodules. However no radiological technique has h
ad acceptable sensitivity in identifying focal cancer especially with nodules sm
aller than 1.0 cm. Together, these studies only emphasize the low sensitivity of
routine prostate biopsy and current radiological techniques in identifying foca
l prostate cancer. The data for focal cryotherapy is scant. In one initial study
, nine patients with biopsy-proven unilateral disease underwent focal cryotherap
y of the prostate, with preservation of the contralateral neurovascular bundle (
79). At a mean follow-up of 36 months, every patient had a stable PSA. Important
ly, seven of the nine patients were able to have intercourse. This potency rate
of 78% appears significantly improved when compared with whole-gland cryotherapy
. Overall morbidity of the procedure was limited as no patient experienced incon
tinence, obstruction, or fistula formation. The largest published series to date
by Bahn et al. followed 31 patients who were treated with focal cryotherapy (80
). At a mean follow-up of 70 months, 92.9% of the patients with postoperative PS
A data showed no recurrence according to ASTRO criteria, and only one patient (4
%) had evidence of cancer on follow-up biopsy. In terms of postprocedure potency
, 48.1% and an additional 40.7% of the patients achieved full potency without an
d with pharmacological assistance, respectively (80). Therefore, potency was pre
served in almost 89% of the patients using pharmacological assistance. These res
ults are very promising; however, additional studies are needed to confirm these
results. Focal HIFU has also been explored in small numbers of patients, again
with limited published data. Although the impotency rate after whole-gland HIFU
does not appear to be high as cryotherapy, it remains a significant side effect.
In an early application of HIFU in 1995, a subset of 10 patients with unilatera
l disease were focally treated with HIFU and subsequent radical prostatectomy (5
1). However, on pathological examination of the prostatectomy specimen, 70% of t
hese patients had viable tumor remaining.
As HIFU moves beyond experimental status in both Europe and the North America, a
pplication of modern HIFU techniques may show improvement on these initial resul
ts.
SUMMARY OF PERTINENT CONCLUSIONS
l
l
l
l
l
Image guided and targeted therapies will assume a larger role in the treatment o
f localized prostate cancer as technology evolves. Cryotherapy and HIFU offer cu
rative, minimally invasive treatment options, previously unavailable to patients
. With continued refinements in technique and machinery, these modalities appear
to offer acceptably low side effects and complications, with adequate cancer co
ntrol. As demand for minimally invasive therapy increases, patients may be willi
ng to potentially sacrifice some of degree of oncological efficacy in pursuing p
artial prostate treatments (male lumpectomy), analogous to women with breast can

cer. Caution must be observed, however, that higher grade, potentially lethal pr
ostate cancers are not missed or undertreated. Focal therapies allow an adequate
window for curative salvage for inadequately treated primary tumors.
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8
The Use of Fiducial Markers for IGRT in Prostate Cancer: The Mayo Clinic Approac
h
BRIAN J. DAVIS, JON KRUSE, CHRISTOPHER C. GOULET, AND MICHAEL G. HERMAN
Department of Radiation Oncology, Mayo Clinic, Rochester, Minnesota, U.S.A.
INTRODUCTION The practice of modern oncology relies in great part on imaging for
screening, diagnosis, staging, therapy, and subsequent assessment. In this rega
rd, the medical management of men with prostate cancer (CaP) does not differ. In
2007, it is estimated that nearly 219,000 men in the United States were diagnos
ed with CaP (1), and approximately two-thirds, or 140,000 will have CaP localize
d to the pelvis. Common treatment options for early-stage CaP as identified by t
he National Comprehensive Cancer Network (nccn.org) include radical prostatectom
y (RRP), external beam radiation therapy (EBRT), and radioactive seed implantati
on, also known as permanent prostate brachytherapy (PPB). Other options exist in
cluding hormonal therapy, high-dose rate (HDR) brachytherapy, cyrotherapy, and t
hermal ablation. The latter two common options, EBRT and PPB, rely on ionizing r
adiation to eradicate the cancer and have been a mainstay in the treatment of Ca
P for decades. At least 40,000 men per year undergo EBRT for therapy of CaP. The
efficacy of these RT options depends on accurate localization of the target and
the delivery of an appropriate dose to this volume. Although many methods are w
idely available to improve the quality of target localization and verification,
the Mayo Clinic Department of
Radiation Oncology has focused primarily on the use of an electronic portal imag
ing device (EPID) with implanted gold markers (fiducials) for prostate localizat
ion. The main goal of this chapter is to describe our approach and experience wi
th this method of target localization as applied to prostate image-guided radiat
ion therapy (IGRT). The rationale for integrating improved techniques of image g
uidance in RT for CaP derives from data supporting the benefits of radiation dos
e escalation for treatment of this disease. One means to assess the outcome of C
aP following therapy is to monitor the serum prostate-specific antigen (PSA). Wh
ile controversy exists regarding the optimal means by which to interpret the PSA
level following therapy, some studies have shown that conventional dose EBRT re
sults in a poorer PSA relapsefree survival as compared to other modalities (2). H
owever, the use of increased dose of EBRT results in comparable outcomes, but in
creased side effects may occur (3). Its tolerance also depends on accurate local
ization and the maximal exclusion of critical normal structures, also known as or
gans at risk, with conformal radiation field arrangements. However, the position
of the target, until recently, was typically inferred only from external referen
ce points, such as skin markings, used to align the patient on the treatment mac
hine. The accuracy of this alignment is monitored
85
86

Davis et al.
with periodic portal imaging of the skeletal structures of the lower pelvis. Alt
hough external reference points should be reproducibly related to skeletal struc
tures, the use of portal imaging during EBRT delivery often reveals displacement
from the intended position, which is commonly referred to as setup variation. In
CaP EBRT, several investigators demonstrated that this error in field alignment
may exceed 10 mm along each of the mutually perpendicular axes of the coordinate
system (49). However, the setup variation only partially accounts for uncertaint
ies in the position of the target relative to the treatment beam. The position o
f the prostate is not fixed relative to the skin marks or the skeletal anatomy o
f the pelvis (5,1013), because the state of bladder and rectal filling may result
in displacement of the prostate (1416). This organ motion adds an additional com
ponent of uncertainty, which may also exceed 10 mm, in localizing the target for
treatment delivery. Both organ motion and setup variation must be taken into ac
count in defining planning target volume (PTV) margins to adequately encompass t
he prostate with the intended dose (1719). Target positional variations over a co
urse of treatment have a number of potential sources, and their statistical desc
riptions as random and systematic processes have been developed to weigh their r
elative influences on targeting accuracy (15,16). As stated, improved biochemica
l (PSA) relapsefree survival (bRFS) has been demonstrated with increased dose of
EBRT (2024). In addition, reduced rates of positive posttreatment biopsies have b
een observed using radiation dose escalation (22). While these improvements in o
utcome are observed with increased radiation dose, increased morbidity may also
result when such doses are delivered. More recently, advances in radiation dose
delivery with intensity-modulated radiotherapy (IMRT) (25) and improvements in t
argeting with daily prostate positioning have led to apparent reduced toxicity.
Consequently, National Cancer Institute (NCI)-supported multiinstitutional prosp
ective trials using radiation dose escalation for CaP have yielded lower-than-ex
pected toxicity rates (2628). While the outcome for most CaPs treated with high-d
ose EBRT appears comparable to other common treatment modalities (2), it appears
feasible that further dose escalation with EBRT is possible without an undue in
crease in normal tissue toxicity. Many trials have investigated the effect of in
creasing dose on late rectal toxicity. Radiation Therapy Oncology Group (RTOG) 9
406 escalated the dose with threedimensional conformal EBRT using five different
dose levels: 68.4, 73.8, 79.2, 74, and 78 Gy. At every dose level analyzed thus
far, the grade 3 or greater gastrointestinal or genitourinary toxicities have b
een significantly reduced when compared to the historical controls of RTOG 7506
and RTOG 7706, which used conventional radiation rather than three-dimensional c
onformal radiation (2628). At the 79.2-Gy dose level, there were no acute grade 3
or greater toxicities (29,30). The late grade 3 toxicities for the rectum and b
ladder were 0.6% and 1.8%, respectively, with no grade 4 or 5 toxicities reporte
d. In the phase III randomized trial of 70 Gy versus 78 Gy from MD Anderson Canc
er Center, the late grade 3 rectal toxicity was 1% in the 70-Gy arm compared to
7% in the 78-Gy arm. However, the radiation was delivered to a conventional four
-field box [11 11 cm2 anteroposteriorly (AP) and 11 9 cm2 laterally] for the fir
st 46 Gy followed by a slight field reduction (9 9 cm2 in both the AP and latera
l dimensions) to 70 Gy. Only after 70 Gy was the six-field three-dimensional con
formal boost added to achieve the final dose of 78 Gy. Over the last several yea
rs, IMRT has been shown to further decrease the rectal toxicity over three-dimen
sional conformal radiation for localized CaP. A recent report of IMRT treatments
from Memorial Sloan Kettering shows only 0.5% grade 3 late rectal and bladder t
oxicity at doses up to 8640 cGy (26). The use of IMRT also showed a decrease in
the grade 2 late toxicities compared to three-dimensional conventional radiation
performed at the same institution (3). Given this level of rectal toxicity, fur
ther dose escalation is likely feasible, especially if localization techniques a
re refined to allow tighter margins. A 2005 study by de Creviosier et al. (31) e
xamined the rectal cross-sectional area on patients treatment plans, and found a
significant correlation between rectal distension and poor biochemical control.

The likely explanation is that for patients with a distended rectum at simulatio
n, the internal position of the prostate did not represent its average position
over the course of treatment. A systematic displacement of the target tissue was
introduced for these patients, leading to a poor outcome. This study emphasized
the value of image guidance in the treatment of CaP. THE MAYO CLINIC TECHNIQUE
FOR PROSTATE IGRT Between 2001 and 2002, a phase I trial was conducted in the De
partment of Radiation Oncology at the Mayo Clinic using an IGRT procedure to ide
ntify intraprostatic gold markers and correct daily variations in target positio
n during external beam radiotherapy for CaP (32). Other centers have also develo
ped this technique, which has become commonplace. An early feasibility study by
Balter et al. described the use of implanted markers to track prostate movement
over the course of routine radiotherapy in 1995. Investigators at the University
of California, San Francisco, recently described their five-year experience
Use of Fiducial Markers for IGRT in Prostate Cancer
87
using implanted markers for prostate radiotherapy. In the approach used at our i
nstitution, pretherapy electronic portal images (EPIs) are acquired with a small
portion of the therapeutic 6-megavolt (6-MV) dose from an orthogonal pair of tr
eatment fields. The position of the intraprostatic gold markers on the EPIs are
aligned with that on the treatment planning digitally reconstructed radiographs
derived from axial computed tomography (CT) images. If the initial three-dimensi
onal target displacement (3DI) exceeds 5 mm or rotations exceed 38, the beam is
realigned before the remainder of the dose is delivered. A custom spreadsheet is
used to calculate three-dimensional translations from the deviations measured i
n each of the two portal images. If the action thresholds are exceeded, the spre
adsheet supplies couch coordinates to correct the prostate position. At the end
of each fraction, the data in the spreadsheet are saved to a database, which can
show deviations and corrective actions taken for each patient over the course o
f treatment. This database is examined after the first five treatment fractions,
and any systematic errors are identified and corrected before imaging for the r
est of the treatment course. In the initial clinical study of 20 patients, field
-only EPIs were acquired for all fields and offline analysis was performed to de
termine the final three-dimensional target placement (3DF). Twenty patients comp
leted protocolspecified treatment, and it was determined that all markers were i
dentified on 99.6% of the pretherapy EPIs. Overall, 53% of treatment fractions w
ere realigned. The mean 3DI was 5.6 mm in all patients (range 3.79.3), and the me
an 3DF was 2.8 mm (range 1.64.0), which was statistically significant (p < 0.001)
. Rotational corrections were made on 15% of treatments. Mean treatment duration
was 1.4 minutes greater for protocol patients than for similar patients in whom
localization was not performed. This study demonstrated that frequent field mis
alignment occurs when external fiducial marks are used for patient alignment as
opposed to using implanted markers. Misalignments can be readily and rapidly ide
ntified and corrected with an
EPID-based online correction procedure that integrates commercially available eq
uipment and software. At the Mayo Clinic, gold fiducial markers are implanted vi
a the transperineal approach with 18-gauge needles using local anesthetic only.
Prior to the procedure, patients are requested to discontinue anticoagulants for
several days, although bleeding is very minimal and, on occasion, patients who
have been on continuous aspirin therapy undergo the procedure without having dis
continued its use. Patients are placed in the dorsal lithotomy position and a st
abilization device is used to maintain probe position. Those experienced with th
is marker placement will typically perform the needle insertion freehand without t
he assistance of a template. Prior to needle placement, a local anesthetic is in
troduced into the perineum and gradually introduced to the level of the prostate
before fiducial marker placement is undertaken. Two needles are typically used
to implant four markers, two markers through each needle. The markers are placed

in a staggered manner so that no overlap occurs on the AP or lateral images. Id

eally, the markers are positioned so that they are maximally displaced from one
another but are not too close (<5 mm) to the prostate periphery in order to limi
t the possibility of migration. From 2002 through mid2007, over 500 patients hav
e undergone this procedure of marker placement and daily prostate localization a
t our institution. In Figure 1, the image on the left is a digitally reconstruct
ed radiograph from the CT scan used to plan the radiation treatment. Four gold i
nterstitial fiducial markers are readily apparent in the middle image. The image
s on the right show the corresponding portal images and treatment field overlay
to those in the image on the far left. In blue, the field pattern produced by th
e multileaf collimation is evident with the small objects enclosed in pink corre
sponding to the fiducial markers. The object in green is the prostate as determi
ned by the initial CT scan of the prostate. The second image on the right shows
the field after it was shifted so that the prostate was
Figure 1 (A) Axial scout view (A-P) of pelvis with 4 implanted fiducial markers,
(B) Digitally-reconstructed radiograph (DRR) of simulation film, (C) correspond
ing electronic portal image showing fiducial markers.
88
Davis et al.
centered in the treatment beam as intended. The field is initially aligned via e
xternal tattoos on the patient. Note also that the prostate position as determin
ed by the implanted fiducial markers has shifted relative to the bony anatomy.
OPTIMIZING IGRT IMPLEMENTATION IN PROSTATE CANCER: IGRT QUALITY CONTROL Quality
assurance (QA) of EPID-based prostate localization starts with QA of the EPID it
self. Specific QA protocols will vary with EPID hardware and clinical utilizatio
n, but general prescriptions for EPID QA can be found in the literature (33), an
d are meant to generally ensure patient safety, image quality, and localization
accuracy. At the Mayo Clinic, the EPID panels are mounted on movable robotic arm
s and collision sensors on the panels are tested daily to ensure that the patien
t will not be harmed if struck by the device. The other patient safety concern w
ith EPID localization is excess radiation dose from imaging procedures. A pair o
f megavoltage portal images may deliver 6 to 8 cGy to the patient per treatment
fractiona dose that can be appreciable with a typically large number of fractions
for prostate treatments. At our institution, the portal images are acquired thr
ough shaped ports, which limit the exposure to the PTV plus a 1-cm margin. The p
atients treatment plan includes a pair of four-monitor unit imaging fields to be
delivered at each treatment session. The dose from the imaging fields is calcula
ted initially and is considered when the influences for the remaining five IMRT
fields are optimized. In this way, the dose from the localization procedure is r
eflected in the treatment plan. EPID image quality is assessed every day as part
of routine linac warm-up, and the imaging panels dark-field and flood-field corr
ections are calibrated every month by Physics staff. Localization accuracy is gu
aranteed through a number of procedures and consistency checks. The first elemen
t of the localization process is for the EPID system to report the position of t
he gold markers in the portal images, relative to their prescribed locations. So
me of the EPIDs at Mayo (Varian Portal Vision v. 7.3) use a gradient search algo
rithm to detect the radiation field edge of the portal image and compare this to
the prescribed field shape, as defined on the reference digitally reconstructed
radiograph (DRR). The location of isocenter is known on the DRR, and so compari
son of the detected and prescribed field shapes enables the EPID software to fin
d the isocenter on the portal image. The detection and alignment of the radiatio
n field edges is crucial for accurate localization of the patient, and it is ver
ified graphically by therapists on every portal image before the location of the
fiducial markers is recorded. On some newer EPID systems (Varian 4D Treatment C
onsole), the robotic support arm reports the location of isocenter

relative to the imaging panel, and this information is used in localizing the ma
rkers. On these machines, the accuracy of the arm calibration is tested daily at
multiple gantry angles by placing a physical graticule tray on the head of the
machine and comparing the image of the central BB to the central axis of the bea
m, as reported by the EPID system. The most critical and robust aspect of the IG
RT QA process is physician review of images acquired during treatment. EPID imag
es can be acquired with the therapeutic fields, showing the positions of the mar
kers during the actual treatment. In the case of IMRT treatments, the EPID panel
will integrate charge continuously through the field delivery and present a sin
gle image representing the entire field delivery. Fiducial markers are visible i
n these integrated images, and physicians review these images after each treatme
nt but before the following session to verify that the prostate is positioned pr
operly during the actual treatment. This review process serves as a comprehensiv
e check on all other components of the localization process, and can be used to
assess other processes such as intrafraction motion.
CLINICAL OUTCOME WITH INTRAPROSTATIC FIDUCIALS AND EPID One research effort has
focused on the estimation of limits of dose escalation using IMRT and precise pr
ostate targeting (35). As described previously, recent reports of dose escalatio
n for CaP indicate minimal toxicity using established dose constraints on normal
adjacent structures. The completed multiinstitutional protocol, RTOG 94-06, enr
olled 1084 patients and did not find the maximum tolerable dose (MTD). This resu
lt suggests that further dose escalation is possible before the MTD is found. In
support of this concept, Zelefsky et al. (3) continued dose escalation for trea
tment of CaP to 8640 cGy with IMRT. At our institution, initial investigations a
ddressed the limits of dose escalation using five-field intensitymodulated radio
therapy (5FIMRT) as a function of PTV margins. CT data was obtained from 18 pati
ents with localized CaP treated with 5FIMRT between September 2003 and February
2005. All patients gave permission for use of their medical records in accordanc
e with the Mayo Clinics institutional review board and Minnesota statutes. Patien
t characteristics are provided in Table 1. The beam angles employed include thos
e described by Burman et al. and used routinely in our clinical practice and els
ewhere: posterior, posterior right and left oblique (P75R, P75L), and anterior r
ight and left oblique (A45R, A45L). The PTV of all patients in the study receive
d 75.6 Gy delivered in 42 fractions. Each patient was treated with interstitial
fiducial markers in place and using daily electronic portal
Use of Fiducial Markers for IGRT in Prostate Cancer Table 1 Patient Characterist
ics and Treatment Details for Simulation Study Examining the Limits of Dose Esca
lation for Treatment of Prostate Cancer Patient characteristics Age (yr) Anterio
r/posterior separation (cm) Lateral separation (cm) Tumor characteristics T stag
e
89 Table 2 Dose Constraints [RTOG Protocol P-0126 (rtog. org)]. <15% Bladder Rec
tum PTV CTV <25% <35% <50%
Avg: 70 Avg: 23.5 Avg: 37.0 T1c T2a T2b 33 34 43 Avg: 6.1
Range: 5780 Range: 12.134.1 Range: 31.144.8 12/18 (67%) 5/18 (28%) 1/18 (6%) 14/18
(78%) 2/18 (11%) 2/18 (11%) Range: 2.413.0 10/18 (56%) 8/18 (44%) 18/18 (100%) 18
/18 (100%) 18/18 (100%) Range: 32103 Range: 62132
V80 V75 V70 V65 V75 V70 V65 V60 <2% less than the prescription dose <2% greater
than 107% of the prescription dose 0% less than the prescription dose
Gleason score
PSA Low risk Intermediate risk Treatment details Dose 75.6 Gy Interstitial Fiduc
ial Markers Five field IMRT Prostate volume on CT (cc) Rectal volume on CT (cc)

Avg: 56 Avg: 97
the PTV, was allowed to receive a calculated dose above the prescription dose. A
ll plans used 6-MV beams, in accordance with the current institutional treatment
policy. Equal priorities were assigned to the rectal V75, and the minimum and m
aximum PTV constraints for every plan. Additional constraints, at the same prior
ity level, were added in the minority of plans, and only when the constraint cou
ld not otherwise be met. Iterative optimizations, adding additional 180-cGy frac
tions, were performed until one or more of these constraints could no longer be
met. The highest dose that met all the constraints was defined as the maximum ac
hievable dose. The relationship between maximum achievable dose and PTV margin,
prostate volume, and PTV overlap with the rectum was examined. Results of Simula
tion Analysis The maximum achievable dose is limited by the rectal V75 dose cons
traint in 61% of cases. The other limiting constraints include the bladder V80 (
19%), PTV coverage (19%), lateral subcutaneous hot spots in the P75 beams (11%),
rectal V70 (4%), rectal V65 (2%), and bladder V75 (2%). Nine of the 54 plans ha
d two criteria that were primarily responsible for limiting the dose. PTV margin
s of 10, 5, and 3 mm yield a mean maximum achievable dose of 83.0 Gy (range 73.81
08.0 Gy), 113.1 Gy (range 90.0151.2 Gy), and 135.9 Gy (range 102.6189.0 Gy), respe
ctively. All comparisons of the maximum achievable dose between margin groups ar
e statistically significant with one-sided p values <0.001 (paired t test). Pros
tate volumes of 30 to 50 cc (n 8) compared with those of 50 to 70 cc (n 7) and 7
0 to 105 cc (n 3) show an inverse correlation with maximum achievable dose (Fig.
2B). Smaller prostates yield significantly higher maximum achievable dose for P
TV margins of 3 and 5 mm (p < 0.05). For plans with 10-mm margins, prostates 30
to 50 cc yield a significantly larger maximum achievable dose than those 70 to 1
05 cc (p 0.009). However, there is no significant difference between any of the
other comparisons of prostate size with maximum achievable dose for the 10-mm ma
rgin groups.
imaging (EPI). All targets were positioned prior to treatment so that the net th
ree-dimensional displacement of the target was less than 5 mm. The prostate to P
TV margin was routinely selected as 7.5 mm uniformly around the prostate except
posteriorly where it was limited to 5 mm. The prostate, rectum, and bladder were
segmented by the treating radiation oncologist in a manner consistent with the
specifications for contouring these structures on RTOG protocols. The clinical t
arget volume (CTV) was defined as the prostate. For each patient, uniform 10-, 5
-, and 3-mm threedimensional expansions of the prostate were used to generate th
e PTV. These definitions of CTV, PTV, and others are nomenclature used in radiat
ion treatment planning and are described in several reports by the International
Commission of Radiologic Units (ICRU). Identical 6-MV 5FIMRT beam arrangements
were used for every plan. The same beam orientations as described above were emp
loyed. Plans were optimized in the Eclipse Planning System (Varian Medical Syste
ms, Palo Alto, California, U.S., version 7.2) in a manner such that the dose con
straints as outlined in RTOG dose escalation protocol P0126 for the rectum, blad
der, PTV, and CTV and as listed in Table 2 were maintained (rtog.org). Hot spots
greater than 107% of the prescription were not allowed in the rectum or bladder
. In addition, no point outside the PTV, except those immediately adjacent to
90
Davis et al.
that by creating an individualized PTV using daily online positioning with inter
stitial fiducial markers and patientspecific respiratory and intrafraction prost
ate motion data, the average PTV margin was 3 mm in the RL and SI directions, an
d 4 mm in the AP direction (34). As such, the lower PTV treatment margin examine
d in this study is 3 mm, whereas those margins employed in RTOG P-0126 are 5 to
10 mm. Intrafraction Motion Many have studied the magnitude of intrafraction mot

ion during prostate radiotherapy with implanted fiducial markers and EPI. Goulet
et al. (35) at our institution studied the estimated increase in dose escalatio
n possible by reducing treatment margins. For example, reduction of the treatmen
t margin from 10 to 3 mm for an intermediatesized prostate gland would theoretic
ally allow an increase of maximum allowable dose from 90 to 128 Gy. Willoughby e
t al. (36) have analyzed the influence of tracking interfraction and intrafracti
on prostate motion with implanted fiducial markers and the margins required to a
ssure that 90% of the patient population receives a minimum dose to the CTV of a
t least 95% of the nominal dose. By this methodology, CTV to PTV margins, mPTV,
are given by: mPTV 2:5 S 0:7s where S is the sum of all of the routine setup erro
rs and s is the random error related to intrafraction motion. For skin-based set
up without and with inclusion of intrafraction motion, prostate treatments would
have required average margins of 8.0, 7.3, and 10.0 mm and 8.2, 10.2, and 12.5
mm, about the left-right, AP, and cranial-caudal directions, respectively. Posit
ioning by prostate markers at the start of the treatment fraction reduced these
values to 1.8, 5.8, and 7.1 mm, respectively. Interbeam adjustment further reduc
ed margins to an average of 1.4, 2.3, and 1.8 mm. Intrabeam adjustment yielded m
argins of 1.3, 1.5, and 1.5 mm, respectively. Consequently, the value of prostat
e localization and intrafraction tracking is substantial when considering the po
tential for dose escalation, reduced cancer recurrence rates, and reduced toxici
ty. In the study by Willoughby et al. (36), 11 patients data were analyzed. Using
the technique of implanted fiducial markers at our institution, we studied the
intrafraction motion of 39 patients receiving 1532 treatments of EBRT. In Figure
3A, the time requirement for the steps involved in delivering one fraction of r
adiation for a patient treated with implanted fiducial markers is given. From mi
nutes 9 through 13, intrafraction motion may occur without compensating for its
occurrence. The position of the
Figure 2 The estimated maximum prescription dose to the prostate using dose cons
traints from the protocol RTOG P-0126 as a function of treatment margin for thre
e different cohorts of prostate size.
Decreasing the prostate to PTV margin size significantly decreases the PTV overl
ap with the rectum (one-sided p < 0.001 between all margin comparisons). The MAD
shows an inverse correlation with the component of PTV volume overlapping the r
ectum. The linear correlation coefficient is 0.72. However, the comparison betwe
en these two variables shows a better logarithmic relationship. The correlation
coefficient between the logarithm of the volume of PTV overlapping the rectum an
d the maximum achievable dose is 0.87 (Fig. 2). Maximum achievable dose declines
from 150 to 100 Gy with 0 to 5 cc of overlap, and thereafter drops slowly to 75
Gy with 5 to 20 cc of overlap. By decreasing the PTV margin while maintaining i
dentical dose constraints, doses significantly greater than those currently pres
cribed for treatment of localized CaP appear feasible. In other words, this simu
lation study shows that the more accurate the treatment delivery and the narrowe
r the margin placed around the prostate, the higher the radiation dose that may
be delivered. The RTOG protocol P0126 specifies a CTV-to-PTV margin of 5 to 10 m
m. However, the use of increasingly accurate target localization methods may per
mit smaller margins, such as 3 mm. Recently, Schallenkamp et al. in a Mayo study
show that using interstitial fiducial markers and daily EPI allow margins of 2.
7, 2.9, and 2.8 mm in the superoinferior (SI), AP, and right-left (RL) axes. The
onedimensional margins were defined so that 95% of patients would receive a min
imum of 95% of the prescribed dose to the CTV, assuming that institutional syste
matic error is zero. On this protocol, patients were shifted only if the fiducia
l markers were greater than 5 mm (3D magnitude of displacement) from their expec
ted position. This method of daily tracking is associated with a reduction in se
tup error by more than 2 mm. Cheung et al. also show
Use of Fiducial Markers for IGRT in Prostate Cancer
91

Figure 3 (A) Time intervals for gold seed localization during EBRT. (B) Localiza
tion errors based on different methods of localization. (C) Number of fractions
versus displacements. (D) Worst 10% of cases, A-P versus absolute displacement.
Abbreviation: EBRT, external beam radiation therapy. Source: From Ref. 37.
prostate after treatment completion is determined without determining the histor
y of this movement. In Figure 3B, the required margin is calculated using the me
thod of Van Herk. The first s represents the systematic error and the second s repre
sents the random error. Based on using skin marks alone, it is estimated that th
e margin required should be from 5.9 to 7.89 mm. If localization by EPID is used
and adjustments of the patient position are performed with no threshold, then t
he margins in all directions need to be 2.7 to 2.8 mm. Likewise, accounting for
the effect of intrafraction motion by itself, required margins are 2.1 to
2.97 mm. In other words, the magnitude of the effect of intrafraction motion on
required margins is essentially similar to that afforded by setting up on the pr
ostate by itself. In the histogram of Figure 3C, the magnitude of the prostate i
ntrafraction motion for each direction and the three-dimensional displacement is
shown. From this chart, it is clear that there is a significant amount of intra
fraction movement. Figure 3D shows the top 10% three-dimensional intrafraction m
otion, along with the corresponding posteroanterior displacement. From this grap
h, it is evident that intrafraction motion is a significant
92
Davis et al. Table 3 IGRT Protocols at the Mayo Clinic Original protocol Imaging
fields Action threshold (3D translation) Action threshold (rotation) Treatment
fields CTV to PTV margin Anterior Right Lateral 5 mm 38 4 Field box 7.5 mm; 5 mm
posterior Present protocol Anterior 608 oblique 4 mm None 5 Field IMRT 7.5 mm;
5 mm posterior
issue, especially in regards to the rectum, the radiation dose limiting structur
e, which lies posterior to the prostate. The average of the top 10% intrafractio
n displacement is 6.5 mm with a standard deviation of 2.6 mm; the top 10% left-r
ight motion averages 3.3 mm with 1.9 mm standard deviation; the inferosuperior i
s 3.8 mm and 1.1 mm, respectively; and the posteroanterior average is 5.0 mm wit
h a standard deviation of 2.6 mm. It is essential to develop a method that bette
r characterizes and substantially reduces the adverse influence of intrafraction
motion on CaP radiotherapy. In this regard, current efforts to develop and test
a continuous tracking device using electromagnetic transponders are worthwhile.
Such a system for accurate target localization and continuous tracking has been
developed by Calypso Medical, Inc. of Seattle, which will be discussed in detai
l in chapter 12. Briefly, the system utilizes miniature, permanently implanted,
wireless electromagnetic transponders and an array to localize them in three-dim
ensional space. The technical design of the system includes the use of nonionizi
ng electromagnetic radiation with negligible tissue interaction. The system is d
esigned for continuous operation during EBRT and provides data at a rate up to 1
0 Hz. A single transponder has an accuracy less than 2 mm rms, whereas for most
configurations tested, the accuracy is submillimeter when using three or more tr
ansponders. The five major components of this system include wireless transponde
rs, a console, an array, a tracking station, and infrared cameras. The transpond
ers are wireless, selfcontained, and do not require an internal power supply. Th
e transponders are encapsulated in biocompatible glass and are designed for perm
anent implantation or surface placement. The array incorporates an electromagnet
ic source to temporarily excite transponders at 300 to 500 kHz and induce resona
nt response signals. Sensors in the array measure transponder signals used to de
termine the positions in three-dimensional space relative to the array. Transpon
der signals are time-multiplexed so that different resonant frequencies may be t
racked independently. The array is registered to the isocenter of the linear acc

elerator through infrared cameras and optical targets mounted on the array. The
user interface displays localization and tracking data inside and outside the ra
diation treatment room. Current Practice at Mayo Clinic The prostate IGRT protoc
ol has evolved over six years of implementation at Mayo Clinic. In the initial p
hase I study in 2001, the patients were treated with a three-dimensional conform
al four-field box technique. The four fields were planned, and then additional a
nterior and right lateral fields were added to the plan for imaging. Monitor uni
ts were
transferred from the anterior and lateral treatment fields to their respective E
PID fields. We found that the patients femurs could occasionally obscure the fidu
cial markers in a lateral image, so the imaging view has been replaced by a 608
anterior oblique. The two EPID fields are planned for four monitor units each, a
nd then five IMRT fields are planned to deliver the remainder of the prescribed
dose. In addition to translational motion, the prostate is often observed to rot
ate in the AP direction, about the left-right axis. This rotation was measured i
n the original protocol, and the rotation of the target was matched by rotating
the collimator angle of the lateral treatment fields. No straightforward correct
ion exists for oblique IMRT treatment fields, so rotational motions of the prost
ate are no longer considered. The action level for translational correction has
been reduced from 5 to 4 mm, although the CTV-to-PTV margins remain at 7.5 mm in
all directions, with the exception of a 5-mm posterior margin. Changes in the I
GRT protocol are summarized in Table 3. SUMMARY OF PERTINENT CONCLUSIONS
l
l
l
l
l
l
Dose escalation in CaP leads to improvement in biochemical control and a reducti
on in positive biopsies following CaP radiotherapy. In order to achieve increase
d dose escalation without a concomitant increase in toxicity, IGRT is essential.
Our approach is to use implanted fiducial markers and daily EPI to achieve rout
ine localization well within 5 mm of the intended positioning. Reduced margins m
ay further allow substantially increased dose escalation based on a theoretical
study presented in this chapter. Our measurements indicate that intrafraction pr
ostate motion is more than 6 mm during approximately 10% of fractions. Tracking
of the prostate during treatment may facilitate more accurate treatment achieved
by the use of implanted electromagnetic beacons.
Use of Fiducial Markers for IGRT in Prostate Cancer
93 14. Roeske JC, Forman JD, Mesina CF, et al. Evaluation of changes in the size
and location of the prostate, seminal vesicles, bladder, and rectum during a co
urse of external beam radiation therapy. Int J Radiat Oncol Biol Phys 1995; 33:1
3211329. 15. van Herk M, Bruce A, Kroes AP, et al. Quantification of organ motion
during conformal radiotherapy of the prostate by three dimensional image regist
ration. Int J Radiat Oncol Biol Phys 1995; 33:13111320. 16. Beard CJ, Kijewski P,
Bussiere M, et al. Analysis of prostate and seminal vesicle motion: Implication
s or treatment planning. Int J Radiat Oncol Biol Phys 1996; 34: 451458. 17. Inter
national Commission on Radiation Units and Measurements. ICRU Report 62: Prescri
bing, Recording and Reporting Photon Beam Therapy (Supplement to ICRU Report 50)
. Bethesda, MD, 1999. 18. Bijhold J, Lebesque JV, Hart AA, et al. Maximizing set
up accuracy using portal images as applied to a conformal boost technique for pr

ostatic cancer. Radiother Oncol 1992; 24:261271. 19. Bel A, Vos PH, Rodrigus PT,
et al. High-precision prostate cancer irradiation by clinical application of an
offline patient setup verification procedure, using portal imaging. Int J Radiat
Oncol Biol Phys 1996; 35:321332. 20. Pollack A, Zagars GK, Starkschall G, et al.
Prostate cancer radiation dose response: results of the M.D. Anderson phase III
randomized trial. Int J Radiat Oncol Biol Phys 2002; 53:10971105. 21. Zietman AL
, Desilvio M, Slater JD, et al. A randomized trial comparing conventional dose (
70.2 GyE) and high-dose (79.2 GyE) conformal radiation in early stage adenocarci
noma of the prostate: results of an interim analysis of PROG 95-09. Int J Radiat
Oncol Biol Phys 2004; 60:S131S132. 22. Zelefsky MJ, Chan H, Fuks Z, et al. Corre
lation of longterm biochemical outcome with post-treatment biopsy results for pa
tients treated with 3-dimensional conformal radiotherapy for prostate cancer. In
t J Radiat Oncol Biol Phys 2004; 60:S170. 23. Michalski JM, Purdy JA, Winter K,
et al. Preliminary report of toxicity following 3D radiation therapy for prostat
e cancer on 3DOG/RTOG 9406. Int J Radiat Oncol Biol Phys 2000; 46:391402. 24. Ryu
JK, Winter K, Michalski JM, et al. Interim report of toxicity from 3D conformal
radiation therapy (3D-CRT) for prostate cancer on 3DOG/RTOG 9406, level III (79
.2 Gy). Int J Radiat Oncol Biol Phys 2002; 54:10361046. 25. Michalski JM, Winter
K, Purdy JA, et al. Toxicity after three-dimensional radiotherapy for prostate c
ancer with RTOG 9406 dose level IV. Int J Radiat Oncol Biol Phys 2004; 58:735742.
26. Zelefsky MJ, Fuks Z, Hunt M, et al. High-dose intensity modulated radiation
therapy for prostate cancer: early toxicity and biochemical outcome in 772 pati
ents. Int J Radiat Oncol Biol Phys 2002; 53:11111116. 27. Zelefsky MJ, Cowen D, F
uks Z, et al. Long term tolerance of high dose three-dimensional conformal radio
therapy in patients with localized prostate carcinoma. Cancer 1999; 85:24602468.
ACKNOWLEDGMENTS The authors would like to thank Drs. Chris Beltran and Thomas M.
Pisansky for their collaboration on projects and publications related to this c
hapter. REFERENCES
1. American Cancer Society. Cancer Facts and Figures 2007. Atlanta: American Can
cer Society, 2007. 2. Kupelian PA, Potters L, Khuntia D, et al. Radical prostate
ctomy, external beam radiotherapy <72 Gy, external beam radiotherapy > or 72 Gy,
permanent seed implantation, or combined seeds/external beam radiotherapy for st
age T1-T2 prostate cancer. Int J Radiat Oncol Biol Phys 2004; 58(1):2533. 3. Zele
fsky MJ, Fuks Z, Hunt M, et al. High dose radiation delivered by intensity modul
ated conformal radiotherapy improves the outcome of localized prostate cancer. J
Urol 2001; 166(3):876881. 4. el-Gayed AA, Bel A, Vijlbrief R, et al. Time trend
of patient setup deviations during pelvic irradiation using electronic portal im
aging. Radiother Oncol 1993; 26: 162171. 5. Vigneault E, Pouliot J, Laverdiere J,
et al. Electronic portal imaging device detection of radioopaque markers for th
e evaluation of prostate position during megavoltage irradiation: a clinical stu
dy. Int J Radiat Oncol Biol Phys 1997; 37:205212. 6. Greer PB, Jose CC, Matthews
JH. Set-up variation of patients treated with radiotherapy to the prostate measu
red with an electronic portal imaging device. Australas Radiol 1998; 42:207212. 7
. Tinger A, Michalski JM, Cheng A, et al. A critical evaluation of the planning
target volume for 3-D conformal radiotherapy of prostate cancer. Int J Radiat On
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accuracy of daily set-ups in prostate radiotherapy using electronic imaging. Br
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ion and predictors of prostate position variability in 50 patients evaluated wit
h multiple CT scans during conformal radiotherapy. Radiother Oncol 1999; 50:22523
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r the course of routine radiotherapy using implanted markers. Int J Radiat Oncol
Biol Phys 1995; 31:113118. 11. Dawson LA, Mah K, Franssen E, et al. Target posit
ion variability throughout prostate radiotherapy. Int J Radiat Oncol Biol Phys 1
998; 42:11551161. 12. Stroom JC, Koper PC, Korevaar GA, et al. Internal organ mot
ion in prostate cancer patients treated in prone and supine treatment position.
Radiother Oncol 1999; 51: 237248. 13. Langen KM, Jones DT. Organ motion and its m
anagement. Int J Radiat Oncol Biol Phys 2001; 50:265278.

94 28. Schallenkamp JM, Herman MG, Kruse JJ, et al. Prostate position relative t
o pelvic bony anatomy base on intraprostatic gold markers and electronic portal
imaging. Int J Radiat Oncol Biol Phys 2005; 63(3):800811. 29. Michalski JM, Winte
r K, Purdy JA, et al. Trade-off to lowgrade toxicity with conformal radiation th
erapy for prostate cancer on Radiation Therapy Oncology Group 9406. Semin Radiat
Oncol 2002; 12:7580. 30. Michalski JM, Winter K, Purdy JA, et al. Preliminary ev
aluation of low-grade toxicity with conformal radiation therapy for prostate can
cer on RTOG 9406 dose levels I and II. Int J Radiat Oncol Biol Phys 2003; 56: 19
2198. 31. de Crevoisier R, Tucker SL, Dong L, et al. Increased risk of biochemica
l and local failure in patients with distended rectum on the planning CT for pro
state cancer radiotherapy. Int J Radiat Oncol Biol Phys 2005; 62:965973. 32. Herm
an MG, Pisansky TM, Kruse JJ, et al. Technical aspects of daily on-line position
ing of the prostate for three-dimensional conformal radiotherapy using an electr
onic portal imaging device. Int J Radiat Oncol Biol Phys 2003; 57(4):11311140.
Davis et al. 33. Herman MG, Balter JM, Jaffray DA, et al. Clinical use of electr
onic portal imaging: report of AAPM Radiation Therapy Committee Task Group 58. M
ed Phys 2001; 28:712737. 34. Cheung R, Tucker SL, Ye JS, et al. Characterization
of rectal normal tissue complication probability after highdose external beam ra
diotherapy for prostate cancer. Int J Radiat Oncol Biol Phys 2004; 58:15131519. 3
5. Goulet CC, Herman MG, Hillman DW, et al. Estimated limits of dose escalation
for localized prostate cancer using intensity modulated radiotherapy, varied pla
nning target volume margins and established dose constraints: correlation with p
rostate size and target volume overlap with the rectum. Int J Radiat Oncol Biol
Phys 2005; 63(suppl 1):S209. 36. Willoughby TR, Kupelian PA, Pouliot J, et al. T
arget localization and real-time tracking using the Calypso 4D localization syst
em in patients with localized prostate cancer. Int J Radiat Oncol Biol Phys 2006
; 65(2):528534. 37. Beltran C, Herman MG, Davis BJ, et al. Planning Target Margin
Calculations for Prostate Radiotherapy Based on Intrafraction and Interfraction
Motion Using Four Localization Methods. Int J Radiat Oncol Biol Phys 2008; 70(1
): 289295.
9
IGRT in Prostate Cancer: Method and Application of Cone-Beam Computed Tomography
DAVID A. JAFFRAY
Radiation Medicine Program, Princess Margaret Hospital/University Health Network
, Departments of Radiation Oncology and Medical Biophysics, University of Toront
o, Toronto, Ontario, Canada
INTRODUCTION The past 10 years has seen radiation therapy of the prostate gland
evolve from a simple techniquebased approach that relied on bony anatomy (1) and
perturbations from the fourfield brick to a modality that is informed from image s
ets derived from a variety of different imaging systems. These images are capabl
e of illustrating the gland and its internal structures, differentiating disease
from normal tissues, and demonstrating the presence of organ motion. As the des
ign phase of prostate radiation therapy continues to advance, there is a need fo
r parallel advancements in the methods used to verify that these planned dose di
stributions are effected within the patient. Recent developments in detector tec
hnology, computational horsepower, and the data-handling systems have permitted
the development of volumetric imaging on the radiation therapy treatment unit us
ing cone-beam computed tomography (CBCT) methods. These are highly integrated so
lutions that permit in-room imaging, alignment, and adjustment of the patient po
sition prior to treatment. ACHIEVING PRECISION AND ACCURACY IN RADIATION THERAPY
FOR PROSTATE CANCER The large number of sensitive adjacent structures (rectum,
bladder, penile bulb, femoral heads), their complex spatial
95
interrelationship, the mobility and distention of the rectum/colon, and the grad
ual filling of the bladder make the conformal treatment of the prostate gland ch
allenging. In this context, the objective of maintaining the prescribed dose to

the prostate gland [gross tumor volume (GTV) and clinical target volume (CTV)] i
s a complex tradeoff of competing dose-volume constraints where the fundamental
limits on the dose gradients in photon beams and the presence of geometric uncer
tainties are in competition. These uncertainties are made complex by the fact th
at the structures can move and change shape both relative to bony anatomy and ea
ch other (26). Imaging methods such as portal imaging or radiographic imaging all
ow localization of bony anatomy or implanted fiducials (713), but do not permit c
haracterization of the changes in shape and relative position of the gland and s
urrounding normal tissues (14). The use of markers in the gland provides a signi
ficant improvement in gland-targeting accuracy and precision over bony anatomy.
However, further advances in imaging are desired to allow visualization of these
structures directly, thereby eliminating the need for marker placement, allowin
g patient-specific dose accumulation, and possibly leading to subprostatic boost
, and adaptive methods to further reduce the planning target volume (PTV) margin
s employed in the design of the treatment plan.
96
Jaffray
CBCT IMAGING Advances in large-area electronics has led to the development of tw
o-dimensional radiographic detectors based upon amorphous silicon technology (15
). The resulting radiographic receptors employ radiation-sensitive phosphors or
scintillators on large (up to 41 cm 41 cm) arrays, free of distortion and reason
ably efficient (>60%) at collecting X-rays and converting the X-ray shadow into
a digital format. Such detectors make it possible to achieve coverage of a large
field of view (FOV) with resolutions that are less than 1 mm. The use of area d
etectors to generate volumetric computed tomography images was pioneered in the
field of single-photon emission computed tomography (SPECT). In this approach, i
t is necessary to collect projection data from multiple directions around the ob
ject of interest. The same is necessary in CBCT, wherein multiple (300600) projec
tions are acquired over a range of angles (*180360) about the patient (16,17). A
sufficiently fast and effective reconstruction algorithm for reconstructing this
type of data was reported by Feldkamp et al. in 1984 (18). The adaptation of th
e flat-panel detector to medical linear accelerators was already underway to all
ow better megavoltage portal imaging quality (19). These systems can now be used
to generate CBCT data directly (20). Alternatively, an additional kV X-rays tub
e can be integrated with the
accelerator, and a kV detector is employed opposite for collection of the multip
le radiographs (21,22). Currently, there are a number of commercial systems avai
lable in the market (Fig. 1). The Elekta SynergyTM System (23), Varian On-Board
ImagerTM (24), and Siemens ArtisteTM offer kV-based CBCT integrated into the tre
atment unit. Siemens is the only company offering a megavoltage (MV) CBCT system
(MVisionTM) (20). There are numerous subtleties in the operation of these syste
ms that determine the relative merits. Factors to be considered include size of
the FOV, the speed of acquisition and reconstruction, the use of correction sche
mes to improve imaging performance, their support for radiographic/ fluoroscopic
modes, the level of integration with the treatment unit, software tools, remote
couch control, and connectivity to the electronic treatment record. Given that
these systems have relatively recently reached the market, there continues to be
significant technical advancement and ongoing development of modes of clinical
use. CBCT SYSTEMS AND ISSUES Image Quality The images generated by the kV CBCT s
ystems have many features that make them attractive for image-guided radiation t
herapy (IGRT) of the prostate. These include
Figure 1 There are four cone-beam CT systems available. These include (A) Elekta
Synergy, (B) Varian Trilogy, and (C) Siemens Artiste offering kV cone-beam CT.
Siemens also offers the MVision adaptation to their accelerator for megavoltage
cone-beam CT (D).

IGRT in Prostate Cancer: Method and Application of CBCT

97
Figure 2 The quality of cone-beam CT images is influenced by
ays scatter, detector nonlinearity, and lag. (A) Axial slice
CT scan of the prostate. (B) Cone-beam CT image acquired at
e image as B with a postprocessing correction applied to the
suppress the influence of scatter.

the presence of X-r

from a conventional
*3 cGy. (C) The sam
projection data to

detection of soft tissue contrast, large FOV, remarkably high spatial resolution
, and relatively low imaging doses. The soft tissue contrast demonstrated with c
urrent systems is not at the level of conventional CT systems. The presence of X
-rays scatter and detector lag in the CBCT systems induces significant shading a
rtifacts such as cupping or streaks (Fig. 2B). These features do not significant
ly reduce the spatial resolution, as a result, very detailed images can be gener
ated with these systems (21). Such a characteristic can be useful when consideri
ng the need to detect and localize implanted fiducials, or in
the case of brachytherapy, distinguish and localize nearly 100 such seeds (25).
However, the shading artifacts negatively affect the ability of these systems to
produce accurate CT numbers, and therefore, accurate electron density estimatio
n. While not critical for image guidance, it does prevent planning directly on t
he CBCT images unless these densities are manually assigned. While early attempt
s at MV CBCT produced images of limited quality (22,26), the utilization of amor
phous silicon detectors has advanced the method significantly. The quality of th
e images produced with these systems has many of the features outlined above for
the kV systems (Fig. 3). While the fundamental limitations on noise per unit im
aging dose will limit soft tissue visualization, the use of the MV beam should r
educe the magnitude of scatter (27,28) and may mitigate some of the artifacts ar
ising from detector nonlinearities and beam-hardening effects. The reduction in
these effects may offset some of the fundamental noise constraints associated wi
th the use of the megavoltage-range X-rays. Currently, the CBCT approach require
s approximately 40 to 120 seconds to generate an image. This time constraint is
due in part to the maximum gantry rotation rate allowed in these systems (1 rpm)
. The reconstruction process assumes a static object over the course of acquisit
ion, and given these long acquisition times, it is highly likely that some motio
n will occur. In terms of image quality for prostate imaging, the most disruptiv
e artifacts arise from the motion of gas in the rectum and colon. The low densit
y of gas makes it a high-contrast material, which can generate streak artifacts
in the images if it is mobile. Efforts to reduce the presence of gas have been i
dentified as a method of improving image quality and the utility of the images (
29,30). Integration with the Medical Linear Accelerator The generation of a CBCT
image is an important feature of the imaging system, however, it is of equal im
portance that these images be easily acquired with the patient
Figure 3 Cone-beam CT images of the prostate and surrounding anatomy generated u
sing (A) kV cone-beam CT (Elekta XVI) and (B) MV cone-beam CT (Siemens MVision).
These are generated with comparable imaging doses.
98
Jaffray
positioned for treatment. This requires that the imaging systems be well integra
ted with the medical linear accelerator. The need for rapid acquisition and seam
less registration is clear; however, the central issues are geometric precision
and accuracy in detection and correction. Accuracy and Precision The adaptation
of CBCT systems to the medical linear accelerator initially raised concerns rega

rding the geometric integrity of the systems, both in terms of CBCT image qualit
y and the registration of the resulting volumes to the coordinate system of the
treatment unit. However, studies are demonstrating that these systems can be rel
iably calibrated and are capable of maintaining calibration over long time inter
vals (24,31). These studies suggest that the intrinsic accuracy and precision ar
e sub-mm for unambiguous targeting tasks (e.g., Lutz-type BB phantoms) (32). The
se results have been verified in patient studies involving markers by comparing
the alignment results of CBCT images to those produced by portal imaging methods
(33). Overall, the geometric precision and accuracy of these systems is proving
to be excellent with monitoring through a suitable quality assurance program (3
4). Image-Guidance System In addition to the geometric performance, there are se
veral additional features that need to be present if volumetric targeting is to
be achieved. These include the ability to overlay reference grayscale images and
reference structures of interest. Automated tools to first detect bony anatomy
and then identify soft tissue target offsets are an excellent way to bound allow
able corrections during the online guidance procedure. The use of this approach
can be used to limit displacements relative to bone in excess of some limit (e.g
., 20 mm) and avoid alignment to the wrong structures. The use of subregions or c
lipboxes to improve the registration to local structures is a valuable addition t
o these systems. This can be taken further with the use of automated prostate-sp
ecific localization methods (29). Once a registration has been performed, a corr
ection must be derived by either adjusting the patient position or altering the
machine parameters. For nonrotational couch corrections, any detected rotation i
n the registration must be accommodated in the translations. This requires the i
dentification of a reference point at which it is desired that the rotation-indu
ced residual error is minimized (35). The appropriate couch correction can then
be transferred to the treatment unit for automated adjustment of the couch. Over
all, these systems are becoming highly integrated, such that, the time required
for CBCT online imaging and correction is
equivalent to the time required for conventional online portal imaging based app
roaches (Princess Margaret Hospital, Internal Study). Dosimetry The use of CBCT
in the guidance of prostate therapy results in a dose penalty that needs conside
ration. In the context of kV CBCT, these doses are distinct from the therapy dos
e. The energy of the kV beam is typically *120 kilovolt peak (kVp) and as a resu
lt, the biological effect of the dose deposited during imaging is not easily int
erpreted in the context of the tumor control and normal tissue toxicity. However
, this does not indicate that imaging doses should not be documented and minimiz
ed. Recent publications have reported on the imaging doses associated with kV CB
CT (3638) and the AAPM Task Group No. 75 describes the dose from a variety of rad
iographic image-guidance systems (39). Chow et al. have recently published a rep
ort describing the combined dose from kV CBCT imaging and therapy for a typical
course of prostate treatment using daily online imaging and soft tissue guidance
(40). The doses were calculated using Monte Carlo methods and accumulated. The
differential dose to the bone and soft tissue structures is evident, however, th
ese doses remain low relative to the dose from the therapy beam. Currently, imag
ing doses delivered for prostate gland visualization in kV CBCT of the prostate
is approximately 3 cGy at isocenter per scan. It should be noted that these quote
d doses are dependent on many variables (energy, FOV, offset geometry, use of bow
-tie filter) and published quantities are no substitute for measurements that co
rrespond to the specific technique. USE OF CBCT FOR IMAGE-GUIDED RADIATION THERA
PY OF THE PROSTATE Online Guidance Models The online guidance approach involves
detecting and (if necessary) correcting the relative position of the prostate an
d treatment isocenter on a fraction-by-fraction basis. Such a method requires th
at all the elements of the process are highly streamlined and sufficient perform
ance is achieved in the imaging to allow accurate and precise estimation of the
gland. At the Princess Margaret Hospital, online IGRT of the prostate using CBCT
has been employed in patients with and without implanted fiducial markers in th
e gland. This is based upon observer studies of online alignment of IGRT of the
prostate with the Elekta Synergy system (33). These studies compared the appropr

iate couch correction for 16 prostate patients imaged and treated on the CBCT-eq
uipped system. These
IGRT in Prostate Cancer: Method and Application of CBCT
99
corrections were derived from either gland-template (i.e., soft tissue) alignmen
t or marker-marker alignment. The soft tissuedetermined corrections did not agree
precisely with that determined from the markers, however, the two systems demon
strated excellent agreement in their mean shifts for any patient. The discrepanc
y between marker and soft tissue results was consistent with the degree of inter
observer variations found in a parallel study. Based on these results, the use o
f CBCT for targeting RT of the prostate is now a consideration. The agreement in
mean position between the marker and soft tissue results suggests that both met
hods are equally good at reducing the systematic errors, which is the dominant s
ource of the margin in these cases. It should be noted that the use of soft tiss
ue alignment at the treatment unit opens the potential for introduction of syste
matic errors of another variety systematic differences in the interpretation of t
he gland outline in the grayscale images. This can be resolved by the physician
(that specified the gland) and the therapist (that align each day) reviewing the
initial alignments to assure consistency between the GTV and target structures
in the CBCT image. The use of MV CBCT for online guidance of prostate RT has not
been reported in the literature, however, it is quite reasonable to pursue this
if fiducials are employed. The high contrast of these objects would allow preci
se and accurate localization at low doses. Soft tissue targeting may require una
cceptably high imaging doses. The inclusion of these doses in the planning proce
ss may permit this to be pursued, provided the imaging dose is appropriately acc
ommodated (41). Further developments in lowerenergy beams using lowatomic number
targets may also serve to further reduce the dose required for soft tissue local
ization. At this point, the use of markers continues for targeting the prostate
gland. The significant adoption of this approach since the development of online
electronic portal imaging devices has produced a clinical practice that is well
equipped to switch to soft tissue targeting. It is not clear how long this appr
oach will persist, but it is likely to transition as image quality in CBCT conti
nues to improve. The development of hypofractionated regimens will also drive th
e use of soft tissue targeting where visualization of the gland and normal tissu
es may be of interest for each fraction. Clearly, this will increase the time re
quired to assess the images and may alter the workflow and delegation issues. Of
f-Line Guidance Models The Beaumont Approach (42,43) is an alternative and matur
e approach that readily adopts CBCT for off-line
image guidance. In the original embodiment of this approach, the systematic and
random characteristics of prostate position and shape are captured through repea
t CT scans in the first week of therapy. This approach has now been adapted to e
mploy CBCT as the source of data (44). In the recent report, the advantages of e
xtending the adaptive process to include online components are described. The Ne
therlands Cancer Institute has recently reported on their adaptive approach usin
g CBCT (30). An average PTV reduction of 29% was achieved with the volume of the
rectum that received >65 Gy reduced by 19% on average. These processes require
high-quality CBCT images for segmentation of the structures of interest (prostat
e, rectal wall), as opposed to isocenter alignment and localization employed in
a simple online approach. Overall, the quality of the images produced with these
types of systems make these approaches feasible (45). The growing interest in h
ypofractionated schedules will place additional pressure on adopting an online s
trategy. This does not mean that the principles of adaptation will not be employ
ed to deal with the issue of intrafraction motion (46). Hybrid Models The distin
ction between online and off-line approaches is somewhat arbitrary. The magnitud
e and frequency of variations in gland and rectal wall configuration that can oc
cur over the course of therapy suggest that a hybrid approach may be beneficial.

Such approaches have been proposed in the past (47). Figure 4 presents a proces
s for which the original CBCT-equipped treatment unit was designed. This process
separated the translational, rotational, and deformational elements of the pros
tate movements to be managed by three different methods: (i) the online couch co
rrection was used to adjust displacements of the prostate apex, (ii) multiple pr
estored plans were indexed to accommodate the presence of rotations, and (iii) a
n ongoing accumulation of the database of fractions allowed patient-specific adapt
ation of deformation to be fed back into an offline process of replanning. While
this approach is still beyond the technical capacity of any commercial radiatio
n therapy management system, all the subcomponents are now available. One exampl
e of the advancements that are at the forefront of this effort has been reported
by Smitsman et al. (29). In their investigations, registration success rates in
excess of 80% were achieved provided rectal gas was managed through an appropri
ate diet. Another example is taken from the same group. Rijkhorst et al. describ
e an alternative approach to correct for prostate rotations employing collimator
and gantry angle rotations (48).
100
Jaffray
Figure 4 Flow chart for a proposed high-precision image-guidance system. The off
-line planning process and preparation of priors produce reference contours and
a constrained plan set. In the online process, the prostate is rapidly delineate
d and registered from a CBCT image, and the prostate rotation and translation de
termine the selected plan and couch adjustment, respectively. An optional repeat
CBCT image acquired immediately prior to and/or after delivery would provide th
e most accurate representation of the treatment. Image data are stored in a data
base for off-line review of prostate and rectal doses and re-evaluation of the p
redefined plan set. Abbreviation: CBCT, cone-beam computed tomography.
Other Issues The ability for soft tissue visualization can also impact the appro
ach taken in planning and image guidance. Two areas of interest that have not be
en employed with CBCT image guidance include rectal avoidance and subprostatic b
oost. Literature on these concepts have been presented for the tomotherapy parad
igm (49) and marker-based approaches (50). It is highly likely that these approa
ches will be pursued using CBCT methods. Another example of novel applications o
f this technology is in the targeting of postprostatectomy patients (51). In thi
s context, the visualization of surrounding normal tissues is more relevant for
image guidance. Showater and Valicenti evaluated some 176 CBCT study sets reveal
ing that rectal and bladder borders were reliably identified in 94% of the image
sets. These results are encouraging and support the application of CBCT methods
in the management of prostate cancer. An important and concerning development a
ssociated with the introduction of soft tissue imaging in the clinical setting i
s in the need for retraining and skill development. Broad use of CBCT will resul
t in the radiation therapists at the treatment units dealing with an order of ma
gnitude
more volumetric with imaging data than their counterparts in the planning proces
s (dosimetrists, planners). These images are open to interpretation and skills n
eed to be developed to properly evaluate these images and maximize their benefit
to the patient. SUMMARY OF PERTINENT CONCLUSIONS
l
l
l
l

l
The development of more volumetric with imaging data in prostate radiation thera
py is maturing at a remarkable rate. In the past five years, we have seen a rapi
d transition from bone-based alignment to online markers, and now to soft tissue
targeting. The broad deployment of CBCT technology assures that this trend will
continue at a rapid pace. The inherent challenge of treating the prostate gland
in its surrounding radiosensitive milieu makes the application of these technol
ogies sensible and appropriate. Further improvements in image quality will make
this a simple and broadly practiced method for assuring precise and accurate tar
geting in prostate radiation therapy.
IGRT in Prostate Cancer: Method and Application of CBCT
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ith a large-area, plat-panel, amorphous silicon imager. Int J Radiat Oncol Biol
Phy 1996; 36:661. 20. Pouliot J, Bani-Hashemi A, Chen J, et al. Low-dose megavol
tage cone-beam CT for radiation therapy. Int J Radiat Oncol Biol Phys 2005; 61(2
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. On-line aSi portal imaging of implanted fiducial markers for the reduction of
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10
IGRT in Prostate Cancer: Focus on BAT Ultrasound
JOSHUA S. SILVERMAN AND ERIC M. HORWITZ
Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, Pennsyl
vania, U.S.A.
INTRODUCTION History of Daily Image Localization at Fox Chase Cancer Center The
development of three-dimensional conformal radiation therapy (3DCRT) and its use
in the treatment of prostate cancer began in the late 1980s at Fox Chase Cancer
Center as Gerald Hanks and colleagues sought to more precisely deliver increasi
ng doses of external beam radiation to the prostate while limiting the toxicitie
s to adjacent structures. Early results were promising as biochemical control in
creased compared with patients treated with conventional techniques and doses as
the radiation dose was increased (1). However, with these increasing control ra
tes came increasing complications, especially in the rectum adjacent to the pros
tate. Initially, a lateral rectal block was added after 5600 cGy to reduce compl
ications (2). As more patients were treated with higher doses of radiation acros
s the country and CT simulators and simulations became more widespread, the issu
e of organ motion was recognized. With this realization came the acknowledgment
that to safely deliver ever higher doses of radiation, this phenomenon would nee
d to be controlled. The development of B-mode acquisition and targeting (BAT) ul
trasound emerged from the goal of obtaining an easyto-use, reproducible, and rob
ust method of accurately localizing the prostate and adjacent critical structure
s in real time prior to daily radiation treatment.
The Rationale for Image Guidance There are several rationales for the use of dai
ly imageguided radiation therapy (IGRT) in the management of prostate cancer. Th
e ability to consistently and reproducibly deliver the prescribed dose of radiat
ion to the target is the most obvious reason. Another reason is the ability to s
afely escalate radiation dose. The first reports illustrating this dose response
appeared in 1995, and since that time, multiple single-institution prospective,
retrospective, and phase III randomized prospective trials have confirmed this
phenomenon in prostate cancer. These studies have consistently demonstrated an i
mprovement in biochemical control as an increasing dose of radiation is delivere
d to the prostate (1,3,4). Finally, IGRT allows for the limiting of dose to adja
cent critical structures and the reduction of early and late complications. Dose
-volume effects have been described in the literature for both the bladder and r
ectum (5,6). Because of dose-volume relations of critical structures, either con
formality must be increased or the planning target volume (PTV) must be reduced
when dose escalation is done, and either of these necessitates more accurate loc
alization and verification of the target (prostate). Movement of the prostate wi
th respect to bladder and rectal filling has been observed and characterized by
several authors and similar patterns have been described (79). Large shifts in pr
ostate position are associated with
103
104

Silverman and Horwitz

rectal gas pockets and can be produced from the introduction of 50 mL of contras
t into the rectum (10,11). Zelefsky et al. report that rectal volume greater tha
n 60 cm3 and bladder volume greater than 40 cm3 are the only independent predict
ors of prostate shifts greater than 3 mm (12). THE FOX CHASE TECHNIQUE FOR ULTRA
SOUND-GUIDED PROSTATE IGRT Simulation In preparation for treatment, patients are
simulated in a supine position in a cast mold of their pelvis with an ankle blo
ck. Patients are simulated with a full bladder (consuming 1216 oz water within se
veral hours of simulation) and empty rectum (magnesium citrate bowel preparation
and, occasionally, an additional Fleets enema). Patients are simulated using CT
and MRI unless there is a contraindication to performing an MRI. A slice thickne
ss of 3 mm is used for both scans. The scans are fused in the planning computer
based on bony and soft tissue anatomy using either chamfer matching or maximizat
ion of mutual information methods. The structures outlined include the prostate,
seminal vesicles, rectum (from ischial tuberosity to the sigmoid flexure), the
entire bladder, and the bilateral femoral heads. The clinical tumor volume (CTV)
is defined as the prostate and the proximal seminal vesicles (the first 9 mm of
the seminal vesicles). The CTV is expanded by 5 to 6 mm posteriorly and 8 mm in
all other directions to produce the PTV. Triangulation tattoos and the use of a
cradle cast ensure efficient and reproducible daily setup and initial alignment
prior to the use of BAT ultrasound daily localization. The radiation dose and t
argets prescribed depend on the stage of cancer (13). Once the planning is compl
ete and the patient is ready for treatment, the contoured images are imported in
to the BAT ultrasound system over a local area network (LAN). Prior to the patie
nt entering the treatment room, the ultrasound system is moved adjacent to the t
reatment couch and is docked to the treatment machine collimator, and the CT- an
d MRI-simulation target images and isocenter are imported into the BAT system. S
ince 2000, intensity-modulated radiation therapy is used exclusively for all pro
state cancer patients treated with external beam radiation therapy. Daily Setup
and Alignment The BAT ultrasound system consists of a B-mode transabdominal arm
attached to a precision tracking arm and connected to a computer (Fig. 1). The c
omputer shows the real-time images from the ultrasound probe and the outlines of
the image-guided contours. The BAT ultrasound
Figure 1 The BAT ultrasound equipment. The ultrasound probe is connected to a co
mputer that enables proper alignment prior to daily treatment. Abbreviation: BAT
, B-mode acquisition and targeting.
system uses the treatment machine isocenter as a reference point to overlay corr
esponding CT contours onto the ultrasound images. Daily setup using BAT ultrasou
nd is accomplished as follows. The patient is aligned to the simulation tattoos.
Weekly portal images are used to verify that the patients
Figure 2 Ultrasound plans. The sagittal and transverse ultrasound planes commonl
y used for BAT ultrasound are depicted in the figure. Abbreviation: BAT, B-mode
acquisition and targeting.
IGRT in Prostate Cancer: Focus on BAT Ultrasound
105
Figure 3 The BAT ultrasound in clinical practice. The ultrasound cabinet is move
d adjacent to the treatment couch prior to the initiation of radiotherapy. Abbre
viation: BAT, B-mode acquisition and targeting.
alignment corresponds to the bony anatomy of the pelvis. The patients CT data set
is chosen from a pull-down menu on the computer console and the suprapubic BAT
ultrasound procedure is initiated. Typical BAT ultrasound settings are 3.5 MHz,

60%
lay
to
The

power, 80% gain, and 14-cm depth. Real-time imaging is available on the disp
screen. The BAT probe recognizes its three-dimensional location with respect
the machine isocenter as transverse and sagittal images are taken (Fig. 2).
BAT

probe is performed with the patient in the treatment position on the treatment c
ouch (Fig. 3). The transverse and sagittal contours are moved by the operator us
ing a touch-screen menu to overlay the images so that the ultrasound and contour
ed CT-derived images are brought into register (Fig. 4A, B) Fine (1.0 mm) and co
arse (1.0 cm) adjustments can be made. The system then calculates the necessary
couch shifts and displays them so that the couch can be adjusted prior to initia
tion of radiotherapy by the radiation therapist. If a large shift is required, t
he BAT ultrasound alignment is repeated to verify the accuracy of the necessary
shift. Morr et al. describes the appearance of the various anatomic structures u
sing BAT ultrasound (14). The bladder is anechoic (dark) and prostate is of inte
rmediate echogenicity. The bladder wall, bladder/prostate interface (difficult t
o determine), prostate, and rectum are hyperechoic. Often, angulation of the ult
rasound probe (approximately 108508) is required in the caudal direction. With BA
T, the apex of the prostate is not easily seen in the sagittal image because of
interference from the pubic bone. Image quality is best when scanning the widest
extent of the prostate in the transverse plane and along the urethral axis in t
he sagittal plane (15). The probe is moved superiorly and angled inferiorly if t
he prostate was overshadowed by the pubic symphysis in the transverse plane. In
the sagittal plane, only the base of the prostate can be fully visualized, even
if the ultrasound probe is moved superiorly.
Figure 4 BAT ultrasound daily treatment alignment. The computer allows the BAT u
ltrasound images to be correlated with contours of the prostate, bladder, and re
ctum. The preshift images (A) illustrate the need for a shift to obtain the prop
er alignment (B). Once a shift is approved, couch shifts are given as an output
and used prior to the initiation of radiotherapy. Abbreviation: BAT, B-mode acqu
isition and targeting.
106
Silverman and Horwitz
Fox Chase Pilot Studies with CT and Ultrasound Guidance At Fox Chase, early stud
ies using image guidance in conjunction with 3DCRT utilized CT imaging (1517). In
the first study, six patients who consented to this prospective study underwent
daily CT simulations to assess setup variations in portal placement and organ m
otion supine position. These patients underwent CT simulation with an alpha crad
le immobilization, intravenous contrast, and urethrograms to determine if improv
ed prostate localization techniques could allow for the reduction of margins aro
und the target to facilitate dose escalation in high-risk patients while minimiz
ing the risk of normal tissue morbidity. Patients received 46 Gy to the initial
planning treatment volume (PTV1) in a four-field conformal technique with the pr
ostate, seminal vesicles, and lymph nodes as the gross tumor volume. The prostat
e or prostate and seminal vesicles (GTV2) then received 56 Gy to PTV2. After fiv
e weeks of treatment (50 Gy), a second CT simulation was performed. The prostate
was contoured and a new isocenter was generated with appropriate surface marker
s. Prostate-only treatment portals were used for the final cone down. The daily
isocenter was recalculated in the anterior-posterior (A-P) and lateral dimension
s and compared to the 50 Gy CT simulation isocenter. The patients were transferr
ed to a stretcher while on the sliding board in the cast and transported to the
treatment room and carefully transferred to the treatment table. The patients we
re then treated to the corrected isocenter. Portal films and electronic portal i
mages were obtained for each field. Utilizing CT-CT image fusions of the daily a
nd 50-Gy baseline CT scans, the isocenter changes were quantified to reflect the
contribution of positional and absolute prostate motion relative to the bony pe

lvis. The maximum daily A-P shift was 7.3 mm. Motion was less than 5 mm in the r
emaining patients, and the overall mean magnitude change was 2.9 mm. The overall
variability was quantified by a pooled standard deviation of 1.7 mm. The maximu
m lateral shifts were less than 3 mm for all patients. With careful attention to
patient positioning, maximal portal placement error was reduced to 3 mm. On the
basis of this study, the authors concluded that prostate motion after 50 Gy was
significantly less than previously reported. Intrapatient and overall populatio
n variance was minimal. With daily isocenter correction of setup and organ motio
n errors by CT imaging, Lattanzi et al. concluded that PTV margins can be signif
icantly reduced or eliminated (16). The second study introduced the BAT ultrasou
nd technology and compared results with those observed with daily CT scans to de
termine the precision of ultrasound localization with respect to CT scans. Thirt
y-five
consecutive men were prospectively studied in a comparison of daily CT and ultra
sound-guided localization at Fox Chase Cancer Center. Daily CT prostate localiza
tion was completed before the delivery of each final boost field. In the CT simu
lation suite, transabdominal ultrasound-based stereotactic localization was also
performed. Sixty-nine daily CT and ultrasound prostate position shifts were rec
orded for 35 patients. The average directed discrepancies between the two techni
ques were extremely small. There were 0.09 2.8 mm in the A-P axis, 0.16 2.4 mm in
the left-right (L-R) axis, and 0.03 2.3 mm in the superior-inferior (S-I) axis. A
nalysis of the paired CT-ultrasound shifts revealed a high correlation between t
he two modalities in all three dimensions. This was the first study to demonstra
te that ultrasound-directed stereotactic localization was safe and accurate comp
ared with CT scanning in targeting the prostate for conformal external beam radi
ation therapy (15). The final Fox Chase ultrasound study quantified the magnitud
e of the patient isocenter shift parameters encountered during clinical implemen
tation of the BAT ultrasound-based targeting system. In this study, 54 patients
underwent a second CT simulation following five weeks of 3DCRT. For each of the
final cone-down treatments (24 fractions), patients underwent ultrasound-based st
ereotactic prostate localization at the treatment machine. One hundred and eight
y-nine daily ultrasound prostate position shifts were recorded. The isocenter fi
eld misalignment between the baseline CT and ultrasound ranged from 26.8 to 33.8
mm in the A-P axis, 10.2 to 30.9 mm in the L-R axis, and 24.6 to 9.0 mm in the S-I
axis. The corresponding directed average disagreements were 3.0 8.3 mm, 1.86 5.7
mm, and 2.6 6.5 mm in the A-P, L-R, and S-I axes, respectively. The magnitudes o
f the misalignments were frequently larger than 5 mm (51%, 31%, and 35% of measu
rements in the A-P, L-R, and S-I axes, respectively) and occasionally larger tha
n 10 mm (21%, 7%, and 12% of measurements in the A-P, L-R, and S-I axes, respect
ively). Similar to the previous study described, there was a high correlation be
tween the ultrasound and CT modalities. This initial Fox Chase experience with t
he BAT system in a large cohort of prostate cancer patients revealed that substa
ntial daily isocenter corrections were encountered in a large percentage of case
s. This data suggested that daily clinical isocenter misalignments are greater t
han would be expected from published data on organ motion and setup variations e
ncountered in the study setting (17,18). The Evolving Use of BAT IGRT versus Alt
ernative Methods BAT ultrasound is one of the several daily localization modalit
ies described for IGRT in prostate cancer (1921).
IGRT in Prostate Cancer: Focus on BAT Ultrasound
107
Other modalities include portal images with a megavolt imager (22), daily CT usi
ng either cone-beam CT or CTon-rails (7,8,12,16,2325), gold seed fiducial markers
(20,2629), rectal balloons, and real-time tracking beacons (30,31). The Use of B
AT Ultrasound in Current Practice at Fox Chase Cancer Center Since 1990, daily l
ocalization has been a mainstay of prostate cancer treatment for clinically loca
lized prostate cancer at Fox Chase Cancer Center. Weekly electronic portal imagi

ng has been used in conjunction with BAT ultrasound to verify accuracy of the in
itial patient setup to bony landmarks of the pelvis prior to ultrasound localiza
tion. More recently, gold seed fiducial markers and realtime Calypso tracking be
acons have been incorporated as modalities for IGRT at Fox Chase. BAT ultrasound
is typically reserved for those who cannot receive Calypso beacons, such as tho
se with an implanted pacemaker or defibrillator and those with a body thickness
greater than 23 cm that precludes beacon detection. Anticoagulation is a relativ
e contraindication for beacon placement. BAT ultrasound cannot be used in obese
men since obesity leads to poor images. Gold seed fiducial markers are used in t
hose for whom Calypso beacons and BAT ultrasound are not appropriate. BAT ultras
ound for daily localization offers advantages that other IGRT modalities do not.
Ultrasound can be used to assess the reproducibility of bladder and rectal fill
ing. The current Fox Chase policy is to treat patients with a full bladder. BAT
ultrasound provides information regarding bladder and rectal filling that other
daily localization modalities do not, except for volumetric imaging methods such
as cone-beam CT. Patients can be coached regarding appropriate fluid intake, an
d an assessment can be made of the degree of rectal filling. Fiducial markers, r
ectal balloons, and Calypso beacons are all invasive techniques.
OPTIMIZING IGRT IMPLEMENTATION IN PROSTATE CANCER: IGRT QUALITY CONTROL In the e
ra of reducing dosimetric margins using 3DCRT and IMRT for prostate cancer, an a
nalysis of BAT ultrasound is critical in determining errors associated with orga
n motion. Attempts at establishing adequate margins must account for inter- and
intrafraction variation. The following discussion includes the various quantitat
ive and qualitative analysis of couch shifts determined by BAT ultrasound and qu
ality control issues related to ultrasoundbased daily localization. Interfractio
n Daily Couch Shifts There are numerous published single institution experiences
documenting interfraction couch shifts using BAT ultrasound (Table 1) (32,33).
Couch shifts are given in the A-P, S-I, and L-R axes. From a review of the data,
several themes emerge. First, couch shifts tend to be greater in the A-P and SI axes in comparison to the L-R axis. This is consistent with a model in which d
aily prostate motion is most affected by the degree and variability of bladder a
nd rectal filling. This suggests that optimal dosimetric margins must not necess
arily reflect uniform expansion of the prostate volume. Interfraction couch shif
ts approximate a Gaussian distribution in frequency-shift histograms (14,3436). D
osimetric margins must account for interfraction variation in couch shifts (whic
h reflect organ motion), if complete coverage of the target (prostate) is desire
d. Comparison of the magnitude and variance in single institution experiences of
BAT ultrasound may be confounded by differences in immobilization technique, im
age modalities used for contouring (CT vs. MRI vs. CT-MRI fusion), and factors a
ffecting the degree of bladder and rectal filling (e.g., protocol for fluid inta
ke and bowel preparation).

Table 1 Interfraction Shifts Using BAT Ultrasound in the Treatment of Prostate C

ancer Study Lattanzi et al. (1999) Serago et al. (2002) Morr et al. (2002) Huang
et al. (2002) Langen et al. (2003) Trichter et al. (2003) Little et al. (2003)
Chandra et al. (2003) Number of patients 23 38 19 20 10 26 35 147 A-P mean SD (m
m) 0.2 4.6 1.3 4.7 4.7 2.7 0.4 4.0 0.7 5.2 0.32 0.6 1.4 6.4 0.49 1.9 S-I m
m) 0.0 5.7 1.0 5.1 4.2 2.8 1.5 3.3 2.7 4.5 0.31 0.7 1.7 6.4 0.59 3.4 R-L me
mm) 0.1 3.4 0.3 2.5 2.6 2.1 0.4 2.2 1.8 3.9 0.32 0.5 0.82 3.2 0.92 3.2
Abbreviations: BAT, B-mode acquisition and targeting; A-P, anterior-posterior; S
-I, superior-inferior; R-L, right-left.
108
Silverman and Horwitz
Table 2 Comparison of BAT Ultrasound Daily Shifts with Shifts Using Other Daily
Treatment Modalities in the Treatment of Prostate Cancer Study Number of patient

s A-P mean SD (mm) 3 1.8 0.09 2.8 0.2 3.7 3.2 3.2 S-I mean SD (mm) 4.6 2.8 0.
3 2.7 3.9 3.3 3.5 R-L mean SD (mm) 2.4 1.8 0.16 2.4 1.6 3.1 2.2 3.7
A. BAT ultrasound shifts compared to daily CT scan Lattanzi et al. (1999) 25 Lat
tanzi et al. (2000) 35
B. BAT ultrasound shifts compared to fiducial marker shifts Langen et al. 2003 1
0 McNair et al. (2006) 26
Abbreviations: BAT, B-mode acquisition and targeting; A-P, anterior-posterior; S
-I, superior-inferior; R-L, right-left.
Comparison with Other Localization Modalities The comparison of ultrasound-based
daily localization to other modalities addresses questions of the reproducibili
ty of daily localization and the possible existence of systematic errors between
treatment modalities. Studies have compared the shifts of ultrasound with daily
CT imaging (Table 2A) and with fiducial markers (Table 2B). A recent study from
Fox Chase examined BAT ultrasound to CTon-rails measurements (37). Two hundred
and eighteen alignments in 15 patients revealed a high level of correlation betw
een BAT ultrasound and CT-on-rails with systematic differences of less than 1 mm
and random differences of about 2 mm. A comparison between the magnitude and va
riation of interfraction couch shifts with ultrasound alone versus alternate mod
ality shifts revealed that the magnitude of shifts was similar between the vario
us daily localization techniques for IGRT. Interuser Variability Interuser varia
bility has also been examined in the literature. McNair et al. report interuser
differences between three observers, with the proportion of readings over 3 mm b
etween the observers being in the range of 25% to 44% (38). The authors did not
feel that there was a significant learning curve required. In this study, one of
the observers was a radiologist specializing in United States, and the results
did not differ in a statistically significantly different way from previous stud
ies or from the other users in the study (38). Another study compared interuser
variability between four radiation oncologists,
two physicists, one radiation therapist, and one urologist who were all novices
at BAT ultrasound (36). The standard deviations between ultrasound- and fiducial
-based alignments of these users were comparable. Interobserver uncertainties ha
ve been characterized in other daily localization modalities, such as CT-based a
lignments (39). At Fox Chase, radiation therapists routinely perform ultrasoundbased daily localization. Therapists are initially trained by those with more ex
perience in BAT ultrasound. Intrafraction Prostate Motion Studies Using BAT BAT
ultrasound localization has been performed both prior to and immediately followi
ng radiation treatment to assess intrafraction variability (Table 3). Sources of
measured intrafraction motion include changes in rectal and bladder volume, pat
ient respiration, patient motion, and intrinsic measurement errors. In one repor
t, 20 men had pre- and post-treatment BAT images analyzed on 10 treatment days f
or a total of 400 BAT alignments (40). The percentage of measurements within 5 m
m in the A-P, S-I, and L-R axes were 99, 99.5 and 100, respectively. No differen
ce in shift was necessary 70% to 80% of the time (depending on the axis). Tricht
er et al. found that shifts of 0.2 cm or greater were only required 17%, 7%, and
10% of the time in the A-P, S-I and L-R axes, respectively (35). The magnitude
and standard error of intrafraction ultrasound shifts are smaller than interfrac
tion shifts (Tables 1 and 3). Intrafraction variability with ultrasound localiza
tion is similar in magnitude to intrafraction variability using gold seed fiduci
al markers (29,35,40).

Table 3 Intrafraction Shifts Using BAT Ultrasound in the Treatment of Prostate C

ancer Study Huang et al. (2002) Trichter et al. (2003) Number of patients 20 26
A-P mean SD (mm) 0.2 1.3 0.0 0.32 S-I mean SD (mm) 0.1 1.0 0.04 0.48 R-L mean S
(mm) 0.01 0.4 0.02 0.28

Abbreviations: BAT, B-mode acquisition and targeting; A-P, anterior-posterior; S

-I, superior-inferior; R-L, right-left.
IGRT in Prostate Cancer: Focus on BAT Ultrasound
109
Reproducibility Over Time Chandra et al. examined the temporal pattern of 3228 c
ouch shifts over a one-year period at MD Andersen Cancer Center (34). Over time,
there was a statistically significant increase in the standard deviation of cou
ch shifts in the A-P and S-I axes, but not in the L-R axis. Although the impact
of this increased variation on outcome is unclear, it seems that the radiotherap
ists performing the BAT measurements became less concerned about setup precision
over time. Periodic quality review should be performed when using BAT ultrasoun
d for daily localization. At Fox Chase, BAT ultrasound images are reviewed by th
e attending physician every day to verify the quality of the daily alignments. T
he importance of initial setup and alignment should be emphasized to BAT ultraso
und operators. The Effect Of Ultrasound Probe Pressure on Prostate Motion Altera
tions in prostate localization resulting from ultrasound probe pressure have bee
n examined. Precedence for ultrasound pressure perturbations of anatomic structu
res exists in the ophthalmology literature (41). McGahan et al. used a specially
designed pelvic phantom with a waterfilled balloon representing the bladder and
rectum and a central, encapsulated sphere representing the prostate to measure
organ motion with differing amounts of probe pressure (42). With increasing amou
nts of pressure, the sphere moved approximately 1 cm, a quantity greater than th
e variation of interfraction couch shifts from BAT ultrasound (Table 1). The aut
hors recommend minimizing ultrasound pressure to prevent large errors in ultraso
undbased daily localization. BAT ultrasound operators should use ample ultrasoun
d gel to guarantee good contact between the probe and skin and reduce the pressu
re needed to obtain high-quality images. The effect of ultrasound probe pressure
has been directly characterized in patients. Trichter and Ennis examined prosta
te motion in two patients with brachytherapy implants before and after BAT ultra
sound (35). Based on implant seed displacement, the largest shifts observed were
0.14 cm in the S-I axis and 0.17 cm in the A-P axis in patient 1 and 0.33 cm in
the S-I axis and 0.17 cm in the A-P axis in patient 2 (35). Artignan et al. obs
erved prostate motion in ten patients with increasing ultrasound probe pressure
(43). With an abdominal displacement of 1.2 cm (the amount of pressure required
for a high-quality image), the average prostate displacement was 3.1 mm. Althoug
h these numbers are small, ultrasound probe pressure can result in shifts in pro
state localization.
CLINICAL OUTCOME The theoretical benefit of daily image guidance with any IGRT m
odality is the ability to maximize dose to the target tissue and minimize dose t
o surrounding critical structures by accounting for variability in organ motion.
No prospective randomized trials have been performed to specifically address th
e role of BAT ultrasound IGRT in the management of prostate cancer. However, the
re is retrospective data that supports ultrasound IGRT as an effective tool to m
aximize target dose while minimizing toxicity to adjacent structures. Kupelian e
t al. at the Cleveland Clinic compared biochemical relapse-free survival (BRFS)
and toxicity in short-course intensity-modulated radiotherapy (SCIM-RT) with 70
Gy in 28 fractions with 3DCRT with 78 Gy in 39 fractions (44). The SCIM-RT patie
nts had daily localization with BAT ultrasound, whereas the 3DCRT patients did n
ot. Although the delivery methods differed (hypofractionated IMRT vs. standard f
ractionated 3DCRT), the results suggest that BAT ultrasound allows for the safe
delivery of high-dose radiotherapy with low levels of toxicity. Five-year BRFS w
as 94% versus 88% in the SCIM-RT and 3DCRT groups, respectively. Grade 23 rectal
toxicity (early and late) was 5% in SCIM-RT (with BAT) versus 12% 3DCRT (without
BAT), and grade 3 late toxicity was 2% in SCIM-RT versus 8% 3D-CRT. One would e
xpect hypofractionated radiotherapy to result in greater toxicity. The extent of
contributions from technique (IMRT vs. conformal RT) and BAT ultrasound localiz

ation cannot be quantified from the Cleveland Clinic experience. However, this d
ata suggests that BAT ultrasound allows for reduced toxicity in dose-intensified
fractionation schemes. Use of external beam radiation fields that conform to th
e shape of the target improves biochemical control in prostate cancer by facilit
ating dose escalation through increased sparing of normal tissue. By correcting
potential organ motion and setup errors, ultrasound-directed stereotactic locali
zation is a method that may improve the accuracy and effectiveness of conformal
technology. The application of this technology to conformal techniques will allo
w the reduction of treatment margins in all dimensions. This should diminish tre
atment-related morbidity and facilitate further dose escalation and improved can
cer control.
SUMMARY OF PERTINENT CONCLUSIONS
l
BAT ultrasound is a noninvasive, reproducible, and time-efficient method of dail
y localization for IGRT in the management of prostate cancer.
110
l
Silverman and Horwitz
l
l
l
l
BAT ultrasound is used to align the patients anatomy prior to treatment with cont
ours designated from CTor MR-simulation images and gives the necessary couch shi
fts. Interfraction variation in couch shifts from BAT ultrasound has been charac
terized in several single institution experiences. The magnitude and standard de
viation of couch shifts between treatments is consistent with studies of prostat
e motion. The robustness and reliability of BAT ultrasound is supported by studi
es of intrafraction and interobserver variations in couch shifts and comparison
studies against other daily localization modalities. Quality control over time s
hould remain an integral part of a clinical program that uses BAT ultrasound for
IGRT in the management of prostate cancer. While no multi-institutional, prospe
ctive, randomized trials have specifically compared treatment with and without B
AT ultrasound using the same fractionation scheme for prostate cancer, a trial b
y Kupelian et al. and other clinical experiences support the use of BAT ultrasou
nd to allow for dose escalation with acceptable toxicities.
8.
9.
10.
11.
12.
13.
14.

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11
Image-Guided Radiation Therapy in Prostate Cancer: William Beaumont Experience
MICHEL GHILEZAN, DI YAN, AND ALVARO MARTINEZ
Department of Radiation Oncology, William Beaumont Hospital Cancer Center, Royal
Oak, Michigan, U.S.A.
INTRODUCTION During the 1980s, significant advances occurred in the planning pro
cess of radiation therapy. Three-dimensional (3D) planning was developed and int
roduced in the clinic as an effort to improve the treatment efficacy. In the ear
ly nineties, at William Beaumont Hospital, we started working on the development
of (i) a system for imaging the target before delivery of radiation treatment to
obtain a high-quality image of the day with soft tissues details versus bony anato
my details, as commonly produced by portal imaging devices and (ii) a process fo
r adapting the beam aperture to correct the individuals daily geometrical and tem
poral variations. The success of 3D conformal and of intensity-modulated radiati
on therapy (IMRT) relies on the accurate delivery of radiation dose. Unfortunate
ly, patient and organ treatment position variation inevitably exists. Errors in
patient positioning, intertreatment and intratreatment variation of organ positi
on, and uncertainty in target localization result in variation of the dose deliv
ered. While it is difficult, if not impossible, to completely eliminate all sour
ces of variation clearly, all efforts must be made to minimize their effects whe
never possible. A major source of treatment uncertainty is the variability in th
e daily setup of the patient. The common practice to minimize setup error is by
evaluating port film
113

weekly, which is helpful but insufficient (1). The use of immobilization devices
is also common and should, in theory, enhance the reproducibility of daily pati
ent setup. However, studies of their effectiveness for the patient treated on th
e thoracic and abdominal regions have produced variable results and are dissatis
fying (25). Two new approaches to compensate for or reduce the magnitude of treat
ment setup error are being developed. The first incorporates generic setup error
, characterized from measurements made of the patient population, into the initi
al treatment planning (69). The variability of the dose distribution due to setup
error is presented to the clinician so that compromises can be made prior to th
e initiation of treatment. In the second approach, decision rules for setup adju
stment are implemented to reduce the magnitude of setup error and to minimize th
e frequency of patient repositioning (1015). The decision rule approach requires
more frequent portal imaging and complements the use of electronic portal imagin
g devices (EPIDs). However, the setup adjustment is not based on treatment plann
ing information, and the potential changes in the dose delivered to the individu
al patient are not considered. More importantly, both approaches employ average
values derived from population study as criteria for plan evaluation or setup ad
justment. With population-averaged parameters, the opportunities to optimize the t
reatment for the individual patient are not exploited.
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Ghilezan et al.

ADAPTIVE RADIATION THERAPY: BASIC PRINCIPLES, PROCESS IMPLEMENTATION, AND WORK F

LOW Optimal treatment delivery is best made on an individual patient basis. Such
assertion can be supported by the following analysis of measured setup error of
a group of individual patients and that of the patient population. When a seque
nce of daily portal images have been acquired for the individual patient, setup
error can be characterized in terms of the mean, mi, and the standard deviation,
si, of the daily setup error. The former represents the systematic error and the
latter the random error of the treatment setup. From a two-dimensional (2D) portal
image, these parameters can be characterized on each of two coordinate directio
ns. For example, for an anterior-to-posterior (AP) treatment, the setup error wo
uld be measured along the lateral (LAT) and superior-toinferior (SI) directions.
If the analysis were performed for a group of individual patients for a specifi
c treatment site, one could further calculate a mean, M (mi), and standard devia
tion, s(si), of the systematic setup errors for these patients. Similarly, M (mi
) and s(si) can be calculated to represent the root-mean-square and standard dev
iation of random setup errors for these patients. When only a few portal images
are available for each patient, as in the case with weekly port film exposure, i
t becomes necessary to pool the setup measurements of each individual patient in
to a larger data sample for analysis. The setup error of the patient populations
for a specific treatment site can then also be characterized on each of two coo
rdinate axes by the mean, Mp, and the standard deviation, sp. It has been shown
that the population mean, Mp, is exactly equal to M (mi), the mean systematic se
tup error for all individual patients. However, the population standard deviatio
n of the setup error, sp is equal to M 2 si s2 mi 1=2 . It includes not only the con
ribution from the root-mean-square of the random setup error of the individual p
atient, but also the standard deviation, or variability, of the systematic setup
error of each patient. The latter once detected, in theory, can be eliminated.
It follows then that sp gives a conservative overestimation of setup error of th
e individual. When it is used to design a margin for setup variability, as in th
e case with port film data, sp is larger than necessary for the individual patie
nt, provided that both systematic and random setup errors could be predicted, an
d the systematic error could be corrected during the course of treatment. The ab
ove analysis shows that a major deficiency in the approach to compensate for set
up error is the lack of incorporation of individual patient uncertainties and var
iability. Setup error of an individual patient could be either overcompensated or
undercompensated in the initial treatment plan. As a result, outcome analyses f

rom clinical
trials of 3D conformal therapy, IMRT, and dose escalation may not achieve the tr
ue potential in terms of increasing local control and decreasing toxicity. It sh
ould not be assumed that every patient would be a suitable candidate for dose es
calation. A patient whose treatment exhibits a highly reproducible setup and tar
get position might be one such candidate. Conversely, a patient with large posit
ion variation might be more suitable for conservative management (15). Unfortuna
tely, much of the patient-specific variation is not known until after initiation
of treatment. Therefore, we have introduced in the mid 1990s a new approach, na
med adaptive radiation therapy (ART), to minimize the deleterious effects of set
up variation, organ motion, and deformation on each individual patient (1517). A
retrospective study was used to demonstrate the feasibility of the ART process.
In the study, treatment plan of the individual patient was adaptively modified p
art way through the treatment based on the time course of setup variation predic
ted from portal measurements made of the earlier treatment fractions (17). By ta
king the advantage of a computer-controlled multileaf collimator (MLC), a second
improved treatment plan was calculated and implemented by adjusting the treatme
nt field shape and prescription dose, simultaneously (18). The ART process is ou
tlined as follows: (i) The patients initial 3D treatment plan is first optimized
and delivered using MLC. (ii) Daily setup error is measured using an EPID and ch
aracterized early on during the treatment course. (iii) The treatment plan is ev
aluated incorporating the characterized setup error to decide if and what modifi
cation is necessary. (iv) The treatment plan is modified accordingly for the rem
aining treatments by reshaping the MLC field and adjusting the prescription dose
(16,17). Results from our first ART study indicate that much could be gained wi
th the patient specific ART approach to improve setup accuracy (17). Our simulat
ed treatments of 30 patients with the ART showed that about one-third of the pat
ients could have their prescription doses escalated by as much as 15%. The maxim
um cumulative dose reduction in the clinical target volume (CTV), as percentage
of prescribed dose, due to internal target motion and organ deformation captured
by serial CT measurements is shown in Figure 1. The first five CT scans were su
fficient to predict the maximum cumulative dose reduction in the CTV. Equally im
portant, we showed that up to 20% of the patients would not be qualified for a h
igher dose escalation because of the large position variation (19). Clinical pro
tocols, which escalate dose with a concomitant generic reduction of the setup ma
rgin, might not produce the desirable proof of the efficacy of high dose 3D conf
ormal therapy including IMRT plans. A more effective approach to implement highdose conformal therapy would be to start off an individual patient with a simple
r treatment strategy, which would then be modified in accordance to
IGRT in Prostate Cancer: William Beaumont Experience
115
Figure 1 The maximum cumulative dose reduction in the CTV, as percentage of pres
cribed dose, due to internal target motion. The calculations are based on a 3D c
onformal four-field box treatment. The CTV is considered, alternately, as the pr
ostate and seminal vesicles (A), and the prostate only (B). The error bars indic
ate the mean dose reduction and one standard deviation thereof for a population
of 30 patients. The dashed lines indicate the bound on the dose reduction achiev
ed for 80% of patients ( p 0.08). Abbreviation: CTV, clinical target volume.
the characteristics of the treatment variation measured during the early phase o
f the treatment course. The systematic and random setup errors of the treatment
after adjustment were compared with those prior to the adjustment. The residual
systematic error after adjustment was used to evaluate the accuracy and reliabil
ity of the ART process. Furthermore, the difference between the random setup err
ors before and after adjustment was used as a measure of the stability of the AR
T process. Recently at the American Society for Therapeutic Radiology and Oncolo

gy (ASTRO) meeting, we presented the quality control results of image-guided ada

ptive radiotherapy (IGART) from 1999 to 2006 for prostate cancer in 1017 patient
s with clinically treated T1-3 tumors. Only 96 patients or 9.4% of patients requ
ired a second modification of the MLC configuration including 63 corrections of
the residual systematic error in one single direction. In addition, only 5 of 10
17 patients required readjustment of the target margins due to a larger random s
etup error (20). Finally, patient-specific setup margins were calculated to demo
nstrate the potential advantages of the ART process in pursuing treatment dose e
scalation. Treatment setup margins for each patient were determined when a confi
dent prediction was achieved. The setup margins were calculated by adding the pr
edicted systematic error to twice the predicted random setup error for each coor
dinate direction of the treatment field if no adjustment was applied, or if an a
djustment was applied, by adding 1 mm (tolerance of the prediction for the syste
matic error) to twice the predicted random setup error. Implementation of the AR
T process requires a new clinical infrastructure in which the clinical treatment
procedures such as treatment
116
Ghilezan et al.
planning, treatment verification, treatment evaluation, and treatment adjustment
are not performed as independent tasks. Instead, a closed-loop treatment proces
s will be used to apply the patient-specific information measured during the tre
atment course to reevaluate and to reoptimize the treatment plan. An optimal way
to implement this feedback process integrates new technologies such as a 3D tre
atment planning system, an online imaging device, and MLC through information an
d control network. With the ART process, the physician could potentially act as
a central controller to manipulate a patients treatment using a computer network.
Modification of the treatment field shape using MLC makes the ART process possi
ble to correct for the systematic error and to compensate for the random setup e
rror by only reshaping the MLC once. Most importantly, reshaping the treatment f
ields allows one to compensate for any target shape change due to a distortion o
f the patient position or internal organ motion. Moreover, adjusting the MLC fie
ld allows accurate adjustment as small as 0.2 mm, minimizes the possibility of un
settling the patient, and reduces the workload of the therapists. However, althou
gh reshaping the treatment field alone can be used to compensate for the target
shape change on the beams eye view plane due to 3D out-of-plane rotation, it may
not be acceptable when a specific setup of conformal therapy has to be precisely
kept to avoid a critical adjacent organ. In this case, adjustments in the ART m
ay also need to include the gantry or treatment couch rotation. On each of the f
irst four days of treatment, a CT scan encompassing the entire bladder to below
the ischium is obtained, and the daily CTV is contoured to assess treatment targ
et motion. In addition, a daily portal image for each treatment field is taken u
sing an EPID, and daily setup error is measured. The patient-specific data of da
ily target motion and setup error are exported to a prediction model to form the
confidence limited planning target volume (cl-PTV). Then, the individual treatm
ent plan is modified to deliver the prescribed dose to the cl-PTV by either cont
inuing the four-field treatment or using IMRT for the remaining treatment (18,19
). To summarize, ART is a treatment process (Fig. 2), which adapts the treatment
plan to patient-specific geometrical and temporal variations. Each patient init
ially undergoes a conventional CT simulation followed by 3D planning. A generic,
but transitory PTV is constructed defined as the CTV plus a 1 cm uniform margin
automatically expanded in 3D. A standard four-field conformal treatment plan wi
th adequate beam aperture is designed to ensure that the generic PTV is covered
by the prescription dose. Daily megavoltage image (MVI) portal and CT images are
acquired with each of the first four days treatments without contrast. These im
ages, documenting daily field placement and spatial organ positions, are used

as treatment feedback and are entered into a prediction model to estimate the sy
stematic variation in daily patient setup and target motion. By taking into cons
ideration the subsequent treatment delivery system (CRT or IMRT), the cl-PTV is
constructed, subject to a predefined targeting dose tolerance, on the basis of t
he random variation of internal target motion and setup error obtained from the
treatment images (18). Using the cl-PTV, a new treatment plan is then generated,
and a prescription dose is selected based on the constraints for dose-limiting
structures in the protocol, currently the rectal wall and bladder, as captured f
rom the initial planning CT images. The subsequent treatment modality in the new
treatment plan could be either a CRT or IMRT. IMRT is selected if the prescript
ion dose could be increased by at least 5% when compared with CRT while meeting
the same normal tissue constraint limits. The new beam apertures, which included
adjustments for the predicted systematic variation, are transferred to the trea
tment machine and applied for the remaining treatments. Our protocol also includ
es weekly CT scans on all ART patients for quality assurance purposes (20). DOSE
ESCALATION USING THE ART PROCESS We performed a prospective study to determine
if the ART process improved the capability of dose escalation. For each patient,
two conformal four-field plans were created, with respect to the generic PTV (C
TV 1 cm) and the cl-PTV, respectively. The corresponding prescription doses were
selected on the basis of the dose-volume constraints on the rectal wall and bla
dder manifested on the initial planning image. In addition, inverse planning was
also performed with both the generic and cl-PTV, respectively, for the first 30
patients treated with the ART process. The minimum and maximum doses in the PTV
(the objective function) were adjusted so that the maximum prescription dose wa
s obtained within the given constraints and the dose heterogeneity (15%) limitat
ion in the PTV. The following constraints had to be met: No more than 5% of the
rectal wall volume receives a dose greater than 75.6 Gy, No more than 30% of rec
tal wall volume receives a dose greater than 72 Gy, No more than 40% of rectal w
all volume receives a dose greater than 65 Gy, No more than 50% of bladder volum
e receives a dose greater than 75.6 Gy, Maximum dose in the bladder is less than
or equal to 80 Gy, and A dose inhomogeneity in the PTV of less than or equal to
10% of the prescription dose for conventional conformal treatment and less than
or equal to 15% for IMRT treatment.
IGRT in Prostate Cancer: William Beaumont Experience
117
Figure 2 (A) Flow chart depicting the ART process. The process performs off-line
analysis of the setup error and organ motion occurring during the first five da
ys of treatment. These characterizations are used to predict variations over the
remaining fractions, and the cl-PTV is designed to provide dosimetric coverage
of the nonsystematic components of these variations. Systematic components are r
emoved via commensurate block aperture shifts during replanning. (B) Illustratio
n of (a) the generic PTV (left) and cl-PTV (right) of a single patient on the sa
gital CT image, and the corresponding treated volume (gray shade) on the beam ey
e view DRR; (b) the corresponding DVHs of PTV (black), rectal wall (dark gray) a
nd bladder (light gray) for the generic PTV (dashed curves) or cl-PTV (solid cur
ves). The solid dots on the curves indicate the dose/volume constraints. Abbrevi
ations: ART, adaptive radiation therapy; cl-PTV, confidence limited planning tar
get volume; DRR, digitally reconstructed radiography; DVH, dose-volume histogram
s.
After completing treatment and reviewing the acute toxicity of the first
ents, these restrictions were extended to: No more than 5% of the rectal
lume receives a dose greater than 82 Gy, No more than 30% of rectal wall
receives a dose greater than 75.6 Gy, No more than 50% of bladder volume
s a dose greater than 75.6 Gy,

40 pati
wall vo
volume
receive

Maximum dose in the bladder is less than or equal to 85 Gy, and A dose inhomogen

eity in the PTV of less than or equal to 10% of the prescription dose for conven
tional conformal treatment and less than or equal to 15% for IMRT treatment. We
analyzed factors predictive of rectal toxicity and Vargas et al. published (21)
on the first 331 consecutively treated patients with image-guided off-line corre
ction with
118
Ghilezan et al.
adaptive high-dose radiotherapy (IGART). The chronic rectal toxicity grade !2 ri
sk was 9%, 18%, and 25% for the rectal wall V70 <15%, 25% to 40%, and >40%, resp
ectively. In addition, the volume of rectum and/or rectal wall irradiated to gre
ater than or equal to 50% was also a strong predictor of rectal damage. The use
of androgen deprivation was not found predictive. We were the first ones to publ
ish that patients experiencing acute rectal toxicity are more likely to experien
ce chronic toxicity (21). In a subsequent publication also from Beaumont, Vargas
et al. (22) reported on prostate cancer patients treated with IGART and the use
of dose-volume constraints to achieve rectal isotoxicity. High doses (79.2 Gy)
to the prostate were safely delivered by our IGART process. Under the rectal dos
e-volume histograms (DVH) constraints for the dose-level selection, the risk of
chronic rectal toxicity is similar among patients treated to different dose leve
ls. Therefore, rectal chronic toxicity rates reflect the dose-volume cutoff used
and are independent of the actual dose level (22). More recently, Harsolia et a
l. (23) from Beaumont published the chronic genitourinary (GU) toxicity observed
on these 331 consecutively treated patients with IGART. They were clinically st
age II to stage III prostate cancer patients and were assessed using the Nationa
l Cancer Institute Common Toxicity Criteria 2.0. The three-year rates of grade !
2 and grade 3 chronic urinary toxicity were 17% and 3.6%, respectively. Acute ur
inary toxicity and bladder wall dose-volume end points are strong predictors for
the development of subsequent chronic urinary toxicity. We recommend to limit t
he bladder wall V30 to less than 30 cc and V82 to less than 7 cc (23). In genera
l, the prescription dose was 92% to 93% of the isocenter dose. Figure 2A depicts
the beam aperture and treated volume for both the generic PTV and the clPTV of
a single patient with conformal four-field plans. The corresponding DVH for the
target (PTVs) and normal organs, as well as the dose/volume constraints are illu
strated in Figure 2B. In addition, for the first 30 patients, the potential for
target miss and normal tissue overtreatment in the conventional treatment proces
s was also evaluated by comparing the conventional generic PTV to the cl-PTV. Th
e first prospective dose-escalation study using the ART process demonstrated tha
t the prescription dose level for the majority of the patients could be increase
d up to 10% (mean 5%) or 1.8 to 7.2 Gy (mean 3.6 Gy) when compared to the conven
tional treatment process. That level could be further increased to 5% to 15% (me
an 7.5%) or 3.2 to 10.8 Gy (mean 5.4 Gy), when the IMRT delivery was combined wi
th the ART process. In that case, a minimum cl-PTV dose of 81 Gy [i.e., 86.7 Gy
at the International Committee on Radiation Units and Measurements (ICRU) isocen
ter] could be prescribed to at
least 50% of the patients. In contrast, there was no clear advantage on the dose
escalation when the generic PTV (CTV 1 cm margin) was applied for the IMRT trea
tment, except when deliberately blocking the rectal wall within the PTV (19). Ou
r study indicated that at least 10% of patients could have potential deficiency
of their treatment volumes, when treated in the conventional treatment process.
In that process, a generic PTV has to be designed with a predefined confidence l
evel regarding the patient/ organ geometric uncertainty, and this confidence lev
el has been commonly selected to be 90%. We expanded the study, and Wloch et al.
(20) presented at the 2007 ASTRO meeting in Los Angeles the result on 263 patie
nts treated with IGART who had six or more CT scans during the course of treatme
nt during 1999 and 2003. Only 12 of 263 patients or 4.6% has a portion of their
seminal vesicle missed with a range of 2 to 22 mm and average of 6.1 mm. Equally

low, 5 of 263 patients or 1.9% had a portion of their prostate missed with a ra
nge of 2 to 9 mm and average of 6.3 mm. The dosimetric effect on missing dose to
the PTV was assessed in these eight patients. In three patients it was less tha
n 1%, in three patients less than 2.0%, in one patient 3.8%, and in the remainin
g patient 7.3% of the prescribed dose (20). DEVELOPMENT OF THE CONE-BEAM-BASED I
GART AT WILLIAM BEAUMONT HOSPITAL Even though in 90% of the patients the prescri
ption dose could be increased safely, the treatment quality of a small group of
patients (510%) cannot be assured in spite of application of ART with or without
IMRT. The ART process identifies this group of patients for which the dose shoul
d not be escalated above conventional levels, due to the large variations in CTV
position observed during the treatment course. Special treatment intervention s
uch as daily-based image guidance was felt as being needed in this group of pati
ents. The identification of these patients is paramount to keep complication rat
es low, and thus improve the therapeutic ratio when very high doses are to be de
livered. Therefore, our research efforts focused on including the position distr
ibution of rectum/bladder in the off-line planning optimization and ultimately o
n real-time, online-based image guidance, where the daily target and normal orga
ns in each patient are assessed immediately prior to treatment. Dose reconstruct
ion and planning optimization are then performed online, based on target and nor
mal tissue positions obtained over time. The desire to further reduce the planni
ng target margins around the CTV has emphasized the treatment-related uncertaint
ies such as, daily positioning and setup errors, interfraction organ motion, and
intrafraction organ motion. Minimizing treatment-related uncertainties has beco
me the intense
IGRT in Prostate Cancer: William Beaumont Experience
119
focus of research in the recent years. Capitalizing on a series of technological
innovations such as the development of amorphous-silicon flat-panel imagers, th
e creation of high-speed cone-beam computed tomography (CBCT) reconstruction har
dware along with the realization of computer-controlled medical linear accelerat
ors, the construction of online CBCT imaging system for IGART has been developed
in our institution (2527,40). Online image guidance should minimize some of the
uncertainties related to routine radiotherapy (positioning and setup errors and
interfraction organ motion). Based on our institutional experience with high-dos
e rate brachytherapy in prostate cancer which appear to indicate a low a/b for p
rostate tumors, IGRT is expected to be used in a hypofractionation regimen with
higher doses per fraction and consequently, longer treatment times. As a prerequ
isite for the translation of the cone-beam image-guidance system into clinical u
se for the treatment of prostate cancer, we studied intrafraction prostate motio
n using serial cine-MR over about 50 minutes in patients in supine radiation tre
atment position. We showed that the motion of the prostate and seminal vesicles
during a time frame similar to a standard treatment fraction is reduced compared
to that reported in interfraction studies. The most significant predictor for i
ntrafraction prostate motion was the status of rectal filling. A prostate displa
cement of less than 3 mm can be expected within 20 minutes of the initial imagin
g for patients with an empty rectum (28). Using 22 image data sets from our ART
prostate database, we analyzed and quantified the theoretical benefit in terms o
f improvement in precision and accuracy of treatment delivery and in dose increa
se with IGART in an ideal setting of no intrafraction motion/deformation. The co
nventional IMRT plan was generated on the basis of pretreatment CT, with a clini
cal target volume to planning target volume (CTV-to-PTV) margin of 1 cm, and the
online IGART plan was created before each treatment fraction on the basis of th
e CT scan of the day, without CTV-to-PTV margin. The inverse planning process wa
s similar for both conventional IMRT and online imageguided IMRT (IG-IMRT). Trea
tment dose for each organ of interest was quantified, including patient daily se
tup error and internal organ motion/deformation. We used generalized equivalent
uniform dose (EUD) to compare the two approaches. The generalized EUD (percentag

e) of each organ of interest was scaled relative to the prescription dose at tre
atment isocenter for evaluation and comparison. On the basis of bladder wall and
rectal wall EUD, a dose-escalation coefficient was calculated, representing the
potential increment of the treatment dose achievable with online IGART as compa
red with conventional IMRT. The average EUDs of bladder wall and rectal wall for
conventional IMRT versus online IG-IMRT were 70.1% versus 47.3%, and 79.4% vers
us 72.2%,
respectively. On average, a target dose increase of 13% (SD 9.7%) could be achie
ved with online IGART based on rectal wall EUDs and 53.3% (SD 15.3%) based on bl
adder wall EUDs. However, the variation (SD 9.7%) was fairly large among patient
s; 27% of patients had only minimal benefit (<5% of dose increment) from online
IGIMRT, and 32% had significant benefit (>1541% of dose increment) (29). Uncertai
nties in Clinical IGART Process The IGART process reduces uncertainties in patie
nt treatment position, but shares rest of uncertainties existing in the conventi
onal treatment simulation, planning, and delivery. Additionally, the specific an
d most significant sources of uncertainties are patient image acquisition and re
gistration, couch or MLC correction, estimation of the systematic and random pos
ition variations, treatment dose construction/evaluation, and adaptive planning
modification, which will be outlined in the following sections. Uncertainties in
Treatment Position Localization The IGART process involves frequent measurement
s of patient anatomical position obtained by comparing treatment images to a ref
erence image of similar modality, i.e., the onboard CBCT image to the reference
planning CT image, or the portal image to the digitally reconstructed radiograph
y (DRR). Deformable organ registration and segmentation have been evaluated usin
g patient image with or without inserting radio markers (30). However, the purpo
se of these studies was rather to validate registration methods instead of clini
cal QA. It would be helpful to have a deformable phantom to test registration me
thod and verify treatment dose construction. Uncertainties in Couch or Beam Aper
ture Correction Couch position and MLC-based beam aperture adjustments have been
the most common means to correct patient treatment position. However, the uncer
tainties associated with each correction method should be determined before a ro
utine application in the clinic. Translational correction accuracy has been achi
eved within 1 mm. However, treatment position corrections using an MLC-based bea
m aperture shift are nontrivial due to the limited leaf width. We recently showe
d in a study of online image guidance and rigid body correction using MLC a maxi
mum of 2% dose discrepancy in the target and a much higher dose discrepancy in n
ormal tissues that could occur in both prostate and head/neck cancer treatment (
31). Beam aperture correction
120
Ghilezan et al.
has also been proposed and tested to compensate for target deformation (18) in c
onformal radiotherapy, and expanded later to IMRT (32). However, so far, the dos
imetric uncertainty caused by multiple leaf segment adjustment has not been full
y explored. Uncertainties in Determining Patient Variation Parameters A unique f
eature of the IGART process is the estimation of the systematic and random error
s of individual treatment position. Since the estimation is performed based on l
imited samples of measurements, uncertainty in parameter estimation is always ac
companied by statistical residuals. The systematic and random errors of treatmen
t setup position have been systematically evaluated for prostate and head/neck c
ancer treatment. Following the estimation, performed at the end of the first tre
atment week, patient setup position has been continuously monitored for addition
al three to five treatment days. If the residual systematic error after the firs
t correction in the prostate cancer treatment was 1 mm (the predetermined cutoff
value) larger than the estimated value, a second correction would be performed.
As we mentioned before, out of 1017 patients only 9.4% required a second correc
tion and 0.07% a third one, which documented how robust the IGART process is (20

). Uncertainties in Treatment Dose Construction and Evaluation At the present ti

me, the individual patient dose tracking and feedback process is under clinical
test and evaluation, instead of routine use. Treatment dose, daily as well as cu
mulative, in organs of interest has been constructed using volumetric image-base
d deformable organ registration (33). Two major factors that have been considere
d in the treatment or 4D dose construction are organ subvolume position displace
ment and variation of patient global density distribution. However, the discrepa
ncy is commonly small (<3%) for dose evaluation in a pelvic region. Treatment do
se in organs of interest can be frequently updated in the IGART process for trea
tment evaluation and planning modification decision. However, uncertainties in i
mage registration and organ delineation affect directly the reliability of using
the dose in treatment evaluation. Study on this aspect, therefore, needs to be
systematically explored. Uncertainties in Adaptive Planning Modification Two typ
ical adaptive planning methods have been explored. One is to determine patient-s
pecific CTVto-PTV margin (18) and then design the corresponding
dose distribution. The other is to design dose distribution to compensate for pa
tient-specific motion directly with adaptive inverse planning (34). Sources of u
ncertainties in the adaptive planning are very similar to those in the conventio
nal planning reported by the Task Group 53. However, the influence could be diff
erent. The influence of organ delineation uncertainty on the accuracy of dose ev
aluation could be reduced because of a potential random effect in the multiple o
rgan delineations. However, the accuracy of dose evaluation becomes more critica
l in the selection of the individual prescription dose. Therefore, IGART quality
control should also include the selection of the most robust planning parameter
s, such as selecting dose/volume constraints on the relative stable portion of D
VH curve for planning evaluation. QA TESTS IN CLINICAL IGART PROCESS Clinical de
velopment of IGART QA should aim at the most significant sources of uncertaintie
s. QA recommendations on image acquisition, anatomical description, dose calcula
tion, and treatment planning have been detailed in American Association of Physi
cists in Medicine (AAPM) Task Group Report 40 and 53. In following recommendatio
ns, we focus only on the additional QA procedures that should be applied to impl
ement a clinical IGART process. QA Verification for Patient Treatment Position A
ssessment and Correction Staff physicists should perform integrated system test
on the image localization and position correction accuracy before starting an IG
ART protocol to determine a baseline reference for routine monthly and daily QA
tests. A therapists/image specialist performs isocenter localization tests daily
for online image guided hypofractionation and monthly for offline image guidanc
e. These tests should also be performed once when corresponding software/ hardwa
re device is updated. Staff physicists perform integrated system tests from the
CT simulation to treatment delivery semiannually. A phantom with embedded radio
markers can be applied for these image localization and position correction test
s. In addition, phantom mounted on a motor-driven motion stage can be used for t
he respiratory correlated CT or CBCT imaging QA. QA Verification for Parameter E
stimation and Treatment Dose Construction The systematic and random errors of th
e individual treatment position are verified in the week after the assessment by
a physicist or dosimetrist and evaluated weekly by a
IGRT in Prostate Cancer: William Beaumont Experience
121 Table 1 Patients Characteristics for 642 Patients Characteristic Age CTV Pret
reatment PSA Gleason score T stage Percent biopsy cores Dose (minimum to PTV) Do
se (isocenter) RT fractions Follow-up PSA nadir Median (mean) 73 yr 50.6 cc 6.7
ng/mL (9.7) 6 (6.3) T1c 27% 75.6 Gy 79.7 Gy 42 4.6 yr 0.6 ng/mL Range 5187 18.3214
.3 0.2120 310 T1aT3c 5%100% 63.079.2 67.085.6 3544 0.17.5 033.5
physician during the entire course of the treatment. It is strongly recommended
that one evaluates the changes in organ volume and the organs center of mass week
ly. Treatment dose to the organs of interest should be evaluated daily for hypof
ractionated radiotherapy and weekly for a normal fractionated treatment by a phy

sicist. If a large dose discrepancy is observed, the corresponding treatment ima

ge and organ contours should be evaluated. QA Verification for Treatment Evaluat
ion and Adaptive Planning The planning CT image should be verified to identify a
nd eliminate the effect of contrast material and unusual events, i.e., bladder c
ontrast and large rectal gas filling, on the dose calculation. The treatment vol
umetric images used in adaptive planning are verified weekly to ensure that they
are appropriated for the 4D treatment dose construction. When a treatment DVH p
arameter is used for treatment evaluation, weekly verification is recommended to
ensure that the parameter is not on the high gradient portion of the DVH curve.
When patient-specific PTV is used in adaptive planning, weekly CT image verific
ation is suggested to avoid unexpected deviation. CLINICAL EXPERIENCE WITH IGART
In 1997 we developed and commenced clinical implementation of the ART process a
t William Beaumont Hospital. In 1999 we initiated an Institutional Review Board
(IRB)-approved phase II prospective dose-escalation/ selection trial in treating
localized prostate adenocarcinoma, taking into account individual patient setup
inaccuracies and internal organ motion (ART process) in the dose prescription.
Hence, a patient-specific PTV was designed, along with the corresponding beam ap
ertures. Each patients dose prescription is selected on the basis of the dose max
ima tolerable according to our specific dosevolume constraints. Patients were el
igible for this protocol if we had tissue confirmation of prostate cancer, all G
leason grades, and clinical stages (T1b-T3, NX-NO, MO). Eligibility criteria for
this protocol included patients with biopsy-proven adenocarcinoma of the prosta
te with a clinical stage of I to III who were diagnosed within 24 weeks of enrol
lment. Patients with clinical or pathologic lymph node positivity were excluded
from the study. For patients with a Gleason score of less than or equal to 6, a
PSA less than 10, and a clinical stage T2a or less, the CTV included the prostat
e only (ART group I). For patients with a Gleason score greater than or equal to
7 and/or a PSA greater than or equal to 10 and/or a clinical stage greater than
or equal to T2b, the CTV include the prostate and proximal 2 cm of
Abbreviations: CTV, clinical target volume; PSA, prostate-specific antigen; PTV,
planning target volume; RT, radiation therapy.
seminal vesicles (ART group II). A pelvis field was never used. For the initial
treatment week the PTV included the CTV plus 1 cm generic uniform margin. A tota
l of 900 cGy (minimal prostate dose) was given in 180 cGy fractions with 18 MV p
hotons for the first week. All acute and chronic side effects were recorded usin
g the Common Toxicity Criteria (CTC) version 3. The worst score seen at any time
was used to score chronic toxicity. Patient characteristics are reported in Tab
le 1. Before reviewing our chronic toxicity data (Tables 24), it is important to
reiterate that using the adaptive process the PTVs are often smaller than those
using standard margins of 0.5 to 1.0 cm about the CTV without compromising CTV c
overage. By decreasing the treatment volume one also treats less of the normal d
ose limiting tissues. This potentially allows a decreased complication rate whil
e maintaining a similar treatment dose or allows escalation of the treatment dos
e while maintaining similar toxicity. In our dose-escalating trial, we elected t
o raise the total dose while maintaining isotoxicity. In the whole-body hyperthe
rmia (WBH) ART trial, the rectal parameters were set so that less than or equal
to 5% of the rectal wall has a dose greater than 82 Gy and less than or equal to
30% of the rectal wall has a dose greater than 75.6 Gy on the basis of the orig
inal CT set. Others have suggested that not only is the maximum rectal dose impo
rtant in predicting for late toxicity, but perhaps equally important is the inte
rmediate volume of rectum receiving lesser dose. While the trial does not impose
a limit on the dose to an intermediate volume of the rectum, it has been our ex
perience that this dose may become unacceptably high when generating IMRT plans,
unless an auxiliary constraint is introduced into the optimization. The bladder
constraints were set so that less than or equal to 50% of the bladder volume ha
s a dose greater than 75.6 Gy, and the maximum allowed dose to the bladder is le
ss than 85 Gy. The minimum prescribed dose to the

122 Table 2 Grade 1 Chronic Toxicity by Dose Level Dose level (Gy) Urinary incon
tinence (%) Urinary retention (%) Increased frequency/urgency (%) Urethral stric
ture (%) Hematuria (%) Diarrhea (%) Rectal pain/tenesmus (%) Rectal bleeding (%)
Rectal ulceration (%) Rectal fistula (%) Proctitis (%) Rectal incontinence (%)
!70.2 to 8 6 2 4 0 6 72.0 (n 109) 22 0 4 20 0 4 >72.0 to 5 5 2 7 0 2
) 21 0 5 18 0 5 >75.6 to 11 2 6 7 0 4
Ghilezan et al.
79.2 (n 232) 25 0 5 25 0 2

Table 3 Grade 2 Chronic Toxicity by Dose Level Dose level (Gy) Urinary incontine
nce (%) Urinary retention (%) Increased frequency/urgency (%) Urethral stricture
(%) Hematuria (%) Diarrhea (%) Rectal pain/tenesmus (%) Rectal bleeding (%) Rec
tal ulceration (%) Rectal fistula (%) Proctitis (%) Rectal incontinence (%) !70.
2 to 0 2 0 2 0 2 72.0 (n 109) 11 0 6 6 2 8 0 0 0 >72.0 to 0 1 0 75
2 7 0 0 2 1 >75.6 to 1 1 1 1 6 79.2 (n 232) 12 0 2 12 0

Table 4 Grade 3 Chronic Toxicity by Dose Level Dose level (Gy) Urinary incontine
nce (%) Urinary retention (%) Increased frequency/urgency (%) Urethral stricture
(%) Hematuria (%) Diarrhea (%) Rectal pain/tenesmus (%) Rectal bleeding (%) Rec
tal ulceration (%) Rectal fistula (%) Proctitis (%) Rectal incontinence (%) !70.
2 to 0 4 3 0 0 0 72.0 (n 109) 0 2 0 8 0 0 2 0 >72.0 to 0 1 0 0 75.6 (
0 0 1 0 0 0 >75.6 to 0 2 0 2 0 1 0 79.2 (n 232) 0 1 0
IGRT in Prostate Cancer: William Beaumont Experience
123 Table 5 Toxicity (CTC version 3) in 728 Prostate Cancer Patients Treated wit
h A-IGRT 3DCRT (grades 2 3) 34% 5% 0.5% 7% 19% 10% 12% 3% 4% 1% 1% 3% 16% IMRT (
grades 2 3) 30% 2% 2% 2% 5% 8% 8% 0.5% 5% 2% 0% 2% 4%
cl-PTV ranged from 70.2 to 79.2 Gy. We want to emphasize that we are not describ
ing central axis (CA) doses, which are generally 5% higher. Our prescription and
delivered doses are specified to be the minimum dose to the patient-specific cl
-PTV. The ART acute and chronic toxicity are similar within the various dose lev
els used and equal or lower to those seen at other institutions that have been d
ose escalating (3537). However, the prostate gland doses selected by the ART proc
ess were on the high end of the spectrum with isocentric ICRU doses being in the
range of 74 to 84 Gy. This high doses may not be necessary for all patients, pa
rticularly those with low-risk features, as it has been suggested by Horwitz et
al. (38), or for some with the intermediate risks factors as reported by Martine
z et al. (39). Until December 2006, we have treated at Beaumont Hospital more th
an 1400 patients with prostate cancer using the IGART process. To allow for a me
aningful follow-up we are reporting on the initial consecutively treated 642 pat
ients with mean follow-up of 4.6 years. The results reported here demonstrate th
at the margin reduction achieved with the ART-PTV, with the corresponding improv
ement in accuracy and precision of dose delivery, has resulted in significant do
se escalation while maintaining acceptable GU and gastrointestinal (GI) isotoxic
ity levels. We compared our 3D conformal ART technique (556 patients) with the A
RT-IMRT technique (172 patients), and the toxicity data was presented at ASTRO 2
007. The distribution of patients in group I or II was similar in both treatment
cohorts. Median follow-up was 4.3 years versus 2.2 years for the three-dimensio
nal conformal radiotherapy (3DCRT) and IMRT groups, respectively ( p < 0.01). Th
ere were more group II patients (larger treatment fields) treated with IMRT (60%
) versus 3DCRT (51%), p < 0.01. 3DCRT patients experienced significantly higher
acute grade !2 urinary retention (7% vs. 2% for IMRT, p 0.03), as well as higher
acute grade !2 rectal pain/ tenesmus (19% vs. 5% for IMRT, p < 0.01). Chronic G
U and GI toxicity were generally low in both groups (Table 5). However, 17 patie
nts (3%) in the 3DCRT group developed grade !2 chronic urinary retention versus
only one patient (0.5%) in the IMRT group ( p 0.05). More importantly, 86 patien

ts (16%) treated with 3DCRT developed grade !2 chronic rectal bleeding versus si
x patients (4%) in the IMRT group ( p < 0.01). The median time to rectal bleedin
g was 1.0 year for 3DCRT versus 0.9 year for IMRT. The actuarial 1, 2, and 3 yea
r grade !2 chronic rectal bleeding was 6%, 17%, and 18% for 3DCRT versus 3%, 3%,
and 5% for IMRT, respectively ( p < 0.01). In terms of clinical outcome, the bi
ochemical control using the Phoenix definition was over 90% at five years. As se
en in Table 6, there are significant differences in
Treated Acute GU frequency/urgency Dysuria GU incontinence Urinary retention Rec
tal pain/tenesmus Diarrhea Chronic GU frequency/urgency Urinary retention Hematu
ria Urethral stricture Rectal pain/tenesmus Diarrhea Rectal bleeding
p value 0.29 0.15 0.04 0.03 <0.01 0.43 0.12 0.05 0.43 0.10 0.16 0.51 <0.01
Abbreviations: A-IGRT, adaptive image-guided radiation therapy; 3DCRT, three-dim
ensional conformal radiotherapy; IMRT, intensitymodulated radiation therapy; GU,
genitourinary.
outcome between group I (favorable-risk) and group II (intermediate/high-risk) p
atients, as expected. However, the five-year outcomes for group II are very enco
uraging. Online Image-Guided ART-IMRT As a natural extension of the ART process,
our efforts were focused on switching from off-line image feedback to real-time
image feedback where the daily target and normal organs in each patient are rea
ssessed immediately before treatment. When required, dose reconstruction and pla
nning optimization could be performed in real time on the basis of target and no
rmal tissue positions obtained over the course of treatment. An online CBCT imag
ing system developed as a bench-top prototype at WBH has been constructed to gen
erate high-resolution, soft-tissue images of the patient
Table 6 ART PSA Control, 5-Year Actuarial Rates All patients (n 642) OS CSS DFS
BF 87.4% 97.9% 90.2% 8.4% Group I (n 342) 90.3% 98.7% 93.9% 5.1% Group II (n 300
) 83.1% 96.8% 85.1% 12.8% p value 0.019 0.170 0.001 0.001
Abbreviations: OS, overall survival; CSS, cause-specific survival; DFS, diseasefree survival; BF, biochemical failure.
124
Ghilezan et al.
at the time of treatment for the purpose of guiding therapy and reducing geometr
ic uncertainties in the process of radiation planning and delivery. The system i
ncludes a kilovoltage (kv) imaging unit capable of radiography, fluoroscopy, and
CBCT that has been integrated with a medical linear accelerator. Kilovoltage Xrays are generated by a conventional X-ray tube mounted on a retractable arm at
908 to the treatment source. A 41 41 cm2 flat-panel X-ray detector is mounted op
posite the kV tube. The entire imaging system operates under computer control, w
ith a single application providing calibration, image acquisition, processing, a
nd CBCT reconstruction. CBCT imaging involves acquiring multiple kV radiographs
as the gantry rotates through 3608 of rotation. A filtered back-projection algor
ithm is employed to reconstruct the volumetric images (27). To assess the potent
ial of increasing the accuracy and precision of prostate radiotherapy delivery b
y using online image guidance through daily onboard CBCT scans, we designed and
activated a clinical protocol with the objective of establishing IGART as a vali
d treatment modality in prostate cancer. Since better targeting and as a result
tighter PTV margins are possible with IGART, the protocol uses a hypofractionati
on regimen that will shorten the total treatment time from eight and a half to f
our weeks, then to three weeks, two weeks, and finally to one week while deliver
ing an equivalent minimal prostate dose of 75.6 Gy. In the first phase, 20 patie
nts from group I (favorable risk) and 20 patients from group II (intermediate/hi
gh risk) will be enrolled in the study and will be treated with a four-week IGAR

T regimen. Toxicity will be evaluated at six-month interval. Target volume defin

ition for patients in group I and II are according to our current ART protocol,
i.e., prostate gland only for patients in group I and prostate plus proximal two
-third of the seminal vesicles for patients in group II. If acute and late GU an
d GI toxicities are not in excess by more than 7% of toxicity seen with our curr
ent ART protocol, we will proceed with step 2. Twenty patients from both groups
I and II will be enrolled on a three-week IGART regimen and will be followed for
six months. Same toxicity criteria applied in step 1 will be used, namely, acut
e and late GU and GI toxicities should not exceed by more than 7% of the toxicit
y seen with our current ART protocol. If toxicity constraint is met, 20 patients
will be enrolled on a twoweek and finally 20 patients on a one-week IGART regim
en. Patients will be followed for six months with the above-mentioned toxicity c
riteria to be met. If the recorded toxicity is greater than 7% higher than the o
ne expected, there will be no progression to the next level. Under local anesthe
sia, three Visicoil markers, 4 cm in length and 0.35 mm in diameter, are implant
ed into the prostate under trans-rectal ultrasound (TRUS) guidance.
Two Visicoils are placed posteriorly in each prostate lobe and the third one is
placed anteriorly close to the anterior fibromuscular area. Visicoil placements
are performed before the start of radiation therapy. Visicoil markers enable tra
cking prostate motion during treatment as well as prostate displacement between
treatments. All patients undergo a pretreatment MRI that will serve as a templat
e for precise location of prostate gland boundaries, seminal vesicles, neurovasc
ular bundles, penile bulb, bladder, rectum, and the Visicoils. The CTV is define
d as the gross tumor volume (GTV) with a 0-mm margin for risk group I and 2 to 4
mm for risk group II patients. Planning target volume (PTV) is defined as the C
TV with a 3-mm margin to compensate for variability in daily treatment setup and
internal target motion due to rectal/bladder motion or other motion (respiratio
n and muscle contractions) during treatment. Of note, this margin is significant
ly smaller than for the conventional 3DCRT and ART because the online correction
component will account for the setup inaccuracies and interfraction organ motio
ns. Treatments are given five days per week. The prescription dose is 3.2 Gy pre
scribed to the isocenter, in 20 fractions, for a total dose of 64 Gy with a norm
alized total dose (NTD) of 79.4 Gy. The total number of fractions is 20, 15, 10,
and 5 in the four-, three-, two-, and one-week treatment groups, respectively.
For the three-, two-, and one-week treatment groups, the prescription dose is 3.
9, 5.1, and 7.8 Gy per fraction, respectively. Seven-field IMRT is used with the
dose prescribed at the PTV (minimum 95% coverage of prescribed dose).
Table 7 Weeks of RT Prostate, a/b 4 Gy Number of fractions Dose/fraction (Gy) To
tal dose (Gy) NTD (Gy) 95% of NTD Bladder, a/b 2.5 Gy <30% volume @ 75.6 Gy 0% v
olume @ 85 Gy
4 20 3.2 64 79.4 75.5 59.4 64.1
3 15 3.9 58.5 79.7 75.7 53.6 57.6
2 10 5.1 51 80.0 76.0 45.9 49.2
1 5 7.8 39 79.3 75.4 34.5 37.0
Before each treatment the patient undergoes a CBCT scan with images reconstructe
d online. This process takes approximately one to two minutes, and the imaging d
ose is under 3 cGy. The registration between the CT image of the day obtained with
CBCT to the planning CT images is made automatically based on Visicoil markers.
Simple positional corrections (rotations and translations) are made to ensure o
ptimal CTV coverage. After the treatment is delivered another CBCT is obtained t
o verify the
IGRT in Prostate Cancer: William Beaumont Experience
l

125
l
On average, a target dose increase of 15% can be achieved with online IG-IMRT ba
sed on rectal wall EUDs and 48% based on bladder wall EUDs. Our excellent result
s in terms of clinical outcome and toxicity profile underline the strong clinica
l impact that image-guidance and the ART process have in the accuracy and precis
ion of radiation therapy delivery.
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SUMMARY OF PERTINENT CONCLUSIONS
l
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A large generic PTV has been the typical way of addressing interpatient and inte
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12
IGRT in Prostate Cancerthe Calypso 4D Localization System
ARUL MAHADEVAN, KEVIN STEPHANS, AND TOUFIK DJEMIL
Cleveland Clinic Foundation, Cleveland, Ohio, U.S.A.
INTRODUCTION Over the past decade, continuous refinement in the extent of irradi
ated tissue has been the focus of development in radiation oncology. The goal ha
s been to irradiate essentially all cancerous cells and exclude surrounding norm
al tissue to maximize the therapeutic ratio. Advances in imaging to facilitate t
arget delineation and enhanced treatment planning techniques like intensity-modu
lated treatment planning (IMRT) have led to the development of enhanced beam del
ivery methods like image-guided radiation therapy (IGRT). Further refinements ar
e now largely limited by the changes in position of target, not just day to day
but also during treatment. Target motion management during the course of radiati
on therapy (RT) has thus become a topic of heightened interest among radiation o
ncologists. Tracking and adjusting to target motion during the delivery of fourdimensional radiation therapy (4D-RT) is a frontier in RT that offers substantia
l advantages for dose escalation and avoidance of normal tissues. 4D-RT offers t
he ability to record and adjust to both interfraction and intrafraction motion.
It adjusts for interfraction variables such as rectal and bladder filling as wel
l as changes in a patients body surface or surface markers on a daily basis. Intr
afraction motion is caused by breathing, bowel motility, and muscle tone. Bowel
motility and gas patterns appear to be affected by rectal filling (1). Furthermo
re,
127
bladder filling and rectal motility are likely altered over the course of RT due
to acute effects of treatment. Following and correcting for these changes over
the course of treatment would allow for more accurate target localization, and a
lso a better understanding of how to design margins for standard, static RT. Ins
ufficient dose to the cancer bearing area of the prostate (2) may occur if targe
t motion is ignored during treatment delivery. In addition, real-time organ trac
king allows for the calculation of the dose actually delivered to the target acc

ounting for any motion that was observed. Thus, dynamic dose reconstruction woul
d allow for fine-tuning of future fractions to compensate for the influence of m
otion on already-delivered fractionsadaptive RT (3). The margins established for
RT often rely on populationbased margins, which attempt to ensure that at least 90
% of patients receive a minimum of 95% of the prescribed planning target volume
(PTV) dose (4). In any normal distribution of patients, this would cause some to
be underdosed while others to be treated to a wider target than necessary. Both
of these shortcomings are improved with four-dimensional radiation delivery. Ev
idence mounts that increased radiation dose improves cancer outcomes. Several re
trospective (5,6) and prospective randomized trials have been reported recently
addressing RT dose escalation in prostate cancer (710). Peeters et al. (8) report
ed their results on a randomized phase III trial conducted in four Dutch
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Mahadevan et al.
institutions that evaluated the role of escalating the RT dose to 78 Gy from 68
Gy in 669 patients with localized prostate cancer. The five-year failure-free su
rvival (FFS) estimate was 64% in the 78 Gy arm and 54% in the 68 Gy arm (p 0.01)
. The Zietman et al. trial (7) reported a 19% improvement in five-year prostatespecific antigen (PSA) FFS rates (FFS of 80.4% for patients who received 79.2 Gy
vs. 61.4% for those who received 70.2 Gy). This benefit to increased radiation
dose was seen for higher-risk patients (44% risk reduction) as well as lowrisk p
atients (51% risk reduction). The Pollack et al. trial (9) reported an improveme
nt in six-year FFS of 6% (FFS of 70% for patients who received 78 Gy vs. 64% for
those who received 70 Gy). There was a preferential benefit to patients with a
PSA >10 mg/L who had an improvement in FFS of 19%. The PSA outcome benefit came
at a cost of modest increase in genitourinary and gastrointestinal toxicity in t
hese trials. For instance, grade 2 or higher rectal toxicity rates at six years
were 12% and 26% for the 70 and 78 Gy arms, respectively (p 0.001) in the Pollac
k trial (9). For patients in the 78 Gy arm, grade 2 or higher rectal toxicity co
rrelated highly with the proportion of the rectum treated to greater than 70 Gy.
Not only does this demonstrate the benefit of dose escalation to prostate tissu
e, but it also suggests significant clinical benefit to restricting rectal doses
below a dose level commonly delivered to the prostate, and thus can positively
influence the therapeutic ratio. Furthermore, the a/b ratio (which describes the
sensitivity of tissue to radiation fraction size) for prostate cancer is estima
ted to be lower (13 Gy) than values normally associated with most neoplasms (!10
Gy) (1113). This has led to the development of hypofractionated regimens (14) tha
t, to date, have yielded encouraging long-term results and are being compared in
randomized studies to conventionally fractionated regimens (RTOG 0415). Hypofra
ctionated regimens thus have the advantage of addressing the potentially low a/b
ratio of prostate cancer as well as improving patient convenience by reducing t
he number of treatment fractions required. A higher dose per fraction, however,
may introduce more potential for late normal tissue effects and also increase th
e length of each treatment allowing more time for intrafraction motion. Both dos
e escalation and hypofractionation have raised the stakes for our ability to del
iver treatment accurately. There is a fine balance between increasing tumor cont
rol and limiting normal tissue toxicity. Increasing the accuracy of treatment de
livery offers advantages on both fronts. In addition, the utilization of IMRT (d
esigned to improve delivery of dose to target and minimize dose to surrounding s
tructures) substantially increases the treatment time (15). This places further
emphasis on the control of intrafraction variables.
A variety of systems can be utilized to provide information about the intrafract
ion motion of the prostate: transabdominal ultrasound, X-ray, CT, and cine-MRI.
These systems have several disadvantages: they provide only snapshots of prostat
e position; they are not available during radiation delivery; most require addit
ional ionizing radiation exposure; they are not efficient and are labor intensiv

e; and they are subjective and operator dependant in acquisition and interpretat
ion. Strategies such as gating afford the ability to turn the beam on and off ba
sed on target location and offer a simple solution to treating only when the tar
get is located within the radiation beam pathway. The downside to gating is that
it may increase treatment time, and in the prostate it may require manual adjus
tment if the target does not quickly move back into position. Hence this technol
ogy may be more applicable to cyclically moving targets (like lung or liver targ
ets) rather than for targets moving unpredictably (like prostate). Other methods
such as real-time localization based on fiducial markers may simplify the calcu
lation of target localization and allow tracking to occur fast enough for the po
sition of the target to be adjusted continuously during radiation delivery. One
such system is the Calypso1 4D Localization System that utilizes implanted Beaco
n1 electromagnetic transponder system (Calypso System, Calypso Medical, Seattle,
Washington, U.S.) will be described in detail below. THE CALYPSO SYSTEM The Cal
ypso System consists of the following components (Fig. 1). Beacon Electromagneti
c Transponders The implantable Beacon1 electromagnetic transponder measures appr
oximately 8 mm in length and 1.85 mm in diameter and consists of a hermetically
sealed biocompatible glass capsule containing a miniature passive electrical cir
cuit made of a small ferrite core and copper coil (Fig. 2). Three transponders a
re implanted in specific regions in the prostate for use as fiducial markers in
RT (Fig. 3). These transponders are passive and echo a signal only when excited
by the nonionizing electromagnetic radiofrequency (RF) radiation generated by th
e system array. The response signal of each transponder is unique and is specifi
c to that transponder. The system uses this signal to determine the location of
the transponder and the location is updated at the rate of 10 Hz. Each Beacon tr
ansponder is packaged in a transfer capsule. Each capsule is color coded and lab
eled for prostate implantation with A for apex of the prostate, L for left base,
or R for right base.
IGRT in Prostate Cancerthe Calypso 4D Localization System
129
Figure 1 (A) Beacon1 transponders; (B) Console; (C) Array; (D) Optical system; (
E) Tracking station.
transponder material, their visibility on megavoltage images depends on the dens
ity of the surrounding anatomic structures. The transponders are MRI safe but wi
ll cause an image artifact around the transponder. The extent of the artifact wi
ll depend on the magnetic strength of the scanner.
Figure 2 Calypso Beacon1 transponder.
4D Console The Calypso 4D Console is a movable unit that is placed in the treatm
ent room next to the treatment table. A mechanical arm attached to the console c
an be extended to allow for manual positioning of the array. It is important tha
t the gantry can rotate freely without collision with the console or array arm.
The computer on the console has a touch screen that provides objective on-screen
graphics and data for QA, calibration, array positioning, and for patient local
ization. Four-Dimensional Electromagnetic Array The array is a flat panel connec
ted to the console by means of an extendable mechanical arm. It contains compone
nts that generate and detect the nonionizing electromagnetic field used to deter
mine the position of the target. The array is positioned over the patient during
setup and is designed to be left in place during treatment. It contains circuit
ry that generates signals to excite the transponders and to receive the response
signal of the transponders. The array also has multiple embedded optical (infra
red) markers for the optical system to determine the position of the array with
respect to the machine isocenter. The array is made of low density, nearly air
Figure 3 (See color insert.) Ideal location of transponders within prostate.

Implanted transponders are visible as opaque structures on kilovoltage X-ray, co

mputed tomography (CT), and ultrasound images. Because of the low density of
130
Mahadevan et al.
equivalent material with peripherally positioned signal and sensor coils so that
the attenuation of the megavoltage treatment beam is equivalent to between 2 an
d 3 mm of water (16). Optical System Three infrared cameras mounted permanently
in the ceiling of the treatment room along with a power supply and a hub constit
utes the optical system. These three cameras provide redundancy in case one of t
he cameras is obscured. As long as half of the optical markers are visible to tw
o of the three cameras, the system has adequate data to continuously monitor the
position of the array without compromising on accuracy. 4D Tracking Station The
4D Tracking Station receives and interprets data from the 4D Console and the op
tical system, which are connected through Ethernet. Information about the target
is presented to the therapist using simple, objective onscreen graphics complem
ented by numerical data and auditory indicators when target moves outside of the
predefined limits. The tracking station is also used for system administrative
functions such as user management, patient records, and daily reports. Treatment
Table Overlay Because of the use of electromagnetic signals by the Calypso Syst
em to locate the target, the system accuracy can be affected by conductive treat
ment tabletops containing metal and/or other conductive materials, including car
bon fiber. Therefore, a table overlay, designed to ensure that the conductive co
mponents are far enough from the transponders so that they do not interfere with
localization, is needed. The tabletop overlay is a nonconductive, rigid surface
and is compatible with standard treatment tables with removable inserts. It con
sists of a headrest, a footrest, and a central gridded part over which the treat
ment area of the patient is positioned. TECHNICAL ISSUES The 4D Electromagnetic
Array consists of four source coils, which generate signals to excite the transp
onders, and 32 sensor coils, which detect the response signals returned by the t
ransponders (17). The source coils generate AC electromagnetic fields in the low
er end of the AM band frequency range (between 300 and 500 kHz). Prior to treatm
ent, the relative polarity of the four source
coils is selected to optimally excite each transponder at its resonant frequency
. Each transponder has a unique configuration and thus is briefly excited at its
distinct frequency by the nonionizing electromagnetic field generated by the so
urce coil. These electromagnetic fields are of long wavelength (low energy) and
thus do not affect treatment delivery. The resonant current amplitude in the tra
nsponder increases during signal excitation and then declines during the respons
e period and is continuously detected by the 32 sensor coils in the array system
. Several hundred repetitive response signals are averaged for each transponder
to increase the signal to noise ratio. The system is able to independently track
each transponder by its distinct resonant frequency. The signal from each trans
ponder at each of the 32 coils is analyzed by the software to solve for the thre
e-dimensional coordinates of each transponder to a measured accuracy of within 0
.5 mm. The relative position of the three transponders can be used to infer the
spatial position and orientation of the target. The 4D Electromagnetic Array is
designed to detect transponders placed within a 14 14 27 cm3 sensing volume dete
rmined to contain the range of common prostate positions from near to the skin s
urface where the array would be positioned on a retrospective review of CT scans
(17). In initial phantom studies, the measured variation in transponder positio
n readout was extremely precise at 80 mm from the source array (a typical distan
ce for a prostate in a thin patient) with a standard deviation of 0.006, 0.01, a
nd 0.006 mm in the x, y, and z directions, respectively (17). Reproducibility de
creased with increasing distance from the array but was still submillimeter at 2
70 mm from the array with a standard deviation of 0.27, 0.36, and 0.48 mm in the
x, y, and z directions, respectively. This introduces a physical limit to the s

ystem. Patients whose dimensions may restrict the array from being placed within
270 mm from the prostate are beyond the approved specifications of the system.
No significant drifts in the location readout were observed in the phantom over
a 20-minute recording period demonstrating stability compared to background elec
tromagnetic interference beyond the time required for real-time organ motion tra
cking. Dynamic accuracy for the Calypso System is defined as the static accuracy
plus the effects of latency, which depend on the target motion. For a typical p
atient having three transponders, the latency to the tracking station screen T i
s less than 0.4 seconds (including all the graphics updates). Therefore, prostat
e moving at velocity V 0.5 cm/sec (although most are slower than this) would hav
e a latency impact of V T *0.2 cm. This is in addition to the static accuracy. S
ubmillimeter accuracy was maintained using the dynamic phantom at speeds of up t
o 3 cm/s (17). The commercial system is optimized
IGRT in Prostate Cancerthe Calypso 4D Localization System
131
for human response times for prostate applications specifically. CLINICAL USE OF
THE CALYPSO SYSTEM Placement of Markers Three transponders are inserted by a ra
diation oncologist or a urologist transrectally or transperineally (under ultras
ound guidance) using a 14-gauge introducer needle assembly, into the apical, lef
t base, and right base regions of the prostate. The technique is similar to obta
ining a prostate biopsy. Similar to gold fiducial placement, midline positioning
of the transponders should be avoided to prevent loss through the urethra. Pati
ents are prepped for implant with antibiotic prophylaxis as well as a same-day e
nema, and the procedure is commonly done using only local anesthesia. Implantati
on generally takes 10 minutes or less from the time of ultrasound probe insertio
n. Similar to gold fiducial placement, patients on anticoagulation or antiplatel
et medications (with the exception of Aspirin) or patients unable to tolerate lo
cal anesthetics due to allergies need medical management before considering the
use of this system. Patients with implanted pacemakers or other signaling medica
l devices should be considered with caution, as the Calypso System utilizes magn
etic fields and compatibility is unknown. Patients with hip replacements or larg
e metal implants like vascular grafts, which are in close proximity to the prost
ate, are contraindications to use of this system. The 4D Electromagnetic Array m
ust be positioned over the region where the transponders are implanted and must
be within 27 cm of the transponders for accurate localization and real-time trac
king. In patients with a significantly large anteroposterior (AP) diameter, the
Calypso System may be used for daily setup localization but not real-time tracki
ng because of geometric constraints between the patients upper surface, the Calyp
so System array, and the gantry head during full gantry rotation. In these patie
nts, the Calypso System is moved away during radiation delivery. This situation
was encountered in 6 out of 41 patients enrolled in a recent study (18). Phantom
studies have shown that target localization is accurate in the presence of eith
er stranded or free radioactive brachytherapy seeds as well as surgical clips (1
9). The accuracy of real-time tracking in the presence of radioactive seeds or c
lips has not yet been demonstrated in patients. Simulation Simulation is commonl
y performed in the supine position as this is associated with increased patient
comfort, increased ease of setup for therapists, and less intrafraction prostate position variation requiring less pretreatment adjustment when com
pared with prone position (20). Institutional standard immobilization devices ma
y be used including a band around the feet, a wedge under the knees, a ring for
the hands, or a Vac-Lock1 bag, though no immobilization devices are required. CT
simulation through the target region should be done with the smallest slice thi
ckness available (1.01.5 mm), as the accuracy of array to transponder localizatio
n is submillimeter (17). Using thicker slice CT acquisition would significantly
decrease the accuracy of any fiducial-based localization system for predicting p
rostate location as accurate fiducial coordinate identification is a critical co

mponent of the localization process. In the case of the electromagnetic transpon

ders, the magnetic center corresponds with the radiographic center of the transp
onders, and a thin slice CT acquisition ensures that the radiographic centers of
the transponders can be accurately identified in the CT images. The image acqui
sition pitch on helical CT scanners must be less than or equal to 2. The skin is
marked in standard fashion (tattoos are commonly used) for initial patient setu
p. Treatment Planning Any treatment planning software can be used to identify th
e transponder and treatment isocenter coordinates. Automatic translation of the
treatment planning coordinate reference frame (CRF) to the Calypso System CRF is
supported for the Philips PinnacleTM, CMS XioTM, and Varian EclipseTM treatment
planning systems (TPS) and for any TPS that uses the IEC-61217 CRF. Coordinates
of the apex, left base, and right base transponders are identified as part of t
he treatment planning process. When the treatment plan is complete, the transpon
der and isocenter positions are recorded and entered into the Calypso System. Pa
tient Setup Prior to the delivery of each fraction of radiation, patients are in
itially set up to their marks (tattoos) from the time of simulation and aligned
to the room lasers. The Calypso System array is then placed over the patient and
activated to localize the position of the transponders relative to the array. T
he infrared cameras detect the position of the array relative to the machine iso
center. The therapist is instructed via a touch-screen PC console which shifts o
f the treatment couch in the AP, lateral, and craniocaudal directions are requir
ed to bring the planned prostate isocenter precisely to the machine isocenter. A
t this point, treatment may begin. During treatment, the array continues to loca
lize the transponders at a frequency of 10 Hz
132
Mahadevan et al.
and reports their positions outside the treatment room on a monitor at the conso
le. The system allows for programming monitoring thresholds and when the target
moves outside of the threshold, the therapist is notified visually and with an a
uditory alert. Predetermined action levels and protocols for corrective action m
ay then be used to accommodate for organ motion during treatment. CLINICAL OUTCO
MES A pilot study involving implantation of the transponders was conducted in 20
patients (21) with a prototype system and prototype transponders. This study in
volved the first human use of the system and evaluated the localization accuracy
of this technique compared with radiographic localization. In addition, the abi
lity to obtain real-time prostate-motion information was also evaluated in an 11
-patient subset of these 20 patients (21,22). Each patient was implanted with 3
transponders and 58 of the 60 implanted transponders were usable for localizatio
n. In one patient, a single transponder was lost postimplantation (likely voided
after urethral implantation), and in another patient one transponder was found
to be unresponsive. A third migrated approximately 3 cm from the apex to the lev
el of the seminal vesicles around the 4th day after implant, presumably migratin
g within the venous plexus, but remained suitable for localization. There were n
o complications from transponder placement. (Post-study processing changes impro
ved the robustness of the transponder, and subsequent clinical studies did not e
ncounter unresponsive transponders. In addition, the commercial system software
supports localization when two of the three implanted transponders are located.)
Changes in distances between transponders (used as a proxy for transponder migr
ation) were minimal in 15 of 20 patients by the 4th day after implantation (mean
of the SD of intertransponder distance was 1.3 mm) and were stable in all patie
nts after 14 days (21). During a mock setup session, 11 patients first underwent
Calypso System localization followed by a kV radiograph and then repeat Calypso
System localization. Eight minutes of real-time tracking was then done followed
by a repeat of Calypso localizations and radiograph as above. Approximately 30
seconds to 1 minute elapsed between Calypso System localizations and the subsequ
ent comparative radiograph and 10 minutes between the two radiographs. Compariso
n of Calypso System localization to radiograph was done to measure the reliabili

ty of the Calypso System, while comparison between the two radiograph localizati
ons demonstrated change in prostate position over a 10-minute period. The differ
ence in three-dimensional vector setup variation between the Calypso System and
kV radiographs for these 44 comparisons was 1.5 0.9 mm. Prostate
motion between radiographs ranged from 0.3 to 6.6 mm, which likely accounts for
some of the Calypso System to radiographic variation, given the 30 second- to 1minute elapsing between the studies. This study demonstrated the high degree of
accuracy in using the Calypso System for patient set up. In a subsequent multiin
stitutional study, 42 additional patients underwent implantation of electromagne
tic transponders (18). Again, implantation was well tolerated and 125 of 126 imp
lanted transponders remained stable and suitable for localization with one trans
ponder being voided without discomfort (that patient later withdrew for unrelate
d reasons leaving 41 patients on study). One patient experienced significant pai
n associated with the ultrasound probe insertion and had an apical transponder p
laced in the prostate-rectal interface without significant associated symptoms a
nd was able to continue with tracking. Transponder stability was again confirmed
when similar comparisons between Calypso System localization to radiographs wer
e made during 1027 treatment sessions (18). The average delay between Calypso Sy
stem localization and X-rays was 152 50 seconds and between the orthogonal X-ray
s was 18 15 seconds. The average differences in lateral, longitudinal, and verti
cal components were 0.1 0.9 mm, 0.4 1.4 mm, and 0.0 1.3 mm. The average vector len
gth difference was 1.9 1.2 mm and the maximum vector difference was 3.3 mm. Thes
e differences are greater than in the phantom studies (17) but likely are accoun
ted for by prostate motion during the delay of two to three minutes from the com
pletion of Calypso System localization to the time of filming X-rays. Because of
the closed loop system and immediate feedback given to the in-room PC console,
the Calypso System is very time efficient in patient set up. Patient setup was t
imed during 1057 treatment sessions (18). Timing began when the patient was set
up to skin marks and the Calypso System console was positioned over the patient.
It ended when the therapist judged that the patient was positioned correctly (t
hus this included activating the Calypso System, reading the PC console instruct
ions, and carrying out the appropriate shifts of the table). The mean setup time
was 104 50 seconds (less than 2 minutes). This compares favorably with other im
age guidance systems such as ultrasound (300 seconds) (23), kV X-rays (250 113 s
econds) (24), and cone-beam CT (>270 seconds) (25). In a single institution inve
stigation, we tracked setup times for both Calypso System and an ultrasound syst
em (RestituTM, Resonant Medical, Montreal, Canada) for daily treatment localizat
ion in a series of 20 consecutive patients. Eleven patients were set up with Cal
ypso System during each treatment fraction (293 observations) and nine with ultr
asound (130 observations). Therapists had prior experience with both
IGRT in Prostate Cancerthe Calypso 4D Localization System
133
systems. Median setup times were 1 minute to setup to tattoos and then an additi
onal 1.6 minutes (SD 0.6 minutes) for Calypso System and 3.7 minutes (SD 1.8 min
utes) for ultrasound (26). The Calypso System appears to be an efficient daily s
etup tool and, because of its simple interface and objective nature, can be used
by therapists of all experience levels. When the Calypso System is used for rea
l-time tracking, the array continues to measure and report prostate position at
a frequency of 10 Hz. Analyzing the tracking data over a full course of radiatio
n therapy in 35 patients (representing 1157 treatment fractions) revealed target
displacement of more than 3 mm from setup location for greater than 30 seconds
in 41% of fractions and more than 5 mm for greater than 30 seconds in 15% of fra
ctions (18). The percentage of fractions in which one particular patient experie
nced motion was quite variable ranging from 3% to 87% of fractions using a 3-mm
cutoff and 0% to 56% using a 5-mm cutoff. If very tight margins are employed for
prostate radiotherapy without continuous real-time tracking, there could be a m

arginal miss in a significant number of patients. The clinical significance of m

arginal misses deserves further study. Graphs of prostate motion were grouped in
to broad categories representing typical patterns of prostate motion: Stable tar
get at baseline, transient excursion, persistent excursion, continuous drift, hi
gh-frequency excursion, and irregular motion (Fig. 4). Stable prostates could be
treated equally well with or without continuous monitoring, if one knew where t
he prostate was located at all times. A transient excursion would result in only
slight underdosing of a proportion of the fraction delivered prior to return to
baseline. Persistent excursion represents the case of most efficient use of rea
l-time monitoring in which a single correction can restore the system to accurat
e treatment. Continuous drift becomes more difficult in that it may not be fixed
by a single correction. However, given the anatomic constraints of the pelvis,
there clearly is a limit to the extent of a continuous drift. Instead, this situ
ation requires waiting for the prostate to settle into a new position prior to c
orrection and is probably just a variant of persistent excursion. Highfrequency
excursion and erratic behavior represent cases that could be identified by realtime monitoring but would be difficult to correct for without simply increasing
the margins of treatment unless the cause of motion can be determined. Of note,
these could be generated by either physical patient motion on the table or motio
n of the patients internal organs. Motion on the table may be correctable by an i
mmobilization device while motion of the patients internal organs perhaps could b
e corrected by medication (i.e., antispasmodics) or a physical device (rectal ba
lloon). Other options could include motion compensation techniques like automati
c
table repositioning (27) or multileaf collimator (MLC) synchronization (28). A f
ew general observations can be made on reviewing the real-time monitoring data.
First and foremost is that the motion is unpredictable, both patient to patient
and, for any patient, from day to day. Secondly, the lateral shifts are minimal
and appear stable over the tracking sessions. This is consistent with the prosta
te and pelvic anatomy. Thirdly, the longitudinal and vertical shifts typically o
ccurred together. Finally, the prostate does not always return to baseline posit
ion after an excursion as noted in some previous cine-MRI studies (29). This und
erscores the clinical importance of real-time tracking as a significant portion
of any long treatment fraction may miss its intended target in these circumstanc
es. With the knowledge of real-time prostate motion, therapeutic interventions t
o observed prostate motion can be implemented. These strategies could include no
action suitable for stable patterns of motion and if the margins used for treatm
ent planning are adequate for the observed range of motion; manual gatingthis can
be achieved by only treating when the target is in alignment; realigning patien
tthis is especially needed when the pattern observed is a persistent shift and ma
y have to be repeated in case of a continuous drift pattern. Realignments betwee
n beams could be easily and efficiently done. Beam interruption to exercise the
later options, especially in the setting of IMRT, can introduce dosimetric conse
quences that have to be further studied. There is sound rationale behind multipl
e strategies for interventions to account for intrafraction motion, and a variet
y of strategies have been employed to date. At the Cleveland Clinic our practice
is that if a translation of 3 mm or more and lasting 30 seconds or longer occur
s, we allow for completion of that individual beam but then briefly hold treatme
nt until alignment is achieved (reposition the patient using the Calypso System,
if needed) prior to the next IMRT segment. SUMMARY OF PERTINENT CONCLUSIONS
l
l
l
l
The Calypso System, as detailed above, represents a simple, straightforward, eff

icient and objective method of accurately localizing the prostate gland. Daily s
etup before initiation of RT can be performed in an efficient manner. Seamless t
ransition to real-time continuous monitoring of prostate location during treatme
nt provides valuable information to potentially prevent inadvertent geographic m
isses of the target. Setup and real-time monitoring throughout treatment deliver
y can be performed without exposure to additional ionizing radiation.
134
Mahadevan et al.
Figure 4 Patterns of prostate motion: (A) Stable at baseline, (B) transient excu
rsion, (C) persistent excursion, (D) continuous drift, (E) high-frequency excurs
ion, (F) irregular motion.
l
l
Prostate motion is largely unpredictable and variable day to day and patient to
patient, hence, exposes the limitations of predictive methods to account for pro
state motion. Strategies for therapeutic intervention can be implemented on the
basis of observed real-time variation in prostate location by therapists of all
skill levels.
l
The use of parameters for position correction due to intrafraction prostate moti
on provides adequate confidence to consider employing reduction in margins. This
, in turn, could either decrease normal tissue toxicity at present dose levels o
r even allows further dose escalation without increased normal tissue toxicity.
IGRT in Prostate Cancerthe Calypso 4D Localization System
135
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. Hypofractionated intensity-modulated radiotherapy (70 gy at 2.5 Gy per fractio
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15. Kry SF, Salehpour M, Followill DS, et al. The calculated risk of fatal secon
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13
Prostate Tomotherapy
RICHARD HUDES AND TIMOTHY HOLMES
St. Agnes Hospital Cancer Center, Baltimore, Maryland, U.S.A.
INTRODUCTION Tomotherapy is a unique form of intensity-modulated radiotherapy (I
MRT) based on a rotating X-ray fan beam (13). The term tomotherapy derives from tom
ographic radio-therapy, literally meaning slice radiotherapy. Tomotherapy treatmen
t delivery is conceptually similar to computed tomographic (CT) imaging where a
threedimensional image volume is acquired by helical irradiation of the patient.

By analogy, a tumor volume can be helically irradiated to achieve a highly conf

ormal threedimensional dose distribution by modulating the intensity pattern of
the incident X-ray beam profile during rotation. The intensity modulation is ach
ieved using a fan-beam multileaf collimator (MLC) whose leaves are pneumatically
driven to achieve near-instantaneous leaf transitions between open and closed s
tates. The Hi-ART IITM helical tomotherapy system (TomoTherapy Inc., Madison, Wi
sconsin, U.S.A.) is a dedicated image-guided IMRT radiotherapy treatment unit bu
ilt on a helical CT ring gantry that incorporates a CT image detector for daily
imaging of the patient prior to treatment (Fig. 1). The ring gantry design of th
e Hi-ART is optimal for CT scanning because it is much more mechanically stable
than the C-arm type gantry used by conventional radiotherapy treatment units. Me
chanical stability minimizes the potential for image artifacts due to flexing of
the source-detector geometry during image acquisition. For
137
comparison, current state-of-the-art C-arm gantry has a mechanical rotation tole
rance of 1 mm whereas the HiART has a tolerance of 0.1 mm similar to the GammaKn
ife and CyberKnife systems, making it suitable for stereotactic radiosurgery app
lications (4). Ideally, mechanical tolerances should be less than the size of an
image pixel for artifacts to be minimized without sophisticated characterizatio
n of the source-detector geometry for image reconstruction. Consistent with this
requirement, Boswell et al. have confirmed that helical tomotherapy is capable
of the subvoxel accuracy for phantom localization when using automatic registrat
ion of megavoltage CT (MVCT) to kilovoltage CT (kVCT) images (5). In the case of
the C-arm gantry used for CT imaging, a sophisticated software model must be us
ed to account for mechanical flexure of the source-detector geometry, and the fl
exure must be reproducible to minimize artifact generation. Another major design
difference between the Hi-ART II and C-arm treatment units is that the latter u
se a kV X-ray source oriented at 90 to the treatment beam to perform CT imaging,
whereas the Hi-ART uses the same MV X-ray system to perform imaging and treatmen
t. The difference between the two states is that the X-ray beam energy is reduce
d from 6 million electron volts (MeV) for treatment to 3.5 MeV for imaging in ad
dition to a reduction in beam current resulting a very low dose of 1 to 2 cGy to
the patient during imaging (6). This low dose is
138
Hudes and Holmes
Figure 1 TomoTherapy Hi-ART II image-guided IMRT treatment unit. Abbreviation: I
MRT, intensity-modulated radiotherapy.
also a consequence of the significantly higher penetration of the Hi-ARTs MV imag
ing beam that results in a higher signal-to-noise ratio at the detector compared
to a kV X-ray source (Fig. 2A). The increased penetration of 3.5MV X-rays reduc
es the likelihood of shadow artifacts in reconstructed images (Fig. 2B). The Hi-AR
T II is designed with a high level of system integration to facilitate image-gui
ded IMRT procedures. For example, the system is designed around a common databas
e that is shared by the inverse treatment planning system, the MVCT imaging subs
ystem, the treatment delivery control system, and the record and verification su
bsystem.
Since the daily MVCT images can easily be accessed by the treatment planning sys
tem, they can be used to recompute an estimate of delivered dose to compare with
the planned dose so that dosimetric errors can be determined and decisions can
be made to adapt future treatments to compensate for these errors (7,8). A geome
trical miss of the target volume constitutes the most important type of treatmen
t error and can be caused by patient weight loss during the treatment course or
internal organ shifts after the planning CT image set is acquired. The daily MVC
T data is, in essence, a mathematical model of the patient that can be used for

planning future treatmentsa process called adaptive radiotherapy (9,10). Helical to

motherapy has been successfully applied to image-guided IMRT at all body sites a
nd target volume sizes ranging from small metastatic lesions in the brain and lu
ng to total body irradiation with organ avoidance for bone marrow transplantatio
n (1120). While IMRT has wide application for curative as well as complex palliat
ive radiotherapy, its largest caseload is for curative treatment of prostate can
cer followed by head and neck and lung cancers. In all curative situations, the
rationale for using CT-guided IMRT is to deliver a high tumoricidal dose (6080 Gy
) to the minimal tumor volume while simultaneously minimizing the risk of shortand long-term complications by reducing unwanted dose to adjacent critical anat
omy. In the case of prostate cancer, the primary dose-limiting organ is the rect
um whose anterior wall sits adjacent to the prostate. The high-dose region typic
ally encompasses the anterior rectal wall making it the region most likely to su
ffer early complications in the form of tissue breakdown and bleeding. In some i
nstances,
Figure 2 (A) Comparison of MV versus kV X-ray penetration in water. Hi-ARTs megav
oltage imaging beam produces a higher signal-to-noise ratio at the detector comp
ared to a kilovoltage X-ray source. (B) CT images of 30 cm diameter image qualit
y test phantom showing the shadow artifact produced by excessive attenuation of lo
w energy kV X-rays compared to MV X-rays. Abbreviations: MV, megavoltage; kV, ki
lovoltage. Source: Courtesy of T.R. Mackie, PhD).
Prostate Tomotherapy
139
the bowel may also need to be considered if it is adjacent to the high-dose volu
me. The bladder and femoral heads are typically included in the planning process
as structures to minimize unwanted dose, albeit at a lower priority than the re
ctum (21). The Hi-ART II is very efficient for prostate radiotherapy where most
procedures are completed within 15 to 20 minutes, with image guidance requiring
less than 10 minutes and treatment delivery completed within 4 to 7 minutes, dep
ending on the size of the target volume (22). A unique feature of the slice-base
d approach is that a tumor slice reaches full dose in one to two minutes regardles
s of overall treatment delivery time. This dosimetric characteristic combined wi
th the accurate targeting of dose makes the Hi-ART II an excellent device for ra
diotherapy treatment of prostate cancer. In the following sections, we will disc
uss the features of the Hi-ART II that make it ideal for CT-guided IMRT of prost
ate cancer. EQUIPMENT DESCRIPTION The Hi-ART II uses a compact electron linear a
ccelerator (linac) to generate X-rays for both treatment and imaging. The linac
is a 30-cm long, 6 MeV, S-band (nominal 3 GHz) magnetron-powered device with a g
ridded gun and a solid-state modulator. An X-ray target is integrated into the b
ody of the linac and is located 85 cm from the gantry rotation axis. Unlike conv
entional treatment units, the Hi-ART II does not need an X-ray flattening filter
. Removal of the filter increases the X-ray output to 8 to 10 Gy/min at the gant
ry rotation axis, and it simplifies the treatment beam modeling and CT image rec
onstruction processes by providing a more monochromatic X-ray energy spectrum an
d reduced scatter outside the field boundary. The treatment head has a unique cla
m-shell design that allows field widths of 5 to 50 mm (nominal). The head shieldi
ng including primary collimators was designed to limit the primary leakage to 0.
01% of the primary beam, or one-tenth the limit (0.1%) used by C-arm radiotherap
y treatment units. This added shielding is required since tomotherapy requires a
larger number of monitor units for an IMRT treatment compared to fixedgantry me
thods. Despite this, the whole-body dose from tomotherapy is lower than that fro
m C-arm linacs, due in part to the low leakage and to the lower scatter outside
the field because of the lack of a flattening filter (2325). Additionally, Lazar
et al. have estimated that the risk of developing secondary cancer from a prosta
te radiotherapy using tomotherapy is lower than that for fixed-gantry IMRT using
a C-arm treatment unit (26). Below the primary collimators resides the binary M

LC module, consisting of 64 leaves made from 10-cm high tungsten with leakage le
ss than 0.5%. A 13-cm thick lead
counterweight is attached to the ring gantry opposite the treatment head that ac
ts as a rotating primary barrier or beam stop. Each binary MLC leaf completely b
locks a portion of the fan beam with a projected shadow of 6.25 mm (nominal) at
the gantry rotation axis. The 64 leaves define a 40-cm diameter treatment fieldof-view, which combined with up to 160-cm (nominal) couch travel enables very la
rge treatment volumes to receive IMRT. Intensity modulation is achieved using pn
eumatic control of the binary MLC leaves by rapidly (*20 milliseconds) switching
the open-closed state of leaves during gantry rotation. The intensity level is
proportional to the time a leaf is open and there are effectively 50 intensity l
evels that that can be delivered. The linac and ring gantry systems of the tomot
herapy system are highly favorable for CT imaging where mechanical stability of
the source-detector positions during rotation and a small source size are desira
ble. An additional benefit of the enclosed ring gantry is that a collision of th
e rotating gantry with the patient is avoided by design unlike the C-arm gantry
of conventional treatment units. The tomotherapy gantry mechanical sag during ro
tation is approximately 0.1 mm, so no sag corrections are required in the CT rec
onstruction algorithm. The size of the electron beam on the target is about 1 mm
so that the resolution is about 1.2 to 1.6 mm, which is comparable to a convent
ional CT scanner for highcontrast objects. Operating at an average dose to the p
atient of 1 to 2 cGy, the images produced have soft tissue contrast of 2% to 3%,
which is poorer than a modern CT scanner, yet are of sufficient quality for ada
ptive radiotherapy processes (6). The tomotherapy units xenon gas detector elemen
ts have tungsten septa separating ionization cavities. In addition to the ioniza
tion collectors, the tungsten plates are embedded photon converters intercepting
the MV photons and yet are thin enough to let an appreciable fraction of the el
ectrons set in motion to deposit energy in the xenon gas. The interception of th
e beam by the tungsten means that the quantum efficiency of the system is about
25%, which is much more than the few percent collection efficiency of modern ele
ctronic portal imaging systems used by C-arm gantry treatment units. Modeling th
e treatment delivery process requires discretization of the continuous motions o
f the gantry and table as well as the continuous intensities of the modulated be
ams. Proper sampling reduces the chance for computational aliasing that can prod
uce streak or thread artifacts in the dose distribution (27). Consequently, each 360
gantry rotation is modeled as 51 beams spaced at 7.06 aparta number chosen to allo
w a 40-cm diameter target volume to be homogeneously treated with a 2.5-cm compl
etely blocked central avoidance structure (28). Following optimization, the inte
nsity levels are
140
Hudes and Holmes
discretized for treatment delivery, with 50 levels chosen to reduce the uncertai
nty in the target dose due to intensity discretization to less than 0.1%. Discre
tization of couch travel is determined by the pitch ratiothe ratio of the couch t
ravel distance per rotation to the field width defined at the axis. In helical t
omotherapy delivery, the pitch is usually set to be less than half to avoid thre
adlike dose artifacts developing near the edge of the field and becoming clinica
lly significant (27). Given a typical pitch of 0.3 for a 25-mm field width, the
table motion is modeled by offsetting adjacent beams by 0.147-mm [e.g., (0.3 x 25
mm)/51] increments parallel to the direction of table motion. PROSTATE TOMOTHER
APY WORKFLOW Since the Hi-Art II design is highly integrated around a common dat
abase and software model, its workflow is efficient to carry out and is the same
for all body sites. The Hi-ART II workflow can be viewed as four distinct proce
sses: (i) treatment planning, (ii) plan verification, (iii) MVCT localization, a
nd (iv) treatment delivery. While it is possible to carry out the full process i
n the same day, treatment planning and plan verification typically occur one to

five days prior to the initial treatment delivery session. IMRT Treatment Planni
ng The treatment planning workflow for the Hi-ART II consists of several process
es that are common to most IMRT systems. A three-dimensional model of the patien
t is required for the planning process, so the first step is to obtain a volumet
ric image set of the patient in the treatment position using a CT simulator. We
place the patient supine on a bean-bag positioner extending under the lower back
for comfort, with their legs slightly bent using a triangular sponge under the
knees, and the feet secured together with a strap to minimize knee and hip movem
ent during the treatment procedure. Thermoplastic pelvic casts can also be used
for immobilization to minimize intrafraction motion of the patient during the pr
ocedure, but they provide no useful improvement in setup accuracy in the daily i
mage guidance, yet add to the overhead of the procedure and require storage spac
e that may be a premium in most clinics. The patients CT image set is transferred
to a thirdparty contouring software tool that supports DICOM-RT data transfers
to the TomoTherapy Planning Station where
CT images are subsequently resampled from their original 512 512 pixels resoluti
on to 256 256 pixels to reduce memory requirements.a Regions of interest (ROIs)
are drawn for the planning target volume (PTV) and organsat-risk (OARs) such as
the bladder, rectum, femoral heads, and bowel. The TomoTherapy Planning System r
equires two additional nontissue structuresthe couch top and the setup marks on t
he patients surface. A 1-cm wide OAR shell is also drawn around the PTV to be used
to force the high-dose region to conform to the PTV. In situations where a metal
hip prosthesis is present, a nonanatomical avoidance ROI can be drawn around th
e prosthesis to prevent beams from passing through the implant. The TomoTherapy
Planning System restricts each voxel to a single ROI; consequently, each ROI is
assigned an overlap precedence ranking so that the optimization software can han
dle structure overlaps properly. In the Optimization panel, the operator defines
the prescription and planning parameters required by the plan optimizer. These
parameters include the dose model, the dose grid resolution, helical pitch, and
the beam width. Three dose models are available in increasing accuracy and compu
tation time: (i) a primary ray trace model (TERMA), (ii) a precomputed pencil be
am model (BEAMLETS), and (iii) a full scatter calculation (FULL SCATTER) that is
performed during each cycle of the optimization. The dose model requires the se
lection of (i) the dose grid resolution (fine: 12 mm; normal: 24 mm; coarse: 48 mm)
, (ii) the beam width (1.0, 2.5, or 5.0 cm), and (iii) the helical pitch (0.11.0)
, all of which set the calculation geometry. Typical values that we use routinel
y for prostate treatment planning are 2.5-cm beam width, 0.25 pitch, and a modul
ation factor of 2.8. The dose-volume (D-V) constraints differ depending on the t
ype of structure. A targets set of D-V constraints define a three-point dose-volu
me histogram (DVH) defined by a minimum and a maximum dose limit and a third poi
nt defining where the shoulder falls between these limits. Only two points are def
ined for OARsa maximum dose limit and a low-dose inflection point located between
zero and the maximum dose limit. In addition, there are D-V penalty factors and
ROI priority factors, which are used in the optimizers objective function to com
pute a numerical score for the current plan estimate. The score allows the optimiz
er to determine if changes in the beam intensities produce an improvement in dos
e conformality. The set of D-V constraints can be saved as a class solution to b
e reused for future cases, if desired. Plan optimization is performed iterativel
y by making changes to the incident intensity pattern for all binary
The reason for this is that the current tomotherapy software (version 2.4) does
not support creation of new contours; the newly released version 3 software prov
ides a complete contouring toolset.
a
Prostate Tomotherapy
141

MLC leaves that intersect the target, also referred to as the sinogram. The name
derives from the fact that each point in the target projects back to a sinusoid
al path in the intensity pattern. The sinogram describes the opening and closing
of the binary MLC leaves as a function of the gantry rotation angle and the tra
nslation position of the couchit is, in effect, the operational instructions for
the Hi-ART treatment delivery. Computation time is affected by the choice of bea
m model and dose grid resolution. For example, if the BEAMLET model is selected
then the system will initiate the computation of three-dimensional dose distribu
tions for all binary MLC leaves that intersect the target volume, typically numb
ering in the thousands of beamlets. This data is computed using a multiprocessor
rack consisting of 32 processors with local memory and disk storage. The result
s are stored locally on the processors so that they can be reused for generating
an alternative plan without the need for recomputation. It takes approximately
10 to 15 minutes to compute the complete set of beamlets using the normal dose g
rid resolution and approximately 1.5 to 2 hours using the fine setting. At this
point the optimization starts, and an iteration completes after 3 to 5 seconds,
with 50 to 200 iterations needed to achieve a good plan. Alternatively, if the F
ULL SCATTER model is used, a single composite three-dimensional dose distributio
n is accurately computed during an iteration, with the computation taking about
two to three minutes per iteration. The optimization can be stopped and restarte
d at any time to review the DVHs and dose distribution and to alter the D-V cons
traints, without loss of any plan data. Once an acceptable result is achieved, t
he numbers of fractions are defined along with the treatment dates in the Fracti
onation panel. A final dose calculation is performed and corrections are applied
to leaf-opening times to account for small changes in output when two adjacent
leaves are opened simultaneously. Final approval of the plan requires the operat
or to enter their username and password, and once approved a plan cannot be chan
ged. On plan approval, the set of daily treatments are recorded in the patients d
atabase record. IMRT Plan Verification All patient treatment plans must be verif
ied by dosimetry measurement prior to the first treatment delivery. Tomotherapy
refers to this as Dosimetry Quality Assurance, or DQA. DQA is typically accompli
shed by using a plastic cylindrical water-equivalent phantom provided with the H
i-ART II as a surrogate for the patient. The DQA phantom supports the use of fil
m and small volume ion chambers for performing measurements. The patients intensi
ty sinogram is used to compute dose in the DQA
phantom for which test procedures are created. The procedures are delivered to t
he phantom with film and ion chambers in place. The film is developed and digiti
zed and the information imported into the DQA analysis panel of the planning sys
tem where it is compared with the computed dose. The ion chamber measurements ar
e converted to dose and input into the DQA analysis panel where it is compared w
ith point data manually sampled from the three-dimensional dose distribution. Th
e data and the comparison results are then stored in the patients database record
. The decline in availability of automatic film processors due to increased use
of digital imaging is driving the adoption of processor-less verification soluti
ons such as radiochromic film, computed radiography, and electronic detector arr
ays. An example of the latter is a matrix ionization chamber having 1020 calibra
ted pixel ion chambers that provide a two-dimensional image of abso lute dose (Ma
triXX, Scanditronix-Welhofer, Germany). This device provides an electronic alter
native to film, albeit at a lower spatial resolution of 7.62 mm between detector
centers. Nevertheless, the example shown in Figure 3 illustrates the excellent
agreement possible using this device for tomotherapy DQA. The reason for this is
that the detector is being translated with the couch through the gantry at a co
nstant speed of less than 1 mm per second while acquiring measurements at a rate
of 1 sample per second. Consequently, the data samples are being acquired at le
ss than 1 mm spacing along the direction of couch motion. Measured and computed
doses are compared by visual inspection of one-dimensional profiles and twodimen
sional isodose distributions or quantified by a color wash two-dimensional g dis
tribution representing how well a pixel in the measured dose image agrees in mag

nitude and spatial position to a neighborhood of pixels in the calculated dose i

mage (29,30). Typically, a 3-mm radius defines the neighborhood of calculated pi
xels used for the evaluation, and difference limits of 3% in magnitude and 3 mm
in spatial position are used in computing the g value at each pixel. A g value o
f less than or equal to 1.0 indicates acceptable agreement, where value of 0.0 i
ndicates perfect agreement. Values greater than 1.0 indicate a discrepancy that
exceeds one or both limits. MVCT Localization Imaging The MVCT detector resoluti
on at the axis of rotation is about 0.6 mm in the transverse direction and equal
to the slice width in the longitudinal direction. The gantry rotation for MVCT
imaging is 10 seconds (6 RPM) and the slice thickness is approximately 4 mm, how
ever, a smaller slice width (i.e., 2 mm) could be used for the fine
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Hudes and Holmes
Figure 3 Tomotherapy DQA dose comparison (isodose, gamma distribution and dose p
rofiles) using the MatriXX to measure absolute dose in a coronal plane through t
he center of the prostate. Abbreviation: DQA, dosimetry quality assurance.
resolution needed for small target volumes. The unit takes about 800 projections
(or views) per rotation with two CT slices obtained per rotation. Helical pitch
es of 1, 1.5, and 2 are available for imaging allowing a typical tumor of 8to 10
-cm length to be imaged in as little as 2 minutes. Longer lengths of tumor and s
maller pitches take more time proportionately. Image reconstruction is carried o
ut in parallel with data acquisition, so there is little delay following acquisi
tion for the images to be analyzed. The Hi-ART II is capable of resolving 1.2- t
o 1.6-mm objects near the edge of a 30-cm diameter phantom. The X-ray energy of
the verification CT is approximately 3.5 MeV. Consequently, the photons interact
almost exclusively by Compton interactions so that the attenuation coefficient
is linear with the electron density of the medium (31). Metal artifacts arise in
conventional CT scanners because the attenuation of the metal is greatly enhanc
ed due to the photoelectric effect. In helical CT, the beam is penetrating enoug
h to eliminate artifacts arising from metal objects like a hip prosthesis; conse
quently, the verification MVCT is a more reliable CT system for patients with me
tal implanted appliances (32). CT imaging is performed prior to each treatment t
o reduce the possibility of a geometrical miss of the target and sensitive struc
tures. An automated comparison of verification and planning image sets is carrie
d out immediately
following image acquisition to guide the adjustment of the patient setup. The pa
tient is assumed to be a rigid object requiring translations and rotations to br
ing the target anatomy and important sensitive structures into alignment with th
e treatment plan. The patient is positioned by aligning the patients skin marks w
ith lasers located outside of the bore of the unit. A sagittal representation of
the patients planning CT is shown on the operator console to aid in selecting th
e slices to be scanned. A verification scan is taken and reconstructed during th
e acquisition. The patient is then transported to the same position outside of t
he gantry bore while the verification image set is fused onto the planning image
set, and the translation and rotation offsets are reported. Typically, the imag
e fusion is first done automatically using a mutual information algorithm (33).
Following automated registration, the patient registration can be fine-tuned man
ually. This allows the operator to take into account, as best as possible, the n
onrigid nature of the transformation. Once the image registration is completed,
the offsets also describe how the patient must be adjusted. Figure 4 is an examp
le of a MVCT localization image registered to the planning CT using the soft-tis
sue anatomy of the prostate and rectum. Bone, while having less contrast than a
conventional CT scan, is still clearly discernable as are the fat-muscle boundar
ies surrounding the prostate gland.

Prostate Tomotherapy
143
Figure 4 MVCT image registration example. The MVCT image is shown as the blue ch
eckerboard color wash overlain on the gray scale kV planning CT. Note that the s
oft-tissue anatomy has been adjusted using the prostate/rectum interface, which
results in a misalignment of the bony pelvis. Abbreviations: MVCT, megavoltage c
omputed tomography; kV, kilovoltage.

The patient can be translated accordingly if adjustment is required. The treatme

nt couch has automated vertical and longitudinal translations and automated gant
ry start angle adjustments to account for patient roll. The couch top can be man
ually adjusted in the lateral direction (x-direction). Yaw and pitch rotations c
an be accommodated using angularly calibrated immobilization/positioning aids, w
hich are especially useful for the head and neck. Moveable CT-simulator lasers a
re used to verify the modified patient position. Fully Dynamic IMRT Treatment De
livery A Hi-ART II treatment delivery of 2.0 Gy typically takes less than 5 minu
tes for small target volumes like a prostate and less than 10 minutes for larger
volumes that include pelvic lymph nodes and seminal vesicles. Overall treatment
delivery time T is a function of target length along the axis of table motion (
L), the beam width (W ), the prescribed dose D, the average dose rate at the tar
get R, and the user-defined modulation factor M defined as the ratio of the maxi
mum leaf-open time of any leaf to the average leaf-opening time of all the nonze
ro values. The parameters are related by (34) T MDL W/WR $ M/W constant Consequentl
, treatment time can be shortened by reducing the intensity modulation or by inc
reasing the beam width in agreement with common sense. Typically, we select a mo
dulation factor of 2.8, with higher values not providing noticeable improvement
in
plan quality. On the other hand, the choice of slit width (1.0, 2.5, 5.0 cm) can
alter treatment time significantly. As an example, a 7-cm prostate volume can b
e adequately treated with a 2.5-cm slit width in approximately 5 minutes, assumi
ng an average dose rate at the target volume of 4 Gy/min and a modulation factor
of 2.5. Yet the time would significantly increase to approximately 20 minutes i
f the 1-cm slit was used with the intent of achieving a more conformal dose dist
ribution. In practice, the smaller slit does not provide much improvement in dos
e conformality because the effective resolution of the helical delivery in the d
irection of table motion is 6.25 mm when using a 2.5-cm slit with a pitch of 0.2
5. Helical tomotherapy is a very efficient method to deliver a tumoricidal dose
to the tumor because of the slice-based nature of the delivery. Fixed-gantry IMR
T techniques used by C-arm gantry treatment units require all beam directions to
be delivered before a tumor subvolume reaches full dose, hence the overall trea
tment time is the relevant parameter when evaluating the average dose rate at th
e tumor for this type of IMRT delivery. Tomotherapy delivery, on the other hand,
continuously irradiates a slice of tissue so that the full dose of 1.8 to 2 Gy
is reached within one to two minutes regardless of the overall length of the tre
atment delivery and body site. Consequently, the average dose rate at the tumor
is typically 1 to 2 Gy/min, and the impact of cell repair during the delivery is
minimized. SPECIAL APPLICATION TOOLS The availability of the CT imaging on the
tomotherapy treatment unit has lead to the development of two new clinical tools
based on this imaging capability. The Plan Adaptive Tool uses daily MVCT images
to evaluate errors in daily treatment delivery, and it can be used to adapt fut
ure treatments to compensate for these errors. The StatRT Tool allows one use th
e Hi-ART II to perform the functions of CT simulation, inverse treatment plannin
g, and treatment delivery within 10 to 15 minutes without moving the patient fro
m the table. Adaptive Radiotherapy: Plan Adaptive Tool Typically, MVCT images ar
e acquired daily for setup localization. Consequently, one has a daily model of
the patient available that can be used for computing a daily estimate of deliver
ed dose. The daily estimates can be compared with the planned dose to determine

the impact of incorrect setup, changes in anatomy, or incomplete treatment deliv

ery. Regions of unacceptable error can be automatically contoured and used to re
plan future treatments to compensate for these errors. This process called
144
Hudes and Holmes
adaptive radiation therapy (ART) is embodied in the Plan Adaptive Tool. The Plan
Adaptive Tool has three functions: (i) it is used for accessing and transferrin
g MVCT images from the patient record to the tomotherapy planner for treatment p
lanning when anatomical changes during treatment or metal artifacts in original
kVCT images warrant their use, (ii) it is used for computing dose using MVCT ima
ges for treatment delivery quality assurance, and (iii) it is used to evaluating
cumulative errors in the delivered dose from which error ROIs are created and u
sed for plan reoptimization. A common problem faced in prostate image-guided rad
iation therapy (IGRT) is the presence of an implanted metal hip prosthesis that
creates significant image artifacts in KVCT images used for planning and treatme
nt setup. Tomotherapy dose computation uses CT numbers to account for tissue den
sity. Consequently, erroneous CT numbers will cause errors in computed dose. Typ
ically, the hip prosthesis casts a shadow of lower CT number in the region of the
prostate. This region of lower CT number can result in significant errors exceed
ing 5% to 10% at the prostate. An extreme example is shown in Figure 5 where the
bilateral prostheses CT artifact resulted in a 10% to 15% error in dose in the
region of the prostate. It is advisable in these situations to use the Plan Adap
tive Tool with a MVCT image set to determine the magnitude of the error and repl
an the case using the MVCT data. Lastly, the Plan Adaptive Tool can be used for
correlating acute side effects with delivered dose estimated using daily MVCT lo
calization images. An example is shown in Figure 6 for the situation where a pla
n was generated for a patient with a full rectum but was treated with an empty r
ectum. The impact is a slightly higher local dose in the anterior rectal wall th
at exceeds 5%.
StatRT StatRT is a new tool that allows the Hi-ART II to perform the functions o
f CT-simulation and treatment planning, in addition to treatment delivery. The i
ntended use is for emergent palliation cases where it is desirable to efficientl
y simulate and treat the patient within 20 minutes and avoid moving the patient
from CT simulator to treatment couch. StatRT implements a fully functional treat
ment planning system at the treatment unit console. Plan optimization is carried
out using the FULL SCATTER model with an acceptable solution achieved in 10 to
20 iterations requiring five minutes of computation time. While this feature may
not be that useful for definitive prostate treatments at normal fractionation,
it may prove useful for hypofractionated prostate radiosurgery applications wher
e it would be desirable to minimize any dose errors at the time of treatment set
up. TOMOTHERAPY IGRT QUALITY ASSURANCE A major difference between helical tomoth
erapy and fixed-gantry IMRT is that the former is a fully dynamic approach where
the gantry, couch, and MLC motions must be properly timed to achieve an accurat
e delivery. In fact, helical tomotherapy is based on the assumption of constant
linac output and constant gantry rotation and couch translation speeds during im
aging and delivery procedures. Conversely, in fixed-gantry IMRT, the MLC is the
only system component that might move during the irradiation; hence, dynamic MLC
quality assurance (QA) methods have been developed to augment the battery of cl
assical QA tests for static treatment delivery using a C-arm linac. On the other
hand, helical tomotherapy QA
Figure 5 Bilateral hip prostheses cause significant artifacts in kVCT images use
d for treatment planning by overcompensating the influence. In this example dose
errors of 5% to 15% occur in the region of the prostate bed. Abbreviation: kVCT
, kilovoltage computed tomography.

Prostate Tomotherapy
145
Figure 6 Pelvis midline-sagittal view showing a comparison of planned versus del
ivered dose where the latter was estimated using the Plan Adaptive Tool. Bowel g
as and stool are present in the planning CT and absent in the MVCT localization
image. The impact of this change are small hot and cold dose regions at the inte
rface between the prostate and the rectum with the remaining region within 0% to
3% agreement. Abbreviation: MVCT, megavoltage computed tomography.
testing has been developed primarily with constancy of output and dynamic motion
s in mind (35,36). Helical tomotherapy QA includes both static and dynamic QA te
sts. Examples of static QA tests are static output calibration and output readou
t calibration, X-ray beam alignment with the jaws and binary MLC, field size geo
metry, and couch offset from the setup position outside the gantry to the starti
ng position inside the gantry. Dynamic tests include gantry rotation speed, couc
h speed, and MLC leaf transitions. These can be tested separately or in combinat
ion. The approach we have developed in our clinic is to perform an imaging and t
reatment delivery procedure that mimics an actual patient treatment during morni
ng warm up. This dynamic QA procedure is specially designed so that constancy of
dynamic output, X-ray energy, couchgantry speed synchronization, couch offset,
movable laser calibration, MVCT localization imaging, and image quality paramete
rs (high- and low-contrast resolutions, CT number constancy) can be evaluated in
a single imaging and delivery procedure (37). Static tests are performed on a m
onthly basis or as necessary following a major servicing of the machine. The mai
n objective of a CT image-guided procedure is to reduce setup uncertainty so tha
t smaller treatment margins can be justified and geometrical misses are minimize
d. It follows that some evaluation of daily setup variation should be monitored,
preferably by body site. In our practice, we tabulate daily position offsets on
all patients by body site for review at our monthly department QA meeting. This
data is useful to evaluate if there is a dominant direction of offset, if the o
ffsets are
random or systematic, and if there are outlier patients who should be monitored
more closely. Another QA measure that is monitored is the discrepancy in DQA dos
imetry measurements performed for plan verification. Two features of the DQA dat
a are monitored: (i) the discrepancies in absolute dose at six points sampled in
the coronal and sagittal planes and (ii) the spatial offsets between measured a
nd calculated relative dose profiles extracted from the sagittal and coronal pla
nes. Systematic discrepancies in the six-point dose samples may indicate that th
e output of the linac has changed from its calibrated value of 858 cGy/min or th
at there is an energy change in the X-ray beam. Positional shifts in the relativ
e dose profiles may indicate that the setup lasers are out of adjustment. Hence
the DQA results are monitored as a secondary indicator of dynamic performance of
the Hi-ART II. PROSTATE TOMOTHERAPY: IN THE CLINIC Target Volume Delineation
Definitive Prostate
Typically, a kVCT simulation is performed with cystourethrogram using 3-mm thick
slices and subsequent contouring of the prostate, rectum, bladder, and femoral
necks (Fig. 7). For high-intermediate- or high-risk patients, the proximal 1- to
1.5-cm seminal vesicle is included and may also require an extra 2- to 3-mm mar
gin posteriorly due to risk of extra capsular extension (38,39). For a subpopula
tion of high-risk patients, pelvic lymph nodes are contoured as part of the init
ial control target volume (CTV),
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Hudes and Holmes

Figure 7 Example of helical tomotherapy for early stage prostate cancer. The arr
ows indicate two clinically relevant evaluation points on the rectum DVH. Abbrev
iation: DVH, dose-volume histogram.
as is the bowel for avoidance (Figs. 8 and 9). Occasionally, MRI fusion may be u
sed for patients with poor kVCT imaging that leads to difficult prostate rectal
interface or apex delineation. Additionally, for patients with artifacts from hi
p prostheses, a MVCT on the Tomotherapy unit will be used as discussed below. In
considering the PTV margin, one needs to consider setup error, interfraction mo
tion, and intrafraction motion. Regarding interfraction movement, using kVCT loc
alization at Fox Chase Center, Lattanzi described an error margin as low as 3 mm
(35). Likewise, using a conebeam CT, a 3-mm margin for setup errors has been re
ported (40). Similar results for a 3-mm margin for setup error have been found f
or MVCT imaging with tomotherapy (41).
(rectum, bladder). Since the TomoTherapy Planning System uses a heterogeneity-co
rrected dose model, the image artifacts will create errors in the delivered dose
as shown in Figure 5 for the case of bilateral hips prostheses. MRI will likewi
se be unable to properly image the ROIs because of the presence of metal and its
distorting effects on the magnetic field of the imager. Consequently, MVCT can
be acquired and used both for contouring and dose planning in this situation wit
h the caveat that the MVCT does have increased inter- and intraobserver variabil
ity in contouring compared to kVCT (42). The MVCT with prostheses can be used fo
r dose calculation since the artifact is minimized due to the increased penetrat
ion of the 3.5 MeV X rays used for imaging (7). Normal Tissue Integral Dose The
integral dose equals the mean dose times the volume irradiated. Aoyama et al. (2
3) investigated treatment planning for prostate cancer with three-dimensional co
nformal radiotherapy (3DCRT), conventional linac IMRT, and helical tomotherapy.
Secondary malignancy estimates showed minimal variations between these technique
s. There was a benefit demonstrated for helical tomotherapy over conventional IM
RT and 3DCRT for localized prostate cancer in regard to dose sparing of rectum a
nd penile bulb without increasing normal tissue integral dose (NTID) and risk of
secondary malignancy.
Postprostatectomy
For patients status post-prostatectomy, the simulation and contouring for patient
s to undergo tomotherapy are generally similar to that of other techniques descr
ibed in chapter 14. The only subset that differs significantly is that with hip
prostheses as discussed below. Hip Prosthesis Artifact A metal hip prosthesis im
aged with kVCT will create significant image artifact that can deleteriously aff
ect the ability to contour the prostate as well as the OARs
Prostate Tomotherapy
147
Figure 8 (See color insert.) Example of a complex prostate case consisting of 45
Gy volume including lymph nodes, 34.2 Gy boost volume, and the total dose.
Target Localization Technique: Daily MVCT Image Guidance
Interfraction Movement
Avoidance of geographical miss has propelled IGRT into the forefront of clinical
importance for prostate cancer since there is a discrepancy between bony anatom
y and prostate position for daily set up. With the TomoTherapy MVCT image guidan
ce system, the images are acquired in the treatment position immediately prior t
o the treatment. The MVCT image is fused and is used for comparison with the pla
nning kVCT for alignment prior to treatment. Alignment is made by shifts in the
x, y, and z coordinates as well as a change in the roll. Automated pitch and yaw
corrections are not currently addressed in the TomoTherapy Hi-Art II system. La

ngen et al. (41) described three different approaches to compare the MVCT and kV
CT images: (i) fiducial markers, (ii) CT anatomy, and (iii) kVCT
contours. In studying 112 alignments from three patients, each of these approach
es was analyzed compared to a reference of the center-of-mass (COM) of the three
fiducial markers. Radiation therapists retrospectively registered the image set
s with anatomy and contour methods. The physician registered all image sets on t
he basis of all three techniques. It was found that the fiducial technique was b
est in looking for a 3-mm difference from the reference COM. Furthermore, the fi
ducial and CT anatomy techniques were similar in looking for a 5-mm difference f
rom the reference COM. It was clear that the contour-based technique was inferio
r than the anatomy or fiducial technique for both the radiation therapists and p
hysician. Interestingly, there was a superiority of physician over therapist in
the 3-mm threshold for anatomy compared to COM alignment, which is attributed to
the physicians experience of CT image viewing (41). The authors concluded that f
iducial placement is preferred, but if it is not used,
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Hudes and Holmes
Figure 9 Dose-volume histograms for the complex case. (A) Initial 45 Gy plan. (B
) 34.2 Gy boost plan. (C) Total dose.
then anatomy-based registration is preferred over contourbased registration beca
use of agreement with a marker-based COM reference alignment and less interuser
variability. The use of daily image guidance results in the ability to use tight
er margins compared to less than daily frequency of image guidance (43).
Intrafraction Movement
Ghilzean et al. (40) evaluated prostate motion using cineMRI and found that a di
splacement of 3 mm can be expected within 20 minutes of the initial imaging for
patients with an empty rectum. They found that the
Prostate Tomotherapy
149
most significant predictor for intrafraction prostate motion is the status of re
ctal filling, with a full rectal state is associated with greater prostate motio
n. Susil et al. (40) performed an intrafraction motion study on eight prostate t
omotherapy patients where they obtained MVCT following treatment to compare with
the pretreatment localization scan. They found that an aggregate margin of 4 mm
encompassed 95% on prostate intrafraction motion for 95% of treatment fractions
examined in their study. Since tomotherapy prostate procedures can be carried o
ut easily in a 15- to 20-minutes time slot, these results would indicate that in
trafraction motion tracking is not needed for the majority of patients if they p
resent with an empty rectum and if a minimal margin of 4 mm is used in defining
the PTV. With these results in mind, typical expansion margins used for prostate
tomotherapy treatment planning are 4 to 5 mm posterior and 6 to 10 mm all other
directions (8,44). The use of real-time electromagnetic tracking (Calypso 4D Lo
calization System, Seattle, Washington, U.S.A.) is now possible, but its use is
just now being studied. Litzenberg et al. (42) used this technology to study 11
patients treated with fixed-gantry IMRT, and they found that only two cases woul
d have benefited from continuous target tracking. Langen et al. (45) found that
there was a minimal dosimetric impact on a cohort of 13 prostate tomotherapy pat
ients when the Calypso intrafraction motion data was used to calculate dose usin
g a four-dimensional dose model and treatment margins of 4 mm posterior and 6 mm
otherwise. The average percentage of treatment time that the prostate was displ
aced by more than 3 mm was 5%, or about 15 seconds out of a 300 seconds treatmen

t. The random nature of day-to-day displacements reduces the cumulative impact s

ignificantly, so that in 93% of the fractions, the intrafraction motion had less
than a 5% impact on the absolute dose to the PTV, and the cumulative impact on
the prostate and PTV D95 was less than 1.2%. On the basis of these works, it wou
ld appear that only a very small cohort of prostate tomotherapy patients would a
ppear to benefit from continuous target tracking.
An example of the impact of rectal filling and deformation on the dose at the pr
ostate rectal interface is shown in Figure 6 where local errors of 5% occur in t
he anterior rectal wall when a plan is generated with gas present in the bowel a
nd subsequently used to treat with an empty rectum. SUMMARY OF PERTINENT CONCLUS
IONS The TomoTherapy Hi-ART II is well suited for imageguided IMRT of prostate c
ancer for the following reasons:
l
l
l
l
l
l
l
l
l
The Hi-ART II is efficient by integrating into one system the processes of treat
ment planning, treatment verification, MVCT image-guidance, IMRT treatment deliv
ery, and adaptive dose recalculation. The Hi-ART II has mechanical accuracy simi
lar to dedicated radiosurgery systems with an accuracy of less than 2 mm for dos
e localization to the target. MVCT image guidance only exposes the patient to ap
proximately 1 cGy per image procedure and can avoid the need for fiducial placem
ent. MVCT imaging allows imaging without artifact for patients with hip prosthes
es. Prostate margins as low as 4 mm posteriorly and 6 mm otherwise are enabled b
y MVCT localization. Fully dynamic IMRT results in maximal sparing of the rectum
and bladder with homogeneous dose to the prostate. Fully dynamic IMRT allows qu
ick delivery of prostate treatments, which are commonly achieved in four to seve
n minutes of irradiation. In adaptive radiotherapy, MVCT images can be used to r
ecalculate delivered dose. StatRT is a new capability for real-time simulation a
nd treatment that may prove useful for daily planning of high-dose stereotactic
prostate treatments while the patient is on the treatment couch in the treatment
position.
Disclosure: Timothy Holmes receives patent royalties on tomotherapy technology.
REFERENCES
1. Mackie TR, Holmes T, Swerdloff S, et al. Tomotherapy: a new concept for the d
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14
Image Guidance in Postprostatectomy Radiation Therapy
TIMOTHY N. SHOWALTER AND RICHARD K. VALICENTI
Department of Radiation Oncology, Kimmel Cancer Center, Thomas Jefferson Univers
ity, Philadelphia, Pennsylvania, U.S.A.
INTRODUCTION Salvage or adjuvant radiotherapy after radical prostatectomy (RP) h
as been shown to improve biochemical control for appropriately selected patients
, with an acceptable level of toxicity. Enhancement in the outcome will depend o
n our ability to assure the safe delivery of high radiation therapy (RT) doses (
>64 Gy). Image-guided radiation therapy (IGRT) is now widely used in the primary
treatment of prostate cancer and proving beneficial in the postprostatectomy se
tting. This new development is mainly due to improved target localization techni
ques and the better characterization of the prostatic fossa (PF) clinical target
volume (CTV). In this chapter, essential data are summarized regarding PF-CTV d
elineation, planning volume definition, and target localization methods for post
prostatectomy IGRT. The Role of Postprostatectomy Radiation Therapy RP is an eff
ective treatment for localized prostate cancer (1), but about 35% of patients ex
hibit biochemical failure as defined by durable rise in prostate-specific antige
n (PSA) (2,3). In a series of nearly 2000 men undergoing RP at Johns Hopkins Uni
versity Hospital, 15% developed a rise in PSA at a follow-up time of 15 years. R

oughly a
third of those patients with biochemical failure displayed distant metastases du
ring the study period (4), but it is presumed that the remaining two-thirds of p
atients with biochemical failure exhibited local recurrence. For such patients w
ith delayed rise in PSA, salvage RT may be offered to provide local control and
prevent subsequent distant dissemination (5). Biochemical failure rates are high
er after RP for patients with adverse pathologic features, such as positive surg
ical margins, seminal vesicle invasion (SVI), or extracapsular extension (ECE) (
6). Adjuvant postoperative RT has demonstrated a biochemical disease-free surviv
al benefit compared with historical controls in retrospective series (711) as wel
l as in three randomized, prospective trials (1214). For patients with adverse pa
thologic factors, adjuvant RT decreased the rate of biochemical failure by 48% a
nd 40% in the randomized trials conducted by the Southwest Oncology Group (SWOG)
and the European Organization for Research and Treatment of Cancer (EORTC), res
pectively (12,13). In the German study of adjuvant radiotherapy (ARO 96-02/ AUO
AP 09/95), which included only those patients with undetectable PSA after RP, RT
provided a 21% absolute advantage in four-year freedom from biochemical progres
sion (14). In summary, the medical evidence is compelling in favor of the routin
e use of adjuvant postoperative RT in appropriately selected patients (Table 1).
153
154 Table 1 Summary of Prospective Trials of
ectomy Study SWOG 8794 (13) EORTC 22911 (12)
8 153 RT technique Conventional Conventional
0 60 0 FFBF (%) 65.1 (5 yr) 36.0 (5 yr) 74.0
yr)

Adjuvant Radiotherapy After Prostat

ARO 96-02 (14) N 172 175 502 503 10
Conventional RT dose (Gy) 6064 0 60
(5 yr) 52.6 (5 yr) 81 (4 yr) 60 (4

Showalter and Valicenti

p value p < 0.001 p < 0.0001 p < 0.0001
For patients with rising PSA, the optimal trigger point for salvage PF RT is con
troversial, as definitions of PSA failure after RP vary. The American Society fo
r Therapeutic Radiology and Oncology (ASTRO) guidelines published in 1999 define
biochemical failure at a PSA level of 1.5 ng/mL (15). Although the range of PSA
level cut points recommended in the literature is wide (9,1626), outcomes may be
improved by instituting RT at a PSA level as low as 0.5 ng/mL (21,27). Clinicop
athologic criteria have been developed to estimate likelihood of local recurrenc
e and appropriateness for adjuvant RT. When considering treatment for presumed l
ocal recurrence, it is important to exclude patients at significant risk of subc
linical metastatic disease. Persistently detectable PSA after prostatectomy rais
es suspicion for possible distant metastasis, and postoperative RT should be off
ered only after correlating the PSA with risk of residual prostate cancer based
on positive surgical margin, ECE, or SVI (5). Distant metastasis after RP may al
so be predicted by time to PSA failure less than 2 years, Gleason score 8 or hig
her, and PSA doubling time less than 10 months (4). These patients are unlikely
to benefit from PF RT, and the careful selection of patients with high likelihoo
d of local-only disease prevents the exposure of patients with metastatic prosta
te cancer to potential toxicity. The distinction of local residual or recurrent
prostate cancer from microscopically disseminated disease is the crucial first s
tep in the radiotherapeutic management of patients with a delayed rise in PSA af
ter RP. Imaging Evaluation of Postprostatectomy Failures Given the limited value
of standard imaging tests as well as clinical examination for the detection of
recurrent disease after RP, the assessment of an elevated PSA after RP presents
a challenge (27,28). For patient with PSA relapse after surgery, the probability
of a positive bone scan or CT scan is low; rates of positive findings on bone s
can are as low as 4.5% for PSA less than 10 ng/mL (28). Therefore, conventional
imaging assessment with CT or bone scan is performed with a low level of

expectation for most patients in this clinical setting, and alternate approaches
should be evaluated.
Magnetic Resonance Imaging
Endorectal-coil magnetic resonance imaging (eMRI) is a promising imaging modalit
y for the detection of local recurrence after RP for patients with isolated rise
in PSA. In an early study of 35 patients with clinical suspicion of PF recurren
ce, specificity of eMRI was 100% and sensitivity was also 100% (29). A more rece
nt study of eMRI with 48 patients showed 95% sensitivity and 100% specificity fo
r identifying local recurrence (30). These results, although with small patient
numbers, suggest that eMRI is the preferred method for imaging PF recurrence fol
lowing RP (31). Additionally, MRI-guided biopsy may be performed for the detecti
on of recurrent disease after RP (32).
Radioimmunoscintigraphy
Functional imaging modalities have been investigated as tools for confirming loc
al recurrence in patients with elevated PSA after RP. Radioimmunoscintigraphy (R
IS) with 111 indium-capromab pendetide (ProstaScint 1 , Cytogen Corp., Princeton
, New Jersey, U.S.) has been used for the detection of local recurrence or resid
ual prostate tissue after RP with mixed results (3335). In a report of 43 men wit
h biochemical recurrence after RP, a negative 111indium-capromab pendetide scan
predicted significantly for a lower rate of biochemical progression (10% vs. 41%
, p 0.026) after salvage RT (36). Chance for durable biochemical control was red
uced by a positive 111 indium-capromab pendetide scan outside the pelvis in a st
udy of 23 patients (37). In contrast, Thomas et al. reported that positive 111in
dium-capromab pendetide findings outside the pelvis were not predictive of bioch
emical control (33). It has also been purported that this test has a positive pr
edictive value of 27% for extrapelvic disease (37), and a sensitivity of 49% for
PF recurrence (38). Thus, the available data does not justify the routine use o
f 111 indium-capromab pendetide scans to determine the site of recurrence in pat
ients with an isolated rise in their PSA after RP (33,39), but technical improve
ments such as the
Image Guidance in Postprostatectomy Radiation Therapy
155
use of CT fusion may increase the diagnostic accuracy of this test (40).
Positron Emission Tomography
Another promising functional imaging approach is positron emission tomography (P
ET). PET frequently uses the tracer, 2-[18F]-fluoro-2-deoxyglucose (FDG), and cu
rrently has low sensitivity for the evaluation of prostate cancer because of mod
est glucose consumption by these tumors and high background activity due to the
proximity of the urinary tract. Local or systemic disease was detected by FDG-PE
T in only 31% of patients in a recent study (41). Due to the low sensitivity of
FDG-PET, other PET tracers have been investigated for the detection of cancer in
the prostate bed. In a retrospective report of patients with elevated PSA after
surgery, 11C-choline PET scans showed a true positive result in 38% of clinical
local recurrences confirmed by ultrasound and/or digital rectal examination (DR
E), but no positive scans were found in patients with PSA levels less than 5 ng/
mL (42). Wachter and colleagues prospectively studied the use of 11 C-acetate PE
T scans fused with MRIs in the setting of post-RP biochemical failure. They foun
d the imaging technique to be feasible, and the functional images revealed local
recurrence not visible on the anatomic imaging in a handful of patients; 11C-ac
etate PET scans affected clinical management in 28% of patients studied (43). PE
T studies with 11C-acetate and 18F-choline successfully identify local recurrenc
e in only about half of the cases with PSA levels less than 1 ng/mL, which is le
ss than that reported with the use of eMRI (43,44). It has been suggested that t

he sensitivity of PET scans may increase with increasing PSA, suggesting a relat
ionship between tumor burden and sensitivity (41). Currently, PET scanning has n
o established role in the early identification of local recurrence or residual t
umor.
clips, bladder neck, and seminal vesicle remnants. Typically, three-dimensional
conformal RT (3DCRT) techniques with four to six fields have been used for postp
rostatectomy RT (5). In the SWOG prospective trial of adjuvant RT, 9 9 cm or 10
10 cm radiation portals were used in a four-field technique (27). In addition to
3DCRT, intensity-modulated radiation therapy (IMRT) has been used in the postpr
osatectomy setting. Bastasch and colleagues delivered IMRT to a target volume de
fined as the prostatic fossa plus periprostatic tissues. This information was ob
tained from the treatment planning CT, influenced by information in the operativ
e report and the location of surgical clips. In their study, a 5-mm margin was a
dded to the CTV for planning purposes (47). Most of prospective and retrospectiv
e studies provide information for RT technique, but are vague in their descripti
ons of the target volume. It is the goal of this section to provide insights int
o PF-CTV definition using both clinical and imaging data, as well as the selecti
on of treatment margins as it applies to target localization techniques in the p
ostprostatectomy setting. Clinicopathologic Principles for Target Volume Definit
ion Principles for the design of the PF-CTV may be derived from an appreciation
of the pathologic and clinical information. It is known that most local recurren
ces after RP occur in the caudal portion of the PF near the surgical anastamosis
(48,49). DRE is 44% sensitive and 91% specific in the detection of local recurr
ence, while transrectal ultrasound is 76% sensitive and 67% specific (48). In a
report of transrectal ultrasound examinations with biopsies for rising PSA after
RP, the most common sites of local recurrence were the urethra-vesicular anasta
mosis (UVA) (66%), the bladder neck (16%), and posterior to the trigone (13%) (5
0), thus indicating the importance of including these locations in the PF-CTV. T
he definition of the PF-CTV may also be based on pathologic findings and the loc
ation of surgical clips. The pathology report provides information regarding SVI
, surgical margin location, and the presence and extent of ECE, and these factor
s should influence the CTV on an individual basis. The use of surgical clips for
delineation of the PF-CTV has not been validated, so the location of the clips
should be correlated with additional pathologic and anatomic data, including dis
cussion with the urologic surgeon. A preferred approach would entail a discussio
n with the surgeon prior to the operation about the strategic placement of the s
urgical clips at the anastamosis, bladder neck, and the space posterior to the b
ladder adjacent to the seminal vesicle remnants.
TARGET VOLUME DEFINITION There is no consensus for target contouring and treatme
nt planning for postprostatectomy RT, although the definition of CTV is often ba
sed on CT imaging data (44). Clinical investigations have revealed significant i
nterobserver variability in defining the PF-CTV with CT, suggesting inadequate c
onsensus and inherent uncertainty in target definition (45). It is not clear whe
ther interobserver variability in target volume delineation for prostate cancer
RT affects normal tissue toxicity in a clinically meaningful way (46), but, none
theless, uncertainty in target definition is suboptimal and further research eff
orts are warranted. The PF target volume at some institutions has been determine
d by the prostate tumor bed, surgical
156
Showalter and Valicenti
Image-Guided Target Volume Definition
Historically, the planning CT simulation has been the primary source of volumetr
ic imaging data for the determination of PF target volume. Although treatment pl
anning using RIS with 111indium-capromab pendetide may influence the identificat
ion of a clinical target when compared with the CT, (51,52), this approach is no

t considered standard. In this section, we will review information pertinent to

anatomic and imaging considerations for optimal PF-CTV delineation. In designing
the PF-CT, alteration in pelvic relational anatomy due to the surgical procedur
e should be considered. Alterations of the prostate CTV by radical retropubic pr
ostatectomy have been studied quantitatively with CT by Sanguineti and colleague
s (52). Six patients were evaluated; each underwent a planning CT scan between o
ne month and one week prior to surgery and clinical target volumes (CTVpre) were
contoured. One to two months after surgery, a planning CT scan was performed in
the same position and target volumes (CTVpost) were contoured. Rectum and bladd
er volumes were also generated and dosimetric and volumetric analyses were perfo
rmed for a standardized four-field 3DCRT plan. Postoperative CTV volumes were 30
% to 50% smaller than preoperative CTV volumes, depending on whether seminal ves
icles were encompassed in the volumes. The reduction in target volume size was h
ypothesized to be related to shifts in adjacent normal organs as such shifts may
alter the anatomic boundaries of the CTV. The imaging data demonstrated that th
e bladder moves posteriorly and caudally after RP, but that the location of the
rectum is not changed significantly due to the procedure. On average, the upper
limit of CTVpost was more caudal than that of CTVpre. The post-RP reduction in v
olume of CTV and alteration in bladder position favorably affected the dose-volu
me histograms (DVHs) for the bladder and rectum. The quantitative description of
surgical alterations in target volumes and organs at risk (OARs) by Sanguineti
et al. provides a framework for considering additional information regarding PF
target volume delineation. Patterns of postprostatectomy locoregional recurrence
have been described by Miralbell et al. using eMRI (53). Their study, which was
modeled after seminal reports that influenced RT field definition guidelines fo
r stomach and rectal cancer (54,55), consisted of 60 consecutive patients who re
ceived salvage radiotherapy at a single institution. All patients underwent both
a diagnostic eMRI and a planning CT prior to RT. The spatial coordinates of the
PF tumor recurrence were transferred from the eMRI to the CT for analysis. A CT
V based on the eMRI information was generated for each patient and compared with
the standard, CT-based CTV designed to encompass the prostate bed. Most sites o
f recurrent or residual prostate tumor
Figure 1 DRR depicting sites of local residual or recurrent disease after prosta
tectomy. Source: Ref. 53.
cells in the PF were located in the inferior and posterior aspects of the UVA. A
scatter plot to show sites of local PF recurrence was produced on orthogonal di
gitally reconstructed radiographs (DRRs) using the spatial coordinates from the
tumor recurrences visualized on eMRI (Fig. 1) (53). Ideal eMRI-based CTVs and pl
anning target volumes (PTVs) were produced using the eMRI information and compar
ed with the standard target volumes for a 3DCRT. In Figure 2, the Beams eye view
(BEV) image on the left depicts a left lateral field with margins around a stand
ard PTV encompassing the PF, while the BEV on the right displays a left lateral
field with a PTV produced used the investigators recommendations. Miralbell et al
. recommend a smaller, cylindrical CTV for postprostatectomy RT, including the U
VA and bladder neck down to the level of the penile bulb. Their recommended CTV
is generally 4-cm long and centered approximately 5 mm posterior and 3 mm inferi
or to the UVA (53). The volumetric data obtained from the above studies with CT
and MRI images depict the anatomic changes resultant from prostatectomy and emph
asize the importance of including the caudal portion of the prostate bed in the
PF-CTV. Interfraction Organ Motion and Planning Margin Fiorino and colleagues ut
ilized weekly CT scans to study rectal and bladder motion during a course of pos
tprostatectomy RT (57). The planning CT scan for each patient was coregistered w
ith subsequent weekly CT scans obtained in the treatment position. The PF-CTV, r
ectum, and bladder were defined by the same observer, and variations in volumes,
DVHs, and organ border position were analyzed. BEV analysis revealed a systemat
ic anterior shift of the anterior rectal wall during RT for six of nine patients
studied, with consequent worsening of the rectal DVH. The anterior rectal wall
shift, which averaged 2.5 mm, was noted only within the cranial half of the

Image Guidance in Postprostatectomy Radiation Therapy

157
Figure 2 Beams eye view projections showing a standard PTV with radiation portal
(A) and the PTV proposed by authors based on sites of recurrence detected by MRI
(B). Source: Ref. 53.
rectum. The anterior wall of the cranial half of the rectum also correlated with
the posterior border of the PF-CTV. The region of rectal wall motion and the re
lationship between CTV and rectum wall are displayed in Figure 3 (57). This stud
y characterizes interfraction pelvic organ motion in the postprostatectomy patie
nt and shows the interrelatedness of the anterior rectal wall and the posterior
PF-CTV border. Researchers at Thomas Jefferson University Hospital (TJUH) have u
sed cone-beam computed tomography (CBCT) images for daily setup correction and t
o study interfraction motion of the bladder and rectum as a method for analyzing
changes in the PF during a course of RT (57). The PF was not contoured on CBCT
images in the study due to reported uncertainty in postprostatectomy target deli
neation on helical CT (45). The rectum and bladder were readily visualized on th
e CBCT images, and the ability to contour the rectum with acceptable
interobserver variability has been reported (56). CBCT images obtained two to fo
ur times weekly for 10 patients in the treatment position immediately prior to R
T were used to determine the interfraction motion of the posterior bladder wall
and anterior rectum wall relative to the reference planning CT (CTref) and to th
e mean organ border position. Measurements were performed at three cranial-to-ca
udal levels titled SUP, MID, and INF. The authors cite a prior report of correlation b
etween PFCTV and anterior rectum wall shifts, which was described only within th
e cranial half of the rectum (57). The authors also proposed CTV-to-PTV margins
that were calculated with the posterior bladder wall motion and anterior rectum
wall motion serving as surrogates for the movement of the anterior and posterior
borders of the PF-CTV. This approach recognizes the uncertainty regarding PF de
finition on CT and assumes that the adjacent pelvic organs contribute to the del
ineation of
Figure 3 Rectal motion occurred within the cranial half of the rectum (A) and re
ctum wall motion correlated with shifts in posterior CTV border over this region
(B). Source: Ref. 57.
158
Showalter and Valicenti
Table 2 Organ Motion Relative to Planning CT as Observed on CBCT Images and Reco
mmended CTV-to-PTV Margins Based on Motion of Adjacent Organ Borders Bladder mot
ion (mm) Observed motion SUP MID 0.4 3.7 2.1 2.8 6.2 INF 1.5 4.0 2.1 2.4 5.9 SUP 2.
6 6.0 3.5 4.0 9.8 Rectal motion (mm) MID 1.6 6.3 3.5 4.5 10.2 INF 2.7 5.8 3.1 3.5
8.6
Relative to CTref ( anterior posterior) Mean 0.1 SD 4.4 Systematic error (S) 2.4 R
andom error (s) 3.3 Calculated PTV margin (2S 0.7s) 7.1
Source: Ref. 58.
the anteroposterior (AP) target volume borders. Table 2 contains results for pel
vic organ motion and for estimated CTV-to-PTV margin recommendations (58). These
values may be considered when determining treatment guidelines for postprostate
ctomy RT. TARGET LOCALIZATION TECHNIQUES Image guidance during the treatment cou
rse is an important aspect of assuring precise RT delivery to the planned PF-CTV
. Imaging at the time of treatment confirms the proper positioning of the target

volume, allows for the correction of setup errors, and provides information reg
arding interfraction organ motion (59). IGRT is most likely to be beneficial whe
n the target volume is immediately adjacent to OARs, and postprostatectomy RT do
se is escalated above 64 Gy (60). Multiple studies support the notion of radiati
on doseresponse for RT after RP (Table 3), with RT doses of 64 Gy or higher lead
ing to significant improvements in biochemical control (15,25,27,61,62), but pot
entially with an increased risk of toxicity. At TJUH, the institutional
Table 3 Summary of Published Studies Supporting Radiation Dose-Response for Trea
tment after Radical Prostatectomy RT doses (Gy) !61.5 <61.5 !64.8 <64.8 !64 <64
>65 65
policy is to deliver 68.4 Gy with IGRT for postprostatectomy patients. The risk
of late rectal bleeding after postprostatectomy RT is reduced by restricting the
amount of rectal volume receiving !50 Gy (63), but only by decreasing the treat
ment volume (64). A small treatment volume that is not localized during the cour
se of RT delivery may lead to underdosing of the PF-CTV and a loss of treatment
efficacy. To assure precise and accurate execution of the RT treatment plan, fre
quent examination of the online CTV during seven- to eight-week course of RT is
now achievable with several IGRT techniques (65). As in the setting of primary R
T for clinically localized prostate cancer, target localization methods may incl
ude the use of transabdominal ultrasound, computed tomography, or electronic por
tal imaging device (EPID) with fiducial markers. Ultrasound Chinnaiyan and colle
agues reported the use of an optically guided, transabdominal ultrasound target
localization system for daily positioning during postprostatectomy 3DCRT. Figure
4 shows a sample ultrasound image from their study, as well as the correspondin
g planning CT. The bladder neck was used as a surrogate anatomic structure for t
he PF, and corrective shifts were determined by the ultrasound-based position of
the bladder neck. Daily localization with transabdominal 3D ultrasound was dete
rmined to be feasible in the radiation therapy clinic. In their experience, dail
y internal motion of the target volume for patients undergoing post-RP RT was si
milar in magnitude to patients with intact prostates, supporting a role for dail
y localization (66). Although the bladder neck was selected as an anatomic surro
gate for the PF in this study, it is unclear whether this structure alone is a r
eliable surrogate for target localization of the PF. Investigators from Fox Chas
e Cancer Center (FCCC) have described an approach for PF target localization
Study Valicenti et al. (Adjuvant pT3) (62) Valicenti et al. (0.2 < PSA > 2.0) (6
2) Schild et al. (salvage) (88) Anscher et al. (salvage) (61)
N 52
FFBF (%) 91 (3 yr) 57 (3 yr) 79 (3 yr) 33 (3 yr) 57 (3 yr) 17 (3 yr) 2.2 yr (med
) 0
p value 0.01
21
0.02
46 89
0.059 <0.001
Image Guidance in Postprostatectomy Radiation Therapy
159
Figure 4 CT (left) and ultrasound (right) images from patient undergoing postpro
statectomy IGRT with rectal balloon in place. Source: From Ref. 66.

using B-mode acquisition and targeting (BAT) ultrasound and CT-on-rails (67). Fo
r patients with intact prostates, the BAT ultrasound system has been demonstrate
d previously to be equivalent to CT for daily localization during definitive RT
(68). In the FCCC study in the postprostatectomy setting, corrective shifts dete
rmined by BAT ultrasound were compared with those obtained by CT scans obtained
in the treatment position prior to RT using CT-on-rails. A total of 90 pairs of
CT and ultrasound images were compared for 9 patients, and the average interfrac
tion PF target volume motions based on CT shifts were 3.0, 3.2, and 5.1 mm in th
e lateral, superoinferior (SI), and AP directions, respectively. Evaluation of t
he accuracy of BAT ultrasound localization of the PF, using CT-based localizatio
n as the standard for comparison, revealed systematic errors in alignment, poten
tially attributable to inherent uncertainties in postoperative target definition
. The disagreement between CT and BAT ultrasound shifts was largely systematic,
rather than random, and the authors noted that the discrepancies could be improv
ed through the use of CT-based templates created using CT scans obtained during
the treatment course. To resolve this discrepancy, the authors proposed a locali
zation technique that combines information from both CT and ultrasound to provid
e reliable target localization (67). Computed Tomography Daily target localizati
on with CT has been shown to allow reduction of PTV margins for patients receivi
ng
3DCRT, and investigators from FCCC have recommended a 3-mm PTV margin for IGRT o
f the intact prostate with daily helical CT (69). Using megavoltage CT for daily
target localization during postprostatectomy RT, Kupelian and colleagues report
ed a small average prostate motion relative to the bony anatomy. Although instan
ces of PF motion greater than 3 mm were infrequent, the study demonstrates a dis
crepancy between alignments based on bony anatomy and the prostate bed (70), sug
gesting a potentially beneficial role for CT as daily target localization. Kilov
oltage CBCT is available for online IGRT and may improve the precision of RT (71
). For patients with intact prostates, an analysis of residual setup errors usin
g online CBCT during 3DCRT showed that a 3-mm margin would be required for patie
nts receiving CBCT target localization (72). Furthermore, when used for online i
mage guidance for RT of the intact prostate, CBCT has been shown to be equivalen
t to the technique of EPID with implanted fiducial markers, but with the added b
enefit of the visualization of adjacent soft tissue structures (73). Since 2005,
kilovoltage CBCT has been used for online image guidance in postprostatectomy r
adiotherapy at TJUH and has been feasible in this capacity (58). Since online CB
CT provides volumetric imaging data as well as the opportunity for setup error c
orrections, this can be a promising option for postprostatectomy IGRT (60). The
computer monitor display shown in Figure 5 contains an example of CBCT image fro
m TJUH used for postprostatectomy IGRT with a commercial software program (Elekt
a AB, Sweden).
160
Showalter and Valicenti
Figure 5 (See color insert.) Computer monitor display for online image registrat
ion and setup error correction using cone-beam CT. Commercial software used at T
JUH (Elekta AB, Sweden).
Fiducials with Electronic Portal Imaging Image-guided postprostatectomy radiothe
rapy has been delivered at the University of California, San Francisco (UCSF) us
ing daily EPID with radiopaque gold markers implanted in the prostate bed (74).
There is extensive experience in using this approach for daily target localizati
on for definitive treatment of patients with intact prostates as described elsew
here in this volume (7578). EPID with fiducial markers has also been shown to be
a valuable method in postprostatectomy IGRT for the correction of interfraction
motion of the prostate bed, with no significant migration of the fiducial marker

s during the RT course. Target positioning errors greater than 5 mm were detecte
d in 14.1%, 38.7%, and 28.2% of RT fractions in the AP, left-right, and SI direc
tions, respectively. Precision was improved with the use of daily target localiz
ation with EPID and implanted fiducial markers (74). OPTIMIZING IGRT IMPLEMENTAT
ION IGRT has the potential to improve the quality of postprostatectomy RT throug
h the definition of the PF target volume and the verification of the target posi
tion during the course of radiotherapy. It should be considered that the success
ful incorporation of complementary imaging modalities in PF target volume deline
ation is dependent on accurate integration of imaging modalities
with optimal image registration (59). In the future, with improvements in resolu
tion, functional imaging may become more important in treatment planning. In the
mean time, CT and eMRI are the most reliable forms of volumetric anatomic imagi
ng. IGRT strategies for target localization have been developed with the goal of
reducing geographic miss of the tumor and reducing dose to adjacent tissues, bu
t it is important to recognize and minimize sources of inaccuracy in treatment v
erification. Anatomic surrogates for the PF have not been validated for target l
ocalization, and interobserver variability may limit the accuracy of targeting s
trategies. Although CT and ultrasound provide volumetric data that may be used t
o study interfraction motion and visualize adjacent normal structures, their rou
tine use requires careful consideration and validation of protocols for image re
gistration including the use of anatomic surrogates for the PF-CTV such as the b
ladder neck and posterior trigone. Daily target localization using EPID with imp
lanted fiducial markers is appealing; the gold seed fiducials do not migrate sig
nificantly during the RT course and are readily identified (74). However, the in
creased information potentially available with volumetric imaging may make daily
ultrasound or CT the preferred options in the future. For patients with intact
prostate glands undergoing definitive IGRT with online kilovoltage CBCT target l
ocalization, the margin required to account for residual setup errors is 3 mm. T
he residual error in this situation
Image Guidance in Postprostatectomy Radiation Therapy
161
may relate to suboptimal image registration or target delineation (72). Patients
undergoing postprostatectomy RT will likely require a similar margin with IGRT
using daily volumetric imaging data. Studies of PF target localization using vol
umetric imaging have shown interfraction internal target motion errors of a magn
itude similar to intact prostates (66), which supports the rationale for applyin
g the image-guidance strategies developed for definitive RT to adjuvant or salva
ge RT of the PF. The correlation between late rectal bleeding and rectal DVH for
patients receiving postprostatectomy RT suggests that toxicity may be reduced b
y efforts to shrink treatment margins with IGRT and to limit the volume of norma
l tissue irradiated using daily imaging. It will be difficult to quantify the be
nefits of IGRT over standard therapy in clinical trials using traditional metric
s, but the improved precision in RT delivery should nonetheless translate into r
educed toxicity and improved tumor control (60). A potential future extension of
IGRT for postoperative RT after prostatectomy may be adaptive, image-guided the
rapy using images obtained in the treatment position. An offline strategy, simil
ar to the approach used for definitive prostate RT (79), may be possible for pos
tprostatectomy RT. With CBCT images, it may be feasible to consider the anterior
rectal wall and posterior bladder wall as surrogates for the AP PF borders for
the purposes of offline adaptive IGRT with conformal avoidance of the adjacent n
ormal structures (58). Further, volumetric imaging may allow online corrective s
trategies that adapt to patient positioning on a daily basis through conformal a
voidance strategies to minimize the volume of rectum and bladder included in the
radiation portals. Although there is uncertainty regarding the definition of th
e PFCTV on CT, the ability to visualize the rectum and bladder on CBCT makes con
formal avoidance strategies attractive. CLINICAL OUTCOME There are no published

reports of biochemical control rates after postprostatectomy IGRT, though it is

reasonable to expect improved biochemical control with IGRT due to image-guided
definition of the PF-CTV and reduced risk of geographic miss with image-guided t
arget localization. Reported biochemical outcome after postprostatectomy salvage
RT varies among series, and the success of RT depends on many disease-related f
actors (5,80,81) and may be improved by the use of higher RT doses (15,25,62,82)
or by androgen suppression in selected patients (83,84). The improvements in PS
A relapse-free survival and in recurrence-free survival obtained with adjuvant R
T in prospective trials were achieved with conventional RT without optimal targe
t
localization techniques. It is anticipated that theoretical improvements in loca
l control provided by IGRT will translate into a greater clinical benefit for po
stprostatectomy patients. No data are available regarding improved normal tissue
toxicity with postprostatectomy IGRT, but clinical gains are expected due to th
e potential to reduce the volume of irradiated normal tissue (60). Overall, curr
ent measures of toxicity show acceptable toxicities with salvage or adjuvant RT
using conformal techniques. Acute toxicities from 3DCRT are tolerable and comple
ted without interruption for 97% of patients in the EORTC 22911 trial (12). Wher
eas conventional RT techniques produced moderate to severe late complications in
7% to 20% of patients undergoing postprostatectomy RT, the rates of late grade
II genitourinary (GU) and gastrointestinal (GI) toxicities after 3DCRT are about
5% and 9%, respectively (16). In a recent report from a large, multiinstitution
al database of patients who received salvage or adjuvant RT after RP, rates of g
rade 2 (10%) and grade 3 (1%) late GU toxicity were low, as were rates of grade
2 (4%) and grade 3 (0.4%) late GI toxicity (85). In a prospective study, adjuvan
t RT was not associated with increased risk for urinary incontinence (85). Patie
nt questionnaires have been used to study urinary and fecal continence for men t
reated with and without adjuvant RT after RP; although incontinence was present
at a higher rate at four to eight months after RT, there was no difference in ra
tes of incontinence at one year between those who did and did not receive RT (86
). In addition to GU and GI side effects, erectile function after adjuvant or sa
lvage RT is important, particularly for patients who chose nerve-sparing procedu
res in hopes of preserving potency. It is unclear if 3DCRT adversely affects pot
ency after RP (87). In one study, IMRT to a mean dose of 69.6 Gy after nerve-spa
ring prostatectomy was shown to preserve potency in all men with intact erectile
function before RT (47). Although favorable toxicity profile has been achieved
with 3DCRT or IMRT, IGRT may improve upon these results through the ability to r
educe RT margins and to minimize the volume of irradiated normal tissue, particu
larly when higher RT doses are administered. QUALITY CONTROL Imaging obtained pr
ior to daily RT fractions in the treatment position provides verification data t
hat are useful for quality assurance procedures. Interfraction organ motion or m
orphologic changes as a result of therapy can also be studied using these images
(59). In this fashion, IGRT may improve the quality of RT through an enriched a
ppreciation of physical variability in the target volume
162
Showalter and Valicenti
and surrounding normal tissues. It is incumbent upon radiation oncologists to pr
ovide data to confirm therapeutic gain from the increased precision of IGRT, but
the intuitive nature of the improvements from this technology make the design o
f meaningful clinical trials essential (60). IGRT may improve the delivery of po
stprostatectomy RT in ways that are difficult to appreciate. The application of
online strategies for target localization may require additional time in the cli
nic, (59) so changes in workflow should be documented when novel IGRT strategies
are implemented. Image-guidance technologies must pass rigorous quality assuran
ce inspection prior to implementation in the clinic. New strategies may be compa
red with other techniques for verification, similar to the comparison of CT to u

ltrasound for daily target localization for intact prostates (68). SUMMARY OF PE
RTINENT CONCLUSIONS
l
based on daily volumetric imaging should include validation of the target locali
zation strategy.
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15
The Use of Image Guidance in Prostate Brachytherapy
CYNTHIA MENARD AND JUANITA CROOK
Department of Radiation Oncology, Princess Margaret Hospital, University Health
Network, University of Toronto, Toronto, Ontario, Canada
INTRODUCTION By virtue of the exponential decline in dose intensity with distanc
e from the source, brachytherapy for prostate cancer requires precise and accura
te source placement. Gains in the therapeutic ratio are largely accomplished wit
h careful spatial selection of target tissues for high-dose radiation exposure.
While modern techniques can achieve precision in delivery, toxicity and local co
ntrol both remain an issue for a subset of patients, and expanding indications a
re placing more demands on the modality. Recent advances in imaging and image gu
idance may address this challenge. The history of prostate brachytherapy illustr
ates a clear benefit to image guidance (1,2). Since Pasteaus publication in 1913
(3), describing insertion of a radium capsule into the prostatic urethra to trea
t carcinoma of the prostate, various techniques have been employed with unsatisf
actory results. In 1917, Barringer first performed transperineal brachytherapy u
nder transrectal tactile guidance (Fig. 1) (4). Decades later, in the early 1970
s, Whitmore described open retropubic implantation guided by both direct intra-o
perative visualization of the prostate and transrectal palpation (5). Poor longterm outcomes were attributed to freehand source placement and inadequate dosime
try. In fact, dosimetry was calculated from plain radiographs, assuming that the
target tissue was accurately
encompassed by the implant (6). The advent of transrectal ultrasound (TRUS), whi
ch permitted direct visualization of needles in relation to prostatic boundaries
, and the subsequent development of the Seattle technique (7) revolutionized bra
chytherapy for prostate cancer. The entire prostate gland has traditionally been
considered the target for prostate brachytherapy for two reasons: (i) prostate
cancer is inherently multifocal, placing the entire gland at risk and (ii) direc
t imaging of foci of cancer within the gland has been elusive. However, in reali
ty, the local extent of prostate cancer is neither confined nor defined by the b
oundaries of the prostate gland. Although modern imaging techniques are still no
t able to map the extent of microscopic disease, they are capable of mapping int
raprostatic sites of macroscopic tumor, knowledge of which may facilitate the st
udy of tumor biology and virulence. These images can now be integrated in prosta
te brachytherapy for selective modulation of dose intensity both within and adja
cent to the prostate gland (8). Given high precision in delivery, image-guided p
rostate brachytherapy may be the ideal platform for investigating intraprostatic
dose modulation. The term image guidance includes both off-line and online guidan
ce techniques. This distinction is important since off-line imaging applies to a
ll image-directed techniques where imaging is performed at a separate
167
168
l
 Menard and Crook
l
Interactive planning: stepwise refinement of the treatment plan using computeriz
ed dose calculation derived from needle positions Dynamic dose calculation: cons

tant updating of dose calculations using deposited seed or dwell positions

Since no single imaging modality embodies all the optimal characteristics for im
age guidance of brachytherapy for prostate cancer, multimodality guidance models
will likely predominate for the foreseeable future. This chapter will place mod
ern prostate brachytherapy techniques in the context of desired image guidance o
bjectives, such that the promise and potential of brachytherapy can be fulfilled
in the spectrum of prostate cancer treatment.
TRANSRECTAL ULTRASOUND TRUS is considered the gold-standard image guidance modal
ity for prostate brachytherapy. It provides goodquality images of the prostate g
land boundary by using a high-frequency (57.5 MHz) intrarectal probe (10). The re
lative ease of mastering the basics of the transrectal sonograhic technique, rea
l-time 2D feedback, and accurate volume assessment of the gland has contributed
to its widespread use for image-guided brachytherapy (11). In fact, the brachyth
erapy community can be credited for initiating 2.5D step-TRUS image acquisition
for reconstructed 3D visualization of the prostate. This technique led to the de
velopment of 3D TRUS using rotation fan scans, where overlay of sequentially acq
uired 2D images results in improved image quality and resolution (Fig. 3) (12).
Substantial rotation (13), translation, and deformation (14) of the prostate wit
h needle insertion highlight the value of real-time TRUS feedback during needle
insertions. One limitation of TRUS in the guidance of therapy for prostate cance
r is its restricted soft tissue resolution. Conventional TRUS is neither suffici
ently sensitive nor specific for visualization of intraprostatic tumor and there
fore has
Figure 1 Transperineal brachytherapy needle placement under transrectal tactile
guidance prior to the advent of image guidance. Source: From Ref. 4.
time from the brachytherapy procedure. In contrast online imaging, or image guidan
ce, is intricately integrated both temporally and spatially in the brachytherapy
procedure. Furthermore, needle guidance should be distinguished from dose guida
nce, with needle guidance referring to the placement of brachytherapy needles or
catheters, and dose guidance referring to the delivery of radiation dose. As de
fined by the American Brachytherapy Society (9), preplanning refers to the creat
ion of a dose plan a few days or weeks prior to the implant procedure, while int
ra-operative planning includes
l
Intra-operative preplanning: creation of a plan as the first step of the implant
procedure
Figure 2 Images acquired after the placement of HDR brachytherapy catheters and
prior to radiation delivery in one patient demonstrating the superiority of MR i
maging in depicting both catheters and prostatic anatomy. (A) TRUS, (B) CT, and
(C) MRI. Source: From Ref. 129.
The Use of Image Guidance in Prostate Brachytherapy
169
limited use for staging purposes (8). Although prostate cancer may present as a
hypoechoic area in the peripheral zone, hypoechoic features are not specific to
prostate cancer and up to 40% of prostate cancers are isoechoic (10). Furthermor
e, implanted seeds cannot be reliably localized on TRUS (15). Seed specularity,
shadowing, and tissue clutter make imaging catheters and seeds difficult using c
onventional ultrasound. Ongoing research in prostate ultrasound is now aimed at
solving these limitations. Color Doppler and its next generation, power Doppler
technology, can show vascular profiles in tissues. Depending on the amplitude of
the signal, the power Doppler image is displayed with varying hue and brightnes

s, with color representing the total energy of the Doppler signal. Although this
technology may help to reduce needle trauma to the peri-prostatic vasculature,
Doppler maps cannot distinguish intraprostatic pathology, and therefore cannot g
uide tumor-targeted brachytherapy (16). Contrastenhanced power Doppler TRUS has
shown promise in this regard and may be useful in defining intraprostatic target
s (10,11,16). Intravascular microbubble contrast agents enhance the back-scatter
ed echo from blood flow (17) and newer bubble agents that resonate at higher fre
quencies may further improve the signal. 4D displays may allow for improved dete
ction and quantification of areas of flow asymmetry (18). Further correlation wi
th pathology is required before this technology can be incorporated in the ident
ification of intraprostatic targets. Ultrasound elastography, or sonoelasticity
imaging, is at an early stage of development, and its potential role in image-gu
ided brachytherapy is promising (8). Mechanical vibration is transferred to the
imaging area, which contains materials of different vibration properties. The di
fferences are detected in Doppler mode ultrasound (9). Sonoelasticity imaging ma
y be used to detect brachytherapy catheters and seeds, and to distinguish tumorbearing prostate tissue. Finally, novel image-processing techniques, such as sin
gular spectrum analysis, have shown promise preclinically in improving seed loca
lization (19), while robotic assistance to catheter and seed segmentation and tr
acking may circumvent some of the limitations of TRUS (20). True interactive pla
nning and dynamic dose calculation under TRUS guidance, will require autosegment
ation of TRUS images (21,22), the subject of which is beyond the scope of this c
hapter. RADIOGRAPHY AND COMPUTED TOMOGRAPHY (X RAY AND CT) By virtue of unparall
eled image resolution of implanted devices, online planar radiography has long b
een employed to augment TRUS in the evaluation of implant
geometry and bladder wall violations. Various techniques have been proposed to o
bjectify this integration with 3D registration of planar radiographs to TRUS ima
ges using fiducial markers attached to the ultrasound probe (23), or implanted n
eedles (24). In this manner, images can be acquired in arbitrary positions to im
prove accurate mapping of seed locations relative to TRUS images. Early clinical
results from the merging of fluoroscopy and TRUS to compute delivered dose intr
a-operatively (25), and guide placement of additional sources to underdosed area
s (26) are promising. Computed tomography (CT) essentially produces a 3D rendere
d image of brachytherapy catheters and seeds. Its current application lies in of
f-line postplanning evaluation of permanent implants, or less frequently in the
treatment planning of temporary high-dose rate (HDR) brachytherapy after TRUS-gu
ided placement of catheters and prior to dose delivery. However, the prostate is
not well demarcated from surrounding structures on CT nor is intraprostatic ana
tomy well demonstrated (Fig. 2) (17). Compared to MR and TRUS, CT displays the l
argest variability and least correspondence when delineating the prostate (27,28
). In general, CT defined volumes are 30% larger than those defined by TRUS (28,
29). Although dynamic contrast enhanced CT can identify high-volume, poorly diff
erentiated prostate cancers, it is unable to map intraprostatic tumor in the maj
ority of patients (10). Attempts to improve soft-tissue delineation accuracy hav
e resorted to the coregistration of alternative imaging modalities to CT. Some i
nvestigators have acquired postplanning CT images in the presence of a TRUS prob
e with subsequent registration of TRUS and CT images (30). A more common approac
h has been to register magnet resonance imaging (MRI) to planning CT. Another we
akness of CT in the brachytherapy scenario is the fact that artifacts from impla
nted material can substantially degrade CT image quality. Techniques to reduce C
T metal artifacts include elegant solutions in post-processing of raw image data
(31). Novel approaches have also been proposed to improve the accuracy of autom
atic 3D localization of seeds from CT scans, reaching sub millimeter accuracy (3
2,33). There are many efforts to bring CT technology from off-line to online gui
dance. Fuller et al. have described a technique in which, following TRUS-guided
brachytherapy, patients proceed immediately to CT for postplanning. The CT image
s are coregistered to TRUS images to map possible dosimetry deficiencies. If nec
essary, the patient is immediately re-prepped for TRUS guidance of remedial sour
ce placement (34). In order to improve efficiency, other investigators have brou

ght diagnostic CT scanners to the brachytherapy suite, performing TRUS-guided br

achytherapy on a modified CT table and thereby circumventing
170
 Menard and Crook
Figure 3 (A) A 3D TRUS prostate image post implantation sliced to visualize a tr
ansverse plane, (B) 3D volume sliced in the transverse and longitudinal directio
ns, and (C) coronal plane. Source: From Ref. 130.
the need for patient transfers (35). A more cost-effective investigational solut
ion involves using cone-beam techniques and amorphous silicon detectors in the b
rachytherapy suite for dual online fluoroscopic and CT guidance (Fig. 4) (9). It
is important to remember that online guidance of prostate brachytherapy using X
-ray technologies risks occupational radiation exposure of health care personnel
. Strategies that increase the use of X-ray in an online-guidance model must car
efully consider and limit such exposures. MAGNETIC RESONANCE IMAGING Unlike TRUS
and X-ray imaging modalities, MRI has not been widely integrated in current pro
state brachytherapy guidance. Image acquisition time, complexity, space constrai
nts, cost, and access to equipment remain as barriers to its universal adoption.
However, MRI guidance of prostate brachytherapy is compelling for a number of r
easons, and the subject of considerable research in both diagnostic and therapeu
tic communities. First and foremost, prostate visualization is best realized wit
h MRI, both for gland boundaries and subglandular architecture, as well as for a
djacent organs at risk (9,36). For tumor-targeted dose intensification, MRI yiel
ds the highest accuracy in identifying the location and extent of both intra- an
d extraprostatic tumor burden. In fact, visualization of extracapsular extension
on
diagnostic MRI is responsible for modification of dose plans in a substantial pr
oportion of patients (37). As prostate MRI acquisition techniques evolve, a comb
ination of MR characteristics, including low T2 (3843), rapid intravenous T1 cont
rast enhancement and washout (44), low diffusivity (45,46), high R2* (4749), and
high cholinecreatine/citrate ratio (5053) will create accurate maps of cancer burd
en. Such tumor-mapping accuracy currently requires the use of an endorectal coil
, and prolonged examination times (4560 minutes). Image voxel resolution varies d
epending on pulse sequence, magnetic field strength, and scan time, achieving 2
0.5 0.5 mm [(T2-weighted fast spin echo (FSE)] to 5 mm3 for (MRSI) (54,55) with
modern equipment. Efforts to improve image resolution to 1 mm3 isotropic voxels
are ongoing. MRI may also provide a window on tumor biology, virulence, and radi
osensitivity. R2* maps from BOLDMRI acquisitions (with rBV correction) have high
sensitivity (95%) and reasonable specificity (70%) for defining intraprostatic
tumor hypoxia, and possibly radioresistance, as defined by pimonidazole staining
(49). MRSI metabolite ratios correlate with Gleason score, since prominent chol
ine spectra correspond to increasing phospholipids cell membrane turnover in mor
e rapidly proliferating aggressive cancers (56). Time to metabolic atrophy measu
red on MRSI after brachytherapy has shown promise as an early measure of respons
e, with metabolic atrophy being seen in 95% of usable voxels in 46% of patients
by 6 months and in 100% at 48 months with a mean time
The Use of Image Guidance in Prostate Brachytherapy
171
Figure 4 Cone-beam CT device (far leftSiemens Powermobil Mobile C-arm) used for se
ed placement verification during a permanent seed brachytherapy procedure. Coron
al CT image (below) shows good resolution of the seeds and surrounding soft tiss
ue anatomy. Source: From Ref. 131.

Figure 5 Fused CT (top) and MR (bottom) images

s based on the seeds as fiducial markers. Both
s on MR, which therefore appear more numerous
ever, the seed pattern on the CT is reproduced
sponding to each seed image.

from mid prostate level. Fusion i

seeds and spacers leave black hole
than the seeds on the CT image. How
on the MR with a black void corre

to atrophy of 29 months (54). Finally, intravenous contrast kinetics may also re

flect angiogenic activity in tumorbearing regions (57). In essence, MRI guidance
may bring us closer to the reality of biologically targeted brachytherapy (58).
FSE MRI provides superior visualization of brachytherapy catheters and seeds co
mpared to TRUS, but is inferior to radiography. Brachytherapy seeds were first r
eported to be visible on MRI in 1997 (59,60). In phantom studies, distortion map
s of different orientations of brachytherapy seeds were generated with susceptib
ility artifact patterns dependent on the orientation of the seeds with the main
magnetic field and phase encode gradient. The highly predictable nature of these
distortion patterns suggested that an automatic localization algorithm could ac
curately locate the implanted seeds using FSE MRI (61). This may be further enha
nced using proton density weighting and IV contrast administration (62). MR elas
tography in a manner akin to TRUS elastography, with
vibration of interstitial needles during brachytherapy procedures, may further i
mprove mapping of instrument geometry (63). Off-line MRI guidance for prostate b
rachytherapy has focused on improving tissue delineation on postplanning CT scan
s. MRI-CT fusion using various image acquisition and registration techniques, ha
s consistently demonstrated improved prostate gland delineation (Fig. 5) (6473).
The incorporation of MRI into postplanning allows assessment of dose to erectile
structures, including the internal pudendal artery (74), neurovascular bundles,
and penile bulb (75). Not surprisingly, performance is dependent on the details
of pulse sequence acquisitions, with T2weighted FSE techniques generally provid
ing optimal soft tissue contrast with minimal susceptibility artifact from impla
nted seeds (76). However, because of the unreliability of extraprostatic source
localization, postplanning based solely on MRI (77) remains problematic. Another
option involves the use of combined X-ray and MRI (XMR) interventional suites f
or postimplant dosimetry. XMR suites have been available for a few years and
172
 Menard and Crook
mainly applied to endovascular interventions. In such an environment, patients a
re transferred between the two imaging systems using a specially designed slidin
g table. Unlike CT/MRI image registration techniques described above, the regist
ration process relies on a combination of calibration and tracking. All sources,
including those in close clusters, are identified by two isocentric radiographs
and transposed to MRI (78). By combining the highest soft tissue (MRI) and seed
(X-ray) image resolution; this approach may achieve the best dosimetric accurac
y in postplanning. The off-line integration of MRI and MRSI for preplanning or i
ntra-operative guidance by deformable registration of MR images to online 3D TRU
S has also been described (79,80). An algorithm has been developed that first pl
aces the MRS positive voxel with respect to the z-axis in US/CT space. This dete
rmines the appropriate axial slice in which the MRS voxel is located. The x and
y coordinates can then be determined. Using an MRS voxel size of 6.25 6.25 3 mm,
the reported positional accuracy was 2.2 1.2 mm (80). The feasibility of this a
pproach for brachytherapy dose intensification to intraprostatic tumor sites has
been demonstrated clinically (81). However, substantial gland deformation intro
duces progressive registration errors through the course of a brachytherapy proc
edure, highlighting the need for more adaptive online MR integration. The use of
an open low-field MRI system for onlineguidance of permanent seed prostate brac
hytherapy was first described by DAmico et al. in 1998 (8285). In an effort to red
uce the toxicity of permanent seed brachytherapy, investigators translated the c

onventional transperineal ultrasound technique to an open MRI scanner architectu

re (85). Even at low field strength, the peripheral zone of the prostate gland c
ould now be distinguished from the central gland and specifically targeted using
custom interactive planning software (Fig. 6). Five-year results now confirm th
e equivalence of partial prostate brachytherapy to radical prostatectomy in bioc
hemical disease-free survival (86). This group has also integrated off-line diag
nostic MRI to online open MRI for the guidance of salvage permanent seed brachyt
herapy to specific subsites of recurrence visible on diagnostic MRI and MRSI (87
). For treatment planning for HDR brachytherapy, investigators at UCSF registere
d previous diagnostic MRI/ MRSI datasets to treatment planning CT or MR images tha
t were acquired after TRUS-guided insertion of brachytherapy catheters (88). Bas
ed on the diagnostic images, intraprostatic tumor sites were identified and targ
eted for dose escalation without exceeding tolerance for the urethra and rectum.
In order to circumvent the error associated with deformable or rigid registrati
on of previously acquired
Figure 6 (Top) Open 0.5-T MR system for performing imageguided brachytherapy pro
cedures. (Below) Prostate gland segmentation identifies peripheral zone (solid a
rrow) and central gland (hollow arrow) in interventional MR images. Source: From
Ref. 132.
images, a technique for transperineal placement of temporary brachytherapy cathe
ters in a 1.5 T scanner was developed (89). To access the perineum under the geo
metric constraint of a 60-cm diameter bore, patients were positioned in the left
lateral decubitus position. Diagnostic images were acquired first, followed by
the placement of brachytherapy catheters using a stereotactically registered per
ineal template. With the template fixed perpendicular to the rigid endorectal co
il, the mean needle targeting accuracy was 2.1 mm. Once the catheters were in pl
ace, final diagnostic-quality T2-weighted images were acquired and used to plan
and optimize radiation delivery (90), constraining radiation dose to the neurova
scular bundle and including extracapsular extension of disease in the high-dose
target volume (91). The disadvantage of this approach is the lateral decubitus p
osition, which is neither stable nor comfortable. Perineal access using a supine
semi-dorsal lithotomy position on the MR table is being explored using a dedicate
d prostate interventional MRI table that provides ample perineal exposure. This
table integrates patient immobilization devices, template hardware attachments,
and can be undocked from the MR scanner for patient transport to an HDR delivery
suite without disturbing the
The Use of Image Guidance in Prostate Brachytherapy
173
resulting in superior image quality and CT registration warrant reevaluation of
the role of ProstaScint in brachytherapy. Case reports (97) and mature series (9
8) demonstrate the feasibility of brachytherapy dose-intensification to lesions
visualized on ProstaScint SPECT/CT. Use of PET imaging for prostate cancer has b
een limited due to the poor sensitivity of FDG PET (99). Newer ligands, such as
F18- or C11-labelled choline, and C11-labeled methionine have recently been eval
uated with promising results. In a pilot study, F18-choline PET accurately discr
iminated positive sextants, with a sensitivity and specificity of 93% and 48%, r
espectively (100). This is consistent with early reports on the diagnostic accur
acy of C11-choline PET (101). Pending further development and validation, these
images have yet to be integrated in image guidance for prostate brachytherapy. I
MAGE GUIDANCE FOR PERMANENT LDR PROSTATE BRACHYTHERAPY Image guidance is essenti
al in every aspect of permanent seed prostate brachytherapy. Accurate definition
of the prostate in planning of the implant, of prostate and needle position dur
ing the execution of the implant, and of prostate and seed positions in the post
implant evaluation are all vital to the success of the procedure. The current st
andard approach is to use TRUS for mapping of the prostate and planning of the i

mplant, a combination of fluoroscopy and TRUS for execution of the implant, and
CT for the post implant evaluation. There are some weaknesses in this standard a
pproach, for which a variety of solutions have been developed. Planning When pre
planning is undertaken two to three weeks prior to the procedure, the plan may n
ot appear to fit when the patient is anesthetized and positioned in the operating
room. With attention to the geometry of the setup including recording of probe a
ngle and hip angles at the time of the mapping, this should be an uncommon occur
rence. However, a popular solution is to perform the mapping intra-operatively a
s the first step in the implant procedure so that the patient only has to be set
up once and there can be no alteration in prostate shape or size between mappin
g and implant execution. Operative Technique Although implants are planned with
ideal coverage, with V150s and D90s within an optimal range, postplan analysis r
eveals that these goals can be difficult to achieve in
Figure 7 Cylindrical 1.5T diagnostic system adapted to prostate brachytherapy in
terventions using a dedicated table (Sentinelle Medical Inc., Toronto, Canada).
Patient is positioned in semilithotomy. Dedicated table provides ample space for
perineal exposure and brachytherapy devices, while undocking from the scanner t
o transport patients to HDR delivery suite without disrupting patient immobiliza
tion (not shown).
patient position and catheter placement (Fig. 7). Others are developing MRI-comp
atible robotics solving the space constraints and real-time image acquisition ch
allenges of MRI guidance in prostate interventions (92,93). Finally, MRI may hav
e a role in the quality assurance of HDR delivery. Because of the dose-varying M
R characteristics of exposed gel polymerization, 3D HDR dose plans can be verifi
ed using phantom polymer gel constructs (94). NUCLEAR IMAGING (SPECT AND PET) Pr
ostaScintTM is a murine monoclonal antibody (capromab pendetide), which binds to
the intracellular domain of the prostate specific membrane antigen (PSMA) and i
s conjugated to 111Indium for SPECT (single-photon emission computerized tomogra
phy) imaging of prostate cancer (95). Unfortunately, problems including restrict
ed access of the antibody to the intracellular compartment, nonspecific binding,
and high blood pool activity causing a low target-to-background ratio have resu
lted in poor sensitivity (60%) and specificity (70%) (96) and limited clinical u
tility. Technical advances in SPECT imaging
174
 Menard and Crook
reality. This can be attributed to either intra-operative or post-operative even
ts.
Intra-operative Events
l l l l
Deteriorating visualization as implant progresses Prostate rotation 28 to needle
insertion Seed rebound/dropping Prostate displacement cephalad
The quality of the TRUS images tends to deteriorate as the implant progresses du
e to prostate and periprostatic edema and bleeding. This can increase the uncert
ainty regarding needle placement with respect to the prostate contour. Every nee
dle insertion applies an off-axis force to the prostate, which is relatively tet
hered at the apex. Rotations in the coronal plane of up to 13.88 have been repor
ted (13), resulting in seed trains that are more closely approximated at the ape
x but diverge at the base. This results in an area of relative under-dose at the
base. Rotational displacement can be minimized by the insertion of multiple nee
dles at a time so that centrally placed needles act as prostatic stabilizers for
the more lateral needles. However, this, in turn, may cause displacement of the
base in a cephalad direction. One study reported an average displacement of 1.5

cm with a range up to 3.0 cm (14). Again this will lead to significant under-do
sing of the base if not recognized. Often, if seed deposition is observed under
US, seeds may appear to rebound when released because of prostatic compression,
or alternatively may drop from the intended position because of placement in a c
ystic space or duct. Even for a preplanned implant, if such misplacements are ob
served, adjustments can be made in the plan to approximate the new position and
determine if remedial action is required in terms of correctional seed placement
. Real-time dosimetry should allow for systematic correction of these misadventu
res by recalculating the dose distribution based on actual seed positions. Unfor
tunately most real-time intra-operative dosimetry is based on needle position ra
ther than seed position (102). Seed visibility is inconsistent on TRUS with only
70% to 80% of the seeds being reliably identified (103). Linkage of seeds impro
ves this situation because the inter-seed spacing is maintained. Individual seed
s cannot drop or rebound as they are part of a strand, and all seed positions within
the strand can thus be interpolated from any two clearly identifiable points in
the strand (15). Other efforts to improve seed localization intraoperatively an
d allow real-time correction include TRUS-fluoroscopy fusion (25), MR-fluoroscop
y fusion, MR-TRUS fusion (79), or intra-operative CT dosimetry (35). Intra-opera
tive dose assessment based on fluoroscopy alone has also been reported using thr
ee fluoroscopic
images taken at 08, 158, and 158. 3D seed coordinates can be computed and a distri
bution calculated in VariSeed. Additional seeds can be added based on deficienci
es in the isodose cloud, although this method does not determine the relation of
the isodose cloud to the actual prostate contour (26). Another option to improv
e visibility intra-operatively is the use of single spectrum analysis, which use
s eigenvalues derived from the diagonalized correlation matrix of envelope-detec
ted radio frequency echo signals to yield a p-value indicative of the likelihood
of a seed specific repetitive signal (19). This has yet to be tested in a clini
cal environment but accurately detects seeds implanted in animal muscle.
Postoperative Events
Another reason for discrepancy between the plan and the final dosimetry can be a
ttributed to postoperative events including seed loss and/or migration as well a
s strand loss and/or migration. Migration of seeds through the blood stream to t
he lungs and occasionally to other organs is well recognized with loose seeds. T
he frequency of occurrence is 1% to 2% of seeds implanted but may affect up to 7
2% of patients (104106). Vascular migration is essentially eliminated with the us
e of stranded preparations (107). Both seeds and strands can be lost through the
urine and can migrate, usually distally, due to action of the perineal muscles
(108,109). The rigidity of some newer stranding materials has led to reports of
strand extrusion into the bladder and subsequent loss through the urinary tract
(110). These events will not be captured by intraoperative dosimetry and may eve
n be underappreciated when postplan imaging is performed the same day as the imp
lant. For this reason, intra-operative dosimetry should be followed with a postp
lan evaluation at a later date. Postplan Evaluation Postplan evaluation by CT-im
aging, whether performed day 1 or day 30, is not straightforward. There is a con
sistent discrepancy between prostate volumes as imaged by CT in comparison to TR
US or MRI, with CT volumes generally being about 30% larger (27). This discrepan
cy arises because the Hounsfield units of the surrounding structures (muscle, ve
ins, bladder neck) are all very similar to those of the prostate itself. This si
tuation is made even more difficult by the presence of metallic seeds and the re
sultant artifacts. Inter-observer variation in prostate contouring and postplan
dosimetric evaluation has been well documented (71,73,111). For an experienced t
eam who produces consistent technically excellent implants, the difference is mi
nimal. Since the seeds are well placed, using their position to guide the contou
rs will yield a result very
The Use of Image Guidance in Prostate Brachytherapy

175
close to the truth. For a beginner, it is perilous, fostering inappropriate conf
idence and leaving the operator unaware of the deficiencies in implant technique
. One solution is to use MR-CT fusion for postplans; the two sets of images can
be fused with millimeter accuracy using the seeds as fiducial markers or matchin
g through the use of mutual information (76). MR improves the soft tissue defini
tion for contouring, while CT is used for identification of the metallic seeds (
Fig. 5). Some experience with MR-CT fusion for postplans may be useful as a lear
ning tool to give a better appreciation of the anatomy of the prostate and its s
urroundings (67). Another reported option is a combination of MR and X-ray, agai
n benefiting from the enhanced soft-tissue visualization of MR for prostate cont
ouring and using X-ray images for seed localization. This method does not have a
n explicit registration or fusion step but relies on system calibration and trac
king (78) for stereoscopic seed localization. A method of TRUS-CT fusion (30,34)
has also been developed. The TRUS probe is inserted with the patient on the CT
couch and the TRUS images are recorded. The probe is then left in situ during th
e CT scan to serve for a registration landmark for the two image sets. We cannot
offer a recommended approach to permanent seed brachytherapy as there is not on
e approach that is clearly superior. Meticulous attention to detail, awareness o
f the various pitfalls and a consistent effort to avoid or circumvent them will
inevitably lead to superior quality implants. IMAGE GUIDANCE FOR TEMPORARY HDR P
ROSTATE BRACHYTHERAPY HDR temporary implant techniques, developed in the 1990s,
offer several advantages in image guidance for high precision brachytherapy. Dos
imetric optimization is performed immediately following catheter placement, perm
itting the treatment plan to be based on the actual geometry of the implant rela
tive to anatomical structures. A single high intensity 192Ir source can be place
d at any position for any length of time within each needle. These two variables
(dwell position and dwell time) can be computer optimized to achieve dose distr
ibutions that conform to the target volume while limiting dose to normal structu
res. The treatment is subsequently delivered with an afterloading technique, and
problems with source migration, inaccurate placement, and post implant edema ar
e largely circumvented. Image Guidance of Needles The burden of precision in nee
dle placement for HDR brachytherapy is considerably less than for permanent
seed brachytherapy, since dwell time is such a powerful variable for dose optimi
zation. Nonetheless, needle guidance and placement is a critical step in meeting
doseplanning objectives. Experts have traditionally advocated equidistant place
ment of initial catheters in the periphery of the target (112). The use of onlin
e, interactive treatment planning tools and subsequent placement of additional c
atheters according to the estimated dosimetry has been explored and found to res
ult in greater uniformity of treatment plans (113). Dynamic estimation of dosime
try during needle placement is feasible for TRUS-guided HDR prostate brachythera
py because it requires imaging the needles, not the individual seeds. If the cho
sen target is the prostate gland, ultrasound guidance of needles with real-time
image feedback is adequate. Visualization of the bladder base can be improved wi
th fluoroscopy and cystography (112). However, the Groupe European de Curiethera
pie recommendations for HDR prostate brachytherapy are somewhat more refined and
include
l l l
CTV1prostate gland, CTV2peripheral zone, CTV3regions infiltrated by macroscopic tumor
(112).
As high-dose targets become more specific to the peripheral zone or intraprostat
ic burden of disease, image guidance for needle placement must evolve to meet th
ese objectives. TRUS guidance can be augmented by using highresolution 3D TRUS t
echnology, Doppler imaging, knowledge of prior biopsy results, and a finer templ
ate grid (3-mm spacing) (114). MRI for needle guidance can also be integrated in
HDR brachytherapy and allows placement of needles within extraprostatic tumor (

115). The use of advanced imaging for needle guidance may also reduce toxicity b
y avoiding needle paths through erectile structures and reducing the total numbe
r of needles (116). Given superior image resolution for mapping zonal anatomy an
d burden of disease, there is a need to explore further the integration of off-l
ine or online MRI for needle guidance in HDR brachytherapy. Image Guidance of Do
se In the modern era dose should be planned and optimized based on 3D patient an
atomy rather than final implant geometry. It cannot be assumed that HDR brachyth
erapy catheters are ideally placed to encompass the target tissues. Anatomy-base
d dwell position and inverse optimization have clearly demonstrated improved dos
imetry compared with more traditional catheter-based planning techniques (88,117
,118). The relative merits of 3D TRUS,
176
 Menard and Crook
CT, and MRI have already been described in the context of target delineation (Fi
g. 2). Precise and accurate dose planning specific to HDR brachytherapy requires
that the catheter dwell positions be determined in reference to target anatomy.
Although best visualized on CT and MRI, the slice thickness (23 mm) of CT and MR
I may introduce small errors in determination of the catheter tip and first dwel
l position (115,119). This small uncertainty as well as uncertainties related to
spatial integrity of MRI in the image-volume of interest must be measured, corr
ected if possible, and accounted for in dose planning (115). Planar X-ray and/or
cone-beam CT may well reduce the uncertainty further in the SI dimensions. Imag
e Guidance of Delivery Maintaining geometric fidelity for the delivery of HDR br
achytherapy in reference to dose planning conditions is essential for accuracy.
This is best accomplished by eliminating patient transfers to maintain the posit
ion of the patient and brachytherapy devices and by reducing the time interval b
etween imaging for planning and subsequent radiation delivery. Despite these eff
orts, catheters can slip in the SI direction due to pelvic motion. Historically,
this problem has been addressed by acquiring orthogonal radiographs of the impl
ant catheters with reference to the contrast-filled balloon of the urethral cath
eter immediately prior to dose delivery (112). Integrating more advanced imaging
and/or tracking methodologies during HDR delivery are desirable to ensure highp
recision therapy. IMPACT OF IMAGE GUIDANCE ON CLINICAL OUTCOMES Disappointment w
ith early historical results and the subsequent success of permanent seed prosta
te brachytherapy with the advent of TRUS and computer planning points to the imp
act of image guidance on clinical outcomes for prostate cancer. Improved freedom
from biochemical failure (FFBF) has been demonstrated with technical maturation
and the implementation of CT-based postplanning dosimetry (Fig. 8) (120). Most
modern publications in this field demonstrate improved dosimetry with further ad
vances in image guidance (26,34,35,7981,85,88, 115,121,122). However, the relatio
nship between dosimetry and clinical outcomes has yet to be fully defined. There
is a strong correlation of 3D dose-volume parameters with local control in pros
tate brachytherapy, with FFBF increasing with higher D90s (dose to 90% of the pr
ostate) for patients with low-risk disease (123126). It is important, however, to
recognize that the accuracy of dose reporting is highly dependent on the accura
cy of
l
Figure 8 Comparison of freedom from prostate-specific antigen (PSA) failure by i
nterval implanted and risk stratum. Image guidance and technical maturation with
the implementation of postplanning dosimetry in 1998 may have led to improved o
utcomes. Source: From Ref. 120.
target delineation, and uncertainties in this regard may have contributed to inc
onsistencies in the observed doseresponse relationship (71,73,111). This problem
holds true for our understanding the relationship between critical organ dose a
nd subsequent toxicity (127,128). SUMMARY OF PERTINENT CONCLUSIONS

l
l
l
Disappointment with early historical results and the subsequent success of prost
ate brachytherapy with the integration of TRUS imaging points to the impact and
importance of image guidance. The entire prostate gland has traditionally been c
onsidered the target for prostate brachytherapy for two reasons (i) prostate can
cer is inherently multifocal, placing the entire gland at risk and (ii) direct i
maging of foci of cancer within the gland has been elusive. In reality, the loca
l extent of prostate cancer is neither confined nor defined by the boundaries of
the prostate gland. Modern imaging techniques, capable of mapping intraprostati
c sites of macroscopic tumor, can now be integrated in prostate brachytherapy fo
r selective modulation of dose intensity both within and adjacent to the prostat
e gland. Since no single imaging modality embodies all the optimal characteristi
cs for image guidance of brachytherapy for prostate cancer, multimodality guidan
ce models will likely predominate for the foreseeable future.
The Use of Image Guidance in Prostate Brachytherapy
l
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The real-time 2D feedback of TRUS, the geometric integrity and brachytherapy dev
ice resolution of X-ray, and soft tissue resolution of the anatomy and biology o
n MRI are highly complementary.
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16
Image-Guided Intensity-Modulated Radiotherapy for Clinically Localized Prostate
Cancer: Targeting Pelvic Lymph Nodes
MACK ROACH, PING XIA, AND JEAN POULIOT
Department of Radiation Oncology, University of California, San Francisco, NCI-D
esignated Comprehensive Cancer Center, San Francisco, California, U.S.A.
INTRODUCTION Why Target Lymph Nodes? Although prophylactic irradiation of lymph
nodes is a routine practice for most cancer sites, the role of prophylactic pelv
ic lymph node irradiation is controversial in men with localized prostate cancer
. Occult lymph node involvement, despite negative imaging is a well-recognized p
roblem in patients with cancers of the head and neck, breast, and many other sol
id tumors. Physicians who do not favor prophylactic pelvic radiotherapy in patie
nts with prostate cancer tend to support their position by making the nihilistic
argument that once the nodes are involved, regional therapy is of no benefit. T
hey also argue that there is increased morbidity with pelvic nodal irradiation,
and even those who favor it concede it is more time consuming and technically mo
re challenging to treat pelvic lymph nodes (1). There are a number of reasons pr
ophylactic pelvic nodal irradiation should be strongly considered in men with in
termediate- to high-risk prostate cancer. This chapter highlights many of these
reasons and discusses technical considerations when doing so. We make the argume
nt that there is no reason to believe prostate cancer is unique among solid tumo
rs in terms of the need to
183
address regional disease when present. We argue that regional disease is common,
and imaging is too insensitive to detect it when present. There is a growing bo
dy of data suggesting that treatment can be effective and well tolerated when pr
operly administered. We explain for whom we recommend prophylactic radiotherapy
and how intensity-modulated image-guided radiotherapy (IGRT) is administered at
the University of California San Francisco (UCSF). How Common is Lymph Node Invo
lvement in Men with Clinically Localized Prostate Cancer? Lymph node involvement
is more common in patients with high- to intermediate-risk disease than general
ly appreciated. Although commonly quoted nomograms give estimates of 2% to 38% f
or such patients, there are a number of reasons to believe that the true inciden
ce is substantially underestimated. The pathological findings from the vast majo
rity of surgical series are based on prostate patients who underwent at most a s
tandard lymph node dissection (SLD) for what was thought to be organ confined di
sease. Lymph node dissections in many of these series were either limited to obt
rurator lymph nodes alone or inclusive of external iliac lymph nodes

184
Roach et al.
chains, although the internal lymph nodes are considered part of the primary lym
phatic drainage. In addition, even the most aggressive node dissection surgeons
tend to ignore presacral and perirectal nodes. A limited number of surgical seri
es reported their findings when a more extensive lymph node dissection was perfo
rmed. For example, Heidenreich et al. performed extended pelvic lymphadenectomie
s (ELD) to assess the incidence of lymph node metastasis in 103 consecutive pati
ents and compared their pathological findings with 100 patients undergoing an SL
D (2). They noted no significant differences in age, preoperative prostate-speci
fic antigen (PSA) or mean biopsy, and Gleason score between the patient groups.
Metastases were diagnosed in 26% of those who underwent the ELD. They concluded
that the use of an ELD was associated with a high rate of lymph node metastasis
outside of the fields of SLD and recommended ELD in all patients with a PSA >10
ng/mL and biopsy Gleason score !7. According to their findings, approximately 40
% of the involved nodes would have been missed by an SLD. Another series include
d 414 patients with a PSA level of 10 ng/mL and a biopsy Gleason score of 6 (3).
According to their findings, greater than 50% of involved nodes were outside SL
D nodal regions. A series of studies conducted by Italian investigators are wort
hy of note (46). First, they constructed a nomogram on the basis of pathological
data obtained from patients who had undergone an ELD. Later they demonstrated th
at there is a critical dependence of the likelihood of detecting positive nodes
on the number of lymph nodes available for analysis. They showed that more than
10 nodes had to be sampled to have a reasonable chance of detecting involved nod
es (5). In keeping with this observation, Schumacher et al. reported the finding
from patients who underwent radical prostatectomy and a bilateral extended pelv
ic node dissection including only patients in whom 10 or more lymph nodes were r
emoved, and who also had a preoperative PSA <10 ng/mL (7). They noted that in 73
% of those with positive lymph nodes, the internal iliac chains were involved ei
ther exclusively or in combination with another area. Thus, it appears that an E
LD is considerably more sensitive than an SLD. It also appears that the true inc
idence of lymph node involvement based on SLD is underestimated by at least 40%
to 50% than when an ELD is performed (Tables 1 and 2). Of note, however, even se
ries based on ELD underestimate the true incidence because they largely ignore p
erirectal (and in some cases presacral) nodes (8,9). On the basis of limited dat
a that is available addressing these areas, the relative risk of lymph node invo
lvement would be 5% to 10% higher if there areas were routinely sampled. Unfortu
nately, even these rates are likely to underestimate the true incidence of lymph
node involvement. In addition to the problem of inadequate lymph node sampling,
the rates are likely to be an additional 10% to
30% higher because of false-negative pathological evaluations and because of the
lack of sensitivity of standard histopathological processing of sampling when c
ompared with more sophisticated methods of detection (1013). Earlier studies assu
med that the gold standard for detecting lymph node involvement was standard cytop
athological evaluation. Table 3 highlights the conclusions of selected studies d
emonstrating that this so-called gold standard lacks in sensitivity. Recent stud
ies have demonstrated that more careful sectioning of the lymph nodes combined w
ith staining for epithelial cells as well as the application of reverse transcri
ptase-polymerase chain reaction (RT-PCR) or PSA nRNA copy number are more sensit
ive methods for identifying cancer in lymph nodes that would otherwise be consid
ered N0 (1012). For example, in a study conducted at the University of Southern C
alifornia, immunohistochemical combined with microscopic sectioning of lymph nod
es also improved the detection of lymph node involvement not recognized at initi
al histological examination. Using this approach, occult lymph node metastases w
ere identified in 13% of patients with pT3N0 prostate cancer and associated with
an increased risk for recurrence and death (10). Investigators from Baylor Univ

ersity reported that RT-PCR is also a more sensitive method than histology for d
etecting occult small lymph node involvement (11). They noted that 20% of patien
ts with T3N0 disease had evidence of involvement using this assay. They used a h
ighly sensitive and specific RT-PCR assay for human glandular kallikrein 2 (hK2)
mRNA to differentially amplify splice variants of hKLK2 gene. They noted that R
T-PCR/hK2 was associated with progression, failure, development of metastases, a
nd prostate cancerspecific mortality (11). Investigators from Mt. Sinai reported
a correlation between the risk of subsequent progression and the number of PSA m
RNA (PSA-N) copies in pathologically N0 patients (12). Patients who were N0 by s
tandard cytopathological evaluation but with a PSA-N ! 100 (17% of patients) app
eared to have a substantially higher failure rate that was an independent predic
tor of failure than patients with a PSA-N <100 (12). Investigators from Japan pe
rformed evaluations on 2215 lymph nodes isolated from 120 patients using quantit
ative real-time RT-PCR and considered positive test as proof of micrometastasis (1
4). They concluded that approximately 30% of patients with clinically localized
prostate cancer had micrometastasis, the presence of which was associated with bio
chemical failure. Thus, it appears that approximately 13.3% to 30% of patients w
ith pathological T3N0 disease have occult pelvic lymph node involvement. With 40
% to 50% of the involved nodes outside of the SLD areas, and an additional 5% to
10% outside of the typical ELD areas and a false-negative rate of up to 30%, it
is plausible that the true estimate of lymph node involvement is as much as 65%
higher than estimated
IGRT for Clinically Localized Prostate Cancer Table 1 Selected Series Assessing
the Risk of Lymph Node Involvement First author, year (reference) Partin, 2001 (
27) Study size N 5079 Type of LND Standard staging pelvic lymphadenectomy Conclusi
ons
185
Cagiannos, 2003 (28)
N 5510
Standard (medial inferior margin of external iliac vein down to internal iliac and
obturator vessels
Touijer, 2007 (41)
N 648
Analysis limited to patients with Partin Table risk of >1%. LND templates of lim
ited pelvic node dissection included the external iliac nodes, standard pelvic l
ymph node dissections included external iliac, obturator, and hypogastric. 2 to
40 nodes (median 14) were removed and examined, and 10.3% were positive. The pos
itivity rate increased with the number of nodes removed. With 2 to 10 nodes: 5.6
% positivity rate; 1014: 8.6% positivity rate; with 1519: 10.2% positivity rate; a
nd 2040: 17.6% positivity rate. The number of positive cores was 119 (median: 4),
and the percentage of positive cores 7.1100% (median: 37.5%).
Briganti, 2007 (5)
N 858
Nomogram grouped patients with PSAs > 10 ng/mL, thus not useful in intermediateto high-risk patients? nomograms predicted most accurately when the risk of posi
tive lymph nodes was low, roughly less than 10%. With higher risk nomograms tend
ed to underestimate the actual positivity Positive lymph node detection rate 7.15
-fold higher for standard vs. limited pelvic lymph node dissection, with the med
ian (mean) number of nodes retrieved was 9 (10) and 14 (15) after limited and st

andard pelvic lymph node dissection, respectively ( p < 0.001). The ROC plot ind
icated that the removal of 28 nodes yielded a 90% ability to detect positive nod
es while 10 or fewer nodes removed was associated with a very low probability of
finding positive nodes.
Briganti, 2007 (6)
N

278

Mean number of lymph nodes examined 17.5; 10.4% positive nodes. Nomogram based o
n clinical stage, PSA, and Gleason score was 79.7% accurate vs. 83% ( p < 0.001)
when percentage of positive cores added. 83.7% vs. 81%; p < 0.001.
Abbreviations: LND, lymph node disease; ROC, receiver operator curve.
Table 2 Examples of Estimates of Risk of Positive Nodes: Roach Equation Vs. Nomo
grams for Intermediate- and High-Risk Patients First author, year (reference) Ro
ach, 1994 (42) Partin, 2001 (27) Cagiannos, 2003 (28) Briganti, 2006 (43)
a
GS

7, PSA 8, T2a 15% 4% 3% *17%

GS

7, PSA 15, T2b 20% *24% 5% *22%

GS

8, PSA 22, T2b 35% 27% 10% 35%

Comments Risk of positive nodes (2/3) PSA [(GS 6) 10]a Only reports data by PSA >
10 ng/mL Standard dissection Data based on extended lymph node dissection
GS 24, 5, 6, 7, 810 (considered as 4, 5, 6, 7, and 8, respectively). Abbreviations
: GS, Gleason score; PSA, prostate-specific antigen.
186
Roach et al.
Table 3 Selected Papers Highlighting More Sensitive Methods for Detecting Pelvic
Lymph Node Involvement in Patients with N0 by Standard Pathological Evaluations
First author (reference) Shariat (11) Study design Archival lymph nodes tissues
evaluated in 199 men with pT3N0 disease using the detection of human glandular
kallikrein 2 (hK2) mRNA in men with histopathologically normal pelvic lymph node
s, removed at radical prostatectomy. 274 patients with pT3 prostate cancer treat
ed by radical prostatectomy and bilateral lymph node dissection. 180 were staged
N0, while 94 were N. Lymph nodes from the 180 patients were evaluated for occult
metastasis by immunohistochemistry. Recurrence and overall survival were compar
ed among patients with occult tumor cells (OTN), with patients whose lymph nodes
remain negative (OLN) and with the 94 N patients. Quantitative measures of PSA mRN
A copies in N0-PLN, defined by PSA-N, and a threshold PSA-N 100 or more vs. PSAN less than 100 copies, were assessed by continuous and categorical multivariate
analyses to be independent. Pathological exams detected tumor cells in 29 lymph
nodes from 11 patients, compared to 143 with micrometastasis using in 32 pN0 pa
tients. In multivariate analysis only micrometastasis independently associated w
ith PSA recurrences. Key findings 20% of patients had positive results, and on m
ultivariable analysis models, the RT-PCR/hK2 result was associated with prostate
cancer progression, development of distant metastases, and prostate cancerspecif
ic survival. Occult tumor cells were found in 24 of 180 N0 patients (13.3%). OLN
was associated with increased recurrence and decreased survival compared with OL
N patients. Outcome for patients with OLN disease was similar to that for patients
with N disease. Immunohistochemistry can detect tumors not recognized at initial
histology.

Pagliarulo (10)
Ferrari (12)
Miyake (2007) (14)
4-yr biochemical failurefree survival of patients with PSA-N ! 100 vs. <100 was 5
5% and 77% ( p < 0.05). PSA-N identifies occult mets in N0-PLN. PSA-N ! 100 is a
n independent molecular marker for identifying patients with occult micrometasta
sis. Approximately 30% of clinically localized prostate cancers shed cancer cell
s to the pelvic lymph nodes, and this fact may explain some of the biochemical r
ecurrences occurring after radical prostatectomy. True incidence of lymph node i
nvolvement is 13.330% higher than estimated based on N0 path status.
Abbreviations: RT-PCR, real-time reverse transcriptase-PCR; PLN, pelvic lymph no
de.
Table 4 Conservative Estimates of the Incidence of Lymph Node Involvement in Int
ermediate- and High-Risk Patients with Clinically Confined Disease Vs. Hypotheti
cal Upper Limits of Incidencea PSA 6.110 and T stage T1c T2a T2b T2c PSA > 10 and
T stage T1c T2a T2b T2c
a
Gleason 3 4 (%) 2 vs. 3.3 3 vs. 5 6 vs. 10 10 vs. 17 Gleason 3 4 (%) 8 vs. 13 14
vs. 23 22 vs. 36 33 vs. 54
Gleason 4 3 (%) 2 vs. 3.3 5 vs. 8 8 vs. 13 13 vs. 21 Gleason 4 3 (%) 10 vs. 17 1
8 vs. 30 27 vs. 45 38 vs. 63
Gleason 4 4 (%) 3 vs. 5 5 vs. 8 8 vs. 13 13 vs. 21 Gleason 4 4 (%) 11 vs. 18 17
vs. 28 27 vs. 45 38 vs. 63
Hypothetical rates are increased by 40% for lack of sensitivity associated with
standard lymph node dissection, by 15% (range 1020%) for false-negative path beca
use of sampling errors, and by 10% for failure to sample prescral and perirectal
nodes (2,812). Thus each nodal positivity rate was increased by 65% to come up w
ith a hypothetical rate. Source: From Ref. 27.
IGRT for Clinically Localized Prostate Cancer
187
on the basis of surgical series on which nomograms have been created. Table 4 su
mmarizes the incidence of lymph node involvement reported by Partin et al. in 20
01 along with the hypothetical incidence of positive lymph nodes if the rates we
re 65% higher. IMAGING AND LYMPH NODE METASTASIS FROM PROSTATE CANCER Ideally, I
GRT addressing pelvic lymph nodes should be designed using abnormalities defined
using imaging modalities that establish the distribution of cancer cells in lym
ph nodes. Magnetic resonance imaging (MRI) provides excellent anatomical detail
and soft tissue contrast but is too insensitive for routine use for the detectio
n of nodal metastasis. Recent studies have shown that by adding lymphotropic sup
erparamagnetic nanoparticles, the sensitivity of MRI can be improved so that lym
ph nodes that would have otherwise been considered normal can be identified as b
eing involved and selectively targeted (15,16). This technology may prove useful
for designing external beam radiotherapy (EBRT) fields in the future, but a lac
k of FDA approval in the United States has hampered the widespread adoption of t
his technology (16,17). Another promising approach for identifying lymph nodes a
t risk for harboring microscopic metastases from prostate cancer involves combin
ing prostate lymphoscintigraphy to identify sentinel lymph nodes (SLNs) (3). Thi

s approach involves directly transrectally applying technetium-99m nanocolloid i

nto the prostate under ultrasonographic guidance one day before lymphadenectomy.
Two hours after the injection, scintigrams were taken and the individual locati
on of SLNs identified (3). Of note, more than half of the patients have positive
lymph nodes outside the region of a standard lymph node, lending further suppor
t to the use of ELD (3). TREATMENT OF PATIENTS WITH PELVIC LYMPH NODES: EVIDENCE
OF EFFICACY Prophylactic Irradiation for Lymph Nodes Although there is no conse
nsus as to the role of prophylactic pelvic lymph node irradiation in men with in
termediate- to high-risk prostate cancer, two randomized trials and a number of
retrospective studies have been conducted addressing this issue. The first prosp
ective trial was conducted in the late 1970s by the Radiation Therapy Oncology G
roup (RTOG) 7706, and it failed to demonstrate a benefit with whole-pelvic radio
therapy (WPRT). However, this study included patients estimated to be at very lo
w risk for lymph node involvement, and PSA data was not
Figure 1 Taken from an analysis of patients treated on arms 1 and 2 from RTOG 94
13 20. Arms 3 and 4 are excluded because they used a different timing for androg
en deprivation therapy (lasted two months later). Whole pelvis (Arm 1) correspon
ded to a superior border placed at the L5-S1 border were compared to subsets of
patients randomized to PO RT (Arm 2). Patients from Arm 2 were dichotomized as P
O with a radiation field size less than the median (i.e., 10 cm 11 cm) versus min
i pelvic (MP) with a field size greater than or equal to the median. The Mini-Pelv
is field was placed roughly at the bottom of the Sacral-iliac joints
available at that time (18). The largest phase III trial completed to date addre
ssing this problem (RTOG 9413) demonstrated that patients with a risk of lymph n
ode involvement of more than 15% might benefit from prophylactic WPRT (19). Figu
re 1 is taken from an analysis of patients treated on arms 1 and 2 from RTOG 941
3 (20). Arms 3 and 4 are excluded because they used a different timing for andro
gen deprivation therapy (lasted two months later). WPRT (arm 1) corresponding to
a superior border placed at the L5-S1 border was compared with subsets of patie
nts randomized to prostate-only radiotherapy (PORT) (arm 2). Patients from arm 2
were dichotomized as PO with a radiation field size less than the median (i.e.,
10 cm 11 cm) versus those dichotomized as mini-pelvic (MP) with a field size grea
ter than or equal to the median. The MP field was placed roughly at the bottom o
f the Sacral-iliac joints (20). Of note, the probability of progression-free sur
vival was critically dependent on the volume of lymph nodes irradiated. In the a
bsence of a larger, more compelling randomized trial, we believe these data sugg
est that this trial establishes neoadjuvant androgen deprivation therapy (NADT)
and WPRT as the standard of care in patients with intermediate- to high-risk dis
ease. Some retrospective studies support the role of WPRT, while others do not (
2123). Investigators from UCSF observed better biochemical control rates when WPR
T was administered with NADT, particularly when the risk
188 Table 5 Selected Papers Highlighting Benefits of Therapy Directed at Pelvic
Lymph Nodes First author (reference) Seaward (21) Study design Retrospective ana
lysis of patients undergoing prostate-only or whole-pelvic radiotherapy with and
without hormonal therapy. Phase III testing whether whole-pelvic radiotherapy i
mproves PFS Retrospective analysis of postoperative patients undergoing prostate
only or WPRT with and without hormonal therapy. Compares the outcomes of two di
fferent surgeons, each performing approximately 2000 radical prostatectomies wit
h pelvic lymph node dissection. One surgeon tended to do extensive while the oth
er tended to perform limited lymph node dissections. Data on all patients underg
oing RP for prostate cancer obtained from the SEER Program (19881991). All surviv
ing patients had a minimal follow-up of 10 yr. Of 57,764 men, 13,020 (22,5%) und
erwent RP. The median survival time was 127 mo. Most tumors were diagnosed as lo
calized stage and SEER grade 2 (Gleason score 5, 6; Gleason pattern 3, moderatel
y differentiated). Conclusions WPRT improves PFS
Roach et al.

Roach (19) Spiotto (22)

WPRT improves PFS WPRT improves PFS in the setting of salvage radiotherapy Exten
ded pelvic lymph node dissections may be of therapeutic value as suggested by hi
gher biochemical control rates
Allaf (25)
Joslyn (26)
There appears to be a relationship between the number of lymph nodes removed and
examined at prostatectomy for prostate cancer and the likelihood of finding lym
ph node metastasis and an increase in the prostate cancerspecific survival even i
n patients who have histologically negative lymph nodes. Patients with more than
one positive lymph node had a significantly greater risk of prostate cancerrelat
ed death. Retrospective data suggest that radiotherapy and surgical resection ma
y favorably impact outcomes in patients at risk for lymph node involvement
Abbreviations: WPRT, whole-pelvic radiotherapy; PFS, progression-free survival;
RP, radical prostatectomy.
of lymph node involvement was between 15% and 35% (21,24). Investigators from St
anford noted benefits associated with WPRT in the setting of postoperative radio
therapy in patients with adverse features (22). In contrast, investigators from
Fox Chase concluded that there was no benefit to the addition of NADT or WPRT to
patients with a risk of nodal involvement greater than 15% (23). However, selec
tion bias, small sample size, failure to consistently use NADT in all patients,
and suboptimal field size can easily explain such findings (20). The optimal ben
efit of WPRT appears to require the use of NADT and WPRT with the superior borde
r placed at the L5-S1 interspace. The point is more comprehensive coverage of ly
mph nodes is associated with a better outcome (20). Table 5 summarizes selected
series supporting the role of prophylactic WPRT. Evidence of Surgical Efficacy f
or Lymph Nodal Disease In addition to the evidence from radiotherapy series, the
re is a growing body of evidence supporting a benefit for the surgical managemen
t of pelvic lymph nodes for men with
prostate cancer (25,26). One study demonstrated a benefit in terms of biochemica
l control, while another suggested that patients who undergo a more extensive ly
mphadenectomy may have a reduction in prostate cancerrelated deaths (25,26). Tabl
e 5 summarizes selected surgical series suggesting a therapeutic value for surgi
cal intervention for pelvic lymph nodes. PATIENTS SELECTION FOR WPRT Given the f
act that imaging is relatively insensitive, the selection of patients for prophy
lactic WPRT is usually based on assumptions about the risk of lymph node involve
ment as suggested by pretreatment parameters. At UCSF, patients who are at low r
isk (e.g., <1015% risk of lymph node involvement) are usually treated only for th
e prostate. Patients with an estimated risk of lymph node involvement greater th
an 15% are usually offered pelvic nodal radiation with the superior border place
d at the L5-S1 junction. Patients with a relative contraindication to pelvic irr
adiation (e.g., prior radiotherapy for seminoma and unilobar disease) are someti
mes offered hemipelvic or even hemi-MP irradiation. At the other
IGRT for Clinically Localized Prostate Cancer
189
extreme, patients with imaging or pathological evidence of lymph node involvemen
t disease are usually offered extended field irradiation that includes the commo
n iliac with or without para-aortic node groups as well. There are a number of d
ifferent methods used to estimate the risk of lymph node involvement. All patien

ts with gross clinical T3 disease (grossly, not transrectal ultrasound, MRI, or

CT) are considered at risk (>15%). For the typical patients with clinically conf
ined disease, the most critical determinant appears to be the Gleason score (!7)
, followed by PSA (>10 ng/mL) and the percent positive biopsies (6,27,28). As di
scussed above, most nomograms are usually based on SLDs and thus are likely to u
nderestimate the true risk of lymph node involvement, particularly if an inadequ
ate number of nodes are taken. RADIOTHERAPY TECHNIQUES FOR THE TREATMENT OF PELV
IC LYMPH NODES Intensity-modulated radiotherapy (IMRT) has been shown to be asso
ciated with significant clinical advantages over conventional and three-dimensio
nal conformal radiotherapy (3DCRT) when treatment is limited to the prostate (29
). There is also a growing body of data suggesting that IMRT provides even great
er advantages when pelvic nodes are being irradiated. The pelvic lymph nodal reg
ions are not adequately covered using a typical fourfield arrangement designed w
ith conventional field borders based on bony anatomy (16,30). For example, WangChesebro et al. reported that the mean dose delivered by four-field box plans to
95%, 80%, and 50% of the pelvic nodal volume (defined on the basis of blood ves
sels with a 0.51.0 cm margin) were 12.7 Gy, 33.6 Gy, and 46.8 Gy, respectively. T
he use of conventional techniques is associated with much larger portions of the
bladder and rectum being included within the high-dose portions of the radiatio
n fields. More than 40% and 95% of the rectum and bladder volumes could receive
greater than 95% of the prescription dose (typically 45 Gy). Table 4 highlights
some of the most significant differences, including
Table 6 Typical IMRT and 3DCRT Dose Volume Comparison Volume of interest Lymph n
odes (D95) Small bowel (V45Gy) Bladder (V40Gy) IMRT 46 Gy 51.5 cm3 20% WP 3DCRT
27 Gy 284 cm3 90% Conclusions
inadequacy of nodal coverage, excessive small bowel and bladder doses with WPRT
compared with IMRT. TREATMENT GUIDELINES At the UCSF, we routinely used pelvic I
MRT to treat patients with intermediate- and high-risk prostate cancer. Prior to
radiotherapy, three markers were implanted in the base, middle, and apex of the
prostate by a urologist. Patients were simulated in supine position with an emp
ty rectum and full bladder, and treatment planning CT scans were acquired with 3
-mm slice thickness. Pelvic lymph node volumes were delineated to include obtrur
ator, external/internal iliac, common iliac, and presacral lymph nodes with 1- t
o 2-cm margins up to the vertebral level of L5-S1 and, in selected cases, perire
ctal nodal groups. The prostate and seminal vesicles were expanded by 2 to 3 mm
for the planning tumor volume (PTV). Rectal volumes were contoured from the anus
at the level of the ischial tuberosities to the sigmoid flexure. The bowel volu
mes (including the colon and large and small bowels) were contoured to include a
bdominal space. Definitive radiation for all patients consisted of a two-phase t
reatment course. The first portion of treatment typically utilizes 45 segments a
nd 7 beam angles (08, 358, 908, 1608, 2008, 2708, and 3158). The second cone-dow
n portion of treatment typically utilizes 25 segments and 7 beam angles (08, 558
, 908, 1358, 2258, 2708, and 3058). UCSF planning objectives used as initial pla
nning guidelines are listed in Table 6. Note that these planning objectives are
used to help set computerized objectives during inverse planning; these objectiv
es are different from final dose-volume histogram (DVH) guidelines used to evalu
ate treatment plans. The first phase concurrently treated the pelvic lymph nodes
and the prostate. For patients treated with external beam radiotherapy in both
phases, the prescription doses to the pelvic lymph nodes and prostate (plus semi
nal vesicle) were 48.6 Gy/1.8 Gy and 54 Gy/2.0 Gy, concurrently (typically to th
e 8591% isodose line). A typical dose distribution for WPRT in the first phase is
shown in
Lymph nodes are covered better with IMRT Small bowel is spared better with IMRT
Urinary bladder is spared better with IMRT
D95, dose in Gy delivered to 95% of lymph nodes; V45Gy, volume of tissue receivi
ng 45Gy; V40Gy, percentage of bladder receiving 40Gy during whole pelvic radioth
erapy. Abbreviations: IMRT, intensity-modulated radiotherapy; WP 3DCRT, whole pr

ostate three-dimensional conformal radiotherapy. Source: Modified from Ref. 30.

190
Roach et al.
Figure 2 (A C) A typical dose distribution for whole pelvic radiation in the fir
st phase is used at UCSF.
Figure 3 (A C) A typical dose distribution for whole pelvic radiation and an IMR
T boost in the second phase used at UCSF. For patients treated with external bea
m radiotherapy in both phases, the prescription doses to the pelvic lymph nodes
and prostate (plus seminal vesicle) were 48.6 Gy/1.8 Gy and 54 Gy/2.0 Gy, concur
rently (typically to the 8591% isodose line).
Figure 2AC. After pelvic IMRT, all patients continued the second phase of treatme
nt to boost the prostate using either brachytherapy (prostate implant or high do
se rate) or IMRT. A typical dose distribution for WPRT and an IMRT boost in the
second phase is shown in Figure 3AC. Figure 4A, B shows typical DVHs of the rectu
m and bladder compared with the DVHs of the same organs if
only the prostate were treated to 72 Gy. From these figures it is clear that the
pelvic irradiation increased the intermediate doses to the rectum and bladder w
hile exposing the similar amount of the rectum and bladder to the high doses. Co
ncurrently treating the prostate and the pelvic lymph nodes poses a technical ch
allenge not previously
IGRT for Clinically Localized Prostate Cancer
191
Figure 4 (A, B) Typical dose volume histograms of the rectum and bladder compare
d with the dose volume histograms of the same organs if only the prostate were t
reated to 72 Gy. From these figures it is clear that the pelvic irradiation incr
eased the intermediate doses to the rectum and bladder while exposing the simila
r amount of the rectum and bladder to the high doses.
addressed in radiotherapy because of the independent movement of the prostate an
d the pelvic lymph nodes. This problem renders the conventional isocenter shifti
ng method inadequate since prostate movement varies from a few millimeters up to
1.5 cm or more relative to the pelvic bones, while pelvic lymph nodes are relat
ively fixed in close proximity to vascular structures. Addressing this problem b
y simply adding a large planning margin to ensure adequate coverage of the targe
t volume unavoidably results in the inclusion of normal structures in the high-d
ose area of the radiation fields, potentially increasing the risk of normal tiss
ue complications. Although some investigators have concluded that this impact is
of limited clinical consequence, this conclusion must be viewed with caution be
cause it is based on fairly conservative assumptions about the magnitude of orga
n movement and setup errors (31). In our experience, it is not uncommon for sign
ificant discrepancies to be associated with ignorance. We have observed several
scenarios in which ignoring these discrepancies would result in rather large and
clinically significant treatment errors, particularly when para-aortic irradiat
ion is employed (see discussion below). The ideal strategy to resolve this chall
enge involves using online imaging and replanning on a daily basis prior to each
treatment. Unfortunately, because of the extended planning time required and th
e potential for prostate and patient movement to occur as time is extended, onli
ne replanning is not currently a practical solution. We have developed a multipl
e adaptive plan IMRT (MAP-IMRT) strategy to address this issue (32). With this s
trategy, we created a set of IMRT plans with a series of presumed prostate posit
ions. According to the position of the prostate detected on a daily basis, relat

ive to the pelvic bony anatomy, an IMRT plan that is closest to the prostate posi
tion of the day, is selected and used for treatment. For example, we recently tre
ated a high-risk prostate cancer patient with a horse-shoe abdominal kidney and
adjacent lymph nodes believed to be involved with MAPIMRT (32). The phase I plan
involved concurrently treating the prostate to 54 Gy and pelvic nodes to 48.6 G
y in 27 fractions. A major dosimetric consideration for this particular patient
was to minimize the dose of radiation received by the kidneys. Using the plannin
g CT, the contour of the prostate was copied and shifted according to the eight
presumed positions: 0.5 and 1.0 cm displacements along the posterior, anterior,
inferior, and superior directions. These displacements were based on the establi
shed pattern of prostate movements. Subsequently, a total of nine IMRT plans wer
e created, including the initial IMRT plan for the prostate at the standard unshif
ted position. The initial IMRT plan for the patient was created on the basis of
our established planning protocol. Since the rectum and bladder are contoured in
the planning CT but are not shifted with the shifted prostate, the anatomic rel
ationships of these two organs with the shifted prostate is ignored. Therefore,
the initial planning dose constraints of the rectum and bladder rendered rough a
pproximations (Table 7). To circumvent this problem, we constructed an artificia
l rind structure around the shifted prostate to create a highly conformal plan,
thus effectively protecting the rectum and bladder. Figures 5 and 6 show the sag
ittal and coronal dose distributions for a patient with the prostate position sh
ifted 1 cm anteriorly (Fig. 5A), unshifted (Fig. 5B, D), shifted 1 cm posteriorl
y (Fig. 5C), shifted 1 cm inferiorly (Fig. 5D), and shifted 1 cm superiorly (Fig
. 5E). To correct for daily displacements, gold marker seeds are used to track t
he prostate position each day prior to each treatment. Note the lymph nodes and
prostate are always covered. Figure 6 shows the axial dose distributions for a p
atient with the prostate shifted 1 cm anteriorly (Fig. 6A), unshifted (Fig. 6B),
shifted 1 cm posteriorly (Fig. 6C), shifted 1 cm inferiorly (Fig. 6D), and shif
ted 1 cm superiorly (Fig. 6E) using gold marker seeds to track the prostate posi
tion each day prior to each treatment. Note
192 Table 7 Typical Dose Constraints for Phase I Planning Organ name Type PTV Ma
x. dose Min. DVH Min dose Max. dose Min DVH Min. dose Max. dose Max. DVH Max. DV
H Max dose Max. DVH Max. dose Max. DVH Max. dose Max. DVH Dose (cGy) >Volume 580
0 5400 5200 5000 4860 4300 5400 4500 3500 5400 4300 5400 3500 4860 4500 95% Weig
ht 3 10 3 3 10 1 4 2 3 1 1 1 1 5 5
Roach et al.
PTV nodes
95%
Rectum
7% 30% 15% 20% 5%
Bladder Bulb Bowel
Abbreviations: PTV, planning tumor volume; DVH, dose-volume histogram.
that regardless of the shift, the prostate is always well covered using the plan
of the day to match the position of the prostate. To accomplish this feat, the ap
propriate plan of the day is chosen to match position of the prostate when shown
. An onboard imaging system is required to implement this approach, which we cal
l MAP-IMRT.
Onboard imaging devices are becoming widely spread and more frequently used for
daily prostate alignment at a number of institutions. Several systems have been

developed to provide a 3D image of the patient in treatment position moments bef

ore radiation delivery including, but not limited to, a CT on rails (33), a kilovo
ltage conebeam CT (kVCBCT) (34), a megavoltage cone-beam CT (MVCBCT) (35), and a
tomotherapy system (36). All these systems can provide a comparison of the imag
e of the day with the planning diagnostic CT. At UCSF, image-guided IMRT (IGRT)
for targeting the pelvic nodes has been developed using the MVCBCT system Mvisio
nTM. An example of such images is shown in Figure 7AC. Registrations on bony anat
omy and on gold markers are sequentially performed to determine the setup error
and the relative prostate displacement. For MVCBCT, projection images are acquir
ed using the linear accelerator (linac) with a low dose rate 6-MV beam. The MVis
ion system consists of a standard linac equipped with an amorphous-silicon flat
panel electronic portal imaging device adapted for MeV photons. An integrated co
mputer workspace provides automated acquisition of projection images, image reco
nstruction, CT to CBCT image registration, and couch shift calculation. These sy
stems demonstrate submillimeter localization precision and sufficient soft tissu
e resolution to visualize structures such as the prostate (37,38). An MVCBCT acq
uisition is performed by rotating the linac gantry in a continuous
Figure 5 (AF) (See color insert.) The sagittal and coronal dose distributions for
a patient with the prostate position shifted anteriorly (A), unshifted (B and D
), shifted posteriorly (C), shifted inferiorly (D), and shifted superiorly (E).
To correct for daily displacements, gold marker seeds are used to track the pros
tate position each day prior to each treatment. Note that the lymph nodes and pr
ostate are always covered. To accomplish this feat, the appropriate the plan of t
he day is chosen to match position of the prostate when shown. An onboard imaging
system is required to implement this approach, which we call multiple adaptive
plan IMRT (MAP-IMRT).
IGRT for Clinically Localized Prostate Cancer
193
Figure 6 (AE) The axial dose distributions for a patient with the prostate shifte
d anteriorly (A), unshifted (B), shifted posteriorly (C), shifted inferiorly (D)
, and shifted superiorly (E) using gold marker seeds to track the prostate posit
ion each day prior to each treatment. Note that regardless of the shift, the pro
state is always well covered using the plan of the day to match the position of th
e prostate. An onboard imaging system is required to implement this approach, wh
ich we call multiple adaptive plan IMRT (MAP-IMRT).
2008 arc. This acquisition procedure lasts for 45 seconds, and the reconstructed
MVCBCT image becomes available within two minutes after the start of the acquis
ition. MVCBCT to CT registration can then be used to determine the proper positi
oning of the patient. This allows a
rapid alignment of the patient before each treatment fraction. We have found tha
t MVCBCT acquisitions of less than 2 MU (1.7 cGy at isocenter) or 4 MU can be us
ed for direct 3D alignment in the presence of gold seeds or with bony anatomy (F
ig. 7B), respectively. Furthermore, Figure 7AC
Figure 7 (A C) (top row) Images generated at UCSF using the MVCBCT system Mvisio
nTM. The MVision system consists of a standard linac equipped with an amorphoussilicon flat panel electronic portal-imaging device adapted for MeV photons. MVC
BCT projection images are acquired using the linac with a low-dose rate 6-MV bea
m. (DF) (lower row) Images from the treatment planning CT. Such images are used s
equentially at UCSF for implementing IGRT to target the pelvic lymph nodes durin
g IMRT. Initially the alignment is based on bony anatomy to determine the setup
error and then adjustments are made on the basis of gold markers placed in the p
rostate. Abbreviations: Linac, linear accelerator; IGRT, image-guided radiothera
py.

194
Roach et al.
shows the potential of using MVCBCT to align the patient on the basis of soft ti
ssue without the need of gold seeds. MVCBCT acquisitions of approximately 9 MU a
re currently required on typical pelvic patients for consistent prostate visuali
zation without the need of gold seeds. To implement MAP-IMRT prior to each treat
ment, an MVCBCT is acquired and aligned with the planning CT twice. One alignmen
t is made with the pelvic bones and the other with the implanted markers. The co
uch shifts obtained from the first alignment (to the pelvic bones) is done to el
iminate setup errors by shifting the treatment couch accordingly. The prostate m
ovement of the day was obtained by the differences between two alignments. Accor
ding to the detected prostate displacements, in the case described above, one of
nine IMRT plans in which the prostate position was close to the prostate positi
on of the day was chosen to treat the patient. Although online replanning may be
the ideal strategy to accommodate independent movement of the prostate and pelv
ic lymph nodes during concurrent treatment, reoptimizing a set of IMRT plans wit
h multiple prostate positions is proven to be clinically feasible and practical.
The use of soft tissue imaging alone appears to be inadequate since there is a
general consensus that registration governed by anatomy outperforms CT-contour-b
ased registration, and that registration of implanted markers has the least inte
ruser variability (39,40).
l
l
resolving conflicting alignment issues using onboard soft tissue imaging (MV or
kVCT scans) are required. IMRT provides clear advantages over 3DCRT when attempt
ing to incorporate all of the nodes at risk while sparing surrounding normal tis
sues such as the rectum, bladder, and penile structures. The guidelines for sele
cting patients for image-guided IMRT directed at pelvic lymph nodes and the magn
itude of the benefit are currently areas of active investigation by the RTOG and
others.
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eason score in men with clinically localized prostate cancer. Int J Radiat Oncol
Biol Phys 1994; 28:3337. 43. Briganti A, Chun FK, Salonia A, et al. Validation o
f a nomogram predicting the probability of lymph node invasion based on the exte
nt of pelvic lymphadenectomy in patients with clinically localized prostate canc
er. BJU Int 2006; 98:788793.
17
Fractionation Schemes with IGRT for Clinically Localized Prostate Cancer

HIMU LUKKA
Department of Oncology, McMaster University, Hamilton, Ontario, Canada
INTRODUCTION The move in modern radiotherapy practice toward highly conformed, h
igh-precision radiotherapy techniques for prostate cancer treatment has reduced
treatment toxicity and has also allowed dose escalation. Dose escalation has now
been shown to improve biochemical disease-free survival in the treatment of pro
state cancer with radiotherapy. As a result of dose escalation, treatment course
s have lengthened from six to seven weeks to eight to nine weeks. This has incre
ased the burden on already stretched radiotherapy services, increased the cost o
f treatment, and placed additional social, emotional, and economic burdens on pa
tients and their families. These problems will increase as more treatment center
s acquire the technical prerequisites for dose-escalated prostatic radiotherapy
and switch to longer treatment schedules. Early evidence suggests that efficacy
and safety similar to that obtained with conventional fractionation schedules ca
n be achieved with a shorter course of radiation treatment using threedimensiona
l conformal radiotherapy (3DCRT) and intensity-modulated radiotherapy (IMRT). If
the safety and efficacy of these hypofractionated regimens are demonstrated in
randomized controlled trials (RCTs), their adoption would reduce the burden of t
reatment for patients and their families, reduce the cost of radiotherapy treatm
ents, and free up strained radiotherapy facilities in
197
some jurisdictions. There are also radiobiological reasons to suggest that hypof
ractionated prostatic radiotherapy may result in improved efficacy while maintai
ning toxicity equivalent to that experienced with conventional fractionation. Th
is chapter will discuss (i) dose and fractionation issues in the treatment of pr
ostate cancer from both radiobiological and clinical perspectives, (ii) normal t
issue tolerance, (iii) evidence from recent RCTs of hypofractionated radiotherap
y, (iv) ongoing trials that will provide additional evidence over the next few y
ears, (v) indications for and contraindications to hypofractionated radiotherapy
in patients with prostate cancer, and (vi) planning and image-guided delivery c
onsiderations particular to hypofractionated radiotherapy. RADIOBIOLOGY Dose and
Fractionation Traditionally, patients with prostate cancer received total doses
of radiation in the range of 60 to 78 Gy, delivered in fractions of 1.8 or 2 Gy
on each of five days per week over six to eight weeks. From the radiobiological
perspective, these schedules may not deliver optimal radiotherapy for prostate
cancer (1). General principles of radiobiology can be used to point to new sched
ules for investigation in clinical trials.
198
Lukka
In very general terms, the probability of eradicating cancer cells improves with
higher doses of radiation, as does the risk and severity of adverse effects. Th
e biological effects of radiation result from the relative level of DNA damage a
nd resultant cell kill in tumor versus normal tissues. The level of tissue-speci
fic cell kill is dependent on a number of factors including activation of cell d
eath pathways (e.g., apoptosis or mitotic catastrophe), cell cycle phase and res
ulting cell cycle arrests, and the appropriate sensing and repair of the damaged
DNA. In addition to intrinsic cellular radiosensitivity, cell survival can also
be modified by the tumor microenvironment and hypoxia (e.g., decreased intracel
lular oxygen tension leads to relative cellular radioresistance). The
radiation survival curve shows the relationship between cell survival after expo
sure and graded doses of radiation, which can vary among tissues. In general, th
e curve has an initial shallow slope (i.e., the shoulder region) at doses up to 4
Gy, followed by a steeper logarithmic slope at higher doses (Fig. 1A). The initi
al shoulder section is thought to reflect the repair of sublethal DNA damage fol

lowing irradiation, and the logarithmic section reflects cell killing in the abs
ence of repair. A linear-quadratic (LQ) model can be used to describe the cell-s
urvival curve, so that the surviving fraction of cells S/S0 is related to the ra
diation dose d as S/S0
exp (ad bd2). Alpha (a) describes the initial slope of the
survival curve and the nonrepairable component of DNA damage (1,2). It is the
Figure 1 Cell survival versus radiation dose. (A) The fractionation sensitivity
of a tissue is described by the a/b ratio. (B) If the a/b ratio for tumor greate
r than normal tissue. (C) If the a/b ratio for normal tissue greater than tumor.
Fractionation Schemes for Clinically Localized Prostate Cancer
199
most variable component among human cell types and best reflects differences in
the intrinsic radiosensitivity of cells. Beta (b) describes the curvature of the
survival curve and represents the transition from repairable to nonrepairable c
ell injury. With the linear component proportional to a and the quadratic compon
ent proportional to b, the degree to which the cell-survival curve bends from th
e shoulder section depends on the ratio of the coefficients alpha and beta (a/b)
. While the single-fraction cell-survival curve is useful for understanding the
radiobiological relationship between dose and cell survival, fractionated radiot
herapy is used in clinical practice. The shoulder and linear components of the c
ell-survival curve are reproduced for each of these fractions. There are several
reasons why fractionated radiotherapy is preferred to single fractions in the r
adical treatment of tumors. Fractionation capitalizes on the difference in a/b r
atios between most epithelial tumors (a/b 10 Gy) and late-reacting normal tissue
(a/b 3 Gy). The ratio of a to b in the LQ equation discussed above also reflect
s the sensitivity of tissue to radiation fraction size (3). The radiosensitivity
of normal cells depends on their relative natural proliferation rate (1). Those
normal tissues with slow cell proliferation rates have long cell cycle times th
at allow for intracellular repair between fractions. In this situation, the rati
o of a to b is low. For example, in the case of bowel tissue, the ratio for late
-reacting effects ranges from 2.5 to 5 and for bladder from 3 to 7 (1). In contr
ast, rapidly proliferating cells have little time for repair and in this scenari
o the a/b ratio is higher. For example, early reactions in skin can be associate
d with an a/b ratio of 9 to 12 and for colon it is 9 to 11 (1). In most rapidly
dividing cancer cell populations, the a/b ratio is approximately 10 Gy. For thes
e tumors, fractionated radiotherapy using fraction sizes of 1.8 to 2.0 Gy is pre
ferred, as it produces improved tumor control while limiting normal tissue damag
e (Fig. 1B). In contrast to the above scenario, there is a growing consensus tha
t the a/b ratio for prostate cancer is low. What is actively debated is the exac
t a/b ratio for prostate cancer. A number of studies have suggested that the a/b
ratio for prostate cancer is between 0.9 and 1.5 Gy (46), indicating relatively
long cell cycle times, but some investigators suggest that the low values report
ed may be because of an artifact resulting from tumor hypoxia (79). This may refl
ect the relative proliferation within a given risk category of the prostate canc
ers being studied (e.g., low- vs. high-risk cancers). The evidence to support a
low a/b ratio is based on basic science and clinical data. Fowler has analyzed t
he biochemical control rates (biochemical no evidence of disease, bNED) from pub
lished radiotherapy series and RCTs and has come to the conclusion that the a/b
ratio for prostate cancer may be as low as 1.5 Gy (1). His analysis of the only
hypofractionation trial that has published efficacy data (10) concludes that the results wo
uld be in keeping with the a/b ratio for prostate cancer being as low as 1.0 to
1.5 Gy (1). The relatively low a/b ratio should make prostate cancer cells very
sensitive to increases in fraction size or dose rate (Fig. 1C). In this setting,
hypofractionated radiotherapy using a higher dose per fraction, lower total dos
e, and shorter overall treatment time would be more efficient at killing tumor c

ells than standard fractionation and should produce equivalent tumor control wit
h reduced toxicity. Some authors suggest that, based on mathematical modeling, s
ome higher dose per fraction hypofractionation regimens could even result in imp
roved tumor control while maintaining the same toxicity as current high-dose 2-G
y fraction regimens (11). Fowler et al. (11) has summarized the radiobiological
implications of hypofractionation in the treatment of prostate cancer as: Estima
tes of the a/b ratio for prostate cancer are around 1.5 Gy, much lower than the
typical value of 10 Gy for many other tumours. The low a/b value is comparable t
o, and possibly even lower than, that of surrounding late-responding normal tiss
ue in rectal mucosa (a/b nominally 3 Gy but also likely to be in the 45 Gy range)
. This lower a/b ratio for prostate cancer than for surrounding late-responding
normal tissue creates the potential for therapeutic gain. Normal Tissue Toleranc
e Radiation effects on normal tissue, which manifest clinically as adverse effec
ts related to tissue injury, are divided into categories often labeled as acute
or late (2). Adverse effects related to acute response to irradiation occur earl
y (i.e., during or soon after radiotherapy), while those related to late respons
e occur weeks, months, or years after completion of radiotherapy and tend to be
chronic. Late adverse effects of radiotherapy can be classified as classic or conse
quential (12). The former are usually related to damage to slowly proliferating t
issue, such as rectum or bladder. This type of late toxicity is considered a dos
e-limiting effect of radiotherapy. Late adverse effects may also occur as a cons
equence of earlier injury due to the acute effects of radiotherapy on skin and m
ucosa (2); these are termed consequential. In a recent analysis of data from a rad
iotherapy dose-escalation study, Heemsbergen et al. investigated the link betwee
n acute and late toxicity and found that acute gastrointestinal (GI) adverse eve
nts were independent predictors of late GI adverse events in men with localized
prostate cancer (12). Mild to moderate urinary, bowel, and sexual side effects a
re common after external beam radiotherapy for prostate cancer. Studies suggest
that late bowel and bladder effects reach a plateau
200
Lukka
between 24 and 36 months after treatment (13). Although all adverse effects can
impinge on patients quality of life and many have the potential to result in seri
ous health problems, late effects can have the added consequence of being perman
ent and progressive. Unlike acute effects, late effects may not heal well and ma
y require surgical intervention. Alteration of radiotherapy fractionation has th
e potential to change the pattern of adverse effects. Acute responses to radioth
erapy generally occur in tissues with rapid turnover, such as GI mucosa and skin
; these rapidly dividing normal tissues have an a/b ratio of approximately 10 Gy
. Consequential late effects could be expected to vary with fraction size in the
same way as acute adverse effects. Classic late effects are usually related to
damage to slowly proliferating tissue such as rectum or bladder, which have a/b
ratios of 2.5 to 5 Gy and 3 to 7 Gy, respectively. Although limited data are ava
ilable for prostate cancer, a model developed for cervix cancer predicts less ac
ute toxicity with hypofractionated regimens than for standard fractionation (14)
. According to this model, 60 Gy in 3 Gy fractions has less effect on acutereact
ing tissue than does 78 Gy given in 2 Gy fractions. Large fractions, especially
those over 2.5 Gy, are usually avoided for most tumor types because late-reactin
g normal tissue is generally more sensitive to large fractions than most tumor c
ells. In prostate cancer, however, tumor cells have an a/b ratio that is estimat
ed to be between 0.9 and 1.5 Gy, making them sensitive to large fractions. Thus,
in theory at least, when combined with imageguided conformal or IMRT radiothera
py, hypofractionated radiotherapy has the potential to produce tumor control and
late classic toxicity rates that are equivalent to conventional fractionation w
ith less acute and consequential late toxicity. While the above discussion has s
ummarized the radiobiological aspects of hypofractionation for prostate cancer,
readers are referred to published papers and reviews for more detailed informati

on on this subject (1,5,6,11). EVIDENCE FROM CLINICAL TRIALS The Role of Clinica
l Studies In addition to radiobiological considerations, clinical experience and
data from clinical trials can also inform choices about fraction size and total
dose of radiotherapy used in clinical practice and randomized trials. Although
improvement in patient survival is the definitive measure of successful cancer t
reatment, the long natural history of prostate cancer presents challenges to des
igning studies with the power to detect statistically significant improvements i
n overall mortality rates. For this reason, surrogate end points are often used
as primary outcome variables in randomized trials in this setting. The most comm
only used
of these surrogate outcomes is biochemical failure. Several definitions for bioc
hemical failure have been described in the literature (1517), but the American So
ciety for Therapeutic Radiology and Oncology (ASTRO) definition has been the mos
t widely accepted. The most recent review of the ASTRO consensus statement at a
joint Radiation Therapy Oncology Group RTOG-ASTRO meeting in 2005 resulted in so
me changes to the definition of biochemical failure to make it generalizable to
patients treated with radiotherapy plus hormonal therapy and to correct for bias
in the original method for determining the date of failure used to calculate es
timates of event-free survival (18). The current consensus statement recommended
that the definition of prostate-specific antigen (PSA) failure be changed to PS
A nadir 2; this definition is now referred to as the Phoenix definition though in
the past it has been referred to as the Houston definition. The Phoenix (RTOG/A
STRO) consensus conference in 2005 also stressed the importance of adequate foll
ow-up in clinical trials and recommended that investigators report results that
reflect data from a point in time two years less than the median follow-up time.
For example, when median follow-up is seven years, freedom from biochemical fai
lure rate should be estimated at five years. Most studies reporting results to d
ate have used the original ASTRO failure definition. It is anticipated that futu
re studies will report outcomes on the basis of the 2005 definition of biochemic
al failure. Evidence of the relative effectiveness and safety of hypofractionate
d radiotherapy comes from randomized trials that compare these regimens with con
ventional fractionation. An understanding of the evidence on total dose used for
conventionally fractionated radiotherapy is important for the design and interp
retation of trials comparing hypofractionated and conventional radiotherapy. The
total dose and mode of delivery used for conventional radiotherapy schedules ha
s evolved as new data emerged from randomized trials. Four randomized trials hav
e compared conventional-dose versus high-dose conformal radiotherapy using fract
ions of 1.8 to 2.0 Gy (1923). Three of four trials reported a benefit for dose-es
calated radiotherapy in terms of freedom from biochemical failure based on PSA l
evels, but none had sufficient numbers or follow-up time to detect differences i
n survival. All four trials reported increased adverse effects with higher doses
of radiotherapy. The trials described above used freedom from failure as their
primary outcome measure. Two ongoing randomized trials, designed to detect stati
stically significant differences in five-year survival rate as the primary outco
me, may clarify the picture on the benefits of high-dose radiotherapy in patient
s with localized prostate cancer (2426). Chapter 1 includes a full discussion on
the evidence and general practice guidelines for imageguided radiation treatment
in prostate cancer.
Fractionation Schemes for Clinically Localized Prostate Cancer
201
HypofractionationCase Series In an attempt to improve freedom from disease and su
rvival rates, the trials described above used higher total doses of radiation wi
th conventional fraction sizes. The net result of this approach is longer overal
l treatment times resulting in more trips to the treatment center by the patient
and a higher burden on the radiotherapy facility. Hypofractionated radiotherapy
, on the other hand, aims to achieve clinical results that are at least equivale

nt to conventional fractionation while shortening overall treatment time and red

ucing the burden on patients and treatment centers. As discussed earlier, the ra
diobiological data suggest that the potential exists for improved tumor control
while maintaining normal tissue toxicity with hypofractionated radiotherapy usin
g even higher dose per fraction in hypofractionated regimens. Evidence on the cl
inical outcomes associated with hypofractionated radiotherapy has been evolving
since the 1980s. Early reports described experiences with radiotherapy for local
ized prostate cancer at centers in Canada, Australia, and the United Kingdom (273
1), but three studies published since 2003 are more relevant to current practice
(3235). The largest was a retrospective case series from the Christie Hospital i
n the United Kingdom where 705 men with localized prostate cancer were treated w
ith conformal radiotherapy to a dose of 50 Gy in 16 daily fractions over 22 days
(fraction size 3.13 Gy) (32). The biochemical relapse-free rates in the low-, m
oderate-, and high-risk groups were 82%, 56%, and 39%, respectively. Grade 2 lat
e bowel toxicity was reported in 5% of patients and grade 2 to 3 late bladder to
xicity in 10% of patients. The Cleveland Clinic in the United States conducted a
prospective study involving 100 men with localized prostate cancer treated to 7
0 Gy at 2.5 Gy/fraction (33). The five-year biochemical relapse-free survival ra
te was 85%, the grade 3 late rectal toxicity rate 3%, and the grade 3 late urina
ry toxicity rate 1%. In a prospective study at the Princess Margaret Hospital in
Canada, 92 patients with prostate cancer received IMRT with a total dose of 60
Gy given as 3.0 Gy fractions over four weeks (34,35). This phase II study was de
signed to assess late toxicity and the feasibility of the hypofractionated regim
en. At the last assessment during a median follow-up of 38 months, there was no
grade 3 or 4 toxicity. Rates of grade 1/2 late adverse events were low, with 7%
of patients reporting GI events and 10% reporting genitourinary (GU) events. Sev
ere acute toxicity occurred in one patient but did not require interruption of t
reatment. The biochemical control rate was 97% at 14 months. In addition, high-q
uality evidence from a randomized trial in breast cancer, which is also postulat
ed to have a relatively low a/b ratio, demonstrated that a 22-day course of radi
otherapy using 16 fractions of 2.66 Gy produced similar rates of local
recurrencefree survival to 50 Gy given in 25 fractions over 35 days, without sign
ificant increases in toxicity (36). HypofractionationRandomized Trials Evidence i
s available from three randomized trials that compared hypofractionated and conv
entionally fractionated radiotherapy regimens for localized prostate cancer (Tab
les 1 and 2). A Canadian study by the Ontario Clinical Oncology Group (OCOG), pu
blished in 2005, was the first randomized trial to evaluate a fractionation sche
dule delivering a dose per fraction higher than 2 Gy for the treatment of locali
zed prostate cancer (10). Between March 1995 and December 1998, 936 men with ear
ly-stage (T1 or T2) disease were randomly assigned to receive 66 Gy in 33 fracti
ons over 45 days (long arm) or 52.5 Gy in 20 fractions of 2.625 Gy over 28 days
(short arm). Patients with PSA levels 40 mg/L were included. They were randomize
d within strata on the basis of PSA level ( or > 15 mg/L), Gleason score (26 or 71
0), method of lymph node assessment (surgical or radiological), and center to en
hance comparability between the long and short arms. The primary study outcome w
as biochemical or clinical failure (BCF), which was based on a cluster of events
that included three consecutive increases in PSA, clinical evidence of local or
distant failure, commencement of hormonal therapy, or death as a result of pros
tate cancer. The study was designed as a noninferiority trial, with statistical
power to demonstrate that the treatment given in the short arm was not worse tha
n that given in the long arm by more than 7.5% in terms of the primary outcome.
The treatment planning in this study was CT-based. This study was undertaken pri
or to the availability of image guidance. The margin around the prostate was 1.5
cm, though posteriorly this could be reduced to 1.0 cm at the discretion of tre
ating physician. Follow-up ranged from 4.5 to 8.3 years with a median time of 5.
7 years. Kaplan-Meier estimates of BCF, based on the 1997 ASTRO definition, were
53% in the long arm and 60% in the short arm at five years [hazard ratio (HR),
1.18; 95% confidence interval (CI), 0.991.41], favoring conventional fractionatio
n over hypofractionation. PSA failure accounted for 83% of BCF events. The obser

ved difference in BCF rate (short minus long arm) was 7.0% (90% CI, 12.6% to 1.4%).
Since the lower bound of the CI on this difference was lower than the predefine
d tolerance of 7.5%, one could not exclude the possibility that the hypofractiona
ted regimen (short arm) was inferior to the standard regimen (long arm). Similar
results were obtained when the Vancouver (17) and Houston (15) definitions for
PSA failure were used. The trial did not detect statistically significant differ
ences between treatment groups in terms of the secondary outcomes of overall
202 Table 1 Randomized Trials of Hypofractionated Radiotherapy in Patients with
Localized Prostate Cancer Trial Source Status as of latest publication Patients
Conventional radiotherapy
Lukka
Hypofractionated radiotherapy
Trial completedresults published OCOG Lukka et al., 2005 (10) (Canada)
Australian
Yeoh et al., 2006 (37,38)
Fox Chase Cancer Center (U.S.A.)
Pollack et al., 2006 (39); clinicaltrials .gov identifier NCT00062309 (40);
Recruitment completed, increased 936 acute toxicity with low, hypofractionation;
no intermediate, significant differences in high risk relapse, late toxicity, o
r survival Recruitment completed, 217 increased GI toxicity in first low, 4 wk w
ith hypofractionation; intermediate, no significant difference in high risk rela
pse and survival rates Recruitment completed, follow- 300 up ongoing, 3-mo toxic
ity data intermediate reported for first 100 patients: and high risk increased G
I toxicity with hypofractionation Recruiting 1204 (target) intermediate risk
66 Gy in 52.5 Gy in 33 fractions, 20 fractions, 2 Gy/fraction 2.62 Gy/fraction
64 Gy in 55 Gy in 32 fractions, 20 fractions, 2 Gy/fraction 2.75 Gy/fraction
76 Gy in 70.2 Gy in 32 fractions, 26 fractions, 2 Gy/fraction 2.7 Gy/fraction
Trial ongoing OCOGclinicaltrials.gov PROFIT identifier (Canada) NCT00304759 (41)
; Study protocol clinicaltrials.gov Institute of identifier Cancer NCT00392535 (
42); Research South et al. (43) (U.K.) RTOG-0415 clinicaltrials.gov (U.S.A.) ide
ntifier NCT00331773 (44); RTOG Web site (45)
a
Recruiting
Recruiting
78 Gy in 39 fractions, 2 Gy/ fractiona 74 Gy in 2577 37 fractions, low, inter2 G
y/ mediate and fractiona some high risk 1067 low risk 73.8 Gy in 41 fractions, 1
.8 Gy/ fractiona
60 Gy in 20 fractions, 3 Gy/fractiona 60 Gy in 20 fractions Or 57 Gy in 19 fract
ions, 3 Gy/fractiona 70 Gy in 28 fractions, 2.5 Gy/fractiona
Trials using three-dimensional conformal radiation treatment techniques, includi
ng intensity-modulated radiotherapy. Abbreviations: RTOG, Radiation Therapy Onco
logy Group; OCOG, Ontario Clinical Oncology Group; GI, gastrointestinal.

Table 2 Published Evidence from Randomized Trials of Hypofractionated vs. Conven

tional Radiotherapy 5-yr Kaplan-Meier estimates (hypofractionated vs. convention
al RT) Trial OCOG (Canada) Lukka et al., 2005 (10) Australian Yeoh et al., 2006
(38) Fox Chase Cancer Center (U.S.A.) Pollack et al., 2006 (39)
a b
Adverse effect rates (hypofractionated vs. conventional RT) Acute GI and GU toxi
city (grade 3 or 4) 11.4% vs. 7.0% P < 0.05 GI toxicity scores higher with hypof
ractionation (P < 0.05) GI: 0% vs. 0% GU: 8% vs. 2% NS Late GI and GU toxicity (
grade 3 or 4) 3.2% vs. 3.2% NS Urgency of defecation and total GU toxicity score
s higher with hypofractionation (P < 0.05) Not reported
Median follow-up 68 mo 48 mo 3b mo
Biochemicalclinical relapse 59.95% vs. 52.95% NSa 57.4% vs. 55.5% NS Not reporte
d
Survival 87.6% vs. 85.2% NS 86.4% vs. 84.1% NS Not reported
Difference between the five-year rates for conventional and hypofractionated arm
s failed to meet the predefined criterion for equivalence. Preliminary results f
rom three-month follow-up of first 100 patients. Abbreviations: NS, difference b
etween groups not statistically significant; RT, radiation therapy; OCGC, Ontari
o Clinical Oncology Group; GI, gastrointestinal; GU, genitourinary.
Fractionation Schemes for Clinically Localized Prostate Cancer
203
survival (estimated 5-year survival rates; 85.2% in the long arm and 87.6% in th
e short arm; HR, 0.85; 95% CI, 0.631.15) or positive prostate biopsy two years af
ter radiotherapy [53.2% in the long arm vs. 51.9% in the short arm; risk differe
nce (RD), 2.3%; 95% CI, 5.1% to 9.8%]. Patients in the long arm experienced signi
ficantly less grade 3 or 4 acute GI or GU toxicity than those in the short arm (
7.0% vs. 11.4%; RD, 4.4%; 95% CI, 8.1% to 0.6%), but rates of severe late toxicity
were similar (3.2% vs. 3.2%; RD, 0.0%; 95% CI, 2.4% to 2.3%). Overall, 3.2% of p
atients experienced grade 3 or 4 GI or GU adverse events more than five months a
fter radiotherapy in both treatment groups, which is consistent with the a/b mod
el for late toxicity. The small but statistically significant increase in acute
toxicity with the hypofractionated regimen is consistent with a higher net rate
of stem cell depletion in the rectal and bladder mucosa resulting from an increa
sed rate of dose accumulation (13.1 Gy/wk in the short arm vs. 10 Gy/wk in the l
ong arm). The dose chosen for the hypofractionated regimen used in the short arm
was based on data from case series published between 1982 and 1993 (2731). The i
ncreased rate of acute toxicity observed in the short arm in the randomized tria
l confirms that further dose escalation would have been impossible with the trea
tment techniques in use during the study period. When the trial was designed in
the early 1990s, a four-field technique was the standard method for delivering r
adiation. Current techniques such as conformal-beam radiation or IMRT together w
ith image-guided radiation therapy (IGRT) should allow the dose per fraction to
be increased to levels that could result in higher tumor-control rates (based on
radiobiological considerations) while maintaining an acceptable incidence of la
te toxicity. Furthermore, the total dose used in the long arm in this study was
low compared with current doses used in high-dose standard fractionation schedul
es. Thus, both the technique and dose of radiation used in this trial limit gene
ralizability of the results to current practice. Preliminary results from two ad
ditional randomized trials provide evidence on the adverse effects of hypofracti
onated radiotherapy for prostate cancer (3740). Yeoh et al. published two reports
on an Australian randomized trial of hypofractionated radiotherapy (37,38). Med

ian follow-up at last report was 48 months (ranging from 6 to 108 months). Two h
undred and seventeen patients with localized early-stage (T1T2) prostate cancer w
ere randomized to receive 64 Gy in 32 fractions over 6.5 weeks (standard fractio
nation) or 55 Gy in 20 fractions over 4 weeks (hypofractionation). As in the OCO
G trial described above, radiotherapy planning used twodimensional computed tomo
graphy (CT) data, and treatment was delivered using a three- or four-field techn
ique. The publication does not state if image guidance was
utilized during the treatment. The primary outcome was late treatment-related to
xicity. The study was designed to detect a difference between groups in the freq
uency of mild late toxicity of 20%. GI and GU symptoms were measured before and
after one month, and then one, two, three, four, and five years consecutively af
ter radiotherapy. Compared to conventional radiotherapy, four measures of GI tox
icity (stool frequency, urgency, mucous discharge, and total GI symptom score) w
ere significantly worse one month after starting hypofractionated radiotherapy,
as were measures of the effect of GI symptoms on daily activities (38). There we
re no statistically significant differences in GI symptoms over the remaining fo
llow-up, with the exception of urgency of defecation at five years, which was wo
rse with hypofractionated radiotherapy. Hypofractionated radiotherapy was an ind
ependent predictor of increased total GI symptoms at two years (risk ratio, 1.32
; 95% CI, 1.071.64) but not at three, four, or five years. Total GU symptom score
s were worse at two and three years follow-up with hypofractionation compared to
conventional fractionation, but this difference was not reflected in the effect
s of GU symptoms on daily activities. No statistically significant differences b
etween treatment groups were detected for the secondary end points of biochemica
l clinical relapse (5-year estimates, 57.4% vs. 55.5% in hypofractionated and co
nventional arms, respectively) and overall survival (5-year estimate, 86.4% with
hypofractionation vs. 84.1% with conventional fractionation). Preliminary resul
ts are available from an American study that had higher total doses in both the
conventional fractionation and hypofractionation arms than the trials described
above. This trial used IMRT in both groups (39,40). Men with intermediate- or hi
gh-risk localized prostate cancer (T1bT3c and Gleason score !5) were randomized t
o receive 76 Gy in 38 fractions (standard fractionation) or 70.2 Gy in 26 fracti
ons (hypofractionation). Details of the planning including simulation, CTV/PTV,
normal tissue delineation and constraints are described in considerable detail i
n the paper (39), however details of the image guidance employed is not outlined
in this paper. The primary end point for the trial, biochemical failure, has no
t yet been analyzed, but a preliminary report on acute toxicity in the first 100
patients was published in 2006 by Pollack et al. Projected accrual is 300 patie
nts (40). There was a significant increase in GI toxicity score during weeks 24 o
f treatment with hypofractionated IMRT compared to conventional IMRT but no diff
erences over the remainder of the three-month follow-up period. There was no gra
de 3 or 4 GI toxicity, but grade 2 adverse GI effects were reported for 18% of p
atients in the hypofractionated arm and 8% in the control arm during radiotherap
y. No statistically significant differences between groups were detected for GU
toxicity.
204
Lukka
Summary of Published Randomized Trials Using Hypofractionation To date, one RCT
has provided data on the efficacy and toxicity of moderate-dose hypofractionated
radiotherapy using older (4-field) treatment techniques (10). Another study usi
ng similar doses and technique has reported toxicity data (37,38). Early results
from a third study comparing higher doses of both conventional radiotherapy (76
Gy in 38 fractions) and a hypofractionated regimen (70.2 Gy in 26 fractions) su
ggested no difference in acute toxicity at three-months follow-up (39). These re
sults are interesting. It is reassuring that late radiation toxicity was found t
o be equivalent and that the hypofractionated regimen has, in general, been foun

d to be well tolerated. Ongoing Trials To evaluate the role of hypofractionated

radiotherapy in the treatment of prostate cancer compared to the current standar
d of high-dose radiotherapy, well-designed RCTs need to be conducted evaluating
hypofractionated regimens versus 76 to 78 Gy using conventional fractionation of
1.8 to 2 Gy. To enable these doses to be delivered, modern conformal and IMRT t
echniques need to be utilized. Three relevant ongoing randomized trials are list
ed in the Web site maintained by the U.S. National Institutes of Health (clinica
ltrials.gov) (Table 1) (41,42,44). All use 3DCRT or IMRT. Doses in the standard
fractionation arm range from 73.8 to 78 Gy, given in fractions of 2 Gy in two st
udies and 1.8 Gy in the third. Two studies use fraction sizes of 3.0 Gy in the h
ypofractionated arm and the third uses 2.5 Gy. The OCOG group is conducting a ra
ndomized trial (known as PROFIT) of hypofractionated radiotherapy (60 Gy in 20 fra
ctions over 4 weeks) versus standard radiotherapy (78 Gy in 39 fractions over 8
weeks) in patients with intermediate-risk prostate cancer (T1T2a, Gleason score 6
, PSA 10.120.0 ng/mL; T2bT2c, Gleason score 6, PSA 20 ng/mL; T1T2, Gleason score
PSA 20 ng/mL) (41). 3DCRT or IMRT is used for both hypofractionated and standar
d radiotherapy. The primary outcome measure is biochemical (PSA) failure defined
by the ASTRO consensus criteria. Secondary outcomes include BCF, death from can
cer, toxic effects of radiotherapy, and health related quality of life. This mul
ticenter trial started recruitment in May 2006. Total expected enrolment is 1204
. A large British trial is comparing two hypofractionated high-dose IMRT schedul
es (60 Gy in 20 fractions over 4 weeks and 57 Gy in 19 fractions over 3.8 weeks)
with high-dose radiotherapy using conventional fractionation (74 Gy in 37 fract
ions over 7.5 weeks) in men with low- to
high-risk-localized prostate cancer (42,43). The study population will include p
atients with T1b to T3a tumors, PSA 40 ng/mL, Gleason score <9, and estimated ri
sk of lymph-node metastases <30% (for Gleason score <6, PSA must be 40 ng/mL; fo
r Gleason score 7, PSA must be <30 ng/mL; for Gleason score
8, PSA must be <15 n
g/mL). Primary outcomes comprise acute and late radiationinduced adverse effects,
as well as recurrence of prostate cancer. Quality of life and economic impact a
re included among the secondary outcomes. Enrolment started in late 2002 with a
target of 2577 patients to be followed for up to 15 years. The RTOG-0415 trial s
tarted in April 2006 (44,45). One-thousand and sixty-seven men with low-risk pro
state cancer (T1T2c, Gleason score 26, PSA <10 ng/mL) will be randomized to conven
tionally fractionated 3DCRT or IMRT (73.8 Gy in 41 fractions over 8.2 weeks) or
hypofractionated 3DCRT or IMRT (70 Gy in 28 fractions over 5.6 weeks). The prima
ry outcome is disease-free survival and secondary outcomes include biochemical r
ecurrence, overall survival, adverse effects, quality of life, anxiety/depressio
n, and cost utility. These studies and other planned trials will provide evidenc
e to help evaluate the role of hypofractionated radiotherapy in the management o
f localized prostate cancer. This, in turn, will help guide future research eval
uating hypofractionated regimens using higher dose per fraction and even shorter
regimens, as will be discussed later in this chapter. BIOLOGICALLY EQUIVALENT D
OSE Comparisons of the biologically equivalent dose (BED) for different conventi
onal and hypofractionated regimens is an interesting modeling exercise that aids
understanding of the regimens used in clinical trials in terms of tumor control
(based on a particular a/b ratio for the tumor), acute toxicity (a/b ratio of 1
0 Gy), and late toxicity (a/b ratio of 3 Gy) (Table 3). While not ideal (cell pr
oliferation is ignored, for example), the BED modeling exercise can be used as a
rough comparator of the regimens used in the randomized trials discussed above.
As an example, randomized trials demonstrate that bNED rates can be improved by
using 78 Gy rather than 70 Gy with conventional fraction sizes of 2 Gy (12,1923)
. If the a/b ratio is 1.5 Gy for prostate cancer, the BED would be 182 with a to
tal dose of 78 Gy compared to 163 with 70 Gy. The BED for acute toxicity (a/b
10
Gy) would be expected to be 93.6 versus 84, respectively, and for late toxicity
(a/b 3 Gy), 130 versus 116.7. This is supported by evidence from clinical studi
es, which found that rates of late rectal toxicity (grade 2 and 3) were slightly
increased with higher doses of radiation.

7,

Fractionation Schemes for Clinically Localized Prostate Cancer Table 3 Biologica

lly Equivalent Dose by a/b Ratio Biologically equivalent dose (BED) assuming dif
ferent a/b ratios Total dose (Gy) 52.5 60.0 66.0 70.0 70.0 70.0 70.2 74.0 78.0 N
umber of fractions 20 20 33 25 28 35 39 37 39 Dose/fraction (Gy) 2.6 3.0 2.0 2.8
2.5 2.0 1.8 2.0 2.0 a/b
1 190.3 240.0 198.0 266.0 245.0 210.0 196.6 222.0 234.0
a/b 1.5 144.4 180.0 154.0 200.7 186.7 163.3 154.4 172.7 182.0 a/b 3 (late effec
ts) 98.4 120.0 110.0 135.3 128.3 116.7 112.3 123.3 130.0 a/b
10 (early effects)
66.3 78.0 79.2 89.6 87.5 84.0 82.8 88.8 93.6
205
However, rates of late bladder toxicity were similar for doses of 70 and 78 Gy.
The bNED rates were improved with the higher dose regimen in keeping with the in
creases in the BED values. Comparison of the regimens used in the published OCOG
hypofractionation trial (66 Gy/33 fractions/6.5 wk vs. 52.5 Gy/20 fractions/4 w
k) would show BEDs of 154 and 144.4, respectively for tumor control, assuming an
a/b ratio of 1.5 Gy (10). These BED values are consistent with the apparent equi
valence of the two regimens in this study. The respective BED values for late tox
icity would be 110 versus 98.4 and for acute toxicity, 79.2 versus 66.3. In the
OCOG trial, while late toxicity rates were equivalent, acute toxicity was slight
ly worse with the hypofractionated regimen (grade 3 or 4 toxicity, 7% vs. 11.4%)
. In this case, the observed acute toxicity results are at variance with the acu
te toxicity BED calculations. This may, in part, be related to the radiation tec
hnique employed in the study. The observation could also arise from a higher net
rate of cell depletion in the rectal and bladder mucosa resulting from an incre
ased rate of dose accumulation. In the ongoing OCOG-PROFIT study that randomizes
patients to 78 Gy in 2 Gy fractions or 60 Gy in 3 Gy fractions, the correspondi
ng BED values are 182 and 180 for tumor control (a/b 1.5 Gy), 130 and 120 for la
te toxicity (a/b 3 Gy), and 93.6 and 78 for acute toxicity (a/b 10 Gy) (41). An
ongoing RTOG trial randomizes patients to 73.8 Gy in 1.80 Gy fractions or 70.0 G
y in 2.5 Gy fractions. According to the study protocol, the BED for tumor contro
l is 162.4 versus 186.7, late toxicity 118.1 versus 128.3, and acute toxicity 87
.1 versus 87.5 (45). These calculations illustrate the comparability between the
hypofractionated and conventional regimens in terms of BED. The modeling exerci
se suggests that hypofractionated regimens have the potential to improve tumor c
ontrol while maintaining late toxicity rates if the a/b ratio for prostate cance
r is as low as is currently thought.
Ultimately, well-designed randomized clinical studies would need to be conducted
comparing these hypofractionated regimens with conventional regimens to assess
their relative efficacy and toxicity (both acute and late). Another common metho
d for comparing regimens is to convert the BED value to the LQ equivalent dose i
n 2 Gy fractions, also referred to as the normalized total dose or NTD2Gy. Fowle
r has recently published on the comparability of hypofractionated regimens to st
andard 2 Gy/fraction regimens (1). Table 4 shows the comparability of hypofracti
onated regimens to conventionally fractionated regimens with regard to tumor con
trol, based on equivalent late rectal toxicity. Based on this model, a regimen o
f 74 Gy in 37 fractions over 7.5 weeks results in a BED for late toxicity of 123
.3 (a/b 3 Gy). In comparison, a regimen of 3.06 Gy in 20 fractions (total dose,
61.11 Gy) will result in a similar BED in late-responding tissue of 123.3, but t
he NTD2Gy would be equivalent to 79.6 Gy. Fowlers calculations suggest that bNED
(biochemical control) would increase from 75.5% to 84% when 74 Gy of radiotherap
y (given in conventional 2 Gy fractions) is changed to a hypofractionated regime
n of 61.11 Gy (in 3.06 Gy fractions). The comparability among other hypofraction
ated regimens, ranging from 25 fractions of 2.63 Gy to 3 fractions of 9.7 Gy, is
also shown in Table 4. It needs to be emphasized that these estimates are based
on modeling and must to be validated by results from wellconducted clinical tri
als. INDICATIONS AND CONTRAINDICATIONS TO HYPOFRACTIONATED PROSTATE RADIOTHERAPY
Ongoing and future studies would need to confirm the role of hypofractionated r
adiotherapy in low-, intermediate-, and high-risk populations. If its role is pr

oven in RCTs,
206 Table 4 Hypofractionated Schedules Calculated by Fowler for Constant Late Re
ctal Complications Assuming a/b 3 Gy Late rectal complications Hypofractionated
schedule 37 F 2.00 Gy 25 F 2.69 Gy 20 F 3.06 Gy 15 F 3.69 Gy 10 F 4.77 Gy 5 F 7.
73 Gy 3 F 9.70 Gy Total dose (Gy) 74.0 65.73 61.11 55.33 47.65 36.16 29.10 BEDGy
3 for a/b 3 Gy 123.3 123.3 123.3 123.3 123.3 123.3 123.3 Late rectal complicatio
ns NTD2Gy for a/b 3 Gy (Gy) 74.0 74.0 74.0 74.0 74.0 74.0 74.0 Prostate tumor ef
fects Calculated NTD2Gy for a/b
1.5 Gy (Gy) 74.0 78.7 79.6 82.0 85.4 90.2 93.1
Lukka
Estimated bNED (%) 75.5 82.8 84.0 87.3 90.0 94.0 95.8
Total dose
74 Gy (1 2/3)/(1 d/3) for the constant BED of 123.3 Gy3. NTD2Gy, norm
alized total dose, the schedule using 2 Gy fractions that would give the same lo
g cell kill. The final two columns also present the consequent prostate tumor ef
fects, as normalized total dose (NTD2Gy) assuming a/b 1.5 Gy; and the estimated f
ive-year percentage bNED. Abbreviations: BED, biologically equivalent dose; bNED
, biochemical no evidence of disease; F, fractions; NTD, normalized total dose.
Source: From Ref. 1.
hypofractionated treatment could be considered in the ongoing treatment of these
subgroups of patients. On the basis of currently available data, it is difficul
t to identify specific indications or contraindications for hypofractionated rad
iotherapy. The inclusion criteria for entry into studies such as the PROFIT tria
l are broad and include any patient eligible for conventional radiotherapy to th
e prostate. Specific indications and contraindications for clinical practice wou
ld need to await the publication of ongoing studies. It is possible that subgrou
p analyses of results from ongoing trials may identify specific sets of patients
who would be more optimally treated with conventional radiotherapy (as opposed
to hypofractionated radiotherapy) because of better tumor control or lower toxic
ity. For example, in an individual patient where there is concern about bowel or
bladder toxicity, it may be preferable to use conventional fractionation regime
ns rather than a hypofractionated regimen. Results of ongoing studies, including
RCTs, are awaited. Finally, there may be selected patients who are more likely
to be at risk for radiotherapy toxicity because of their inherent genetics; but
further prospective studies are required to confidently predict the incidence an
d existence of these patients within large radiotherapy cohorts (46). IMAGE-GUID
ED PLANNING AND DELIVERY Following publication of a randomized trial that demons
trated reduced toxicity with conformal radiotherapy compared to traditional four
-field techniques, conformal techniques were adopted even when moderate doses of
radiotherapy were used (47). These conformal techniques reduce dose to both rec
tum and bladder resulting in reduced toxicity in these organs. IMRT, which allow
s
further dose shaping around rectum and bladder, is now increasingly being used i
n clinical practice. IGRT now forms an important aspect of treatment to ensure t
hat treatment is delivered as planned. TECHNICAL FACTORS Treatment Planning Conf
ormal radiotherapy, and particularly, IMRT techniques require careful attention
be paid to definition of tumor volume and normal tissue to evaluate the dose dis
tribution against accepted standards. These standards include definitions of max
imum and minimum doses allowed within the planning target volume (PTV), as well
as a set of dose-volume histogram specifications for both target and organs at r
isk. Examples from the RTOG and OCOG protocols defining the PTV parameters are s
hown in Table 5. Similarly, dose constraints on normal tissue need to be careful
ly evaluated with hypofractionated treatments to minimize toxicity. Studies corr
elating normal tissue constraints to toxicity with specific hypofractionated reg
imens need to be published to help confirm the safety of these regimens. Once ef
ficacy and safety are confirmed, clinicians using such regimens need to ensure t

hat due care and attention is paid through the planning process with regard to t
he PTV and clinical target volume (CTV) dosimetry, and that the doses delivered
to normal tissue are within acceptable normal tissue constraints. Two different
ways of defining normal tissue have been outlined for two of the ongoing RCTs ev
aluating hypofractionated regimens (RTOG and OCOG) (41,44). The RTOG method is a
lso used in other RTOG studies such as protocol 0126 (26). In the RTOG studies,
the rectum is defined from the anus to the recto-sigmoid junction as a volume (i
ncluding the rectal contents,
Fractionation Schemes for Clinically Localized Prostate Cancer Table 5 Planning
Parameters and Dose Constraints for OCOG and RTOG Trials OCOG-PROFIT Dose prescr
iption CTV definition CTV dose PTV definition PTV dose Bladder constraint Minimu
m Maximum Contoured bladder wall: 50% to receive less than 70% to receive less t
han Bladder volume: 15% to receive more than 25% to receive more than 35% to rec
eive more than 50% to receive more than Contoured rectal wall: 50% to receive le
ss than 70% to receive less than Rectal volume: 15% to receive more than 25% to
receive more than 35% to receive more than 50% to receive more than <5% to recei
ve Minimum Maximum 60 Gy in 20 (3 Gy) fractions 78 Gy in 39 (2 Gy) fractions RTO
G-0415 70 Gy in 28 (2.5 Gy) fractions
207
73.8 Gy in 41 (1.8) Gy fractions

Contoured prostate and, if applicable, Prostate and first 1 cm of seminal vesicl

e seminal vesicle 60 Gy 78 Gy 70 Gy 73.8 Gy 64.2 Gy 83.5 Gy Expansion 7 mm poste
riorly (toward the 510 mm around CTV rectum) and 10 mm in all other planes 57 Gy
74.1 Gy 70 Gy 73.8 Gy 63 Gy 81.9 Gy 74.9 79 37 Gy 46 Gy 37 Gy 46 Gy >43 Gy 53
71 Gy 53 Gy 71 Gy >53 Gy 74 69 64 59 79 74 69 64 Gy Gy Gy Gy Gy Gy G
65 60 80 75 70 65 Gy Gy Gy Gy Gy Gy Gy Gy
Rectum constraint
Femoral head and neck constraint Penile bulb constraint
Mean dose

51 Gy
52.5 Gy
Abbreviations: CTV, clinical target volume; PTV, planning target volume. Source:
From OCOG-PROFIT and RTOG-0415 protocols.
feces, and air). In the case of the bladder, the whole bladder volume is delinea
ted (including the urine volume) in the RTOG protocols (Fig. 2A). In contrast, t
he OCOGPROFIT protocol defines only the rectal wall (and not its contents) and b
ladder wall thickness (not urine volume) (Fig. 2B). In the OCOG-PROFIT protocol,
the bowel and bladder walls are contoured 18 mm (to beam edge) beyond the most
inferior and superior contoured prostate slices (or seminal vesicles when they a
re included in the CTV). In each protocol, the acceptable dose constraints are b
ased on clinical experience from previous studies. Table 5 summarizes the constr
aints for rectum and bladder for each of these protocols. There has not been a d
irect comparison of these methods of normal tissue delineation or normal tissue
constraints. Both methods have advantages and disadvantages. Ultimately, it woul
d be reasonable to expect that as long as the normal tissue delineation are adhe

red to and normal tissue constraints

respected, toxicity would be acceptable. The importance of ensuring this for hyp
ofractionated regimens cannot be overemphasized. The current PTV and normal tiss
ue constraint definitions in hypofractionated regimens are based on initial stud
ies. The ongoing RCTs evaluating hypofractionated regimens would need to confirm
the efficacy and safety of these regimens before they are routinely used in cli
nical practice and outside a clinical trial. Note should be made that, given tha
t a large variety of hypofractionated regimens are potentially available for use
, the efficacy and toxicity would vary by regimen. With differences in normal ti
ssue toxicity depending on the dose per fraction and total dose, the normal tiss
ue dose constraints will vary depending on the hypofractionated regimen used and
may not be transferable to other hypofractionated regimens. It is, therefore, i
mportant that regimens and normal tissue constraints from good published hypofra
ctionated regimens be used. Where
208
Lukka
Figure 2 (See color insert.) Organs at risk. (A) RTOG definition for rectal thic
kness and bladder thickness. (B) PROFIT definition for rectal thickness and blad
der thickness.
high-dose treatments are utilized, it would be extremely important to ensure tha
t the doses within the PTV and normal tissues are in keeping with the definition
s of these criteria established in protocols conducted by good clinical trial gr
oups. With hypofractionated regimens (and higher doses per fraction), it is impo
rtant to ensure that tumor and normal tissue definition and planning parameters
(such as the use of convolution/superposition algorithms for heterogeneity corre
ction, the use of appropriate margins, or the accounting of portal imaging dose)
are in keeping with published and accepted data. Treatment Delivery High-dose p
rostate radiotherapy using conformal or IMRT techniques requires the monitoring
of prostate position at the time of treatment delivery and adjusting the treatme
nt parameters for accurate targeting. Several techniques are available to ensure
accurate positioning for IGRT, including placement of fiducial markers, utiliza
tion of cone beam CT, and use of B-mode acquisition and targeting (BAT) system u
ltrasound. It is extremely important that one of these localization techniques b
e used as part of IGRT for prostate cancer in light of the tight margins employe
d to minimize the dose to normal tissue. With hypofractionated radiotherapy, it
is especially important to ensure acceptable dose distribution particularly to no
rmal tissue. With fewer treatments being given in hypofractionated regimens, ine
xact targeting of the tumor during radiotherapy for even one treatment has a lar
ger impact on total dose delivered to the tumor than would be the case with conv
entional, more protracted radiotherapy involving more fractions. Suboptimal IGRT
could potentially result in a higher proportion of geographic misses and conseq
uent poorer tumor control. Conversely, the larger dose used for each treatment h
as the potential to produce more damage to normal tissue and thus more adverse e
ffects to bladder and rectum, if treatment is not delivered as planned. For both
these reasons, the importance of IGRT with hypofractionated regimens cannot be
overemphasized.
THE FUTURE Radiobiological principles indicate that doses per fraction beyond th
ose employed in completed or ongoing trials warrant investigation. Fowler et al.
used standard LQ modeling to determine a set of hypofractionated regimens that
could be tested in randomized trials (1,11). Using data from dose-escalation stu
dies, they developed a doseresponse curve for biochemical control with bNED in i
ntermediate-risk prostate cancer patients treated with external-beam radiotherap
y alone given in conventional 2 Gy fractions. The investigators then went on to
calculate the dose per fraction and total doses for regimens that would be predi

cted to have the same level of late toxicity as conventional radiotherapy, assum
ing that prostate tumors have an a/b ratio of 1.5 Gy and rectal tissue a ratio o
f 3 Gy (Fig. 3). The end result was a series of hypofractionated regimens that t
heoretically keep the rate of late complications constant while causing more dam
age to tumors as fraction size increases and the number of fractions decreases.
Fowler et al. also considered the effect of each of the proposed hypofractionate
d regimens on prostate tumors by calculating the NTD, which is a measure of the
total dose given in 2 Gy fractions that would give the equivalent biological eff
ect to the hypofractionated regimen in question. For example, the effect of 60 G
y given as 20 fractions of 3 Gy corresponds to 77.1 Gy given in 2 Gy fractions.
Ten fractions of 4.4 Gy should produce the same level of tumor control as 75.3 G
y given in 2 Gy fractions, but with the same late complication rate as 66 Gy in
2 Gy fractions. In the latter example, the estimated bNED would increase, in the
ory, from 51.6% to 77.1%. This hypothesized 25% increase in bNED could be tested
against conventional radiotherapy in a randomized trial with only 72 participan
ts in each arm. Fowler et al. warned against using treatment times of less than
five weeks because of a possible risk of acute rectal toxicity, or treatments wi
th fewer than five fractions, which might limit the possibility of re-oxygenation
or redistribution of tumor cells into more sensitive phases of cell cycles.
Fractionation Schemes for Clinically Localized Prostate Cancer
209
Figure 3 Estimated increase in bNED as total dose is decreased with fewer and la
rger fractions. Each of the three graphs shows the curve for 2 Gy rising to the
right and another curve rising to the left for hypofractionated regimens with to
tal doses calculated to keep late rectal complications constant at 66 Gy NTD2Gy
(A), 72 Gy (B), and 78 Gy (C). The number of fractions and size of each fraction
is noted for each point on the curve. Abbreviations: bNED, biochemical no evide
nce of disease; NTD, normalized total dose. Source: From Ref. 1.
regimen of either 20 fractions of 3 Gy or 10 fractions of 4.7 Gy, compared to 36
fractions of 2 Gy, while resulting in equivalent late toxicity. Other hypofract
ionation regimens and the impact on tumor control while maintaining the same lev
el of late toxicity are also shown. On the basis of recent data, it would appear
that 78 Gy in 2 Gy fractions is the preferred dose for intermediate-risk patien
ts. The additional benefit, in terms of tumor control, of using hypofractionated
regimens rather than conventionally fractionated treatment with a total dose of
78 Gy may not be as great as the benefit compared to lower doses such as 72 Gy
in 2 Gy fractions. On the basis of Fowlers graphs, the change from 78 (2 Gy and 3
9 fractions) to 49.2 Gy (4.92 Gy and 10 fractions) would result in improvement i
n tumor control from 83% to 96%. In comparison, a change from 72 (2 Gy and 36 fr
actions) to 47 Gy (4.7 Gy in 10 fractions) would result in improvement in tumor
control from 69% to 89%. Current RCTs are evaluating hypofractionated regimens i
n the 2.5 to 3 Gy/fraction range. If these trials confirm the role of hypofracti
onated regimens, higher doses per fraction with fewer fractionsprovided the overa
ll treatment time is no shorter than five weeks could be investigated. One would
need to await the results of current and ongoing hypofractionated radiotherapy t
rials before embarking on the next generation of studies evaluating shorter, hig
her dose per fraction regimens. These regimens, which are based on radiobiologic
al modeling, are interesting but would need to be carefully evaluated before the
y can be adopted as part of clinical practice. Phase I/II studies would be neede
d to confirm tolerability. If toxicity is found to be acceptable, these regimens
then need to be compared with current standard treatments in well-conducted ran
domized trials. It is interesting to note that a phase III study has been propos
ed to compare 78 Gy (39 fractions of 2 Gy) to 42.7 Gy (7 fractions of 6.1 Gy in
4 weeks) (1). This regimen has a rectal-equivalent late-toxicity NTD2Gy of 78 Gy
(assuming a/b
3 Gy), but the BED values show that the hypofractionated regimen
(42.7 Gy in 7 fractions of 6.1 Gy) has a tumor-equivalent dose of 92.7 Gy in ter

ms of NTD2Gy (assuming a/b ratio of 1.5 Gy for prostate cancer). SUMMARY OF PERT
INENT CONCLUSIONS
l
l
Fowlers estimates show improved tumor control using some hypofractionated regimen
s compared to some conventional fractionation regimens, while maintaining equiva
lent late toxicity (1). For example, as shown in Figure 3B, there would be impro
ved tumor control using a
Current high-dose treatments using 1.8 to 2 Gy fractions are more efficacious th
an doses used in the past and have acceptable toxicity. These conventional regim
ens may involve treatment in up to 39 fractions with associated social, emotiona
l, and economic burdens for patients and their families. Use of these regimens h
as also increased the cost of treatment and increased the burden on stretched ra
diotherapy resources in some jurisdictions.
210
l
Lukka
l
l
l
l
l
l
l
l
l
l
Hypofractionated radiotherapy has potential advantages if found to be efficaciou
s and to have acceptable toxicity compared to conventional radiotherapy. Radiobi
ological modeling suggests that hypofractionated prostatic radiotherapy may resu
lt in improved efficacy without increased toxicity. The a/b ratio for prostate c
ancer is likely between 0.9 and 1.5 Gy, whereas the a/b ratio is 3.0 Gy for late
toxicity. This should make prostate cancer cells more sensitive to large dose f
ractions than to conventional fraction sizes. Well-designed randomized trials ne
ed to be conducted comparing hypofractionated regimens to conventionally fractio
nated high-dose regimens to assess their relative advantages and disadvantages.
Early evidence suggests that efficacy and safety similar to that obtained with c
onventional fractionation schedules can be achieved with a shorter course of hyp
ofractionated radiation treatment using 3DCRT and IMRT; however, the results of
ongoing randomized trials are awaited. Ongoing and future studies need to confir
m the role of hypofractionated radiotherapy in low-, intermediate-, and high-ris
k subgroups. Radiobiological principles suggest that doses per fraction beyond t
hose employed in completed or ongoing trials warrant investigation. If the safet
y and efficacy of hypofractionated regimens are demonstrated, their adoption wou

ld reduce the burden of treatment for patients and their families, reduce the co
st of radiotherapy treatments, and free up strained radiotherapy facilities in s
ome jurisdictions. When hypofractionated regimens are used, conformal radiothera
py, and particularly, IMRT techniques require careful attention be paid to plann
ing protocol: definition of tumor and organ-at-risk volumes, dose constraints, m
argins, method of heterogeneity corrections, dose grid resolution, and account o
f daily portal imaging dose. The normal tissue dose constraints need to be confi
rmed for particular hypofractionated regimens through clinical trials before use
in patients off-study. The importance of IGRT with hypofractionated regimens ca
nnot be overemphasized.
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ACKNOWLEDGMENTS The author wishes to thank Dr. Charles Catton, Dr. Rob Bristow,
Dr. Orest Ostapiak, and Mary Johnston for assistance with the manuscript. The au
thor also wishes to thank RTOG and OCOG (Ontario Clinical Oncology Group) for th
eir assistance in providing details of the ongoing hypofractionation studies (04
15 and PROFIT, respectively).
Fractionation Schemes for Clinically Localized Prostate Cancer assessment of fol
low-up lead-time bias. Int J Radiat Oncol Biol Phys 2003; 57(1):1118. Pickles T,
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17.
33.
18.
34.
35.
19.
36.
20.
37.
21.
22.
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29.
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31.
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18
Image-Guided Proton Beam Radiation Therapy for Prostate Cancer
CARLOS E. VARGAS, JATINDER R. PALTA, DANIEL INDELICATO, AND ZUOFENG LI

Department of Radiation Oncology, University of Florida, Proton Therapy Institut

e, Jacksonville, Florida, U.S.A.
INTRODUCTION Advances in technology and understanding of tumor characteristics h
ave improved our ability to provide patients with better treatments and minimize
d side effects. However, these changes so far have been largely confined to real
m of ionizing electromagnetic wave radiation. In this manner, radiation oncology
has progressed from twodimensional (2-D), 3-D, intensity-modulated radiation th
erapy (IMRT), image-guided radiation therapy (IGRT), to adaptive radiation thera
py. Improved conformality is an extension of our increased ability to shape the
high-dose area within the patient. We recognize, however, that our target will o
ften vary in position, shape, or even volume during the course of therapy. Diffe
rent strategies have evolved to address this problem, including image-guided rad
iation, consisting of daily target localization and methods of patient-specific
treatment planning, so-called adaptive radiotherapy. Similar to conventional radio
therapy, proton therapy has to incorporate the principles of image guidance and
adaptive therapy to optimize the therapeutic ratio. This is particularly true in
prostate cancer, where the large interfraction positional variation makes image
guidance critical.
OVERVIEW OF PROTON THERAPY AND IMAGE GUIDANCE A comprehensive review of current
proton therapy applications is beyond the scope of this chapter. However, to pro
vide a framework of proton issues specific to prostate cancer, we will introduce
some key general elements of proton therapy planning and delivery. Proton thera
py is particulate ionizing radiation. Like any charged particle, protons have a
characteristic Bragg peak at the end of their ionization track. Because of their
large mass (1.6726 1027 kg), protons follow a relatively linear path through tis
sue, which allows for easier dosimetry than electrons and a predictable Bragg pe
ak position. Basically, a proton will start depositing its energy as soon as it
enters tissue but will deposit the majority of the energy at the end of its traj
ectory (Fig. 1). The range or the most distal penetration of the proton beam in
a patient is determined by the energy of proton beam, which is continuously vari
able. The proton energy and spread-out Bragg peak (SOBP) are selected to cover t
he entire thickness of the target along the beam axis. The result is a highly ho
mogenous dose distribution within the target as well as a sharp dose falloff bey
ond the target. The use of fewer beams decreases the integral dose with a
213
214
Vargas et al.
Figure 1 Diagram representing proton beam spread-out Bragg peak. Source: UFPTI b
eam data.
minimal impact on target coverage. This provides an advantage over photon therap
y, where the high-dose gradient area can only be shaped with multiple beam angle
s, therefore increasing the low-dose volume. Proton therapy, with its huge poten
tial, is in its infancy. The state-of-the-art in proton therapy is to deliver pr
oton beams that are termed as scattered proton beams. These require the fabricat
ion of patient-specific bolus material and apertures. Even though these beams sp
are a significant amount of normal tissue compared with X-ray radiation therapy,
they do not provide very conformal radiation dose to the tumor. Specific limita
tions of proton therapy are because of the differences in the creation of the hi
gh-dose gradient. For IMRT, multiple beams and segments will shape high dose to
the target, relatively independent of tissues transverse. For proton therapy, ea
ch beam will create a high-dose area that will be defined by the changes in stop
ping power over the path of the incident proton. Thus, the target itself will he
lp define the high-dose gradient area and changes in the tissues transverse will

have a large impact on SOBP localization. For this reason, the beam angles for
image-guided proton therapy must be carefully selected. For example, in prostate
cancer, anterior or posterior beams, where the radiological path length may dra
matically vary, are avoided. The result is treatment plans limited to lateral or
lateral oblique fields. This constraint is defined by the physical properties o
f the particle and will not vary with proton delivery technique such as double s
cattering, uniform scanning, and intensity-modulated proton therapy (IMPT). The
first problem can be summarized as the limitation in the beams angles that can b
e employed for optimal results with minimal delivery uncertainty. The second pro
blem is related to the smearing function. To assure target coverage, each beam p
lan has to be optimized to cover the target (i.e., the prostate). Each
voxel in the prostate must be expanded to a radius equivalent to the potential t
arget motion during an imageguided approach. This information, although not used
to define target size per se, is used to define the range necessary to cover di
stal edge of the target as the beam is realigned for each new prostate position.
In other words, it represents a convex hull of all potential prostate positions
encountered during treatment. The outcome is distal high-dose edge that is less
conformal compared to the shape of the prostate at the time of simulation. The
practical result is that improved target coverage from an image-guided approach
will be limited if it is based on the initial prostate position alone. A third p
roblem is related to uncertainty in correlating CT numbers to the stopping power
in CT-based proton treatment planning. The practical implications are that the
distal and proximal margins have to be increased to provide adequate target cove
rage. One result is that adequate target coverage will necessarily decrease the
conformality index. Another implication is that the Bragg peak cannot be stopped
in front of a dose-limiting structure if it is the only beam employed or if it
has a disproportional high weight among multiple beams. Thus, with improper plan
design, the use of the sharp edge of the Bragg peak to limit the dose to the re
ctum or bladder may lead to a completely opposite result. Currently, delivering
proton therapy has been looked upon as a problem of degrading a pristine Bragg p
eak and pencil beam such that it becomes a clinically useful broad field. Howeve
r, a narrow pencil beam, with a near monoenergetic Bragg peak, can be viewed as
a single spot of dose, well confined in three dimensions. If it were possible to d
ynamically position such Bragg peaks in three dimensions throughout the target v
olume, then it would be possible to use this dose spot to paint the dose to the ta
rget as required. This is the idea behind the active scanning approach to proton
therapy, which is now gaining a certain amount of popularity within the proton
therapy community. With active scanning techniques, the concept of an SOBP, a ce
ntral component of the passive scattering approach, is abandoned. Instead, it re
lies on its ability to individually place and weight spots with full flexibility
, and under computer control. From the treatment planning aspect, this requires
algorithms for determining the optimal weight of each Bragg peak in three dimens
ions. Active scanning can be accomplished by either mechanical or magnetic means
, or a combination of the two. Through computer steering, it is possible to posi
tion a Bragg peak anywhere in three dimensions within the target volume. In addi
tion, it is also necessary to be able to individually modulate the dose delivere
d at each Bragg peak. As with most dynamic delivery systems, the dose at any giv
en point is controlled by the length of time a spot
Image-Guided Proton Beam Radiation Therapy for Prostate Cancer
215
dwells at each position, with the number of delivered protons being controlled b
y a fast monitor just before the patient. Once the desired number of monitor uni
ts has been delivered, the beam at this point is switched off using a fast magne
tic deflection of the beam away from the treatment room. This fast switching off
of the beam is an important aspect of the spot scanning method where the beam i
s switched off when the steering elements are being altered, and is only applied

to the patient when all elements are stable. We believe that we have only scrat
ched the surface of possible technologies with protons to improve outcomes for p
atients. Through the development of IMPT, we should be able to deliver highly co
nformal radiation to the tumor while sparing the healthy normal tissue. IMPT wil
l rival any advanced technologybased conventional radiation therapy treatment. Th
e full potential advantage of proton therapy can be realized only if, uncertaint
ies in the localization of the distal dose gradient region in the patient becaus
e of uncertainties in the dose calculation, biological considerations, setup and
anatomical variations, and internal movement of high-density/low-density organs
in the beam are minimized. Therefore, image/dose guidance, managing inter-/intr
aorgan motion and its effects, and minimizing residual uncertainties are essenti
al for proton therapy. In this chapter, we describe our strategy to deal with th
ese issues in the treatment of prostate cancer with proton beam. RECOGNIZING REC
TAL ANATOMY Proton therapy can achieve a conformal dose distribution with one or
two beam angles, but optimizing beam arrangements is crucial. For prostate canc
er, we employ lateral oblique field arrangements, which provide clear access to
the prostate and maximize sparing of the doselimiting normal structures. As ment
ioned earlier, in the first problem, the limitation of useful beam angles and th
e ability to create highly conformal plans is also an opportunity. If the anatomy can be optimized for those limited angles, the doses to t
he normal structures can be minimal. A rectal balloon (RB) has the theoretical a
dvantage of distending the rectal wall and displacing it from the highdose area
anteriorly (15). It therefore decreases the relative volume of rectum or rectal w
all radiated to intermediate and high doses. For photon therapy, it also has the
relative advantage of increasing the rectal air volume and decreasing the dose
at the inner rectal wall (5). Although an RB may likewise decrease the relative
volume of the rectum or rectal wall radiated in proton therapy, this may provide
only a small absolute advantage because of the low rectal doses being delivered
. Furthermore, for proton therapy water is used in the RB to better control the
proton dose distribution and decrease inhomogeneities because of the air-tissue
interface. Thus, proton therapy will not necessarily have the additional advanta
ge of decreasing the inner rectal wall dose seen with photons in the build-up re
gion. However, water alone in the rectum may adequately distend the rectal wall
during the course of proton therapy and obviate the need for an actual balloon.
We do not routinely use an RB for our prostate proton plans. As mentioned above,
the absolute benefit of an RB may be clinically insignificant when low rectal d
oses are already achieved through highly conformal radiation. Vargas et al. repo
rted only 9% grade 2 or higher rectal toxicity and 0.8% grade 3 toxicity if the
rectal dose V70 was below 25% (6). In our study, only 17.5% of the rectum receiv
ed 50 Gray Equivalent (GE) and 10.2% received 70 GE. At these V50 and V70 parame
ters, a respective 4.7% and 2.7% absolute benefit was observed with an RB. Furth
ermore, the RB will not modify the shape of the dose-volume curve; it will only
shift it. As a result, the relative benefit, although similar at different dose
levels (V10V80), will be associated with a constantly decreasing real benefit (Ta
bles 24). In a detailed analysis of the topic, we found a significant benefit for
the use of RB only in selected cases (Table 1 and Fig. 2). A relative improveme
nt greater than
30 sc
Table 1 Statistically Significant Dose-Volume Parameters for the Rectum (n
ans) RB mean (SD) (%) Rectum Rectum Rectum Rectum Rectum Rectum Rectum Rectum V1
0 V20 V30 V40 V50 V60 V65 V70 29.7 (7.4) 24.6 (6.8) 20.9 (6.0) 17.9 (5.3) 15.0 (
4.7) 12.2 (3.9) 10.6 (3.9) 8.8 (3.5) Water alone mean (SD) (%) 37.7 31.7 27.2 23
.3 19.7 16.0 14.2 11.5 (10.3) (9.0) (8.0) (7.2) (6.2) (5.4) (5.0) (4.4) p value
0.02 0.02 0.02 0.03 0.03 0.04 0.04 0.07 Absolute difference (%) 8.0 7.1 6.3 5.4
4.7 3.8 3.6 2.7
Abbreviations: RB, rectal balloon; SD, standard deviation.
216

Vargas et al.
should be preserved; and (iii) In intermediate- or highrisk cases, the proximal
seminal vesicles should not saddle around the rectum. If all three criteria are
met, the patient will likely benefit from an RB instead of water alone. DECREASI
NG DOSE TO NORMAL STRUCTURES The physical and biological properties of proton th
erapy are desirable for many reasons (7). In addition to reducing dose to normal
structures through strategic placement of the Bragg peak, protons have a biolog
ical effect similar to photons, and therefore familiar tumoricidal doses and nor
mal tissue dose constraints can be employed (8,9). In the past, radiation doses
have been limited because of the risk of chronic rectal and bladder toxicity (101
9). Proton therapy has been proposed as a means to deliver elevated doses while
limiting potential toxicity to normal surrounding structures (2025). Cella et al.
contrasted IMRT, double-scattered protons, and IMPT; however, only one case was
analyzed and the IMRT and double-scattered proton plans were not actually desig
ned for treatment purposes (26). This could have biased the IMRT plans over the
double-scattered proton plan. In that study, the double-scattered proton therapy
rectal dose-volume curve was superior to the IMRT plan to doses up to 60 GE or
75% of the prescribed dose; beyond this point, however, IMRT had lower doses to
the rectum. For the bladder, the dose-volume curves for proton therapy and IMRT
crossed over twice making interpretation difficult. Zhang et al. published the r
esults for 10 patients treated with IMRT and created 10 companion double-scatter
ed proton plans for retrospective analysis (27). They found that the rectal dose
-volume curves favored proton therapy in the low-dose range but after a dose of
40 Gy, or 50% of the target dose, IMRT achieved a better rectal dose distributio
n. For the bladder dose, they found a similar proton advantage in the lowdose re
gion (<50%) after which both curves basically overlapped. Trofimov et al. found
comparable rectal dose with IMRT or proton therapy (28). Furthermore, they found
lower bladder doses with IMRT at V60 and higher. In contrast to the previous st
udies, we were able to demonstrate statistically significant improvements for re
ctal dose-volume parameters in the low-, intermediate- and high-dose areas as se
en in Figure 4. This translated into a 59% improvement in the rectal mean dose o
ver IMRT (Table 2). For the bladder, the improvement was smaller but nonetheless
translated into a 35% relative decrease in the mean dose (Table 3) and curves s
uggested a benefit up to a dose of 65 GE (Fig. 5). The greater benefit seen in o
ur study is likely because of the independent optimization of dose distribution,
beam angle, and aperture margins for
Figure 2 Line-dose graph for all rectal dose-volume parameters between V10V82 wit
h RB or water alone (n
30). The boxes represent 95% standard error. Abbreviation
: RB, rectal balloon.
5% at V50 was seen in 5 of 15 cases [9.2 2.3% compared to 2.4 1.3% for the remai
nder 10 cases ( p < 0.001)]. For these same five cases, the improvement at 70 Gy
persisted: 5.8 2.2% compared to 1.2 1.8% ( p < 0.001). For the remaining cases,
however, the difference between water alone versus an RB was less than 1% or ev
en had a detrimental effect. Thus, patient selection is critical in realizing th
e role of an RB in proton radiotherapy. We found that best predictor of an RB be
nefit is a sagittal view of the patient on imaging as seen in Figure 3. Specific
ally, we found the following three reference details to be useful in our decisio
n: (i) The posterior rectal wall at the prostate level should be displaced poste
riorly; (ii) If present, the rectal wall-prostate interface space
Figure 3 Sagittal projection of
d with water alone (right). The
rostate is lost with the RB and
patient did not benefit from an

the planning volumetric CTscan with RB (left) an

plane between the anterior rectal wall and the p
no posterior rectal displacement was seen. This
RB. Abbreviation: RB, rectal balloon.

Image-Guided Proton Beam Radiation Therapy for Prostate Cancer

217
Figure 4 Combined rectal dose-volume curves for proton therapy and IMRT (n
20 pl
ans) (error box shows 95% SE). Abbreviations: RB, rectal balloon; IMRT, intensit
y-modulated radiation therapy.
Figure 5 Combined bladder dose-volume curves for proton therapy and IMRT (n 20 p
lans) (error box shows 95% SE). Abbreviation: IMRT, intensity-modulated radiatio
n therapy.
each beam combined with our compensator design. The penumbra and dose homogeneit
y observed in our prostate plans could also play an important role. Since our pr
ostate technique uses lateral oblique beam arrangements, the ability to treat th
e target volume with small margins from planning tumor volume (PTV) to aperture
(block) edge
significantly decreased our dose to surrounding doselimiting normal structures a
s seen in Figure 6. Thus, for our proton plans, the distance of the 98% isodose
line (IDL) to the 80% IDL was 4.4 mm for a 4.5% dose falloff per millimeter in t
he posterior rectal aspect compared to 5.5 mm for our IMRT plans. Although the i
nitial dose
Table 2 Percent Volume of the Rectum Receiving Doses Between 10 GE/Gy to 80 GE/G
y and Mean Dose (n
20 plans) Protons (SD) (%) Rectum Rectum Rectum Rectum Rectum
Rectum Rectum dose V10 V30 V50 V70 V78 V80 mean 29.8 20.7 14.6 7.9 2.9 0.1 14.2
(5.6) (3.9) (3.0) (1.8) (1.2) (0.3) (3.7) GE IMRT (SD) (%) 72.1 55.4 31.3 14.0
5.0 1.8 34.8 (7.6) (5.7) (4.1) (2.9) (1.2)% (1.8) (3.0) Gy p value <0.001 <0.001
<0.001 <0.001 0.01 0.01 <0.001 Relative benefit (%) 58.7 62.7 53.4 43.6 42.0 94
.4 59.2 Absolute benefit (%) 42.3 34.7 16.7 6.1 2.1 1.7 20.1 GE/Gy
Abbreviations: IMRT, intensity-modulated radiation therapy; SD, standard deviati
on.
Table 3 Percent Volume of the Bladder Receiving Doses Between 10 GE/Gy to 80 GE/
Gy and Mean Dose (n 20 plans) Protons (SD) (%) Bladder Bladder Bladder Bladder B
ladder dose V10 V20 V30 V35 mean 36.4 31.4 27.7 26.0 18.4 (13.2) (12.1) (11.1) (
10.6) (6.2) GE IMRT (SD) (%) 60.0 50.8 42.8 38.2 28.4 (20.1) (18.0) (15.1) (13.2
) (9.4) Gy p value 0.007 0.01 0.02 0.04 0.01 Relative benefit (%) 39.3 38.2 35.3
31.9 35.2 Absolute benefit (%) 23.6 19.4 15.1 12.2 10.0
Abbreviations: IMRT, intensity-modulated radiation therapy; SD, standard deviati
on.
218
Vargas et al.
Figure 6 Sagittal (left) and axial (right) projection for the same patient as in
Figure 1 including IDLs with water alone. The green line represents the 50% IDL
that includes less than half the rectal circumference. Abbreviation: IDL, isodo
se line.
falloff is similar between IMRT and proton plans, the gradient beyond this regio
n is where the advantage of proton therapy is most evident: With our proton plan
, the distance between the 80% to the 20% IDL was only 7 to 9 mm in the posterio
r rectal aspect. This is in contrast to the >2 cm falloff seen in an optimized I
MRT plan. With similar target doses of 79.2 GE at Massachusetts General Hospital
(MGH) and 78 GE at out institution, the rectum V50 was 34.4% versus 31.3%, rect
um V70 14.5% versus 14.0%, rectum mean dose 39.4 GE versus 33.2 GE, bladder V50

23.7% versus 25%; bladder V70 11.4% versus 14%; and bladder mean dose 29.9 GE an
d 28.4 GE, respectively. Overall, the doses to normal tissues for the MGH IMRT p
lans and our IMRT plans were similar with minor variations. Since doses to norma
l structures were similar for the different IMRT plans, we have concluded that t
he relative advantage in overall dosimetry is related to the steep gradients ach
ieved with proton therapy.
IMAGE GUIDANCE FOR PROTON TREATMENTS Interfraction prostate motion has been an a
rea of major concern over the last few years in prostate cancer therapy (2934). L
arge variations in daily prostate position have been described in various studie
s (29,30,32,34). Furthermore, studies have found a poor correlation between the
location of the prostate and skin marks or bony anatomy (33,35). Ultrasound syst
ems cannot reliably define the daily prostate position (32,33). As a result, whe
n daily treatment setup is based on skin marks, bony anatomy, or ultrasound guid
ance, large margins are necessary to ensure accurate delivery of the prescribed
dose (30,32,33). Several different approaches have been used in an attempt to so
lve the problem of prostate motion and to reduce treatment margins. Yan et al. u
sed multiple CT scans to define prostate motion and electronic daily
images to quantify setup inaccuracies (34). They were able to derive patient-spe
cific margins through an off-line adaptive process. PTV size was greatly reduced
when compared with margin definition via class solution. A second approach, onl
ine image guidance, relies on daily information to derive the daily prostate pos
ition (30,32,33,35,36). Different approaches have been used including cone-beam
CT, CT-on-rails, fiducial markers, or beacon transponders (29,30,32,33,3539). The
accuracy of these systems relies on the accuracy of the measurement and the app
ropriate patient translation adjustments in relation to the treatment machine is
ocenter. We have adopted the use of Visicoils as fiducial markers in conjunction
with orthogonal X-rays. Excellent spatial resolution is possible. Fiducial disp
lacement is minimal and thus permits a reproducible and reliable strategy for pr
ostate positioning. There are fundamental differences between IMRT and proton th
erapy treatment plans. With IMRT, the confluence of multiple beam segments arran
ged into five to seven beam angle directions create a high-dose area. The moveme
nt of this high-dose area will have small repercussions in the dose and shape of
this high-dose area. Thus, for image guidance, few modifications are necessary
when implementing an IMRT plan. For proton therapy the same is not true. First a
nd foremost, variations in the tissues traversed by the beam path will have larg
e repercussions in the shape and position of the proton high-dose gradient. Seco
nd, variation in the shape of the surface and relative distance changes secondar
y to variable external anatomy will have large impacts in the treatment plan. Th
us, the plan should be modified to account for these variations, but more import
antly, certain beam angles should be avoided. A proton plan should be evaluated
in the light of the movements of the patients external contours, internal organs,
and prostate position. In summary, image-guided proton plans should not be eval
uated on the basis of a single static target position; instead plans should be o
ptimized and evaluated on the basis of the ability of maintaining target coverag
e for various different prostate positions encountered during the course of ther
apy. Practical experience and plan evaluations has shown us that anterior, poste
rior, or lateral oblique beams more than 208 from horizontal should not be emplo
yed for double scattered, uniform scanning, or IMPT for prostate cancer unless d
aily cone-beam CTs can be performed and immediate range variations can be utiliz
ed. Bladder filling or rectal filling will vary from day to day modifying the ra
diological path length of the beam. Therefore, a particular beam can underdose t
he target, and more importantly, increase the dose to the rectum and bladder. Fu
rthermore, since every radiobiology paper for proton therapy has found an increa
sed relative biological effectiveness (RBE) at the end of the beam path, the dos
e-volume histogram
Image-Guided Proton Beam Radiation Therapy for Prostate Cancer

219
(DVH) advantage of a sharp distal Bragg peak falloff may be outweighed by a 10%
to 30% increase in the RBE at the beams distal margin. Another reason we generall
y avoid anterior and posterior beam arrangements is because the variability of t
he stopping power can affect the distal range for any given beam. Each beam util
ized should have an additional proximal and distal margin to account for stoppin
g power variability. Thus, a target volume that should stop at the surface of th
e rectum may need to extend a few millimeters into the rectum, thereby increasin
g the rectal dose. Furthermore, if daily prostate depth cannot be strictly maint
ained, the distal edge of the beam will have to take into account potential vari
ations in prostate depth. The variability in prostate position from day to day a
nd during each fraction will mandate that the distal margin of the beam is exten
ded even further into the rectum. For our institutional image-guided approach, w
e use a distal and proximal edge margin between 5 and 9 mm as defined by the dis
tance between the 98% IDL and the PTV. Alternately, a general formula [(target d
istance 0.018) 2 mm] can be employed. We chose to use both the formula and IDL a
s a reference because we found variations in the distance to the distal edge are
relatively small for our prostate patient population. However, larger variation
s can be seen because of bone density differences and cortical thickening across
the PTV that cannot be completely characterized by a general formula for indivi
dual patients. In employing the IDL reference and the formula we can use the ave
rage calculated distance [(25 cm 0.018) 2 mm] and customize it for the individua
l patient, adding additional margin when necessary; ultimately, our minimal dist
al or proximal margin is 5 mm. Our calculations in water phantoms have demonstra
ted excellent agreement (2%) with our predicted beam profiles. Our approach to s
mearing has been also based on the objectives of our image guidance approach. Sm
earing is an expansion of the each voxel perpendicular to the beam angle to acco
mmodate for variation in target position and/or depth. The smearing margin was c
onstructed based on the following formula: q
TM between fractions, and to a lesser extent, intrafraction motion. The distal
target position averages 25 cm with an additional 2 mm compensator thickness at
the point of maximum depth. No modifications were made for setup error since wit
h image guidance the residual setup error at our institution has been measured t
o be 0.3 mm (y), 0.15 mm (x), and 0.03 (z) mm. The resulting radius will be 18.8
mm. For
practical purposes, the smearing margin will allow for an image-guidance approac
h equal to the smearing minus the residual error of the image-guided approach. P
roton radiation dose distribution relies more on tissue stopping power character
istics than IMRT. The depth of each individual Bragg peak, and therefore the res
ulting SOBP, depends on the energy of the beam and its interaction with tissue (
40). As previously mentioned, the SOBP position will vary if changes occur in th
e proton stopping power of the tissues along the beam path. This raises the ques
tion of whether daily image-guided beam realignment can be safely implemented in
proton therapy to account for physiological inter- and intrafraction target dis
placement from original simulation (41). Specifically, if the beam is realigned
relative to a new target position, the beam path will likely include different b
ony and soft tissue structures. Changes in prostate dose with defined changes in
prostate position are important in developing treatment strategies for proton t
herapy. For our treatments, we prescribe a minimum dose to the PTV of 74.1 GE an
d prescribe 78 GE to cover 95% of the PTV. Thus, the doses within the PTV are no
t completely homogeneous, and different clinical target volume (CTV) positions w
ithin the PTV may have different minimum doses and prescribed dose coverage. As
a result, we may face a fundamental paradox when we use image guidance for proto
n therapy: In attempting to deliver a more accurate radiation dose, is it possib
le that we compromise target coverage through realignment of proton beam positio
ns? For the purpose of answering this question, we quantified CTV coverage for s
everal prostate cancer patients on the basis of the initial plan compared with d
ifferent target CTV locations. To fully evaluate the differences of dose distrib

ution inside the PTV and outside the PTV, we repositioned the prostate employing
unidirectional vectors of 5 and 10 mm, and multidirectional vectors of 10 mm. O
ur data for alignment with motion within the PTV are shown in Table 4. With beam
realignment, target coverage increased, and the entire CTV received the prescri
bed dose. Even without beam realignment, however, coverage greater than or equal
to 99% was seen regardless of the direction of the vector of motion, when displ
acements were within the PTV expansion. The minimum prostate dose was decreased
by a maximum of only 1.6 GE. This difference, although statistically significant
, is likely not clinically relevant (Fig. 7). These findings suggest that small
intrafraction motions accounted for in the PTV expansion will have negligible im
pact on dose delivered to the prostate with proton therapy. Our data for alignme
nt with motion outside the PTV is shown in Table 5. Moderate changes in coverage
and minimum CTV dose were observed in the absence of
220 Table 4 Prostate (CTV) Coverage With and Without Image Guidance for CTV Move
ments Within the PTV (5 mm) No image guidance (SD) 5-mm anterior Prostate V78 (%
) Prostate minimum 5-mm inferior Prostate V78 (%) Prostate minimum 5-mm posterio
r Prostate V78 (%) Prostate minimum 5-mm superior Prostate V78 (%) Prostate mini
mum 99.6% (0.5)% 76.52 (1.17) GE 99.6% (0.5)% 78.03 (0.34) GE 99.4% (0.8)% 76.75
(1.49) GE 99.9% (0.02)% 78.10% (0.30) GE Image guidance (SD) 100% (0.03)% 78.15
(0.27) GE 100% (0.03)% 78.19 (0.23) GE 100% (0.007)% 78.29 (0.30) GE 100% (0.00
4)% 78.27 (0.32) GE p value 0.04 0.001 0.04 0.3 0.05 0.008 0.2 0.3
Vargas et al.
dose
dose
dose
dose
Abbreviations: CTV, clinical target volume; PTV, planning tumor volume; SD, stan
dard deviation.
Figure 7 Dose-volume curves for 5 mm prostate shifts with (~ . &) and without (
) beam realignment. The curves for all prostate positions overlap each other, re
gardless of whether beam realignment was utilized.
Table 5 Prostate (CTV) Coverage With and Without Image Guidance for CTV Movement
s Outside the PTV (10 mm) No image guidance 10-mm inferior Prostate V78 (%) Pros
tate minimum dose 10-mm posterior Prostate V78 (%) Prostate minimum dose 10 mm s
uperior Prostate V78 (%) Prostate minimum dose 96.5% (1.2)% 72.47 GE (0.90) GE 8
9.8% (3.9)% 64.75 GE (5.90) GE 94.4% (2.0)% 72.78 GE (0.70) GE Image guidance 10
0% (0.1)% 78.07 GE (0.27) GE 100% (0.1)% 78.31 GE (0.53) GE 100% (0.3)% 78.28 (0
.41) GE p value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
Image-Guided Proton Beam Radiation Therapy for Prostate Cancer
221
Figure 8 Dose-volume curves for 10 mm prostate shifts with (~ and high target co
verage are seen with beam repositioning.
.) and without ( ) beam realignment. Moderate improvements
beam realignment. Clinically and statistically significant improvements in CTV c
overage were seen with imageguided beam realignment (Fig. 8). Coverage was 99.9%
or better with beam realignment, and the minimum dose to the CTV was similar to

the initial plan. These findings suggest that beam realignment is necessary for
target displacement greater than accounted for by the PTV expansion, but that t
he impact of minor changes in the length and composition of the proton beam path
are likely accounted for using the smearing approach utilized in our treatment
planning protocol.
Prostate displacements of 10 mm in the three axes are often seen with both inter
- and intrafraction prostate motion. Our data regarding 10 mm multidirectional t
arget displacement is seen in Table 6, which characterizes changes in dosimetry
related to CTV position, with and without beam realignment. As illustrated, sign
ificant changes in mean, minimum, and maximum dose, as well as overall CTV cover
age are noted without image-guided beam realignment (Fig. 9). CTV coverage impro
ved considerably with beam realignment for most displacements.
Table 6 Prostate (CTV) Coverage With and Without Image Guidance for CTV Movement
s Compounding Inter- and Intrafraction Movements No image guidance Point A Prost
ate Prostate Prostate Point B Prostate Prostate Prostate Point C Prostate Prosta
te Prostate Point D Prostate Prostate Prostate V78 (%) mean dose minimum dose V7
8 (%) mean dose minimum dose V78 (%) mean dose minimum dose V78 (%) mean dose mi
nimum dose 83.56% (4.7) % 78.48 GE (0.39) GE 52.92 GE (4.89) GE 85.57% (3.3) % 7
8.66 GE (0.31) GE 54.34 GE (4.57) GE 82.6% (4.2) % 78.39 GE (0.41) GE 52.19 GE (
5.58) GE 86.53% (3.9) % 78.73 GE (0.42) GE 54.93 GE (4.47) GE Image guidance 98.
49% (2.8) % 79.51 GE (0.34) GE 77.59 GE (1.27) GE 90.16% (23.5) % 79.28 GE (0.38
) GE 77.15 GE (0.77) GE 99.2% (1.9) % 79.57 GE (0.29) GE 77.54 GE (1.09) GE 97.3
9% (3.4)% 79.31 GE (0.36) GE 76.60 GE (0.83) GE p value <0.001 <0.001 <0.001 <0.
001 0.002 <0.001 <0.001 <0.001 <0.001 <0.001 0.006 <0.001
222
Vargas et al.
Figure 9 Dose-volume curves for 10 mm prostate shifts with (~ . &) and without (
) beam realignment. Large improvements are seen including large shifts in for t
he DVH curve with beam realignment. Abbreviation: DVH, dose-volume histogram.
Minimum CTV dose with realignment was better or equal to 77 GE (>98%) for all mu
ltidimensional shifts. Greater benefits were documented when 10 mm changes in CT
V position were made in the superior, inferior, or posterior dimension. No trans
lations of 10 mm were performed anteriorly as they would extend the CTV unrealis
tically inside the symphysis pubis for all cases studied. These larger translati
onal vectors may better characterize the potential variations in prostate positi
on that can be encountered after the initial laser-based patient alignment for p
rostate cancer therapy. As seen in Table 3, without image guidance, moderate cha
nges in CTV V78 and minimum CTV dose were observed. However, with beam realignme
nt, we demonstrated clinically relevant and statistically significant improvemen
ts in CTV V78 and minimum CTV dose. According to multiple studies showing that c
hanges of 8 Gy or higher will have a significant impact on prostate cancer outco
mes, this cutoff was used for defining clinically relevant changes (18,21,4246).
Although large changes were seen in CTV V78 and CTV minimum dose, the maximum CT
V dose and mean dose did not change substantially when compared with the initial
CTV. This finding questions the utility of mean dose and maximum dose in descri
bing changes resulting from CTV positional variation. A difficult problem arises
when we try to quantify improvements in dose distribution for motion in the vec
tor of the proton beam direction (47). If the lateral change is because of setup
error, the beam will not need to be realigned as the depth for the dose deposit
ion will not vary and will cover the target adequately. In other words, if the p
rostate position changes in the beam direction in relation
to the isocenter without varying its position within the pelvis, the beam will t
raverse a similar distance, and no variation in dose coverage location should be

seen. However, if the lateral shift is within the pelvis, changes in the CTV po
sition and variation in depth will be accompanied by corresponding changes in th
e proton path length. Therefore, the dose-deposition area may miss the CTV. Scha
llenkamp et al. described the relationship between bony anatomy and prostate mot
ion (35). Although they found that prostate motion was independent of bony align
ment (setup position) for most translational vectors, in the lateral direction s
etup corrections may improve target localization. Nederveen et al. also found th
at bony anatomy setup correction can decrease the error of the prostate position
in the lateral direction by half (48). Related to this, Van Herk et al. found m
inimal lateral prostate motion in relation to the pelvis with cone-beam CT (0.9
mm SD) (49). Thus, although lateral prostate shifts are possible, they are mostl
y related to changes in position of the whole pelvic anatomy, which will not aff
ect the proton beam depth deposition, meaning that most motion relevant to chang
es in dose distribution for proton prostate therapy will be described by shifts
that are in the superior-inferior (SI) or anterior-posterior (AP) direction rath
er than the lateral direction. For this reason, it has been proposed to employ P
TV margins that are dependent on the beam angle rather than anisotropic CTV expa
nsions. To quantify the impact of movement in the lateral direction and other po
tential changes resulting from interand intrafraction motion, we quantified CTV
dose-volume variations with shifts of 10 mm in the three directions.
Image-Guided Proton Beam Radiation Therapy for Prostate Cancer
223
Although, as mentioned earlier, lateral shifts of the prostate in relation to pe
lvic anatomy will rarely reach 10 mm, we used this degree of displacement to mim
ic coverage for the potential worst-case scenario (35,48,49). For points A to D
large changes in CTV V78 and minimum dose were seen. With beam realignment, we a
chieved clinically significant benefits in CTV V78 for all prostate positions. F
or points A, C, and D, CTV V78 was better or equal to 97%. Even for point B, whe
re the CTV V78 was only improved to 90%, CTV minimum dose was 77.2 GE. In other
words, although 10% of the CTV was not covered by the prescribed dose, the entir
e CTV did receive at least 99% of the prescribed dose. Image guidance of daily t
reatment is theoretically possible with proton therapy. With appropriate target
volume delineation, identification and quantification of uncertainties in treatm
ent planning, and appropriate dose prescription, image guidance can be used to m
aximize the benefits of improved dose distributions achievable with proton thera
py. ACTION LEVEL THRESHOLD FOR IMAGE GUIDANCE Accurate prostate localization for
prostate cancer treatment can improve cure rates and minimize doses to doselimi
ting normal structures (5054). More reliable prostate localization will permit th
e delivery of higher doses to the target, and through tighter margins, reduce th
e normal tissue exposed to intermediate and high doses (5558). Target margins to
account for variations in the actual prostate position have been defined for dif
ferent image guidance systems (32,33,59). Serago et al. compared image guidance
systems with fiducial-based megavoltage (MV) imaging (33). Since MV images are p
roduced by the beam itself, they represent the true position of the markers in r
elation to the treatment axis. This method is limited by accurate representation
of the crosshairs with respect to the true machine isocenter and gantry-specifi
c isocenter variations with different beam angles ( 2 mm). Disagreements between
the locations of reference fiducials have been observed between ultrasound posi
tion and MV position of 7.0 4.6 mm, and kilovoltage (kV) imaging and MV position
of 1.9 1.5 mm. Furthermore, an additional disparity of 1.9 1.2 mm has been repo
rted between the Calypso system and kV images that theoretically should actually
be added to the aforementioned MV and kV errors (60). Cone-beam CT has also sho
wn differences between MV and CT information with linear vectors of 1.05 to 1.0 0
.58 to 1.29 mm (61). When evaluating an image guidance system, it is important t
o identify the limitations of the system in applying corrections. After image gu
idance, the average prostate position

variation should be close to zero and the residual difference will define the ac
curacy of the whole system including its systematic errors (62). In our prostate
cases, the kV images are acquired via an orthogonal pair X-ray system where one
of the sources is within the nozzle, representing the beams eye view. Images are
acquired in-line with each beam prior to treatment, reducing disagreement arisi
ng from variations in gantry isocenter diameter, intrafraction error, and error
in repositioning the patient. For our treatments, orthogonal pairs are acquired
and single or multiple shifts based on action level threshold (ALT) of 2.5 mm in
all three axes are performed as necessary. Since our verification films are tak
en at the patients final treatment position and the X-ray is directly along the b
eam line, no correction for couch errors or incongruence within the imaging syst
em is necessary. Our process, therefore, has accuracy similar to a MV image for
each field. In a recent study to confirm the feasibility and performance of our
prostate localization strategy, a total of 772 treatments were delivered and 311
0 images acquired and analyzed employing the methods described by van Herk (6365)
and Yan et al. (6669). Patients were treated for every fraction using image guid
ance and an ALT of 2.5 mm. No treatment was delivered without reaching and confi
rming a prostate position less than 2.5 mm from the predetermined ideal. To reac
h our ALT, most patients required only zero to one corrections and no patient re
quired more than three corrections (Table 7). The high probability of a true tra
nslation (prostate in stable position) within our action level (2.5 mm) implies
that the intrafraction error seen over the 3 to 4 minutes required for the corre
ction and verification is smaller than the ALT. If the ALT was smaller than the
intrafraction error for the same time period, the overall number of corrections
necessary would have been higher and directly proportional to the difference. In
the same study, the average error for the AP dimension (z), SI dimension (y), a
nd right to left dimension (x) was close to 0 (Tables 810). The margins for each
patient were very heterogeneous and ranged between 0.5 mm to 2.2 mm (p < 0.001).
As proposed by Van Herk, the theoretical mean error for the preparation should
approach zero. Furthermore, any disagreement with zero will describe the systema
tic error of the approach (70). Figures 10 to 13 show the final prostate positio
n in relation to isocenter. They illustrate positional errors for each individua
l patient (Figs. 10 and 12) and for all cases (Figs 1113). The small intrafractio
n error found our study is in agreement with published literature based on multi
ple X-rays or cinefluoro. For a study involving longer treatment durations, Kott
e et al. identified small intrafraction errors that did not require corrections
between treated fields (71).
224 Table 7 Number of Corrections Needed Within Action Level Threshold of 2.5 mm
for 20 Patients and All Final 772 Prostate Positions 2.5-mm action level Patien
t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Total Fractions 39 38 38 39
35 38 39 39 37 41 37 39 41 37 41 39 41 36 39 39 0 corrections 10.3 7.9 5.3 0.0
2.9 0.0 10.3 0.0 8.1 0.0 8.1 2.6 4.9 0.0 0.0 53.8 51.2 0.0 5.1 0.0 8.7 (67/772)
1 correction 89.7 92.1 89.5 89.7 94.3 94.7 79.5 92.3 81.1 97.6 78.4 87.2 85.4 94
.6 73.2 41.0 43.9 83.3 74.4 84.6 82.1 (634/772) 2 corrections 0.0 0.0 5.3 10.3 2
.9 5.3 7.7 7.7 10.8 2.4 10.8 7.7 7.3 2.7 26.8 5.1 4.9 13.9 17.9 15.4 8.3 (64/772
) 3 corrections 0.0 0.0 0.0 0.0 0.0 0.0 2.6 0.0 0.0 0.0 2.7 2.6 2.4 2.7 0.0 0.0
0.0 2.8 2.6 0.0 0.9 (7/772)
Vargas et al.
Table 8 Final Prostate Position After All Corrections With Online Image Guidance
for the Anterior-Posterior (z) Axis With an Action Level of 2.5 mm for 20 Patie
nts and All 772 Final Prostate Positions 2.5-mm action level threshold Patient 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 All cases
a b
Mean (SD) cm 0.004 (0.061) 0.021 (0.063) 0.017 (0.060) 0.003 (0.071) 0.012 (0.067) 0
.002 (0.072) 0.018 (0.103) 0.011 (0.104) 0.015 (0.093) 0.020 (0.070) 0.003 (0.134) 0
.018 (0.062) 0.001 (0.098) 0.008 (0.078) 0.019 (0.136) 0.058 (0.110) 0.028 (0.087)

0.019 (0.126) 0.012 (0.120) 0.005 (0.081) 0.003 (0.094)

Range (cm) 0.330 0.260 0.320 0.370 0.270 0.370 0.430 0.430 0.450 0.290 0.460 0.2
80 0.440 0.380 0.470 0.470 0.360 0.450 0.450 0.410 0.470
Marginsa 0.0527 0.0966 0.0845 0.0572 0.0769 0.0554 0.1171 0.1003 0.1026 0.0990 0
.1013 0.0884 0.0711 0.0746 0.1427 0.2220 0.1309 0.1357 0.1140 0.0692 0.0733
Marginsb 0.120 0.123 0.118 0.139 0.131 0.141 0.202 0.204 0.182 0.137 0.263 0.122
0.192 0.153 0.267 0.216 0.171 0.247 0.235 0.159 0.184
2.5S 0.7 @. 95% of prostate positions for all cases. Abbreviation: SD, standard
deviation.
Image-Guided Proton Beam Radiation Therapy for Prostate Cancer Table 9 Final Pro
state Position After All Corrections With Online Image Guidance for the Superior
-Inferior (y) Axis With an Action Level of 2.5 mm for 20 Patients and All 772 Fi
nal Prostate Positions 2.5-mm action level threshold Patient 1 2 3 4 5 6 7 8 9 1
0 11 12 13 14 15 16 17 18 19 20 All cases
a b
225
Mean (SD)
011 0.032
) (0.052)
) (0.076)

cm 0.002 0.029 0.031 0.014 0.015 0.023 0.023 0.042 0.041 0.035 0.068 0.
0.005 0.028 0.024 0.040 0.059 0.055 0.050 0.028 (0.052) (0.064) (0.068
(0.081) (0.046) (0.093) (0.058) (0.094) (0.082) (0.080) (0.036) (0.060
(0.041) (0.075) (0.097) (0.053) (0.089) (0.064) (0.073)

Range (cm) 0.270 0.400 0.320 0.220 0.390 0.260 0.430 0.240 0.380 0.350 0.420 0.1
60 0.280 0.360 0.230 0.330 0.430 0.280 0.360 0.280 0.430
Marginsa 0.0414 0.1173 0.1251 0.0714 0.0942 0.0897 0.1226 0.1456 0.1683 0.1449 0
.2260 0.0527 0.1220 0.0657 0.0987 0.1125 0.1679 0.1846 0.1998 0.1698 0.1211
Marginsb 0.102 0.125 0.133 0.102 0.159 0.090 0.182 0.114 0.184 0.161 0.157 0.071
0.118 0.149 0.080 0.147 0.190 0.104 0.174 0.125 0.143
2.5S 0.7 @. 95% of prostate positions for all cases. Abbreviation: SD, standard
deviation.
Furthermore, Nederveen et al. have found small variations in prostate position o
f only 0.3 0.5 mm and 0.4 0.7 mm during 3 to 4 minutes employing cine-MV (7274). B
ecause of the short treatment times (7080 seconds) for our prostate cancer treatm
ents, the total time between the last verification X-ray and treatment completio
n is 3 to 4 minutes. In this setting, an accurate image guidance and treatment s
ystem theoretically allows very small margins. PELVIC RADIATION AND PROTON THERA
PY Situations arise in high-risk prostate cancer where a clinician may feel comp
elled to irradiate the adjacent lymphatics. We compared seven-field IMRT to the
pelvic lymph nodes (LNs) and prostate with a three isocenter, six-field double-s
cattered proton plan. Significant reductions in dose were seen for the rectum, b
ladder, and small bowel favoring the proton plan. Furthermore, the LN target dos
e of 46 GE/Gy was delivered in 23 fractions for the proton treatments while it w
as delivered over 39 fractions with IMRT. Although it is possible to have two pl
ans where the LN and prostate are treated with an IMRT plan followed by a proton
boost, the dose with IMRT to a
central target (i.e., prostate) will still receive a higher dose per fraction th
an a peripheral target (i.e., LN). As can be seen in Figures 1416, doses to rectu
m, bladder, and small bowel can be decreased employing proton therapy. The impro
vement is larger for pelvic radiation than when treating the prostate alone. How

ever, the integration of image guidance for pelvic radiation is not well defined
. The movement of the LN vasculature in relation to the prostate and pelvic stru
ctures remains unclear. INTENSITY-MODULATED PROTON THERAPY Most IMPT plans seen
in the literature show an advantage for IMPT over double-scattered proton therap
y. Cella et al. found a substantial improvement with IMPT over proton therapy (7
5). However, multiple beams angles were employed including angles that used the
distal falloff to decrease doses to the rectum and bladder. Since these angles c
annot be used for an image-guided approach, the results are not practical in our
current setting. Trofimov likewise found a small advantage with IMPT over doubl
escattered proton therapy (76). In this study, since the angles used for IMPT we
re similar to the angles employed
226 Table 10 Final Prostate Position After All Corrections With Online Image Gui
dance for the Left to Right (X) Axis With an Action Level of 2.5 mm for 20 Patie
nts and All 772 Final Prostate Positions 2.5-mm Action level threshold Patient 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 All cases
a b
Vargas et al.
Mean (SD) cm 0.017 0.041 0.033 0.005 0.009 0.036 0.022 0.015 0.053 0.000 0.013 0.004 0
.004 0.016 0.028 0.038 0.058 0.027 0.023 0.023 0.013 (0.063) (0.072) (0.063) (0.070) (
0.049) (0.056) (0.080) (0.071) (0.077) (0.064) (0.066) (0.049) (0.071) (0.065) (
0.060) (0.093) (0.088) (0.079) (0.091) (0.063) (0.074)
Range (cm) 0.340 0.260 0.340 0.270 0.180 0.260 0.410 0.310 0.320 0.320 0.290 0.2
20 0.280 0.230 0.310 0.370 0.360 0.370 0.460 0.270 0.460
Marginsa 0.0866 0.1529 0.1266 0.0615 0.0568 0.1292 0.1110 0.0872 0.1864 0.0448 0
.0787 0.0443 0.0597 0.0855 0.1120 0.1601 0.2066 0.1228 0.1212 0.1016 0.0843
Marginsb 0.123 0.141 0.123 0.137 0.096 0.110 0.157 0.139 0.151 0.125 0.129 0.096
0.139 0.127 0.118 0.182 0.172 0.155 0.178 0.123 0.145
2.5S 0.7 @. 95% of prostate positions for all cases. Abbreviation: SD, standard
deviation.
Figure 10 Final prostate positions for each case in the superior to inferior (y)
and anterior to posterior (z), curves correspond to the 95% CI for each case.
Figure 11 Final prostate positions for all cases in the superior to inferior (y)
and anterior to posterior (z). Curves correspond to the 95% CI for all cases an
d histograms are seen in the border.
Image-Guided Proton Beam Radiation Therapy for Prostate Cancer
227
Figure 12 Final prostate positions for each case in the superior to inferior (y)
and anterior to posterior (z). Curves correspond to the 95% CI for each case.
Figure 14 Mean relative rectal dose for IMRT and proton therapy. Square boxes re
present the standard error. Abbreviation: IMRT, intensity-modulated radiation th
erapy.
Figure 15 Mean relative bladder dose for IMRT and proton therapy. Square boxes r
epresent the standard error. Abbreviation: IMRT, intensity-modulated radiation t
herapy.
Figure 13 Final prostate positions for all cases in the superior to inferior (y)

and anterior to posterior (z). Curves correspond to the 95% CI for all cases an
d histograms are seen in the border.
for double-scattered proton therapy, it is likely that the advantage in rectal a
nd bladder dose was because of a sharper penumbra. Supporting this, their IMPT D
VH curves are similar to the DVH curves obtained in our institution with doublescattered proton therapy. The penumbra in the lateral aspect of the beam is rela
ted to the scatter. Theoretically, an IMPT beam can achieve slightly better late
ral dose profile than a double-scattered beam but the lateral penumbra is primar
ily governed by scatter found as the result of the depth, the composition of tis
sues traversed, and inherent physical characteristics of a
Figure 16 Mean small bowel absolute volume for IMRT and proton therapy. Square b
oxes represent the standard error. Abbreviation: IMRT, intensity-modulated radia
tion therapy.
228
Vargas et al.
particle. Thus, a well-done double-scattered plan should have a similar lateral
dose profile as an IMPT plan employing similar beam arrangements. The benefit of
IMPT may translate into a slightly sharper lateral falloff and a superior confo
rmality index. If an image-guided approach and a smearing algorithm are employed
with IMPT, the conformality index will be similar to a doublescattered image-gu
ided plan for the prostate alone. However, IMPT may be superior when complex sem
inal vesicle shapes are encountered or the clinical situation mandates the inclu
sion of at-risk pelvic LNs into our target. In this case, IMPT will be superior
because it allows customization of both the distal and proximal edges, rather th
an just the distal edge alone (as currently possible with double scattering).
sites. These concepts can be further investigated for other treatment sites. REF
ERENCES
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ose escalation study of image-guided adaptive radiotherapy for prostate
SUMMARY OF PERTINENT CONCLUSIONS
l
l
l
l
l
l
l
l
Protons sharp distal falloff of dose is potentially a two-edged sword. On one han
d, it allows the delivery of dose to the tumor while completely sparing distal t
issues. On the other hand, an underestimation/overestimation of the path length
for any reason could cause overshoot/undershoot of the beam with consequent comp
lete miss of a distal portion of the tumor or irradiation of normal tissue to th
erapeutic doses. Inter-/Intrafraction motion, setup variations, bladder and rect
al filling, weight loss, uncertainties in CT numbers, and stopping powers derive
d from approximations of dose computation models, especially in the presence of
complex heterogeneities, have far more consequences in the treatment of prostate
cancer with proton therapy than conventional radiation therapy. Understanding o
f the dose-deposition characteristics seen with proton therapy and the potential
areas of variability of the high-dose gradient area are sources for error and o
pportunity with correct implementation. The sharp dose falloff of the lateral as
pect of the beam allows lower doses to the rectum and bladder in properly design
ed treatment plans. Image guidance is possible with proton therapy. However, spe
cial considerations are necessary. Proton systems are highly accurate allowing f
or very small margins within an image-guided process. Complex treatment plans an
d beam arrangements can be optimized for the treatment of large, complex shape s
tructures as seen for nodal pelvic proton therapy. The concepts explained in thi
s chapter outline our efforts to understand potential uncertainties in our treat
ment preparation and execution. The problems and solutions outlined are not uniq
ue to prostate cancer but common to all nonfixed anatomy cancer
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19
The Testing of Prostate IGRT in Clinical Trials
VINCENT S. KHOO
Urology Oncology Unit, Royal Marsden NHS Foundation Trust and Institute of Cance
r Research, Chelsea, London, U.K.
DAVID P. DEARNALEY
Academic Department of Radiotherapy, Institute of Cancer Research and Royal Mars
den NHS Foundation Trust, Sutton, Surrey, U.K.
HISTORY OF CLINICAL TRIALS FOR RADIOTHERAPY TECHNOLOGY Testing of radiotherapy t
echnology is as old as the field of radiation itself. However, the application o
f formal clinical trials in radiotherapy technology is a relatively newer activi
ty compared with the numerous clinical studies undertaken in this arena. Part of
the issue lies in the defining what is meant by testing within clinical studies v
ersus clinical trials. The term clinical study is a relatively loose and a general i
diom that is often used to group any form of clinical or technological assessmen
t and evaluation. It does not necessarily encompass the methodological rigors re
quired for a formal clinical trial. Clinical studies can include a large spectru

m of assessments that range from case reports to feasibility studies or retrospe

ctive reviews to prospective but noncomparative assessments. Critically, such cl
inical studies may be flawed by sources of data uncertainty such as systematic e
rrors or biases and random errors. These sources of data uncertainty can severel
y limit the strength of the evidence produced by the study and restrict the vali
dity of the studys outcomes.
233
A clinical trial should have formal methods to address and minimize these source
s of systematic and random uncertainty to ensure appropriate validity of the tri
al results. These sources of uncertainty will be discussed in more detail in sec
tion Systematic and Random Errors in Trial Design. It is important for investiga
tors to understand that these sources of study error can be controlled by approp
riate and proper trial design but cannot be accounted for subsequently by statis
tical analysis alone. Thus, it is critical to have a well-designed clinical tria
l that addresses these issues at the outset to ensure authenticity of trial outc
omes. It is also important to consider the motivations involved in undertaking c
linical research and subsequently in performing clinical trials in radiotherapy.
The incentive to carry out clinical radiotherapy research is to discover approp
riate evidence and to provide relevant information for the project. Good clinica
l research is about asking important and well-defined questions and obtaining re
liable answers. This is an opportunity to assess current or new therapies, to co
mpare and contrast different image-guided strategies, and to provide new directi
ons in research or strategies. A clinical trial is one of the means whereby the
value or effectiveness of a new radiotherapy
234
Khoo and Dearnaley
strategy such as image-guided radiation therapy (IGRT) can be established. Clini
cal trials should be prospective and controlled to provide results that can be v
alidated. It should provide evidence produced with scientific rigor to rationali
ze decision making rather than merely plausible reasoning to support the studys c
onclusions. It is evident that there have been numerous advances made in medicin
e and radiation therapy without conducting formal clinical trials. This is usual
ly possible when the treatment effects, differences, or advantages are very larg
e, and when these treatment effects remain significant despite the potential pre
sence of bias and uncertainty when using less than formal methods of assessment.
Good examples of this in medicine include the introduction of penicillin and in
radiotherapy technology, the application of cross-sectional imaging information
using computed tomography (CT) for radiotherapy treatment planning. Other advan
ces in radiotherapy technology include the replacement of cobalt-60 and betatron
machines by computer-controlled linear accelerators (Linacs) and the use of mul
tileaf collimators (MLCs). The use of CT together with Linac hardware developmen
ts, such as MLCs and electronic portal imaging devices (EPIDs), as well as the c
omputer revolution has led to the foundation of modern radiotherapy. The next lo
gical technological development for precision radiotherapy is the introduction o
f image-guided strategies. It is evident that previous technological advances in
radiotherapy have been undertaken in numerous small steps but few, if any, of t
hese steps have been formally evaluated within randomized controlled trials to f
ully justify its clinical value or have been deemed to have achieved level 1 evi
dence as judged by modern contemporaneous criteria used in evidence-based medici
ne (EBM) (1). In brief, EBM is the conscientious, judicious, and explicit use of
current best clinical evidence in making decisions regarding the management of
patients. EBM involves integrating the best available external clinical evidence
from systematic research with individual or peer group expertise and incorporat
es the process whereby different types of clinical evidence are categorized and
Table 1 Evidence-Based Medicine: Levels of Evidence Level schema Level I Level I
I-1 Level II-2 Level II-3 Level III

Source: From Ref. 2.

ranked according to the strength of the trials/studies that are free from the va
rious biases, which may limit their findings. In this hierarchy of evidence-base
d levels, level 1 is the strongest evidence level for a particular therapeutic i
ntervention and is provided by systematic review of appropriate randomized compa
rative trials. There are several systems in use, and they are based on similar p
rinciples. Two common examples of EBM systems are provided from the United State
s (Table 1) and from the United Kingdom (Tables 2A and 2B) describing their evid
ence categorization (2,3). In the history of clinical trials for radiotherapy te
chnology, one important historical example is the use of CT in radiotherapy. It
is fair to say that the integration of CT has revolutionized radiotherapy practi
ce, but yet there have been no randomized clinical trials defining its introduct
ion in radiotherapy. Its relative value in radiotherapy has been inferred from t
he many studies undertaken when CT was initially introduced in the diagnostic fi
eld. Some examples of clinical studies defining the utility of CT in both diagno
stic imaging and radiotherapy practice are illustrated below and some of the lim
itations of these studies are discussed. One of the earliest imaging studies of
CT assessed the detection of brain metastasis by comparing the standard imaging
method used at that time which was radionuclide scanning with CT (4). This clini
cal study reported similar diagnostic rates using radionuclide brain scanning an
d contrast enhanced CT. However, only just over half (53%) of the patients in th
is 47-patient study underwent both radionuclide scanning and CT. Obvious limitat
ions of this study are that not all cases were imaged with both imaging modaliti
es, the small patient cohort, and potential patient selection bias in recruitmen
t. In this initial setting to define the utility of CT for the detection of brai
n metastasis, it is clear that a larger better-designed comparative study was ne
eded. Subsequently, a National Cancer Institute (NCI) multicentre study of 2928
patients compared radionuclide brain scanning with CT, plain radiography, and an
giography (5). This NCI study also evaluated the relative comparative ability to
diagnose intracranial
Description of U.S. Preventive Services Task Force This would arise from evidenc
e obtained from at least one properly designed randomized controlled trial. This
would arise from evidence obtained from well-designed controlled trials without
randomization. This would arise from evidence obtained from well-designed cohor
t or case-control analytic studies, preferably from more than one center or rese
arch group. This would arise from evidence obtained from multiple time series wi
th or without the intervention. Dramatic results in uncontrolled trials might al
so be regarded as this type of evidence. This would arise from opinions of respe
cted authorities, based on clinical experience, descriptive studies, or recommen
dations from expert based committees.
The Testing of Prostate IGRT in Clinical Trials Table 2A Oxford Center for Evide
nce-Based Medicine Levels of Evidence (May 2001) Differential diagnosis/ symptom
prevalence study SR (with homogeneitya) of prospective cohort studies
235
Therapy/prevention, Level etiology/harm 1a SR (with homogeneitya) of RCTs
Prognosis SR (with homogeneitya) of inception cohort studies; CDRb validated in
different populations Individual inception cohort study with !80% follow-up; CDR
b validated in a single population
Diagnosis SR (with homogeneitya) of level 1 diagnostic studies; CDRb with 1b stu
dies from different clinical centers Validatingj cohort study with goodh referen
ce standards; or CDRb tested within one clinical center Absolute SpPins and SnNo
utsg SR (with homogeneitya) of level >2 diagnostic studies

Economic and decision analyses SR (with homogeneitya) of level 1 economic studie

s
1b
Individual RCT (with narrow confidence intervalc)
Prospective cohort study with good follow-upl
1c
All or noned
All or none case series SR (with homogeneitya) of either retrospective cohort st
udies or untreated control groups in RCTs Retrospective cohort study or follow-u
p of untreated control patients in an RCT; derivation of CDRb or validated on sp
lit samplef only Outcomes research
All or none case series SR (with homogeneitya) of 2b and better studies
2a
SR (with homogeneitya) of cohort studies
Analysis based on clinically sensible costs or alternatives; systematic review(s
) of the evidence; and including multiway sensitivity analyses Absolute better-v
alue or worse-value analysesi SR (with homogeneitya) of level >2 economic studie
s
2b
Individual cohort study (including lowquality RCT; e.g., <80% follow-up)
Exploratoryj cohort study with goodh reference standards; CDRb after derivation,
or validated only on split samplef or databases
Retrospective cohort study, or poor follow-up
2c 3a
3b
Outcomes research; ecological studies SR (with homogeneitya) of case-control studi
es Individual case-control study
Ecological studies SR (with homogeneitya) of 3b and better studies Nonconsecutiv
e study; or without consistently applied reference standards SR (with homogeneit
ya) of 3b and better studies Nonconsecutive cohort study, or very limited popula
tion
4
Case series (and poor quality cohort and case-control studiese)
Case series (and poor quality prognostic cohort studiesl )
Case-control study, poor, or nonindependent reference standard
Case series or superseded reference standards

Analysis based on clinically sensible costs or alternatives; limited review(s) o

f the evidence, or single studies, and including multiway sensitivity analyses A
udit or outcomes research SR (with homogeneitya) of 3b and better studies Analys
is based on limited alternatives or costs, poor quality estimates of data, but i
ncluding sensitivity analyses incorporating clinically sensible variations Analy
sis with no sensitivity analysis
(Continued )
236 Table 2A Oxford Center for Evidence-Based Medicine Levels of Evidence (May 2
001) (Continued) Differential diagnosis/ symptom prevalence study Expert opinion
without explicit critical appraisal, or based on physiology, bench research or f
irst principles
Khoo and Dearnaley
Therapy/prevention, Level etiology/harm 5 Expert opinion without explicit critic
al appraisal, or based on physiology, bench research, or first principles
Prognosis Expert opinion without explicit critical appraisal, or based on physio
logy, bench research or first principles
Diagnosis Expert opinion without explicit critical appraisal, or based on physio
logy, bench research or first principles
Economic and decision analyses Expert opinion without explicit critical appraisa
l, or based on economic theory or first principles
Users can add a minus sign to denote the level of that fails to provide a conclusi
ve answer because of either a single result with a wide confidence interval (e.g
., an ARR in an RCT is not statistically significant but whose confidence interv
als fail to exclude clinically important benefit or harm) or a systematic review
with troublesome (and statistically significant) heterogeneity. Such evidence i
s inconclusive, and therefore can only generate grade D recommendations. a Homog
eneity mean a systematic review that is free of worrisome variations (heterogene
ity) in the directions and degrees of results between individual studies. Not al
l systematic reviews with statistically significant heterogeneity need be worris
ome, and not all worrisome heterogeneity need be statistically significant. As n
oted above, studies displaying worrisome heterogeneity should be tagged with a at
the end of their designated level. b Clinical decision rule. (These are algorith
ms or scoring systems, which lead to a prognostic estimation or a diagnostic cat
egory.) c See note #2 for advice on how to understand, rate, and use trials or o
ther studies with wide confidence intervals. d Met when all patients died before
the Rx became available, but some now survive on it; or when some patients died
before the Rx became available, but none now die on it. e Poor quality cohort s
tudy failed to clearly define comparison groups and/or failed to measure exposur
es and outcomes in the same (preferably blinded), objective way in both exposed
and nonexposed individuals and/or failed to identify or appropriately control kn
own confounders and/or failed to carry out a sufficiently long and complete foll
ow-up of patients. Poor quality case-control study failed to clearly define comp
arison groups and/or failed to measure exposures and outcomes in the same (prefe
rably blinded), objective way in both cases and controls and/or failed to identi
fy or appropriately control known confounders. f Split-sample validation is achi
eved by collecting all the information in a single tranche, then artificially di
viding this into derivation and validation samples. g An Absolute SpPin is a diagnosti
c finding whose Specificity is so high that a Positive result rules-in the diagn
osis. An Absolute SnNout is a diagnostic finding whose Sensitivity is so high that
a Negative result rules-out the diagnosis. h Good reference standards are indep
endent of the test, and applied blindly or objectively to all patients. Poor ref
erence standards are haphazardly applied, but still independent of the test. Use

of a nonindependent reference standard (where the test is included in the referenc

e, or where the testing affects the reference) implies a level 4 study. i Better-valu
e treatments are clearly as good but cheaper, or better at the same or reduced c
ost. Worse-value treatments are as good and more expensive, or worse and the equ
ally or more expensive. j Validating studies test the quality of a specific diag
nostic test, based on prior evidence. An exploratory study collects information
and trawls the data (e.g. using a regression analysis) to find which factors are
significant. k Poor quality prognostic cohort study is one in which sampling was
biased in favor of patients who already had the target outcome, or the measureme
nt of outcomes was accomplished in <80% of study patients, or outcomes were dete
rmined in an unblinded, nonobjective way, or there was no correction for confoun
ding factors. l Good follow-up in a differential diagnosis study is >80%, with a
dequate time for alternative diagnoses to emerge (e.g., 16 months acute, 15 years
chronic). Produced by Bob Phillips, Chris Ball, Dave Sackett, Doug Badenoch, Sha
ron Straus, Brian Haynes, Martin Dawes since November 1998. Source: From Ref: 3.
Table 2B Evidence-Based Medicine: Recommendation Grading Based on Oxford Center
for EBM Recommendation grades Grade Grade Grade Grade
a
Description of Oxford Center for EBM, United Kingdom Consistent level 1 studies
Consistent level 2 or 3 studies or extrapolationsa from level 1 studies Level 4
studies or extrapolationsa from level 2 or level 3 studies Level 5 evidence or t
roublingly inconsistent or inclusive studies of any level
A B C D
This is where data is used in a situation, which has potentially clinically impo
rtant differences than the original study situation. Abbreviation: EBM, evidence
-based medicine. Source: From Ref. 3.
lesions depending on the order in which CT and radionuclide scanning was perform
ed. This study reported that CT with and without contrast enhancement was more a
ccurate than radionuclide brain scanning and plain radiography but was as accura
te as angiography in detecting brain masses. It also reported that randomizing f
or the imaging
order was not a crucial determinant of the results and concluded that randomizat
ion for the order of imaging was not necessary (6). The important feature here i
s to blind the reporting of the imaging tests. It is clear that this study only
evaluated diagnostic ability for metastasis detection and did not address the CT
value of providing cross-sectional
The Testing of Prostate IGRT in Clinical Trials
237
internal anatomy information that will not be available from radionuclide or ang
iographic imaging. For modern radiotherapy including stereotactic radiotherapy,
crosssectional anatomical information is vital for threedimensional spatial loca
lization and treatment planning. In radiotherapy practice, the utility of CT com
pared with conventional methods of radiotherapy planning which used imaging with
radiographic examinations, xerograms, lymphangiograms, arteriograms, air contra
st studies, isotopic studies, and ultrasound was prospectively assessed in this
early study (7). This study revealed that 44 of 77 (52%) of the cases had their
conventional treatment changed as a result of using CT. Inadequate coverage of t
he target volume was noted in 32 of 77 (42%) treatment plans, and 24 of 77 (31%)
cases had some portion of the target outside of the 50% isodose region. Compare
d with CT, it was demonstrated that planning volumes of interest (VOI) clinicall
y estimated from plain radiographs could not accurately define tumor extent. Usi

ng an empirical predictive model, Goitein suggested that the improved tumor volu
me localization using CT could result in an increase in local tumor control prob
ability by an average of 6% with the chance of five-year cancer survival increas
ing by an average of 3.5% (8). It is clear that CT has permitted more accurate t
umor/target volume delineation and provided the foundation for precise treatment
planning. Another important issue in radiotherapy is the proper assessment of t
he spatial relationship of the target volume with its surrounding critical organ
s at risk (OAR). This assessment is needed to limit and reduce the likelihood of
treatment related complications and remains a crucial aspect to maintain the th
erapeutic ratio in radiotherapy. Despite the lack of randomized clinical trials
for the use of CT in radiotherapy treatment planning, the integration of CT in r
adiotherapy remains the current standard of care and is the basis for conformal ra
diotherapy (CFRT) and intensity-modulated radiotherapy (IMRT). The use of CFRT a
nd IMRT techniques will also permit the treatment paradigm of conformal avoidance
of OARs to be achieved once the three-dimensional treatment space is known. It h
as also instigated the establishment of treatment volume nomenclature from the I
nternational Commission for Radiation Units (ICRU) Reports 50 and 62 (9,10) that
allow for uniformity in treatment planning and delivery as well as in consisten
cy in reporting. In addition, the application of cross-sectional imaging is also
one of the means through which IGRT is implemented. In the clinical assessment
of conformal planning technology for prostate radiotherapy, a phase III external
beam radiotherapy UK clinical trial randomised prostate cancer patients to unsh
aped treatment fields (conventional) or shaped treatment fields (conformal) with
the same dose and dose per fraction in each arm (11). In this
trial of 225 men, a significant reduction in late rectal toxicity, which is the
dose limiting constraint for prostate radiotherapy, was reported for the conform
al treatment arm. Level 1 evidence from this U.K. trial eventually provided the
basis for its countrys advisory body National Institute for heath and Clinical Ex
cellence (NICE) to support the widespread implementation of conformal techniques
for prostate radiotherapy. It is good quality evidence of this type that can in
fluence and change national policies and lead to better outcomes for our patient
s. Another example for the testing of radiotherapy technology is in the clinical
application of IMRT. There is a rapidly growing and almost voluminous amount of
published reports demonstrating the superiority of IMRT compared with CFRT tech
niques in dosimetric terms with better coverage of the nominated target volume a
nd with substantially less dose to the adjacent OAR or critical structures. Some
of these planning studies have been comparative physics evaluations but remain
theoretical exercises. Many of these planning studies have been followed by nume
rous clinical studies undertaken in almost every tumor site ranging from the cen
tral nervous system to soft-tissue sarcomas. In particular, irradiation of the p
rostate gland has been extensively studied. Most of the clinical IMRT studies in
prostate cancer have been either feasibility studies assessing the potential of
delivering IMRT (12) or noncomparative studies assessing patient cohorts for do
se escalation using IMRT (13). Most of the reported randomized controlled trials
in prostate cancer are evaluating the benefits of CFRT dose escalation with con
ventional 1.8 to 2 Gy dose per fraction schedules (1417) or testing the potential
radiobiological advantage of hypofractionation (18,19). It is clear that these
clinical trials are not testing the value of the IMRT technique itself in a comp
arative study. The dosimetric assessments from these clinical studies do appear
impressive for IMRT usage. Although nonrandomized clinical studies may provide e
arly evidence of the potential benefit of IMRT, the true clinical value of the I
MRT technique on treatment related morbidity, local tumor control, and disease s
urvival will need to be assessed in formal prospective and randomized clinical t
rials. Such randomized trials are currently being undertaken in a few clinical c
ancer subsites (e.g., head and neck and breast) and are easier to perform in som
e countries than in others for a number of reasons that include clinician bias,
popular patient beliefs and local advocacy groups, and the restructuring of reim
bursement for radiotherapy. This is a crucial pragmatic issue for if there is a
lack of objective observer or participant equality about the treatment arms of a

randomized trial, it would be difficult to initiate uptake and completion of th

e trial either from investigators or patients alike. Such a trial would be
238
Khoo and Dearnaley
doomed from the outset irrespective of the appropriateness or importance of the
therapeutic question under investigation. This issue has plagued randomized comp
arative studies of IMRT to standard treatment methods in some countries and may
also limit similar studies in the assessment of IGRT for prostate cancer. Some s
tudies, for example the U.K. multicentre randomized control trial CHHiP of conve
ntional or hypofractionated high-dose IMRT permit either more simple forward-pla
nned or inverse-planned solutions (20,21). This trial will provide high quality
but nonrandomized comparative data [U.S. level 21 evidence (Table 1) and U.K. lev
el 2 evidence (Table 2)] relating planning and delivery techniques to dosimetry,
treatment related sequela using physician, and patient-based instruments as wel
l as disease control end points. In a similar fashion, level 2 evidence for the
use of MLCs rather than low melting point alloy blocks and portal imaging rather
than film verification was generated from the Medical Research Council (MRC) RT
01 trial by detailed analysis of patients receiving treatment using different co
mbinations of techniques (17,22). There is emerging evidence that IGRT is import
ant and relevant for prostate radiotherapy. The standard method of using a singl
e planning CT scan to determine the threedimensional spatial location and shape
of the prostate position can lead to systematic errors in treatment planning. A
consequence of such systematic errors is a loss of biochemical prostate-specific
antigen (PSA) control rates in external beam prostate radiotherapy, as reported
recently from retrospective analyses from two randomized studies of prostate do
se escalation (23,24). ISSUES OF CLINICAL TRIAL DESIGN FOR PROSTATE IGRT The stu
dy components of any well-executed clinical trial for prostate IGRT will need to
include close attention to trial design, IGRT method implementation and executi
on, quality control, collection and analysis of data, and interpretation of resu
lts. These components are summarized in Table 3. The initiation for the developm
ent of any clinical trial in prostate IGRT is to answer a relevant or important
therapeutic or management question that deals with minimizing or limiting the te
mporal-spatial uncertainty in target volume definition, treatment delivery, or v
erification. This question will need to be carefully considered and refined on t
he basis of a careful analysis of the current standard of care in prostate radio
therapy, the known limitations in treatment delivery, the potential advantage of
the image-guided technique to current radiotherapy methods, as well as a review
of previous research in this area. It is important for the investigators to inv
olve all key radiotherapy health professionals at the outset of any
clinical trial design for prostate IGRT. This group should include clinicians, p
hysicists, radiation therapists, dosimetrists, radiotherapy nurses, and other re
levant ancillary staff. It is imperative that this investigative group should cr
itically evaluate the current evidence concerning the radiotherapeutic managemen
t of prostate radiotherapy with respect to the new image-guided strategy being i
nvestigated. In general, this should involve a review of the published literatur
e and current ongoing clinical trials on the topic. This will provide a sound kn
owledge base to determine whether any preliminary studies needed to evaluate a p
rostate IGRT strategy is feasible and promising. This is particularly important
if a comparative study to determine the value of IGRT for relevant patient outco
mes is being contemplated. Types of Clinical Trials The type and the intention o
f clinical trial need to be clarified and defined at the outset. The conventiona
l phases of clinical trials are described in Table 4. This description pertains
mainly to clinical drug or biological therapy trials. The situation for radiothe
rapy technology is slightly different but the principles are broadly similar and
can be translated over to trials in radiotherapy technology. The initial incent
ive for clinical drug trials may either be an observational/case study report or

laboratory evidence of anticancer activity using a particular drug agent. In ra

diotherapy, particularly for IGRT strategies, initial study incentive may arise
from clinical observational or case studies demonstrating variations in temporal
-spatial variation of the tumor target and theoretical planning or mechanical st
udies outlining a potential IGRT method that may deal with this uncertainty. Suc
h studies only provide passive observational data with no control of the observe
d parameters and substantial inherent biases. It cannot define the appropriate p
atient group, the IGRT methodology may not be feasible or practical, and the imp
act on treatment outcomes is unknown. A phase I drug trial would be designed to
determine the appropriate drug dose for use in phase II. For clinical trials in
IGRT, this could be a feasibility study to assess the methodology and practicali
ty of the IGRT process. This could be undertaken in a manner similar to drug tri
als where initial cohorts of three patients are tested with the IGRT method and
if successful, a further three patient cohort is tested up to a total of 9 or 12
patients. Clear and relevant thresholds or limits for the success of the proces
s will need to be defined to either enable stoppage or continuation of the study
. If the phase I or feasibility study for the IGRT method is successful, then a
phase II study can be conducted whereby the IGRT method is applied to larger coh
ort of patients to further define its usage, safety, as well as potential effect
iveness. Finally, a
The Testing of Prostate IGRT in Clinical Trials Table 3 Steps in the Design of a
Successful Clinical IGRT Trial Contents Aims/goals of the trial Description of
the steps
239
Trial methods
Regulatory trial approval
Study execution
Data collection and preparation
Quality control and trial monitoring
Dissemination of results Other issues
General l To formulate an important and relevant therapeutic or management IGRT
question that can be addressed through a clinical trial Specific l To define the
primary and secondary end points for the trial General l To develop the study m
ethodology and IGRT study protocol Specific l To define the patient population t
o be studied l To provide clear indications for patient inclusion and exclusion
l To obtain informed patient consent l To define the method of radiation treatme
nt and image guided strategy involved l To establish the process of image guidan
ce l To address statistical consideration with respects to the power and signifi
cance levels for the study l To predetermine interim analysis for the trial with
stopping or continuation rules where appropriate General process l All clinical
trials will need approval by the relevant authority or regulatory body for the
country in which the trial is being performed l The regulatory approval process
for clinical trials will differ in different countries. In general, a clinical t
rial will have to undergo an independent scientific review or peer review follow
ed by approval by a research ethics committee Specific process l In the United S
tates, this will involve the Institutional Review Board (IRB) that may be centra
l and local l In the United Kingdom, this will involve both independent peer and
ethics review. If the trial involves more than 1 center then a multicenter rese
arch ethics committee (MREC) approval is needed. Otherwise a local research ethi
cs committees (LREC) approval will suffice l To ensure the logistics and mechani
cs for efficient and seamless execution of the study l To have adequate trained

personnel for patient recruitment, assessment, and radiotherapy treatment l To h

ave sufficient quality assured radiotherapy facilities to enable trial completio
n l To provide for complete data collection l To quality assure the collected da
ta l To make full notes on any inconsistencies in the collection of the data l T
o provide for analysis of the trial data l To ensure quality assurance for the r
adiotherapy and image-guided methodology. This is a particularly important aspec
t for any radiotherapy technology trial l To have a system in place for the repo
rting of any serious unexpected adverse events (SUSARS) l Data Monitoring Commit
tee (DMC). If the trial is substantial or multicenter, then a DMC is likely to b
e convened. The DMC, together with the ethics committee will monitor any serious
unexpected side effects and they can stop the trial at any time if there are an
y concerns about the welfare of the participating patients l To present trial re
sults to local, national, and international meetings/conferences l To submit a m
anuscript for peer review and publication in a recognized journal. l To obtain a
dequate funding for the conduct and completion of the clinical trial l To declar
e any potential conflict of interest from the investigators and minimize this is
sue l To provide participants with understanding of the trial rationale and desi
gn; adequate time to digest the trial information and opportunity to clarify any
concerns in participation l To ensure that all investigators are appropriately
trained to conduct the study according to best clinical practice
240 Table 4 Conventional Description for the Phases in Clinical Trials Type of t
rial Phase I Phase II Phase III Description of trial
Khoo and Dearnaley
Dose finding studies: An experimental drug or therapy is tested for the first ti
me in a small group of patients (2080) to evaluate a safe dose range and identify
side effects Safety and activity studies: The experimental drug or therapy is u
sed in a larger cohort of patients (100300) to see if it is effective and to furt
her evaluate its safety Comparative studies: The experimental drug or therapy is
given to larger groups of patients (5003000) and compared to standard or commonl
y used therapies to confirm its effectiveness, monitor side effects, and collect
other information on its use
Table 5 Evidence-Based Medicine: End Points Grading schema A B C D Description o
f end points (direct and indirect in descending order of strength) Total mortali
ty Cause specific mortality Quality of life Indirect surrogates Disease-free sur
vival Progression-free survival Survival or tumor response rate
clinical randomized trial can be conducted to compare the experimental or IGRT a
rm with the current radiotherapy standard of delivery looking at appropriate cli
nical end points (Table 5) such as superior local tumor control rates, reduction
in the incidence of treatment related morbidity, or improved quality of life me
asures. Systematic and Random Errors in Trial Design The strength and validity o
f a clinical trial result is dependent on methods to control and minimize system
atic and random errors. In principle, these sources of errors will be the same i
rrespective of the method of image guidance under investigation for prostate IGR
T. They are important issues in the design for any prostate IGRT clinical trial
and will be discussed below. In clinical trials, any unaccounted for bias can pr
oduce a systematically higher or lower incorrect estimate of the true treatment
effect. Methods to reduce bias include the use of appropriate eligibility criter
ia and randomization, which will reduce the probability of selection bias in the
estimation of any treatment differences arising from the clinical trial. More i
mportantly, significant biases may arise after entry of patients into the clinic
al trial by (i) subsequent exclusion of patients after randomization; (ii) loss
of patient data that may be due to inappropriate censoring or noncompliance; (ii
i) use of different outcome assessments for different treatment groups; and (iv)
applying retrospective definitions or thresholds in the protocol.

Another aspect of uncertainties in clinical trials is the errors attributable to

chance or random errors. In traditional terms, there are two categories of rand
om errors. Type 1 error is a false positive in which there is no treatment effect
or difference noted in the clinical trial but where the investigators have wrong
ly concluded there is. Type 1 error must be carefully considered by the trial in
vestigators as this aspect is often under the control of the investigators when
either designing the clinical trial or analyzing it. Type 1 errors can occur whe
n multiple statistical tests are performed, especially, when examining accumulat
ing data or during sequential interim data reviews. It can be normally controlle
d by selecting the appropriate level of significance to be used in the statistic
al tests. Type II error is a false negative whereby the investigators fail to dete
ct a treatment effect or difference when it is actually present. This type of er
ror can be controlled by selecting the appropriate sample size for the study and
relates to the power of the clinical trial. By having a large enough sample siz
e, the clinical trial is then adequately powered to note a treatment effect of a
specified size to be statistically significant when it occurs. This type of err
or cannot be controlled by any statistical analysis. While a small study may hav
e power to detect a very large treatment difference, it may be unable to detect
smaller or modest treatment differences, which can be still clinically relevant
to clinicians. This is an extremely important issue to consider in the design of
clinical trials. It would not be logical to undertake a trial when the chance o
f missing a clinically relevant finding is larger than the chance of finding it.
Other Trial Issues Other issues that may arise in the conduct of any clinical t
rial include the motivation of its participants and addressing the trial resourc
e issues (Table 3). Motivation for trial participation applies to research inves
tigators as well as to the patients themselves. This can play a subconscious but
important part for both investigators and patients. It can have a negative or p
ositive effect on the trials success or failure. If there is a
The Testing of Prostate IGRT in Clinical Trials
241
belief that the experimental prostate radiotherapy arm utilizing IGRT is superio
r, then investigators will not be in a position to conduct the trial while patie
nts will be unwilling to be randomized to the standard radiotherapy arm which wi
ll be perceived to be a less effective treatment. Patient beliefs and perception
s can be greatly influenced by manufacturing advertising as well as impartial pa
tient advocacy groups. Greater understanding for the image-guided technology and
current radiotherapy standards in prostate radiotherapy are needed in these sit
uations. Careful objective explanation of the prostate IGRT rationale and design
will need to be provided by the trial investigators. Adequate resources are nee
ded for the initiation, development, efficient management, and timely completion
of any clinical trial. This aspect is often underestimated and is particularly
important when undertaking a radiotherapy technology clinical trial such as that
with prostate IGRT. This involves confirmed commitment of time and finances fro
m investigators and their staff. Infrastructure for the trial and all its indivi
dual steps remain crucial. CURRENT CLINICAL TRIALS TESTING IMAGE-GUIDANCE TECHNO
LOGY Listings of Current Clinical Trials Testing Prostate IGRT Although there ar
e numerous reports of centers testing technology for IGRT in prostate radiothera
py, there are few randomized clinical trials formally testing the utility of IGR
T or the impact of prostate IGRT on patient outcomes. Most published studies rem
ain initial technology assessments or feasibility studies. A search (August, 200
7) was performed of the trial databases for the NCI, National Cancer Institute o
f Canada (NCIC), National Cancer Research Network (NCRN), and European Organizat
ion for Research and Treatment of Cancer (EORTC) reflecting the past and current
listing of prostate radiotherapy clinical trials in the United States, Canada,
United Kingdom, and Europe, respectively. This search will not be a complete lis
ting of all worldwide prostate IGRT trials but provides a relatively comprehensi
ve review of the current state of affairs for clinical trials in prostate IGRT.

There will be some overlap between these databases as some trials will be listed
in several databases. In the search undertaken of the NCI web database for clin
ical trials in prostate cancer, two searches were performed for active and close
d trials (25). The following search filters were used: cancer typeprostate cancer
; trial typetreatment and methods development; stage of cancerstages 1 to 3; statu
s of trialactive or closed; phase of trialall; sponsor of trialall, and special
categoryall. This search revealed 178 active clinical trials and 219 closed clini
cal trials. Each of the trials listed was manually reviewed online. There was on
ly one phase III trial listed with the aim of examining the role of IGRT from Fr
ance. Although there were no web/online details for this French comparative stud
y, details for this trial will be outlined in the next section. There are no oth
er open or closed prostate cancer clinical trials listed on the NCI database tha
t is expressively designed to evaluate the utility of prostate IGRT. In the sear
ch undertaken of the Clinical Trials Group of the NCIC database (26), there are
19 clinical trials in prostate cancer listed in their website, of which 4 are op
en, 10 are closed, 3 are planned, and 2 have been withdrawn. Most of these liste
d trials are randomized phase III clinical trials, but again none are addressing
the value of IGRT in prostate radiotherapy. In the search of the NCRN clinical
trial portfolio database (27), there are 32 listed prostate cancer trials of whi
ch 16 are open, 16 are closed, and 2 are planned. None of the listed trials are
addressing the issue of IGRT in prostate cancer. In the search of the EORTC prot
ocols database (28), there are 26 trial protocols listed for prostate cancer of
which 2 trials are open. In the section for radiotherapy, there are 109 trials o
f which 11 are open, 97 are closed, and 1 is being planned. There are again no l
isted protocols or trials that are examining the utility of prostate IGRT. Pract
ical Assessment of IGRT in Prostate Cancer Image-guided strategies and its relat
ed technology is such a new field that this term can be broadly used for every a
spect in the radiotherapy management chain, as each link or step in the radiothe
rapy process involves some form of imaging from the definition of volumes of int
erest for radiotherapy treatment planning to the determination of field placemen
t accuracy and subsequently radiotherapy margins. Thus, image guidance for prost
ate radiotherapy may have a different definition depending whether it is taken f
rom the clinicians or physicists perspective. In this section, we will provide exa
mples and methods of clinical trials/studies to evaluate each of these important
steps. Assessment of Target Volumes for Prostate IGRT CT has been the standard
imaging modality used to determine VOI for the prostate and seminal vesicles as
well as surrounding tissue structures or OAR positions for radiotherapy treatmen
t planning. However, CT is of no value in defining areas of cancer within the pr
ostate gland. Magnetic resonance imaging (MRI) techniques including T2-weighted
sequences, dynamic contrastenhanced images, diffusionweighted images, and MR spec
troscopy (MRS), all hold the
242
Khoo and Dearnaley
promise of defining significant tumor nodules within the prostate itself (29). R
adiotherapy planning case studies have shown, in principle, that it is certainly
possible to selectively boost regions of apparent tumor or single tumor nodules
using IMRT methods with dose distributions, which are likely to maintain low to
xicity profiles (3032). How should this prostate IGRT approach be tested? The ini
tial and fundamental requirement is that the imaging modality to be used needs t
o precisely identify appropriate regions of cancer nodules within the prostate g
land. To achieve this, there must be confirmation that the imaging modality used
, such as MRI or MRS, correlates accurately and reliably with the histopathology
. The type of study needed will be a technical evaluation using patients pre-ima
ged and undergoing radical prostatectomies. There can be many potential problems
that will need to be overcome before clinical testing of this prostate IGRT can
be appropriately undertaken. Firstly, there needs to be accurate spatial alignm

ent between the whole mount histopathological prostate sections and correspondin
g MR images. This type of procedure is not usually performed in routine practice
. We have developed a customized prostate holder and slicing device specifically
for the purpose of processing and orientating whole mount histopathological sli
ces with their corresponding MR images (Fig. 1) (33). Other tissue processing is
sues that will need to be dealt with include the problem of tissue deformation d
uring the slicing process and shrinkage due to fixation in formalin so that subs
equent three-dimensional spatial alignment and localization of the tumor regions
with the imaging remains accurate. There are also other issues with MR imaging
whereby reliability of the MR parameters will need to be defined and thresholds
established to distinguish imaging criteria best associated with benign or malig
nant prostate tissue. Once the most promising parameters are defined, then sensi
tivity and specificity can be reviewed to see whether or not this imaging assess
ment is likely to be useful in clinical practice. For example, using dynamic con
trastenhanced MRI, our preliminary studies have suggested a very high specificit
y (93%) with a sensitivity of approximately 60% for prostate adenocarcinoma that
increases to 73% for cancer nodules that are greater than 1 cm3 (34). The next
appropriate set of studies would be to model this IGRT strategy of creating a de
liberately heterogeneous dose distribution by escalating dose (8690 Gy) to the ca
ncer nodule(s) while maintaining a standard dose to the rest of the prostate gland
. These studies can be performed on a small number of patients, in the order of
6 to 12 cases, to evaluate the distribution of intraprostatic nodule(s) location
s that would be best treated using this IGRT strategy and the treatment techniqu
e. In addition, estimations of the potential tumor control probabilities (TCP) a
nd normal tissues complication probabilities
Figure 1 A study method to correlate radical prostatectomy histopathological fin
dings with magnetic resonance imaging (MRI). (A) Mounting of the resected prosta
te gland in the same orientation as the imaging procedure. (B) Slicing of the mo
unted prostate gland. (C) Sliced prostate sections to correspond to the imaging
slices. (D) An example for one of the macroscopic whole mount slides of the pros
tate. (E) Corresponding macroscopic whole prostate section. (F) The image sectio
n of the prostate gland using MRI.
(NTCP) can be profiled to determine the appropriate tumor boost or even to indiv
idualize boost doses for each patient. It is evident that substantial studies of
this nature are needed before clinical testing of this prostate IGRT strategy s
hould be undertaken. For these studies outlined above, randomized trials are ind
eed not appropriate as these studies are designed to establish the methodology.
Subsequently, following confirmation of accurate histopathological imaging corre
lation and the radiotherapeutic design of boost strategies with attention to fou
r-dimensional delivery issues, feasibility studies can be initiated to determine
the safety of this method and its potential impact on clinical patient outcomes
.
The Testing of Prostate IGRT in Clinical Trials
243
For the example of the IGRT prostate strategy given above to treat single or mul
tiple dominant intraprostatic nodules, this can be undertaken as a phase I/II fe
asibility study. Acceptable clinical parameters for both acute and late toxicity
will need to be defined. It is also important to ensure that adequate follow-up
time is provided to capture any untoward late side effects. In this situation,
study follow-up at a minimum time of two years is often mandatory to make a prel
iminary assessment of late morbidity before progressing to the next stage of cli
nical trials. If toxicity end points are being considered, then the number of pa
tients needed for these feasibility studies will be dependent on the nominated t
oxicity grades and thresholds. For example, if we wished to exclude a level of g
rade 2 Radiation Therapy Oncology Group (RTOG) toxicity rate at 25%, then approx

imately 30 patients will be needed with 90% confidence assuming that the real toxi
city rate is 10%. A more realistic assessment would be to exclude RTOG grade 2 t
oxicity rates of 20% or more, and this lower threshold will now require approxim
ately 130 patients. In such a study, the outcomes will be strongly dependent on
radiotherapy technique, planning margins used, and methods of treatment delivery
and verification. In consideration of the definitive randomized trial, there is
a strong argument to perform a randomized phase II type study following the fea
sibility studies whereby patients will be randomized between the experimental trea
tment and standard arm. Several trial comparisons may be undertaken in this rega
rd. One clinical trial option is to randomize between the experimental boost dos
e and the standard dose (which may range from 74 to 78 Gy) to the whole prostate
(1417,35). Using the patient numbers quoted above, the power to detect differenc
es between the treatment groups may be relatively weak, but the advantage of thi
s approach is that it can act as a good pilot for the definitive randomized cont
rol trial, which will need to assess treatment efficacy. Treatment efficacy may
be considered in terms of local disease control or treatment related morbidity.
In prostate cancer trials, control of biochemical PSA levels is commonly chosen
as the principal end point, although there is no convincing data that this is a
good proxy for clinically relevant end points such as development of metastases,
use of salvage therapy (hormonal therapy), or causespecific or overall survival
. For a phase III clinical trial intending to evaluate an improvement in PSA bio
chemical control levels from 70% to 85%, the required number of cases for 80% po
wer and a two-sided a value of 0.05 would be 175 per randomized arm. If a smalle
r improvement in biochemical PSA levels was anticipated, for example from 70% to
80%, then for 90% power and a two-sided a value of 0.05, the number needed per
randomized arm would be 445 cases.
Another trial option would be to assess the utility of image-guided technology b
y comparing radiotherapy delivery given with or without the use of the new inroo
m methods of daily target guidance but with daily EPID verification in both arms
as the standard. This will be discussed in more depth in the following section.
Assessment of Treatment Planning Margins, Target Tracking, and Treatment Verifi
cation Some of the issues relevant to studies of IGRT include the determination
of treatment planning margins, target tracking, and treatment verification. We h
ave performed three small randomized control trials relating to these aspects of
treatment delivery. They will be discussed to illustrate the types of methodolo
gy that can be adopted to evaluate other aspects of radiotherapy technology. The
determination of radiotherapy treatment margins for prostate radiotherapy remai
ns a crucial issue. There have been many different methods used to assess the ma
gnitude of prostate motion such as repeated CT scanning during radiotherapy to i
mplanted intraprostatic markers (36,37). The aim of these methods is to rational
ize the design of treatment margins for patients receiving high doses of radiati
on. On the basis of population statistics, margin recipes have been developed to
aid the determination of treatment margins (38). However, irrespective of the m
argin selected, clinical relevance and potential impact on patient outcomes can
only be determined through clinical trials. One example of a clinical trial asse
ssing the appropriateness of a set of treatment margins is illustrated here. In
the United Kingdom, in the mid-late nineties, most radiotherapy centers were usi
ng uniform margins around the prostate of 1.0 to 1.5 cm. The question posed was
which of these treatment margins was most appropriate. In a randomized pilot stu
dy of dose escalation comparing 64 Gy versus 74 Gy (35), we added a second rando
mization. A 2 2 factorial design randomized patients to radiotherapy with either
a 1.0-cm or 1.5-cm margin. One hundred and twenty five men were included in the
study and this was enough to show a statistically significant increase in the R
TOG ! 2 rate of late bowel toxicity (21% vs. 13%, p 0.005). The biochemical PSA
control rate of disease was identical between the treatment arms suggesting no l
oss of local tumor control using the 1-cm margin. However, although this is the
only randomized control trial at any tumor site, to our knowledge, to compare a
radiotherapy margin, the evidence falls far short of proving equivalence in effi
cacy between the two treatment groups. To do this, adequately, a noninferiority

clinical trial design would be required. If a clinical trial is aimed at determi

ning noninferiority between the trial arms, then the number of patients needed w
ill depend on the level of
244 Table 6 Allocation of Patients Numbers Needed Within Clinical Trials That Ai
m for Noninferiority Number per group Difference allowed 5% 6% 7% 8% 9% 10% 80%
power 1039 721 530 406 321 260 90% power 1439 999 734 562 444 360
Khoo and Dearnaley
difference allowed and the confidence limits needed. Examples of this are listed
in Table 6 where if a difference of 10% is allowed, the number of patients requ
ired per randomized arm for 80% and 90% power is 260 and 360 cases, respectively
. However, if only a difference of 5% is allowed, then the number of patients re
quired per randomized arm for 80% and 90% power increases to 1039 and 1439 cases
, respectively. Clearly, such a trial is impractical, and the evidence from this
small pilot study was sufficient to convince the U.K. investigators that the 1.
0-cm margin was more appropriate. This planning margin was then used in the subs
equent larger international MRC RT01 phase III trial (17). Compared with the cli
nical trials described above, a technical trial may require considerably less pati
ents. For example, our initial assessment of an EPID in the early 1990s evaluate
d 12 patients with portal imaging at the beginning and end of their treatments f
or hypofractionated (6 Gy/fraction) radiotherapy in prostate and bladder cancer
and assessed the value of portal imaging by reimaging the patient at the end of
their treatment (39). Each patient had the order of their treatment randomized to
be either corrected on the basis of the portal image or not. This study showed t
hat at the end of therapy in the conventionally managed treatment fractions, the
re was a displacement of 4.9 2 mm compared with 2.1 1.5 mm ( p < 0.01) for the c
orrected treatments. Furthermore, the proportion of treatments delivered with a
field-placement error of !5 mm decreased from 69% to 7%. This study verified the
value of interventional portal imaging to improve treatment accuracy. More impo
rtantly, this study demonstrates that this format of intrapatient randomization
can be used effectively to assess the value of a technical innovation in radioth
erapy and has definite applications for the evaluation of IGRT in prostate cance
r. However, not all technical studies will produce the expected benefits (and de
sired) result. We have also undertaken a small randomized control trial evaluati
ng the use of the Vacfix pelvic immobilization device (40). In this study, 30 pati
ents were recruited and treated
radically with prostate radiotherapy. All patients were double planned both using
our standard immobilization system (knee support and ankle stocks) or the same w
ith the addition of a Vacfix pelvic immobilization device. In this study, a simi
lar intrapatient randomization was used for patients being treated either with o
r without the Vacfix bag for weeks 1 to3 or 4 to 6 of treatment. Neither random
nor systematic errors were improved by the pelvic immobilization device. In cont
rast, the accuracy of treatment setup was marginally better with the standard se
tup technique despite the patients reporting that the use of the pelvic immobili
zation device was more comfortable during treatment. This is a valuable finding
as many departments may have assumed that new technology will improve treatment
outcomes, and this clinical trial of radiotherapy technology was salutary in sho
wing the opposite! Accuracy, however, was very good in both groups of treatment
and was deemed improved on our previous experience, which we judged to be becaus
e of the introduction of electronic portal imaging. We are using a broadly simil
ar clinical design to test the value of a new prostate localizing obturator call
ed ProSpare (patent pending). This endorectal device containing radiopaque markers
has been designed to sit comfortably within the anorectum and be self-positione
d by the patient. The intention of this device is to stabilize the position of t
he prostate by maintaining a constant rectal volume as the device is vented, to of
fer some normal tissue sparing as the anterior and posterior rectal walls are se
parated, as well as to provide markers for treatment localization. The accuracy

of treatment will be judged by comparing fiducial markers implanted into the pro
state with those within the ProSpare device. Again, patients will act as their o
wn controls being double planned with and without the ProSpare device, and weeks 1
and 2 of treatment will be randomized to be either with or without the device.
An initial cohort of 25 patients will assess acceptability and tolerability usin
g an actual device tolerability rate of 85%. This will exclude a lower limit of
tolerability of 50% with 90% power. This study will also allow a paired standard
ized difference of 0.67 mm (2-mm difference with standard deviation of 3 mm) bet
ween treatments with and without ProSpare to be detected (89% power, two-sided a
0.05 using a paired t test). By illustration of the trial methodologies, these
small detailed technical studies reveal how innovative and technical methods of
treatment delivery can be tested in an appropriate fashion in small numbers of p
atients. However, these clinical trials do not prove that they are clinically of
benefit. As outlined above, many more patients are needed to show benefits in t
erms of clinical efficacy of treatment albeit lower numbers may be adequate to a
ppropriately measure benefits in terms of reduction of toxicity.
The Testing of Prostate IGRT in Clinical Trials
245
The French multicentre trial of IGRT in prostate cancer mentioned earlier has tw
o parts (Dr. R. de Crevoisier, personal communication). The first part aims to e
valuate the accuracy of conventional portal imaging for the localization of the
prostate gland by also incorporating daily inroom cone-beam imaging during prost
ate external beam radiotherapy. This part of the study aims to recruit 80 patien
ts. The second part of the trial randomizes 404 patients to two different freque
ncies of prostate position verification, which is either for daily cone beam ima
ging pre-treatment or for cone beam imaging during the first 3 days of treatment
followed by weekly cone beam imaging. For this study, the main end point is bio
chemical disease-free survival with the intention of showing a 12% benefit in th
e five-year biochemical disease-free survival rates for the use of daily cone-be
am imaging. This is calculated to require 202 patients in each arm (power 80%, a
0.05 using 2 sided log-rank test).
It is also clear that sophisticated treatment planning technique such as IMRT wi
ll need to incorporate some form of IGRT so that the potential benefits for the
closely shaped high-dose regions can be directed to the target volume accurately
and reliably in time and space to maintain the expected patient outcomes. Again
, for clinical efficacy, this can only be properly validated in comparative rand
omized controlled trials. SUMMARY OF PERTINENT CONCLUSIONS
l
l
l
FUTURE DIRECTIONS Current studies in prostate IGRT have been exploratory investi
gations assessing methodology of the IGRT process, quantifying the IGRT mechanis
m, or early feasibility or phase I/II studies. There are many groups that may be
lieve it is not ethical to randomize patients into randomized control trials com
paring the utility of IGRT with standard delivery methods for prostate radiother
apy whether it be for dose escalation, boost strategies, or hypofractionation re
gimes. However, similar to the situation that currently exists for the value of
IMRT, it is clear that randomized controlled trials are the only method to truly
determine the utility of IGRT procedures or strategies on patient outcomes whet
her it is to reduce treatment related toxicity or to improved local tumor contro
l rates. Irrespectively of the IGRT question under trial, it is important for al
l radiotherapy trials to incorporate adequate and appropriate quality assurance.
Another important understanding is that while IGRT for treatment delivery may s

ubstantially reduce the magnitude of systematic error and limit random error in
treatment setup and target localization, this does not necessarily mean that tre
atment planning margins can be zero. Random errors in treatment delivery by natu
re cannot be completely eliminated, and thus some form of margin for treatment u
ncertainty will always remain. As IGRT methods for fourdimensional treatment del
ivery are validated and assessed in clinical trials, attention will need to be d
irected to the definition of target volumes, as this will remain the main and la
rgest source of uncertainty. This will both influence intra- and interobserver v
ariability and can be a major concern for multicentre clinical trials.
l
l
l
l
l
Clinical trials in prostate IGRT need to be well designed and executed to reliab
ly separate treatment effects from study uncertainties such as bias and random e
rror. Clinical trials for prostate IGRT will need to define appropriate end poin
ts for the image-guided strategy under investigation. The magnitude of anticipat
ed difference together with the confidence limit will determine the number of pa
tients required for any clinical trial. Small clinical trials ( 30 patients) usi
ng intrapatient randomizations can be designed to determine if new technologies
improve treatment accuracy. Relatively small randomized controlled trials can be
designed to assess if a new technique reduces treatment related side effects co
mpared with standard treatment (<300 patients). Large randomized controlled tria
ls are required to show equivalent efficacy of new and standard techniques (>100
0 to 2000 patients). The prospective randomized comparative clinical trial in pr
ostate IGRT will be the only method of logically confirming the true value for p
atient outcomes whether it is a reduction in treatment related toxicity or impro
vement in biochemical PSA control rates. The successful conduct of any clinical
trial on radiation technology will need to include (i) the development of approp
riate study protocols and technology methodology; (ii) the definition of suitabl
e study end points; (iii) obtaining the necessary regulatory approvals; (iv) the
provisions of suitable resources for study execution and quality assurance; (v)
data collection and analysis; and (vi) dissemination of the study results.
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20
Future Developments I: Online Dosimetric Verification with Fiducial Dosimeter Pl
anning and Verification
MARK WIESMEYER, HABEEB SALEH, MARTIN MURPHY, AND MITCHELL S. ANSCHER
Department of Radiation Oncology, Virginia Commonwealth University School of Med
icine, Richmond, Virginia, U.S.A.
GLORIA P. BEYER AND CHARLES W. SCARANTINO
Sicel Technologies, Morrisville, North Carolina, U.S.A.
JOANNA E. CYGLER
Department of Medical Physics, Ottawa Hospital Regional Cancer Centre, Ottawa, O
ntario, Canada
INTRODUCTION The tolerance of normal tissues limits the dose of radiation that c
an be delivered in the treatment of malignancy (1). Nevertheless, advances in tr
eatment delivery technology, combined with improvements in imaging that facilita
te better tumor targeting, have enabled the radiation oncologist to deliver high
er doses using external beam radiation therapy than had been previously thought
possible. Usually, the strategy employed to deliver high radiation doses depends
on the ability to reduce the margin of normal tissue around the target that rec
eives full-dose irradiation. In this situation, it is incumbent upon the radiati
on oncologist to demonstrate that the planned dose is actually being delivered t
o the target. The treatment of prostate cancer with external beam radiation ther
apy (EBRT) is one of the few situations in oncology in which level-one evidence
exists to support radiation dose escalation in selected patient populations (2).
Equally compelling is the evidence of a dose-volume
249
relationship for late toxicity in the pelvic organs surrounding the prostate, es
pecially the rectum (2,3). Despite its location in the floor of the pelvis benea
th the peritoneum, the prostate can demonstrate considerable movement between tr
eatment fractions, and to a lesser extent, during a treatment fraction (4,5). Th

us, accurate dose delivery, with narrow margins around the target, can present a
technical challenge. Even more challenging can be accurate dose verification wi
thin the target. Until recently, it was not possible to measure dose in vivo, ex
cept on the skin surface and in easily accessible regions of body cavities (6),
and a true in situ dose measurement, for most cancers, was impossible, since the
dose measuring devices had to be removed from the body in order to be interroga
ted. Currently, it is now possible, with certain limitations, to implant a dosim
eter into or adjacent to a tumor and to determine the actual dose delivered to t
he target telemetrically throughout a course of treatment. Herein, we will revie
w the history of in vivo dosimetry, describe the design and operation of a new i
n situ dose verification system,
250
Wiesmeyer et al.
review the available animal and human data that display its capabilities, and of
fer practical guidelines for implementation of in situ dosimetry into routine cl
inical practice. HISTORY OF IN VIVO DOSIMETRY During the first decades of practi
ce in radiation therapy, there were no standardized quantitative units to measur
e the patients radiation exposure. Dose was typically expressed in terms of some
physiological response, e.g., the erythema dose (7), which would have been the amo
unt of radiation needed to induce skin reddening. The corresponding dose to inte
rnal treatment sites would then have been extrapolated from the skin dose. One c
ommon practice used to calibrate an applicator for radium therapy involved holdi
ng it to the skin of a healthy subject until a skin reaction appeared (8). This
procedure established a reference exposure level for use during the therapy. Thu
s, early dosimetry was in vivo by definition, insofar as it measured dose to tis
sue directly, but more specifically, it was entrance dosimetry that required cal
culation and inference to estimate the dose to internal organs. Subsequently, va
rious film and electronic systems have replaced the skin as the radiosensitive e
lement for entrance and exit dosimetry. A comprehensive historical survey of the
large literature on this subject is beyond the scope of this chapter, but some
specific examples to illustrate the various methods and their place in the devel
opment time line will be presented below. Contemporary in vivo dosimetry methods
are distinguished by the type and placement of the radiosensitive elements. The
detectors can be film or discrete element sensors such as diodes and MOSFET (me
tal oxide semiconductor field effect transistor) sensors, or electronic detector
systems such as an electronic portal imaging device (EPID). They can be placed
on the skin at the point of beam entry (entrance dosimetry) or behind the patien
t (exit dosimetry). In these geometries, the inference of the dose at the treatm
ent site is an extrapolation problem. Transit dosimetry measures both the entran
ce and exit dose. This changes the extrapolation problem into an interpolation i
ssue, which generally is better constrained and thus will give more accurate res
ults. Finally, placement of the sensor directly at the treatment site minimizes
calculation and inference, while giving the most direct measurement. In 1978, Ha
ssan and Pearce (9) described a technical study of a swallowable pill containing
a mercuric iodide detector and radiotransmitter that could potentially be used
for in situ measurements of ionizing radiation. However, the modern era of in vi
vo dosimetry using external detectors began circa 1990. Initially, entrance and
exit dose measurements were made with diodes and thermoluminescent detectors (TL
Ds) (1012). By the mid 1990s,
commercial diode systems were available for skin dosimetry. These provided multi
ple diode detectors coupled to a multichannel readout unit (13). Portal films pr
esent the opportunity to measure exit dose radiographically (14). In vivo dosime
try using EPIDs was introduced somewhat later (15,16). The MOSFET was first conc
eived by Lilienfeld in the 1920s (17), but its first practical realization was n
ot until 1960 by Kahng and Atalla at Bell Labs. The major technical advances req
uired to realize Lilienfelds idea were the silicon planar process developed in th

e late 1950s by McCaldin and Hoerni and the integrated circuit developed by Kilb
y and Noyce. The notion of MOSFET-based dosimetry was introduced in 1978 by Holm
es-Siedle in the form of a space-charge measuring device (18). Since then, MOSFE
Ts have mostly been used to monitor radiation encountered by man-made earth-orbi
ting satellites. Perhaps, Thomson et al. (19), in 1984, published the earliest d
escription of a MOSFET dosimeter that might be used in a radiation therapy setti
ng. Subsequently, the detector was introduced into the discussion (20) as an alt
ernative to the TLD for skin placement. Although all of these methods are common
ly called in vivo dosimetry, they still require inference of the dose at the int
ernal tumor and critical structures from remote measurements. If one considers t
he more explicit form of in situ dosimetry, where one measures the dose directly
at the internal sites of interest, one finds only a very small number of histor
ical and recent examples. An early and somewhat theatrical attempt at in situ do
simetry was motivated by the development of rotation therapy in the early 1950s.
The use of rotation therapy to increase the dose to deep tumors (an early appro
ach to three-dimensional therapy) made dose calculations significantly more comp
licated and greatly increased the importance of dose verification measurements a
t the time of treatment. To demonstrate in situ dosimetry for rotation therapy,
Webb employed a professional sword swallower to insert a long rod with an ioniza
tion chamber at its tip through his esophagus into the thorax (21). The typical
patient, though, would probably balk at this procedure. Endoscopic insertion of
dosimeters, however, can provide a more conventional access to some internal sit
es. This has motivated the development of scintillator crystals that can be pass
ed through an endoscope while optically coupled via fiber to an external photomu
ltiplier tube or photodiode (22). High dose rate (HDR) brachytherapy provides so
me opportunities for approximately in situ dosimetry by attaching dosimeters to
the applicator. For example, Pai et al. (23) described use of radiochromic film
taped to the outside of the applicator for cervical HDR brachytherapy. However,
because of the extreme difficulty of getting dose-measuring sensors into most in
ternal treatment sites, using external detectors has largely
Future Developments I: Online Dosimetric Verification
251
satisfied the measurement of in vivo dose during external beam radiotherapy. Muc
h of the incentive to develop in situ dosimetry has come from radioimmunotherapy
(RIT), which is not amenable to the use of external detectors. In 1994, Gladsto
ne et al. (24) described a miniature MOSFET dosimeter designed to be inserted in
a 16-gauge needle and thence directly into tissue. This dosimeter was tested in
mice during RIT (25). Yorke et al. (26) addressed the particular issues that ar
ise in the use of TLDs for in situ dosimetry in RIT. TLDs are sensitive to tempe
rature, chemical environment, time delays between exposure and readout, and othe
r factors. In contrast to external beam (i.e., entrance) dosimetry, where the TL
Ds are exposed to air at room temperature and read out within 48 hours, a TLD im
planted in situ for RIT is in a tissue environment at mammalian temperature for
up to two weeks. Such significant differences would be expected to alter their i
n situ performance relative to their better-documented external characteristics.
These issues were pursued further in studies by Demidecki et al. (27), which me
asured the effects on TLDs of prolonged immersion in in vitro media intended to
simulate a tissue environment. However, these were feasibility studies that did
not extend to animal or human subject testing. Molecular imaging techniques offe
r promise as a means to measure dose deposition at the target site directly and
noninvasively. For in situ dosimetry during proton therapy, Paans and Schippers
(28) proposed in a feasibility study to use positron emission tomography (PET) i
maging of the resulting proton-induced activity at the treatment site. Finally,
in 2002, Ishikawa et al. (29) patented an internal dosimetry system using small
radiosensitive transponders injected directly into the tumor. The transponders i
ncluded electronics to communicate dose data with a remote external receiver. Th
is device operates, in principle, in a manner similar to a more recently develop

ed commercially available MOSFET-based system for in situ dosimetry. MOSFET AS A

PATIENT DOSIMETER Structure and Operation MOSFETs are a type of transistor, and
as such, they can be used to control the flow of electrical charge (current) in
electronic circuits. There are two basic types of MOSFETs: n-type and p-type. T
he n in n-type and p in p-type refer to the fact that semiconductors such as silicon
can be doped (i.e., trace atomic impurities can be added to pure silicon). By dop
ing, imperfections are formed in the silicon matrix that has either a surplus of
electrons (n-type) or shortage of them (p-type). A shortage
Figure 1 Cross section of a p-type MOSFET. Abbreviation: MOSFET, metal oxide sem
iconductor field effect transistor.
of electrons is often called a hole. The doped silicon acquires the property that
its electrical conductance can be modulated by an external electric field. Trans
istors, in general, can be thought of as charge faucets. Figure 1 shows the schema
tic of a p-type MOSFET, which shows that this analogy is reflected in the naming
of some MOSFET components (e.g., source, gate, drain, channel). By convention,
MOSFETs are categorized according to the doping of their source and drain or, eq
uivalently, by the type of channel that is formed between source and drain. Thus,
the schematic of an n-type MOSFET would be identical in structure to the p-type
MOSFET shown in Figure 1, differing only in the doping of the source, drain, and
substrate. Most dosimetry applications use p-type MOSFETs. When a negative volt
age with respect to the body electrode is applied to the gate of a p-type MOSFET
, an electric field is produced that attracts holes from the body substrate, sou
rce, and drain to the region immediately below the oxide insulator. With a suffi
cient gate voltage, VG, a sufficient number of holes may accumulate forming a ch
annel that allows current to flow between the source and drain. The minimum volt
age required allowing flow of current between the source and drain is known as t
hreshold voltage (VTH). The operation of an n-type MOSFET is precisely the same
as that of a p-type, except that the activating gate voltage polarity is positiv
e and the role of holes and electrons is reversed. MOSFETs make suitable radiati
on dosimeters, because, with irradiation, the relationship between gate voltage
and current flow changes in a predictable manner. They have the added features o
f small size, simple interface circuitry, low power during readout, and the abil
ity to measure radiation while in a powered down (passive) state. When a metal o
xide semiconductor device is irradiated, several things occur within the insulat
ing oxide: trapped charge builds up in the oxide, the number of interface traps
increases, and the number of bulk oxide traps increases (19). Electron-hole pair
s are created within the oxide insulator by the incident ionizing radiation. Ele
ctrons, having about four orders of magnitude greater mobility than holes in SiO
2 tend to move quickly toward
252
Wiesmeyer et al.
positively biased contacts and exit the oxide layer (30). Depending on the appli
ed electric field, some fraction of electrons and holes will recombine. Holes th
at escape initial recombination are relatively stable and remain near their poin
t of generation in the oxide layer. Thus, when a p-type MOSFET is exposed to rad
iation, the threshold voltage magnitude increases. When MOSFET dosimeters are un
biased, their radiation sensitivity is lower, less linear, and they may exhibit
less signal drift, also known as fade. If the change in threshold voltage due to a
known radiation dose can be characterized, then the MOSFET may be used to accur
ately measure radiation dose. The sensitivity of MOSFET devices (i) increases wi
th a positive gate bias during irradiation, which increases long-term survival o
f holes by decreasing electron-hole recombination, and (ii) increases with an in
crease in the thickness of the oxide layer, which promotes overall electron-hole
generation. Supporting Electronics For any p-type MOSFET, the VTH that is requi
red to sustain a specific drain-to-source current (IDS) increases with irradiati

on. Electronics supporting the use of these devices are designed to adjust, meas
ure, and report the input voltage required to obtain a specific current flow. Th
e change in VTH before and after irradiation to sustain this current flow multip
lied by a calibration factor yields dose for the irradiation fraction. For an in
dividual MOSFET detector, the relationship between VTH and IDS can be especially
susceptible to temperature and the dose to which the detector has been exposed
(other dependencies will be discussed later). Temperature changes can affect VTH
by as much as 4 to 5 mV/8C; exposure to high irradiation levels can affect the
linearity of dose response. Dual bias dual MOSFET detector circuits were devised
to limit these dependencies (31). They have the additional benefit of enabling
the sensitivity of the detector to be modulated. Figure 2 shows a dual bias dual
detector circuit with two MOSFETs and their immediate supporting circuitry; the
y are typically fabricated on the same silicon substrate allowing the MOSFETs to
be very near to each other. Because of their proximity and very small size, the
MOSFETs are assumed exposed identically when irradiated. The voltage applied to
the MOSFET gates is different, as implied by the circuits name. Target drain-tosource currents may be the same or different when dosimeters are read. As with t
he single dosimeter discussion above, the VTH is adjusted to obtain a specific
a
Figure 2 Readout circuitry for dual bias, dual MOSFET. Abbreviation: MOSFET, met
al oxide semiconductor field effect transistor. Source: From Ref. 31.
IDS. The reported dose is equal to k(DVTH1 DVTH2), where k is a constant of prop
ortionality with units Gy/V. Patient Dosimetry The use of MOSFETS for dosimetry
of EBRT is relatively recent. Thomson first proposed the idea in 1984 (19). This
paper discussed the use of these devices in a phantom in the presence of electr
on and photon beams. Most published research has used commercial devices and app
aratus, some of them prototypes. Table 1 shows a samplea of the available litera
ture on the use of MOSFETs in photonbased EBRT. As can be seen from the table, t
here is very little published research discussing the use of these dosimeters in
vivo. Commercial vendors of MOSFET dosimeters include Best Medical Canada, Ltd.
(Ottawa, Ontario, Canada), Sicel Technologies, Inc. (Morrisville, North Carolin
a, U.S.), and REM Oxford, Ltd. (Eynsham, Oxford, U.K.). Best Medical produces MO
SFETs and supporting apparatus formerly marketed by Thomson-Nielsen (Ottawa, Can
ada). The Best dosimeters are primarily intended for entrance and exit dosimetry
, whereas Sicel Technologies produces MOSFET for entrance dosimetry (OneDoseTM)
and for implantable dosimetry [Dose Verification System (DVS1) dosimeters that a
re wireless and can be implanted directly into the tumor volume, then read telem
etrically]. Details of the operation and clinical application of the DVS implant
able dosimeter will be presented later in this chapter.
We only present relatively new papers on the use of MOSFET dosimeters in high-en
ergy external photon beams. Older papers are not discussed because their results
, although interesting with respect to the evolution of MOSFET dosimeter technol
ogy, may not be clinically applicable.
Future Developments I: Online Dosimetric Verification Table 1 A Sample of the Cu
rrent MOSFET Dosimeter Literature Reference Beddar et al. (32) Bharanidharan et
al. (33) Black et al. (34) Briere et al. (35) Butson et al. (36) Cheung et al. (
37) Chuang et al. (38) Halvorsen (39) Jornet et al. (40) Marcie et al. (41) Rama
ni et al. (20) Ramaseshan et al. (42) Rowbottom (43) Scalchi 1998 (44) Scalchi e
t al. (45) Scarantino et al. (46) Scarantino et al. (47) Soubra et al. (31)
a
253
Company Sicel Best Sicel Sicel REM REM Best Sicel Best Best Best Best Best Best
Best Sicel Sicel Best

Model DVS TN502RD DVS DVS TOT500 TOT500 TN502RD OneDose TN502RD TN502RD TN502RD
TN RDM 502 TN RDM 502 TN502RD TN502RD DVS DVS TN502RDa
Phantom Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes
In vivo No No Yes No No No No No No Yes No No No No No Yesa Yes No
Source/Energy
60 60
Co Co, 6 MV, 15 MV 6 MV, 18 MV, electrons 60 Co 6 MV, 18 MV 6 MV 6 MV 6 MV 18 MV
6 MV 60 Co, 6, 18, 25 MV, electrons 60 Co, 4, 6, 10, 18 MV, electron 6 MV 6 MV
6 MV 6 MV, 18 MV 6 MV, 18 MV 60 Co, electrons
In vivo measurements were done in canines. Abbreviations: MOSFET, metal oxide se
miconductor field effect transistor; DVS, Dose Verification System.
In Vivo Usage The following discussion is limited to the use of MOSFET dosimeter
s in high-energy, photon-based radiation treatments. MOSFET dosimeters have been
used to monitor kilovoltage (4850), electron (5153), and brachytherapy (24,5458) t
reatments. The use of MOSFET dosimeters in the context of brachytherapy will be
discussed later in this chapter. In vivo dosimetry can be divided into several s
ubareas: surface, transit, intraluminal, and implanted. The term in situ has bee
n used for intraluminal and implanted dosimetry. Surface dosimetry involves the
use of dosimeters on the patient surface at well-specified entrance and exit poi
nts along the path of a beam. Transit dosimetry requires the use of an external
measurement device, like an EPID, not in contact with the patient surface. Intra
luminal dosimetry involves placing the dosimeter inside a lumen, that is, in pro
ximity to the area to be treated; in the case of prostate irradiation, measureme
nts have been done with dosimeters placed in the urethra (58,59) and rectum (6).
Implanted dosimetry involves the placement of the dosimeter directly into the t
issue at a point of interest in either target or organ at risk. Implanted dosime
ters can be either permanent (46,47) or temporary (24,25). MOSFET dosimeters can
be used for all types of in vivo dosimetry, except transit dosimetry. In both s
urface and transit dosimetry, an external dose measurement is used to infer corr
ectness of treatment dose in the patient. If the dose measurements at the patien
t surface or at a detector panel agree with predicted values
from a treatment planning system (TPS), then the dose delivered to target or nor
mal tissues is potentially correct. If predicted and measured values do not agre
e, dose to target or normal tissues is probably incorrect. With intraluminal dos
imetry used in prostate treatments, the dosimeter is placed in tissues that are
of interest (i.e., rectum, urethra) and dosimetry to the prostate is inferred. T
he dosimeter may be volumetrically imaged during CT simulation, and dose to the
dosimeter can be calculated by the TPS. If the dose measured by the dosimeter du
ring treatment agrees with predicted dose, then the delivered dose is potentiall
y, but not guaranteed to be, correct. The only direct means of measuring dose at
an arbitrary point in target or normal tissue is through dosimeter implantation
. Because of their small size and the fact that they do not need an immediate ex
ternal connection to a power supply or reader, MOSFETs are extremely versatile a
nd ideal for implantation. They can also be used for almost any type of measurem
ent in which TLDs and diodes have been used, including very low energy radiation
treatments where photoelectric interactions predominate. Because of their high
atomic number silicon-based composition, diodes and MOSFETs exhibit overresponse
to very low energy photons. It has been shown for some MOSFET detectors that th
e maximum overresponse is on the order of about fourfold for energies of 33 keV.
No energy dependencies are seen for energies greater than 120 keV for some MOSF
ET dosimeters (60), yet others have shown an overresponse by a factor of two to
three at 120 keV. Possibly because of these photoelectric effects, lower

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Wiesmeyer et al.
energy applications may require calibration more often than those employing high
er energy (61). Dosimeter Placement For surface dosimeters, placement on the cen
tral axis of a beam or along a weight point ray is the norm. Typically, doses me
asured at the surface of the patient are used to estimate dose at the depth of m
aximum dose by multiplying raw dosimeter readings by calibration factors, field
size correction factors, dose rate factors, and so forth (TG62) (62). Intralumin
al or implanted dosimeter placement must be more thoughtfully considered. There
are issues of patient comfort and safety, dosimeter location stability, as well
as dosimetric considerations. One source of guidance for dosimeter placement in
target tissues is the International Commission on Radiation Units and Measuremen
ts (ICRU) reference point recommendations (62,63). The ICRU recommends point place
ment in tissue that is clinically relevant and representative of the dose distri
bution throughout the planning target volume (PTV), easy to define in a clear an
d unambiguous way, and in a location where the dose can be accurately determined
. Additionally, the point should be in a region where there are no large dose gr
adients. A point located at the center (or central part) of the PTV generally fu
lfills these requirements and is recommended as the ICRU reference point. Dosime
ter placement in normal tissue may be challenging, as the clinician is often mos
t interested in dose to normal tissues near the edge of the PTV, where dose grad
ients are likely to be significant. Characterization and Dependencies What follo
ws is an overview of important MOSFET dosimeter characteristics and dependencies
. In general, all commercial devices have properties that make them a good choic
e for high-energy photon dosimetry applications that have previously employed TL
Ds and diodes. Before using MOSFET dosimeters in the clinic, the medical physici
st should validate them under the conditions in which they will be used. Accurac
y MOSFET dosimeters have shown an achievable accuracy of 3% to 5% with respect t
o phantom measurements (3436,38,39,41,42,47). Practical accuracy during patient t
reatments (the difference from TPS estimation) may
b
be affected by cumulative errors from patient setup, patient motion, and dose ca
lculation (34,41,47). With implantable dosimeters, migration after implantation
has been reported (46,47), which may be an additional source of error.b Signal d
rift, known as fade, is present in all MOSFET dosimeters and must be taken into
account by reading dosimeters in the time frame recommended by the manufacturer.
Temperature Independence Some MOSFET dosimeters have been shown to be practical
ly free of temperature dependency over a wide range of temperatures (e.g., 20408C
) (31,44). Other dosimeters show modest dependencies that can be controlled by t
aking preirradiation and postirradiation readings at the same temperature (37).
This can be accomplished by placing the dosimeter on the patient and allowing it
s temperature to equilibrate to that of the patient over a period of about two m
inutes. This is probably good clinical practice, in general, for dosimeters. Ang
ular Variation Angular variation in dosimeter readings is due to interactions be
tween beam orientation and asymmetries in dosimeter design or construction. The
clearest example of a design asymmetry is with regard to surface dosimeters that
have a bulb side for build up and a flat side meant to be in contact with the p
atients skin. In practice, it is difficult to make a dosimeter that is totally sy
mmetric from both a design and construction standpoint, and angular variation ha
s been seen from 2% to over 27%, depending on dosimeter model (33,38,4245). Most
dosimeters have angular dependencies in the 2% to 8% range. Angular variation ef
fects may be difficult to avoid both in surface applications, where an unavoidab
le sloping patient surface may exist, and in implanted applications, where multi
ple beam angles make it impossible to eliminate angular variations between devic
e orientation and beam incidence. Implantable dosimeters may exhibit reduced ang
ular dependency due to full buildup provided by surrounding tissue. Dose and Ene
rgy Effects A critical aspect of a good dosimeter is that it exhibits a predicta

ble response to a specific quantity of dose, its response is independent of dose

rate, and it is relatively independent of energy spectrum changes within the en
ergy range of clinical interest. The response of modern
Dosimeter migration with respect to the DVS dosimeter can be eliminated if the d
osimeter is used according to updated manufacturers guidelines.
Future Developments I: Online Dosimetric Verification
255
MOSFET dosimeters has been shown to be linear with dose (31,33,3840,42) and indep
endent of dose rate (33,39,42). Chuang et al. (38) found no systematic deviation
from depth doses measured with an ion chamber for a 6 MV beam indicating that t
here may be no significant energy spectrum effects for the type of dosimeter tha
t was tested. There are two methods for achieving linear response: use of the du
al bias MOSFET circuit (e.g., Thomson-Nielsen/Best Medical) or use of a nonlinea
r calibration curve to effectively linearize the dosimeter response (e.g., Sicel
Technologies). Calibration Factors A calibration factor is a proportionality co
nstant with units of Gy/mV required to convert raw voltage readings in millivolt
s to dose. Some dosimeters may come precalibrated from the manufacturer (e.g., S
icel Technologies), while others are calibrated by the physicist (e.g., Best Med
ical). The dose response of some MOSFET dosimeters is affected by field size and
energy spectrum and may need to be individually calibrated for specific applica
tions (33,44). For others MOSFET dosimeters, a single calibration factor may suf
fice with a maximum of 5% error over a broad range of field sizes, energies, ori
entation, and depths (42). MOSFETs have a limited lifetime because of the increa
se of trapped charge in the oxide layer. Saturation occurs after a specific amou
nt of dose. For example, DVS implantable dosimeters can be used up to a cumulati
ve dose of approximately 80 Gy. THE USE OF MOSFET DOSIMETRY IN IMAGEGUIDED RADIA
TION THERAPY The use of implantable MOSFET dosimeters with imageguided radiation
therapy (IGRT) systems can serve two purposes. The first purpose is to investig
ate the precision, accuracy, and possible performance issues of IGRT systems. Th
e second purpose is to use the MOSFET dosimeter as a fiducial marker. For exampl
e, the DVS is known as a SmartmarkerTM because of its ability to be used both as
a dosimeter and a marker for certain IGRT treatments. The IGRT technique introd
uces an X-ray imaging system integrated to medical linear accelerators, allowing
patient imaging at the time of treatment. IGRT imaging systems that are in use
today may include: (i) megavolt EPID, (ii) Megavolt computed tomography (MVCT),
(iii) accelerator-mounted kilovolt (kV) imagers, (iv) kilovolt cone-beam compute
d tomography (CBCT), (v) ultrasound imaging systems, and (vi) imageless localiza
tion via beacons (53). In IGRT, the errors due to movement between imaging and dos
e delivery are minimized because of the imaging
system being available just before irradiation. This new technique requires new
quality assurance (QA) procedures to examine the precision and overall targeting
accuracy of the integrated system, which is related to uncertainties in leaf pl
acement and linear accelerator, isocenter stability with gantry, and coach rotat
ion (43,64). An ideal detector for IGRT should be visible under radiological exa
minations and should have a very small active volume in order not to perturb the
dose distribution. It should be possible to easily compare the measured dose wi
th point-dose calculations in TPSs. Moreover, immediate readout and reuse of the
detector should allow for repeated measurements without disturbing the experime
ntal setup (43,64). The imaging capabilities, overall precision, and accuracy of
an IGRT system must be known before safe clinical implementation can take place
. The investigation of IGRT system accuracy and precision requires the use of a
dosimeter, which can be seen in images produced by the system. Such dosimeters m
ust be very small, exhibit minimal angular sensitivity, and be capable of detect
ing small doses of radiation (~1 cGy). They must also have measurement precision
higher than that of the imaging and the delivery system (43). It has been repor

ted that MOSFETs are clearly visible on CBCT image (43,65). Other IGRT systems,
which are capable of visualizing MOSFETs, are MVCT, megavolt EPID, kilovolt EPID
, and ultrasound (34). Currently, in the prostate, placement of MOSFET dosimeter
s is most commonly performed with aid of ultrasound imaging. Rowbottom et al. us
ed MOSFET dosimeters in a phantom to study the precision and accuracy of CBCT (6
4). Their study focused on the capability of the CBCT system to determine the ef
fects of parameters such as gantry rotation accuracy and multileaf collimator (M
LC) position accuracy. These parameters, in turn, can affect the size of the sma
llest margin required for treatment delivery. The stationary unambiguous phantom
that was used means that patient positioning and movement were not taken into c
onsideration. The authors concluded that with the aid of image guidance, imaging
and delivery can be achieved with submillimeter accuracy in the anterior-poster
ior (AP) and lateral directions and to within a millimeter in the superior-infer
ior direction. The uncertainty in the delivery of dose was approximately 0.2 mm
in the axial plane and 1.0 mm for the superior-inferior plane. These additional
margins may be added to PTV margins to account for systematic uncertainty. In ad
dition to being able to be used for dose verification, MOSFET dosimeters have gr
eat potential of being employed as a fiducial marker. If so used, they may elimi
nate the need for conventional fiducial markers, such as gold seeds, for target
localization. The challenge of using a MOSFET dosimeter as a fiducial maker lies
in its relatively small size. For example, typical dimensions of the
256
Wiesmeyer et al.
Figure 3 Megavolt EPID image of a phantom containing 2-MOSFET dosimeters. With E
PID imager used in clinical mode exposed to 2 monitor units (MU), it was not possi
ble to visualize any of the dosimeter. Abbreviations: EPID, electronic portal im
aging device; MOSFET, metal oxide semiconductor field effect transistor.
DVS dosimeter, including structural material (the capsule), are 20- to 30-mm lon
g and 1 to 3 mm in diameter; however, the metal oxide or the active area is less
than 1 mm2 (34) and may not be in the region that is best imaged (e.g., the coi
l). The use of MOSFETs as fiducial markers is relatively recent, and there is ve
ry little published research on the subject. Because literature on using MOSFET
as fiducial marker is so scarce, the following experiment was designed to invest
igate the possibility of visualizing MOSFETs using different IGRT imaging system
s (Figs. 35). Two DVS MOSFET dosimeters were placed in a phantom. The phantom is
a polystyrene cylinder measuring 20 cm in diameter and 20-cm long. With EPID ima
ger used in clinical mode with 2 monitor unit (MU), it was not possible to visuali
ze any of the dosimeter as shown in Figure 3. However, using the imager in servic
e mode with high quality image option being selected, the dosimeters were poorly
visible. Figure 4 shows a kilovolt onboard imager (OBI) image of the same phanto
m. Both
Figure 4 Kilovolt OBI image of the same phantom in Figure 3 containing 2-MOSFET
dosimeters. Both dosimeters are visible in the image. Abbreviations: OBI, on-boa
rd imager; MOSFET, metal oxide semiconductor field effect transistor.
dosimeters are visible in the image. Figure 5 is CBCT image of the phantom. The
dosimeters are clearly visible in all three views: axial, sagittal, and coronal.
Suffice it to say that in both the kilovolt EPID image and the kV CBCT image th
e whole MOSFET dosimeters were visible rather than the active volume only. QA US
ING MOSFET DOSIMETERS MOSFET dosimeters can be used as a QA tool in radiation th
erapy. Their use may include both patient-specific QA and equipment QA. Patient
QA refers to practice of comparing measurements of predicted TPS dose to patient
dose measurements. These measurements may either occur on a daily basis or one
to several times during treatment.

Figure 5 Kilovolt CBCT image of the same phantom in Figures 3 and 4, containing
2-MOSFET dosimeters. The dosimeters are clearly visible in all three views: axia
l, sagittal, and coronal. Abbreviations: CBCT, cone-beam computed tomography; MO
SFET, metal oxide semiconductor field effect transistor.
Future Developments I: Online Dosimetric Verification
257
There are several factors that make MOSFET dosimeters an attractive choice for i
ntensity-modulated radiation therapy (IMRT) QA. These factors are: fast and simp
le dose reading procedures, the small size of the detector especially when used
in small homogenous dose regions, and dose linearity. The disadvantage of using
MOSFET for IMRT QA lies in the fact that many dosimeters would be necessary to o
btain a complete isodose distribution. Chuang et al. used MOSFET dosimeters for
clinical IMRT dose verification concurrently with ion chambers (38). It was repo
rted that the difference between calculated dose and measured dose is 5% when us
ing MOSFET dosimeters, while the difference is 3% using ion chamber measurements
. The microMOSFET (TN-502RDM) has been characterized for its application to inte
gral system tests for IGRT (43,64). The position of peak response to a 0.08-mm s
lit of radiation was determined, and the dosimeter was found clearly visible on
the CBCT images with no added artifacts (43). The ability to locate the MOSFET a
ctive volume to within 0.2 mm by CBCT allows their use in an integral system tes
t in a phantom containing an array of microMOSFETs, to investigate the precision
of the IGRT system components in absence of patient-related errors (64). The pr
actically isotropic angular response of microMOSFETs allows their application fo
r noncoplanar beams (42). Highsensitivity TN microMOSFETs (TN-1002RDM) were used
to measure dose on patients undergoing CBCT using the X-ray volume imaging (XVI
1) system integrated with a medical accelerator (Synergy, ELEKTA). The average a
greement between measured and estimated skin doses of five patients were found t
o be within 5% (66). MicroMOSFETs with radiopaque markers have been introduced in
tomotherapy patient QA (67) to verify the skin doses and to check the skin spar
ing approach achieved by a new treatment planning technique. MOSFET dosimeters c
an also be used to perform QA measurements for linear accelerator output. The ve
rsatility and accuracy of these dosimeters facilitate a quick check of the outpu
t of the accelerator, which might be important if machine output is reported to
be out of specification during daily QA. Normally, absolute output measurements
require elaborate and time-consuming equipment setup (ionization chambers) and m
easurements using the TG-51 calibration protocol (63). With MOSFET dosimeters, a
rapid QA check of output measurements might be performed much more easily. A ty
pical measurement setup would be a 10 10 cm2 beam, 100-cm source-to-surface dist
ance, water or solid phantom as medium, and 200-cGy dose. Dosimeters would be pl
aced at depth of maximum dose, which is the typical calibration point for linear
accelerators. Briere et al. reported the use of MOSFET dosimeters for performin
g accelerator output QA (35). The measurements were performed for both 6 and 18
MV. Halvorsen also used MOSFET dosimeter to
measure absolute outputs for both 6 and 18 MV beams. They observed an average diff
erence of less than 2% compared to manufacturers calibration when measured at the
depth of maximum dose (39). In summary, IGRT systems alone cannot detect misadm
inistrations that might occur, such as improper dose calibration, incomplete IMR
T QA, or MLC leaf sequence errors. However, when IGRT systems are used in combin
ation with an implantable dosimeter, such as MOSFET, this may help to facilitate
more accurate dose delivery. DESIGN AND OPERATION OF AN IN SITU DOSIMETER SYSTE
M Until recently, it has been impractical to measure actual delivered dose for e
ach treatment session at a tumor site below the skin surface. Now, however, the
first wireless, permanently implantable dosimeter has been developed to measure
external beam radiation dose received at the target tissue. This dosimeter has b
een clinically implemented for prostate and breast cancer treatments, with other
target sites likely to follow. The implantable dosimeter, DVS, uses a p-channel

MOSFET as the radiation-sensing element. MOSFETs require minimal power to operat

e making it possible to provide enough power by inductive coupling from an outsi
de coil directly to an antenna contained within the dosimeter. The DVS implantab
le dosimeter can be used as an adjunct to treatment planning and radiation deliv
ery techniques to verify that the actual dose received at the tumor volume on a
daily basis is within the acceptable dose range prescribed. Dosimeter Design The
DVS dosimeter is encapsulated in a hermetic, biocompatible glass capsule (Fig.
6). The implantable dosimeter consists of an antenna and a microelectronic hybri
d
Figure 6 Implantable dosimeter DVS and internal components.
258
Wiesmeyer et al.
The dosimeter RFID system transmits radiation dose data that is utilized by the
reader to convert the voltage shift into absorbed dose-to-tissue at the MOSFET l
ocation using dosimeter specific calibration values. Telemetric Data Acquisition
System Although the DVS dosimeter requires power to operate, it is often referr
ed to as a passive device since it contains no battery. As with many RFID produc
ts, dosimeter power is derived from the low frequency magnetic field generated b
y the DVS reader antenna. The magnetic field is coupled to the ferrite core ante
nna inside the dosimeter capsule, which generates a voltage proportional to the
magnetic field intensity. To maximize the antenna voltage and improve reading ra
nge, the antenna is tuned to the frequency generated by the reader. In addition
to providing dosimeter power, the magnetic field generated by the reader provide
s the bidirectional communications interface between the reader and dosimeter. T
o facilitate communication with multiple dosimeters, the reader uniquely address
es each dosimeter via a 32-bit identification (ID) number. Once the DVS reader b
roadcasts the ID to all dosimeters in the magnetic field, only the dosimeter wit
h a matching ID number responds with sensor data. The reader can also request da
ta from multiple radiation sensors and an onboard temperature sensor. The reader
transmits packets to the implantable dosimeter by amplitude modulation of the m
agnetic field. The amplitude modulation is decoded by the dosimeter ASIC receive
r circuitry. After each valid bit is received, the ASIC receiver determines if t
he dosimeter is being addressed. Once a dosimeter is addressed, the ASIC state m
achine begins the data acquisition and packet transmission process. The ASIC con
tains a 14-bit sigma delta analog-to-digital converter (ADC) to digitize the MOS
FET radiation sensor threshold voltage. Once the analog-to-digital conversion pr
ocess is complete, the ASIC state machine creates a transmit packet consisting o
f the 32-bit ID, 14-bit sensor data, and a 16-bit CRC (cyclic redundancy check s
um) value to ensure packet transmission integrity (68). The dosimeter data packe
t is transmitted to the reader using a method commonly referred to as backscatte
r modulation. Unlike a traditional radio transmitter that produces a modulated r
adio frequency signal to transmit information, backscatter modulation utilizes t
he magnetic field generated by the reader to transmit data. The link between the
reader antenna and the dosimeter antenna can be modeled as a loosely coupled tr
ansformer with the reader antenna forming the primary winding and the dosimeter
antenna forming the secondary winding. The dosimeter transmits data by loading a
nd unloading the antenna circuit. The load variations are reflected to
Figure 7 Radiographic image showing two DVS dosimeters. Source: From Ref. 68.
assembly that contains the MOSFET radiation sensors, customized circuitry, and s
everal passive components (Fig. 6). After the antenna is connected to the hybrid
, the antenna/hybrid assembly is inserted into an epoxyfilled capsule prior to l
aser sealing. To prevent moisture ingress into the capsule, the open end of the
capsule is laser-sealed and the hermetic seal is tested using a helium leak dete
ction process. The dosimeter is commercially available in a capsule dimension of

2.1 mm in diameter and 20 mm in length. Clinical trials on patients were perfor

med with a larger dosimeter design (3.25 mm 25 mm). The dosimeter and components
are visible on kV images, CT, and ultrasound (Fig. 7). The detailed structure o
f the MOSFET as well as the technical aspects of the dosimeter system has previo
usly been described (34,46,47). The DVS system consists of an implantable, telem
etric radiation dosimeter and an external reader system to communicate with the
dosimeter (Fig. 8). Dose is communicated to the external reader using a reader w
and (reader antenna) by radio frequency identification (RFID) technology. A cust
omized application-specific integrated circuit (ASIC) was developed to digitize
the MOSFET voltage readings and transmit the data to a reader system.
Figure 8 DVS reader system and reader wand. Abbreviation: DVS, dose verification
system.
Future Developments I: Online Dosimetric Verification
259
the transformer primary (reader antenna) where sensitive receiver circuitry loca
ted in the reader can detect and demodulate the data transmitted by the dosimete
r. The dosimeter-induced voltage perturbation on the reader receive antenna is u
sually in the range of 10 to 100 mV depending on transmit power and the distance
between the dosimeter and the reader antenna. The reader antenna voltage that p
roduces the magnetic field is greater than 600 V, which mandates the use of a se
nsitive receiver to detect the 10 to 100 mV data modulation signal superimposed
in the 600 V drive voltage. The reader receive circuitry contains filters to mit
igate the effect of external noise sources from electronic equipment. Dosimeter
Calibration Since calibration of the implanted dosimeter cannot be accomplished
once it is inside a patient, precise methods for precalibrating these devices ha
ve been developed and tested. The DVS dosimeter is currently calibrated for use
with typical EBRT doses and irradiation protocols: doses of 150 to 250 cGy once
per day. The calibration protocol takes into account the variables that can affe
ct this type of dose delivery to increase the performance and accuracy of the do
simeter response in a patient. New calibration protocols are being developed for
use of the dosimeter with hypofractionated doses and other radiation sources (I
r-192). Since the amount of trapped charge is proportional to the ionizing radia
tion dose, the MOSFET threshold voltage shift may be used to measure dose. A MOS
FETs radiation sensitivity is the term used to describe the change in threshold v
oltage caused by a given amount of radiation. More precisely, the radiation sens
itivity of a MOSFET is defined as the change in threshold voltage per unit of do
se applied, expressed typically in mV/cGy. Ionizing radiation affects a MOSFET b
y changing the threshold voltage required to enable current flow from the source
to the drain of the MOSFET (Fig. 1). To determine the radiation sensitivity (Ra
dsens) of the MOSFET, the threshold voltage is measured before and after dose is
applied (Eq. 1): Radsens Vpost Vpre , D 1
Figure 9 Relationship between radiation sensitivity and cumulative dose. Source:
From Ref. 68.
where Vpost is the threshold voltage after the dose is applied (mV), Vpre is the
threshold voltage before the dose is applied (mV), and D is the dose applied (c
Gy). As radiation exposure increases, more charge is trapped in the gate oxide.
The positive space charge resulting from the trapped charge repels holes and thu
s decreases the chance of the positively charged holes being trapped near the Si
/SiO2 interface. The net effect is a reduction in voltage radiation sensitivity
(VTH/D) for increasing
cumulative dose. This change in threshold voltage of the MOSFET is cumulative as
a function of dose. As a result, the radiation sensitivity is not constant for
the life of the MOSFET and becomes a nonlinear function of the threshold voltage

(Fig. 9). The DVS dosimeter saturates at around 80 Gy. A 60Co source is used for
calibration because of its consistent dose. As part of the manufacturing process
, the nominal radiation sensitivity of each DVS dosimeter is measured using 60Co
. These values are used to correct the voltage response of each individual dosim
eter. This process allows each dosimeter to be fine-tuned to improve its accuracy.
The calibration of the DVS also requires knowledge of the dose response curve u
p to 80 Gy to determine the relationship between the radiation dose applied and
the change in threshold voltage. This response curve is determined for each dosi
meter lot production, since small changes in production can affect the dosimeter
response. Sample dosimeters from each lot are tested over the full-dose range t
o develop this dose response curve. An independent radiation calibration laborat
ory verifies the resulting calibration for each lot. Once verified, the calibrat
ion coefficients are applied to each dosimeter within a lot. Each dosimeter has
a calibration certificate detailing the specific accuracy as a function of cumul
ative dose based on its lot production. The dosimeter performance has been valid
ated by in vitro studies (32,35,68). The factory calibration protocol determines
the calibration curve for each dosimeter used in a typical external beam radiat
ion treatment regime. In addition, the curve characterizes the response of the M
OSFET, including fade effects, for daily radiation delivery. Fade is the term us
ed to describe the decrease in the trapped charge in a MOSFET as a function of t
ime after radiation exposure. Test measurements on the MOSFET yield less than 2%
fade over 20 minutes. The dosimeters are read within two to three minutes after
exposure during calibration. Since
260
Wiesmeyer et al.
the MOSFET response can be affected by temperature, the dosimeters are calibrate
d for use at 378C (human body temperature). Each DVS dosimeter also contains a t
hermal sensor. This sensor is calibrated during the manufacturing process and ca
n be used to monitor the temperature during the calibration process. To measure
absorbed dose by the dosimeter, a predose and postdose reading is acquired. The
predose reading is acquired prior to radiation delivery using the hand-held read
er wand, which contains the antenna (Fig. 8). Similarly, the postdose reading is
acquired after radiation delivery. To minimize fade effects, the postdose readi
ngs need to be acquired within 10 minutes after irradiation. These readings are
then used to calculate the daily dose fraction that is reported for each treatme
nt session by relating the voltage shift in the calibration curve. The daily fra
ctional dose values are stored in a database and are summed to calculate a cumul
ative dose. Radiation Characteristics The dosimeters radiation characteristics ha
ve been set forth in several published studies. A summary of the basic response
of the dosimeter as a function of angular incidence, temperature, attenuation, a
nd energy is provided in this section. Additional details on the relevant method
ology and test data can be found in each of the references provided below. The d
osimeter angular dependency has been found to be very small (<1.5%) for radiatio
n incident perpendicular (radial) to the dosimeter long axis. The maximum angula
r dependence of approximately 6% is obtained for radiation that is incident para
llel to the dosimeter axis traveling through the coil and electronics (Fig. 10).
This larger deviation is expected since the coil and electronics are in the dir
ect path between the radiation beam and the MOSFETs and can be eliminated for tr
aditional clinical radiation treatments by implanting the dosimeter in close par
allel alignment with the body axis (<308 off axis, as has been done in the pilot
studies and clinical trial patients) (46,47,68). Previous in vitro studies for
the implantable dosimeter have shown a large sensitivity to temperature variatio
ns during irradiations (32). This relationship did not pose a problem during tem
perature controlled in vitro testing or in vivo applications since the body regu
lates temperature to a constant 378C. The previous DVS dosimeter used a MOSFET w
ith a large threshold voltage temperature coefficient, resulting in approximatel
y an 8 to 20 cGy variation for each 18C difference in predose and postdose readi

ng temperature during a 200-cGy irradiation (46). The new commercially available

DVS utilizes a MOSFET with a low threshold voltage temperature coefficient,
Figure 10 DVS radiation (A) radial and (B) longitudinal angular dependency. Sour
ce: From Ref. 68.
resulting in approximately 1.0 to 3.3 cGy variation for each 18C difference duri
ng a 200-cGy irradiation. Due to the lower temperature variation dependency of t
he current dosimeter, it is possible to perform measurements in a well-insulated
phantom at room temperature. Testing has shown that the DVS dosimeter is approx
imately 3.32% more sensitive (higher dose reading for same applied dose) when ir
radiated at 378C versus 238C (68). The testing methodology used to develop this
correction factor was validated up to 8 to 10 sessions of radiation dose (1620 Gy
). Additional studies would have to be performed to validate the room temperatur
e correction and simulate a complete course of radiation therapy (2050 sessions)
at room temperature. Nevertheless, this correction factor provides a useful mech
anism to perform testing in a simple water equivalent phantom at room temperatur
e. The components of the DVS dosimeter (ferrite, copper coil, analog circuit boa
rd) could cause some attenuation of radiation dose on the side of the dosimeter
opposite the radiation field (68). It has been found that the attenuation is sim
ilar to surgical suture or gold seed markers. Testing showed that the maximum do
se reduction for 6 MV radiation occurs right next to the dosimeter, as with any
other metallic surgical marker or implant. At 2 mm below the dosimeter, a maximu
m dose reduction of 3.7% is encountered directly under the antenna. The dose rec
overs to less than 1% reduction at 7.5 mm below the dosimeter. At the depth of 9
.4 mm, no dose perturbation was measured. This data was the result from only one
beam of radiation. In clinical practice, radiation is directed to the body from
Future Developments I: Online Dosimetric Verification
261
different angles, further reducing the overall effect of dose attenuation around
the dosimeter. The dosimeter has shown small energy dependency within the clini
cal MV energy range if implanted at depths beyond dmax. The ratio of sensitiviti
es between 60Co and 6 MV has also shown a small energy dependency (~0.5%). The D
VS has decreased sensitivity (~1%) when irradiating with 18 MV as compared with
6 MV. Because of this small energy dependence, the calibration of the dosimeter
is set such that the 6-MV dose response is biased to read 0.5% higher while the
18-MV dose response is biased 0.5% lower. Phantom testing has been performed to
confirm the performance of a calibrated dosimeter for different energy ranges (6
8,69). Studies have shown that MOSFETs have a stronger energy dependency below 1
30 kV, increasing three to four times as the energy decreases below the 100 kV r
ange (70). A recent report demonstrated that the DVS MOSFET dosimeter can overre
spond by a factor of 2 to 3 in the kV energy range due to the predominance of th
e photoelectric effect (71). It has been documented that pelvic kV CBCT images c
an deliver additional dose to patients in the range of 3 to 6 cGy (72). This dos
e can be detected and measured by the DVS dosimeter, resulting in additional dos
e reading in the range of 12 to 16 cGy to the center of a 30 cm square shaped ph
antom (68). Additional data has showed that standard two-dimensional kV portal i
mages deliver negligible or nonmeasurable dose to the DVS. Therefore, if CBCT is
used on a frequent basis for patient treatment, this dose can be considered for
the final dose calculation or can be eliminated by taking the predose reading a
fter the CBCT imaging is performed. For MV CBCT or MV planar images, the dosimet
er could easily track the additional dose delivered to a patient due to its line
ar response in this energy range. ANIMAL STUDIES OF IN SITU DOSIMETRY Relatively
little in situ dosimetry has been performed in animal models. Gladstone et al.
(25) tested a miniature implantable MOSFET dosimeter in mice during RIT trials.
More recently, Scarantino et al. (46) have tested an implantable telemetric MOSF
ET dosimeter in vitro and in 10 dogs. Each canine subject presented with a spont
aneous, malignant tumor to be treated in 16 fractions with EBRT. One telemetric

sensor was implanted in each tumor. In three dogs a second sensor was implanted
in nearby normal tissue. The experiments were designed to observe (i) stability/
mobility of the implanted devices in vivo, (ii) successful readout, and (iii) bi
ocompatibility with a tissue environment. During the experiments, two of the sen
sors in two different subjects were observed to
migrate by 3.5 and 7 cm, respectively. This underscores the importance of anchor
ing the devices securely. Dosimetric measurements showed instances of daily vari
ation in excess of 10%, which the authors attributed to inaccuracies in the cali
bration procedure used for the tests. In one of the three canine subjects with t
wo redundant dosimeters, an instance of daily fractional underdosage on the orde
r of 20% to 30% was indicated by both dosimeters. The authors suggested that the
coincident measurement of underdose in both sensors was indicative of a true do
se delivery error, but in the absence of corroborating entrance/exit dose measur
ements, this would not have been provable. HUMAN STUDIES OF IN SITU DOSIMETRY Al
though the ability to measure radiation doses in vivo has been in existence for
many years, it has only recently become possible to measure actual doses at the
tumor/ target site. Thus, for the purposes of this discussion, it may be helpful
to distinguish in vivo dosimetry from in situ dosimetry. Historically, in the f
ormer case, dose measurements are determined with detectors, which are placed on
the skin to measure entrance and/or exit dose of a beam, and the dose to the ta
rget within this beam is calculated using dose reconstruction algorithms (7376).
The reports claim an accuracy of less than or equal to 5%, but utilizing the met
hod of entrance and exit can be time consuming and may not be able to identify t
he effect of specific uncertainties such as organ movement or deformation and tr
eatment or patient setup errors. More recently, EPIDs were developed for verific
ation and correction of daily radiotherapy treatment alignment (7780). The portal
images can be used to verify two-dimensional dose distributions (77,78), since
the pixel signals are related to the amount of radiation transmitted through the
patient. The available in vivo methods described do not provide an actual measu
red dose, but rather an extrapolation of dose. In situ intracavitary dosimetry,
involving the placement of probes into the bladder and/or rectum prior to treatm
ent, has recently been described (6,81,82). The probes may contain diodes, which
can transmit normal tissue dose data electronically (6,81), or they may contain
ionization chambers (82). Accurate repositioning of the probe prior to each tre
atment fraction is critical and requires onboard imaging capabilities for maximu
m accuracy (82). These approaches may be useful for normal tissue dose measureme
nts immediately adjacent to the prostate, but do not provide information on dose
to the malignant target volume. They typically have been utilized during only a
few fractions per patient, rather than throughout an entire course of treatment
. Weber et al. (81) assessed the dose delivered to the anal canal using thermolu
minescent dosimetry (TLD) during
262
Wiesmeyer et al.
the initial dose of external beam irradiation in 31 patients. TLD values differe
d by a mean of 5.8%; however, differences of at least 10% were noted in eight (2
6%) patients and 15% in three patients. Hayne et al. (6) measured the in situ do
se using a rectal probe placed in the anorectum in nine patients (during the fir
st five fractions in five patients and two fractions in four patients) with canc
ers of the prostate, bladder, cervix, or uterus. The probe contained five n-type
photon-detecting diodes placed at 2-cm intervals from the anal verge. The avera
ge measured doses in the target volume (center) were all within 7% of predicted
doses, whereas the dose at the edge of the target volume varied significantly (68
% to 68%). Outside the target volume, doses up to 0.3 Gy were measured, which wer
e not predicted by their planning system. One of the difficulties encountered wi
th these methods was to insure consistency in the daily placement of the dosimet
ers. More recently, Wertz et al. (82) addressed this problem by using CBCT and a

natomical landmarks to insure consistent placement of ionization chambers in the

rectum. They measured the in situ dose in seven patients (21 dose measurements)
undergoing IMRT for prostate cancer. They found that there was good agreement (
1.4 4.9%) between measured doses and predicted doses when the dose was compared
with a point relative to the isocenter. However, the mean dose deviation at corr
esponding anatomic positions was 6.5 21.6%. The daily use of probes described ab
ove can be problematic, since it is possible, due to rectal irritation developin
g as treatment progresses, that probe insertion may become too uncomfortable for
some patients. Therefore, a more convenient method, as described below, would a
ppear more suited to daily use, especially considering the influences of long tr
eatment times with IMRT and the potential for intrafractional variation in deliv
ered dose. In recent years, high dose per fraction EBRT has experienced resurgen
ce, and several early trials have been published using this approach in the trea
tment of prostate cancer. High-dose single-fraction EBRT has not been reported,
to our knowledge, as a treatment for prostate cancer. It has been used in the in
traoperative setting for the treatment of several different malignancies. Ciocca
et al. (83) reported on the use of in situ dosimetry using radiochromic film pl
aced in the operative bed in a series of 54 patients with early stage breast can
cer, who received 21 Gy in a single fraction to the surgical bed following quadr
antectomy. In general, they found good agreement between measured and expected d
ose (mean deviation 1.8 4.7%), with deviations larger than 7% in 23% of cases. T
hey used dosimetry information to define an action plan if errors of greater tha
n or equal to 7% were detected in two consecutive patients, or greater than or e
qual to 10% in one patient. Comparable results were recently reported using a MO
SFET system (51). These
authors reported the results of in situ dosimetry in a group of 12 patients trea
ted with single fraction intraoperative electron beam radiotherapy for breast ca
ncer. Measured values were within 5% of calculated doses in all but one case, an
d the one discrepancy was felt to be due to improper positioning of the dosimete
r. The characteristics of dual MOSFETs such as small size, full buildup isotropy
, instant readout, waterproof design, and temperature independent response make
these detectors suitable for measurements in high-dose gradient fields present i
n both low dose rate (LDR) and HDR brachytherapy treatments. The high sensitivit
y microMOSFET dosimeter (TN-1002RDM) with the high sensitivity bias supply was u
sed for in vivo dosimetry during LDR prostate brachytherapy (58). The MOSFET has
been inserted inside a urinary catheter placed in urethra. The dosimeter sensit
ivity was found to be about 31 to 33 mV/cGy for a low-energy 125I source. Patien
t data demonstrated that the maximum initial dose rate at the urethra varied bet
ween 10 and 16 cGy/hr, corresponding to a total absorbed dose of 205 to 328 Gy.
The intraurethra initial dose rate measurements with MOSFETs provided evaluation
of the overall quality of the implant, by analyzing the maximum dose received b
y the urethra, the prostate base and apex coverage, and the length of the prosta
tic urethra being irradiated. The standard sensitivity 5-MOSFET linear array (TN
-252LA5) was also used for patient urethral dose verification in prostate HDR br
achytherapy for single and multiple fractions (600 cGy per fraction) (84). An ex
cellent correlation of 2.8% was found between the MOSFET readings and the treatm
ent planning dose calculations. In vivo dosimetry with MOSFETs in brachytherapy
has been proven effective to indicate possible treatment complications due to ex
cessive dose to the urethra. Scarantino et al. (46) reported the first permanent
ly implantable in situ dosimeter. This device uses a MOSFET dosimeter and is rea
d telemetrically each day after treatment as described in the section Design and
Operation of an in situ Dosimeter System. The design and operation of the device
are reviewed above. Its use in patients was first reported by Scarantino et al.
(47) in 2005 and was updated by Black et al. (34) that same year. Initial FDA-ap
proved pilot studies contained a total of 18 patients who underwent implantation
of one or two dosimeters into the gross tumor volume and/or surrounding normal
tissues. Sites implanted were lung (n
3), rectum (pelvis) (n 4), prostate (n
4),
breast (n 6), and thigh (n 1). A total of 31 sensors were implanted. Treatment
planning CTs were obtained after implantation, and the dosimeters were included

as anatomic structures in the treatment plans for the purpose of calculating the
expected dose. The accuracy of the system was confirmed via in vitro phantom ex
periments, and it was determined that under idealized
Future Developments I: Online Dosimetric Verification
263
conditions, an uncertainty level of 5%, consistent with machine commissioning, is
achievable. Considerable variability, however, between observed versus expected
dose was observed in the patients. In two-thirds of the patients, dose deviatio
ns of more than 5% were recorded for at least 40% of treatments. Since significa
nt movement of the dosimeter was noted in only one patient in whom the device wa
s implanted in necrotic tissue, this was not felt to be the cause of the dose de
viations. Patients were not treated using image-guided techniques, but rather we
re aligned using skin tattoos and bony landmarks. The nearGaussian distribution
of dose readings suggested random rather than systematic error as the cause. On
the basis of the results of the pilot studies, a pivotal study was instituted (8
5). The objectives as well as study parameters were similar to the pilot study e
xcept that the study was limited to prostate and breast cancer patients. A total
of 59 patients were entered and 119 dosimeters implanted. The protocol recommen
ded that each patient receive two dosimeters, one associated with the tumor (pro
state capsule) and one in normal tissue (12 cm from capsule in prostate). A trans
perineal approach, employing a specially designed canula and trochar, was used w
hen inserting the DVS into the prostate. A total of 1749 daily readings were obt
ained from prostate cancer patients; 1308 during treatment of the primary field
and 441 during reduced field. A variability of greater than 7% between observed
and expected daily readings was noted in 23% of patients during large field irra
diation and in 35% during treatment of the boost field. IMRT was utilized during
the boost field. More importantly, a difference of greater than or equal to 7%
variability in the cumulative dose occurred in 27% (8 of 29) of the patients dur
ing large field and in 38% (7 of 18) during boost-field irradiation. Finally, a
total of 18 out of 29 patients were treated with IGRT techniques; two with trans
abdominal ultrasound (BAT, B-mode acquisition and targeting), three with CBCT, a
nd 13 with implanted gold fiducials and MV imaging. The use of IGRT techniques d
id not result in any appreciable difference in the degree of variability between
measured and expected dose (86). Further, in most patients the pattern of dose
measurements was consistent (either overdose or underdose), which was consistent
from day to day and observed throughout the treatment, especially during irradi
ation of the boost field. The pattern in the first three to five fractions was i
ndicative of the treatment course. The high variability in dose observed during
irradiation of the boost field is probably related to both the tight fields and
organ movement and could be compounded if patient is not properly positioned for
treatment. Errors in treatment delivery can occur for several reasons including
organ movement and deformity, which are difficult to control, and patient posit
ioning and treatment planning errors, which are more controllable. Regardless of the cause, unle
ss there is documentation of the dose measurement, the physician will not be awa
re of the variability and its possible impact on patient treatment. PRACTICAL GU
IDELINES FOR IMPLEMENTATION OF IN SITU DOSIMETRY IN THE CLINIC The DVS is intend
ed for use in radiation therapy to verify treatment planning and radiation dose
to target tissues and organs in or near the irradiated areas of a patient. The a
ccuracy of the DVS is less than or equal to 5.5% (2%) up to 20 Gy and less than o
r equal to 6.5% (2%) up to 74 Gy. The accuracy decreases slightly for doses beyon
d 74 Gy. The lot-specific accuracy is detailed in the calibration certificate in
cluded with each dosimeter. Readings deviating from the prescribed dose by more
than the specified accuracy could indicate a trend that should be noted. The phy
sician should decide the maximum acceptable percent discrepancy between the pres
cribed and measured dose. On the basis of the inherent accuracy of the device, g

uidelines have been developed to provide the physician a frame of reference chec
klist to help with the clinical implementation and data analysis. The guidelines
for clinical implementation detailed below should act as a reference point only
since each clinical site should review and implement their own guidelines for D
VS clinical data analysis. Guidelines for clinical implementation: I. Dose measu
rements should be made after every dose of radiation within 10 minutes of dose d
elivery (optimally 23 minutes after dose delivery). a. Daily dose measurements he
lp identify a pattern for the patients radiation treatment. II. The recorded dose
measurements should be reviewed by the physician at least once every week. a. T
he medical physicist should also include a review of the DVS data as part of the
ir weekly chart checks. b. It is recommended to carefully review the data obtain
ed during the first three to five fractions of a new patient treatment. III. Sys
tematic dose deviations of greater than or equal to 7% over a number of fraction
s should result in a reevaluation of the treatment plan, patient setup, and equi
pment function to determine the reason for the variation. Replanning and/or resi
mulation of the patient should be considered after careful evaluation of the abo
ve-mentioned parameters. The following steps can be referenced if a systematic e
rrant daily dose reading pattern is obtained:
264
Wiesmeyer et al.
a. Treatment plan: 1. Verify that the MOSFET area of the DVS is properly identif
ied on the treatment plan. Use diagnostic film, kV CT scout images to verify exa
ct position of the DVS. 2. Verify that the appropriate predicted dose value has
been entered into the DVS system. 3. Verify that the prescription isodose line h
as been incorporated in the predicted dose value. 4. Include and/or review effec
ts of heterogeneity corrections. 5. Evaluate dose gradient around the DVS (~3 mm
). 6. Verify plan transfer to record and verify (R&V) system. 7. Review IMRT QA
results, if applicable. b. Patient setup: 1. Verify that the patient setup posit
ion corresponds to patient simulation position. 2. Evaluate patient source-skin
distance (SSD) parameters, AP separation, patient weight changes. 3. Verify digi
tally reconstructed radiographs (DRRs) and port films. 4. Verify appropriate blo
cks or MLC shape are being used, wedges, etc. 5. Evaluate patient treatment prep
aration (bladder/ bowel filling if applicable). 6. Evaluate treatment margins an
d replan, if needed. 7. If using IGRT: i. Verify that shift or patient adjustmen
t did not exceed department limits. ii. Compare distance of adjustment with dist
ance to agreement on treatment plan. iii. Identify if there is a correlation bet
ween adjustment of patient and significant variance. c. Equipment function: 1. V
erify any previous machine repairs performed. 2. Review linear accelerator daily
output and beam performance records. 3. Verify that the treatment and verificat
ion system are working properly. 4. Verify IGRT equipment QA and functionality.
d. If necessary, repeat CT simulation: 1. Verify DVS position is within same iso
dose region as planned (include heterogeneity factors, if appropriate). 2. Compa
re current DVS position with original pretreatment position. IV. Random variatio
ns of greater than or equal to 7% observed over a number of fractions should res
ult in
an evaluation of positioning consistency, patient movement and/or organ movement
during treatment. Replanning and/or resimulation of the patient should be consi
dered after careful evaluation of the abovementioned parameters. The following s
teps can be referenced if a random errant daily dose reading pattern is obtained
: a. Evaluate patient positioning consistency. 1. Verify that treatment position
corresponds to patient position during simulation. 2. Verify day-to-day patient
skin SSD parameters and anatomic changes because of weight. 3. Verify DRRs and
port films. It is recommended to repeat port films as an initial troubleshooting
maneuver. 4. Evaluate patient daily treatment preparation (bladder/bowel fillin
g, if applicable). 5. For IGRT: i. Verify shift or patient adjustment daily for
consistency and operator accuracy. ii. Verify that patient adjustment did not ex

ceed clinic limits. b. Evaluate patient movement during treatment. 1. Evaluate i

mmobilization devices used. 2. Evaluate: deep breathing or coughing during treat
ment, irritable patient. 3. Evaluate treatment margins. c. Organ movement during
treatment. 1. Reevaluate treatment margins. 2. Review treatment plan. 3. Re-CT,
if necessary. V. A single error reading of greater than or equal to 10% should
be immediately reported to the radiation oncologist, and it is recommended that
an evaluation of the patient setup, treatment plan, setup images, and equipment
should be undertaken prior to the next patient treatment. a. Notify the physicia
n in case error readings exceed a certain threshold (10% recommended). VI. Durin
g highly conformal radiation therapy, significant variation in measured dose may
be observed with dosimeters placed outside the prescription dose area due to po
ssible high dose gradients around the dosimeter. a. If the dosimeter is within o
r very close (<3 mm) to the field edge or in normal tissue outside the field, th
e DVS readings could vary due to the field dose gradient. b. Dosimeters placed i
n dose gradient regions should be evaluated with a similar process as that used
for
Future Developments I: Online Dosimetric Verification
265
l
IMRT QA evaluation, that is, taking into account that very small positioning err
ors (<3 mm) could lead to high dose reading variations (gamma analysis function:
local dose difference, and the distance to agreement).
FUTURE DEVELOPMENTS Internal in situ dosimetry presents two new opportunities: (
i) the direct verification of dose delivery for those patients amenable to the p
rocedure, and (ii) more accurate validation of conventional entrance/exit dosime
try techniques. Once there is sufficient confidence in the accuracy of implanted
telemetric sensors, they can be used as a ground truth to evaluate external dosim
etry. This would extend some of the benefits of internal dosimetry to those pati
ents who are not suited to the invasive procedure. Recently, a novel concept of
MOSFET-based fourdimensional in vivo dosimetry in radiotherapy has been conceive
d (87). A prototype of a new system capable of simultaneous measurement of dose
and spatial position is being developed. The device, controlled by a computer, c
onsists of a probe combining two technologies: a MOSFET radiation detector coupl
ed with a magnetic positioning device. Special software that provides sampling p
osition and dose in user-defined time intervals has been developed. Tests conduc
ted in a 6 MV beam indicate that there is no interference from the linear accele
rator electromagnetic field on the performance of this new fourdimensional dosim
etry system. The new system can be used in IGRT to complement the information no
t only about the dosimeter position during entire treatment time, but also about
the dose accumulation as a function of irradiation time. SUMMARY OF PERTINENT C
ONCLUSIONS
l
l
l
Target dose measurements can provide the final confirmation of the dose received
in the target by measuring the net effect of all the variables that can affect
accurate dose delivery, such as organ motion and patient movement during treatme
nt. This information can assist the radiation oncologist in optimizing the radia
tion treatment for each patient. Daily in vivo dose measurements may be especial
ly valuable as radiation oncologists develop plans with tighter margins, higher
total doses, or hypofractionation, requiring increasingly complex delivery metho
ds, for which localization devices such as ultrasound or CBCT may not provide op
timal pretreatment visualization. Future versions of these devices will likely c

ombine simultaneous measurement of dose with spatial position transmission, whic

h should be available in real time.
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intensity modulated radiation therapy (IMRT) in prostate cancer. Radiother Oncol
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lity throughout prostate radiotherapy. Int J Radiat Oncol Biol Phys 1998; 42:115
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21
Future Developments II: Dose Painting with Functional Imaging, Online Dose Track
ing and Adaptation, IMRT with Robotic Brachytherapy, and Other Nonradiation Moda
lities
YING XIAO, TARUN PODDER, ADAM P. DICKER, AND YAN YU
Department of Radiation Oncology, Kimmel Cancer Center, Thomas Jefferson Univers

ity, Philadelphia, Pennsylvania, U.S.A.

DOSE PAINTING WITH INTENSITY-MODULATED RADIATION THERAPY: THE RADIOBIOLOGICAL TA
RGET The latest available technology in radiation therapy, intensity-modulated r
adiation therapy (IMRT), offers more precise radiation delivery with high dose t
o target volume while sparing nearby critical structures. IMRT is defined as the
radiation delivery technique that uses radiation fields of various sizes whose
intensity could be varied to control the dose distributions that could be tailor
ed to fit complex geometric arrangement of targets and for avoidance of critical
structures. By allowing the intensity within the target to vary opened the door
to the possibility of protecting critical structures that were positioned near
the target and fell within one or more of the different treatment fields, as wel
l as paint or sculpt high-/low-dose regions within the target. IMRT delivery technol
ogy has advanced rapidly over the recent years. Figure 1 depicts various multile
af collimator (MLC)-based IMRT delivery technologies (1). Other IMRT delivery te
chniques include physical attenuators and robotic delivery with collimators. Wit
h the ability of IMRT to deliver, by design, nonuniform
269
dose across the target, the question arises as to how to optimize the radiation
delivery, taking into account all the information available to us regarding the
tumor location, tumor extent, and the response of tumor to radiation dose to ach
ieve maximum kill of tumor cells and maximum protection of normal structures. Th
e advent of imaging capabilities in the area of nuclear magnetic resonance imagi
ng (MRI) and spectroscopy, positron emission tomography (PET), and molecular ima
ging promises to provide physiology and functional information, as well as tumor
biology at the genotype and phenotype levels. The significance of MRI in cancer
detection and staging lies in its soft tissue contrast because of the differenc
es in the T1 and T2 relaxation parameters of normal and abnormal states. Develop
ments of contrast-enhancement techniques can yield physiological and microenviro
nmental information such as blood flow, water molecule mobility, oxygen concentr
ation, etc. Nuclear magnetic resonance (NMR) can provide ample biological inform
ation in the level of biomolecules. Proton (1H) spectroscopy is one of the NMR s
pectroscopy techniques that offers sensitivity, spatial resolution, sufficient s
ignal-to-noise ratio, and reasonable image acquisition time. MR spectroscopy (MR
S) is able to detect signals from low molecular weight
270
Xiao et al.
Figure 1 MLC-based IMRT delivery techniques. Abbreviations: MLC, multileaf colli
mator; IMRT, intensity-modulated radiation therapy; DMLC, dynamic multileaf coll
imation; SMLC, segmental multileaf collimation; IMAT, intensity-modulated arc th
erapy. Source: From Ref. 1.
metabolites such as choline and creatine that are present at concentrations of a
few millimeters in tissue. The contrast achieved with MRS provides a promising
alternative for identifying tumor extent and regions of high metabolic activity.
Spectra may be acquired from single voxels or from a two-dimensional or three-d
imensional array of voxels using spectroscopic imaging. The current state of the
art achieves a spatial resolution of 6 to 10 mm in a scan time of about 10 to 1
5 minutes. Coregistered MR images can be acquired in the same examination. MR sp
ectroscopic imaging (MRSI) provides a noninvasive method of evaluating metabolic
markers of prostate cancer or healthy prostatic tissue. Multiple studies have s
howed the incremental role of MRSI combined with the anatomical information prov
ided by MRI for assessment of cancer location and extent within the prostate, st
aging, and cancer aggressiveness. Further developmentsincluding new 3T technologyw
ill likely provide improved spectral resolution for better prostate cancer detec
tion and characterization. It is anticipated that MRS will become an essential t

ool for treatment planning where other modalities lack the necessary contrast (24
). T2-weighted MRI for imaging prostate has much superior contrast to CT, transr
ectal ultrasound, and digital rectal examinations. However, the sensitivity and
specificity are still 83% and 50%, respectively by MRI alone (5). The use of 1H
MRSI in the prostate is needed to aid target definition. The main spectral peaks
observed in normal prostate are those of the choline-containing compounds, crea
tine and phosphocreatine, and a large peak from citrate. Choline compounds are a
ssociated primarily with membrane synthesis, while creatine is involved in energ
y metabolism. Citrate is a product of normal epithelial
cell metabolism in the prostate, where high levels of zinc inhibit the enzyme ac
onitase and hence prevent the oxidation of citrate in the Krebs cycle that occur
s in other cells. In prostate cancer, choline is elevated and the normal product
ion of citrate is reduced. Benign prostatic hyperplasia, on the other hand, is c
haracterized by high levels of citrate. Hence, the choline/citrate ratio is a fa
irly reliable measure of the presence of cancer. A strong correlation has been f
ound between negative MRSI and negative biopsy findings and between positive MRS
I and positive biopsy findings (6). However, there is only a weak correlation be
tween the concentration of prostate-specific antigen (PSA, the current gold stand
ard) and either biopsy or MRSI findings (6). Step-section pathological examinatio
n of radical prostatectomy specimens demonstrated that MRI combined with MRSI yi
elded a significant improvement in cancer localization to a prostate sextant (le
ft or right; base, mid-gland, or apex) compared with MRI alone (7). There is str
ong evidence that MRS has a valuable role to play in radiotherapy treatment plan
ning. MRSI has been used in combination with MRI to define regions for dose esca
lation within the prostate (811), permitting a dose of greater than 90 Gy to the
high-risk region while treating the remainder of the prostate to about 70 Gy. 1
H MRSI data may be acquired from the prostate using an external phased-array coi
l. However, the best signalto-noise ratio is achieved using an endorectal coil.
The main disadvantage of the endorectal approach is slight deformation of the pr
ostate, which needs to be allowed for in using the images for radiotherapy treat
ment planning. Deformation will be discussed in the following section. Another c
oncern with MRS is that voxel sizes of typically 8 to 10 mm are required to achi
eve an adequate signal-to-noise ratio,
Future Developments II
271
with a large part of this volume needing to be occupied by abnormal tissue for a
change in signal to be detected. Thus, small infiltrating lesions are unlikely
to be detectable. While some improvement in sensitivity and spectral specificity
is expected with higher field scanners and improved sensitivity coils, this is
likely to yield only a small improvement in spatial resolution. However, while i
n principle both MRI and CT have much better spatial resolution, MRS has the pot
ential to improve identification of the gross tumor volume and hence improve tre
atment using radiotherapy. Fluorodeoxyglucose (FDG) is shown not to be a suitabl
e PET tracer for diagnosing prostate cancer. Unlike other tumor types, prostate
cancer often does not display increased glucose metabolism (12). Studies showed
little difference between FDG uptake in prostate cancer and benign prostatic hyp
erplasia (13,14), and that there is limited additional information from FDG-PET
in diagnosis and staging of prostate cancer (1518). However, due to the fact that
tissue uptake of radiolabeled choline corresponds to an increase in membrane li
pid synthesis, [C-11]-choline-PET have comparable or even better sensitivities t
han with MRI and MRS for primary lesions (19). Initial preclinical and clinical
studies showed that [F-18]-fluorocholine is a promising tracer for the evaluatio
n of primary and metastatic prostate cancer (2022). The coregistered functional a
nd anatomical information of PET/CT appears to be particularly helpful in the ev
aluation of PET tracers in the abdomen and pelvis. [C-11]-choline-PET/CT reveale
d a sensitivity of 83% for localization of nodules greater than 5 mm, which was

comparable to transrectal ultrasound-guided biopsy (23). Because of the limited

resolution of PET imaging, information regarding the detection of extraprostatic
tumor extension could not be found. The same group performed a direct compariso
n of [C-11]-choline-PET/CT with sextant results of step-section histopathology a
nd found a sensitivity, specificity, and accuracy of 66%, 81%, and 71%, respecti
vely (24). Choline PET and acetate PET are promising tracers in the diagnosis of
prostate cancer, but their validity in local tumor demarcation, lymph node diag
nosis, and detection of recurrence has to be defined in future clinical trials (
25). Combined radiotherapy and gene therapy is a novel therapeutic approach for
prostate cancer (2628). There are various potential benefits in combining ionizin
g radiation with gene therapy to achieve enhanced antitumor effects: (i) ionizin
g radiation improves transfection/transduction efficiency, transgene integration
, and possibly, the bystander effect of gene therapy; (ii) gene therapy, on the ot
her hand, may interfere with repair of radiationinduced DNA damage and increase
DNA susceptibility to radiation damage in cancer cells; and (iii) radiotherapy a
nd gene therapy target at different parts of the cell cycle. Preclinical data ha
ve demonstrated the enhanced antitumor
effects of this combined approach in local tumor control, prolongation of surviv
al, as well as systemic control. The goal of this combined approach is to enhanc
e cancer cure without an increase in treatment-related toxicity. This approach a
lso offers a new paradigm in spatial cooperation, whereby two local therapies ar
e combined to elicit both local and systemic effects. Early clinical results sho
wed the safety of this approach. ONLINE DOSE TRACKING AND ADAPTATION Deformable
Target and Structures Image-guided radiation therapy (IGRT) for the treatment of
cancer has become a reality with the availability of imaging capabilities in th
e radiation treatment room with the patient in the actual treatment position, wh
ere volume images can be acquired on a daily basis. Commercially available in-ro
om image devices now include CT/volumetric CT. It is reasonable to expect even m
ore sophisticated functional imaging modalities, e.g., PET, available for the pa
tient in treatment position, in the future. These images can potentially guide r
adiation therapy in the following two aspects. First, the direct reference of th
e images obtained with the patient in the treatment position to the treatment ma
chine coordinates can substantially reduce the geometric uncertainty of internal
structures. With the additional real-time tracking and adjustment capabilities,
more precise radiation will then be delivered with high dose to target volume w
hile sparing nearby critical structures, taking advantage of the latest availabl
e technology in radiation therapy, i.e., IMRT. Second, with the images available
on a more frequent basis, analysis will yield tumor response during radiation t
reatments in the early stages that will allow adjustment of treatment techniques
(fraction size, radiation modality, radiation treatment plan modification, etc.
). IGRT has limited implementation, mostly for localization for the time being,
especially for the radiotherapy of the cancer of the prostate. However, mainly d
evelopments are made in research of strategies and tools for image analysis and/
or for adjusting treatments when image/dose analysis indicates the need for chan
ge. Fully integrated package that includes image/dose analysis methods and radia
tion treatment adjustment tools are being developed to make these new imaging ca
pabilities truly helpful. Among the developments for efficient image analysis, i
nitially for target localization with the potential for target delineation and c
ritical structure definition for the images, some of the tools acquired images o
nline with patient in the treatment position daily (e.g., daily cone-beam CT) an
d adopted rigid body gray-scale registration (2931) and deformable imageregistrati
on approach (32,33). These tools and approaches are reported to improve upon the
272
Xiao et al.
efficiency and to reduce the variation among observers, which is an issue in pro
state radiotherapy (34). Among the most widely implemented deformable registrati

on algorithms is Thirions demons algorithm (35). In its implementation, each image

is viewed as a set of isointensity contours. The main idea is that a regular gri
d of forces deforms an image by pushing the contours in the normal direction. Th
e orientation and magnitude of the displacement is derived from the instantaneou
s optical flow equation that contains the information from the fixed image and t
he moving image to be registered and the displacement or optical flow between th
e images. For registration, the projection of the vector on the direction of the
intensity gradient becomes unstable for small values of the image gradient, res
ulting in large displacement values. To overcome this problem, Thirion renormali
zed the equation. Reconstruction of the deformation field is an ill-posed proble
m where matching the fixed and moving images has many solutions. For example, si
nce each image pixel is free to move independently, it is possible that all pixe
ls of one particular value in the moving image could map to a single image pixel
in the fixed image of the same value. The resulting deformation field may be un
realistic for real-world applications. An option to solve for the field uniquely
is to enforce an elastic-like behavior, smoothing the deformation field with a
Gaussian filter between iterations. In Vivo Dose Verification and Reconstruction
Electronic Portal Imaging Device Dose Verification and Reconstruction
Electronic portal imaging device (EPID) have been widely used for verification o
f patient positioning in place of film because of its efficiency in obtaining in
stantaneous digital images of the patient anatomy, and the treatment field can b
e compared with digital reconstructed radiographs (DRRs) from the planning syste
m. Besides utility in positioning verification, studies have been ongoing to obt
ain dosimetric information from the EPID for treatment delivery verification wit
hout (pretreatment) or with (in vivo) patient (3644). In vivo EPID dosimetry has
the following advantages: the hardware is already fixed to the linear accelerato
r, providing instantaneous high-resolution digital images. EPID image acquisitio
n can be part of the actual patient treatment procedure, which does not require
additional time and dose to the patient. If one is able to obtain three-dimensio
nal reconstructed delivered dose information, adaptive plans can be generated on
the basis of the delivered dose to the patient. EPID images were acquired eithe
r with camera, ion chamber matrix, or with solid-state (e.g., a-Si) flat panel i
mager. They generally have large enough detection area
that can contain most radiation treatment field sizes with submillimeter resolut
ions. Verification of the delivered dose with EPID falls into two categories. On
e compared the dose at the EPID plane (40,45) and the other made comparisons bet
ween the reconstructed dose to the voxels delivered inside the patient and the d
ose predicted by the treatment planning system (38,46). With the second approach
, however, it is potentially feasible to adjust dose based not just on the anato
my variation but also delivered dose variation during the treatment, especially
with the three-dimensional reconstructed dose information (4749). There are a num
ber of methods applied to derive twodimensional planar dose for a certain plane
in the patient intersecting the beam (46,50). The two-dimensional planar back-pr
ojected dose reconstruction method starts from conversion of segment images to a
n absolute twodimensional dose distribution in the plane of the patient, defined
as the plane perpendicular to the beam axis intersecting the isocenter. Pixel v
alues of the transit dose image are processed using scatter kernels (for scatter
within the EPID and scatter from the patient to the EPID), the scatter-to-prima
ry ratio (for scattered radiation within the patient), the inverse square law, a
nd the measured transmission to obtain the absolute dose distribution in the iso
centric plane of the patient. The measured transmission of the beam through the
patient is determined from images acquired for each field (or segment), both wit
h and without the patient. The location of the reconstruction plane is arbitrary
, so a correction is required to account for attenuation of the beam from the is
ocentric plane to the exit surface. The external contour of the patient CT scan
is used to obtain the ratio of geometrical path lengths, which is used to calcul
ate the attenuation per pixel. The density of the transmission medium is assumed
to be homogeneous; therefore, the dose may be incorrect for areas on the plane

where the beam passed through media of nontissue equivalent density. Reconstruct
ed two-dimensional dose distributions for each field are then the sum of the rec
onstructed dose distributions of all segments belonging to that field. The secon
d two-dimensional planar dose derivation method first calculates the two-dimensi
onal contribution of the primary and scattered dose component at the exit side o
f the patient or phantom from the measured transmission dose. Then, a correction
is applied for the difference in contribution for both dose components between
exit side and midplane, yielding the midplane dose. These two-dimensional method
s have been adopted for clinical in vivo dosimetric verifications, especially wh
en IMRT is involved. In one example of using this backprojection algorithm (46),
two-dimensional dose distributions inside a phantom or patient are reconstructe
d from portal images. The method requires the primary dose
Future Developments II
273
component at the position of the EPID. A parameterized description of the latera
l scatter within the imager was obtained from measurements with an ionization ch
amber in a mini phantom. In addition to point dose measurements on the central a
xis of square fields of different size, dose profiles of those fields are used a
s reference input data. This method yielded a better description of the lateral
scatter within the EPID, which resulted in a higher accuracy in the back-project
ed, two-dimensional dose distributions. The accuracy was tested for pretreatment
verification of IMRT plans for the treatment of prostate cancer. All segments a
re evaluated by comparing the back-projected, two-dimensional EPID dose distribu
tion with a film measurement inside a homogeneous slab phantom. The g-evaluation
method was used with a dose-difference criterion of 2% of dose maximum and a di
stance-to-agreement criterion of 2 mm. Excellent agreement was found between EPI
D and film measurements for each field, both in the central part of the beam and
in the penumbra and low-dose regions. Simple two-dimensional dose reconstructio
n has been extended to three dimensions in the vicinity of the isocenter plane w
ith reasonable accuracy (47). However, with the availability of online CT data f
rom cone-beam and other techniques, more sophisticated and potentially more accu
rate three-dimensional reconstruction methods can be applied. In one concept ori
ginally presented by McNutt et al. (49), an iterative superposition/convolution
method was used to obtain the primary energy fluence. The three-dimensional pati
ent dose was then calculated applying predefined scatter kernels. Another effort
by Patridge et al. (48) also used iterative method for primary energy fluence p
rediction. Instead of a direct dose calculation using predefined scatter kernels
, they proposed Monte Carlo method or the calculation engine from a treatment pl
anning system. Three-dimensional dose reconstruction is an area that deserves a
great deal of research efforts. An accurate three-dimensional in vivo dose const
ruction for radiotherapy delivery is essential for any successful dose-adaptive
treatment protocol. In order for the dose information to be derived from EPID si
gnals, properties of the EPID response behavior need to be well understood. Stud
ies have identified characteristics of EPID that need to be considered and corre
ction factors applied to obtain dose accuracy from EPID measurements to be withi
n 1% (51). The overresponse of the EPID signal can be significant over the patie
nt-detector distance. Addition of a certain thickness of material could reduce t
his over response. The response of EPID varies over dose per pulse, pulse repeti
tion frequency, and number of monitor units. Ghosting effects, in which under-re
sponse is found for shorter beam times, also contribute to the variation of EPID
response. The long-term stability of some of the EPID panels are studied
and found to be suitable for dosimetry acquisition after applying a dynamic corr
ection (52).
Three-Dimensional In Vivo Dosimetry with PET-CT
With the increasingly wider implementation of heavy particle therapy, along with

high energy photon radiotherapy, it is possible to take advantage of the photon

uclear and direct nuclear interactions in tissue for in-vivo dosimetric imaging
with PET-CT. Ion beams produce PET emitters through nuclear interactions. Radioa
ctive beams could also consist of intrinsic PET emitters. These would allow dire
ct imaging of Bragg peak distribution which is related to the absorbed dose. The
high energy photons, on the other hand, produce positron emitters with energies
above 20 MeV. The intensities are proportional to the photon fluence which is i
ntimately related to absorbed dose. With the prior delivered dose information, i
t is then possible to adapt the future treatment plan basing upon not only the t
arget and structural variation, but also the exact dose still needed for target,
adding more to or subtracting from the delivered dose. With the help from multi
ple image modalities, future re-planning process would include information from
numerous aspects: radiation responsiveness (resistance) considering hypoxia, pos
sible planning and delivery errors, general deformation of patient anatomy throu
ghout the treatment course (53). RADIOTHERAPY TREATMENT PLANNING WITH TARGET MOT
ION Online Imaging, Dose Tracking, Real-Time Planning, Parallel Optimization Wit
h online target definition and prior in vivo dose derived, it is conceivable for
real-time planning to be used as an adjustment strategy. Accuracy and precision
are the qualities of the treatment plans that have improved immensely in the pa
st 20 years, especially since the advent of IMRT. Yet, new developments in imagi
ng technologies provide us with a possibility to augment, quantitatively and qua
litatively, the treatment planning process in a way that was not previously clin
ically viable. However, concomitant to that is also the growth of complexities o
f reported solutions and their implementations. Treatment planning computation (
TPC) is a mathematical inverse problem predicated on the prescription expressed
either as physical and/or biological stipulations. In principle, the latter are
more relevant but they are not mature enough to replace the former constraints.
However, the biological goals preponderance makes it imperative for a modern TPC
to accommodate a relevant component, at least in terms of the research capabilit
ies
274
Xiao et al.
or the extensibility of the system. Another tacit facet of the TPC is the techno
logical constraints that impinge indirectly on the domain of realizable treatmen
ts. These constraints may address issues that are transparent for therapeutic go
als, for instance, the modeling of the resultant intensity map that could be com
putationally amenable to the given leaf sequencing algorithm or in general to th
is phase of plan preparation (54). TPC employs some optimization schemes or rela
ted methods. The computation of beam weights includes dose-based voxel dependent
and dose-volume constraints [dosevolume histogram (DVH) equivalent] as articula
ted by prescriptions. Another concept used in modeling is the equivalent uniform
1=N N Da 1=a (56) can be viewed a
dose (EUD) (55). Its most popular P version EUD
s a i i handy modeling construct because of the mathematical characteristics of
the function. However, there is still a nexus with the biological underlining [e
.g., tumor control probability (TCP) and normal tissue complication probability
(NTCP)] via the power law behavior of the effective dose used for the reduction
of the DVH (5761); a is a structure-dependent parameter, negative for targets and
positive for organs at risk (OARs). The EUD addresses the inhomogeneity of the
dose distribution in structures. It is important to model the computation with c
riteria that are also used for the plan evaluation. Another aspect that we want
to control is the complexity of the intensity patterns. This can be addressed by
either the exogenous way of adding a smoothing constraint (62) to the optimizat
ion or endogenously using smoothing component (63) within the optimization. Thes
e solutions allow us to keep the therapeutic aspect of the sought dose distribut
ion in sync with the technological limitations of the dose delivery. With the on
line acquired image data available and the dose reconstructed via one method or
another, ideally one would be able to optimize the radiotherapy treatment plan o

n a day-to-day basis. This planning problem poses a rather stringent request on

the speed of the computation. The algorithms that could accommodate the above mo
deling and computational characteristics and could be implemented with paralleli
zation to take advantage of the ever-increasing computing power are investigated
. Among those are the subgradient projection method and the component averaging
projection technique (6474). The usefulness of these algorithms is outlined in co
mputational context (7074). They are fast and inherently parallel, which allows u
s to use modern multiprocessor computers. They can provide solutions within one
to two minutes. The findings also indicate that they inherently strive to produc
e relatively smooth patterns (71), yet the explicit constraint of this type can
be applied as well. The dose-based and dose volume-based constraints (70,71) wer
e prototyped. The EUD a-sublevel set (75)-based constraints
can be defined as convex for OARs and targets. The proposed iterative algorithms
must use convex functions as constraints. The accommodation of the EUD for targ
et and critical structures will be realized with the EUD supra- and sublevel set
s (75) that comply with the convexity requirement. The other methods are the (qu
asi) Newton method and conjugate gradient methods (76,77) that are often reporte
d in the IMRT optimization (7881). They are fast and amenable to accommodate cons
traints. They might be combined with the feasibility search methods for hybrid o
ptimization approach. Robust Optimization Another treatment planning optimizatio
n approach that addresses the target motion is incorporating motion information
into the initial formation of the optimization problem. The recent development o
f planning optimization involves the idea of a motion pdf (8286), which uses a prob
ability density function (pdf) to define what proportion of time the tumor spend
s in each breathing phase (in reality, discretization leads to a probability mas
s function or pmf). Similar pmf models exist for interfraction motion (87). This
pmf approach requires that the motion is reproducible and stable during the tre
atment delivery (82). Using a pmf to account for tumor motion produces acceptabl
e dose distributions, provided the motion does not deviate significantly from wh
at is expected. The uncertainty associated with pmf however can also be modeled
and formulated with mathematical approaches (88). These solutions are found to d
eliver fewer doses to nearby critical structures than the conventional margin so
lutions, which typically overcompensate for the target motion (89,90). It is als
o possible to vary the level of concern for various structures and for individua
l patient cases. IMAGE- AND DOSE-GUIDED ROBOTIC DELIVERY The word robot was introd
uced in English language in 1921 by the play writer Karl Cape. Now we use it ver
y commonly in our daily life. But what is really meant by a robot? The most comp
lete definition is A robot is a reprogrammable multifunctional manipulator design
ed to move materials, parts, tools, or specialized devices through variable prog
rammed motions for performance of a variety of tasks (91). Of the four branches o
f robotics, namely, medical robotics, land-based or industrial robotics, space r
obotics, and underwater robotics, the medical robotics is the latest one and now
gaining significant momentum. Presently, there are various applications of robo
ts in the medical field. The application of robotic technology has undergone rap
id growth in the field of
Future Developments II
275
endoscopic surgery in the past several years, particularly in robotic-assisted r
adical prostatectomy. Increasingly, robotic devices are coming into the radiothe
rapy clinic as potential solutions to online adaptive treatment delivery. When c
oupled with close-loop control strategies, image and physiological sensor guidan
ce, predictive and feedforward tracking algorithms, and real-time dose-guided IM
RT, robotic assistance promises to catalyze the next paradigm change in high-pre
cision radiation delivery. In prostate brachytherapy, accuracy of radioactive se
ed placement is important for optimal dose delivery to the target tissue while s
paring the critical organs and structures. But accurate placement of needles in

soft tissue is challenging because of tissue heterogeneity and elastic stiffness

, tissue deformation and movement, unfavorable anatomic structures, needle bendi
ng, inadequate sensing, and poor maneuverability. In contemporary prostate brach
ytherapy procedures, the needles are inserted manually using ultrasound guidance
through fixed holes of a physical template. Therefore, flexibility and maneuver
ability of needle insertion is severely limited. Sometimes it is difficult to av
oid pubic arch (especially for patients with larger prostates) because the needl
es can only be inserted straight through the templates holes. The consistency and
efficiency of the treatment procedure are highly dependent on the skills of the
clinicians. A robotic system can potentially improve the accuracy and consisten
cy of operation by assisting the clinicians. In the past several years, research
ers have been developing ultrasound-guided robotic systems for prostate brachyth
erapy (9299). Fichtinger et al. (92) and Stoianovici et al. (93) have developed a
needle placement robotic system that is comprised of a 3 degree-of-freedom (DOF
) Cartesian bridge over the patient, a 2DOF remote center of motion (RCM), and a
1DOF needle insertion with friction transmission. They have employed a 7DOF pas
sive arm between the Cartesian stage and the remaining two modules, namely RCM a
nd needle inserter, to position and orient the needle in imaging instruments suc
h as CT imager. In this system, the brachytherapy seeds can be deposited manuall
y. Using a 5DOF industrial robot, Wei et al. (94,95) were successful in position
ing and orienting a single hole template for prostate brachytherapy. Through thi
s accurately positioned template a needle can be inserted manually. A motorized
needle rotation module was integrated with the robot and ultrasound probe rotati
on was motorized for 3D image reconstruction. Kronreif et al. (96,97) developed
a 4DOF needle placement robotic system which consisted of two offset x-y stages
allowing positioning and orienting the needle over the perineum. In this system
the needles can be manually inserted through a needle guiding attachment. Needle
insertion depth is monitored in ultrasound images. Meltsner et al. (98) develop
ed a custom-built 6 DOF robotic system for
prostate brachytherapy and reported about 2.1 mm (about 7 mm without rotation) a
ccuracy in seed delivery into gel phantom at 10-cm depth. Kennedy et al. (99) de
signed a 4 DOF prototype, which was intended to replace the conventional fixed t
emplate. The commercially available robotic system for prostate seed delivery is
the Nucletrons FIRST system (100). This system has two motorized motions: one fo
r rotating the transrectal ultrasound (TRUS) probe and the other for pushing the
seeds and spacer from the cartridges. All other motions including needle placem
ent using conventional template are manual. Recently, Yu et al. (101) have devel
oped a fully automated robotic system for prostate seed implant (Figs. 2 and 3).
Numerous techniques such as needle rotation and angulation during insertion, fo
rce-torque detection, which were perfected by a variety of proof-ofprinciple exp
eriments, have been implemented in the system design. Experimental results revea
led that velocity modulation, especially rotation [partial rotation (102) or con
tinuous (103,104)], significantly improves needle insertion and targeting accura
cy. Reduction in force also minimizes tissue/organ deformation and target deflec
tion (Figs. 4 and 5) and thus is expected to improve seed implantation accuracy.
During automated robotic mode of operation, in the absence of human tactile sen
sing, the needle insertion force must be monitored for safety. To measure and mo
nitor force profiles during the operational procedures, two single-axis force se
nsors (model M13, Honeywell Sensotech, Columbus, Ohio, U.S.) were
Figure 2 Prototype robotic system.
276
Xiao et al.
Figure 3 Assembled surgery module: drawing (top) and fabricated prototype (botto
m).

Figure 4 Reduction of needle deflection (bevel-tip brachytherapy needle in PVC p

hantom). Abbreviation: PVC, polyvinyl chloride.
Figure 5 Reduction in insertion force on needle (diamond-tip brachytherapy needl
e in bovine liver phantom).
Future Developments II
277
Figure 6 Needle driver.
installed each at the proximal ends of the stylet and cannula, and one six-axis
force-torque sensor (model Nano17, ATI Industrial Automation, Apex, North Caroli
na, U.S.) was installed at the distal end of the cannula (Fig. 6). Monitoring of
these forces is useful in detecting pubic arch interference (PAI) and will help
in assessing needle bending due to transverse forces. Preliminary seed implant
experiments using tissue equivalent soft material phantom prepared from polyviny
l chloride exhibited (Fig. 7) seed placement error (root mean square) of about 0
.5 mm, which is quite encouraging. Robot-assisted therapeutic delivery system is
attractive for several reasons. The main advantages are increased accuracy, red
uced human variability, and possible reduction of operation time and clinicians f
atigue. There can be two methods of robotic needle insertion and seed deposition
: (i) singe-channel technique and (ii) multichannel technique. In single-channel
mode, one needle
Figure 7 Seed delivery in soft material (PVC) phantom. Abbreviation: PVC, polyvi
nyl chloride.
can be inserted at a time and typically, two to five seeds along a needle track
are deposited in the prostate according to the dosimetric plan. Drawbacks of thi
s modality are (i) it is a slow process because one needle is used at a time (ty
pically, 1525 needles are required); (ii) every time the needle is inserted, the
prostate and other organs are deformed and displaced, thereby incurring seed pla
cement inaccuracy; (iii) there is a chance of a needle colliding with already de
posited seeds and displacing the seed; and (iv) needle deflection and its relati
ve position in the prostate cannot be predicted in advance for interactive (or d
ynamic) dosimetric planning. In contrast, the multichannel system is capable of
placing several needles or even all needles at a time, and thereby, it will be f
aster to deliver the seeds required for the treatment. A multichannel delivery s
ystem can effectively avoid the problem of gradual prostate swelling (i.e., edem
a) and deformation, which occurs while depositing the seeds by a single needle.
Since the prostate is not rigidly mounted, it can move and rotate as well as def
orm quite unpredictably each time a needle is inserted. However, when several ne
edles are inserted concurrently, the prostate will be uniformly pushed back symm
etrically to a more stable position and the deformation can better be estimated
for precise delivery of seeds. Thus, the multichannel system can overcome the dr
awbacks that may be encountered by the single-channel systems. Currently, Podder
et al. (105) are developing a multichannel robotic system that will be, unlike
a single-channel robotic system or conventional manual technique, capable of ins
erting a large number of needles concurrently (Figs. 8 and 9). This system posse
sses several potential added advantages such as reduced target displacement, red
uced edema, and less operating time as compared with single-needle insertion tec
hnique. This multichannel system has been designed to insert multiple
278
Xiao et al.
Figure 8 Needle insertion module of the multichannel robotic systemsimultaneous r

otation and insertion of multiple needles. (A) Front-end and side view. (B) Back
-end and side view.
needles simultaneously into the prostate. As shown in Figure 8, all 16 needles c
an be inserted simultaneously while all the needles can be rotated by using a si
ngle motor (Fig. 8B). This provision of needle rotation will reduce the insertio
n force as well as organ (or target) deflection and deformation. After inserting
the needles at the desired positions and depths, they will be fixed into positi
on using a locking mechanism in the template. Then,
Figure 9 x-y table and seed applicator for multichannel robotic system. (A) Fron
t-end and side view. (B) Back-end and side view.
the insertion module will be replaced by the x-y table module (Fig. 9) using the
dove-tail joint, while the applicator is used to deliver the radioactive seeds
needleby-needle at the desired coordinates based on the dosimetric plan. The x-y
table moves in the transverse plane to position the seed applicator at the desi
red location while the main translational actuator activates the stylet to push
the seed into the prostate. The entire procedure will be performed under ultraso
und image guidance. It is known that among the available imaging modalities, MRI
is the best modality for differentiating the soft tissue boundaries between the
prostate and its surrounding anatomy. Therefore, by utilizing MRI in prostate b
rachytherapy, the ability of delivering a precisely shaped treatment may be impr
oved, and it may be possible to achieve more targeted therapy while sparing norm
al tissues and critical structures. Robot-assisted brachytherapy system can enha
nce needle placement accuracy and consistency of treatment. Thus, a robotic syst
em guided by MRI has the potential to further improve the delivery of radiation
treatment for prostate cancer. Recently, Muntener et al. (106) and Patriciu et a
l. (107) reported an MR compatible robotic system for prostate seed implant. Thi
s system was able to deliver seeds in tissue-mimicking phantom under 0.5T to 3T
MR environment with an accuracy of 0.72 0.36 mm. As high-dose-rate (HDR) brachyt
herapy is attracting increased attention in clinical circles, an MR-guided HDR r
obotic system aided by powerful optimization engine like the genetic algorithm (
GA) would be the next state-of-the-art technology in this frontier field. Furthe
r improvement may be possible if adaptive dosimetric planning is integrated with
patientspecific predictive models (108) of tissue deformation and displacement
aided by artificial neural networks. However, in the near future, ultrasound may
remain as
Future Developments II
279
Figure 10 HexaPOD robotic couch. (A) External isometric view. (B) Internal isome
tric view. (C) Schematic of the leg.
the common modality for imaging because of its cost and convenience. Various int
ernal organs including prostate move because of respiratory and cardiac motions.
Among different available techniques, the MLC gating is commonly used to compen
sate the undesired motion of the target. However, MLC-gating technique has sever
al disadvantages. For example, use of internal fiducials for motion monitoring r
equires kilovoltage X-ray, which delivers unwanted radiation dose to the patient
. Gating suffers from severely low duty cycle (only 3050%) and IMRT efficiency (o
nly 2050%). All these lead to a 4- to 15-fold increase in delivery time over conv
entional treatment (109). Presently available commercial robotic systems for ext
ernal beam radiation therapy include the CyberKnife Radiosurgery System (Accuray
, Sunnyvale, California, U.S.), and 6 DOF HexaPOD patient positioning system (El
ekta AB, Stockholm, Sweden). These systems have the potential to enable position
and motion compensation with promising speed and accuracy. Coordinated control
between high-speed MLCs and robotic couch offers another intriguing opportunity.

Recently, a group of researchers have investigated the feasibility of using the

treatment couch (HexaPOD and Dynatrac) for intrafraction motion tracking (110).
Their study concluded that the achievable speed was much less than that require
d for compensating respiration-induced tumor motion in the realm of clinically r
elevant motion amplitudes. Such a robotic system, which is subjected to a large
load and operating at moderate frequency and amplitude, requires not only robust
electromechanical design but also stable closed-loop dynamic control that can p
roduce a high degree of repeatability and accuracy with varying external perturb
ations. The movement of target-volume (internal organ/tumor) can be measured eit
her using a combination of various sensory systems such as (i) surrogate breathi
ng motion signals from external markers, (ii) implanted electromagnetic transpon
ders, and (iii) four-dimensional CT (and/or X-ray) images of implanted fiducial
markers. In the former
case, a robust three-dimensional model correlating the tumor motion and the exte
rnal markers signal can be developed. In the later cases, the coordinates of the
tumor volume can be periodically extracted from the direct measurement of the t
ransponders position. A closed-loop dynamic control of the 6 DOF robotic couch ca
n be employed to continuously reposition the patient reciprocatively to the tumo
r motion so that the treatment beam always finds the target-volume stationary. R
ecently, Podder et al. (111) reported a decentralized closed-loop dynamic contro
ller for adjusting the MLCbank and robotic couch on the basis of their dynamic r
esponse bandwidth, so that the target-volume appears to be stationary to the rad
iation beam and that the beam can be delivered close to 100% duty cycle. To trac
k the tumor for optimal dose delivery to the clinical target volume (CTV) with m
inimal planning target volume (PTV), it was proposed to control both the MLC-ban
k and the HexaPOD (which is a special type of Stewart platform, i.e., a parallel
robotic manipulator, Fig. 10) simultaneously (112,113). These two subsystems (M
LC-bank and HexaPOD couch) have vastly different dynamic responses, i.e., natura
l frequency bandwidths. The trajectory of the tumor movement due to respiratory
and cardiac motions is decomposed into two segments (high- and low-frequency seg
ments) using the wavelet technique. The highfrequency component is assigned to t
he lighter subsystem, i.e., the MLC or MLC-bank, and the low-frequency component
with larger amplitude is allocated to the heavier subsystem, i.e., the couch (F
ig. 11). Simulated tumor motion was decomposed using wavelet technique into two
segments, where the low-frequency component was allocated to the HexaPOD couch a
nd the high-frequency component was allocated to MLC-bank, as shown in Figure 12
. Significant improvement in the tracking of tumor was observed when the decompo
sed trajectories were allocated to both the MLC-bank and the HexaPOD couch (Fig.
13, blue line), whereas when only the HexaPOD couch was used, large residual fl
uctuating error in tumor tracking was found (Fig. 13, green line).
280
Xiao et al.
Figure 11 Block diagram of the decentralized coordinated dynamics-based closed-l
oop controller for HexaPOD robotic couch and linear accelerators MLC-bank. Abbrev
iation: MLC, multileaf collimator.
Thus, it appears in numerical simulation that decentralized dynamics-based contr
oller can track the tumor motion more accurately. Implementation of the proposed
technique can potentially improve real-time tracking of the tumor-volume to del
iver precise radiation dose at almost 100% duty cycle while minimizing irradiati
on to surrounding normal tissues and sparing critical organs. This, in turn, wil
l potentially improve the quality of patient treatment by lowering the toxicity
level and increasing survival. In this study, a relatively simple but elegant cl
osed-loop proportional integral derivative (PID) control algorithm has been depl
oyed, and the results are quite promising. However, more sophisticated control s
trategies such as adaptive learning control aided by artificial neural network m

ay yield the next higher level solution that can potentially accommodate the dyn
amics of surgical and therapeutic environment. One of the main challenges and re
search opportunities is to synchronize the respiratory motion with the robotic c
ouch and/or IMRT delivery considering system latency, patients physiodynamics, an
d target-volume deformation in addition to target movement.
Figure 12 Wavelet decomposition of the tumor motion. (A) Resultant motion. (B) L
ow-frequency component. (C) Highfrequency component.

1 Introduction
Adam P Dicker, Gregory S Merrick, Frank M Waterman, Richard K Valicenti and Leon
ard G Gomella Prostate cancer poses significant biologic, economic and personal
burdens on our healthcare system and society in general. Because of an aging pop
ulation and the implementation of routine PSA screening, the incidence of prosta
te cancer has increased dramatically with the number of new cases projected to r
each approximately 200000 per year within the next decade. Fortunately, since th
e mid-1990s prostate cancer causespecific mortality has decreasedlikely due to ea
rlier diagnosis and better therapeutic options including prostate brachytherapy.
Since the mid-1980s, prostate brachytherapy has been utilized increasingly as a
potentially curative treatment for patients of all ages with clinically localiz
ed prostate cancer. This resurgence of interest in brachytherapy was primarily d
ue to the routine availability of transrectal ultrasonography, the development o
f a closed transperineal approach and sophisticated treatment planning software.
These imaging and planning advances dramatically improved the accuracy of seed
placement. In addition, computerized tomography (CT)-based postoperative dosimet
ry provided the ability to evaluate implant quality and proactively influence ou
tcome. Prostate brachytherapy represents the ultimate three-dimensional conforma
l therapy, permitting dose escalation far exceeding other radiation modalities w
ith cancericidal treatment margins substantially larger than those obtainable wi
th radical prostatectomy. Although the majority of the brachytherapy literature
has demonstrated biochemical results and morbidity profiles that compare favorab
ly with radical prostatectomy and external beam radiation therapy series, it has
become increasingly apparent that efficacy and morbidity are highly dependent o
n implant quality. Sophisticated dosimetric analyses have demonstrated that cure
rates, urinary and rectal complications and potency preservation are related to
specific source placement patterns and the subsequent dose gradients produced.
Our upcoming challenges include ensuring that high-quality brachytherapy is tran
slatable from the subspecialist to the community practitioner, the development o
f intraoperative planning and dosimetry to maximize optimal dose distributions,
improved intraoperative technique to include better delivery systems and imaging
capabilities, and the development of evidence-based algorithms for patient sele
ction and supplemental therapies including external beam radiation therapy and a
ndrogen deprivation therapy. Although other prostate brachytherapy textbooks are
available, to date none have been written exclusively for physician education.
In this book, we present an overview of prostate brachytherapy to include ration
ale, patient selection, technique, dosimetry, morbidity and biochemical outcome.
In order to provide a balanced view of the currently available knowledge of pro
state brachytherapy outcomes and controversies, physicians
Basic and advanced techniques in prostate brachytherapy
2
from multiple prostate cancer disciplines with varying opinions of brachytherapys
role in the mainstream prostate cancer armentarium have been included. We belie
ve the future of prostate brachytherapy is bright and that time will definitivel
y document long-term biochemical durability along with a favorable quality-of-li
fe profile. Additional developments in the field will continue to require close

interaction between genitourinary oncologists (both urologic and radiation) and

medical physicists to further refine patient selection, technique and evaluation
. This prostate brachytherapy textbook, with its varying opinions, provides inte
llectual stimulus for fruitful discussion, examines the advantages and shortcomi
ngs of brachytherapy, and helps establish guidelines to improve the general prac
tice of prostate brachytherapy.
Part I Understanding the problem: fundamentals of pathology and implications for
brachytherapy
2 Effect of radiotherapy on prostate histopathology and assessment of residual c
ancer
Kenneth A Iczkowski and David G Bostwick Introduction There are about thirty art
icles in the published literature about the pathology of irradiated prostate can
cer. This represents 0.3% of all Medline citations on prostate cancer over the p
ast thirty years. Changes after brachytherapy resemble those after external beam
therapy, although some findings are peculiar to brachytherapy (see below). The
rate of postradiation therapy positive biopsy varies widely, ranging from 19% to
93% based on patient selection factors, the interval from treatment, the number
of biopsy samples obtained, the use of other therapies, and, perhaps most impor
tantly, histologic interpretation.1 Factors that determine the likelihood of a p
ositive biopsy include pretreatment clinical stage, cancer grade, posttreatment
serum, prostate-specific antigen (PSA), and digital rectal examination. There ar
e three main problems with interpretation: (1) false-negative biopsies resulting
from sampling variation; (2) false-positive biopsies due to slow regression of
tumor; and (3) biopsies showing residual tumor of indeterminate viability. In th
is chapter we evaluate the diagnostic criteria for a positive biopsy after radio
therapy and the prognostic significance of these findings. Consideration of the
effects of irradiation on the benign prostate serves as a baseline for interpret
ing changes in cancer. Pathologic findings following radiotherapy The diversity
of histopathologic changes in the prostate after radiotherapy have been well-des
cribed,26 but treated specimens continue to challenge the surgical pathologist. T
he difficulty of biopsy interpretation after treatment is multifactorial and inc
ludes separation of carcinoma from its many mimics, identification of small foci
of carcinoma, and separation of treatment effects in normal tissue from recurre
nt or persistent carcinoma.2,3,713 As more patients choose radiotherapy, particul
arly brachytherapy, and as these patients are observed for longer intervals, pat
hologists bear an increasing burden to discriminate irradiated benign acini from
irradiated adenocarcinoma.1,14,15
Effect of radiotherapy on prostate
7
Benign tissue, including hyperplasia The degree of histologic change caused by r
adiation in benign or hyperplastic acini varies with the dose and duration of ir
radiation and interval from therapy onset.11,16 Changes include acinar atrophy,
distortion with loss of cytoplasm, and decreased ratio of acini to stroma (Figur
e 2.1). Nuclear changes include nuclear enlargement (86% of cases) and prominent
nucleoli (50%).3 Acinar secretory cells are more sensitive to irradiation necro
sis than basal cells; the basal cell layer is the proliferative compartment in b
enign acini. Consequently, atypical basal cell hyperplasia is seen in 57% of cas
es (Figure 2.2),3 defined as basal cell proliferation with prominent nucleoli in
>10% of cells. Stroma may be fibrotic, with paucicellular scarring, and vascula
r changes include intimal thickening and medial fibrosis (Table 2.1).2 Pathologi
sts must be aware of these changes because they preclude the usual reliance on n
uclear and nucleolar size to help identify prostate cancer. More atypia of benig
n glands was noted after brachytherapy than after external beam therapy in a com
parative study of 44 cases, and our experience verifies this. This atypia seems
to persist longer after brachytherapy as well. With external beam therapy, there

was less atypia in men biopsied 48 months after treatment compared to those bio
psied at a shorter interval after treatment. (In some cases, however, abnormal f
indings persisted to a variable degree for 10 years.) In contrast, no decrease i
n atypia over time was noted in men treated with brachytherapy.17
Figure 2.1 Compared to untreated glands (left), benign irradiated prostate (righ
t) shows glandular shrinkage, with cells showing loss of cytoplasm.
Basic and advanced techniques in prostate brachytherapy
8
Figure 2.2 Atypical basal cell hyperplasia in irradiated glands (right). Nuclei
are larger than those from the same patient pretreatment (left). This cytologic
atypia can also occur in secretory cells and can exceed the degree of atypia com
monly used as a criterion for cancer. Table 2.1 Histopathologic findings in beni
gn prostatic tissue in postirradiation needle biopsies at the time of PSA (bioch
emical) failure
Hispathologic Endings
Inflammation Atrophy Postatrophic hyperplasia Acinar distortion Decreased acinsr
/stromal ratio Basal cell hyperplasia Atypical basal cell hyperplasia Hyperplast
ic (proloferative) change Squamous metaplasia Eosinophilic metaplasia Stromal ch
anges Stromal fibrosis Stromal edema Stromal calcification
Percentage of cases
39 79 18 54 86 68 57 11 0 21 93 21 21
Effect of radiotherapy on prostate
Hemosiderin deposition Atypical fibroblasts Necrosis Granulation tissue formatio
n Myointimal proliferation Cytologic changes Nuclear pyknosis Nuclear enlargemen
t Prominent nucleoli Bizarre nuclei Cytoplasmic vacuolization Intraluminal conte
nts Crystalloids Mucin Eosinophilic granular secretions Corpora amylacea
9
0 25 0 0 11 75 86 50 54 29 0 4 39 32
High grade prostatic intraepithelial neoplasia After radiotherapy, prostatic int
raepithelial neoplasia (PIN) retains characteristic features of untreated PIN an
d is readily recognized in biopsy and prostatectomy specimens (Figure 2.3). The
salient microscopic features include nuclear crowding, nuclear overlapping and s
tratification, nuclear hyperchromasia, and prominent nucleoli. The basal cell la
yer is present but often fragmented. The most common patterns of PIN after treat
ment, the tufting and micropapillary patterns, are similar to those reported in
untreated prostates.18 The only radio therapy-related observations were occasion
al cytoplasmic vacuolation or sloughing of epithelium into the lumen.16,19 The p
revalence of high grade PIN accompanying cancer is 82100% of non-irradiated radic
al prostatectomy specimens.18,20 It was noted in only 62% of cases after radioth
erapy,19 a decreased prevalence, similar to that seen after
Figure 2.3 Cells of irradiated high grade prostate intraepithelial neoplasia (PI
N) retain nuclear stratification, but
Basic and advanced techniques in prostate brachytherapy
10
have nuclear enlargement and hyperchromasia.
androgen ablation (50%).20 Volume of PIN without radiotherapy,19 averaged 1.32 c
m3 compared to 0.12 cm3 after radiotherapy.18 One study paradoxically noted a hi
gher prevalence (70%) of PIN after radiotherapy than expected,21 but these inves

tigators failed to employ accepted diagnostic criteria for PIN, so their results
are not comparable with those of the authors,19 or others. High grade PIN was r
eported in 9% of posttherapy biopsies,22 but sampling limitation underestimates
the prevalence. It is possible that radiation alters the phenotype of PIN such t
hat recognition is obscured. No significant correlation was seen between PIN in
postirradiation salvage prostatectomy specimens and cancerspecific survival or o
ther clinicopathologic data.19 For isolated high grade PIN in needle biopsies, t
he general recommendation has been to perform repeat biopsies in order to rule o
ut cancer. Use of 12-core sampling rather than sextant sampling, however, dimini
shes the positive predictive value of isolated high grade PIN for cancer, possib
ly obviating the need for repeat biopsy unless clinical suspicion is high.23 Ade
nocarcinoma Just as most prostate cancer grows slowly, it is slow to regress, wi
th histologic changes evolving at least 12 months after the completion of irradi
ation therapy. Needle biopsy is of limited value prior to about 1218 months owing
to ongoing tumor cell death (Table 2.2).2 Slow tumor death is attributed to the
fact that radiotherapy causes necrosis only after a prostate cell has gone thro
ugh cell division,24 and to long tumor doubling time. After this period, biopsy
is a good method for assessing local tumor control,
Table 2.2 Histopathologic findings in prostatic adenocarcinoma in postirradiatio
n biopsies
Hispathologic findings
Gleason score <7 7 >7 Percentage of cancer involvement 10 1140 4180 81100 Number of
cancer foci 1 24 >5 Combined score of radiation effect* 02 (minimal)
Percentage of cases
17 48 35 31 28 35 6 36 50 14 52
Effect of radiotherapy on prostate
11
34 (moderate) 38 56 (severe) 10 Infiltrative growth 100 Perineural invasion 31 Atr
ophic change 10 Nuclear pyknosis 72 Nuclear enlargement 93 Prominent nucleoli 79
Percentage of cytoplasmic vacuolization <10 45 1050 45 >50 10 Inflammation 0 Str
omal desmoplasia 76 Necrosis 0 Intraluminal contents Crystalloids 3 Mucin 21 Eos
imophilic secretions 24 Corpora amylacea 0 Concomitant high-grade PIN 7 * Radiat
ion effect was quantified using the scoring system described by Crook and co-wor
kers.22 (Crook JM, Bahadur YA, Robertson SJ, Perry GA, Esche BA, Evaluation of r
adiation effect, tumor differentiation, and prostate specific antigen in sequent
ial prostate biopsies after external beam radiotherapy for patients with prostat
e carcinoma. Cancer 1997; 79:8189,)
but complete histologic resolution of cancer may take 23 years.22 Sampling variat
ion is minimized by obtaining multiple specimens.2,19,22,2530 The therapeutic suc
cess of radiotherapy for prostate cancer requires complete or nearcomplete eradi
cation of tumor. Conventional external beam radiotherapy misses 20% to 35% of th
e target volume when compared with three-dimensional conformal planning with dos
e escala tion.31 Brachytherapy techniques will probably improve local cancer con
trol and prolong survival.32 Evaluation of local tumor control is assisted by di
gital rectal examination and transrectal ultrasound. Posttherapy serum PSA corre
lates with posttherapy biopsy results, including degree of radiation effect.11 C
rook et al diagnosed postradiotherapy biopsies as indeterminate in 33% of first
biopsies (median 13 months), 24% of second biopsies (28 months), 18% of third bi
opsies (36 months), and 7% of fourth biopsies (44 months).22 These figures are h
igher than the 1.59.0% of biopsies with atypical indeterminate findings in unsele
cted non-irradiated series,7,12 highlighting the increased diagnostic challenge
after radiotherapy. The identification of cancer in needle biopsy specimens afte
r radiotherapy has a significant impact on patient management; positive needle b
iopsies portend a worse prognosis.1,15,3338 The histologic diagnosis of cancer wi

thout radiation effect relies on both architectural and cytoplasmic atypia. In s

implest terms, radiotherapy causes cytologic atypia of benign glands, forcing th
e pathologist to discriminate cancer almost totally on architectural findings. C
hanges vary widely among patients.11 Radiotherapy causes shrinkage of
Basic and advanced techniques in prostate brachytherapy
12
cancer glands and loss of cytoplasm (Figure 2.4). Features most helpful for the
diagnosis of cancer after radiotherapy are mostly architectural: infiltrative gr
owth (Figure 2.5), perineural invasion, intraluminal crystalloids, blue mucin se
cretions, the absence of corpora amylacea, and the presence of concomitant high
grade PIN (Table 2.2). Paneth cell-like change can be seen in 32% of biopsies.11
Occasionally, cytologic findings such as double nucleoli in a secretory cell, c
an be helpful (Figure 2.6). Cancer grade and DNA ploidy after radiotherapy Posti
rradiation Gleason grade and DNA ploidy are independent prognostic factors in pa
tients with prostate cancer who fail radiotherapy.26 There is a slight shift aft
er therapy toward nondiploid cancer, higher Gleason grade, and high tumor stage,
indicating increasing biologic aggressiveness and cancer dedifferentiation afte
r radiation.26,30,35,39 Particularly in grade 4 cancer, radiotherapy may cause d
isappearance of glandular lumina, resulting in grade 5 morphology (Figure 2.7).
The authors found a good correlation of Gleason grade between postirradiation sa
lvage prostatectomy and treated biopsy specimens.26,40 Needle biopsies underesti
mated prostatectomy Gleason grade in 35% of cases and overestimated grade in 14%
of cases, similar to the findings in studies of patients who were not treated b
y radiotherapy.38,4045 By comparison, in 316 patients who underwent radical prost
atectomy without prior androgen deprivation or radiotherapy, Gleason grade in ne
edle biopsies under
Figure 2.4 Irradiated cancer glands (right) retain an angulated, infiltrative pa
ttern and luminal secretions, as seen pretreatment (left). The degree of cytolog
ic atypia is paradoxically less than in some benign irradiated glands.
Effect of radiotherapy on prostate
13
Figure 2.5 The infiltrative pattern is characteristic of cancer in this irradiat
ed gland. Cytoplasm is moderate and clear to finely granular in this case.
Figure 2.6 In this irradiated cancer, the finding of double nucleoli (upper left
) together with an infiltrative pattern helps establish the diagnosis of residua
l cancer.
estimated prostatectomy grade in 40% of cases and overestimated grade in 25% of
cases.40 Siders and Lee evaluated matched preradiation and postradiation specime
ns from 58 patients and found a 24% increase in the number of poorly differentia
ted cancers (Gleason score 810) and a shift toward aneuploid cancer in 31% of pre
treatment diploid cancers.5 Similarly, others found an increase in tumor grade f
ollowing irradiation,1630 suggesting that the higher grade cancer frequently foun
d after treatment was related to a process of clonal evolution that resulted in
cancer progression and tumor dedifferentiation. Some investigators recommend gra
ding of cancer in specimens after radiotherapy, recognizing that the biologic si
gnificance of grade may be different from that in untreated cancer.26 The author
s believe that Gleason grade in postirradiation
Basic and advanced techniques in prostate brachytherapy
14

needle biopsy specimens provides useful predictive information and recommend its
use in this setting,3 despite suggestions to the contrary.46
Figure 2.7 High grade prostate cancer pretreatment (left) loses any remnant of g
landular lumina after irradiation (right), consistent with evolution of a higher
grade tumor clone.
Clinical significance of postradiation biopsy results Digital rectal examination
for the detection of radiation failure is imprecise unless there is gross cance
r recurrence.47 Consequently, some clinicians favor postirradiation biopsy for t
he preclinical detection of recurrence, thereby allowing earlier intervention wi
th salvage therapy; others consider routine postirradiation biopsy justifiable o
nly in a research setting. Studies suggest that if prostatic carcinoma is not hi
stologically ablated by radiotherapy after 12 months, it is probably biologicall
y active.2,28,40 The rate of positive findings on biopsy varies from 20% to 93%
following external beam radiotherapy,22,27,29,4851 and from 5% to 55% following b
rachytherapy.46 This wide variation is attributable to selection of patients wit
h broad ranges of pretreatment serum PSA, stage and grade of tumor, number of bi
opsy cores taken (more in contemporary studies), and radiation dosage. Interobse
rver variability may be an extra source of variation, as discussed below. A posi
tive biopsy result within 12 to 18 months of external beam radiotherapy may cont
ain cancer in regression, and 30% of patients show eventual clearance of tumor a
t a mean time of 30 months after radiotherapy.52 Kuban and Schellhammer have sho
wn that a positive biopsy result after 1218 months predicted clinical recurrence
in approximately
Effect of radiotherapy on prostate
15
80% of patients; remarkably, approximately 20% had no evidence of cancer at 10 y
ears follow-up.1 However, one of us (DGB) has reviewed the histopathologic findin
gs from that study and noted an original diagnostic error rate of about 10%, cal
ling the results into question. Crook et al extended this interval to 24 months,
eliminating biopsies prior to that from their study because delayed tumor progr
ession was seen in 30% of patients.22 At 2436 months, the biopsy result was one o
f two independent predictors of outcome, along with PSA nadir. Perineural invasi
on of cancer, however, was not an independent prognosticator in patients undergo
ing brachytherapy.53 Conversely, 30% of patients with local or distant failure h
ad negative findings on biopsy.48 An identical 30% positive rebiopsy rate was fo
und in men suspected of having cancer but whose initial TRUSguided biopsy was ne
gative.54 This underscores the role of sampling variation: the falsepositive rat
e of biopsy is 23% based on repeat biopsies in untreated men with prior positive
biopsy.55 Interobserver reproducibility in the diagnosis of cancer in postradia
tion biopsies varies moderately. Miller et al found a false-positive rate of 15% (
4/26 specimens) and a false-negative rate of 3% (2/70 specimens).56 Jones et al cl
assified 107 cases signed out by non-subspecialty pathologists and found 1 false
-positive and 9 false-negative cases.57 However, 5 of 6 cases classified as susp
icious by nonsubspecialty pathologists were negative according to at least two o
f a panel of three specialty urologic pathologists, again showing some tendency
toward overdiagnosis. Urologic pathologists disagreed with each other in 3% (3/1
07) cases; two of three agreed with 23% of cases and all agreed with 74% of case
s. Mean Kappa value was 0.66, indicating only moderate reproducibility. Radiothe
rapy combined with androgen ablation Neoadjuvant androgen deprivation therapy (A
DT) appears to have an additive or synergistic effect with external beam radioth
erapy. In one study, 31 patients were treated with ADT before radiotherapy, and
only 3 (10%) had cancer on posttherapy biopsy compared to 44 of 106 men (41%) tr
eated with radiotherapy alone (p= 0.004).16 Androgen ablation probably also pote
ntiates brachytherapy. Scoring radiation effect in the benign prostate To determ
ine whether the severity and extent of radiation changes in the prostate are of
prognostic value, Crook and colleagues graded nuclear and cytoplasmic changes in

biopsy specimens following external beam radiotherapy.22 Cytoplasmic and nuclea

r changes were each graded on a 03 scale, and added together for a score of 06. Th
ey found that grading of radiation effect in the noncancerous prostate correlate
d with serum PSA nadir, immunoreactivity for proliferating cell nuclear antigen
(PCNA), and local cancer recurrence.22 Patients did poorly if there was little o
r no evidence of radiation change in the needle biopsy, suggesting incomplete co
verage of the prostate by the therapeutic field or radiation-resistant foci as t
he source of local failure. Goldstein and co-workers consider grading nuclear an
d cytoplasmic changes useful in a threeyear
Basic and advanced techniques in prostate brachytherapy
16
prospective study of patients receiving brachytherapy.46 They also noted that th
e presence of adenocarcinoma on posttreatment biopsy was an important predictor
of failure. Salvage radical prostatectomy specimens, conversely, demonstrated gr
eat discrepancy with biopsies in the scoring of radiation effect after external
beam radiotherapy.26 In needle biopsy specimens, 48% had moderate or severe radi
ation effect compared with only 6% of radical prostatectomy specimens. These fin
dings suggest that scoring of radiation effect in needle biopsies may also overe
stimate the effectiveness of brachytherapy and could be misleading. This discrep
ancy could also explain why cytologic atypia in benign glands was observed in 98
% of posttatectomy specimens after radiotherapy for urothelial irradiation biops
ies,11 and 77% of prostates in cystoproscarcinoma.58 Quantification of radiation
effect is of questionable relevance in patients who fail to be cured by radioth
erapy. Differential diagnosis of prostate cancer after radiotherapy In the autho
rs experience, atypical basal cell hyperplasia most frequently mimics treated can
cer following irradiation. Atypical basal cell hyperplasia is defined as basal c
ell proliferation with more than 10% of cells exhibiting prominent nucleoli. The
se cells were present in 57% of cases in the authors recent study of salvage pros
tatectomies and seemed to represent a nonspecific host response to radiation inj
ury. Immunohistochemical and other findings after radiotherapy Prostatic acid ph
osphatase, prostate-specific antigen, and keratin 34E12 No definite method exists
for the assessment of tumor via
ility after irradiation. Presence of secretory
cells can
e documented
y reactivity for prostatic acid phosphatase (PAP), lead
ing one group of investigators to suggest that tumor cells capa
le of protein pr
oduction pro
a
ly retain the potential for cell division and consequent metastat
ic spread.59 Expression of prostate-specific antigen (PSA) (Figure 2.8) and pancytokeratin often persists after therapy. In a recent small study, residual carc
inoma was present in 6 of 14 cases after
rachytherapy. PSA reactivity was noted
to
e decreased in glands that show radiation effect.60 Basal cell cytokeratin
(34E12) expression also persists after radiotherapy in
enign and atrophic glands
, helping to visualize treated adenocarcinoma (Figure 2.9). Some authors report
an indeterminate rate of 33% on first posttherapy
iopsy, decreasing to 7% on fo
urth
iopsy.53 However, in our experience, indeterminate findings occur in fewer
than 10% of cases with the use of this immunostain on serial sections.25 Partic
ularly with use of the steam-EDTA optimized method,61
asal cell cytokeratin hel
ps exclude the cancer mimics mentioned a
ove: atypical
asal cell hyperplasia, a
typical adenomatous hyperplasia, sclerosing adenosis, and postatrophic hyperplas
ia.
Effect of radiotherapy on prostate
17
Proliferation markers MIB-1 (Ki-67) immunoreactivity in pretreatment needle
iop
sies independently predicts postirradiation recurrence,62 and helps determine op
timal radiation dose. In

Figure 2.8 No immunostain can prove via
ility of residual cancer,
ut when via
i
lity is in question (left), prostatespecific antigen (PSA) indicates that secret
ory cells are present, suggesting via
ility (right).
Basic and advanced techniques in prostate
rachytherapy
18
Figure 2.9 A
sence of immunoreactivity for
asal cell cytokeratin 34E12 can confi
rm that small atypical glands are cancer (left, 60; right, 160).
postradiotherapy prostate
iopsy specimens, retention of proliferating cell nucl
ear antigen (PCNA),52,63 or Mi
-1 (Ki-67)26,52 immunoreactivity correlates with
local cancer recurrence (p=0.004). After
rachytherapy, residual carcinoma that
shows radiation injury also has a minimal (<5%) Ki-67 reactivity.60 Furthermore,
prostate cancer in salvage prostatectomies is proliferative in 96% of cases, sh
owing increased Mi
-1 immunostaining.26 The mean Ki-67 la
eling index in recurre
nt prostate cancer after radiation is increased (mean: 7.0%) when compared with
the index in prostatectomy series without prior radiotherapy (mean: 2.7%) (unpu

lished data, DG Bostwick, MD). Oncogenes and tumor suppressor genes Prostate can
cer after radiotherapy has increased p53 nuclear accumulation, although some oth
er results suggest no significant difference.64 Prendergast and co-workers studi
ed 18 patients with locally recurrent prostate cancer after radiotherapy and fou
nd that 72% had p53 nuclear immunoreactivity.65 Of five patients for whom the re
sults of preradiation
iopsies were availa
le, all had p53 immunoreactivity. The
immunohistochemical findings correlated with single strand conformational polym
orphism and DNA sequencing analysis.65 These findings suggest that p53 alteratio
ns are present
efore radiotherapy and may serve as a pretherapy factor predicti
ve of cancer recurrence. Glutathione S-transferase pi (GST-) is a detoxifying enz
yme that inactivates reactive oxygen free radical secies by conjugation with gl
utathione. Most rostate cancers do not
Effect of radiotheray on rostate
19
exress GST-, and loss of GST- exression is considered as a henotye associated
with malignant transformation.66 21WAF1 and 27Ki1 are members of the KIP fami
ly of cell cycle roteins and inhibit several cyclin-deendent kinase comlexes.
Functional loss of the cycle-deendent inhibitors has been imlicated in carcin
ogenesis and cancer rogression. Loss of 27Ki1 exression in rostatic and non
rostatic malignancies is 21WAF1 function has been imlicated in the failure of
associated with a more aggressive henotye.67 Loss of irradiation resonse, an
d 21 has been shown to be an indeendent rognostic factor in rostate carcinom
a. The authors detected 21WAF1 nuclear immunoreactivity in cancer cells in 39 (
75%) of 52 atients (median nuclear immunoreactivity, 5%; range: 080%); 27Ki1 n
uclear immunoreactivity was detected in all 52 atients (median nuclear immunore
activity, 50%; range: 590%). Five-year distant metastasis-free and cancer-secifi
c survival rates were 71% and 82% for atients with low exression of 21 (5%), c
omared with 94% and 100% for atients with high exression of 21 (>5%) (=0.02
and 0.01, resec tively).67 Five-year distant metastasisfree survival and cance
r-secific survival rates were 91% and 82% for atients with low exression of 
27 (<50%), comared with 88% and 96% for atients with high exression of 27 (50
%) (=0.06 and 0.01, resectively). Antiaotosis genes Early growth resonse-1
(Egr-1) gene is an early resonse gene, in the family of c-jun and c-fos. Egr-1
activation is required for the cellular resonse to radiation injury. The author
s noted overexression of Egr-1 in rostate cancer, which increased with Gleason
grade.68 Ahmed et al later found that Egr-1 immunohistochemical exression corr
elated with treatment failure. The overexressed Egr-1 is in a mutant form which
does not transactivate the usual target genes TP53, RB, and Bax.69 Egr-1 may c
ome to be used as art of a anel with a roliferation marker to redict rognos

is. Microvessel density In a study by Hall and co-workers,70 microvessel density

was higher in cancer secimens from atients who failed radiotheray than in a
tients who did not fail; however, the results were not analysed indeendently of
cancer grade. Nuclear morhometry The degree to which nuclei deviate from circu
larity redicts the rognosis of atients with stage 2 rostate cancer. This obs
ervation was alied to biosies from men treated with external beam irradiation
. A rognostic factor score incororating two arameters, subotimal circle fit
and feretdiameter ratio, redicted cancer-free survival (=0.0014).71 Summary Su
bstantial and characteristic changes occur in the microscoic aearance and imm
unohenotye of the hyerlastic rostate and adenocarcinoma following androgen
Basic and advanced techniques in rostate brachytheray
20
derivation theray and radiotheray. These changes are rarely seen in untreated
cancer, and in the authors oinion, the combinations of features following thera
y are sufficiently distinctive that athologists can usually recognize them. Pa
thologists must be aware of these distinct changes because of the reliance lace
d on nuclear and nucleolar size in the identification of rostate cancer, artic
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the management of rostate cancer. J Urol 1992; 147:917921. 34. Kuban DA, Schellh
ammer PF. Prognostic significance of ost-irradiation rostate biosies. Oncolog
y 1993; 7:2938. 35. Pontes JE, Montie J, Klein E, et al. Salvage surgery for radi
ation failure in rostate cancer. Cancer 1993; 71:976980. 36. Prestidge BR, Kala
n I, Cox RS, et al. Predictors of survival after ositive ost-irradiation rost
ate biosy. Int J Radiat Oncol Biol Phys 1993; 28:1722. 37. Prestidge BR, Kalan
I, Cox RS, et al. The clinical significance of a ositive ost-irradiation rost
ate biosy without metastasis. Int J Radiat Oncol Biol Phys 1992; 24:403408. 38.
Zagars GK, von Eschenbach AC, Ayala AG, et al. The influence of local control on
metastatic dissemination of rostate cancer treated by external beam megavoltag
e radiation theray. Cancer 1991;68: 23702377. 39. Ahlering TE, Lieskovsky G, Ski
nner DG. Salvage surgery lus androgen derivation for radioresistant rostatic
adenocarcinoma. J Urol 1992; 147:900902. 40. Bostwick DG. Gleason grading of ros
tatic needle biosies: Correlation with grade in 316 matched rostatectomies. Am
J Surg Pathol 1994; 18:796803. 41. Bostwick DG. Grading rostate cancer. Am J Cl
in Pathol 1994; 102 (4 sul 1):538556.
Basic and advanced techniques in rostate brachytheray
22
42. Mills SE, Fowler JE. Gleason histologic grading of rostatic carcinoma: Corr
elations between biosy and rostatectomy secimens. Cancer 1996; 57:346349. 43.

Sires SE, Cibull ML, Wood DP, et al. Gleason histologic grading in rostatic ca
rcinoma: Correlation of 18-gauge core biosy with rostatectomy. Arch Pathol Lab
Med 1994; 118:705 708. 44. Steinberg DM, Sauvageot J, Piantadosi S, et al. Corre
lation of rostate needle biosy and radical rostatectomy Gleason grade in acad
emic and community settings. Am J Surg Pathol 1997; 21:566576. 45. Thickman D, S
eers WC, Philott PJ, et al. Effect of the number of 1core biosies of the rost
ate on redicting Gleason score of rostate cancer. J Urol 1996; 156:110113. 46.
Goldstein NS, Martinez A, Vicini F, Stromberg J. The histology of radiation ther
ay effect on rostate adenocarcinoma as assessed by needle biosy after brachyt
heray boost: correlation with biochemical failure. Am J Clin Pathol 1998; 110:7
65775. 47. Letran JL, Brawer MK. Management of radiation failure for localized r
ostate cancer. Prostate Cancer Prostatic Dis 1998; 1:119127. 48. Freiha FS, Bagsh
aw MA. Carcinoma of the rostate: results of ostirradiation biosy. Prostate 19
84; 5:1924. 49. Forman JD, Oenheim T, Liu H, et al. Frequency of residual neol
asm in the rostate following three-dimensional conformal radiotheray. Prostate
1993; 23:235243. 50. Herr HW, Whitmore WF. Significance of rostatic biosies af
ter radiation theray for carcinoma of the rostate. Prostate 1982; 3:339350. 51.
Scardino PT, Frankel JM, Wheeler TM, et al. The rognostic significance of ost
-irradiation biosy results in atients with rostatic cancer. J Urol 1986; 135:
510516. 52. Crook J, Malone S, Perry G, et al. Postradiotheray rostate biosies
: what do they really mean? Results for 498 atients. Int J Radiat Oncol Biol Ph
ys 2000; 48:355367. 53. Merrick GS, Butler WM, Galbreath RW, et al. Perineural in
vasion is not redictive of biochemical outcome following rostate brachytheray
. Cancer J 2001; 7:404412. 54. Fleshner NE, OSullivan M, Fair WR. Prevalence and 
redictors of a ositive reeat transrectal ultrasound-guided needle biosy of th
e rostate. J Urol 1997; 158:505508. 55. Rabbani F, Stroumbakis N, Kava BR, et al
. Incidence and clinical significance of false-negative sextant rostate biosie
s. J Urol 1998; 159:12471250. 56. Miller EB, Ladaga LE, El-Mahdi AM, Schellhammer
PF. Reevaluation of rostate biosy after definitive radiation theray. Urology
1993; 41:311316. 57. Jones EC, Srigley J, Daya D, et al. The assessment of ostradiation rostatic needle biosies for residual adenocarcinoma: a study of inte
robserver agreement. Mod Pathol 2002; 15:166A. 58. Sheaff MT, Baithun SI. Effect
s of radiation on the normal rostate gland. Histoathology 1997; 30:341348. 59.
Mahan DE, Bruce AW, Manley PN, et al. Immunohistochemical evaluation of rostati
c carcinoma before and after radiotheray. J Urol 1980; 124:488492. 60. Srouse J
T, Smathers SL, Wallner K, True LD. Histologic and immunohenotyic features aft
er rostate brachytheray. Am J Clin Pathol 1999; 112:548. 61. Iczkowski KA, Che
ng L, Crawford BG, Bostwick DG. Steam heat with an EDTA buffer and rotease dige
stion otimizes immunohistochemical exression of basal cell-secific antikerati
n 34E12 to discriminate cancer in prostatic epithelium. Mod Pathol 1999; 12:14. 62
. Scalzo DA, Kallakury BVS, Gaddipati RV, et al. Cell proliferation rate
y MIB1 immunohistochemistry predicts postradiation recurrence in prostatic adenocarci
omas. Am J Clin Pathol 1998; 109:163168. 63. Crook J, Ro
ertson S, Esche B. Proli
ferative cell nuclear antigen in postradiotherapy prostate
iopsies. Int J Radia
t Oncol Biol Phys 1994; 30:303308. 64. Rakozy C, Grignon DJ, Sarkar FH, et al. Ex
pression of
cl-2, p53, and p21 in
enign and malignant prostatic tissue
efore
and after radiation therapy. Mod Pathol 1998; 11:892899.
Effect of radiotherapy on prostate
23
65. Prendergast NJ, Atkins MR, Schatte EC, et al. p53 immunohistochemical and ge
netic alterations are associated at high incidence with post-irradiation locally
persistent prostate carcinoma. J Urol 1996; 155:16851692. 66. Brooks JD, Weinste
in M, Lin X, et al. CG island methlyation changes near the GSTP1 gene in prostat
ic intraepithelial neoplasia. Cancer Epidemiol Biomarkers Prev 1998; 7:531536. 67
. Cheng L, Lloyd RV, Weaver AL, et al. The cell cycle inhi
itors p21WAF1 and p27
KIP1 are associated with survival in patients treated
y salvage prostatectomy
after radiation therapy. Clin Cancer Res 2000; 6:18961899. 68. Eid MA, Kumar MV,

Iczkowski KA, et al. Expression of early growth response-1 genes in human prosta
te cancer. Cancer Res 1998; 58:24612468. 69. Ahmed MM, Chendil D, Lele S, et al.
Early growth response-1 gene: potential radiation response gene marker in prosta
te cancer. Am J Clin Oncol 2001; 24:500505. 70. Hall MC, Troncoso P, Pollack A, e
t al. Significance of tumor angiogenesis in clinically localized prostate carcin
oma treated with external
eam radiotherapy. Urology 1994; 44:869 875. 71. Hurwit
z MD, DeWeese TL, Zinreich ES, et al. Nuclear morphometry predicts disease-free
interval for clinically localized adenocarcinoma of the prostate treated with de
finitive radiation therapy. Int J Cancer (Pred Oncol) 1999; 84:594597.
3 What should you ask your pathologist when contemplating minimally invasive the
rapy?
Ro
ert O Petersen Introduction Central to the choice of definitive therapy for p
rostate cancer whether surgery, radiation, hormonal, or watchful waiting, is the
prediction of tumor stage. Protocols designed to detect possi
le distant and/or
local progression have
een standardized. In addition, the 1990s witnessed the
development of numerous models or nomograms designed to provide pretherapy predi
ction of organ containment or extraprostatic extension. Foremost among these nom
ograms are the Partin Ta
les introduced in 1993 with multiple su
sequent revisions
and updates.14 These nomograms com
ine the information of: (1) clinical stage de
termined
y digital rectal examination (DRE); (2) serum prostate-specific antige
n (PSA) level; and (3) Gleason score of the needle
iopsy. The American Brachyth
erapy Society (ABS) utilizes these same three parameters to form the
asis of sui
ta
ility criteria in evaluating patients for this form of definitive therapy.58 An
analysis of these parameters has repeatedly demonstrated that DRE-determined cl
inical stage and serum PSA used individually or in com
ination, have limited dis
criminatory value. Only the Gleason grade has a measure of o
jectivity and has p
roven predictive value. The dramatic change in the patient population during the
mid and late 1990s, characterized
y a significant decrease in patient age, ser
um PSA levels and recorded tumor volumes has added stress to the system. Patients
are frequently identified in screening programs
y elevations of serum PSA witho
ut accompanying a
normal DRE (stage T1c). Recognizing a minimal PSA level elevat
ed a
ove a previously esta
lished
aseline with all determinations less than 4 n
g/mL, is o
served in increasing num
ers of patients. The predictive value of nom
ograms constructed on a patient population not characterized
y these presentati
ons is currently under study. Throughout this past decade the predictive value o
f the needle
iopsy Gleason score has
een repeatedly demonstrated. This chapter
will address the contri
utions, and
Ta
le 3.1 Prognostically important information from prostate needle
iopsies
1. Gleason score 2. Num
er of positive
iopsies 3. Tumor volume in needle
iopsi
es 4. Site of positive
iopsies
What should you ask your pathologist
25
5. Perineural invasion 6. Histologic variants of prostate carcinoma
indeed, the limitations of Gleason grading, and six additional morphologic param
eters (also provided
y analysis of prostate needle
iopsies) are listed in Ta
l
e 3.1. Future nomograms will pro
a
ly incorporate those additional features prov
ing to increase their sensitivity and positive predictive value. Gleason grading
of prostate carcinoma in needle
iopsies The grading of prostate carcinoma date
s
ack approximately 80 years. During this period, as many as thirty proposed tu
mor grading protocols have appeared in the literature. Among all the proposed pr
otocols, only the Gleason grading system introduced in 1966 has emerged to achie
ve world-wide acceptance during the past decade.1113 Exclusive of tumor stage, it
is the only morphologic parameter to
e of proven prognostic importance.13 The
Gleason grade of the tumor identified in needle
iopsies has demonstrated value

in pretherapy prediction of capsular penetration, seminal vesicle involvement, p

ositive surgical margins, pelvic lymph node metastases, and posttherapy
iochemi
cal failure and cancer-specific survival following definitive therapy. Multiple
nomograms, foremost the Partin nomograms, for predicting pathologic stage from p
reoperative clinical and pathologic parameters, utilize the Gleason grading syst
em.1,3,4,14 Gleason grading recognizes five histologic patterns of tumor prolife
ration which were formalized and illustrated
y Dr Gleason. Cytologic features,
although contri
uting to the diagnosis of prostate carcinoma, are not considered
in the Gleason grading criteria. Recognizing the typical heterogeneity of tumor
patterns, the grading system recognizes a predominant and minority pattern. Thu
s, the final Gleason score results from the addition of the two num
ered pattern
s, for example, 3+2(5), 4+3(7). The potential range of Gleason scores is 1+1(2)
to 5+5(10). Cumulative experience has demonstrated the majority of prostate carc
inomas have Gleason scores 57, with fewer than 10% of cases Gleason 24, and approx
imately 20% of cases Gleason 810. In recent studies, the significance of tertiary
patterns contri
uting <5% of the tumor volume in needle
iopsies has
een evalu
ated.15,16 The results suggest prognostic importance, a finding to
e confirmed
in future studies. Currently, it is recommended that this additional pattern inf
ormation
e included in a note, and not in a formal modification of the Gleason
score in the diagnosis. A similar notation of a tertiary score that represents t
he highest Gleason pattern, regardless of the volume contri
ution, should
e inc
luded
ecause it has possi
le prognostic implications.15,16 Reflecting the signi
ficant difference in clinical aggressiveness of Gleason 6 and Gleason 7 (the lat
ter containing a component of Gleason pattern 4), multiple studies have addresse
d the significance of Gleason pattern 4 and 5 in needle
iopsies. Loch et al (19
95) demonstrated that the largest tumor in the prostatectomy specimen could
e p
redicted
y the side of the greatest volume of Gleason 4/5 pattern in the diagno
stic
iopsies.17 Conrad et al (1998) o
served significant increase in risk of pe
lvic node metastases when Gleason 4/5 patterns were o
served in needle
iopsies.
18 Taneja et al (1999) reported among patients with
ilaterally positive
iopsie
s, a significant association
Basic and advanced techniques in prostate
rachytherapy
26
of the
iopsy site positive for Gleason 4/5 and the site of extraprostatic exten
sion of tumor in the prostatectomy specimen.19 Most recently, Noguchi et al (200
1) reported the presence of Gleason 4/5 significantly reduced the pro
a
ility th
at the prostate tumor was clinically insignificant (<0.2 cc).20 The Brachytherap
y Quality Assurance Group of the American Brachytherapy Society recognized the s
ignificant increased aggressiveness of pattern 4, recommending
achytherapy, as
monotherapy,
e reserved only for patients with
iopsy Gleason scores <8.6 Altho
ugh Gleason grading of prostatic needle
iopsies is the cornerstone of therapeut
ic decision making, a discussion of its contri
ution would
e incomplete without
considering its limitations demonstrated in daily practice. Grading, in the fin
al analysis, is an exercise of interpreting morphologic features and the applica
tion of esta
lished Gleason criteria. The su
jectivity is always present,
ut fr
equently difficult to quantitate. Intra- and intero
server varia
ility of Gleaso
n grade assignment is regretta
ly not infrequent. Intero
server varia
ility is c
urrently commonly encountered when prostate
iopsies are reviewed in referral pa
thology departments.21 Indeed, varia
ility of interpretation among experienced u
rologic pathologists has
een documented.22 If the experts disagree, it is not a m
ajor surprise that Gleason grading errors
y less experienced pathologists commo
nly occur. The most frequent error of
iopsy interpretation of prostatic cancer
is undergrading with frequencies of 2657% (Ta
le 3.2).2335 Overgrading occurs, alt
hough less frequently (526%). Studies of the clinical implications of the spectru
m of Gleason scores, originating from the same major institutions reporting conc
ordance of
iopsy and prostatectomy Gleason scores ranging from 26% to 58% (mean
: 42%) is so
ering. The tendency of undergrading is not uniform throughout the G

leason grade spectrum. The highest frequency of undergrading occurs in the range
of Gleason 24. Epstein has stated that the diagnosis of Gleason score 24 should n
ot
e made on needle
iopsies, acknowledging the high frequency of undergrading
associated with this diagnosis.21 The lowest frequency occurs in the spectrum of
Gleason 810. The extent of the non-concordance of Gleason grading reported in th
e literature, has
een acknowledged,
ut not emphasized. Equal emphasis has
een
placed on the su
stantially higher concordance if it is com
ined with plus and
minus one Gleason score. This form of concordance produces a range of 6295%, clearl
y more accepta
le. However, on closer inspection, a different interpretation eme
rges. The clinical significance of undergrading a carcinoma found to
e Gleason
5 or 6 in the prostate specimen has marginal clinical significance. However, whe
n a Gleason 7 is interpreted as Gleason 5 or 6 on
iopsy, the consequences are n
ot trivial. The frequency of such undergrading is 3990%. Ta
le 3.2 reviews 15 Gle
ason grading concordance studies demonstrating that prostate cancers interpreted
as Gleason 5 and 6 represented undergrading of Gleason 7 neoplasms in 31%, and
45% of cases, respectively. When undergrading of Gleason 7 occurs, the inherent
increased aggressiveness of Gleason pattern 4 is not
eing recognized preoperati
vely, with the resultant flaw in the preoperative predictive power of accurate G
leason grading. Explanations of this non-concordance include: (1) nonrepresentat
ive sampling of the tumor: (2) small volume of tumor in the needle
iopsy; and (
3) failure to recognize invasive features (pattern 3) or gland fusion (pattern 4
). In recent studies, documented tumor volumes appear to
e decreasing in the er
a of PSA screening, with the anticipated
What should you ask your pathologist
27
effect on this component of grading error. With increasing experience, and expos
ure to computer teaching programs
Ta
le 3.2 Biopsy/prostatectomy concordance studies
Spires Bost Kojima Paul Hump Thick Stein 94 wick 95 son hrey man
erg U. 94 MDA 94 95
96 97 Kent Mayo Duke Wash Comm JHH ucky Clinic U unity Total cases Conco rdance 1
2 Under grade (%) Over grade (%) Clust ered Conco rdance Bx 36 % Glea son 3 6 Bx
7 10 % Glea son 710 Bx 6 % Glea son >6 Bx 5 5 % Glea son >6 Bx 7 % Glea son <7 6
7 316 135 734 50 124 499 Cook Danz Dja son iger van 97 97 98 MSSK NYMC U Texas 226
100 34% 72% 88% 46% 17% 415 Kok Ega sal, vad 00 01 Istan Upp
ul sala U. 144 121 A
ltay 01 U EGE Nog Pete uchi rson* 01 TJUH Stan ford 222 223
57
58% 34% 48%
42% 26% 28%
58% 31% 95% 90% 100% 94% 36% 54% 7% 14%
93% 74% 94% 67% 76% 62% 97% 93% 100% 79% 96% 85% 26% 40% 46% 43% 48% 57% 14% 25%
5% 14% 26% 15%
37% 45% 45% 47% 36% 84% 26 42% 58% 74% 89% 93% 77% N.I. 90% 91% 98% 100% 100% N.I
. 100% 50.1% 45% 39% 42% 46% 36% 26 43% 57% 12.8% 10% 16% 18% 18% 10% 5 15% 26%
61% 50% 18%
41% 81% 65%
64% 61%

64%
72% 75% 75% 71% N.I. 67% 18 58% 75% 73% 75% 92% 62% N.I. 80% 67 67% 98% 46% 40% 38
.5% 25% N.I. 34% 25 45% 90% 24% 17% 4% 56% N.I. 39% 0 31% 87%
92% 79% 98%
84% 60% 67%
88% 80%
87%
50% 61% 90%
40% 29% 50%
38% 50%
45%
0
46% 80%
29% 6%
37%
29% 39%
21%
6%
22% 2%
15% 33% 27%
13% 21%
16%
29% 26% 11% 71% N.I. 22% 2 16% 27%
* Unpu
lished study
y author
of Gleason grading, diagnostic accuracy will improve. The component of Gleason n
onconcordance attri
uta
le to non-representative sampling will pro
a
ly always

e present with potential for undergrading. In summary, the predictive value of t
he Gleason score of prostate carcinoma identified in needle
iopsies is esta
lis
hed and has
een incorporated into virtually all nomograms designed to preoperat
ively predict tumor pathologic stage. The predictive value of the Gleason score
is enhanced when com
ined with other preoperative parameters. The pro
lem of und
ergrading is significant, and underscores the importance of relia
le and accurat
e interpretation of the pathologic features contri
uting to Gleason grade assign
ment in needle
iopsies.
Basic and advanced techniques in prostate
rachytherapy

28
Num
er of positive
iopsies As a measura
le preoperative parameter it is intuiti
ve that the higher the num
er of positive
iopsies, the larger the prostate carc
inoma, and the higher the pro
a
ility of local and distant spread (higher pathol
ogic stage). The formal studies pu
lished testing this hypothesis are numerous a
nd reflect continuing refinement of
iopsy protocols during the 1990s. The intro
duction of the sextant
iopsy protocol
y Hodge et al occurred in 1989.36 A signif
icant improvement in diagnostic yield was achieved compared to previously employ
ed directed
iopsies. The effectiveness of the sextant protocol was confirmed in l
ater studies.3740 Indeed, its effectiveness prompted studies to evaluate the freq
uency of detection of clinically insignificant tumors.41,42 Clinically unimporta
nt tumors were variously reported to constitute 27%, 4% of the cases with two or
more, and one positive
iopsy, respectively.41,42 The limitations of the sextan
t protocol soon appeared to dominate reported studies. The adverse influence of
increased gland size on the diagnostic yield was reported.39,40 Numerous studies
investigated the frequency of false-negative
iopsies when the sextant protocol
was used.4348 In vitro sextant
iopsies performed on prostatectomy specimens kno
wn to har
or adenocarcinoma, second sextant
iopsies performed immediately at th
e time of initial presentation, or
efore prostatectomy were reported. False-neg
ative
iopsies with a frequency of 2045% were o
served employing the a
ove protoc
ol designs.4348 This reported rate of false-negative
iopsies prompted the next g
eneration of studies designed to include previously unexamined intraprostatic lo
cations, including the transition zones and the lateral su
capsular areas.4951 Ag
ain, increasing the num
er of
iopsies significantly increased the diagnostic yi
eld.4951 Initially the 5-zone region protocol introduced
y Eskew et al (1998), was
followed
y 10-
iopsy and 12-
iopsy protocols.49 Concern a
out the possi
le res
ultant increase in identification of clinically insignificant tumors was again p
ut to rest. Appropriately designed studies, measuring the tumor volume and patho
logic stage of prostatectomy specimens, demonstrated no significant increased de
tection of clinically unimportant tumors.5153 Throughout this period of nomogram
development and refinement, increasing num
ers of patients were coming to clinic
al attention via prostate-specific antigen (PSA) screening programs, as a group
har
oring smaller neoplasms (clinical stage T1c). The percentage of patients wit
h significantly elevated PSA (>20 ng/mL) decreased while the frequency of patien
ts with PSA <10, indeed, <4 progressively increased. The contri
ution of clinica
l staging (DRE) and serum PSA has correspondingly diminished and the need to exp
loit information from the needle
iopsies intensified. Within this historical
a
ckground, studies demonstrated that the num
er of positive
iopsies significantl
y related to the frequency of: (1) positive surgical margins; and (2) positive p
elvic lymph nodes.5457 The relationship was reported as linear in many studies, o
thers showing cut-off num
ers of three or more positive
iopsies.5860 Importantly,
the presence of tumor in only one
iopsy in a sextant series did not significant
ly relate to the su
sequent identification of insignificant tumor in prostatecto
my specimens.61 A similar lack of predictive value to identify insignificant tum
or was
eing reported in studies of
iopsy tumor volume.
What should you ask your pathologist
29
Tumor volume in needle
iopsies The predictive value of
iopsy tumor volume has

een the su
ject of many studies, the majority involving the sextant
iopsy prot
ocol.50,54,58,60,62,63 Various endpoints were examined, including pathologic sta
ge and tumor volume,
oth determined after prostatectomy. Clinically significant
tumors were reported to
e relia
ly identified
y
iopsies containing >50% tumo
r volume and >3 mm of tumor in as few as one or two
iopsies.42,6567 Extraprostat
ic tumor extension is relia
ly predicted
y tumor volume in
iopsies, as determi
ned
y multivariate analysis.50,62 In several studies, Gleason grade, num
er of
positive
iopsies, and
iopsy tumor volume, used in com
ination, proved to
e th

e most relia
ly significant predictor of extraprostatic extension of tumor.42,60

,68 Finally, similar to the failure of low num
er positive
iopsies failing to r
elia
ly detect insignificant tumors, minimal (<3 mm) tumor volume in
iopsies di
d not relia
ly identify these patients.65,66,69,70 Site of positive
iopsy Limit
ed studies addressing the clinical significance of the site of positive
iopsies
have
een reported.17,19,48,56,58,71,72 The importance of positive
iopsies in
the prostate
ase has
een o
served. Badalament et al (1996) demonstrated that

ilateral positive prostate
ase
iopsies were found to significantly relate to t
he risk of extraprostatic extension.58 Tigrani et al (1999) reported the same as
sociation of
ilateral positive
iopsies in the prostate
ase associated with el
evated risk of positive surgical margins in prostatectomy specimens.56 Taneja et
al (1999) o
served the com
ination of positive prostate
ase
iopsies,
iopsy t
umor volume >50% and Gleason score >8 significantly predicted extraprostatic ext
ension.19 In contrast, Epstein et al (1999) reported that no pattern of positive

iopsies relia
ly predicted risk of extraprostatic extension.47 Perineural inva
sion The clinical significance of perineural invasion o
served in prostate needl
e
iopsies is currently undecided.13,6264,7377 Initial interest in perineural inva
sion is appropriately attri
uted to the studies of Villers et al (1988).78 These
authors studied the anatomical details of the prostatic neurovascular
undle, a
nd reported that 50% of cases accomplished capsular penetration exclusively via
perineural space invasion and extension. Additional cases demonstrated
oth peri
neural invasion and direct capsule invasion. Prostatectomy specimens demonstrate
d perineural invasion in excess of 80% of cases, including examples with organ-c
onfined tumor. Perineural invasion o
served in the apex was assocaited with an e
levated risk of positive surgical resection margins.78 The implications of this
form of prostatic carcinoma spread were o
vious in the era of nerve-sparing pros
tatectomies. Nervesparing on the ipsi-side of perineural invasion identified in
needle
iopsies would
e inappropriate.74
Basic and advanced techniques in prostate
rachytherapy
30
The reported frequency of perineural invasion in prostate needle
iopsies in fiv
e studies ranged from 16.8% to 47% (mean: 27%).54,63,74,75,79 Numerous studies a
ttempted to determine if this morphologic feature in needle
iopsies was an inde
pendent predictor of extraprostatic extension, or alternatively, a predictor of
PSA failure following radiation therapy or prostatectomy.54,62,8082 The results a
re decidedly mixed. Five studies reported perineural invasion to
e a significan
t predictor on univariate analysis of: (1) pathologic stage; (2) PSA failure fol
lowing definitive therapy; and (3) risk of positive surgical margins.54,62,8082 I
ndependent predictive value on multivariate analysis has
een reported in four s
tudies, all related to PSA failure following definitive therapy (radiation thera
py or prostatectomy).73,74,76,77,83 Currently, acknowledging that the matter req
uires further study, it is recommended that the presence of perineural invasion

e reported in needle
iopsy specimens.84 Histologic variants of prostate carcin
oma Uncommon histologic variants of prostate carcinoma, some with
iologic chara
cteristics impacting on patient management are listed in Ta
le 3.3. Virtually al
l are reported in the literature in the form of case reports and the current cum
ulative knowledge of these variants is incomplete. Clinically significant inform
ation a
out: (1) clinical
ehavior; and (2) response to various therapy protocol
s have
een documented. Basal cell carcinoma/adenoid cystic carcinoma These pros
tate carcinomas have
een reported in fewer than 30 patients, mostly in the last
decade.8589 These patients most commonly present with
ladder outlet o
struction
, importantly, without accompanying elevation of serum PSA. Limited availa
le in
formation affords that most are at low pathologic stage and demonstrate limited
aggressive tendencies. One reported patient died with tumor metastases.89 The tu
mor is PSA-negative, and therefore posttherapy monitoring of serum PSA is not ap
propriate. No reported cases have
een treated with radiation therapy. Carcinoid
tumors of the prostate These tumors have
een reported in approximately 20 pati

ents, only five demonstrating pure examples of this

Ta
le 3.3 Histologic variants of prostate
1. Basal cell/Adenoid cystic carcinoma 2.
oepithelial carcinoma 5. Signet-ring cell
ed (neuroendocrine) carcinoma 7. Squamous
rcinoma

carcinoma
Carcinoid 3. Ductal carcinoma 4. Lymph
carcinoma 6. Small cell undifferentiat
cell carcinoma 8. Transitional cell ca

What should you ask your pathologist

31
neoplasm.9096 The majority of prostatic carcinoid tumors are a histologic compone
nt admixed with typical prostatic adenocarcinoma. None of the patients demonstra
ted the carcinoid syndrome. Follow-up is limited,
ut five patients were reporte
d to have died of tumor-related causes with metastases. These neoplasms are to

e differentiated from the more frequent and aggressive small cell undifferentiat
ed (neuroendocrine) carcinomas (see discussion
elow). Ductal carcinoma of the p
rostate This is currently the most controversial variant of prostate carcinoma.
Originally reported in the 1960s as endometriaP carcinoma, an interpretation now
a
andoned in favor of the overwhelming evidence of prostatic origin (these neopl
asms are PSA-positive).9799 Having settled this aspect of histogenesis, the curre
nt de
ate centers on whether there is a nosologic entity of ductal carcinoma, or w
hether these neoplasms represent extensive intraductal spread of a typical (acin
ar) prostatic adenocarcinoma.100106 This matter unresolved aside, the greatest cl
inical significance of neoplasms demonstrating extensive ductal involvement is t
hat they demonstrate an elevated frequency of high stage at presentation and inc
reased aggressiveness.102,103 Mention should
e made of the diagnostic challenge
these cases frequently pose when encountered in needle
iopsies. The differenti
al diagnosis of cri
riform high grade prostatic intraepithelial neoplasia (PIN)
or intraductal spread of prostate carcinoma (as a component of invasive malignan
cy) may
e unresolva
le without additional
iopsies.106,108 Immunohistochemical
staining for
asal cells (high molecular weight cytokeratin) does not resolve th
e issue. Definitive therapyprostatectomy or radiation therapyshould not
e institu
ted
efore the diagnosis of invasive carcinoma is esta
lished. Lymphoepithelioma
-like prostatic adenocarcinoma One example of this has
een reported. The patien
t was treated
y radical prostatectomy, the pathologic stage determined to
e pT
3c, and was reported alive without evidence of progression at 15 months.107 Expe
rience with greater num
ers of cases will determine if this prostatic neoplasms i
nherent
iologic
ehavior is similar to lymphoepithelioma-like carcinomas in oth
er sites of origin. Signet-ring cell carcinomas of the prostate These appear in
the literature as case reports and a few small series.108110 The limited informat
ion distilled from the literature indicates that most are aggressive neoplasms w
ith a high stage at initial presentation. All are PSApositive, and the majority
of these neoplasms are mucin-positive. Their
ehavior reflects the high Gleason
grade (pattern 4 or 5). Small cell undifferentiated neuroendocrine carcinomas (S
CUCa) These are the most frequent histologic variants of prostate carcinoma with
80% of the 220 cases reported since 1990.111120 The majority of the reported cas
es are present at the
Basic and advanced techniques in prostate
rachytherapy
32
time of initial presentation, with approximately 20% of cases evolving from typica
l prostatic adenocarcinomas, frequently following hormonal and/or radiation ther
apy. SCUCa are commonly high stage at initial presentation, with fewer than 5% o
f reported patients surviving two years after the diagnosis. These neoplasms are
frequently associated with only modest, or no elevations of serum PSA.115 Appro
ximately 10% are associated with paraneoplastic syndromes.118120 No standardized

effective therapy has evolved, several reports outline chemotherapy protocols.11

4,116 The neoplasms are hormone-refractory. Squamous cell carcinoma This is rare
ly primary in the prostate gland. In the literature, 36 cases have
een located
half of which have
een reported since 1980.121,122 Like transitional cell carci
nomas of the prostate, these patients frequently present with urinary retention
without elevations of serum PSA. Spread
eyond the prostate is common at the tim
e of initial presentation. A wide spectrum of primary therapies has
een employe
d with limited success. Of 19 patients with provided follow-up information, 15 w
ere alive with tumor or had died of tumor-related causes. Primary transitional c
ell carcinoma of the prostate This is rare, the overwhelming majority representi
ng secondary involvement of a urinary
ladder primary tumor.123125 Prostatic invo
lvement
y transitional cell carcinoma is not associated with elevations of seru
m PSA. The neoplasm is not responsive to hormonal therapy. Radical prostatectomy
, with or without postoperative radiation, appears to
e the most effective ther
apy in the limited studies providing therapy information. Adjuvant chemotherapy
has recently
een introduced, the therapeutic
enefit of which awaits further ev
aluation in this clinical setting.125 Finally, currently ongoing and future stud
ies may clarify the clinical utility and predictive value of: (1) DNA ploidy; (2
) microvessel density; and (3) proliferation markers. To date, these potential a
djunct parameters do not contri
ute to therapy decisions
ecause of the prelimin
ary state of data collection. Summary In summary, the cornerstone of therapeutic
decision making rests on models or nomograms developed in the early 1990s. Incr
easing experience has supported multiple revisions and updates of the widely uti
lized Partin Ta
les. Most nomograms are
ased on evaluations of clinical stage (di
gital rectal examination: DRE), serum prostatespecific antigen (PSA), and
iopsy
Gleason score. The characteristics of the patient population have dramatically
changed with significantly greater num
ers of cases identified
y PSAscreening p
rograms. The mean age, serum PSA level, and tumor size have all progressively an
d significantly decreased in recent years. The inherent insensitivity of clinica
l stage determination
y DRE and the serum PSA have corresponding diminishing di
scriminatory properties in the stage T1c patient population. By default, the
io
psy
What should you ask your pathologist
33
Gleason score assumes a greater role in preoperative therapeutic decisions. Addi
tional morphologic parameters derived from prostate needle
iopsies, supplementi
ng the Gleason score, include: (1) the num
er of positive
iopsies; (2) the volu
me of tumor in the needle cores; (3) the site of positive
iopsies; (4) the pres
ence of perineural invasion; (5) certain histologic variants of prostatic carcin
oma. The future will further clarify their value in preoperative stage predictio
n, and one hopes,
e supplemented
y additional parameters, such as DNA ploidy,
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et al. Ectopic secretion of corticotropin-releasing factor as a cause of Cushin
gs syndrome. A clinical, morphologic and
iochemical study. N Eng J Med 1984; 311
:1320. 119. Smith DC, Tucker JA, Trump DL. Hypercalcemia and neuroendocrine carci
noma of the prostate: A report of three cases and a review of the literature. J
Clin Oncol 1992; 10:499505. 120. Haukaas SA, Halvorsen OJ, Nygaard SJT, Paus E. C
ushings syndrome in prostate cancer. Urol Int 1999; 63:126129. 121. Kahler JE. Car
cinoma of the prostate gland; a pathologic study. J Urol 1939; 41:557574. 122. Na

i G, Ansari MS, Singh II, et al. Primary squamous cell carcinoma of the prostat
e: A rare clinicopathological entity. Report of 2 cases and review of literature
. Urol Int 2001; 66:216 219. 123. Cheville JC, Dundore PA, Bostwick DG, et al. Tr
ansitional cell carcinoma of the prostate. Clinicopathologic study of 50 cases.
Cancer 1998; 82:703707. 124. Matzkin H, Soloway MS, Hardeman S. Transitional cell
carcinoma of the prostate. J Urol 1991; 146:12071212. 125. Takashi M, Sakata T,
Nagai T, et al. Primary transitional cell carcinoma of prostate: case with node
metastasis eradicated
y neoadjuvant methotrexate, vin
lastine, doxoru
icin and
cisplatin (M-VAC) therapy. Urology 1990; 36:9699.
4 Detailed mapping of prostate cancer: implications for
rachytherapy
Michael E Chen, Dennis A Johnston, and Patricia Troncoso Introduction Study of t
he distri
ution of tumor foci within the prostate may have implications for opti
mizing locally directed therapies for prostate cancer, such as
rachytherapy, cr
yotherapy, or, in the future, direct intraprostatic injection of antineoplastic
agents. However, prostate carcinoma is well known to exhi
it considera
le histol
ogic and anatomical heterogeneity within the gland. Such heterogeneity makes it
difficult to summarize the distri
ution of prostate cancer foci. Previous studie
s have
een limited to written descriptions of tumor distri
ution, or to schemat
ic diagrams of a small num
er of cases superimposed on prostate crosssections.14
We have developed a computer-
ased methodology that graphically summarizes the t
umor distri
ution of a large num
er of prostate cancer cases on to sections of a
paradigm prostate. This system allows the display of relative tumor concentrati

ons in different regions of the prostate. We have used such plots of tumor distr
i
ution to devise improved
iopsy strategies to detect prostate cancer. Material
s and methods A total of 180 radical prostatectomy specimens from 1990 to 1996 w
ere serially sectioned and regions of tumor mapped for each gland as previously
descri
ed.6 Cases were non-consecutive. Sections of the apex and
ase were not m
apped. The outlines of each prostate gland and the tumor foci within each gland
were then digitized into the computer. The
oundary
etween the peripheral zone
and transition zone of each prostate was also entered. The central zone was not
outlined
ecause it generally lacked welldefined anatomic
oundaries, and was pr
esent predominantly at the prostate
ase, a region not completely included in ou
r model. Only 0.5% of all tumor foci (Ta
le 4.1) were found in the central zone.
The peri
Ta
le 4.1 Num
er and zonal distri
ution of cancer foci in 180 radical prostatect
omy specimens
Num
er of foci per specimen One focus Greater than one focus Range Mean Median 3
1 (17%) 149 (83%) 110 3.37 3.0
Basic and advanced techniques in prostate
rachytherapy
40
Num
er of cancer foci
y zone Peripheral zone (PZ) 448 (74%) Transition zone (TZ
) 141 (23%) Central zone (CZ) 3 (0.5%) Zone indeterminate 15 (2.5%) Zonal distri

ution of cancer foci per specimen Foci in PZ only 67 (37%) Foci in PZ and TZ 93
(52%) Foci in TZ only 3 (2%) Foci in PZ and CZ 2 (1%) Foci of indeterminate ori
gin 15 (8%) Dominant focus origin in cases with PZ and TZ foci Peripheral zone 6
0 (65%) Transition zone 33 (35%) Indeterminate cancers were generally large canc
ers occupying multiple zones, whose zone of origin could not
e determined.
urethral region was also not mapped
ecause its small size, poor anatomic defini
tion, and lack of tumor. A technique of three-dimensional (3D) histograms was de
veloped to demonstrate the aggregate locations of prostate tumors. A paradigm pr
ostate was used to map the tumor foci. This prostate was chosen
ecause it repre
sented a typical prostate shape. This paradigm prostate was then used to standar
dize the height and width of other
Figure 4.1 Summary distri
ution of all cancer foci plotted in a 256 level graysc
ale scheme (a), and in a pseudocolor scheme (
) for the same
Detailed mapping of prostate cancer
41
paradigm prostate section. The corresponding grayscale and pseudocolor ta
les ar
e shown to the right of the respective prostate sections. For details regarding
interpretation of the grayscale and pseudo-color schemes, see the text.
prostates at a given length. All prostates were normalized to a 100% length scal
e. Thus, the dimensions of a given prostate were normalized to the paradigm pros
tate model. For any normalized pixel that contained tumor, an increment of one w
as added to a 18014019 matrix. The 3D histograms were then plotted as contour plot
s with 256 levels of gray or pseudo-color (Figure 4.1). Black pixels indicate a

sence of tumor, and white pixels indicate maximum tumor frequency, with shades o
f gray or pseudocolor indicating intermediate frequencies. The plots thus contai
n quantitative information. For the pseudocolor scheme, pixels plotted in red to
white thus represent areas where tumor occurred from 50% to 100% of the maximum
rate. Results Of the 180 cases, 24 (13%) were T1c classification. The median pr
ostate-specific antigen (PSA) for these cases was 7.45 ng/mL, median gland weigh
t was 38.0 g, 26 prostates (14%) had tumors with Gleason scores of 6 or less. A
total of 108 (60%) prostates had a Gleason score of 7, and the remainder of glan

ds (46 cases, 26%) had Gleason scores of 8 or more. Median total tumor volume wa
s 1.39 cm3. Most prostates contained more than one tumor focus (149 cases, 83%,
see Ta
le 4.1). Most tumor foci (448 foci, 74%) were located in the peripheral z
one. There were only three prostates (2%) that contained tumor foci located excl
usively in the transition zone. The computer plot of the aggregate distri
ution
of all tumor foci for all cases is shown in Figure 4.2. As can
e seen tumor foc
i are concentrated in the posterolateral peripheral zone of the prostate. Periph
eral zone foci tended to
e concentrated from apex to midgland. At the
ase, per
ipheral zone foci tended to diverge laterally. An additional concentration of tu
mor is found in the anterior transition zone, reflecting the predominant locatio
n of these transition zone tumors. A plot of an individual prostate with tumor e
xclusively located in the transition zone (Figure 4.3) again emphasizes the typi
cal anteromedial location of such tumors. Cases were stratified
y patient prost
ate-specific antigen (PSA) levels. Increased PSA levels correlated with increase
d total tumor volume, peripheral zone tumor volume, and transition zone tumor vo
lumes (data not shown). The computer plots of tumor distri
ution stratified
y P
SA levels reflect this correlation (Figure 4.4). As shown in Figure 4.5, cases w
ith Tlc classification (non-palpa
le tumor on digital rectal examination) exhi
i
ted relatively more tumor in the transition zone. This appeared to
e due to a r
elative decrease in peripheral zone tumor volume; transition zone tumor volume f
or Tlc cases was not significantly different than other cases (data not shown).
Cases with
Basic and advanced techniques in prostate
rachytherapy
42
a Gleason score of 6 or less (Figure 4.6) also exhi
ited relatively more tumor i
n the transition zone. For these cases, peripheral zone tumor volume was decreas
ed and transition zone tumor volume was increased (data not shown). In contrast,
large glands (greater than 50 g) had relatively less transition zone cancer (Fi
gure 4.7). In addition, in large glands it was not uncommon to see peripheral zo
ne tumors pushed peripherally
y prominent transition zone adenomatous tissue (Fig
ure 4.8). Discussion Our studies confirm the heterogeneous nature of prostate ca
ncer. Our overall results are generally consistent with previous reports.1,2 Pro
state cancer is typically multifocal and multizonal. The predominant location of
tumors is in the posterolateral peripheral zone (PZ) of the prostate. PZ tumors
were preferentially concentrated from apex to mid-gland, and diverged laterally
as they neared the
ase. Transition zone (TZ) cancers were focused anteriorly,
near the midline. Cases with non-palpa
le tumors (T1c classi-fication) had incre
ased TZ tumors. Other studies have also reported this finding.7,8 Cases with a G
leason score of 6 or less also had relatively more TZ tumors. Unlike T1c cases,
this appears to
e due to
oth a decrease in PZ tumor volume and an increase in
PZ tumor volume. TZ tumors tend to
e well-differentiated and usually have a Gle
ason score equal to or less than that of any PZ tumors in the same gland. We sug
gest that the development of large TZ tumors occurs primarily in the context of
well-differentiat
Figure 4.2 Top row: Summary distri
ution of all cancer foci: 19 virtual cross-se
ctions from the 18014019 pixel matrix are displayed. The first section on the
Detailed mapping of prostate cancer
43
upper left represents the
asal most section. The last section on the lower righ
t represents the section near the apex. Outlines of the urethra and the
oundary

etween the peripheral zone (PZ) and transition zone (TZ) are also traced. Tumo
r distri
ution is superimposed on the outlines of a chosen paradigm prostate. Fo
r interpretation of the pseudo-color scheme see Figure 4.1 and the text.

Figure 4.3 Serial sections of a prostate with tumor exclusively present in the t
ransition zone. Tumor foci are in red. For this case, the classification was Tlc
. The PSA level was 9.9 ng/mL The specimen Gleason score was 6, and the gland we
ight was 61 g. Tumor volume was 6.22 cm3.
Basic and advanced techniques in prostate
rachytherapy
44
Figure 4.4 The distri
ution of tumor foci for cases stratified
y patient prosta
te-specific antigen (PSA) level. Slices 3, 7, 11, 15, 19 from the original 19 sl
ice matrix are displayed. See Figure 4.1 for the pseudo-color key, and Figure 4.
2 for the slice locations.
Figure 4.5 Distri
ution of tumor in cases stratified
y Tlc (non-palpa
le) versu
s other classifications. Slices 3, 7, 11, 15, 19 from the original 19 slice matr
ix are displayed. See Figure 4.1
Detailed mapping of prostate cancer
45
for the pseudo-color key, and Figure 4.2 for the slice locations.
ed PZ tumors. If the PZ tumors are more poorly differentiated, it is pro
a
le th
at such tumors would grow rapidly and
e detected prior to the development of la
rge TZ cancers. In large glands with large TZs secondary to
enign prostatic hyp
erplasia, it was not unusual to see PZ cancers compressed toward the edge of the
prostate. Large glands also had less TZ cancer. We have reported previously tha
t large glands have a higher frequency of small volume cancers (<0.5 cm3).9 We h
ave suggested that for at least some of these large prostates, small incidental
cancers were detected
ecause of an elevated PSA level caused largely
y the enl
arged adenomatous tissue, and not
y significant cancer. We emphasize, however,
that the findings are statistical summaries. For a given individual case, the di
stri
ution of prostate cancer foci within the gland cannot
e predicted with cer
tainty from clinical parameters. The findings of digital rectal examination (DRE
), or the location of
Figure 4.6 Distri
ution of tumor in cases stratified
y the Gleason score of the
prostate specimen. Slices 3, 7, 11, 15, 19 from the original 19 slice matrix ar
e displayed. See Figure 4.1 for the pseudo-color key, and Figure 4.2 for the sli
ce locations.
Basic and advanced techniques in prostate
rachytherapy
46
Figure 4.7 Tumor distri
ution
y gland weight. Slices 3, 7, 11, 15, 19 from the
original 19 slice matrix are displayed. See Figure 4.1 for the pseudo-color key,
and Figure 4.2 for the slice locations.
Figure 4.8 Serial sections of a large prostate with a prominent transition zone.
Tumor foci are in red. A tenth apical section is not displayed. For this case,
the classification was T1c. The PSA level was 11.6 ng/mL The
Detailed mapping of prostate cancer
47
specimen Gleason score was 6, and the gland weight was 85 g. Tumor volume was 0.

06 cm3.
prostate cancer in
iopsy cores, do not correlate a
solutely with final patholog
ic findings.1012 Hypoechoic regions noted on transrectal ultrasound may suggest a
reas of cancer,
ut are not completely specific.13,14 Given the unpredicta
le di
stri
ution of cancer foci in an individual case, it is prudent, indeed imperativ
e, that all regions of the prostate
e sufficiently treated, with the exception
of the periurethral region. Indeed, commonly used modified peripheral loading sc
hemes aim to accomplish these two goals. Nevertheless, knowledge of the pro
a
il
ity of cancer within any given region of the prostate may
e useful in developin
g the treatment plan. Within the
ounds of wellesta
lished criteria for implant
design (dose conformation to the prostate, relative implant uniformity, and redu
ction of dose to the urethra), we suggest that the
rachytherapist may also wish
to consider the likelihood of cancer within certain regions of the prostate. Ce
rtainly, careful attention should
e given to the posterolateral and anteromedia
l prostatic regions in the course of planning and implantation. The
oundary
et
ween the peripheral zone and the transition zone is usually readily identifia
le
on transrectal ultrasound. We suggest that in cases where there is a prominent
transition zone, that the
rachytherapist take into account the likely displacem
ent of the peripheral zone tumors toward the outer edge of the prostate. A degre
e of inhomogeneity is inherent to any
rachytherapy treatment plan, with hot spot
s centered around each seed implant. Some have suggested that doses 20% higher th
an prescription may have some increased
iologic effect.15 Although it is a matt
er of speculation how such high doses (a
ove prescription and centered around ea
ch individual source) ultimately affects tumor, the
rachytherapist may wish to
consider incorporating areas of high cancer pro
a
ility (the posterolateral pros
tate and the anterior prostate near the midline) within higher isodose regions,
after all other primary considerations have
een met. References
1. McNeal JE, Redwine EA, Freiha FS, Stamey TA. Zonal distri
ution of prostatic
adenocarcinoma. Correlation with histologic pattern and direction of spread. Am
J Surg Path 1988; 12:897906. 2. Wheeler TM. Anatomic considerations in carcinoma
of the prostate. Urol Clin North Am 1989; 16:623634. 3. Ba
aian RJ, Troncoso P, A
yala A. Transurethral-resection zone prostate cancer detected at cystoprostatect
omy. A detailed histologic analysis and clinical implications. Cancer 1991; 67:1
4181422. 4. Tiguert R, Gheiler EL, Tefilli MV, et al. Racial differences and prog
nostic significance of tumor location in radical prostatectomy specimens. Prosta
te 1998; 37:230235. 5. Ba
aian RJ, Toi A, Kamoi K, et al. A comparative analysis
of sextant and an extended 11-core multisite directed
iopsy strategy. J Urol 20
00; 163:152157. 6. Chen ME, Johnston DA, Tang K, et al. Detailed mapping of prost
ate carcinoma foci:
iopsy strategy implications. Cancer 2000; 89:18001809.
Basic and advanced techniques in prostate
rachytherapy
48
7. Elgamal AA, Van Poppel HP, Van de Voorde WM, et al. Impalpa
le invisi
le stag
e T1c prostate cancer: characteristics and clinical relevance in 100 radical pro
statectomy specimensa different view [See comments]. J Urol 1997; 157:244250. 8. S
tamey TA, Sozen TS, Yemoto CM, et al. Classification of localized untreated pros
tate cancer
ased on 791 men treated only with radical prostatectomy: common gro
und for therapeutic trials and TNM su
groups. J Urol 1998; 159:20092012. 9. Chen
ME, Troncoso P, Johnston D, et al. Prostate cancer detection: relationship to pr
ostate size. Urology 1999; 53:764768. 10. Mueller EJ, Crain TW, Thompson IM, et a
l. An evaluation of serial digital rectal examinations in screening for prostate
cancer. J Urol 1988; 140:14451447. 11. Cupp MR, Bostwick DG, Myers RP, Oesterlin
g JE. The volume of prostate cancer in the
iopsy specimen cannot relia
ly predi
ct the quantity of cancer in the radical prostatectomy specimen on an individual

asis. J Urol 1995; 153:15431548. 12. Wang X, Brannigan RE, Rademaker AW, et al.
One core positive prostate
iopsy is a poor predictor of cancer volume in the r
adical prostatectomy specimen. J Urol 1997; 158:14311435. 13. Carter HB, Hamper U
M, Sheth S, et al. Evaluation of transrectal ultrasound in the early detection o

f prostate cancer. J Urol 1989; 142:10081010. 14. Flanigan RC, Catalona WJ, Richi
e JP, et al. Accuracy of digital rectal examination and transrectal ultrasonogra
phy in localizing prostate cancer. J Urol 1994; 152:15061509. 15. Ling CC, Roy J,
Sahoo N, et al. Quantifying the effect of dose inhomogeneity in
rachytherapy:
application to permanent prostatic implant with 1251 seeds. Int J Radiat Oncol B
iol Phys 1994; 28:971978.
5 Defining permanent prostate
rachytherapy target volumes from evaluation of wh
olemount prostatectomy specimens
Brian J Davis, Thomas M Pisansky, John C Cheville, and Torrence M Wilson Introdu
ction The goals of treatment with primary radiotherapy are cancer cure with orga
n preservation. The treatment goals are achieved, in part,
ecause cancer cells
are generally more sensitive to the cytotoxic effects of ionizing radiation as c
ompared to normal tissue. Nevertheless, normal tissue tolerance to radiotherapy
may limit the radiation dose that may
e delivered to a given anatomical site. T
herefore, information regarding cancer location as it relates to normal adjacent
tissue is relevant to the appropriate delivery of radiation therapy. Such infor
mation may
e determined
y analysis of pathologic data from primary surgical th
erapy. In cancer of the prostate, detailed histopathologic study of prostatectom
y specimens and analysis of patient pretreatment prognostic factors has led to t
he development of various nomograms for predicting the presence of adverse patho
logic features. Such nomograms may then
e used to influence treatment approache
s
ased on predicting the extent and location of cancer. In the radiotherapeutic
management of prostate cancer, the key issues revolve around determining the ri
sk of lymph node involvement (LNI), seminal vesicle involvement (SVI), and extra
prostatic extension (EPE). The terminology, extraprostatic extension (EPE), is pre
ferred instead of extracapsular extension (ECE),
ecause the prostate does not hav
e a complete capsule around it.1,2 As such, prostate cancer penetrating
eyond t
he margin of the prostate at a location where a capsule is a
sent would not
e a
ppropriately termed ECE, a misnomer in such a circumstance,
ut rather EPE. Curren
t standards for defining the
rachytherapy treatment volume While many groups ha
ve reported their practice regarding defining treatment volumes for prostate
ra
chytherapy, one may consider two sources as representative of current standards
in this area: (1) Radiation Therapy Oncology Group (RTOG) clinical trials; and (
2) an American College of Surgeons Oncology Group (ACOSOG) clinical trial. Prere
quisite to a discussion of treatment volume definitions is knowledge of the Inte
rnational Commission on Radiation Units and Measurement (ICRU) report num
er 58.
3 The relevant terminology from this ICRU report includes the gross tumor volume
(GTV), clinical target volume (CTV), and planning target volume (PTV). In prost
ate cancer radiotherapy, the CTV is usually defined as the GTV, which is the pro
state itself,
Basic and advanced techniques in prostate
rachytherapy
50
with or without the seminal vesicles. A noted exception is the situation where a
specifically defined nodule within the prostate or a dominant intraprostatic le
sion is treated or
oosted to a higher dose.4 The rationale for treatment of the
entire prostate is that prostate cancer is usually found to
e multifocal appro
ximately 5080% of the time.58 Therefore, treatment of the entire prostate is a rat
ional approach. RTOG trial 9805 was a multi-institutional Phase I/II trial for tr
eatment of men with early stage and low risk prostate cancer that accrued 98 pat
ients. The first results of this trial were reported in 2002.9 The CTV included
preimplant transrectal ultrasound (TRUS) definition of the prostate. The PTV inc
luded an enlargement of the CTV
y 23 mm in the lateral and anterior dimensions a
nd 5 mm in the superior (cephalad) and inferior (caudad) dimensions,
ut no enla
rgement posteriorly near the rectum. Thus, the prostate dimensions are increased
1.0 cm in the superior-inferior direction, 46 mm in the lateral dimension, and 23
mm in the anterior-posterior dimension. In two su
sequent trials, P0126 and P-0

232, treatment volumes are defined in a similar manner. The ACOSOG trial uses an
identical set of criteria for treatment volumes as the RTOG trials. Consequentl
y, these descri
ed treatment margins may
e viewed as a standard for permanent p
rostate
rachytherapy
ut not necessarily a universal one in that some experienc
ed practitioners have reported using treatment margins that vary from those desc
ri
ed. Serial sectioning of wholemount prostatectomy specimens A num
er of repor
ts descri
e the process of whole-mounting a prostate specimen harvested from a r
adical prostatectomy.10,11 While variations exist in institutional practice, com
mon aspects of these approaches to whole-mounting include specimen weighing, mea
suring, fixation in
uffered formalin, and inking, along with separate removal o
f the apex and the
ase. Seminal vesicles are sectioned from their
ase and pres
erved in their entirety from their proximal to distal ends. As illustrated in Fi
gure 5.1, the prostate is sectioned contiguously in 35 mm sections from
ase to a
pex in planes roughly perpendicular to the rectal wall and urethra. Most section
s appear similar to those o
served
y axial TRUS imaging. Seminal vesicles
Figure 5.1 Schematic of the wholemounting process of radical prostatectomy speci
mens. EPE, extraprostatic extension.
Defining permanent prostate
rachytherapy
51
may
e sectioned in axial or sagittal sections. All sections are mounted on glas
s slides and frequently require the use of slides larger than those typically em
ployed in routine histopathology. Tumor maps are duplicated for each slide and s
how critical features including locations of EPE, positive margins, and tumor fo
ci. In the Mayo series, specimens were o
tained during the period 19911993 as rep
orted
y Bostwick et al.10 The following discussion is organized first
y consid
ering prostate cancer location and prosatectomy findings as they pertain to the
urethra, the chosen anatomical center of the prostate, and then progressing outw
ards. Issues related to the risk of SVI or LNI have
een addressed elsewhere and
will not
e reviewed within the scope of this chapter.12 Urethra-cancer distanc
e The location of cancer immediately adjacent to the urethra is relevant in perm
anent prostate
rachytherapy (PPB) in considering the degree of peripheral loadi
ng and urethral sparing that is accepta
le. Detailed measurement of the proximity
of cancer immediately adjacent to the urethra was first descri
ed
y Lei
ovich e
t al in 2000.13 The method of measurement is illustrated in Figures 5.2a,
. A t
otal of 350 specimens were evaluated in this series. The urethra-cancer distance
was determined
y measuring the radial distance
etween the urethral mucosa and
the nearest focus of cancer. No linear shrinkage factor due to tissue processin
g was used in the study as it was estimated to
e only 4.357.7%.14,15 Urethra-can
cer distance was correlated with clinical, pathologic, and la
oratory factors
y
univariate and multivariate analysis. In 17% of the patients, the cancer a
utte
d the urethra and in 84% of the cases, the nearest focus of cancer was within 5
mm of the urethra. A decreasing urethra-cancer distance was associated with an i
ncreased rate of cancer recurrence. Multivariate analysis revealed that decreasi
ng urethra-cancer distance was associated with increased serum prostate-specific
antigen (PSA), Gleason score in the
iopsy specimen, and percent of
iopsy spec
imen with Gleason pattern 4 or 5. Characteristics of the study cohort were such
that a considera
le portion of patients would have
een candidates for PPB monot
herapy, if judged on clinical stage, preoperative PSA, and Gleason
iopsy score
alone. A total of 39.7% of patients were clinical stage T2a or less, 54.5% had s
erum PSA of 9.9 or less, and 46% had Gleason
iopsy score of 6 or less. Two su
s
equent studies have also examined the urethra-cancer distance. Ru
in et al exami
ned 52 specimens associated with low risk features and determined the frequency
of cancer within the transitional zone.16 The study
Basic and advanced techniques in prostate
rachytherapy

52
Figure 5.2 (a) Illustration of wholemount section showing the method of measurem
ent of the urethracancer distance. X identifies the location of the rethra. The d
arkened region of the sectioned prostate illustrates prostate cancer, and the me
asurement A shows the minimal urethracancer distance. (
) Section of prostate ill
ustrating the urethracancer distance measurement.
found that even among specimens with peripheral zone cancer only, periurethral c
ancer was common. The closest urethra-cancer distance per specimen ranged from 0
.07 mm to 1.9 mm with a median of 0.6 mm for all cases. In 86% of the cases, the
urethra-cancer distance was 1 mm or less. Rukstalis et al examined 112 patients
treated from 1998 to 2000
y radical perineal prostatectomy with median preoper
ative PSA of 7.0 (range: 0.7 200 ng/mL), and a median Gleason score of 6 (range:
410).17 The location of cancer foci with respect to the urethra were evaluated
y
zonal anatomy including the transition and peripheral zdnes along with location
s in the apex, mid and
ase of the prostate. In all sets, the median minimum dis
tance from cancer foci to the urethra was 1 mm or less. The mean distance ranged
from 0.5 mm to 1.6 mm. In all of these three series, the minimum urethracancer
distance was frequently less than 1 mm and a majority of the measurements were l
ess than 5 mm. No studies have suggested that the volume of periurethral cancer
is small, although, to our knowledge, detailed data on the cancer volume as a fu
nction of radial distance from the urethra have not
een pu
lished. Clearly, the
resources to generate such data exist,
ut it is dou
tful in reviewing presentl
y availa
le data that the findings will reveal small, yet close, amounts of canc
er adjacent to the entire course of the urethra. Therefore, the implication of t
hese
Defining permanent prostate
rachytherapy
53
findings with respect to the practice of PPB is that treatment of the urethral w
all and mucosa is necessary to facilitate eradication of the cancer. Furthermore
, there is no suggestion that significant reduction in radiation dosage to the u
rethra and periurethral region is warranted
ased on these histopathologic data
alone. It appears evident from these data that there is not a reasona
le
asis t
o argue in favor of significant urethral dose de-escalation in the present day p
ractice of PPB. Also relevant to these considerations are that a num
er of studi
es have attempted to correlate urethral radiation dosimetry with urinary mor
idi
ty,18
ut few have found a clear association. In a study
y Merrick et al19 an a
nalysis of urethral dosimetry of 13 patients of 425 undergoing implantation who
developed urethral strictures was performed. It was determined that dose to the
prostatic urethra was not predictive of stricture,
ut the extent and magnitude
of the high dose regions within the prostate were predictive. In view of these f
indings, it is therefore considered accepta
le practice that the urethra remains
part of the CTV in treatment planning and should receive the prescription dose,

ut should not
e unnecessarily overtreated as would
e typical of a uniform so
urce loading pattern. These data and studies may
e reasona
ly interpreted as su
pporting the current approach of using a peripherally loaded seed distri
ution w
ith the urethra receiving the prescription dose. A similar interpretation of the
se data has also
een espoused
y Merrick et al.20 Intraprostatic tumor volume a
nd multifocality A num
er of investigators have examined intraprostatic tumor vo
lume and the extent of multifocality. Intraprostatic tumor volume has
een shown
to correlate with outcome in a radical prostatectomy series.21 It is an accepte
d concept that with all other factors
eing equal, an increased intraprostatic t
umor volume is associated with a decreased rate of tumor eradication with radiot
herapeutic management. Empiric data from other cancers including head and neck c
ancer,22 and
rain metastases,23 demonstrate that larger tumors require greater
doses of radiation to achieve local control than do smaller ones. Therefore, kno
wledge of data on intraprostatic tumor volume is relevant in considering definit

ive treatment of prostate cancer
y PPB. In the Mayo series of whole-mount prost
atectomy specimens, intraprostatic tumor volume was determined in those patients
with clinical factors associated with eligi
ility criteria for PPB monotherapy
or
rachytherapy com
ined with external
eam radiotherapy (EBRT). These criteria
have
een set forth
y Nag et al24 in the American Brachytherapy Society (ABS)
recommendations for patient selection and include serum PSA <20, Gleason sum 7,
and clinical tumor classification <T3.24 A total of 313 patients were included i
n the study,15 with their characteristics given in Ta
le 5.1. The mean and media
n intraprostatic tumor volumes were found to
e 7.7 cc and 5.6 cc, respectively,
with a range of 038.1 cc. More recently, Se
o et al examined intraprostatic tumo
r volume and its predictors in 454 prostatectomy specimens from 1995 to 1998.25
Over 90% of the patients would have met the ABS eligi
ility criteria for PPB mon
otherapy in this study.
Ta
le 5.1 Clinical characteristics in 313 patients treated
y radical prostatect
omy. 15
Clinical stage (AJCC 1997) T1a 3
Basic and advanced techniques in prostate
rachytherapy
T1
8 T1c 40 T2a 91 T2
171 Preoperative PSA (ng/mL) 03.9 63 49.9 95 1019.9 155 Spe
cimen Gleason score 3 2 4 14 5 107 6 21 7 169 AJ ICC, American Joint Committee o
n Cancer; PSA, prostate-specie antigen.
54
Mean tumor volume was found to
e 6.7 cc, whereas the median tumor volume was fo
und to
e 2.6 cc. Other studies have demonstrated that tumor volume less than 0.
2 cc or less than 0.5 cc is frequently found in patients with PSA < 4, T1c tumor
stage and Gleason sum of 6 or less.26 Such patients have a low rate of
iochemi
cal failure,27 with prostate
rachytherapy and a low rate of prostate cancer spe
cific mortality with primary surgical or radiotherapeutic management.28 The radi
al distance and volume of extraprostatic extension Prior studies have examined e
xtraprostatic extension (EPE) in terms of the area of capsular perforation,29 or
the linear extension along or parallel to the prostatic capsule.30 These data d
o not provide the critical measurement applica
le to prostate
rachytherapy
eca
use the steepest radiation dose gradient is in the direction away from the cente
r of the implanted prostate and not in a direction circumferential or tangential
to it.31 Consequently, more recent studies have examined whole-mount prostatect
omy series to determine the radial extent of EPE. This distance measures the rad
ial extent of prostate cancer from the prostate capsule, if present at that loca
tion, or from the prostate margin roughly perpendicular to the edge of the prost
ate. The urethra is used as the anatomical origin of this radial measurement whe
never possi
le. The method of measurement is shown in Figures 5.3a,
. Two serie
s provide detailed measurements of radial EPE, the results of which are summariz
ed in Ta
le 5.2. In the Mayo Clinic series a total of 376 specimens were evaluat
ed from patients with clinically organ-confined prostate cancer and no prior hor
monal or radiation therapy.32 Detailed measurements of EPE were made and informa
tion regarding extraprostatic tumor density and presence of positive surgical ma
rgins at the EPE measurement site were noted. In this series, a total of 78 pati
ents with T2 or less clinical stage and no prior history of hormonal or radiatio
n therapy had EPE measurements at sites without positive margins. In the series

y Sohayda et al from the Cleveland Clinic,33 38 patients met such criteria. The
results of these two studies for patients with clinically organ confined prosta
te cancer are remarka
ly alike. In the Mayo
Defining permanent prostate
rachytherapy
55
Clinic series, the range of radial EPE was 0.044.0 mm in such patients and in the

Cleveland Clinic series, it was 0.15.0 mm. Median EPE distances in the two serie
s were 0.5 mm and 1.1 mm,
ut the Cleveland Clinic series also included patients
with clinical T3 tumors. Mean EPE distances in the two series in patients with
EPE were 0.8 mm and 1.7 mm, and 90% of low risk patients had EPE that was within
2.03.3 mm of the outer edge of the prostate.
Figure 5.3 (a) Schematic representation of the prostate from which the radial ex
traprostatic extension (EPE) distance was measured with inset shown.32 (
) Schem
atic representation of the radial EPE distance corresponding to the inset in Fig
ure 53a.32 (Reproduced with permission from Cancer 1999, 85:26302637. 1999)
Basic and advanced techniques in prostate
rachytherapy
56
Ta
le 5.2 Comparison of results of two pu
lished studies on the radial distance
of extraprostatic extension (EPE).32,33
Characteristic Mayo Clinic32 Cleveland Clinic33
265 92 38 More recent Yes (6%) Yes (6%) Yes Yes 0.15.0 mm 1.1 mm 1.7 mm 3.3 mm No
. patients 376 No. with EPE 105 No. with <T3, neg, margins, and no HT 78 Time pe
riod 19911993 T3 patients included? No Prior RT included? No HT patients included
? No Radial distance specified? Yes Range of radial EPE if <T3 0.044.4 mm Median EP
E for specimens with EPE 0.5mm Mean EPE for specimens with EPE 0.8 mm 90% percen
tile for favora
le patients <2.0 mm HT, hormonal therapy; RT, radical prostatect
omy.
The implications of these studies are that a treatment margin of 35 mm from the p
rostate edge will encompass the vast majority of EPE of tumor in prostate cancer
patients deemed appropriate for permanent prostate
rachytherapy (PPB) monother
apy. The dose
eyond this 35 mm margin typically extends another 5 mm
eyond this

oundary
efore it declines
y 50% when iodine-125 (125I) sources are employed.
15,31 Treatment of EPE within this dose fall-off region may well
e effective
e
cause treatment of EPE in the posterior peripheral zone is constrained
y the pr
esence of the rectum. Indeed, RTOG clinical trial 9805 specified a posterior tre
atment margin of 0 mm from the CTV to the PTV. Other series with a long follow-u
p also report using a 0 mm treatment margin posteriorly at the mid-gland.27,34 B
utz
ach et al performed a detailed examination of 22 patients treated with palla
dium (103Pd) PPB monotherapy and a 35 mm margin and determined from postimplant C
T-
ased dosimetry that the dose margin met these criteria.35 Also of relevance i
s the fact that the amount of cancer in extraprostatic locations is small compar
ed to the volume of intraprostatic cancer. In a study
ased on the Mayo Clinic w
hole-mount series,15 the volume of extraprostatic cancer excluding seminal vesic
le involvement (SVI) was estimated from a volume formula using the known area of
capsular penetration, radial EPE distance, num
er of EPE sites on the specimen
and the estimated cancer density. The formula and illustration of this calculati
on are shown in Figure 5.4. Extraprostatic cancer volume ranged from 0 cc to 4.6
cc with a mean of only 0.06 cc. The ratio of extraprostatic to intraprostatic c
ancer volume ranged from 0% to 18% with a mean ratio of only 0.4%. The interpret
ation of these data suggests that the dose required to treat this small extrapro
static cancer volume may not
e as great as that
Defining permanent prostate
rachytherapy
57
Figure 5.4 Schematic representation of the calculation of extraprostatic extensi
on (EPE) cancer volume.15 (Reproduced with permission from Tech Urol 2000, 6:7076
. 2000)
required to treat intraprostatic cancer. Consequently, it is likely that within
the region of dose fall-off at the periphery of the prostate small amounts of cl

inically occult prostate cancer are effectively treated if they are even present
at all. However, one should not infer from this supposition that the dose margi
ns set for the PTV should
e altered
ut rather that satisfactory results report
ed with PPB monotherapy for local control relate to the margin incorporated into
defining the PTV. Portions of the PTV margin may
e identical to the prostate m
argin and yet still effectively treat small amounts of extraprostatic cancer wit
hin this dose fall-off region. Other factors likely play a more important role i
n effective treatment of EPE. The most relevant factor relates to the accuracy a
nd resolution of the imaging modalities employed in guiding and documenting PPB
source placement. Intra- and intero
server varia
ility of postimplant segmentati
on of the prostate from CT images occurs
etween experienced practitioners. In a
study
y Du
ois et al36 this varia
ility in segmentation resulted in difference
s in prostate dimensions
y CT that were typically 5 mm. Statistically significa
nt differences were also found in determination of prostate volume. Similarly, s
tudies
y Lee et al37 and Al-Qaisieh,38 have demonstrated that this intero
serve
r varia
ility results in differences in estimation of postimplant dosimetry that
are significant in terms of judging the adequacy of an implant. Another factor
that may play a role in treatment of extraprostatic extension includes accuracy
of seed placement, which, on average, is typically no
etter than 2.55 mm.3941 Fur
thermore, seed migration typically occurs in approximately 1% of all loose seeds
implanted,42,43 and has
een correlated with planned placement of extraprostati
c seeds.43 Such migration of seeds
Basic and advanced techniques in prostate
rachytherapy
58
placed in the periphery of the prostate may work to decrease the extent of the p
rescription dose in few areas. Although addressed elsewhere in detail in this te
xt, these data also deserve interpretation in the context of giving com
ined EBR
T with PPB. While some advocate treatment of all PPB patients with supplemental
EBRT, Potters et al performed a detailed multivariate analysis on over 600 patie
nts treated with either PPB monotherapy alone or com
ined with EBRT.44 These ana
lyses demonstrated that the radiation dosimetry parameter D90 is the third most
important predictor of
iochemical failure following Gleason score and serum PSA
. Com
ining EBRT with PPB was insignificant in terms of offering a reduction in

iochemical recurrence. Nevertheless, a learning curve for clinicians newly engage
d in PPB has
een descri
ed and is associated with marginal dosimetry in early c
ases.45 Consequently, EBRT given as a supplement to a prostate implant having a
su
optimal seed distri
ution may serve to treat regions of occult EPE at the pro
state periphery. Conclusions Detailed histopathologic study of whole-mount prost
atectomy specimens from patients with clinically organconfined cancer has provid
ed data useful for evaluating treatment volumes as they relate to the contempora
ry radiotherapeutic management of prostate cancer. The current practice of perma
nent prostate
rachytherapy includes treatment of the entire urethra to the pres
cription dose and use of a treatment margin that readily allows for effective th
erapy of the radial distance and volume of extraprostatic cancer. References
1. Sakr WA, Wheeler TM, Blute M, et al. Staging and reporting of prostate cancels
ampling of the radical prostatectomy specimen. Cancer 1996; 78(2):366368. 2. Ayal
a AG, Ro JY, Ba
aian R, et al. The prostatic capsule: does it exist? Its importa
nce in the staging and treatment of prostatic carcinoma. Amer J Surg Path 1989;
13(l):2127. 3. International Commission on Radiation Units and Measurements. In:
Chassagne D, Dutreix A, eds. Dose and volume specifications for reporting inters
titial therapy. ICRU Report No. 58, 1997. 4. Xia P, Pickett B, Vigneault E, et a
l. Forward or inversely planned segmental multileaf collimator IMRT and sequenti
al tomotherapy to treat multiple dominant intraprostatic lesions of prostate can
cer to 90 Gy. Int J Radiat Oncol Biol Phys 2001; 51(1):244254. 5. Djavan B, Susan
i M, Bursa B, et al. Predicta
ility and significance of multifocal prostate canc
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TH, McLeod DG, Mostofi FK, et al. Prostate-specific antigen-detected prostate c

ancer (stage T1c): an analysis of whole-mount prostatectomy specimens. Prostate

1997; 32(1):5964. 7. Epstein JI, Stein
erg GD. The significance of low-grade pros
tate cancer on needle
iopsy. A radical prostatectomy study of tumor grade, volu
me, and stage of the
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8. Smith DS, Catalona WJ. The nature of prostate cancer detected through prostat
e specific antigen
ased screening. J Urol 1994; 152(5 Pt 2):17321736.
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9. Lee WR, Scott C, Lawton C, et al. Health-related quality of life (HRQOL) in m
en treated with prostate
rachytherapy alone on radiation therapy oncology group
(RTOG) Trial 9805. Int J Radiat Oncol Biol Phys 2002; 54(2S):4748. 10. Bostwick D
G, Myers RP, Oesterling JE. Staging of prostate cancer. Semin Surg Oncol 1994; 1
0(1):6072. 11. Miller GJ, Cygan JM. Diagnostic correlations with whole mounts of
radical prostatectomy specimens. Monogr Pathol 1992; 34:183197. 12. Pisansky TM,
Blute ML, Hillman DW, et al. The relevance of prostatectomy findings in
rachyth
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ov
ich BC, Blute ML, Bostwick DG, et al. Proximity of prostate cancer to the urethr
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on factor in the calculation of prostate cancer volume. Am J Surg Pathol 1996; 2
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static cancer in clinically organ confined prostate cancer
y permanent intersti
tial
rachytherapy: Is extraprostatic seed placement necessary? Tech Urol 2000;
6:7077. 16. Ru
in MA, Bagiella E, Ennis RD. Urethral sparing techniques in prosta
te
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l Biol Phys 2002; 54(2)S:3940. 17. Ruckstalis DB, Goldknopf JL, Crowley EM, Garci
a FU. Prostate cryoa
lation: a scientific rationale for future modifications. Ur
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tler WM, Tollenaar BG, et al. The dosimetry of prostate
rachytherapy-induced ur
ethral strictures. Int J Radiat Oncol Biol Phys 2002; 52(2):461468. 20. Merrick G
S, Wallner KE, Butler WM. Permanent interstitial
rachytherapy for the managemen
t of carcinoma of the prostate gland. J Urol 2003; 169(5):16431652. 21. Stamey TA
, McNeal JE, Yemoto CM, et al. Biological determinants of cancer progression in
men with prostate cancer. JAMA 1999; 281(15):13951400. 22. Million RR, Cassisi NJ
, Mancuso AA, et al. Management of the neck for squamous cell carcinoma. In: Man
agement of head and neck cancer: a multidisciplinary approach, 2nd edn. Million
RR, Cassisi NJ, eds. Philadelphia: JB Lippincott, 1994. 23. Nieder C, Ber
erich
W, Nestle U, et al. Relation
etween local result and total dose of radiotherapy
for
rain metastases. Int J Radiat Oncol Biol Phys 1995; 33(2):349355. 24. Nag S
, Beyer D, Friedland J, et al. American Brachytherapy Society (ABS) recommendati
ons for transperineal permanent
rachytherapy for prostate cancer. Int J Radiat
Oncol Biol Phys 1999; 44:789799. 25. Se
o TJ, Cheville JC, Riehle DL, et al. Pred
icting prostate carcinoma volume and stage at radical prostatectomy
y assessing
needle
iopsy specimens for percent surface area and cores positive for carcino
ma, perineural invasion, Gleason score, DNA ploidy and proliferation, and preope
rative serum prostate specific antigen: a report of 454 cases. Cancer 2001; 91(1
1):2196 2204. 26. Krumholtz JS, Carvalhal GF, Ramos CG, et al. Prostate-specific
antigen cutoff of 2.6 ng/mL for prostate cancer screening is associated with fav
ora
le pathologic tumor features. Urology 2002; 60(3):469473. 27. Blasko, J, Grim
m P, Sylvester J, et al. Palladium-103
rachytherapy for prostate carcinoma. Int
J Radiat Oncol Biol Phys 2002; 46:839850. 28. DAmico AV, Moul J, Carroll PR, et a
l. Cancer-specific mortality after surgery or radiation for patients with clinic
ally localized prostate cancer managed during the prostate-specific antigen era.
J Clin Oncol 2003; 21(11):21632172. 29. Stamey TA, McNeal JE, Freiha FS, Reedwin
d E. Morphometric and clinical studies on 68 consecutive radical prostatectomies

. J Urol 1988; 139:12351241.

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30. Epstein JI, Carmichael JM, Pizov G, Walsh PC. Influence of capsular penetrat
ion on progression following radical prostatectomy: a study of 196 cases with lo
ng-term follow-up. J Urol 1993; 150:135141. 31. Dawson JE, Wu T, Roy T, et al. Do
se effects of seed placement deviations from pre-planned positions in ultrasound
guided prostate implants. Radiother Oncol 1994; 32:268270. 32. Davis BJ, Pisansk
y TM, Wilson TM, et al. The radial distance of extraprostatic extension of prost
ate cancer: implications for prostate
rachytherapy. Cancer 1999; 85:26302637. 33
. Sohayda C, Kupelian PA, Levin HS, Klein EA. Extent of extracapsular extension
in localized prostate cancer. Urology 2000; 55(3):382386. 34. Grimm PD, Blasko JC
, Sylvester JE, et al. 10-year
iochemical (prostate-specific antigen) control o
f prostate cancer with I-125
rachytherapy. Int J Radiat Oncol Biol Phys 2001; 5
1(1):3140. 35. Butz
ach D, Waterman FM, Dicker AP. Can extraprostatic extension

e treated
y prostate
rachytherapy? An analysis
ased on postimplant dosimetry.
Int J Radiat Oncol Biol Phys 2001; 51:11961199. 36. Du
ois DF, Prestidge BR, Hot
chkiss LA, et al. Intrao
server and intero
server varia
ility of MR imaging- and
CT-derived prostate volumes after transperineal interstitial permanent prostate

rachytherapy. Radiology 1998; 207(3):785789. 37. Lee WR, Roach M 3rd, Michalski
J, et al. Intero
server varia
ility leads to significant differences in quantif
iers of prostate implant adequacy. Int J Radiat Oncol Biol Phys 2002; 54(2):45746
1. 38. Al-Qaisieh B, Ash D, Bottomley DM, et al. Impact of prostate volume evalu
ation
y different o
servers on CT-
ased post-implant dosimetry. Radiother Oncol
2002; 62:267273. 39. Yu Y, Waterman FM, Suntharalingam N, et al. Limitations of
the minimum peripheral dose as a parameter for dose specification in permanent 1
25I prostate implants. Int J Radiat Oncol Biol Phys 1996; 34(3):717725. 40. Ro
er
son PL, Narayana V, McShan DL, et al. Source placement error for permanent impla
nt of the prostate. Med Phys 1997; 24(2):251257. 41. Davis BJ, Herman MG, LaJoie
WN, et al. Supplemental implantation for su
optimal permanent prostate
rachythe
rapy: a prostate phantom study. Radiother Oncol 2000; 55S:9192. 42. Tapen EM, Bla
sko JC, Grimm PD, et al. Reduction of radioactive seed em
olization to the lung
following prostate
rachytherapy. Int J Radiat Oncol Biol Phys 1998; 42:10631067.
43. Eshleman JS, Davis BJ, Pisansky TM, et al. Radioactive seed migration to th
e chest following transperineal interstitial permanent prostate
rachytherapy: e
xtraprostatic seed placement correlates with migration. Int J Radiat Oncol Biol
Phys 2004; 59(2):419425. 44. Potters L, Cao Y, Calugaru E, et al. A comprehensive
review of CT-
ased dosimetry parameters and
iochemical control in patients tre
ated with permanent prostate
rachytherapy. Int J Radiat Oncol Biol Phys 2001; 5
0(3):605614. 45. Lee WR, deGuzman AF, Bare RL, et al. Postimplant analysis of tra
nsperineal interstitial permanent prostate
rachytherapy: evidence for a learnin
g curve in the first year at a single institution. Int J Radiat Oncol Biol Phys
2000; 46(1):8388.
6 Prostate cancer staging: PSMA-
ased serum assays and radioscintigraphy
Ganesh V Raj and Thomas J Polascik Introduction With the advent of the serum pro
state-specific antigen (PSA) assay, men are
eing diagnosed with prostate cancer
at earlier stages. Statistical algorithms, such as the Partin and Kattan nomogr
ams, that incorporate multivariate analyses of several pretreatment varia
les, p
rovide the clinician with valua
le data regarding the likelihood of extraprostat
ic disease.1,2 For example, a man with newly diagnosed prostate cancer, with a s
erum PSA of 5.0 ng/mL, a Gleason score of 6 (3+3), and a clinical stage T1c lesi
on has, according to the Partin Ta
les, an 80% (7883) chance of organ-confined di
sease, 19% (1621) pro
a
ility of extraprostatic extension, and 0% (01) chance of l
ymph node invasion.1 With such a high likelihood of organconfined disease, this
patient may
e a candidate for
rachytherapy, external
eam radiation, or radica
l prostatectomy as definitive local treatment for prostate cancer. Recent multi-

institutional reports of over 4100 patients treated with radical prostatectomy d

emonstrated that only 61% of early, non-palpa
le, PSA-detected tumors (clinical
stage T1c, TNM staging, AJCC 2002 guidelines) were organconfined.35 However, for
any individual patient, the disease is either organ-confined (0%) or not organ-c
onfined (100%). Accurate detection of clinically significant extraprostatic exte
nsion (EPE) of prostate cancer at the time of diagnosis would
oth avoid unneces
sary non-curative local therapy as well as identify a su
set of patients who cou
ld
enefit from more aggressive therapeutic interventions. Several approaches in
cluding reverse transcriptase polymerase chain reaction (RT-PCR) amplification t
echnologies and alternative prostate-specific markers, such as prostatic acid ph
osphatase (PAP), have not demonstrated a higher sensitivity and specificity of d
etection of EPE of newly diagnosed prostate cancer.67 Computed tomography (CT) an
d magnetic resonance imaging (MRI) have
een largely ineffective for demonstrati
ng lymph node metastases due to low sensitivity and high false-negative rates. S
everal attempts were made initially to increase the sensitivity of imaging studi
es to detect prostate cancer, using radiola
eled monoclonal anti
odies (indium-1
11; 111I) directed against antigens unique to the prostate. Radiola
eled anti
od
ies against
oth PSA and prostatic acid phosphatase (PAP) were shown to have poo
r sensitivity in limited early clinical trials.8 However, a US Food and Drug Adm
inistration (FDA) approved radiola
eled anti
ody against the prostate-specific m
em
rane antigen (PSMA), caproma
pendetide, marketed under the name ProstaScint (
Cytogen, Princeton, NJ), showed significant promise to detect soft tissue metast
ases in prostate cancer patients. The
Basic and advanced techniques in prostate
rachytherapy
62
ProstaScint scan has
een shown to detect metastatic lesions as small as 5 mm an
d hence is associated with a higher sensitivity to detect prostate cancer.9,10 T
he sensitivity, specificity, and overall accuracy of this radionuclide scan has

een reported to
e 62%, 72%, and 88% in early analyses,11 and up to 75%, 86%, a
nd 81% in more recent studies, respectively.1214 In this chapter, we examine the
utility of serum and radiological assays directed against PSMA in accurately sta
ging prostate cancer. Serum PSMA assays PSMA, a 100 kD type II transmem
rane gly
coprotein originally characterized
y the monoclonal anti
ody (mA
) 7E11 (Figure
6.1), has a high tissue-specificity, with expression primarily restricted to th
e prostate (PSMA references). PSMA is expressed in
enign prostatic epithelium,

enign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN), a
nd prostate cancer.1519 Interestingly, immunoreactivity of PSMA has
een shown to

e elevated in PIN and prostate cancer relative to
enign prostatic epithelium,
with further increased expression of PSMA noted in higher Gleason score tumors,
metastatic, and
Figure 6.1 PSMA (prostate-specific mem
rane antigen) protein structure. PSMA is
a transmem
rane protein, with distinct extracellular, transmem
rane, and intrace
llular domains. Location of the epitope targeted
y the ProstaScint anti
ody is
also shown. (Reproduced with the permission of Cytogen Corporation, Princeton, N
J.) Ta
le 6.1 Characterisitics of PSMA as a marker for prostate
1. Expression is highly specific for prostatic tissue 2. Expression level increa
sed in most prostate cancers 3. Expression level increased with higher Gleason s
core
Prostate cancer staging
63
4. Expression level increased with tumor-associated neovasculature 5. Expression
level increased at sites of metastatic spread, especially
ones 6. Expression l
evel increased with androgen-independent prostate cancer

hormone-refractory disease.1519 PSMA is also expressed on the surface of tumorass

ociated
ut not normal
lood vessels. Although the physiologic role of PSMA rema
ins unknown, its expression profile of PSMA makes it an excellent marker for the
detection of prostate cancer (Ta
le 6.1). Circulating PSMA in the serum of pati
ents with prostate cancer can
e detected using enzyme-linked immunoa
sor
ent as
says (ELISA) and Western
lots. Serum PSMA levels have
een noted to
e elevated
in patients with prostate cancer.20 In some patients with hormone-refractory tu
mors, in whom the serum PSA levels are low, elevated levels of serum PSMA have

een noted.20 However, initial hopes that serum PSMA assays could serve an adjunc
t role in the detection of prostate cancer have not materialized. Serum PSMA ass
ays have
een plagued
y an inconsistent detection of serum PSMA levels, with so
me investigators reporting an ina
ility to detect serum PSMA.19 Most of the init
ial studies with serum PSMA were performed with the 7E11 anti
ody, which is dire
cted against an intracytoplasmic domain of PSMA. Other anti
odies directed again
st extracytoplasmic domains of PSMA have
een developed; however their utility i
n detecting serum PSMA or staging prostate cancer remains unproven. Additional s
erum assays incorporating RT-PCR to amplify PSMA in circulating cells have
een
explored. RTPCR allows for a 109-fold amplification of the signal and may detect
a single cell expressing the signal RNA in a
ackground of 109 cells that do no
t express the targeted sequence.21 If the targeted sequence is highly specific f
or a tumor or tissue type, then identification of expression of that sequence ou
tside the primary tumor site would indicate that tumor cells have spread
eyond
the primary site. In other words, detection of the target tissue-specific RNA ex
pression in sites of known clinical metastasis or routes of dissemination sugges
ts micrometastatic deposits or circulating tumor cells. The tissue-specific expr
ession of PSMA in prostatic epithelial cells offers the possi
ility of using PSM
A gene expression as a marker for the detection of occult metastases of prostate
cancer. Initial reports utilizing nested primers against PSMA detected circulat
ing cells expressing PSMA in 48/77 patients with prostate cancer, compared to on
ly 7/77 in whom PSA gene expression could
e detected.21 However, additional res
ults have
een disappointing. Ghossein concluded that while RT-PCR of PSMA was a

le to detect circulating cells expressing PSMA in up to 63% of patients, it was
not sensitive enough to warrant changes in clinical management.22 Currently, th
is RT-PCR assay for PSMA does not have a proven utility in staging prostate canc
er. Future advances in molecular staging may involve quantitative real-time RT-P
CR.6 Overall, despite initial promise, serum PSMA assays have
een plagued
y in
consistency in detection. At the moment, these assays should
e considered inves
tigational and not yet ready for clinical application.
Basic and advanced techniques in prostate
rachytherapy
64
ProstaScint scan A scintigraphic 111I-radiola
eled murine monoclonal anti
ody re
active with PSMA forms the
asis for the caproma
pendetide imaging study (Prost
aScint, Cytogen Corporation, Princeton, NJ). The FDA-approved ProstaScint scan ut
ilizes an immunoglo
ulin G (IgG) murine monoclonal anti
ody 7E-11-C5.3 (directed
against an intracellular N-terminal epitope) conjugated with the linker-chelato
r glycyl-tyrosyl-(N, diethylenetriaminepentaacetic acid)-lysine (GYK-DTPA) (Figu
re 6.2). Prior investigations have demonstrated 111In-caproma
pende-tide immuno
scintigraphy to
e safe with mild adverse effects and minimally elevated human a
ntimouse anti
ody (HAMA) levels on rare occasions.10,11,24 Patients who undergo
radioimmunoscintigraphy receive an intravenous injection containing 5.0 mCi of 1
11In-radiola
eled monoclonal anti
ody followed
y
Figure 6.2 Schematic of the ProstaScint anti
ody. An immunoglo
ulin G (IgG) muri
ne monoclonal anti
ody 7E-11-C53 (directed against an intracellular Nterminal ep
itope) is conjugated with the linker-chelator glycyl-tyrosyl-(N, diethylenetriam
inepentaacetic acid)lysine (GYK-DTPA) and a gammaemitting radionucleotide indium

-111 (111In). (Reproduced with the permission of Cytogen Corporation, Princeton,

NJ.)
planar and cross-sectional, single-photon emission computed tomography (SPECT) i
mages (Figure 6.3). Due to the kinetics of anti
ody
inding, imaging with this a
gent is
est performed approximately 72120 hours after administration of the isot
ope. The 2.8 day half-life of 111In facilitates this imaging time interval. Repe
at studies are performed 72120 hours after injection to allow for clearance from
the vascular and intestinal
Prostate cancer staging
65
structures. (Note. imaging times reflect clinical trial experiences and package
insert.) Clinical studies have shown that asymmetrical vessels or
one marrow di
stri
ution can lead to a false interpretation of the scan without initial and de
layed images. Expertise is required for proper interpretation of these scans, ma
king it imperative that the nuclear medicine physician undergo training to
ecom
e adept at interpretation and to understand the need for proper image acquisitio
n. More details on ProstaScint image acquisition and analyses can
e found in th
e excellent review
y Blend and Sodee.25 In a preliminary Phase I study, 40 men
with known distant prostate cancer metastases underwent a ProstaScint
Figure 6.3 Normal
iodistri
ution of the caproma
pendetide uptake is shown at d
ay 0 and day 4 after administration of the radiola
eled anti
ody in different pl
anes. (Reproduced with permission from Cytogen Corporation, Princeton, NJ.)
scan. There were no adverse reactions. The ProstaScint scan detected
one metast
ases in 55% of men, including 12 of 14 men receiving hormonal therapy. Soft tiss
ue lesions detected
y immunoscinitigraphy were su
sequently confirmed in 4 of 6
men.26 In another report, 19 men with
iopsy-proven prostate cancer underwent a
preoperative ProstaScint scan and pelvic CT or MRI, prior to
ilateral pelvic l
ymph node dissection. The ProstaScint scan detected four of eight men with posit
ive lymph nodes, with a detection threshold in respect to nodal size of 5 mm or
greater. Two false-positive scans were noted. The overall sensitivity and specif
icity of the ProstaScint scan were 44% and 86%, respectively. The authors conclu
ded that ProstaScint scan is safe and capa
le of detecting soft tissue nodal dis
ease.27
Basic and advanced techniques in prostate
rachytherapy
66
Although the ProstaScint scan has not
een used to formally assess metastatic sp
read to the
ones, initial data suggest that traditional
one scintigraphy is mo
re specific for detection of osseous prostate cancer. In addition, caproma
pend
etide accumulates in fracture sites and areas of inflammation resulting from art
hritis,
ursitis, or tendonitis. Thus, the ProstaScint scan is not useful for de
tection of disease spread to the
ones. Two possi
le clinical uses for ProstaSci
nt include the detection of lymph node disease and occult metastases prior to pr
imary therapy, or the site of relapse in those with a detecta
le PSA after defin
itive local therapy. ProstaScint scan in preoperative staging In many men presen
ting with localized prostate cancer and high-risk criteria (eg PSA10 ng/mL, Gleas
on score 7, clinical stage T2
), a thorough preoperative lymph node staging is imp
ortant since there is a strong association
etween lymph node involvement and th
e presence of distant metastasis. With lymph node involvement, cancerspecific su
rvival is decreased and surgery or radiotherapy alone is likely not curative. In
a recent series of men with clinically localized prostate cancer treated with r
adical prostatectomy, the actuarial 10 year metastasis-free survival was 99% for
patients with organ-confined disease and 68% for those with micrometastases to
the pelvic lymph nodes.5 Most prostate cancers are thought to spread through the

lymphatic channels in a sequential manner, usually first involving the o
turato

r, then internal iliac followed
y the external iliac lymph nodes. Later, it pro
gresses distally to the common iliac, retroperitoneal, and mesenteric lymph node
s, and eventually into the mediastinal and supraclavicular lymph nodes. Occasion
al skip metastases may
e noted. The ProstaScint scan can simultaneously evaluat
e the entire
ody for lymph node spread (Figures 6.4 6.6). Although pelvic lympha
denectomy is considered to
e the gold standard to determine lymphatic metastase
s, this procedure largely only samples the middle chain of the external iliac ly
mphatic vessels and does not provide information a
out other nodal groups or alt
ernative lymphatic drainage patterns. In a study of over 750 autopsy cases of pr
ostate cancer with metastases, Saitoh et al determined that there was an alterna
te lymphatic drainage pattern of prostate cancer involving the paraaortic lymph
node chain alone.28 These authors found that over 50% of 23 cases with metastase
s confined to the lymph nodes involved the paraaortic nodes in the a
sence of pe
lvic nodal involvement. In a large autopsy study of 1589 patients with prostate
cancer, 415 of whom had concurrent lymphatic involvement, paraaortic nodal disea
se (75%) was seen more often than cancer in the pelvic lymph nodes (55%).29 The
metastatic pattern of prostate cancer from these autopsy studies is surprisingly
similar to that seen with the ProstaScint scan. In one study, 23% of patients s
howed uptake of caproma
pendetide in paraaortic nodes and 22% in pelvic lymph n
odes (Figure 6.6).13 In a separate report, 12 of 152 evalua
le patients had evid
ence of soft tissue metastases
y 111In-caproma
pendetide immunoscintigraphy in
areas outside the field of the pelvic lymph node dissection and were negative f
or malignancy within the field of the pelvic lymph node dissection.30 Thus, the
a
sence of pelvic lymph node involvement
y histologic examination does not excl
ude the presence of metastatic soft tissue disease. Results from
oth autopsy an
d ProstaScint scans suggest
Prostate cancer staging
67
that prostate cancer has an alternative lymph node pattern of metastasis. In thi
s respect, 111 In-caproma
pendetide immunoscintigraphy may
e a more sensitive
indicator of soft tissue involvement since it can evaluate the whole
ody for me
tastatic disease. In another clinical trial, investigators scanned radical prost
atectomy candidates
efore surgery who were considered to
e at relatively high
risk (a Gleason score 7, PSA 20 ng/mL, clinical T3 disease, or equivocal CT or MRI
suggesting evidence of lymph node metastasis) for lymph node metastasis.31,32 Of
the 64 patients with surgically confirmed pelvic lymph node metastases, 40 Pros
taScint scans were interpreted as positive, giving the ProstaScint scan a superi
or (62%) sensitivity compared to CT (4%) or MRI (15%) in the same group. Logisti
c regression analyses demonstrated that the ProstaScint scan was the most powerf
ul single predictor of metastatic disease when compared with any other varia
le,
including PSA or Gleason score. For patients with a PSA40 ng/mL and a Gleason su
m 7, the positive predictive value of radioimmunoscintigraphy was greater than 80
%.31 In their analyses of 48 men undergoing pelvic lymph node dissection and
io
psy of scan-positive areas prior to radical prostatectomy, Hinkle and colleagues
showed that the ProstaScint scan had a 75% sensitivity and 86% specificity.14,3
3 Further, in a study of 275 patients receiving
Figure 6.4 Normal
iodistri
ution of caproma
pendetide. Serial sections of the
whole
ody imaging showing normal caproma
pendetide uptake at days after admini
stration of the radiola
eled anti
ody. (Reproduced with permission from Drs Tere
nce
Basic and advanced techniques in prostate
rachytherapy
68

Wong and Edward Coleman, Duke University Medical Center, Durham, NC)
treatment for prostate cancer with nodal disease or metastatic disease determine
d
y either surgery or
one scan, respectively, Murphy et al found that incorpor
ation of ProstaScint, PSA level, and
one scan results into artificial neural ne
tworks indicated that ProstaScint results were a significant prognostic varia
le
for non-localized cancer.3436 Another study of 198 patients with high risk preop
erative parameters (mean PSA: 57.2 ng/mL, mean
iopsy Gleason score: 7.1) examin
ed the utility of the ProstaScint scan in comparison to or in com
ination with s
everal clinical algorithms to evaluate the pro
a
ility of lymph node involvement
.30 This study used histologic verification of the pelvic lymph nodes o
tained

y surgical lymphadenectomy as a
asis to compare the utility of the clinical alg
orithms and 111In-caproma
pendetide immunoscintigraphy for predicting lymph nod
e involvement. PSMA expression correlated with histologic findings, and 88% of P
SMA-positive lymph nodes had histologic evidence of metastatic prostate cancer.
In contrast, none of the PSMA negative lymph nodes were found to have cancer pre
sent. Comparing surgical lymphadenectomy specimens with PSMA staining and the Pr
ostaScint scan is the
est availa
le model for such a comparison despite the fac
t that as many as 33% of patients with clinically localized prostate cancer have
isolated metastases in lymphatic chains distinct from the
oundaries of the sta
ndard pelvic lymph node dissection.1,2 For example, a man with a clinical classi
fication T3a tumor, PSA level of 21 ng/mL, and
iopsy Gleason score of 8 has the
following pro
a
ilities of lymph node metastases according to the various algor
ithms: 33% (Roach et al),37 42% (Partin et al),1 43% (Bluestein et al),38 and 70
% (Sands et al).39 The positive predictive value (PPV) of the clinical algorithm
s ranged from 40.5% to 46.6% with an area under the receiveroperating-characteri
stic (ROC) curve of 0.52 to 0.61. The
Figure 6.5 Detection of metastatic disease. Whole
ody imaging shows
Prostate cancer staging
69
uptake of caproma
pendetide at various locations outside the prostate in a pati
ent with metastatic spread of disease after a radical prostatectomy. (Reproduced
with permission from Cytogen Corporation, Princeton, NJ.)
In-caproma
pendetide scan had a sensitivity of 67%, a specificity of 80%, a pos
itive predictive value (PPV) of 66.7%, and a negative predictive value (NPV) of
73%, with an area under the ROC curve of 0.71. Com
ining the results of the 111I
n-caproma
pendetide scan with one or more clinical algorithms using logistic re
gression analysis increased the PPV to 72%.30 Since the PPV is the most importan
t measure to determine whether a patient would
enefit from staging lymphadenect
omy prior to definitive local therapy, this study suggests that 111In-caproma
p
endetide immunoscintigraphy is a strong independent predictor and that com
ining
the results of the nuclear scan with clinical algorithms increases the predicti
ve power to determine the likelihood of lymph node metastases. However, data fro
m other investigators are not congruent with these findings.40 In a smaller seri
es of 22 patients undergoing staging pelvic lymphadenectomy and radical surgery,
the preoperative ProstaScint scan was evaluated with definitive histological da
ta. Of nine areas of ProstaScint uptake, only one was noted to
e a true positiv
e, while five areas showing no ProstaScint uptake were noted to
e false negativ
e, giving this scan a 17% sensitivity, 90% specificity, 94% NPV, and 11% PPV. In
summary, in men newly diagnosed with prostate cancer and with high risk of lymp
h node involvement, ProstaScint immunoscintigraphy may detect lymph node metasta
ses not identified
y CT or MRI. Currently, ProstaScint does not appear to
e an
important part of the initial assessment of most patients with low risk patholo
gical features (low serum PSA, Gleason score 6 ng/mL, clinical stage T2a). The Pro
staScint scan may help to provide more accurate staging of clinically localized
prostate cancer prior to definitive therapy and may help guide clinical decision
making in patients with intermediate risk and high risk characteristics. Howeve

r, the significance of an incongruent positive or negative result is not current

ly clear. Further, long-term data on the clinical utility of the ProstaScint sca
n is lacking at the present time and needs to
e evaluated in randomized control
led trials. ProstaScint scan in detection of recurrent disease Several studies h
ave demonstrated that over 30% of men will experience
iochemical recurrence (el
evation in the serum PSA level a
ove the limit of detection) of prostate cancer
after definitive treatment with long-term (greater than 10 years) follow-up.35,414
3 Cancer recurrence may
111
Basic and advanced techniques in prostate
rachytherapy
70
Figure 6.6 Detection of cancer spread to lymph nodes, (a) Biopsy confirmed cases
of spread to periaortic lymph nodes and (
) internal iliac lymph nodes in patie
nts with newly diagnosed prostate cancer prior to definitive local intervention.
(Reproduced with permission from Cytogen Corporation, Princeton, NJ.)
occur locally in the prostatic fossa, or may
e seen in the regional nodes, or o
ther distant sites. The site of cancer recurrence may potentially identify the u
tility of local salvage interventions or systemic therapy (i.e. patients with lo
calized recurrence may
e candidates for salvage local therapy), while those wit
h distant recurrence should consider systemic therapy. Classification of recurre
nt prostate cancer into locally recurrent or systemic disease has
een limited

y availa
le radiologic imaging techniques. Traditionally, most patients evaluate
d for an elevated postsurgical serum PSA level undergo a CT scan or
one
Prostate cancer staging
71
scintigraphy. CT scans of the a
domen and pelvis require tumor deposits, in gene
ral, to
e greater than 1015 mm prior to detection, and further, the finding of r
adiographically enlarged lymph nodes is not diagnostic of cancer.44,45 Bone scan
s are limited to the potential detection of
one metastasis, and often are assoc
iated with a high serum PSA level (>40 ng/mL).46,47 Thus, the sensitivity of CT
and
one scans is technically limited and ineffective for differentiating local
from distant disease for patients with early evidence of
iochemical progression
(detecta
le serum PSA).47,48 Thus, the requirement of a sufficiently large tumo
r
urden prior to detection
y conventional radiographic studies or digital rect
al examination, and
iopsies of the prostatic fossa precludes their use in direc
ting treatment of patients with early recurrent prostate cancer. Here again, we
examine the utility of radioimmunoscintigraphy directed against PSMA to accurate
ly stage prostate cancer prior to and after definitive treatment. Preliminary re
sults from the ProstaScint Study Group suggest that ProstaScint imaging can help
differentiate
etween local and distant recurrence in patients with prostate ca
ncer in whom the only evidence of disease after definitive therapy is a detecta

le PSA level (average 28.7 ng/mL, median: 13.8). In this study of 48 men, the Pr
ostaScint scan was positive in 38 men, with 3 showing uptake localized to the pr
ostatic fossa only and 35 showing evidence of disease
eyond the prostatic fossa
.49 What is the sensitivity of detection of the ProstaScint scan in recurrent pr
ostate cancer? Or, put another way, how early after
iochemical failure can the
ProstaScint scan show uptake? In one study, 68% (23/34) of patients with
iochem
ical failure after surgical intervention and serum PSA 4.0 ng/mL showed evidence
for caproma
pendetide uptake, in comparison to 75% (12/16) with serum PSA leve
ls >4.0 ng/mL.50 In a large multicenter study of 877 patients with a mean serum
PSA of 8.9 ng/mL after surgical resection of the prostate, a 10-fold increase in
serum PSA was associated with a 1.23-fold higher risk of a positive caproma
pe
ndetide uptake in the prostatic fossa.51 Additional studies have suggested that
a low serum PSA level after primary treatment of prostate cancer may
e associat

ed with a negative ProstaScint scan.11,34,45 In a pu
lished series of 255 patien

ts with
iochemical evidence of disease after radical prostatectomy and having a
mean postoperative serum PSA level of 1.1 ng/mL, 185 (73%) demonstrated uptake
of the monoclonal anti
ody.52 Localized uptake (prostatic fossa only) (Figure 6.
7) was detected in 78 patients (30.6%), regional uptake (regional nodes  prostati
c fossa, with no evidence of distant uptake) in 109 patients (42.8%), and distan
t uptake (any uptake in distant nodes and/or
one) in 75 patients (29.4%). No mi
nimum serum PSA level was necessary for
Basic and advanced techniques in prostate
rachytherapy
72
Figure 6.7 Detection of recurrent disease. Biopsy confirmed case of recurrence i
n the prostatic fossa in a 54-year-old patient with PSA 2.4 ng/mL after radical
prostatectomy. (Reproduced with permission of Cytogen Corporation, Princeton, NJ
.) Ta
le 6.2 Utility of 111In-caproma
pendetide scintigraphy (ProstaScint) for d
etecting disease recurrence in patients with serum PSA4.0 ng/mL
study serum PSA cutoff No. patients Positive scans Local uptake
8 38 34 255 7(88%) 4 (50%) 30 (79%) 14(37%) 23 (68%) Not reported 185 (72%) 78 (
30.6%) Sodee et al51 3.6 ng/mL Elgamal et al34 1.0 ng/mL Petronis et al50 4.0 ng
/mL Raj et al52 4.0 ng/mL
monoclonal anti
ody uptake, either locally, regionally, or distantly (Ta
le 6.2)
. Overall, the data indicate that the true utility of the ProstaScint scan lies
in the detection and localization of early recurrent disease after definitive lo
cal intervention. If uptake of the monoclonal anti
ody represents cancer recurre
nce, the implication is that the ProstaScint scan may help to clinically stratif
y patients with
iochemical failure after definitive local therapy into those wi
th local, regional, or distant recurrent disease.36,53 Pelvic radiation given to
all patients with
iochemical failure demonstrates a sustained remission in a
o
ut half over a 12 month period.54 However, the utility of salvage therapy is con
troversial, with other reports indicating less favora
le outcomes.55 57 The utili
ty of the ProstaScint scan in differentiating local from distant recurrence afte
r radical prostatectomy is to
etter select patients for possi
le salvage therap
y to the prostatic
ed and avoid unnecessary salvage treatment in those with met
astatic disease.43,49,58,59
Prostate cancer staging
73
The clinical importance of localized ProstaScint uptake in directing therapeutic
options was explored in one interesting study.60 This study involved ProstaScin
t scanning of 32 men who had
iochemical evidence of recurrence (PSA >0.3 ng/mL)
following definitive local intervention and prior to salvage radiation therapy.
Dura
le complete response to radiation therapy (PSA<0.3 ng/mL) for more than 6
months was achieved in 70% (16/23) of men with no evidence of radiola
eled anti

ody uptake outside the prostatic fossa. A follow-up study indicated that 61% sti
ll had dura
le complete response at longer follow-up (>35 months), suggesting th
at the treatment effectively a
lated the recurrent tumor. In contrast, of nine m
en who had evidence of radiola
eled anti
ody uptake outside the prostatic fossa,
only 2 (22%) had dura
le complete response to salvage radiation therapy. In ano
ther study, in a su
set of 95 patients for whom the site of recurrence was esta

lished, a positive ProstaScint scan was found to have 73% sensitivity, 53% speci
ficity, and an 89% positive predictive value (PPV) of the site of disease.52 Reg
ional uptake of the anti
ody (n=60) was found to correlate with disease with 76%
sensitivity, 54% specificity, and 90% PPV. Distant uptake of the anti
ody (n=46
) was found to correlate with disease with 69% sensitivity, 58% specificity and
90% PPV. In comparison, a positive CT and/or
one scan result (n=20) was associa

ted with 21% sensitivity, 63% specificity, and 65% PPV of disease detection. The
se data taken together suggest that ProstaScint imaging can
e used to different
iate
etween patients who may respond to salvage local therapy after failed defi
nitive local therapy. However, in a recent report, for patients with postprostat
ectomy
iochemical relapse who received salvage radiation therapy (RT), presalva
ge RT In-ma
scan findings outside the prostate fossa were not predictive of
io
chemical control after RT.61 Using an American Society of Therapeutic Radiation
and Oncology (ASTRO) definition of PSA failure, in men with a positive scan in a
t least one location (n=14), the cumulative 2 year PSA control after salvage RT
was 0.380.13 compared with 0.310.13 for men with a normal anti
ody scan in and out
side the prostate fossa (n=15). Clearly, randomized controlled prospective trial
s are needed to evaluate the true utility of the ProstaScint scan as an adjunct
to clinical decision making. Limitations of the ProstaScint scan The primary lim
itation of our and other studies is a lack of histological confirmation of the P
rostaScint signal. Previous studies have indicated that a positive ProstaScint s
can correlates well with histologic evidence of metastatic prostate cancer.10,32
,33,62,63 Clinical follow-up data in a few patients with disease progression in
one study suggest a correlation
etween regions of uptake and metastatic lesions
,
ut the num
ers of patients are too small for meaningful analyses.52 The limit
ed and varia
le follow-up also does not allow for computation of the true clinic
al utility of this scan to detect recurrence. Further, the followup data may hav
e a selection
ias and may enhance the apparent effectiveness of this scan. Long
er follow-up is needed to determine whether ProstaScint immunoscintigraphy corre
lates with clinical outcomes. Second, since the ProstaScint scan employs a murin
e monoclonal anti
ody, consideration must also
e given to human antimouse anti

ody (HAMA) reaction.
Basic and advanced techniques in prostate
rachytherapy
74
Although rare, this host response against the anti
ody may present a risk for a
hypersensitivity reaction, as represented
y urticaria,
ronchospasm, or even hy
potension and anaphylaxis. HAMA formation occurred in 8% after the initial and 1
9% following the secondary infusion of anti
odies. However, the HAMA reaction wa
s transient and mild, and not associated with any adverse outcomes. Administrati
on of the ProstaScint scan to patients, who have HAMA to a prior mouse anti
ody
exposure, may elicit a stronger immune HAMA response and should
e used with cau
tion. Hyper
iliru
inemia, hypotension, and hypertension occurred in 1% of patien
ts in clinical trials. To overcome this, murine anti
odies have now
een humanize
d with recom
inant DNA technology. Third, as previously mentioned, the target of
the ProstaScint scan with the 7E-11 anti
ody is an epitope on the intracellular
domain of PSMA. In general, anti
odies that target an intracellular epitope are
thought to
e at a distinct disadvantage in comparison to those targeting extrac
ellular epitopes. If the antigen is intracellular, targeting with anti
odies sho
uld theoretically only react with those tumor areas at which cell necrosis perme
a
ilizes the cell mem
rane to the anti
ody. These may
e relevant in rapidly gro
wing tumors that outgrow their
lood supply and thus have areas of cell death. G
enerally, prostate cancer grows slowly and it does not have areas of necrosis, u
nless visualized after androgen deprivation therapy. However, the true in vivo s
ituation with monoclonal anti
ody directed against 7E-11 is not known. Fourth, p
ooling of radiola
eled anti
odies in a wellvascularized tumor can give the appea
rance of a positive scan when compared to
ackground. Thus, it is not surprising
that the occasional hypervascular renal tumor may show a false-positive ProstaS
cint uptake, reflecting more the temporal pooling of the anti
ody rather than re
action with PSMA epitope. Similarly, asymmetric
lood vessels, aneurysms, varice
s, and other vascular malformations may give false-positives: however, careful c
orrelation with
lood-pooled images should eliminate these diagnoses. Further, c
aproma
pendetide may nonspecifically accumulate in inflammatory lymph nodes. Fo
cal uptake in an a
normal location, like cervical lymph nodes, without correspon

ding uptake in the a
dominal or pelvic lymph nodes, should
e cautiously interpr
eted and confirmed with clinical, radiological, or tissue findings. Lastly, the
skill required to interpret nuclear scintigraphy is paramount in the analysis of
the ProstaScint scan. Trained radiologists must
e familiar with the detailed a
natomy of the human pelvis to interpret normal and a
normal caproma
pendetide u
ptake. Cytogen Corporation requires training and certification in a special trai
ning program (Partners in Excellence) so that the nuclear medicine radiologist c
an accurately interpret the scan. The correlation with three-dimensional (3D) im
aging and superimposition of ProstaScint images with CT, positron emission tomog
raphy (PET), or MRI may ultimately
e required to delineate the true utility of
radiola
eled monoclonal anti
odies (Figures 6.9 and 6.10). ProstaScint and
rach
ytherapy Although most of the pu
lished data examine the utility of ProstaScint
scan
efore and after radical prostatectomy, many conclusions are also applica
l
e to
rachytherapy. Fusion of pelvic CT and ProstaScint scans has
een used to i
ndividualize
rachytherapy
Prostate cancer staging
75
implantation, with placement of additional seeds in areas of high ProstaScint up
take, while at the same time decreasing the dose to normal structures, where pos
si
le.64 One important caveat to note for post-
rachytherapy recurrence, in cont
rast to the data presented for radical prostatectomy series, is concerns that af
ter
rachytherapy, residual normal prostate tissue expressing PSMA may
e presen
t. Following
rachytherapy, uptake of caproma
pendetide in the periprostatic or
perirectal soft tissue may
e seen due to chronic inflammation. This situation
can persist for years after treatment, making it more difficult to diagnose resi
dual or recurrent disease within the radiation field. However, in a majority of
cases, there was less epithelial atypia in periprostatic tissue
iopsied >48 mon
ths after treatment compared with those with a shorter interval
etween
iopsy a
nd treatment.65 The true utility of ProstaScint scinitigraphy in these patients
is to examine the lymph nodes for any evidence of metastatic spread of disease (
Figure 6.8). Clearly, as evidenced for patients after radical prostatectomy, the
ProstaScint scan can detect lesions in lymph nodes with a greater degree of sen
sitivity than traditional imaging modalities of CT and
one scanning. Fusion ima
ging com
ining MRI with the ProstaScint scan may have a higher sensitivity and s
pecificity (Figure 6.9). Future developments In 1997, new monoclonal anti
odies
reactive to the extracellular domain of PSMA were identified. The coupling of on
e of these anti
odies, J591, with indium-111 has shown promise in
oth radioimmu
nolocalization and radioimmunotherapy of recurrent disease in preliminary trials
.66 Newer scans using dual isotope imaging involving simultaneous indium-111 mon
oclonal anti
ody and
Basic and advanced techniques in prostate
rachytherapy
76
Figure 6.8 Detection of recurrent disease after
rachytherapy. Whole
ody imagin
g showing caproma
pendetide uptake in a patient with rising PSA 6 months after

rachytherapy for localized prostate cancer. (Reproduced with permission from Dr
Samuel Kipper, Pacific Coast Imaging, Irvine, CA.)
Figure 6.9 Detection of recurrent disease after
rachytherapy. Fusion imaging wi
th a CT scan demonstrating
Prostate cancer staging
77

localized caproma
pendetide uptake in a patient with rising PSA 6 months after

rachytherapy for localized prostate cancer. (Reproduced with permission from Dr
Samuel Kipper, Pacific Coast Imaging, Irvine, CA.)
Figure 6.10 Co-registration with CT scanning. Co-registration with CT scanning s
hows extraprostatic uptake of caproma
pendetide in a patient with newly diagnos
ed prostate cancer. (Reproduced with permission from Drs Terence Wong and Edward
Coleman, Duke University Medical Center, Durham, NC.)
technetium-99 RBC (red
lood cell) SPECT acquistion help su
tract the vascular c
omponent of the scans and help minimize the false-positive signals seen in highl
y
Basic and advanced techniques in prostate
rachytherapy
78
vascular tissues.67 Further, three-dimensional image reconstruction and CT/MRI o
verlay may improve the accuracy of the ProstaScint scan (Figure 6.10).68,69 Conc
lusions Although PSMA appears to
e an optimal marker for prostate cancer, serum
PSMA assays have
een plagued
y inconsistency in detection and are not yet rea
dy for clinical application. PSMA-
ased immunoscintigraphy appears to have a sma
ller threshold for detection of extraprostatic soft tissue spread of prostate ca
ncer
efore and after definitive local intervention. Enhanced sensitivity of the
ProstaScint scan over traditional imaging modalities may
e related to the incr
eased detecta
ility of the radiola
eled anti
ody.7072 Fewer prostate cells and th
us smaller lesions may
e needed for a positive signal on a ProstaScint scan. In
men newly diagnosed with prostate cancer and with a high risk of lymph node inv
olvement, 111In-caproma
pendetide immunoscintigraphy may detect lymph node meta
stases not detecta
le
y CT or MRI. The ProstaScint scan may help provide more a
ccurate staging of clinically localized prostate cancer prior to definitive ther
apy in patients with intermediate risk and high risk pathological features. Howe
ver, long-term data on the clinical utility of the ProstaScint scan needs to
e
evaluated in randomized controlled trials. Many studies have shown that the Pros
taScint imaging can help differentiate
etween local and distant recurrence in p
atients with prostate cancer in whom the only evidence
Ta
le 6.3 Current indications for ProstaScint scans
For primary prostate cancer Staging patients who are at high risk (Gleason score
>7, PSA>10, stage>cT2
) for metastatic disease When evaluating patients with dis
cordant PSA, Gleason score, and clinical stages For recurrent prostate cancer Fo
r staging patients with early as with early
iochemical failure after definitive
local theraphy to help guide clinical decision making
of disease after definitive therapy is a detecta
le prostatespecific antigen (PS
A) level. Several preliminary reports indicate that ProstaScint imaging may
e u
sed to differentiate
etween patients who may respond to salvage local therapy a
fter failed definitive local therapy. The current indications for the ProstaScin
t scan are outlined in Ta
le 6.3. While this radioimmunoscintigraphy scan holds
much promise in elucidating the
iology of prostate cancer, its utility in clini
cal decision making has not
een clearly proven. Large scale randomized controll
ed studies are needed to esta
lish the prognostic significance of a positive Pro
staScint scan.
Prostate cancer staging
79
References
1. Partin AW, Kattan MW, Su
ong MS, et al. Com
ination of prostatespecific antig
en, clinical stage and Gleason score to predict pathological stage of localized

prostate cancer: a multiinstitutional update. JAMA 1999; 277:14451451. 2. Kattan

MW, Eastham JA, Stapleton AMF, et al. A preoperative nomogram for disease recurr
ence following radical prostatectomy for prostate cancer. J Natl Cancer Inst 199
8; 90:766771. 3. Pound CR, Partin AW, Epstein JI, Walsh PC. PSA following anatomi
cal radical retropu
ic prostatectomy: patterns of recurrence and cancer control.
Urol Clin North Am 1997; 24:395 406. 4. Pound CR, Partin AW, Eisen
erger MA, et
al. Natural history of progression after PSA elevation following radical prostat
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al (prostate specific antigen) recurrence pro
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prostate specific antigen in the management of prostate cancer. J Urol 1997; 15
8(2):326337. 7. Polascik TJ, Oesterling JE, Partin AW. Prostate specific antigen:
a decade of discoverywhat we have learned and where we are going. J Urol 1999; 1
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aian RJ, Lamki LM. Radioimmunoscintigraphy of prostate cancer
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ning in prostatic cancer patients. Prostate 1997; 33:281285. 10. Haseman MK, Reed
NL, Rosenthal SA. Monoclonal anti
ody imaging of occult prostate cancer in pati
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pendetide imaging of prostate cancer. Cancer Biother Radioph
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pendetide in the evaluation of patients with residual or r
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14. Hinkle GH, Burgers JK, Neal CE, et al. Multicenter radioimmunoscintigraphic
evaluation of patients with prostate carcinoma using indium111 caproma
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Expression of prostate-specific mem
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enign and malignant
prostate tissues. Urol Oncol 1995; 1:1828. 16. Sweat SD, Pacelli A, Murphy GP, B
ostwick DG. Prostate-specific mem
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e adenocarcinoma and lymph node metastases. Urology 1998; 52(4):637640. 17. Silve
r DA, Pellicer I, Fair WR, et al. Prostate-specific mem
rane antigen expression
in normal and malignant human tissues. Clin Cancer Res 1997; 3(1):8185. 18. Lapid
us RG, Tiffany CW, Isaacs JT, Slusher BS. Prostate-specific mem
rane antigen (PS
MA) enzyme activity is elevated in prostate cancer cells. Prostate 2000; 45(4):3
50354. 19. Chang SS, Heston WD. The clinical role of prostate-specific mem
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20. Douglas TH, Morgan TO, McLeod DG, et al. Comparison of serum prostate specif
ic mem
rane antigen, prostate specific antigen, and free prostate specific antig
en levels in radical prostatectomy patients. Cancer 1997; 80(1):107114. 21. Raj G
V, Moreno JG, Gomella LG. Utilization of polymerase chain reaction technology in
the detection of solid tumors. Cancer 1998; 82(8):14191442. 22. Ghossein RA, Osm
an I, Bhattacharya S, et al. Detection of prostatic specific mem
rane antigen me
ssenger RNA using immuno
ead reverse transcriptase polymerase chain reaction. Di
agn Mol Path 1999; 8(2):5965. 23. Su SL, Boynton AL, Holmes EH, et al. Detection
of extraprostatic prostate cells utilizing reverse transcription-polymerase chai
n reaction. Semin Surg Oncol 2000; 18(1):1728. 24. Sodee DB, Conant R, Chalfant M
, et al. Preliminary imaging results using In-111 la
eled CYT356 (ProstaScint) in
the detection of recurrent prostate cancer. Clin Nucl Med 1996; 21:759 767. 25.
Blend MJ, Sodee DB. ProstaScint: An update. In: LM Freeman ed. Nuclear medicine
annual. Philadelphia: Lippincott, Williams & Wilkins, 2001:109138. 26. Wynant GE,
Murphy GP, Horoszewicz JS, et al. Immunoscintigraphy of prostatic cancer: preli

minary results with 111In-la
eled monoclonal anti
ody 7E11-C5.3 (CYT-356). Prost

ate 1991; 18(3):229241. 27. Ba
aian RJ, Sayer J, Podoloff DA, et al. Radioimmunos
cintigraphy of pelvic lymph nodes with 111indium-la
eled monoclonal anti
ody CYT
-356. J Urol 1994; 152(6):19521955. 28. Saitoh H, Yoshida K, Uchijima Y, et al. T
wo different lymph node metastatic patterns of a prostatic cancer. Cancer 1990;
65(8):18431846. 29. Bu
endorf L, Schopfer A, Wagner U, et al. Metastatic patterns
of prostate cancer: an autopsy study of 1,589 patients. Hum Pathol 2000; 31(5):
578583. 30. Polascik TJ, Manyak MJ, Haseman MK, et al. Comparison of clinical sta
ging algorithms and 111indium-caproma
pendetide immunoscintigraphy in the predi
ction of lymph node involvement in high risk prostate carcinoma patients. Cancer
1999; 85(7):15861592. 31. Manyak MJ, Hinkle GH, Olsen JO, et al. Immunoscintigra
phy with 111In-Caproma
Pendetide: Evaluation
efore definitive therapy in patie
nts with prostate cancer. Urology 1999; 54:1058 1063. 32. Manyak MJ. Clinical app
lications of radioimmunoscintigraphy with prostate-specific anti
odies for prost
ate cancer. Cancer Control 1998; 5(6):493499. 33. Hinkle GH, Burgers JK, Olsen JO
, et al. Prostate cancer a
dominal metastases detected with Indium In-111 Caprom
a
Pendetide. J Nucl Med 1998; 39:650653. 34. Elgamal AA, Troychak MJ, Murphy GP.
ProstaScint scan may enhance identification of prostate cancer recurrences afte
r prostatectomy, radiation, or hormone therapy: analysis of 136 scans of 100 pat
ients. Prostate 1998; 37(4):261269. 35. Murphy GP, Elgamal AA, Troychak MJ, Kenny
GM. Follow-up ProstaScint scans verify detection of occult soft-tissue recurren
ce after failure of primary prostate cancer therapy. Prostate 2000; 42(4):315317.
36. Murphy GP, Snow PB, Brandt J, et al. Evaluation of prostate cancer patients
receiving multiple staging tests, including ProstaScint scintiscans. Prostate 2
000; 42(2): 145149. 37. Roach M III, Marquez C, Hae-Sook Y, et al. Predicting the
risk of lymph node involvement using the pre-treatment prostate-specific antige
n and Gleason score in men with clinically localized prostate cancer. Int J Radi
at Oncol Biol Phys 1994; 28:3337. 38. Bluestein DL, Bostwick DG, Bergstralh EJ, O
esterling JE. Eliminating the need for
ilateral pelvic lymphadenectomy in selec
t patients with prostate cancer. J Urol 1994; 151:13151320. 39. Sands ME, Zagars
GK, Pollack A, von Eschen
ach AC. Serum prostate-specific antigen, clinical stag
e, pathologic grade, and the incidence of nodal metastases in prostate cancer. U
rology 1994; 44:215220. 40. Ponsky LE, Cherullo EE, Starkey R, et al. Evaluation
of preoperative ProstaScint scans in the prediction of nodal disease. Prostate C
ancer and Prostatic Diseases 2002; 5(2):132135.
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41. Partin AW, Pound CR, Clemens JQ, et al. Serum PSA after anatomical radical p
rostatectomy: the Johns Hopkins experience after 10 years. Urol Clin North Am 19
93; 20:713725. 42. Partin AW, Pearson JD, Landis PK, et al. Evaluation of serum p
rostate specific antigen velocity after radical prostatectomy to distinguish loc
al recurrence from distant metastasis. Urology 1994; 43(5):649659. 43. Moul JW. P
rostate specific antigen only progression of prostate cancer. J Urol 2000; 163(6
): 16321642. 44. Kramer S, Goricj J, Gottfried HW, et al. Sensitivity of computed
tomography in detecting local recurrence of prostatic carcinoma following radic
al prostatectomy. Br J Radiol 1997; 70(838):986991. 45. Seltzer MA, Bar
aric Z, B
elldegrun A, et al. Comparison of helical computerized tomography, positron emis
sion tomography and monoclonal anti
ody scans for evaluation of lymph node metas
tases in patients with prostate specific antigen relapse after treatment for loc
alized prostate cancer. J Urol 1999; 162(4):13221328. 46. Cher ML, Bianco FJ, Lam
JS, et al. Limited role of radionuclide
one scintigraphy in patients with pros
tate specific antigen elevations after radical prostatectomy. J Urol 1998; 160(4
):1387 1391. 47. Manyak MJ, Javitt MC. The role of computerized tomography, magne
tic resonance imaging,
one scan, and monoclonal anti
ody nuclear scan for progn
osis prediction in prostate cancer. Semin Urol Oncol 1998; 16(3):145152. 48. Temp
any CM, Zhou X, Zerhouni EA, et al. Staging of prostate cancer: results of Radio
logy Diagnostic Oncology Group project comparison of three MR imaging techniques

. Radiology 1994; 193:4754. 49. Levesque PE, Nieh PT, Zinman LT, et al. Radiola
e
led monoclonal anti
ody indium 111la
eled CYT-356 localizes extraprostatic recur
rent carcinoma after prostatectomy. Urology 1998; 51:978984. 50. Petronis JD, Reg
an F, Lin K. Indium-111 caproma
pendetide imaging to detect recurrent and metas
tatic prostate cancer. Clin Nucl Med 1998;23(10):672677. 51. Sodee DB, Malguria N
, Faulha
er P, et al. Multicenter prostascint imaging findings in 2154 patients
with prostate cancer. Urology 2000; 56:988993. 52. Raj GV, Partin AW, Polascik TJ
. Clinical utility of indium 111caproma
pendetide immunoscintigraphy in the det
ection of early, recurrent prostate carcinoma after radical prostatectomy. Cance
r 2002; 94(4):987996. 53. Fang DX, Stock RG, Stone NN, et al. Use of radioimmunos
cintigraphy with indium-111la
eled CYT-356 (ProstaScint) scan for evaluation of
patients for salvage
rachytherapy. Tech Urol 2000; 6(2):146150. 54. Anscher MS,
Clough R, Dodge R. Radiotherapy for a rising prostatespecific antigen after radi
cal prostatectomy: the first 10 years. Int J Radiat Oncol Biol Phys 2000; 48(20)
:369375. 55. Cadeddu JA, Partin AW, DeWeese TL, Walsh PC. Long-term results of ra
diation therapy for prostate cancer recurrence following radical prostatectomy.
J Urol 1998; 159(1):173177. 56. Vicini FA, Ziaja EL, Kestin LL, et al. Treatment
outcome with adjuvant and salvage irradiation after radical prostatectomy for pr
ostate cancer. Urology 1999; 54(1):111117. 57. Peschel RE, Ro
nett TJ, Hesse D, e
t al. PSA
ased review of adjuvant and salvage radiation therapy vs. o
servation
in postoperative prostate cancer patients. Int J Cancer 2000; 90(1):29 36. 58. L
am
HM, Faulds D. Caproma
pendetide. A review of its use as an imaging agent in
prostate cancer. Drugs Aging 1998; 12(4):293304. 59. Burgers JK, Hinkle GH, Hase
man MK. Monoclonal anti
ody imaging of recurrent and metastatic prostate cancer.
Semin Urol Oncol 1995; 13:103112. 60. Kahn D, Williams RD, Haseman MK, et al. Ra
dioimmunoscintigraphy with In-111-la
eled caproma
pendetide predicts prostate c
ancer response to salvage radiotherapy after failed radical prostatectomy. J Cli
n Oncol 1998; 16(1):284289.
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61. Thomas CT, Montie JE, Sandler HS, et al. Evaluation of agreement rates
etwe
en radionuclide
one scintigraphy and radioimmunoscintigraphy with Indium-111-Ca
proma
Pendetide (ProstaScint) in patients with rising PSA after definitive pros
tate cancer treatment. J Clin Oncol 2003; 21(9):17151721. 62. Anderson RS, Eifert
B, Tartt S, King P. Radioimmunoguided surgery using Indium-111 Caproma
Pendeti
de (ProstaScint) to diagnose supraclavicular metastasis from prostate cancer. Ur
ology 2000; 56(4):66. 63. Ellis RJ, Kim EY, Conant R, et al. Radioimmunoguided i
maging of prostate cancer foci with histopathological correlation. Int J Radiat
Oncol Biol Phys 2000; 49(5):12811286. 64. Ellis RJ, Sodee DB, Spirnak JP, et al.
Feasi
ility and acute toxicities of radioimmunoguided prostate
rachytherapy. In
t J Radiat Oncol Biol Phys 2000; 48(3):683687. 65. Magi-Galluzzi C, Sanderson H,
Epstein JI. Atypia in nonneoplastic prostate glands after radiotherapy for prost
ate cancer: duration of atypia and relation to type of radiotherapy. Am J Surg P
athol 2003; 27(2):206212. 66. Yao D, Tra
ulsi EJ, Kostakoglu L, et al. The utilit
y of monoclonal anti
odies in the imaging of prostate cancer. Semin Urol Oncol 2
002; 20(3):211218. 67. Carroll MJ, El-Megadmi H, Elnaas S, et al. P18. Prostate c
ancer: com
ined Prostascint SPET/CT/
lood pool imaging. Nucl Med Commun 2003; 24
(4):473. 68. Quintana JC, Blend MJ. The dual-isotope ProstaScint imaging procedu
re: clinical experience and staging results in 145 patients. Clin Nucl Med 2000;
25(1):3340. 69. Sodee DB, Ellis RJ, Samuels MA, et al. Prostate cancer and prost
ate
ed SPECT imaging with ProstaScint: semi-quantitative correlation with prosta
tic
iopsy results. Prostate 1998; 37:140 148. 70. Freeman LM, Krynyckyi BR, Li Y
, et al. National Prostascint study group. The role of (111)In Caproma
Pendetid
e (Prosta-ScintR) immunoscintigraphy in the management of prostate cancer. Q J N
ucl Med 2002; 46(2):131137. 71. Lange PH. PROSTASCINT scan for staging prostate c
ancer. Urology 2001; 57(3):402406. 72. Sartor O, McLeod D. Indium-111-caproma
pe
ndetide scans: an important test relevant to clinical decision making. Urology 2

001; 57(3):399401.
Part II Treatment choices: perspectives from the physician and patient
7 Treatment decisions: surgery versus
rachytherapy. A urologists perspective
Michael Perrotti and Leonard G Gomella Introduction Prostate cancer is the most
common cancer in men in the United States, and is the second most common cause o
f mortality. An estimated 230 110 men will
e diagnosed with prostate cancer in
the year 2004, accounting for approximately 33% of incident cancer cases in men,
with 29 900 expected deaths from this disease.1 We have learned that prostate c
ancer is not a disease unique to the elderly. In 1991, prostate cancer claimed t
he lives of 12 306 men aged 55 to 74 years and 20 909 men a
ove age 75.2 Availa

le information regarding the anticipated natural history of disease, as well as
availa
le pu
lished prognostic nomograms,3,4 may serve as a reference when couns
eling patients regarding their newly diagnosed prostate cancer, and may provide
assistance in clinical decision making to
oth physician and patient. It is gene
rally recognized that to reduce the risk of death from prostate cancer in the ma
le with clinically localized disease and life expectancy of at least 10 years, a
n effective treatment must
e employed. As there are several availa
le therapies
, disease outcome prognostication, as well as information regarding therapy spec
ific health-related quality of life outcomes have
ecome increasingly important.
Efforts have
een directed not only toward patient education regarding availa
l
e treatment modalities,
ut anticipated outcome with regard to disease control a
nd side effect profile. In this chapter, the cogent issues related to the modali
ties of radical prostatectomy and prostate
rachytherapy will
e discussed as th
ey relate to the patient with newly diagnosed clinically organ-confined prostate
cancer. Natural history of prostate cancer We have learned a great deal a
out t
he natural history of prostate cancer.57 In one widely referenced study, investi
gators utilizing the Connecticut Tumor Registry provided estimates of survival

ased on a competing risk analysis for men diagnosed with clinically localized pr
ostate cancer managed conservatively.5 Patients were stratified
y age at diagno
sis and primary tumor histology using the Gleason scoring system,8 and followed
for up to 10 to 20 years after diagnosis. These investigators reported that alth
ough the risk of death from prostate cancer was low in men diagnosed with Gleaso
n score 24 disease, men with Gleason score 5 or 6 tumors faced a modest risk of d
eath, and men with Gleason score 710 disease faced a high risk of death (Ta
le 7.
1) when
Treatment decisions
85
managed conservatively. Lu-Yao and Yoa reported on a population-
ased study comp
rised of 59 876 cancer registry patients aged 5079 years.9 The purpose of their s
tudy was to ascertain overall and prostate-cancer-specific survival in men treat
ed with prostatectomy, radiotherapy, or conservative management. For patients ma
naged with surveillance, 10 year prostatecancer-specific survival for grade 1 (G
leason 24), grade 2 (Gleason 57), and grade 3 (Gleason 810) cancer were 93%, 77%, a
nd 53%, respectively.
Ta
le 7.1 Risk of death at 15 years in men with newly diagnosed prostate cancer
managed conservatively
Gleason score Risk of death from prostate cancet at 15 yrs
24 5 6 7 810 47% 611% 1830% 4270% 6087%
Evidence for the effectiveness of PSA screening As we are
arely 10 years
eyond
the development and wide utilization of the serum prostate-specific antigen (PS
A) test, it is too early to assess the a
ility of PSA screening to reduce prosta
te cancer mortality
y altering the unfavora
le natural history of this disease.
There are, however, other measures to determine the effectiveness of this scree

ning test.10 Since the introduction of PSA screening in the late 1980s, investig
ators utilizing the National Cancer Institutes Surveillance Epidemiology and Ends
Results (SEER) Data
ase, have reported changes in the diagnosis of prostate can
cer that are consistent with an effective screening test.11 These changes includ
e a significant downward trend in the age at diagnosis, concomitant with a downw
ard shift in stage of disease at diagnosis. The majority of cancers diagnosed in
the PSA era are moderately differentiated (International Classification of Dise
ases of the World Health Organization grade 2; Gleason 5, 6, 7) and organ-confin
ed.11 These findings would indicate the detection of potentially lethal cancers
while amena
le to definitive local therapy. In a separate study, International C
lassification of Diseases of the World Health Organization grade 3 (Gleason 8, 9
, 10) prostate cancers were shown to
e less likely metastatic at diagnosis, and
more likely to
e treated definitively
etween 1990 to 1994 compared with 1980
to 1984.12 Given the known natural history of moderately and poorly differentiat
ed tumors (see Ta
le 7.1), and the complications of metastatic prostate cancer,1
3 this evidence for the effectiveness of PSA screening is encouraging as we awai
t mature prostate cancer mortality data. We also await the results of the Scandi
navian Prostate Cancer Group Study and the Prostate Cancer Intervention Versus O

servation Trial (PIVOT).14 These two large prospective randomized controlled st
udies compare radical prostatectomy and watchful waiting as treatment modalities
for localized prostate cancer.
Basic and advanced techniques in prostate
rachytherapy
86
Radical prostatectomy PSA non-progression rates Radical retropu
ic prostatectomy
gained widespread popularity in the early 1980s after the introduction
y Dr Wa
lsh of a series of technical modifications
ased on an improved understanding of
the prostatic and periprostatic surgical anatomy.15,16 Further refinement of th
e operative technique has continued into the contemporary era,17,18 and several
large series,1922 now provide actuarial PSA
ased 5-, 7-, and 10-year PSA non-prog
ression rates following radical prostatectomy for patients with localized
Ta
le 7.2 PSA non-progression (
NED) rates in contemporary radical prostatectomy
series for localized disease
Study No patients 5yrs
NED rate 7 yrs 10 yrs
Hopkins 1623 80% 68% Wash U 1778 81% Baylor 1120 76% 71% Mayo 2518 77% 68% PSA,
prostate-specific antigen;
NED,
iochemical with no evidence of disease.
Ta
le 7.3 PSA non-progression (
NED) rates at 7 years following radical prostate
ctomy stratified
y preoperative serum PSA
Preoperative PSA {ng/mL} 7 yr
NED rate
<2.5 93% 2.64.0 88% 4.19.9 76% >10 49% See Ta
le 7.2 for a

reviations.
disease (Ta
le 7.2). In the series
y Catalona and Smith,20 PSA non-progression
following radical prostatectomy was found to
e favora
ly influenced
y lower pr
eoperative serum PSA level, nonpalpa
le localized clinical stage (i.e. T1c), low
er tumor grade, and localized pathologic stage (i.e. pT2). In their report, 1778
men with localized disease underwent radical retropu
ic prostatectomy. Preopera
tively, the serum PSA was 2.5 or less in 124 men (7.7%), 2.64.0 in 127 men (7.9%)
, 4.19.9 in 924 men (57.2%), and 10 or greater in 440 men (27.2%). Preoperative s
erum PSA correlated with 7 year
NED (
iochemical with no evidence of disease) r
ate (Ta
le 7.3), with resultant influence in the
NED for the entire cohort. Gle
ason score similarly impacted on
NED rate. A total of 218 men (12%) had well-di
fferentiated (Gleason 24) tumors, 1375 men (77%) had moderately differentiated (G
leason 57) tumors, and 185 men (10%) had poorly differentiated (Gleason 810) tumor
s. Seven-year PSA non-progression rate was correlated with tumor grade (Ta
le 7.
4). The PSA non-progression rate of 68% and 48%

Treatment decisions
87
for moderate and poorly differentiated tumors, respectively, is encouraging give
n the recognized aggressiveness of
Ta

le 7.4 PSA non-progression (
NED) rates at 7 years following radical prostat
ectomy stratified
y Gleason score
Gleason score 7 yr
NED rate
24 84% 57 68% 810 48% See Ta
le 7.2 for a

reviations.
Ta
le 7.5 Prostate-cancer-specific 10 year survival rate from diagnosis in a pop
ulation-
ased study of 59 876 men with localized prostate cancer
Group Prostatectomy Radiotherapy Surveillance
1 2 3 94% 87% 67% 90% 76% 53% 93% 77% 45%
these tumors. Such results, from large single center patient cohorts with interm
ediate to long term follow-up duration, illustrate the effectiveness of radical
prostatectomy in the management of localized prostate cancer. Evidence for the e
ffectiveness of radical prostatectomy also comes from the population-
ased study
reported
y Lu-Yao and Yao.9 This study comprised 59 876 cancer registry patien
ts aged 5079 years, the prostate-cancer-specific survival in men treated with pro
statectomy and radiation therapy was reported (see Ta
le 7.5). By the intentiont
o-treat approach (i.e. included even if prostatectomy a
andoned secondary to pos
itive lymph nodes), 10 year prostate-cancer-specific survival in the prostatecto
my cohort (n=24 257) for grade 1 (Gleason 24), grade 2 (Gleason 57), and grade 3 (
Gleason 810) tumors was 94%, 87%, and 67%, respectively. The 10 year prostatecanc
er-specific survival for the cohort treated with radiation therapy for grade 1,
2, and 3 tumors, was 90%, 76%, and 53%, respectively. However, in the a
sence of
a prospective study design, these data should not
e used to compare outcomes f
ollowing radical prostatectomy and radiation therapy. Prostate
rachytherapy PSA
non-progression rates Prostate
rachytherapy, or permanent interstitial implant
ation, for the management of prostate cancer has undergone dramatic changes sinc
e its first report in 1910.27 Since that initial approach using a radium source
inserted through a urethral catheter, advances in this field have
een influence
d
y the availa
ility of newer isotopes, and more accurate access to the prostat
e gland. A renewed interest in permanent interstitial implantation was seen in t
he 1960s via the open retropu
ic approach,24,25
ut it was realized that this
Basic and advanced techniques in prostate
rachytherapy
88
approach was flawed
y technical limitations, and is truly considered of histori
cal interest in the modern era of prostate cancer therapy. In the present era, p
rostate
rachytherapy is administered percutaneously via a perineal route accord
ing to a computerized preplan template. Ongoing improvements in template design,
imaging, prostate sta
ilization, and technique have improved the accuracy of th
is procedure. Prostate
rachytherapy as monotherapy in modern practice utilizes
either palladium103 (103Pd) or iodine-125 (125I). The doses prescri
ed for these
two isotopes are different. 103 Pd has a half-life of 17 days and a dose-rate o
f 1820 cGy per hour, whereas 125I has a half-life of 59 days and a dose-rate of 7
cGy per hour.26 Based on these differences, a dose of 145 Gy for 125I is felt t
o
e equivalent to a dose of 115 Gy for 103Pd.27 The actual dose delivered to th
e prostate gland, or postimplant dosimetric isodose curve, likely impacts on the

iochemical relapse rate, and the American Brachytherapy Society (ABS) recommen
ds that postimplant dosimetry
e performed on all patients. Using 125I, Stock an
d associates have shown that when the dose delivered to 90% of the prostate (D90

) was >140 Gy, a significantly improved relapse-free rate was o
served compared
to lower D90 levels.28 Ragde and associates have reported their results using pr
ostate
rachytherapy in 551 patients.29 In this series, 320 patients (group 1) w
ere treated with implant alone, whereas 231 patients (group 2) who were felt to

e higher risk also received 45 Gy of external
eam radiotherapy in addition to
implant. Among group 1 patients, those with a Gleason score 25 received 125I to a
matched peripheral dose (MPD) of 160 Gy, those with a Gleason score 710 received
103Pd to an MPD of 115 Gy, and those with a Gleason score 6 were treated with e
ither isotope. The pretreatment clinical parameters for this cohort are displaye
d in Ta
le 7.6. The median follow-up was 55 months, with 152 patients followed f
or 5 years and 28 patients followed for 7 years. The reported 7 year actuarial f
reedom from
iochemical failure, defined as a serum PSA level 1.0ng/mL, was 80%.
That the 5 and 7 year actuarial freedom from
iochemical failure correlated with
pretreatment serum PSA level is illustrated in Ta
le 7.7, similar to radical pr
ostatectomy. Grimm and associates recently reported on a cohort of 125 men under
going prostate
rachytherapy as monotherapy.30 Brachytherapy as monotherapy in t
his study was limited to men with Gleason scores of 6 or less. In the reported c
ohort, 86% were clinical stage T2a or less, and
Ta
le 7.6 Pretreatment clinical stage, serum PSA and Gleason score, in a total o
f 1006 patients undergoing prostate
rachytherapy
Status No. patients (total 551)
Pretreatment clinical stage T1a 6 T1
10 T1c 59 T2a 207 T2
87 T2c 4 Serum PSA
Treatment decisions
04 4.110 10.120 >20 Gleason score 24 56 710
89
88 159 57 16 130 161 22
Ta
le 7.7 Influence of pretreatment serum PSA (ng/mL) on
iochemical freedom fro
m disease (
NED) rate in 320 patients undergoing prostate
rachytherapy as monot
herapy
Pretreatment PSA 5 yrsa 7 yrs

04 95% 4.110 87% 10.120 77% >20 65% a 152 patients at risk.
28 patients at risk.
87% 83% 72% 49%
77% had pretreatment PSA value of 10 ng/mL or less. Using a definition similar t
o that of the American Society of Therapeutic Radiation and Oncology (ASTRO), th
e 10 year actuarial
iochemical progression-free survival was 87%. A widely refe
renced study is that reported
y DAmico and colleagues.31 In this retrospective i
nvestigation, actuarial freedom from PSA failure was evaluated in 1874 men with
prostate cancer treated with radical prostatectomy (n=888), external
eam radiot
herapy (EBRT), (n=766) or interstitial implant with or without neo-adjuvant andr
ogen deprivation therapy (ADT) (n=218). The median follow-up duration for the
r
achytherapy cohort was 41 months. Biochemical failure was defined according to t
he ASTRO 1996 consensus statement for all study patients.32 This defined
iochem
ical failure as three consecutive rising PSA values each o
tained at least 3 mon
ths apart and the time of PSA failure defined as the midpoint
etween the time o
f PSA nadir and the time of the first rising PSA value. The relative risk (RR) o
f PSA failure in low risk patients (i.e. clinical state T1c/T2a and pretreatment
PSA10 ng/mL and Gleason6) undergoing
rachytherapy alone or
rachytherapy plus ne
o-adjuvant ADT, was 1.1 and 0.5, respectively compared with radical prostatectom
y. The RR of PSA failure in intermediate risk (i.e. clinical stage T2
or Gleaso
n score 7 or PSA>10 ng/mL) and high risk (i.e. clinical stage T2c or Gleason8 or
PSA >20 ng/mL) patients treated with
rachytherapy alone were 3.1 and 3.0, respe
ctively, compared to radical prostatectomy. No significant
enefit was seen with
a short course of neo-adjuvant ADT administered in the
rachytherapy plus andro
gen deprivation cohort, and this is in accordance with other reports.33,34 Brach
man and associates reported freedom from
iochemical progression after prostate


rachytherapy monotherapy of 53% for pretreatment PSA levels of 1020 ng/mL

Basic and advanced techniques in prostate
rachytherapy
90
and 28% for Gleason score 7.35 Other investigators, however, have reported more
favora
le results for
rachytherapy as monotherapy in intermediate and high risk
disease. For patients with Gleason score of at least 7 or PSA greater than 10 n
g/mL, Blasko and colleagues reported a nine-year freedom from
iochemical progre
ssion of 82% with 103Pd monotherapy.36 For patients with pretreatment PSA levels
>20 ng/mL the 9 year freedom from
iochemical progression was reported to
e 65
%.37 Dattoli and colleagues reported that 62% of patients with Gleason score 89 t
umors and 70% of patients with pretreatment PSA>20 ng/mL were
iochemically dise
ase free (PSA <1.0) at five years after moderate doses of external
eam radiatio
n therapy followed
y 103Pd
oost.37 Quality of life measures after local therap
y It is difficult to define the incidence and quantify the degree of toxicity in
men undergoing definitive therapy for prostate cancer, in part due to a lack of
conformity in reporting these data in the literature. The Prostate Cancer Outco
mes Study is a population-
ased longitudinal cohort study which has evaluated th
e changes in urinary and sexual function in men who have undergone radical prost
atectomy for clinically localized prostate cancer.38 In this population-
ased st
udy, patients randomly identified through the Surveillance, Epidemiology, and En
d Results cancer registries were offered the opportunity to complete a 6 and/or
12 month survey and a 24 month survey related to demographics, medical history,
urinary,
owel, and sexual function at
aseline and during the past month, satis
faction with treatment and quality of life. Men diagnosed with localized prostat
e cancer
etween 1 Octo
er 1994 and 31 Octo
er 1995 and who underwent radical pr
ostatectomy within 6 months of diagnosis were eligi
le, and the reported analysi
s included 1042 men aged 3989 years. At the time of the 24 month survey, 11.9% of
patients experienced incontinence more than twice daily, and 3.3% required thre
e or more pads per day. Level of
other due to urinary symptoms improved over ti
me, with 8.7% of patients reporting that incontinence was a moderate-to-severe p
ro
lem at 24 months. Age was related to the level of urinary control, frequency
of incontinence, and
other. Interestingly, only 56.4% of men were younger than
65 years at diagnosis in the present study. Among men under 60 years of age at d
iagnosis, 0.7% reported no urinary control at 24 months and 10% reported over tw
o episodes of incontinence per day. For men aged 6574 years, these values were 0.
9% and 15.7%, respective-ly, and for men aged 7579 years, these values were 13.8%
and 40.8%, respectively. With regard to sexual function, 72.7% of men reported
erections that were satisfactory for intercourse at
aseline prior to radical pr
ostatectomy. The proportion with erections firm enough for sexual inter-course a
t 24 months was influenced
y age at diagnosis (<60yrs: 39%; 6064 yrs: 21.7%; 6574
yrs: 15.3%; 7579 yrs: 19.1%) and whether a nerve-sparing procedure was attempted
(34.4% of non-nerve sparing; 41.4% of unilateral nerve sparing; 44% of
ilatera
l nerve sparing). Younger men reported more frequent sexual activity at each sur
vey period, and more often reported that sexual function was a moderate-to-sever
e pro
lem compared to older men. Approximately half the patients were delighted
or pleased with their surgery, and 4% were dissatisfied. At 18 or more months fo
llowing surgery, 71.5% reported that they would make the same choice again, and
7.3% reported that they would not choose radical prostatectomy again.
Treatment decisions
91
In a report from the Prostate Cancer Outcomes
radical prostatectomy and EBRT were compared
years after treatment, men undergoing radical
an men receiving EBRT to
e incontinent (9.6%

Study,39 health outcomes following

in a population-
ased cohort. Two
prostat-ectomy were more likely th
vs 3.5%), to have higher rates of

impo-tence (79.6% vs 61.5%), greater rate of needing treatment for urinary stric
ture (17.4% vs 7.2%), and lower incidence of proctitis (1.6% vs 18.7%). To our k
nowledge, a population-
ased study evaluating prostatic
rachytherapy as monothe
rapy has not
een reported. Wei and associates have reported on the healthrelate
d quality of life (HRQOL) following prostate
rachytherapy, utilizing a crosssec
tional survey from a single academic institution administered to patients underg
oing radical prostatectomy, prostate
rachytherapy, or EBRT, and to age-matched
controls.40 In this investigation, general HRQOL was evaluated with the RAND SF36, general cancer-related QOL was assessed using FACT-G, and general prostate c
ancer-related QOL was measured using the FACT-P. Instruments utilized to measure
domains specific to localized prostate cancer and therapy were the AUASI, and a
modified expansion of the UCLAPCI (EPIC). Among 1355 eligi
le patients, 1014 co
nsented to participate and completed required questionnaires (response rate: 74.
8%). General HRQOL measures did not detect significant differences
etween thera
py groups and controls. Radical prostatectomy was associated with worse urinary
incontinence (p<0.0001) and sexual HRQOL (p<0.0001) than controls. External
eam
radiotherapy was associated with worse
owel (p<0.0001) and sexual (p<0.0001) H
RQOL than controls. Prostate
rachytherapy was associated with significantly wor
se urinary irritative (p<0.0001), urinary o
structive (p<0.0001),
owel (p<0.000
1), and sexual (p<0.0001) HRQOL, and showed marginal adverse urinary incontinenc
e (p=0.01). As this is a single institution study, and not population-
ased, it
should simply serve to represent that centers experience, and generalizations sho
uld
e made with caution. Other investigators have indicated the potential for i
ncreased urinary toxicity after prostate
rachytherapy compared to external
eam
radiotherapy.41 The rates of urinary retention and 5 year risk of urethral stri
cture following prostate
rachytherapy vary widely in the literature, and have

een reported to
e 222%,4150 and 512%,41,43,51,52 respectively. Merrick and colleag
ues have reported that urinary symptoms peak at 2 weeks postimplant and return t
o
aseline at a median of 6 weeks,42 and there is evidence that the use of alpha
-
lockers results in a faster return to
aseline pre-
rachytherapy urinary sympt
om scores.53 These same investigators from the Schiffler Cancer Center recently
evaluated long-term urinary quality of life after prostate
rachytherapy utilizi
ng the EPIC and AUASI questionnaires.54 At a median follow-up duration of 64 mon
ths, no significant difference in urinary symptoms
etween treated men and men i
n a demographically matched control group was o
served. Rectal complications pri
marily consist of mild proctitis. Rectal
leeding will
e seen in 411%, and long
term dysfunction is uncommon.55,56 The incidence of erectile dysfunction followi
ng prostate
rachytherapy has
een reported over a wide range (690%), with Stock
and colleagues recently reporting an erectile dysfunction rate of 41% in patient
s potent prior to treatment.57
Basic and advanced techniques in prostate
rachytherapy
92
Conclusions Prostate cancer is a lethal disease, and significant differences in
disease specific outcome in large population
ased series now availa
le are seen

etween treatment and surveillance groups, particularly in those cohorts with mo
derately and poorly differentiated tumors. There is evidence from these same ser
ies to support the effectiveness of radical prostatectomy in treating localized
disease. The mor
idity of this effective therapy has
een reported from investig
ators utilizing the National Cancer Institutes SEER cancer registry, the results
of which would indicate an accepta
le mor
idity profile, particularly in the mal
e 65 years of age at diagnosis, and that level of satisfaction with treatment is
high. The allure of prostate
rachytherapy as monotherapy is its effectiveness i
n cancer control in reported series, and its once time administration, often on
an outpatient
asis. Prostate
rachytherapy as monotherapy has
een reserved, pr
imarily, for the patient with favora
le clinical stage, low serum PSA, and a Gle
ason score of 6, although some investigators have reported favora
le results in i
ntermediate and high risk patients. Reports are accruing that would indicate a m

or
idity profile associated with prostate
rachytherapy with regard to urinary a

nd
owel
other and erectile function that is not negligi
le. It is anticipated
that disease outcome prognostication, as well as information regarding therapy-s
pecific healthrelated quality of life outcomes, will remain paramount in counsel
ing the patient with newly diagnosed clinically localized prostate cancer. The a
vaila
le information to date should assist the clinician and patient in this end
eavor. References
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review: 19731991 (NIH Pu
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for disease recurrence following radical prostatectomy for prostate cancer. J Na
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agde H, et al. Does
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30. Grimm PD, Blasko JC, Sylvester JE, et al. 10-year
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rachytherapy. Int J Radiat O
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eam radiation therapy, o
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rachytherapy. J Clin Oncol 2000; 18:11871192. 35. Brachman DG, Tho
mas T, Hil
e J, et al. Failure-free survival following
rachytherapy alone or ex
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rom a single practice. Int J Radiat Oncol Biol Phys 2000; 48:111117. 36. Blasko J
C, Grimm PD, Sylvester JE. Palladium-103
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ased radiati
on for prostatic carcinoma. Int J Radiat Oncol Biol Phys 1999; 45:853856. 38. Sta
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40. Wei JT, Dunn RL, Sandler HM, et al. Comprehensive comparison of health relat
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Clin Oncol 2002; 20:557566. 41. Zelefsky MJ, Wallner KE, Ling CC, et al. Compari
son of the 5-year outcome and mor
idity of three dimensional conformal radiother
apy versus transperineal permanent iodine-125 implantation for early stage prost
ate cancer. J Clin Oncol 1999; 17:517522. 42. Merrick GS, Butler WM, Lief JH, et
al. Temporal resolution of urinary mor
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rachytherapy. I
nt J Radiat Oncol Biol Phys 2000; 47:121128. 43. Zelefsky MJ, Hollister T, Ra
en
A, et al. Five-year
iochemical outcome and toxicity with transperineal CT-plann
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50. Kang SK, Chou RH, Dodge RK, et al. Acute urinary toxicity following transper
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, Tollenaar BG, et al. The dosimetry of prostate
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8 Treatment decisions: which therapy? A patients perspective
William J Hilsman Introduction This chapter descri
es my personal experience in
dealing with the issue of decision making for prostate cancer, and how I used av
aila
le information on the disease to go a
out making my decision whether to hav
e com
ined hormone therapy, external
eam radiation, and
rachytherapy. Many res
ources are availa
le to patients today thanks to advances in technology. The gui
dance and information provided
y my physicians was an essential element in my t
reatment decision,
ut I stress that the final decision was mine. How it all
eg
an I had just landed in San Diego, California. It was just past 5:30 pm on a war
m May day in 1994. I was checking my office answering machine while waiting for
my luggage. There was a message from my doctor Colonel Ken Torrington, at Walter
Reed Army Medical Center (WRAMC) asking me to call him. A signal went off in my
head. I had just completed my annual physical at WRAMC a few days
efore, and D
r Torrington had said he felt a little nodule on the prostate during the digital
rectal examination. He said he wanted to see what my prostate-specific antigen
(PSA) was
lood had
een taken the morning of my physicaland he would call me. I kn
ew there was such a thing as a PSA test,
ut I never paid much attention to it.
When I got to him on the phone, he said my PSA had increased from 3.7 to 4.9, an
d said I should call my urologist Dr David McLeod to schedule a
iopsy. For the
next few weeks
efore the scheduled
iopsyI admitI was just a little more than anx
ious. I went to the li
rary and found
ooks on prostate cancer. I wanted to get
as much information as I could
efore B day
iopsy day. On 7 June, 1994,1 reported t
o WRAMC for my
iopsy. Again, I will admit that I was a little scared. I was pre
pared for the
iopsy
y Dr McLeod, and the procedure
egan. I was watching the T
V screen and listening to Dr McLeod as he performed the transcrectal ultrasound
(TRUS). He measured the prostate gland as
eing 39 mL in volume, explained to me
what he was doing, and then gave me the good news! I
elieve we are looking at a
calcium deposit that is causing the rough feeling. I am not going to do the
io
psy. I just do not
elieve in any action like a
iopsy unless I think it is nece

ssary. He then said, Lets wait and check the PSA again in 6 months. I was elated,
u
t I was also a lot
Treatment decisions: which therapy?
97

smarter than I was a month ago. I now knew something a
out prostate cancer and w
as sure I would get a lot smartera lot, lot smarter very quickly. Since I was liv
ing in the Philadelphia area and WRAMC was in Washington, DC, I decided to consu
lt with a local urologist at Thomas Jefferson University Hospital. This
egan my
association with Dr Leonard Gomella. As Dr McLeod said to me, You now have two o
f the
est urologists in the nation, Dr Gomella and me. I agreed. In Novem
er 199
4, the PSA went up to 5.3. Six months later, it was still 5.3. Then we started t
o see a decrease: July 1995, 5.0; May 1996, 4.7. Meanwhile, I was getting more e
ducated. I read as many pu
lications as I could find and joined many we
sites to
get information. Then came my PSA result in July 1997. One remem
ers some event
s with the greatest of clarity. I was on my way to Washington in my car when I c
alled on my cell phone to Dr Gomellas office: 6.2 said the nurse. No signals went
off this timejust damn. I knew that a jump from 4.7 to 6.2 in one year was a stron
g indicator for cancer. Dr Gomella ordered a free PSA, which I did. This was goo
d news, or so I thought. My free PSA was 24.9%. Below 14%, I was told, was
ad.
However, at the same time, Dr Gomella suggested it was time to do a
iopsy. His
digital rectal examination told him there was a change. For the most part, the

iopsy was a non-event: six sticks. Again I watched. I asked for the volume as th
ey finished79 cc. Again, no signals, just damn. I remem
ered the 39 cc reading from
1994.1 knew enough from my reading that I was getting closer to what I did not
want to hear. I could pro
a
ly never tell you a
out any department store experie
nce I ever had, except at Nordstrom in Alexandria, Virginia. As I was leaving th
e store, I called my answering machine and had a message to call Dr Gomella. I w
as just leaving to get in my car, so I called him from my car phone just as I wa
s going
y the Washington monumentyou do remem
er these things. Your
iopsy is
ac
k, and you do have cancer. That was the message. Again, I was very calm, and I kn
ew what I wanted to know next. In how many cores? I asked. Two of the six, he replie
d. One side or two? I asked. One side, he said. What was the Gleason? I wanted to know
+3) 6, he said. Okay. I knew with a PSA of 6.2, a Gleason of 6, and the way we ha
d followed the case, that I had a high pro
a
ility of having caught the cancer e
arly. When do we schedule the
one scan and CAT (computed tomographic) scan? I ask
ed. I was just a
out to go on a two week vacation with my familywife, four kids,
and six grandkids. When you get
ack, he said. I felt OK with that. Now I knew I h
ad reached the stage of laying out a plan that would lead me to making my decisi
on on the procedure I would follow. I knew that if the
one scan, and CAT scan w
ere negative, I had some alternatives for treatment, and I knew what they were.
Just to
e sure of where I was, I took my slides to WRAMC for a second reading:
(3+3) 6 in one, (3+2) 5 in the other. Basically the same result. Informing the f
amily I knew one issue I had to face early was on informing my family. Cancer! O
h, my God! One hardly needs to say that announcing cancer to family mem
ers can

e very
Basic and advanced techniques in prostate
rachytherapy
98
traumatic. In my case, it was not so
ad, for one reason a
ove all. I knew a gre
at deal a
out my condition. I knew I had it early. I knew what options were out
there, and I knew I had a good medical team
ehind me. As I discussed the diagno
sis with my family, I was calm, knowledgea
le, and prepared. It made all of the
difference in the world. The analysis phase My reading and discussions with indi
viduals who had
een recently diagnosed and my many, many inputs from different
Internet we
sites, had focused me on the different options to include radical pr

ostatectomy (RP), external
eam radiation therapy (EBRT),
rachytherapy (seeding

), com
ined hormone therapy (CHT), cryosurgery, and watchful waiting.1 I discard
ed cryosurgery early on. I felt that it was just too experimental for me at that
time.24 A little further into the analysis, I dismissed watchful waiting.5 I had
, in fact, done that for 3 years. I was still comparatively young65 years old. I
pretty well knew that if I chose
rachytherapy, I would have to do CHT to
ring
the size of my prostate down.6 Therefore, I laid out a strategy to get informed
opinions from my doctors. I
egan with Dr Gomella, and to his credit, he was imm
ediately open and suggested that I get different opinions. He staged me at T2
a
nd suggested that I was indeed a candidate for RP.7 He went on to say that he wa
nted me to meet Dr Adam Dicker, in the Department of Radiation Oncology at Jeffe
rson Hospital, and get a second opinion from Dr Dicker. He also suggested that I
see Dr McLeod at WRAMC for his opinion. Dr Dicker staged my cancer at T2
, and
suggested that I was also a candidate for
rachytherapy.8 Dr Dicker explained hi
s
ackground. He had worked under Dr Kent Wallner at Sloan Kettering, one of the
pioneers in
rachytherapy. He went on to say that on the Jefferson team was Dr
Frank Waterman, the physicist who shared in the planning, also from Sloan Ketter
ing. I decided I wanted to know more a
out the team, so I spent 2 to 3 hours at
Jefferson working with the physicist team to understand the role they play in th
e procedure. I understood how important and critical the planning was, and under
stood how they did it at Jefferson. I liked what I saw. Meanwhile, I joined an i
nternet site, seedpods@rattler. cameron.edu. On the site were patients and docto
rs who had had experience with
rachytherapy. I learned more a
out the procedure
and the decision process, especially the question as to seeding alone;9,10 seed
ing with EBRT;11 and seeding, EBRT, and CHT. I also found different approaches t
o
rachytherapythe high dose radiation method practiced in Seattle and Tulsa,12 a
nd the seeding followed
y EBRT as practiced
y the Radiotherapy Clinics of Geor
gia (RCOG) versus the EBRT followed
y seeding more commonly used at other hospi
tals. I studied types of seedsiodine or palladium.13 I felt very good after my ed
ucation, especially that I understood the procedure and the risks. I then went f
or my third and fourth opinions at WRAMC in Washington. Dr David McLeod, like Dr
Gomella, one of the
est urologists in the nation in my mind, also suggested th
at I was a candidate for RP,
ut he also arranged for me to meet with Dr John Ha
lligan, their radiation oncology team leader. Dr Halligan spent 3 hours with me
explaining all the pros and cons. I was extremely impressed
y Dr Halligan and h
is total dedication, professionalism, and knowledge of the treatments. He also s
uggested that I get a fifth opinion from one of the nations
est known
rachyther
apists, Dr Dan Clark, at
Treatment decisions: which therapy?
99
the Northern Virginia Center in Alexandria, Virginia. Dr Clark had one more prot
ocol he used in diagnosing whether or not the cancer was contained. He used an e
ndorectal coil magnetic resonance imaging (MRI). This procedure showed that the
cancer was near the extremity of the gland. Dr Clark staged my cancer at T2c. He
suggested from his analysis and the size of my prostate that I was a candidate
for CHT plus EBRT plus seeding. He also felt that I was a candidate for RP. Deci
sion time I now had my analysis phase complete. I first had to decide on radical
prostatectomy or radiation. This was not an easy choice
ecause I saw merit in

oth approaches. As Dr McLeod said, If I take it out, Ill
ottle it in a jar, and
you can take it home and
e rid of it. But, he also said that he felt he could on
ly save nerves on one side
ecause of his feel of the gland and the
iopsy repor
t. I knew that incontinence and impotence were potential side effects. I really
made my decision against RP
ased on a philosophy I had used throughout my life.
If I have a choice, I will do the less invasive procedure unless it makes no se
nse at all. Although RP had a good 15 years of track record statistics, and seed
ing had only 8 years at the time, I was very comforta
le with the 8 year reports
(1997) coming out of Seattle.9 So my first decision was made. RP was eliminated

. My next choice, did I want to do EBRT alone, as recommended
y some of my frie

nds who have had prostate cancer. I decided I did not. I did not like the odds.
Did I want the
rachytherapy alone? I did not. Again, I went to the Partin ta
le
s, that had
een developed at Johns Hopkins University, looked at the oddsPSA 6.2
, Gleason (3+3) 6, and a staging of T2
/T2cand decided I wanted to go with the pr
otocol recommended
y Dr Clark. I understood that the com
ination therapy was no
t widely practiced and could have the risk of increased side effects. I then met
with Drs Gomella and Dicker at Jefferson and CHT, EBRT, and
rachytherapy were
agreed on. So, another decision had
een made. Now, for the final decision: Wher
e to have it done? I considered strongly Dr McLeod and the team at WRAMC, RCOG i
n Georgia, Dr Clark in Alexandria, and Drs Dicker and Gomella at Jefferson. I ch
ose the Jefferson team for the following reasons: (1)1 felt very comforta
le wit
h Dr Adam Dicker and the radiation oncology team; (2) I also knew Dr Gomella wou
ld
e in the operating room as a part of the team; and (3) Dr Watermans physics t
eam was
ehind me: in other words, I felt I had a great team working with me; (4
) the Kimmel Cancer Center of Thomas Jefferson University Hospital was an excell
ent center to have
ehind me if something went wrong or I had a reccurrence; and
(5) I really liked the Jefferson protocol, which kept the patient overnight to
help assure that the catheter, when it came out, would not have to go
ack. (Mos
t other hospitals have patients in
y 1:00 pm and out
y 5:00 pm.) I had made my
decision. I knew it was right. I never looked
ack.
Basic and advanced techniques in prostate
rachytherapy
100
Let the process
egin Com
ined hormone therapy (CHT) was first. I went in for my
first 90 day Lupron shot and
egan a flutamide regime in Septem
er 1997.14 Flut
amide was stopped after 90 days
ecause of liver issues. My aspartate aminotrans
ferase (AST) and alanine aminotransferase (ALT) readings increased significantly
. Lupron was continued. In January 1998, the original time planned for external

eam radiotherapy (EBRT), we still did not like the size of the prostate nor its
closeness to the
ladder, so a 2 month delay was imposed. I
egan a 28 session
EBRT (six directional, not four) in March, which was completed in April. On Thur
sday, 28 May, I was seeded at the Bodine Cancer Center of Thomas Jefferson Unive
rsity Hospital in Philadelphia, Pennsylvania. I was administered 68 iodine125 se
eds
y Dr Adam Dicker who was assisted
y Dr Leonard Gomella. My team (a good th
oughtcmy team) recommended general anesthesia
ut would have done a spinal if I ha
d insisted. Again research! I went with the general anesthesia, mostly on the ad
vice of my team who wanted to
e a
le to
e as precise and accurate as they coul
d and felt this was a
etter procedure for them. I had no trou
le then or later.
While I was asleep, the team implanted the seeds and checked to see if any seed
s had gone to the
ladder. Then I went
ack to the CAT scan equipment so my team
could see how we did. Quoting Dr Dicker, the plot was gorgeous. Im not sure I woul
d have used this term on my prostate
ut I loved it! As I mentioned earlier, my te
am also kept their seed implants (SI) patients overnight with the catheter in pl
ace. I already knew I liked the idea from my own research on the internet and in
puts from previous patients. I was not hungry that evening,
ut I ate a little a
nd drank lots of water. I am sure I passed at least two
lood clots during the n
ightmay
e more. The catheter came out at 6:00 am the next day. I did enjoy
reakf
ast. My first attempt at urination was not a stream, was not a dri

le,
ut a sp
rayeither sit down or use the
ottle provided. I walked
ack to the hotel at 11:0
0 am. I had a lot of urgency and
urning until 4:00 pm when I passed a dime-size
d
lood clot. Things got
etter at the end of day one. I treated my wife and mys
elf to dinner at one of Philadelphias finest restaurants. I still had some urgenc
y, some mild
urning, and had to make lots of visits at night
ut otherwise I felt
great. For these pro
lems, my team had given me Flomax ta
lets, 0.4 mg (tamulos
in hydrochloride) if I felt I needed it. I chose not to use it at that time. But
I did take some i
uprofen. O
servations and lessons learned Stay tuned to the r
est of your
ody with your general practitioner as you enter into this treatment

. In my early research, when I found that WRAMC and Bethesda Naval Medical Cente
r were doing
rachytherapy, I knew we were past the experimental stage. I feel t
hat the triple hit of CHT, EBRT, and SI was
est for my future even though it to
ok a long time.
Treatment decisions: which therapy?
101
Although there is controversy as to whether magnetic resonance imaging (MRI) is
necessary due to the cost, in my case it was used to lay out my plan of attack i
n August 1997 and for the SI plan in 1998. It was good for me. Two weeks
efore
my diagnosis in 1997, my free PSA/ ratio came
ack at 24.9% reference range less
than 14% indicating cancer. The free PSA really missed it in my case. I feel tha
t the overnight stay with catheter was a good decision. I feel that confidence i
n the teammy teamis critical to the program. I feel that good diet/nutrition are e
ssential for success. For the rest of my lifelow fat, lots of vegeta
les and frui
t, a planned vitamin and her
program, lots of soya, two glasses of the finest w
ine with dinner, and a good exercise programthat is where I will
e. The flutamid
e issue, which can
e seen with any of the non-steroidal antiandrogens, could ha
ve
een serious if we were not monitoring the AST and ALT at my quarterly
lood
tests. In the small print of flutamide information, it suggests some patients co
uld have this adverse liver reaction, as I did. My doctors did follow this and m
y advice to others is to watch it. I handled almost 9 months of Lupron with no d
ifficulty. In my opinion, soya was very important in this process. I had major h
ot flashes,
ut not real pro
lems. I did find myself a
it quick tempered. I kne
w of the weight gain pro
lem with CHT, and fought it successfully with diet and
exercise. (I did a 2 week get healthy with diet and exercise program at the
eginn
ing to
e as healthy as possi
le.) I weighed 182 pounds in July 1997 and 177 pou
nds on seeding day. Some people may not find a 5 pound loss significant,
ut mos
t people are reporting weight gains during CHT. Thus, in my opinion, diet and ex
ercise are an important aspect of the process. I experienced no difficulty durin
g the 28 sessions of EBRT totaling 50.4 Gy. I was impressed with the physics of
six-dimension EBRT practiced at the Kimmel Cancer Center as I studied the plots
and computer graphics and the way the team went a
out the procedure. I am convin
ced that having researched the issue and o
taining as much information as I had,
allowed me to operate under what we would call the Doctrine of No Surprises a grea
t
enefit.
And now for the rest of the story As the time of writing, I am six years past se
eding day, and my PSA continues to
e less than 0.100. I did have some side effe
cts. Lupron was continued 3 months past seeding. My weight went up to 193 pounds
. I am
ack to 180 today. Eight months past seeding, I started to have trou
le w
ith urinating. I started using Flomax and have this pro
lem under control now. I
started with one ta
let a day. I then went to one ta
let every other day, then
one ta
let every third day. Today, I no longer need Flomax. I did have one more
nasty complication, which lasted 4 to 5 months, January through May of 1999. In
January, 1 started rectal
leeding every 2 or 3 daysheavy clotting. This stopped in
Fe
ruary. Then, in April, I took a 10 minute amusement park ride, like
Basic and advanced techniques in prostate
rachytherapy
102
riding fast on a horse with no previous experience. That night was a
solute mise
ry urgency
urning. Things got worse for 2 or 3 more weeks. I was experiencing re
ctal
leeding (clots) every 3 days. By the end of May, I was improving. We decid
ed to take a look anyway since there had
een some
leeding. I had a flexi
le si
gmoidoscopy. Lying on the ta
le and watching the TV screen was an experience. Wh
at I clearly saw was the result of radiation sun
urn. When I saw it I could unde

rstand why I felt the
urning. In my case, the
urning was most intense while I
was urinating with the most intensity in the rectum. It was so intense that at t
imes it
rought tears to my eyes. It also seemed to get right to my nerves and r
attle me from my fingertips to my toes. The flexi
le sigmoidoscopy itself had ca
used the
urning to get worse. I have no idea or proof as to whether the protoco
l I followed made any difference, or whether I would have healed doing nothing.
All I can say is I have
eaten the pro
lem. The
urning and
leeding stopped in
a
out 2 months. First, I followed a natural nutritional regimen of Seacure, a wh
ite fish dietary protein supplement with omega-3 fatty acids, and L-glutamine, a
n amino acid, suggested
y a few individuals on my internet site. Second, I adde
d colostrum, another dietary supplement from sheeps milk, to the program. Third,
I kept taking my regular supplements that included Saw Palmetto, E, C, Q-10, zin
c, selenium, green tea, and my multivitamins. Because I could feel some swelling
in the rectal area, I also went
ack to my successful treatment immediately aft
er seedingi
uprofen. Today and tomorrow As all prostate cancer survivors know, th
ere is no free lunch in any choice we make to fight the disease. As of today and
this chapter, I can only say that side effects are not an issue, and most impor
tant, a PSA of less than 0.100 has to lift anyones spirits. But, I also know the
fight will continue for the rest of my life. There will
e some anxiety as I do
my annual PSA. I continue to educate myself on what others are doing, and what e
lse is new in the fight for the cure. May
e most importantly, those of us who ar
e as fortunate as I, have the opportunity to help others who are just
eginning
the fight, or even those who might never have to face the pro
lem. I had input a
nd recommendations from outstanding urologists and radiation oncologists,
ut th
e final decision was minethe patientas I
elieve it should
e. I used resources av
aila
le, such as seedpods on the internet. Today, I dedicate myself to helping o
thers as they meet the challenge of surviving prostate cancer, as others helped
me to meet the challenge. I dedicate myself to help others as I have
een helped
in the past. My door is always open. I am a telephone call or an email away. Re
ferences
1. Drachen
erg DE. Treatment of prostate cancer; Watchful waiting, radical prost
atectomy, and cryoa
lation. Semin Surg Oncol 2000; 18:37. 2. Porter MP, Ahaghotu
CA, Leoning SA, et al. Disease-free and overall survival after cryosurgical mon
otherapy for clinical stages B and C carcinoma of the prostate: A 20-year follow
up [See comments]. J Urol 1997; 158:1466.
Treatment decisions: which therapy?
103
3. Badalament RA, Bahn DK, Kim H, et al. Patient-reported complications after cr
yoa
lation therapy for prostate cancer. Urology 1999; 54:95. 4. Gould RS. Total
cryosurgery of the prostate versus standard cryosurgery versus radical prostatec
tomy: Comparison of early results and the role of transurethral resection in cry
osurgery. J Urol 1999; 162:1653. 5. Kattan MW, Cowen ME, Miles BJ. A decision an
alysis for treatment of clinically localized prostate cancer [see comments]. J G
en Intern Med 1997; 12:299. 6. Blank KR, Whittington R, Arjomany B, et al. Neoad
juvant androgen deprivation prior to transperineal prostate
rachytherapy: Small
er volumes, less mor
idity. Cancer J Sci Am 1999; 5:370. 7. Jacqmin D. Indicatio
ns and results of radical prostatectomy. Cancer Radiother 1997; 1:418. 8. Bey P.
Indications and results of exclusive radiotherapy in early prostatic adenocarci
noma. Cancer Radiother 1997; 1:431. 9. Ragde H, Blasko JC, Grimm PD, et al. Brac
hytherapy for clinically localized prostate cancer: Results at 7- and 8-year fol
low-up [Pu
lished erratum appears in Semin Surg Oncol 1998; 14:185]. Semin Surg
Oncol 1997; 13:438. 10. Nag S, Beyer D, Friedland J, et al. American Brachythera
py Society (ABS) recommendations for transperineal permanent
rachytherapy of pr
ostate cancer. Int J Radiat Oncol Biol Phys 1999; 44:789. 11. Andreopoulos D, Pi
atkowiak M, Krenkel B, et al. Com
ined treatment of localized prostate cancer wi
th HDR-Iridium 192 remote
rachytherapy and external
eam irradiation. Strahlent
her Onkol 1999; 175:387. 12. Mate TP, Gottesman JE, Hatton J, et al. High dose-r

ate after loading 192Iridium prostate
rachytherapy: Feasi
ility report. Int J R

adiat Oncol Biol Phys 1998; 41:525. 13. Sharkey J, Chovnick SD, Behar RJ, et al.
Outpatient ultrasoundguided palladium 103
rachytherapy for localized adenocarc
inoma of the prostate: A preliminary report of 434 patients [See comments]. Urol
ogy 1998; 51:796. 14. Migliari R, Muscas G, Murru M, et al. Antiandrogens: A sum
mary review of pharmacodynamic properties and tolera
ility in prostate cancer th
erapy. Arch Ital Urol Androl 1999; 71:293.
Part III Pretreatment and real-time planning for permanent, low dose rate prosta
te
rachytherapy
9 Brachytherapy from the urologists perspective
Phuong N Huynh and Howard J Korman Historical perspective The goal of prostate

rachytherapy is to induce cytotoxic events
y placing radioactive sources into t
he tumor to mediate an irreversi
le radiation-induced
reak in the dou
le strand
DNA through free radicals.1 This technique, first descri
ed
y Pasteau in 19132
and Barringer in 1917,3 involved placement of radium-containing needles into th
e prostate (Ta
le 9.1). Young performed transperineal
rachytherapy in the Unite
d States
y the 1920s.4 By the 1950s, Flocks et al were placing permanent seeds
using radioactive gold-198 (198Au).5 In the 1960s Whitmore et al6 at Memorial Sl
oan Kettering Cancer Center popularized the open retropu
ic implantation of radi
oactive iodine125 (125I) into the prostate
y the free hand technique com
ined wit
h pelvic lymphadenectomy. Although this resulted in su
optimal distri
ution of s
eeds and poor dosimetry,7,8 this method remained popular throughout the 1980s. C
omplications attendant to this method included lymphocele, a
scess and hematoma
formation, pelvic cellulitis, impotence and rectourethral fistulae. The latter c
omplication was most commonly seen in patients who required su
sequent transuret
hral resection of the prostate (TURP) or external
eam radiotherapy (EBRT).9
Ta
le 9.1 Timeline of prostatic
rachytherapy
Early 1900s Radium needles, transperineal approach 1950s Permanent gold-198 seed
s 19601980s Permanent iodine-125 seeds, retropu
ic approach (free hand) 1980s Tra
nsrectal ultrasound and template guided approach 1986 Introduction of PSA Early
1990s High dose rate iridium-192
rachytherapy 1996 ASTRO Criteria
Prostate
rachytherapy was further revolutionized
y Holm et al who detailed clo
sed transperineal implantation with transrectal ultrasound (TRUS) and template g
uidance in 1983.10 This clearly improved the accuracy of radioactive seed placem
ent
y allowing transperineal visualization of the needle and
y guiding the ins
ertion of seeds into the prostate as compared to the free hand open retropu
ic a
pproach. Ragde
rought this concept to the United States when maturing open retr
opu
ic implantation results showed
Basic and advanced techniques in prostate
rachytherapy
106
a higher recurrence rate than treatment with EBRT or radical prostatectomy (RP).
11 A conceptually similar approach using computed tomography (CT) was developed,
12
ut the ultrasound-guided approach was easily disseminated to other instituti
ons and therefore
ecome the mainstream approach. By 1987, retropu
ic implantati
on was essentially a
andoned. The technique descri
ed
y Carlton et al13 of impl
antation of 198Au seeds com
ined with EBRT resulted in 5-year survival rates of
71%, 59%, and 46% and 10-year survival rates of 54%, 26% and 40% for stages B1,
B2 and C1 respectively.14 Better outcomes were achieved with the open retropu
ic
approach. The initial open retropu
ic prostate
rachytherapy results from the 1
970s demonstrated overall 5-year survival rates of 95%, 64%, and 59% for stage B
1, B2 and C lesions, respectively,
ut the tumor-free survival rates were noted
only to
e 66%, 30% and 21% for the respective stages.1517 Su
sequent results ref
lecting changes in technology showed
etter survival data. Advances in imaging w

ere essential to the resurgence of
rachytherapy. The
iplanar mechanical sector

endocavitary pro
es were not introduced until the late 1980s which Stone used t
o perform real-time ultrasound implantation of seeds.18 By 1996, Bruel & Kjaer h
ad developed the superior
iplanar linear array endorectal pro
e
y designing ne
w components.19 This pro
e was superior to the mechanical sector pro
e
ecause i
t allowed clear visualization of the anterior rectal wall in the sagittal view.
Current software allows live monitoring of each seeds position and radiation fiel
ds created. Although a dosimetric treatment plan can
e calculated using preimpl
ant TRUS images,20 intraoperative dosimetric planning (real-time technique)21 ha
s also
een performed with software advances. This uses a nomogram or reference
ta
le to order the sources once the patient has
een placed in position. Intraop
erative planning is now the preferred means of prostate
rachytherapy as recomme
nded
y the American Brachytherapy Society.22 Another development is the use of
CT images in conjunction with a three-dimensional (3D) software program to study
postimplantation dosimetry and to generate dose-volume histograms, providing an
understanding of seed placement in the prostate gland while avoiding toxicity t
o the rectum and urethra.23 This quality check allows essential measurements of
delivered doses to the prostate gland and surrounding areas. Postimplantation CT
is usually not performed until 2 to 6 weeks after the procedure due to postimpl
antation edema, depending on the kind of seeds used.24 Impact of PSA testing The
treatment of prostate cancer has
een significantly changed
y the use of prost
atespecific antigen (PSA) testing. The introduction of PSA testing in the 1980s
for early detection and monitoring of prostate cancer dramatically increased the
diagnosis of prostate cancer with a peak incidence in 1992. The disease-specifi
c mortality peaked in 1993 and has continued to decline since that time.2527 Cart
er et al were a
le to quantify the diagnostic lead time from PSA testing.28 Usin
g a PSA cutoff of 4.0 ng/mL, they found that 78% of localized prostate cancer co
uld
e diagnosed a median of 4.9 years earlier than
y clinical diagnosis alone.
Additionally, metastatic disease could
e detected with an elevated PSA level

y more than 11 years
efore clinical diagnosis alone. Gann et al found similar r
esults for men with ten years of follow-up.29 Using a PSA cutoff value of
Brachytherapy from the urologists perspective
107
4.0 ng/mL, they found the diagnostic lead time for all prostate cancer diagnoses
to
e 5.4 years and for fatal prostate cancer to
e 3.6 years. The spurious eff
ect of lead time causes an increase in 5-year survival, resulting in a larger nu
m
er of patients who are likely to survive at least 5 years, which may skew data
on the impact of treatment for prostate cancer.30 Therefore, the effectiveness
of prostate cancer treatment is more accurately measured in prostate cancer mort
ality rates.28 Although mortality rates have
een declining, this appears to
e
due to multiple factors and not solely due to improvement in treatment. Most not
a
ly, PSA screening has led to stage migration, resulting in increased organconf
ined prostate cancer detection,
etween 70% and 80% of those screened.3134 Simila
rly, the incidence and mortality rates of metastatic prostate cancer have steadi
ly declined, suggesting that PSA testing may
e a factor in the decrease in pros
tate cancer mortality rates.34 Furthermore, the year of diagnosis was found to

e an independent predictor of the likelihood for cure.30,35 This o
servation sug
gests that the increased rate of organ-confined disease has resulted in increase
d disease-free survival. Finally, widespread use of androgen deprivation for ear
ly tumor recurrence may delay disease progression allowing other competing comor

idities to
e the cause of death. In summary, it appears that the effect of PSA
testing has caused stage migration towards detecting locally confined, low volu
me prostate cancer. This has likely contri
uted to
etter survival statistics re
gardless of treatment modality. Results Since the introduction of serum PSA in 1
986, treatment outcomes have
een measured in terms of
iochemical disease-free
periods. However, a standardized definition of
iochemical failure after radiati
on therapy was not developed until the 1996 American Society of Therapeutic Radi

ology and Oncology (ASTRO) Consensus Conference.36 Failure was defined as three
consecutive increases in the PSA levels (3 to 4 months apart in the first 2 year
s, then every 6 months thereafter) with the date of failure
ackdated to midway

etween the PSA nadir and the date of first increase. According to implant monot
herapy studies
efore the ASTRO definition of
iochemical failure, the 5- and 10
year actuarial rates of freedom from
iochemical relapse were 87% to 95% for pat
ients with low risk features (Gleason 6 or less, PSA 10 ng/mL or less, and clini
cal stages T2a or less).3739 Those definitions of
iochemical failure varied from
an a
solute posttreatment PSA value
eing greater than 0.5 ng/mL to posttreatme
nt PSA
eing greater than pretreatment PSA. Grimm et al reported a learning curv
e
y demonstrating differences in progression-free survival in patients of low r
isk
efore and after 1988.40 Certainly, definitions of
iochemical failure diffe
r
etween surgery and radiotherapy. Standardized definitions allowing accurate c
omparisons of the two most commonly used curative treatment modalities must
e a
greed upon to give any real hope of meaningful comparative studies.
Basic and advanced techniques in prostate
rachytherapy
108
Isotopes Several choices of isotopes for permanent seed implantation exist. Whil
e external
eam radiation delivers high energies from linear accelerators (615 Me
V), low energies are emitted from 125I and palladium-103 (103Pd). 125I has a hal
f-life of approximately 60 days and emits 27 keV of energy. Introduced in 1986,
103Pd has a half-life of 17 days and emits 21 keV of photon energy. Theoreticall
y, there is a more rapid fall-off at the
orders of the implantation when 103Pd
seeds are used which reduces the dose to normal surrounding structures
ut is le
ss forgiving in terms of tumor aim. No randomized pu
lished trials comparing out
comes and toxicity rates of 125I and 103Pd exist,
ut clinical data that have
e
en reported do not indicate any difference in
iochemical control or outcome.41,
42 As recommended
y the American Association of Physicists in Medicine, the dos
es for 125I and 103Pd are 145 Gy and 124 Gy, respectively.43 Treatment options F
or men with low risk features of prostate cancer permanent interstitial
rachyth
erapy is a proven and effective treatment option. Using the standardized ASTRO d
efinition of
iochemical failure, the 5 to 10-year
iochemical control rates of
low risk patients ranged from 65% to 93%4446 using 125I and 103Pd seeds. The Amer
ican Brachytherapy Society recommends monotherapy47 for men with favora
le risk
prostate cancer. 125I and 103Pd permanent radioactive seeds are also known as Lo
w Dose Rate (LDR)
rachytherapy. High Dose Rate (HDR)
rachytherapy is
eing use
d to circumvent many pro
lems inherent with LDR, and it is also
eing used as mo
notherapy to treat favora
le risk prostate cancer.48,49 This development has
ee
n possi
le with computer and software advancements. HDR
rachytherapy uses high
intensity iridium-192 (192Ir) sources stored in a computer-controlled lead safe
called a remote afterloader. 192Ir emits gamma radiation of 400 keV which has fa
r more penetrating energy than 125I and 103Pd. Martinez et al48 descri
ed this t
echnique. After transperineal placement of needles into the prostate gland via u
ltrasound guidance, an intraoperative dosimetric treatment plan is developed
y
using an online optimization software program.49,50 A CT scan confirms needle po
sitions. The 192Ir sources are transferred to the needles
y the computer-contro
lled remote afterloader for a prescri
ed treatment time period (typically 1015 mi
nutes), delivering a precise dose depending on the dwell times. Since the source
s are retrieved after the prescri
ed treatment times, no radioactive sources rem
ain in the patients. In summary, their smart seed technique48 of conformal HDR pro
vides several
enefits over conventional LDR
ecause the treatment period is red
uced from weeks to minutes. In addition, the source position and dwell times are
more accurately controlled resulting in a
etter dose distri
ution. Also, realtime technique allows
etter source targeting, and the patients go home without

eing radioactive. Furthermore, the risk of seed migration is eliminated. Prelim
inary 3-year actuarial
iochemical disease free rates

Brachytherapy from the urologists perspective

109
Ta
le 9.2 Treatment strategies for unfavora
le risk or locally advanced prostate
CA
Brachytherapy with supplemental EBRT Dose escalating 3-D conformal radiotherapy
Conforms! HDR
rachytherapy
oost plus EBRT Neoadjuvant hormonal therapy plus EB
RT Neoadjuvant and adjuvant hormonal therapy plus conformal HDR
rachytherapy
were 94% for low risk features and 55% for high risk features (PSA>20 ng/mL, Gle
ason>7, or StageT3) as reported
y Yoshioka et al51 for conformal HDR
rachythera
py monotherapy. For patients with unfavora
le risk or locally advanced prostate
cancer, the optimal treatment modality remains unclear (Ta
le 9.2). The survival
rates with RP and low dose EBRT (monotherapy or adjuvant) are su
optimal.5254 Br
achytherapy has
een com
ined with EBRT in these patients. This strategy is empl
oyed to treat the periprostatic tissues also. There are no randomized studies co
mparing
rachytherapy monotherapy to EBRT with
rachytherapy
oost. But in multi
variate analysis, there were no statistical differences in treatment outcomes wh
en supplemental EBRT was added to
rachytherapy for this group of patients.44,55
,56 Treatment strategies now include dose escalating conformal radiotherapy and
neoadjuvant hormonal deprivation with EBRT. Dose escalating conformal radiothera
py is accomplished
y 3-D conformal radiotherapy,57 or
y conformal HDR
rachyth
erapy
oost plus EBRT.49,58 In theory, dose escalation is needed to achieve tumo
ricidal radiation doses in tumor cells radioresistent at conventional dose level
s. 3-D conformal radiotherapy developed with CT advancement in the early 1990s.
3-D imaging of the target gland was possi
le, allowing radiation
eams to confor
m to the shape of the target gland. This allowed more accurate delivery of radia
tion to the target gland with lower doses to the surrounding normal tissues. Pat
ients were, therefore, a
le to
e treated with higher doses safely. It appears t
hat doses greater than 75 Gy are needed for accepta
le tumor control,54,59 achie
ving a greater than 85% 5-year
iochemical control rate for prostate cancer with
an unfavora
le risk factor. The
oost method of dose escalation using conformal
HDR
rachytherapy is interdigitated at the end of weeks 1 and 3 of the 5-week r
adiotherapy course. Martinez et al58 have shown a 5-year actuarial
iochemical c
ontrol rate of 87% (high dose group receiving 92 Gy or greater) for patients wit
h Gleason 7 or greater, or PSA 10 ng/mL or greater. Mate et al49 showed similar
results of a
iochemical control rate of 84% at 5 years for those with pretreatm
ent PSA levels of greater than 10 ng/mL. The role of hormonal therapy in conjunc
tion with radiotherapy remains unclear. Possi
ly, hormonal a
lation and radiothe
rapy can produce a synergistic cytotoxic effect to the tumor cells for local con
trol, and hormonal a
lation can su
clinically control micrometastases outside of
the irradiated areas.60 Bolla et al61 showed in a randomized, prospective trial
that there is a survival advantage in those with locally advanced prostate canc
er treated with neoadjuvant hormonal therapy at the initiation of EBRT and conti
nued for 3 years. This is consistent with other study findings.62,63 Patients wi
th intermediate and high risk features who undergo
rachytherapy often receive h
ormonal
Basic and advanced techniques in prostate
rachytherapy
110
therapy as a result of extrapolation from the EBRT data,
ut there remains no de
finitive evidence of
enefit from hormonal manipulation for
rachytherapy.42,64
The Radiation Therapy Oncology Group (RTOG) has investigational trials to addres
s questions of duration and
enefits of hormonal therapy. RTOG 9408 was designed
to determine if hormonal a
lation played a role in low and intermediate risk pro
state cancers treated with EBRT with 4 months total of hormonal therapy. RTOG 991
0 compares 28 weeks to 8 weeks of neoadjuvant hormonal therapy followed
y EBRT

for intermediate risk prostate cancer. Based on the a
ove data, we routinely use
neoadjuvant and adjuvant hormonal therapy (1 versus 2 years) in com
ination wit
h conformal HDR for high volume, high grade disease as part of an in-house, rand
omized protocol at our institution. Side effects Acute and late complications fo
r permanent interstitial
rachytherapy have
een descri
ed (Ta
le 9.3). Some acu
te complications that can occur include perineal pain, ecchymosis and swelling.
Temporary urinary retention can occur at reported rates ranging from 2 to 34%.65
,66 This trou
lesome complication can
e su
stantially ameliorated with administ
ration of prophylactic alpha
lockers for up to 6 weeks.67 Late complications inc
lude urethral stricture disease, hemorrhagic cystitis, radiation proctitis, impo
tence, and urinary incontinence, especially in those who had prior TURP.9,68,69,
70,71 The 5-year actuarial risk of urethral stricture has
een reported to
e 5%
to 12%,11,72,73 likely correlating with the amount of urethral dosage received.
Some pro
lems were resolved with urethral dilation alone while others required
more invasive treatment. Seed migration to the pulmonary vasculature is approxim
ately 1022%,74
ut no detrimental events have
een reported in the literature. Th
e standard tool most commonly used to descri
ed complications from radiotherapy
is the RTOG mor
idity scoring scheme which consists of grades 0 to 4 to characte
rize acute and late gastrointestinal and genitourinary complications. For confor
mal HDR
rachytherapy the rates of RTOG grade 3 and 4 late gastrointestinal comp
lications were less than 5%, and 0% to 0.5%, respectively as reported
y Yoshiok
a et al51 and Martinez et al.58 Rectal complications were usually self-limiting
and consisted of mild proctitis with rectal
leeding peaking at a median of 8 mo
nths with an incidence of 4% to 11%.75,76 Rectal
leeding usually correlates wit
h rectal dose,
ut studies have failed to correlate prostate size with rectal co
mplications.75,76 The rates of grade 3 and 4 late genitourinary complications we
re 8% and 0%,58 respectively. There have
een wide ranging reports of erectile d
ysfunction after
rachytherapy,
ut generally a rate of 50% within 5 years of im
plantation can
e expected.77 Erectile dysfunction after radiotherapy tends to h
ave a gradual onset with loss of function that may not peak until 23 years after
treatment. This contrasts with RP where erectile dysfunction is immediate with g
radual return of function with time if nerve-sparing procedures have
een
Brachytherapy from the urologists perspective
111
Ta
le 9.3 Side effects of radiotherapy
Genitourinary Gastrointestinal Others
Urinary retention Constipation Secondary malignancies Urinary incontinence Fecal
urgency Ecchymosis Hemorrhagic cystitis Fecal incontinence Seed migration Ureth
ral stricture disease Hematochezia Perineal pain and swelling Urethral necrosis
and o
struction Fistulae Fistulae Erectile dysfunction
performed. Fortunately, erectile dysfunction after radiotherapy is often respons
ive to sildenafil citrate.78 Preimplantation erectile dysfunction is a strong pr
edictor of su
sequent pro
lems as shown
y Stock et al.79 At 6 years they found
that there was a 70% potency rate for men without pretreatment erectile dysfunct
ion versus only a 34% potency rate for men with su
optimal pretreatment erection
s. Similar findings were reported
y Merrick et al.80 In another study without p
retreatment erectile function assessment, the 5-year actuarial impotency rate re
ported with conformal HDR
rachytherapy
oost was 51%. Impotence occurred at a m
edian interval of 0.7 year, and no difference in impotence rates was found with
different
rachytherapy
oost doses.58 Comparative studies Several studies have
compared outcomes of RP to permanent interstitial
rachytherapy,11,81,82
ut the
se retrospective and inter-institutional studies had confounding varia
les inclu
ding pathologic interpretations of
oth
iopsies and prostatectomy specimens, cl
inically staging, pretreatment PSA, and level of expertise in delivering treatme
nt. It is dou
tful that the perfect randomized, prospective trial will ever
e p
erformed given the difficulty of finding patients who would
e willing to
e ran

domized and given the different definitions of treatment failure. Most recently
reported outcomes for favora
le risk prostate cancer treatment have
een similar
for RP, permanent interstitial
rachytherapy alone, and EBRT alone.83 However,
surveys continue to show that for men with moderately differentiated, clinically
localized prostate cancer and a life expectancy of 10 years or greater, urologi
sts heavily favor RP while radiation oncologists strongly prefer radiotherapy as
the primary treatment modality.84,85 Several works have shown that the life exp
ectancy of men with prostate cancer is directly related to their tumor grade.8689
From a urological perspective, most favora
le risk prostate cancer patients are
likely to have a long life expectancy. Surgical extirpation theoretically can e
liminate the risk of late cancer-related failure that may occur from either pers
istence or new occurrence of prostate cancer after radiotherapy. Furthermore, pa
thologic staging is o
tained with surgery and follow-up is more straight forward
as the PSA should
ecome undetecta
le and remain so if the patient is disease f
ree. One small
ut significant disadvantage to radiotherapy is the risk of radia
tion-induced secondary malignancies. Two large studies have descri
ed these even
ts in detail.90,91 The risk of any second solid tumor was approximately 6% overa
ll. This rate increases
Basic and advanced techniques in prostate
rachytherapy
112
proportionately to the num
er of years survived after initial treatment, especia
lly after 10 years of survival. Most frequent second solid organ malignancies we
re
ladder, lung and rectal carcinoma. Compared to patients treated surgically,
irradiated patients had an 85% increase in risk of developing sarcoma in the rad
iated field although they found no increase in the rate of leukemia. These facts
contri
ute to the
ias of urologists toward surgery for younger men with a grea
ter than 20 year life expectancy. Hence, younger men are more commonly treated w
ith RP and older men with radiotherapy. Contraindications There are many contrai
ndications to consider in selecting patients for
rachytherapy (Ta
le 9.4). Men
with prostate glands of 50 cm3 are often not candidates for
rachytherapy due to
the higher inherent risk of urinary mor
idity and technical difficulties with pu

ic arch interference. This pro
lem, however, can
e overcome using neoadjuvant
hormonal therapy for cytoreduction and
y using the realtime implantation method
.9294 Traditionally, prior TURP was a contraindication to
rachytherapy due to it
s inherent risks of urinary incontinence,
ut su
sequent studies have shown impr
oved rates of urinary incontinence in these men using peripheral loading techniq
ue with realtime seeding placement to avoid placing sources too close to the ure
thra.95,96 Still, at our institution we tend to avoid
rachytherapy for patients
who have undergone TURP. Prostatitis and significant o
structive symptomatology
are relative contraindications to
rachytherapy. These patients may fare
etter
with prostatectomy especially in voiding status. Voiding symptoms can
e quanti
fied
y the international prostate symptom score (IPSS)97 which allows comparati
ve studies of outcomes of different treatment modalities. Although there were no
pretreatment IPSS data, Krupski et al98 found that the radical retropu
ic prost
atectomy group had significantly lower posttreatment total and o
structive IPSS
symptom scores than the
rachytherapy monotherapy group. Furthermore, the irrita
tive and o
structive symptoms were much more pronounced in the
rachytherapy wit
h supplemental EBRT group than the RP group.
Ta
le 9.4 Relative contraindications for
rachytherapy
Prostate glands 50 cm3 Prior TURP Prostatitis Significant voiding symptoms Inflam
matory
owel disease
Men with inflammatory
owel disease may
e treated with
rachytherapy,
ut we pre
fer to avoid radiotherapy, whether
rachytherapy or EBRT, due to potential incre
ased
owel toxicity. However, RP is not contraindicated for these patients. O
es
e men can
e effectively treated for favora
le risk prostate cancer with permane

nt interstitial
rachytherapy.100 If an RP is planned, we prefer the perineal ap

proach over the retropu
ic approach.
Brachytherapy from the urologists perspective
113
Conclusions There have
een dramatic improvements in radiation delivery systems.
These advancements along with prostate cancer stage migration have led to a res
urgence of
rachytherapy
eing performed separately and together
y radiation on
cologists and urologists. As with all prostate cancer treatments, appropriate pa
tient selection will lead to the
est results and should
alance
oth disease fr
ee survival and quality of life issues. Selection should
e
ased upon age, over
all health, patient attitudes and concerns, disease stage and grade, and prostat
e size. Neoadjuvant and adjuvant androgen a
lation can
e used to help facilitat
e technical aspects of
rachytherapy and may improve longterm efficacy of treatm
ent. Hopefully, long-term randomized studies will help guide
oth radiation onco
logists and urologists in providing the most case appropriate care for patients
with prostate cancer. References
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15. 83. DAmico AV, Whittington R, Malkowicz SB, et al. Biochemical outcome after
radical prostatectomy, external
eam radiation therapy, or interstitial radiatio
n therapy for clinically localized prostate cancer. JAMA 1998; 280:969974. 84. Mo
ore MJ, OSullivan BO, Tannock IF. How expert physicians would wish to
e treated
if they had genitourinary cancer. J Clin Oncol 1988; 6:17361745. 85. Fowler FJ Jr
, McNaughton Collins M, Al
ertsen PC, et al. Comparison of recommendations
y ur
ologists and radiation oncologists for treatment of clinically localized prostat
e cancer. JAMA 2000; 283:32173222. 86. Johansson JE, Holm
erg L, Johansson S, et
al. Fifteen-year survival in prostate cancer. A prospective, population-
ased st
udy in Sweden. JAMA 1997; 277:467471. 87. Chodak GW, Thisted RA, Ger
er GS, et al
. Results of conservative management of clinically localized prostate cancer. N
Eng J Med 1994; 330:242248. 88. Aus G, Hugosson J, Norlen L. Long term survival a

nd mortality in prostate cancer treated with noncurative intent. J Urol 1995; 15

4:460465. 89. Lu-Yao GL, Yao SL. Population-
ased study of long-term survival in
patients with clinically localised prostate cancer. Lancet 1997; 349:906910. 90.
Brenner DJ, Curtis RE, Hall EJ, Ron E. Second malignancies in prostate carcinoma
patients after radiotherapy compared with surgery. Cancer 2000; 88:398406. 91. N
eugut AI, Ahsan H, Ro
inson E, Ennis RD. Bladder carcinoma and other second mali
gnancies after radiotherapy for prostate carcinoma. Cancer 1997; 79:16001604. 92.
Kucway R, Vicini F, Huang R, et al. Prostate volume reduction with androgen dep
rivation therapy
efore interstitial
rachytherapy. J Urol 2002; 167:24432447. 93
. Whittington R, Broderick A, Arger P, et al. The effect of androgen deprivation
on the early changes in prostate volume following transperineal ultrasound guid
ed interstitial therapy for localized carcinoma of the prostate. Int J Radiat On
col Biol Phys 1999; 44:11071110. 94. Stone NN, Stock RG. Prostate
rachytherapy i
n patients with prostate volumes >/=50 cm3: dosimetric analysis of implant quali
ty. Int J Radiat Oncol Biol Phys 2000; 46:11991204. 95. Wallner K, Lee H, Wasserm
an S, Dattoli M. Low risk of urinary incontinence following prostate
rachythera
py in patients with a prior transurethral resection of the prostate. Int J Radia
t Oncol Biol Phys 1997; 37:565569. 96. Stone NN, Ratnow ER, Stock RG. Prior trans
urethral resection does not increase mor
idity following real-time ultrasound gu
ided prostate seed implantation. Tech Urol 2000; 6:123127. 97. Barry MJ, Fowler F
J Jr, OLeary MP, et al, for the Measurement Committee of the American Urological
Association. The American Urological Association symptom index for
enign prosta
tic hyperplasia. J Urol 1992; 15491557. 98. Krupski T, Petroni GR, Bissonette EA,
Theodorescu D. Quality-oflife comparison of radical prostatectomy and interstit
ial
rachytherapy in the treatment of clinically localized prostate cancer. Urol
ogy 2000; 55:736742.
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118
99. Grann A, Wallner K. Prostate
rachytherapy in patients with imflammatory
ow
el disease. Int J Radiat Oncol Biol Phys 1998; 40:135138. 100. Merrick GS, Butler
WM, Wallner K, et al. Permanent prostate
rachytherapy-induced mor
idity in pat
ients with grade II and III o
esity. Urology 2002; 60:104108.
10 Sonographic anatomy of the prostate
Ethan J Halpern Introduction This chapter explores the anatomy of the prostate g
land with particular emphasis on those aspects of the sonographic appearance tha
t are relevant to the radiation oncologist. Sonographic anatomy is demonstrated
and correlated with the histologically defined zonal glandular anatomy. Common a
natomical variants are illustrated, as well as the pathologic
asis for the grow
th patterns of prostate cancer. Gross anatomy The normal prostate is a well-defi
ned, smoothly marginated gland situated
elow the
ladder and a
ove the symphysi
s pu
is. Although the prostate may
e visualized through a full urinary
ladder

y transa
dominal sonography, it is more clearly visualized with transrectal son
ography. The transrectal approach places the ultrasound transducer immediately a
djacent to the prostate and provides high resolution images with transmit freque
ncies in the range of 610 MHz. Sonographic measurements of the prostate should
e
o
tained in two orthogonal planes (Figure 10.1). As the urethra exits from the

ladder it courses caudally and anteriorly along the long axis of the prostate.
The urethra is visi
le on the sagittal midline imaging plane used for the anteri
or-posterior and cranio-caudal measurements. The cranial aspect of the prostate
just under the
ladder is denoted as the
ase of the prostate. The
ase of the p
rostate is clearly defined against the
ladder. The apex of the prostate is adja
cent to the muscles of the pelvic floor. The inferior margin of the prostate at
the apex may
e difficult to define sonographically from the adjacent periurethr
al tissues. The symphysis pu
is is usually visi
le anterior to the region of the
prostatic apex (Figure 10.1c). Mean dimensions of the young adult prostate are
3.3 cm in-height, 2.4 cm in thickness, and 4.1 cm in width,1 with a sonographic

volume of 12.937.1 cc.2 The seminal vesicles and vasa deferentia are paired struc
tures on either side of midline just a
ove the prostate (Figure 10.2). These str
uctures extend cranial to the prostate and posterior to the
ladder. Often,
oth
seminal vesicles may
e imaged together on a single axial image. However, in so
me cases the orientation of the seminal vesicle requires independent imaging of
each side. The two seminal vesicles should
e relatively similar in size, and ea
ch seminal vesicle should taper in cali
er as it approaches the midline, produci
ng a
eak sign.3 The typical size of a seminal vesicle is 2750 mm in length and 1215
mm in thickness.4,5 The vas deferens is generally visi
le just superior to the

ody of the ipsilateral seminal vesicle. The ampullary portion of the vas, adjac
ent to the
Basic and advanced techniques in prostate
rachytherapy
120
midline, is the largest and most easily visualized portion of the vas deferens.
The distal vas deferens courses medial to the seminal vesicle, and joins with th
e ipsilateral seminal vesicle to form an ejaculatory duct as it enters the
ase
of the prostate. The two ejaculatory ducts course through the
ase of the prosta
te to enter the urethra at the verumontanum. Although there is no true epithelia
l capsule around the prostate, a thin layer of fi
romuscular stroma (no more tha
n half a millimeter in thickness) surrounds the prostate, resulting in a smooth
glandular contour. The fascial planes surrounding the prostate are a caudal cont
inuation of the hypogastric sheath and contain many neurovascular structures. So
nographically, these fascial planes appear as welldefined echogenic tissue aroun
d the prostate. Vascular structures are often visi
le within these planes with c
olor Doppler imaging. The venous plexus of Santorini is contained within the fas
cia of Zukerkandl anterior to the prostate. The prostatic arteries are within th
e lateral pelvic fascia on either side of the prostate. The neurovascular
undle
s responsi
le for male potency usually lie
etween the prostate and the rectum,
along Denonvilliers fascia. Small perforating vessels and neural structures ente
r the
Figure 10.1 Sagittal (a) and transverse (
) transrectal images o
atined for meas
urement of the prostate. Transverse view (c) near the apex of the prostate. The
cranio-caudal and anterior-posterior dimension are measured on the sagittal imag
e. The
Sonographic anatomy of the prostate
121
transverse dimension is o
atined in the axial plane at the level that demonstrat
es the largest transverse diameter. Arrows mark the hypoechoic internal sphincte
r that surrounds the proximal prostatic urethra. A solid line parallels the uret
hra and demonstrates the anterior angulation at the level of the verumontanum. T
ransverse view near the apex of the prostate (c) demonstrates the pu
lic
one on
each side of the pu
lic symphysis (arrows). It may
e difficult to determine th
e presise smooth sphincter
lends with the tissues of the external sphincter nea
r the apex of the prostate.
Figure 10.2 Transrectal sonography of the seminal. Transverse midline view just
a
ove the prostate (a) demonstartes a seminal vesicle on each side with a normal
tapered appearance toward the midline. Arrows demonstrate the ampullary portion
of the vas deferens on either side of the
Basic and advanced techniques in prostate
rachytherapy
122

midline, just superior to the medial aspect of the seminal vesicle. Transverse v
iew at a slightly more cranial level (
) again demonstrates the vas deferens (ar
rows) medial to each seminal vesicle. In a different patient (c), the two semina
l vesicles could not
e imaged in a single plane. An angled transverse view of t
he right side is presented to demonstrate the right seminal vesicle and associat
ed vas deferns (arrow).
prostate from the neurovascular structures in the adjacent fascial planes, and r
epresent a potential pathway for spread of prostate cancer through the prostatic
capsule.6 Zonal anatomy of the prostate For the purposes of zonal anatomy, we c
onsider the young adult prostate prior to the onset of
enign prostatic hyperpla
sia. The anterior one third of this ideal prostate is composed of non-glandular
tissue: the anterior fi
romuscular stroma. This fi
romuscular stroma is stretche
d around the lateral and posterior margins of the gland as a thin capsule. The r
emaining two thirds of the prostatic parenchyma are glandular tissue. The zonal
anatomy of this tissue has
een descri
ed in detail
y McNeal.7,8 Zonal anatomy
of the prostate is defined with respect to the prostatic urethra. The verumontan
um is a raised ridge of tissue along the posterior aspect of the prostatic ureth
ra. The young adult urethra measures approximately 15 mm proximal to the verumon
tanum and 15 mm distal to the verumontanum. The urethra is angled forward
y a
o
ut 35 degrees at the verumontanum (Figure 10.1). The upper portion of the prosta
tic urethra is surrounded
y muscular fi
ers from the
ladder that form the inte
rnal sphincter. These fi
ers are often sonographically visi
le as a hypoechoic r
egion around the proximal prostatic urethra (Figure 10.1). The internal sphincte
r ends a
ove the verumontanum. A thin muscular layer called the distal smooth sp
hincter surrounds the urethra from the verumontanum to the prostatic apex, and m
erges with the external striated sphincter
elow the prostate. The zonal anatomy
defined
y McNeal divides the glandular tissue of the prostate into four zones:
Periurethral glands with limited glandular development empty into the urethra t
hrough lateral line ducts along the course of the prostatic urethra. The glands
of this periurethral zone are confined within the muscular tissue that surrounds
the urethra. This muscular tissue may limit glandular development in this zone.
The periurethral zone accounts for 1% of the glandular tissue in the prostate.
Transition zone tissue is composed of two lo
es of glandular tissue on either si
de of the urethra. The transition zone glands drain into the urethra at the veru
montanum, just
Sonographic anatomy of the prostate
123
a
ove the ejaculatory duct openings. In the a
sence of
enign prostatic hyperpla
sia, transition zone glands are found only a
ove the level of the verumontanum,
and the transition zone accounts for no more than 5% of the glandular tissue in
the prostate. Central zone glands surround the ejaculatory ducts as they course
toward the verumontanum. The central zone exists only a
ove the verumontanum, an
d ducts from this zone empty into the urethra at the verumontanum. The central z
one accounts for 25% of the glandular tissue in the prostate. Peripheral zone gl
ands surround the central zone at the
ase of the gland, and also form the
ulk
of the glandular parenchyma
elow the level of the verumontanum. These glands dr
ain into the urethra on either side of the verumontanum as well as
elow the ver
umontanum along the crista urethralisa posterior midline crest that extends along
the urethra. The peripheral zone accounts for 70% of the glandular tissue in th
e prostate. For sonographic purposes, the periurethral and transition zones comp
rise the inner gland, while the central and peripheral zones comprise the outer
gland. The inner gland and outer gland can
e distinguished from each other
y d
ifferences in their echotexture. The individual zones within the inner gland and
the individual zones within the outer gland are not sonographically distinguish
a
le. The normal outer gland is homogeneously echogenic, and is usually more ech
ogenic than the inner gland. The inner gland is often heterogeneous in its appea

rance due to the presence of
enign prostatic hyperplasia. The inner gland and t
he outer gland are separated
y the surgical capsule which defines the plane of
enucleation for suprapu
ic prostatectomy. The surgical capsule is not a true ana
tomic structure,
ut is often defined
y linear deposition of corpora amylacea o
r calcification that is sonographically visi
le. Benign prostatic hyperplasia Th
e classic zonal anatomy as defined
y McNeal is o
served only in the young adult
prostate. As a male ages, the glandular distri
ution changes due to
enign pros
tatic hyperplasia.9 Benign prostatic hyperplasia results in enlargement of the i
nner gland due to an increase in the num
er of glandular elements as well as hyp
ertrophy of the tissues.10 Benign prostatic hyperplasia is most marked in the tr
ansition zone,
ut is also noted in the periurethral zone. Since prostate cancer
is a disease of older men, most men with prostate cancer demonstrate some degre
e of
enign prostatic hyperplasia. Benign prostatic hyperplasia results in a nod
ular enlargement of the inner gland (Figure 10.3). Often the two lo
es of the tr
ansition zone are seen to enlarge on the two sides of midline. Enlargement of th
e transition zone stretches and thins the outer gland tissue, and may stretch th
e urethra and deviate it posteriorly. The enlarged transition zone often extends

elow the level of the verumontanum. When the periurethral glands are involved,
the resulting hyperplasia may result in an enlarged median
Basic and advanced techniques in prostate
rachytherapy
124
Figure 10.3 Transverse (a) and sagittal (
) views of the prostate in a patient w
ith
enign prostatic hyperplasia. The hypoechoic inner gland is surrounded
y th
e more echogenic outer gland. Inner gland enlargement is demonstrated on
oth si
des of midline on the transverse view (a). The urethra (not visi
le due to shado
wing in the midline) is compressed and stretched
etween the two lo
es of the tr
ansition zone. The sagittal view (
) demonstrates inner gland enlargement
ulgin
g into the
ase of the
ladder. Calipers mark the cranio-caudal and anterior-pos
terior dimensions.
lo
e. Although there is no true median lo
e, this term is used to refer to midlin
e enlargement of the prostate that
ulges into the
ase of the
ladder. Enlargem
ent of the prostate with
enign prostatic hyperplasia may result in a marked cha
nge in the overall shape and position of the prostate. The enlarged transition z
one may
ecome the dominant glandular component of the prostate. As the gland
e
comes more glo
ular in shape, it may extend anteriorly over the symphysis pu
is.
Among patients referred for
rachytherapy,
enign prostatic hyperplasia is an i
mportant cause of pu
lic arch interference to the introduction of seeds through
a perineal approach. In addition to the glandular enlargement associated With
e
nign prostatic hyperplasia, this process also results in a more heterogeneous ap
pearance of the gland. Hypoechoic hyperplastic nodules visi
le within the inner
gland may
e indistinguisha
le from hypoechoic masses of cancer. Due to the high
prevalence of
enign prostatic hyperplasia in middle aged to elderly males, it
is impossi
le to use grayscale sonographic criteria to detect cancer within the
inner gland.
Sonographic anatomy of the prostate
125
Prostatic calcifications are often related to the presence of
enign prostatic h
yperplasia. Calcifications are commonly seen along the surgical capsule as well
as in the periurethral tissues (Figure 10.4),
ut dystrophic calcification may

e present throughout the hyperplastic inner gland. Acoustical shadowing from the
se calcifications may complicate the visualization and placement of seeds for
r
achytherapy. Ductal ectasia of the seminal vesicles is commonly associated with

enign prostatic hyperplasia (Figure 10.5). The enlarged inner gland compresses
the ejaculatery ducts as they course to the verumontanum, resulting in relative

o
struction. The tu
ules within the seminal vesicles as well as the vasa deferen
tia may dilate secondary to this o
struction. In a patient with cancer of the pr
ostate, it is often impossi
le to determine whether o
struction of the seminal v
esicles is related to
enign prostatic hyperplasia or to the cancer. Cystic chan
ges are often found in areas of
enign hyperplasia (Figure 10.6). Although this
finding is most commonly found in the inner gland, cystic areas of hyperplasia m
ay
e seen within the outer gland as well. Both cystic change and calcification
of the glandular parenchyma may also
e related to prostatitis. In my experience
, cystic changes in the prostate are overwhelmingly associated with
enign disea
se. Finally,
enign prostatic hyperplasia is associated with a distortion of the
normal pattern of
lood flow within the prostate. The young adult prostate demo
nstrates capsular flow with perforating
ranches that radiate toward the urethra
at the center of the gland (Figure 10.7). Enlargement of the inner gland associ
ated with
enign prostatic hyperplasia distorts this radial pattern of flow, and
results in increased flow to the hyperplastic inner gland. Areas of increased D
oppler flow in the inner gland may mimic the increased flow associated with pros
tate cancer. Cysts of the prostate Cysts of the prostate are relatively common a
nd should
e recognized
y their characteristic location. Mllerian duct
Figure 10.4 Benign prostatic hyperplasia. Transverse view of the prostate (a) de
monstrates enlargement
Basic and advanced techniques in prostate
rachytherapy
126
of the inner gland with calcification along the left side of the surgical capsul
e (arrows). Transverse view in a second patient (
) demonstrates periurethral ca
lcification (arrow).
Figure 10.5 Ductal ectaisa of the seminal vesicles. A transverse midline image o
f the seminal vesicles (a) demonstrates mild
ilateral ductal ectasia. The dialt
ed ducts appear slightly hypoechoic to the remainder of the seminal vesicle. In
another patient with more o
vious ductal ectasia,
oth the right (
) and left (c
) seminal vesicles demonstrate o
vious fluidfilled tu
ular structures (arrows).
The ipsilateral vas deferens is visi
le a
ove each seminal vesicle.
cysts are the most common congenital cyst of the prostate. The Mllerian duct cyst
is a remnant of the Mllerian tu
ercle, and is commonly seen in the midline, near
the
ase of the prostate. Mllerian duct cysts may extend or arise a
ove the pros
tate. Ejaculatery duct cysts are common acquired cysts that arise along the cour
se of the ejaculatory ducts on either side of the midline,
ut may appear to lie
in the midline. Ejaculatory duct cysts are
Sonographic anatomy of the prostate
127
often associated with ejaculatory duct o
struction,11
ut Mllerian cysts may also
result in ejaculatory duct o
struction.12 Aspiration of an ejaculatory duct cys
t should demonstrate the presence of spermatozoa. Sperm is not present in a
Figure 10.6 Cystic changes associated with
enign hyperplasia. Transverse image
through the mid-gland (a) demonstrates a large area along the right peripheral z
one with multiple small cystic structures, a single cyst in the inner gland and
several tiny cysts in the left peripheral zone (arrows). Transverse image throug
h the apex in a different patient (
) demonstrates larger cystic spaces on
oth
side of the apex of the prostate (calipers). These may arise in the outer gland,
or may represent extension of inner gland material to the apex secondary to
en
ign prostatic hyperplasia.
Mllerian cyst. The distinction
etween these two entities is rarely of clinical i

mportance. Nonetheless, it is important to recognize these common cystic entitie

s. Other cysts of the prostate are less common. Cysts of the seminal vesicle are
much less common than ductal ectasia of the seminal vesicles (Figure 10.5). The
seminal vesicles, vas deferens, and ejaculatory duct are Wolffian structures. C
ongenital anomalies and true cysts of the seminal vesicle may
e associated with
other Wolffian anomalies.13 Cysts of the seminal vesicles are associated with i
psilateral renal agenesis in two thirds of cases.14 The prostatic utricle is a t
iny pouch along the verumontanum. Dilatation of the prostatic utricle to larger
than 4 mm with loss of epithelial papillations results in a utricular cyst.5 Utr
icular cysts communicate with the urethra and may result in post-void dri

ling.
15 Utricular cysts are associated with other anomalies of the genitourinary syst
em including hypospadias, incomplete testicular descent, and renal agenesis.16
Basic and advanced techniques in prostate
rachytherapy
128
Sonographic appearance of prostate cancer Adenocarcinoma of the prostate arises
most frequently in the outer gland.1719 It is generally accepted that cancer of t
he prostate appears hypoechoic on sonography. Unfortunately, only a
out half of
all cancers within the prostate are sonographically visi
le. This may
e related,
in part, to the growth pattern of adenocarcinoma of the prostate. Prostate canc
er infrequently presents as a solitary round mass. Rather, prostate cancer tends
to
e multifocal with infiltrative growth along a path that is parallel to the
capsule of the gland. The growth pattern tends to respect
oth the surgical caps
ule and the prostatic capsule as
arriers to spread. Although many cancers of th
e prostate are not visi
le, a recent study suggests that sonographically visi
le
tumor on grayscale imaging is more likely to
e hypervascular on Doppler imagin
g, and to have a significantly higher Gleason score.20 The classic sonographic a
ppearance of prostate cancer is that of a hypoechoic mass (Figure 10.8). The hyp
oechoic nature is
est appreciated on transverse images which allow a side to si
de comparison of the prostatic parenchyma. Other sonographic features include a
focal
ulge of the prostate contour, or an irregularity of the margin of the gla
nd. The thin fi
romuscular stroma surrounding the prostate is responsi
le for th
e smooth appearance of the margin of the gland. When this capsule is invaded
y
tumor, the smooth contour of the gland is lost and the margin may appear irregul
ar. The prostatic capsule invaginates around the ejaculatory ducts, and is incom
plete in the region of the prostatic apex. These two areas are relative weak poi
nts for extracapsular spread of prostate cancer.5 Spread of cancer to a seminal
vesicle may
e visualized as asymmetry of the seminal vesicles or as loss of the
normal tapered appearance of the medial aspect of a seminal vesicle. Color Dopp
ler evaluation of the normal prostate gland demonstrates the presence of
lood f
low in a symmetric radial pattern extending from the capsule toward the urethra.
21 Cancer may
e associated with focal areas of increased flow (Figure 10.9).2224
Recent studies suggest that power Doppler offers an extended dynamic range,25
Sonographic anatomy of the prostate
129
Figure 10.7 Transverse color (a) and power (
) Doppler images demonstrate normal
flow at the
ase to mid-gland of the prostate. Symmetric flow along
oth sides
of the gland is present with perforating vessels oriented toward the center of t
he prostate. In two patients with
enign prostatic hyperplasia (c and d) increas
ed flow is present within the hyperplastic inner gland. There is distortion of t
he normal radial flow pattern.
28
and may
e even more sensitive than color Doppler for the detection of prostate
cancer.26 Nonetheless, at this time, neither grayscale nor Doppler techniques are

sufficiently accurate to direct radiation therapy to limited portions of the gl

and. Posttreatment appearance of the prostate
After prostatectomy, the urethrovesical anastomosis should present a smooth, tap
ered appearance from the
ladder neck to the urethral sphincter (Figure 10.10).2
9 Tissue related to the prostate and seminal vesicles is removed during radical
prostatectomy.
Basic and advanced techniques in prostate
rachytherapy
130
Nonetheless, post prostatectomy scans often demonstrate portions of the seminal
vesicles that have not
een removed. The more superior and lateral portions of t
he seminal vesicles may not
e removed at surgery. After radiation therapy the p
rostate appears shrunken, and may
e difficult to visualize. Brachytherapy seeds
are visi
le as
right foci with acoustical shadowing. After external
eam thera
py, the gland may appear quite hypoechoic and heterogeneous. Recurrence of prost
ate tumor in the prostate
ed can present as a focal solid nodule or as a comple
x solid and cystic lesion.30 After radical prostatectomy, locally recurrent mass
es are found most frequently in the perianastomotic area,
ut can also
e found
in adjacent structures.31 Local recurrence after radiation therapy may
e very d
ifficult to identify within the shrunken, heterogeneous
Figure 10.8 Transverse sonography of the prostate at the mid-gland level. A foca
l hypoechoic area at the posterolateral aspect of the right peripheral zone (arr
ows) corresponds to a Gleason score 8 cancer growing along the capsule of the pr
ostate. There is a less well-defined hypoechoic appearance on the left side,
ut
all
iopsy specimens from this area returned
enign tissue.
prostate. The presence of recurrent disease can only
e confirmed
y
iopsy (Fig
ure 10.10). Conclusions The prostate gland surrounds the urethra as it courses o
ut of the
ladder. Anteriorly, the prostate is composed of fi
romuscular tissue
which is rarely involved
y disease
Sonographic anatomy of the prostate
131
processes. The glandular portion of the prostate is located laterally and poster
iorly. The glandular prostate is divided into four zones: the periurethral, tran
sition, central, and peripheral zones. Benign prostatic hyperplasia occurs exclu
sively in the inner gland, primarily in the transition zone,
ut also in the per
iurethral glands. Adenocarcinoma of the prostate arises most commonly in the per
ipheral zone or central zone, is often multifocal and grows parallel to the caps
ule of the gland. Both the surgical capsule and the prostatic capsule serve as r
elative
arriers to the spread of prostatic cancer. The seminal vesicles and vas
a deferentia are located a
ove the prostate. Due to local defects in
Figure 10.9 Gray sclae (a), color Doppler (
), and power Doppler (c) transverse
images through the midgland level of the prostate. A hypoechoic mass on the left
side of the glands is marked
y arrows (a). Both color and power Doppler studie
s demonstrate hypervascularity (arrows) that extends along the peripheral zone a
nterior to the mass identified
y grayscale sonography. Targeted
iopsy cores of
the left mid-gland
Basic and advanced techniques in prostate
rachytherapy
132
demonstrated that
oth the gray scale and Doppler findings corresponded to a Gle

ason score 7 cancer. Normal prostate tissue was identified on sextant
iopsy of
the right mid-gland.
Figure 10.10 Post-radical prostatectomy. The normal postprostatectomy appearance
(a) demonstrates a smooth taper at the vesicourethral junction. Although the an
astomosis was normal, a small focal nodule was found (
) and measured (calipers)
. Biopsy of this nodule (c) demonstrated recurrent adenocarcinoma.
the prostatic capsule, local spread of prostate cancer may invade directly into
the seminal vesicles or out the apex of the gland. An understanding of these ana
tomical considerations and their sonographic appearance is critical for adequate
treatment of adenocarcinoma of the prostate.
Sonographic anatomy of the prostate
133
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t al. Transrectal ultrasound of the seminal vesicles and ejaculatory ducts: clin
ical correlation. Radiology 1988; 168:626628. 12. Halpern EJ, Hirsch IH. Sonograp
hically guided transurethral laser incision of a Mllerian duct cyst for treatment
of ejaculatory duct o
struction. Am J Roentgenology 2000; 175:777778. 13. Carval
ho HA, Paiva JLB, Santos VHV, et al. Ultrasonic recognition of a cystic seminal
vesicle with ipsilateral renal agenesis. J Urol 1986; 135:12671268. 14. Heaney JA
, Pfister RC, Meares EM. Giant cyst of the seminal vesicle with renal agenesis.
Am J Roentgenology 1987; 149:139140. 15. Sha
sigh R, Lerner S, Fishman IJ, Kadmon
D. The role of ultrasonography in the diagnosis and management of prostatic and
seminal vesicle cysts. J Urol 1989; 141:12061209. 16. Nghiem HT, Kellman GM, San
d
erg SA, Craig BM. Cystic lesions of the prostate. Radiographics 1990; 10:635650
. 17. McNeal JE. Origin and development of carcinoma in the prostate. Cancer 196
9; 23:2433. 18. McNeal JE, Price HM, Redwine EA, et al. Stage A versus stage B ca
rcinoma of the prostate: Morphologic comparison and
iologic significance. J Uro
l 1988; 139:6165. 19. McNeal JE, Redwine EA, Freiha FS, Stamey TA. Zonal distri
u
tion of prostatic adenocarcinoma. Correlation with histologic pattern and direct
ion of spread. Am J Surg Pathol 1988; 12(12):897906. 20. Cornud F, Hamida K, Flam
T, et al. Endorectal color doppler sonography and endorectal MR imaging feature
s of nonpalpa
le prostate cancer: correlation with radical prostatectomy finding
s. Am J Roentgenol 2000; 175(4):11611168. 21. Neumaier CE, Martinoli C, Derchi LE
, et al. Normal prostate gland: examination with color Doppler US. Radiology 199
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erg M, et al. Prostatic cancer
: role of Doppler imaging in transrectal sonography. Am J Radiol 1998; 171:205210
. 23. Shigeno K, Igawa H, Shiina H, et al. The role of colour Doppler ultrasonog
raphy in detecting prostate cancer. BJU Int 2000; 86:229233.
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134
24. Cornud F, Hamida K, Flam T, et al. Endorectal color Doppler sonography and e
ndorectal MR imaging features of nonpalpa
le prostate cancer: correlation with r
adical prostatectomy findings. Am J Roentgenol 2000; 175:11611168. 25. Ru
in JM,
Bude RO, Carson PL, et al. Power Doppler US: A potentially useful alternative to
mean frequency-
ased color Doppler US. Radiology 1994; 190:853856. 26. Downey DB
, Fenster A. Three-dimensional power Doppler detection of prostatic cancer. Am J
Roentgenol 1995; 165:741. 27. Sakarya ME, Arslan H, Unal O, et al. The role of
power Doppler ultrasonography in the diagnosis of prostate cancer: a preliminary
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, Midi T. Transrectal power Doppler imaging in the detection of prostate cancer.
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ransrectal US in evaluation of patients after radical prostatectomy: I. Normal p
ostoperative anatomy. Radiology 1992; 185:361366. 30. Wasserman NF, Kapoor DA, Hi
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11 What to look for when choosing treatmentplanning software for prostate
rachy
therapy
Yan Yu Introduction Computerized treatment planning and dosimetry are an essenti
al component in modern radiation therapy, including
rachytherapy. With the wide
spread use of tomographic imaging such as computed tomography, magnetic resonanc
e imaging, and ultrasound, three-dimensional image-
ased, anatomy-specific treat
ment planning is now commonplace in external
eam radiotherapy. Continued techni
cal advancement such as intensity modulated radiation therapy (IMRT) and image-g
uided therapy are also
eginning to come into routine clinical use. At the same
time, significant advances in
rachytherapy planning and guidance have taken pla
ce, especially in the treatment of prostate cancer. The transrectal ultrasound (
TRUS) imaging format lends itself naturally to 3D anatomy-
ased planning and dos
imetry. Interstitial seed placement under TRUS guidance affords remarka
le flexi

ility in terms of customizing the dose distri
ution to target the known disease
and to spare critical structures in real time. Indeed, it may
e argued that mo
dern prostate
rachytherapy is so far the most comprehensive image-guided therap
y with dose intensity modulation. Continued evolution of treatment planning soft
ware plays an important role in the growth of this field. The choice of treatmen
t planning software functionalities can directly influence the practice style of
the users
rachytherapy program. For example, the American Brachytherapy Society
(ABS) categorized prostate
rachytherapy planning into three distinctive levels
(Ta
le 11.1), ranging from idealized planning to dynamic dosimetry.1 The functi
onal requirements of the corresponding planning software increase in complexity.
It is important that the
rachytherapy team is familiar with the capa
ilities a
nd limitations of the planning system selected. The requisites Image acquisition
Ultrasound equipment in common use for prostate
rachytherapy usually has one o
r more video output formats, including real-time, live video images. The most di
rect method of image acquisition and import to the
Basic and advanced techniques in prostate
rachytherapy
136
Ta
le 11.1 American Brachytherapy Society (ABS) terminology for elements of intr
aoperative planning
Level Terminology
0 1 2 3 Preplanning Intraoperative preplanning Interactive planning Dynamic dose
calculation

Definition
None Creation of a plan in the operating room just
efore the implant procedure
with immediate execution of the plan Stepwise refinement of the treatment plan u
sing dose calculations derived from image-
ased needle position feed
ack Constan
t updating of dose calculations of implanted sources using continuous seed posit
ion feed
ack
treatment planning system is via frame-gra

ing, a method to convert video frames
into computer image formats, such as
itmap or JPG. Either an internal video cap
ture/framegra

er card or an external USB (sometimes fire wire) connection is need
ed on the treatment planning computer. If the prostate volume study is performed
at equal slice intervals, it suffices to
e a
le to define the starting z-posit
ion, the z-increment, or decrement after each image capture, and the direction o
f the scan (
ase to apex or vice versa). Note that if x increases from patient r
ight to patient left, y increases from posterior to anterior, then z needs to in
crease from
ase to apex to ensure a right-handed coordinate system. If intraope
rative treatment planning and dosimetry is not anticipated
y the
rachytherapy
team, then only still image import is required. Bitmap or JPG image formats can

e conveniently transferred and manipulated across computer platforms, although
DICOM image transfer is increasingly availa
le, such as from ultrasound PACS sys
tems. In the case of DICOM transfer, care should
e taken to verify that the z (
or
aseapex) positions are correctly identified to the treatment planning system
. Image segmentation Image segmentation is often referred to as contouring. In p
rostate
rachytherapy, the anatomical structures that need to
e contoured are t
he prostate, rectum, and urethra. Other structures of potential relevance includ
e the
ladder, pu
ic arch, penile
ul
, neurovascular
undles, and tumor foci. A
t a minimum, the treatment-planning software should permit the user to interacti
vely draw each selected contour over the grayscale image. When using TRUS images
, it is necessary to esta
lish the on-screen grid locations so as to correctly r
egister the segmented versus the actual anatomy. This is usually accomplished
y
identifying two known template holes on the TRUS image. It is also useful to al
low fine adjustment in positioning the software template grid, which may
e used
to account for any systematic shift of, for example, the sta
ilizing needles ve
rsus the ultrasound grid. Realigning the software grid is analogous to shifting
the
rachytherapy template; it invalidates any dosimetry plan previously generat
ed.
What to look for when choosing treatment
137
TG-43 formalism As the National Institute of Standards and Technology (NIST) sta
ndardizes the cali
ration of each type of clinical
rachytherapy sources, the se
ed manufacturers will need to specify their activity range in terms of the air k
erma strength according to the American Association of Physicists in Medicine (A
APM) Task Group 43 (TG-43) formalism.2 The traditional unit of activity in mCi i
s replaced
y the air kerma strength in units of Gy/h m2, or U. A simple method of
estimating dose at distance r away from a seed is then: 1.44(half-life)(air kerma
strength) (dose rate constant)(radial dose function) (anisotropy constant)/r2, usin
g consistent units of time. The dose rate constant, radial dose function, and an
isotropy constant are specific to each design of the radioactive seed, and are c
ommonly determined
y two independent dosimetry studies (measurement and/or calc
ulation). The treatment-planning system must therefore allow definition of diffe
rent seed designs and user input/modification of the dosimetry parameters. Conve
rsion factors
etween the air kerma strength and mCi activity of different seeds
can
e found in text
ooks or calculated from the dose rate constant; however, t
he reader is cautioned that such conversion factors may
e su
ject to change as
the cali
ration standard or the dose rate constant is revised. Isodose and dosevolume histogram Isodose display as color overlay over grayscale images is a sta

ndard feature of most treatment-planning systems. A useful additional functional

ity is numerical display of the dose at any location pointed to
y the cursor on
the image. The user is therefore a
le to quickly determine the dose delivered

y the dosimetric plan to any anatomical structure of concern. The user interface
should also allow rapid paging through the different image planes (e.g.
y provid
ing thum
nail panels that may
e
rowsed/ magnified). The dose-volume histogram
(DVH) is a standard method for evaluating the volumetric irradiation of a given
anatomical structure previously segmented. The cumulative volume (either in cm3
or as percent of total volume) of the anatomical structure receiving greater tha
n or equal to a given dose (either in Gy or as percent of the prescri
ed dose) i
s plotted versus the dose. In prostate
rachytherapy, certain parametric evaluat
ors of implant quality have evolved into routine clinical use. For example, the
AAPM,3 as well as the ABS,4 recommended the use of the following parameters: 1.
The values of D100, D90, and D80 (the dose that covers 100%, 90%, and 80% of the
prostate, respectively). 2. The values of V200, V150, V100, V90, and V80 (the f
ractional volume of the prostate that receives 200%, 150%, 100%, 90%, and 80% of
the prescri
ed dose, respectively). These DVH values should
e conveniently o
t
aina
le
y ta
le look-up or interpolation from the treatmentplanning output.
Basic and advanced techniques in prostate
rachytherapy
138
Calculation of the DVH essentially involves sampling the dose at points distri
u
ted within the anatomical structure. Treatment-planning software generally uses
one of two methods for selecting these pointsrandom sampling or uniform sampling.
In either method, sufficient points must
e calculated to generate relia
le DVH
parameters, which need to
e
alanced with the speed of calculation. The dose-s
urface histogram (DSH) is similar to the DVH in concept except that the dose is
calculated
y sampling points distri
uted on a surface instead of within a volum
e. The DSH is a more suita
le measure of dose to the anterior rectal wall
ecaus
e it should
e less dependent on the rectal content at the time of imaging and l
ess distorted
y the much lower dose to the posterior rectum. The DSH is also co
nceptually more suited to the urethra, where Volume may
e considered meaningless
. Postimplant computed tomography dosimetry The current technique for evaluating
individual implants as recommended
y the ABS is computed tomography (CT)-
ased
postimplant dosimetry.4 For this purpose, the treatment planning system should

e a
le to import DICOM image sets, display CT images with window/ level control
, and facilitate contouring of the anatomy and identification of seeds. A partic
ularly useful feature of the software is automatic identification of seeds on CT
. This is readily achieva
le using standard imageprocessing techniques; however,

ecause a single seed may
e imaged on two or more adjacent CT slices, an algor
ithm must
e provided to eliminate redundant identification. A simple algorithm
is to let the user specify the num
er of resulting seeds desired, and then to co
m
ine the nearest neigh
ors iteratively until the desired seed count results. Th
e one shortcoming of this method is the assumption of a specified num
er of seed
s: the user must independently determine the num
er of seeds in the patient (e.g
. using plane films), which may
e different from that implanted due to migratio
n and seed loss. More sophisticated algorithms involve image analysis in two and
three dimensions (in-plane and adjacent plane thresholding and region growth) u
sing the expected size of the seed as prior knowledge to determine the actual nu
m
er of seeds present in the volume of interest. It is also possi
le to determin
e the orientation of some of the seeds using such an algorithm, although the eff
icacy of incorporating anisotropic dose distri
ution around seeds has not
een e
sta
lished in prostate
rachytherapy. Advanced tools Auto-segmentation Automatic
contouring of the prostate and rectum on TRUS is a very useful tool in prostate

rachytherapy planning, particularly intraoperative planning. Traditionally, th
e prostate is outlined on the planning computer
efore the preplan is generated,
and again outlined on the TRUS screen in the operating room after patient posit
ioning. There is therefore significant timesaving if the autosegmentation tool p

roduces consistent and accurate results. In one method,5 a generic 3D model of t

he prostate is initially incorporated into the algorithm
y prior training
ased
on experts manual segmentation
What to look for when choosing treatment
139
of a variety of TRUS series. For a given TRUS series to
e segmented, the unique
properties of the prostate morphology are first determined
y image processing
and used to individualize the 3D model. Using a technique called principal compon
ent analysis, a
est fit
etween
oundary points found
y image processing and kn
own prostate morphologies is solved
y the algorithm, which then translates into
prostate contours on each TRUS slice. The entire process takes approximately 1
minute on a computer. Inverse planning In inverse planning, the user of the trea
tment planning system specifies the planning goals and constraints, such as the
prescri
ed dose to the target volume, dose limits to organs at risk, dose unifor
mity, etc., and the planning software automatically generates solutions (i.e. do
simetric plans, that completely or partially satisfy the goals and constraints).
In external
eam radiation therapy, inverse planning is increasingly
eing reli
ed upon to produce optimized conformal and intensity modulated radiotherapy (IMR
T) plans. In prostate
rachytherapy, inverse planning is so far primarily used i
n computer-aided intraoperative planning with optimization.68 The a
ility to rapi
dly (within 1 minute) produce a host of dose plans, each selected for optimizing
aspects of the treatment intent (goals and constraints), is an attractive featu
re in a planning software. If the optimization algorithm em
edded in the inverse
planning engine is multi-o
jective, it is then not uncommon to include dose esc
alation to previously identified tumor foci (e.g. from magnetic resonance spectr
oscopy, sonoelastography, or
iopsy results), mapped on to intraoperative TRUS u
sing image registration or
y reference to anatomical landmarks. Pu
ic arch inte
rference may
e avoided
y
locking out the availa
le optimization space anterio
r to the pu
ic arch projection. A novel capa
ility of inverse planning in
rachy
therapy is its a
ility to incorporate sensitivity analysis on the dosimetric pla
ns when the radioactive sources are su
ject to random displacements simulating w
hat is typically found in actual implants.9 As expected, the extent of tolerance
to seed misplacement is not equal among different dosimetric plans, and should
depend on the locations of the seeds relative to areas of likely underdosage. A
delicately planned seed distri
ution will tend to suffer greater underdosage in
the hands of inexperienced practitioners compared to aro
ust plan taking into acco
unt potential placement uncertainties. The sensitivity analysis functionality si
mulates this effect
y assuming a Gaussian distri
ution of displacements in the
seed distri
ution using a standard error reflective of the practitioners experien
ce. Needle tracking Whereas the preplan relies on accurate reproduction of the p
rostate geometry
etween the preplanning and the intraoperative volume studies,
intraoperative preplanning allows any interval volume changes as well as changes
in the relationship
etween prostate, rectum, and urethra to
e accounted for.
In
oth techniques, however, the dosimetry reflects an idealized (i.e. perfect)
needle and seed placement in their planned locations. The technique of needle tr
acking was designed to provide realistic (rather than idealistic) dosimetry eval
uation in real time
y accounting for needle splaying (i.e. deviation from the p
lanned grid location, as o
served on TRUS as the needle is
eing placed). The ac
tual
Basic and advanced techniques in prostate
rachytherapy
140
hyperechoic spot of the needle is identified
y the user in one mouse click on the
planning computers screen that displays the live ultrasound in its transverse vi
ew. If one assumes that the needle deviation is zero at the mid-thickness of the

template, then a straight line can
e drawn from that point to the needle tip a
t known depth in the prostate. Then for each z-location corresponding to an inte
nded seed deposition, a set of x and y for the leftright and anterior-posterior de
viations can
e calculated
y linear interpolation, which is different for each
seed (greater deviations towards the
ase). The dose distri
ution is immediately
updated to show any changes in target coverage and/or critical structure sparin
g. All of the a
ove steps may
e completed even
efore the seeds are deposited i
nto tissue, therefore this tool can
e used to help the clinicians decide if it
is necessary to reposition the needle
efore committing to the seed placement. I
ntraoperative seed detection Although the needle tracking technique turns an ide
alized dosimetry plan to a realistic dose distri
ution reflective of the actual
needle positions, it is not meant to replace postimplant dosimetry
ecause the a
ctual seed positions can
e pertur
ed
y the withdrawal of the needle or tissue
elasticity. Intraoperative seed detection will overcome this limitation. A set o
f closely spaced 2D TRUS scans of the prostate containing implanted seeds is acq
uired, resulting in a 3D volume ultrasound dataset. Electromagnetic (EM), optica
l, or mechanical sensors can
e used to map the 2D ultrasound scan planes into t
he 3D ultrasound volume. Among these, EM positional sensors are the most commonl
y used method in radiological 3D ultrasound. Its accuracy for intraoperative
ra
chytherapy was first esta
lished
y Watana
e and Anderson.10 A small EM emitter
is attached to the handle of the ultrasound pro
e, and an EM receiver is positio
ned 50100 cm away in a stationary location. Linear and rotational movement of the
EM emitter relative to the receiver is tracked in real-time and translated into
pro
e motion, which is registered with the corresponding image capture. On the
treatmentplanning system, image-processing techniques involving statistical and
texture analysis as well as neural networks are then applied to the image set to
distinguish actual seeds from such artifacts as air gaps,
leeding, and calcifi
cation.11 Dosimetry is recalculated
ased on the actual seed positions detected
from postimplant TRUS, which also displays the prostate anatomy. These procedure
s may
e completed in 12 minutes from the 3D ultrasound scan to dose recalculatio
n. It is therefore possi
le to perform intraoperative seed detection not only at
the end of the implant when all the planned seeds have
een deposited,
ut also
incrementally after a group of needles is delivered. In the latter case, opport
unity still exists to compensate for any imperfections in previous seed placemen
t
y re-optimization of the remaining plan or additional seed insertion. This me
thod of intraoperative postimplant dosimetry directly overcomes the dilemma in C
T-
ased postimplant dosimetry where the patient usually cannot
enefit from the
knowledge gained from the dose assessment.
What to look for when choosing treatment
141
Summary The field of prostate
rachytherapy planning is now sufficiently mature
that the requisite functionalities are widely availa
le in any software system s
pecifically designed for this purpose. Advanced features, such as the tools disc
ussed in the previous section, are designed to present a great variety of real-t
ime data to the
rachytherapist during the procedure for making more informed de
cisions regarding dosimetry. Although the practice style of the
rachytherapy te
am can
e significantly altered
y treatmentplanning technology, the ultimate go
al is to allow increased dosimetric knowledge to directly
enefit the patient un
der treatment. This is the driving force in the continued evolution of treatment
-planning software for prostate
rachytherapy. References
1. Nag S, Ciezki JP, Cormack R, et al. Intraoperative planning and evaluation of
permanent prostate
rachytherapy: Report of the American Brachytherapy Society.
Int J Radiat Oncol Biol Phys 2001; 51:14221430. 2. Nath R, Anderson LL, Luxton G
, et al. Dosimetry of interstitial
rachytherapy sources: Recommendations of the
AAPM Radiation Committee Task Group No. 43. Med Phys 1995; 22:209234. 3. Yu Y, A
nderson LL, Li Z, et al. Permanent prostate seed implant
rachytherapy: Report o
f the American Association of Physicists in Medicine Task Group No. 64. Med Phys

1999; 26:2054 2076. 4. Nag S, Bice W, DeWyngaert K, et al. The American Brachyth
erapy Society recommendations for permanent prostate
rachytherapy postimplant d
osimetric analysis. Int J Radiat Oncol Biol Phys 2000; 46:221230. 5. Liu H, Cheng
G, Ru
ens D, et al. Automatic segmentation of prostate
oundaries in transrecta
l ultrasound (TRUS) imaging. Proc SPIE 2002; 4684:412423. 6. Yu Y, Zhang JB-Y, Br
asacchio RA, et al. Automated treatment planning engine for prostate seed implan
t
rachytherapy. Int J Radiat Oncol Biol Phys 1998; 43:647652. 7. Messing E.M, Zh
ang JB-Y, Ru
ens DJ, et al. Intraoperative optimized inverse planning for prosta
te
rachytherapy: Early experience. Int J Radiat Oncol Biol Phys 1999; 44:801808.
8. Zelefsky MJ, Yamada Y, Cohen G, et al. Postimplantation dosimetric analysis
of permanent transperineal prostate implantation: improved dose distri
utions wi
th an intraoperative computer-optimized conformal planning technique. Int J Radi
at Oncol Biol Phys 2000; 48:601 608. 9. Yu Y. Multio
jective decision theory for
computational optimization in radiation therapy. Med Phys 1997; 24:14451454. 10.
Watana
e Y, Anderson LL. A system for nonradiographic source localization and re
al-time planning of intraoperative high dose rate
rachytherapy. Med Phys 1997;
24:20142023. 11. Cheng G, Liu H, Liao L, Yu Y. Dynamic
rachytherapy of the prost
ate under active image guidance In: WJ Niessen, MA Viergeva (eds) Medical Image
Computing and ComputerAssisted InterventionMICCAI 2001, 4th International Confere
nce, Utrecht, The Netherlands, Octo
er 1417, 2001, Proceedings. Lecture notes in
Computer Science 2208. Berlin, Springer, 2001.
12 Treatment planning for low and high dose rate
rachytherapy
Marco Zaider and Eva K Lee Introduction Treatment planning in
rachytherapy cons
ists of a sequence of steps that include the following: Selection of appropriate
sources Localization of potential source positions Dose prescription Treatment plan
design and verification. In this chapter we shall discuss these topics as appli
ed to the
rachytherapy of prostate cancer. Although the methodology descri
ed

elow reflects (unavoida
ly) accepted practice at Memorial Sloan-Kettering Cancer
Center (MSKCC), the emphasis is on those aspects thatat least in our viewshould d
efine standard of care in prostate
rachytherapy. Currently, for instance, a maj
ority of prostate implants appears to
e performed using preplanned source distr
i
utions, while here we strongly advocate the use of intraoperative computer-opt
imized planninga technique used routinely at MSKCC and at several other instituti
ons. Selection of appropriate sources In
rachytherapy, radioactive isotopes are
selected
ased on two criteria: (1) the energy of the ionizing particle; and (2
) the decay rate of the radionuclide. Low energy sources are preferred
ecause o
f their evident advantage in terms of radiation protection (see Ta
le 12.1). The
y also offer
etter flexi
ility in designing conformal plans, as well as avoidin
g excess irradiation of healthy tissues that surround the target. The main
enef
it of high energy sources is that (dosimetrically) they cover a larger volume an
d thus fewer sources may
e needed. The decay rate of the radionuclide may have
radio
iological implications
ecause it determines the initial dose rate, shortlived radioactive sources having, for the same total dose, a larger initial dose
rate. Indeed, for a temporary implant of duration t, the total dose delivered,
D(t), is given
y: (1)
Treatment planning for low and high dose
143
Ta
le 12.1 Physical characteristics of radioactive sources currently used in
ra
chytherapy
Isotope E/MeV E(max)/Mev T1/2
192
Air kerma rate coastant/cGy/h cm2/mCi cm2/mCi
HVL (P
)/cm

74.2 4.11 0.3 days 125 I 0.028 60.2 1.32 0.002 days 103 Pd 0.021 17 days 1.296 0
.002 HVL represents half-value layer and indicates the thickness of material tha
t will reduce the fluence of unchared particle to half.
Ir
0.380
0.67
where is the decay constant and T1/2 (=0.693/) is the haf-ife of the isotope. I
n particuar, for a permanent impant: (2) It goes without saying that ong-ive
d radionucides are not appropriate for permanent impants. Another important is
sue in the context of source seection is the reative bioogica effectiveness
(RBE) of the radiation fied. Low energy sources tend to have higher RBE vaues.
1,2 For instance, the RBE of iodine-125 (125I) has been extensivey studied and
resuts of 1.22 have been reported for the dose rate range of 0.039 Gy/h.3 Simiar
y, for paadium-103 (103Pd) a study performed at 0.070.8 Gy/h reported RBE vau
es of 1.90.7.36 Tabe 12.1 ists the main physica characteristics of radiation so
urces used in prostate brachytherapy: iridium-192 (192Ir) is used for high dose
rate (HDR) treatments, and 125I and 103Pd are used for ow dose rate (LDR) perma
nent impants. The air kerma rate constant (), relates the activity, A, of a raio
nuclie emitting photons to its kerma rate. Specifically, is the quotient of by A,
where is the air kerma rate ue to photons of energy greater than , at a istanc
e 1 cm in vacuo from a point source of this nuclie:7 (3) is measure in units of
m2y s1 Bq1. Localization of potential source positions In permanent prostate impl
ants potential source positions are localize with respect to a template that is
place in a fixe position relative to the treatment region (the prostate glan
). The template, shown in Figure 12.1, has a rectangular pattern of holes; neel
es are inserte through the template gri an sees are place along each neele
at positions (typically, in multiples of 0.5 cm) etermine by the treatment pl
an. A series of parallel
Basic an avance techniques in prostate brachytherapy
144
ultrasoun (US) images is taken through the prostate an firmware in the ultraso
un unit overlays a gri of ots on to these images that correspon to the templ
ate holes (Figure 12.2). The gri coorinates on the template an the istance o
f the US image away from the template uniquely ientify the 3D coorinates of ea
ch potential see position relative to the glan anatomy. It is often the case t
hat, as they penetrate into the prostate, inserte neeles eviate from the init
ial
Figure 12.1 Template use in a prostate permanent implant.
Figure 12.2 The gri superimpose on the ultrasoun image provies the (x, y) co
orinates of the inserte neeles. Potential see positions (x, y, z) are along
these neeles. The thir
Treatment planning for low an high ose
145
coorinate (z) is etermine by the position of the ultrasoun probe (equivalent
ly, by the image number).
gri coorinates. However moern planning systems have provisions for taking thi
s into account in osimetric calculations. High ose rate (HDR) prostate treatme
nt (aministere at MSKCC as a boost before external raiation therapy) is perfo

rme with remote afterloaer machines.

Figure 12.3 Template an flexiguie tubes use in high ose rate (HDR) prostate
treatment.
A template, similar to that use for permanent low ose rate (LDR) implants, is
employe to place flexiguie tubes into the prostate (Figure 12.3). Potential st
opping positions for the 192Ir source are obtaine via compute tomography (CT)
of the prostate with the sourceguiing neeles inserte in it. A ifficulty in
this approach is the fact that the catheters relative positions insie the target
are not the same as the original perineal gri
Basic an avance techniques in prostate brachytherapy
146
coorinates an thus their coorinates may vary from one slice to the next (Figu
re 12.4). In our treatmentplanning system, catheters are igitize on each CT s
lice (Figure 12.5), their trajectory fitte to a thir egree polynomial (spline
fitting) an then equiistant stopping positions are calculate analytically an
 entere in the planning software (Figure 12.6).
Figure 12.4 Scout image of a high ose rate (HDR) prostate implant.
Figure 12.5 In each compute tomography (CT) slice the location of catheters is
igitize (green or yellow circles). The trajectory of each catheter is then rec
onstructe an stopping positions, 0.5 cm apart, are calculate an store in th
e planning software.
Treatment planning for low an high ose
147
Dose prescription In prostate brachytherapy, the ose prescription is mae relat
ive to the clinical target. Specifically, the recommene proceure is to use th
e minimum peripheral ose (mPD), which is the largest ose isoose surface that
completely surrouns the target (Figure 12.7).8 Often, this is foun to be too r
estrictive (an perhaps unrealistic in terms of being able to implement the plan
) an a ifferent type of prescription makes use of D90, which means that one st
ipulates that a ose equal to or larger than the
Figure 12.6 Reconstructe catheter geometry an stopping positions.
Figure 12.7 Dose prescription for a permanent prostate implant. In this 2D ultra
soun image the white line elineates the prostate an the 100%
Basic an avance techniques in prostate brachytherapy
148
isoose line (mPD=144 y) is shown in green. reen ots inicate sees an re 
ots show unuse see locations along neeles.
prescription ose (144 y for permanent implants, 56 y/fraction for HDR treatmen
ts) be elivere to at least 90% of the target volume. The etermination that th
e treatment plan achieves the require mPD or D90 can be mae with the ai of o
sevolume histograms (DVH), which plot as a function of ose, D, the probability
that a ranomly selecte voxel volume receives a ose of at least D (this is, o
f course, the cumulative probability istribution of ose in the target volume).
An example is given in Figure 12.8, which shows the DVH as planne for an HDR t
reatment of the prostate. In this case the prescription ose, 5.5 y, covers 96%
of the target.

Figure 12.8 Dosevolume histogram (DVH) for a HDR treatment plan (prescription 
ose: 5.5 y).
Treatment plan esign an verification In brachytherapy, planning means fining
a pattern of sources (of given strength) that is consistent with osimetric cons
traintstypically, a minimum ose for the target an a maximum ose for the health
y tissues ajacent to the target. The search for this optimal source istributio
n may be performe using iterative (trial an error) methoology oras escribe h
ereusing computerbase optimization. For the latter, a mathematical moel is usu
ally evelope that inclues the essential osimetric constraints, an an object
ive function (often userspecific). The objective function is a mathematical exp
ression that measures the quality of the ose istribution. This metric can be s
electe accoring to the esire of the planner in the characteristics of the res
ulting plan. The moel is then solve
Treatment planning for low an high ose
149
by some algorithms. Search algorithms use in brachytherapy treatment planning i
nclue exact algorithm, such as branchanboun, or heuristic approaches as in
simulate annealing,9 an genetic algorithm. Branchanboun algorithm is a tree
search approach that works by searching through the set of all feasible plans (
those which satisfy all the input constraints in the moel) an returns an optim
al plan that provies the best objective value. When allowe to run to completio
n, this approach will return a provenoptimal plan. Heuristics proceures work so
mewhat ifferently. Depening on the esign, the search oes not necessarily ret
urn plans that satisfy all the impose constraints, rather, the search attempts
to obtain see patterns which provie the least violation to the impose constra
ints. Furthermore, there is no information on whether one obtains an optimal see
 configuration or not. Instea, the termination of heuristics algorithms is bas
e on the number of iterations that the users input. At each iteration the algor
ithm obtains a plan an will evaluate its associate objective function value. I
f the objective function is better than the incumbent plan, it will be upate.
Treatmentplanning moels The planning problem consists of etermining if each p
ossible source location shoul be implante with a raioactive source or not. He
nce, the ecision variables are the location of the gri position. Mathematicall
y, this can be moele as an integer programa mathematical moel that consists of
integer ecision variables.1012 In our case, it is the yes or no ecision of placing
a see or not at each possible location. Mathematically, let xj be a 0/1 ecisi
on variable for recoring the placement of a source at gri position, j. The tot
al ose, D(P), at point P is given by: (4) where n is the number of potential so
urce positions, R. is a vector that gives the coorinates of gri position j, ||
PRj|| is the Eucliian istance between P an R, an D(r) is the ose contributio
n to P from a source at istance r away (within the point source approximation).
For each point of interest one can efine upper (UP) an lower (LP) ose limits
that D(P) must satisfy: (5)

enerally, it is not possible to have all points P satisfy these constraints, in

which case there will be no feasible solution [xj] to this problem. Instea, on
e attempts to maximize the number of points that satisfy these inequalities. Thi
s is achieve in the following manner. Let UP+MP enote the absolute maximum acc
eptable ose at point P, an similarly LPNP for the absolute minimum ose. Furthe
r, let, WP, VP be binary (0/1) variables that inicate whether Eqs (5) are satis
fie (when equal to 1) or not (when equal to 0). With this, the constraints, Eqs
(5), become:
Basic an avance techniques in prostate brachytherapy
150

(6)
an the sum: (7) which epens on the configuration [xj], gives the total number
of points P that satisfy the original constraints, Eqs(5). The optimization pro
blem consists of maximizing the objective function, F. All points P nee not hav
e the same clinical importance; for instance, avoiing urethral toxicity (a comm
on sie effect) may be more important than satisfying the conition of ose unif
ormity across the target. This is aresse by assigning ifferent weights, P nd
P, to vP nd wP, respectively, nd m ximizing inste d: (8) The pro
lem descri
ed

y the expression, Eq (8), nd constr ints, Eq (6), is known s line r intege
r progr ming (IP) pro
lem
ec use the o
jective function is line r in the unknow
n v ri
les nd since these v ri
les c n t ke only integer (here 0 or 1) v lues
. Other o
jectives c n lso
e employed. For ex mple, inste d of m ximizing the
num
er of points which s tisfy the origin l constr ints Eqs (5), one c n employ
non-neg tive continuous v ri
les yP nd ZP to c pture the devi tions of the dos
e level t given point from its t rget lower nd upper
ounds, respectively. I
n this c se, Eqs (5)
ecome: (9)

nd the sum F()
ecomes: (10) When the t rget
ounds LP nd UP re expressed s
multiples of t rget prescription dose, TP, nother n tur l ppro ch is to c pt
ure the devi tions from TP directly.13 In our model, this c n
e chieved
y rep
l cing constr ints Eq (9) with: (11) where yP is continuous v ri
le, unrestri
cted in sign. In the o
jective, one c n then minimize the q norm of the vector y
of ll devi tions, th t is, minimize:
Tre tment pl nning for low nd high dose
151
(12) In this c se, the pro
lem
ecomes qu dr tic 0/1 integer progr m. Algorith
ms commonly used for solving this pro
lem re now descri
ed. Algorithms Br nch-
nd-
ound The cl ssic l ppro ch to solving line r 0/1 mixed integer progr ms (MI
P) is
r nch- nd
ound. This is tree se rch ppro ch where, t e ch node of the
tree, cert in
in ry v ri
les re fixed to zero or one, nd the rem ining
in
ry v ri
les re rel xed (i.e., llowed to ssume ny v lue
etween zero nd one
). This results in line r progr m (LP)
eing ssoci ted with e ch node of the
tree. The LP t the root node is simply the origin l 0/1 MIP inst nce with ll o
f the
in ry v ri
les rel xed. The tree is constructed such th t the
in ry v r
i
les fixed in p rent node will
e fixed identic lly in ny of its children,
nd e ch child will h ve n ddition l
in ry v ri
le fixed to zero or one. Typ
ic lly, children re formed in p irs s follows. Assume th t the LP t given n
ode is solved, nd one or more of the rel xed
in ry v ri
les is fr ction l in
the optim l solution. One selects such fr ction l
in ry v ri
le nd
r nches
on it. Th t is, two child nodes re formed; one with the selected
in ry v ri

le fixed to zero, nd the other with the selected
in ry v ri
le fixed to one.
Of course, e ch child lso inherits ll of the fixed
in ry v ri
les of its p r
ent. Note th t the o
jective v lue of child node c n
e no gre ter (in the c s
e of m ximiz tion) th n the o
jective v lue of its p rent. If the line r progr m
t given node is solved nd the optim l solution h ppens to h ve integr l v l
ues for ll the rel xed
in ry v ri
les, then this solution is fe si
le for the
origin l 0/1 MIP. Once fe si
le solution for the origin l pro
lem is found, t
he ssoci ted o
jective v lue c n
e used s lower
ound (in the c se of m xim
iz tion) for the o
jective v lues of LPs t other nodes. In p rticul r, if n LP
t nother node is solved, nd its o
jective v lue is less th n or equ l to the
lower
ound, then none of its children could yield fe si
le solution for the
origin l MIP with gre ter o
jective v lue th n the one lre dy o
t ined. Hence
, no further explor tion of this other node is needed, nd the node is s id to

e f thomed. Two other criteri for f thoming node re o
vious: if the ssoci t
ed LP is infe si
le, or if the optim l solution of the LP h s integr l v lues fo

r ll rel xed
in ry v ri
les, then no further explor tion of the node is requi
red. In the l tter c se, the optim l o
jective v lue of the LP will
e comp red
with the current lower
ound, nd the lower
ound will
e upd ted if needed. The
tree se rch ends when ll nodes re f thomed. A v riety of str tegies h ve
een
proposed for intelligently selecting
r nching v ri
les nd nodes to process.
However, no str tegy st nds out s
eing
est in ll c ses. Wh t h s
ecome cle
r from recent rese rch in comput tion l MIP is th t
r nch- nd
ound is most effe
ctive when coupled with other comput tion l devices, such s pro
lem preprocessi
ng, prim l heuristics, glo
l nd loc l reduced-cost fixing, nd cutting pl nes.
The re der c n refer to the rticle
y Lee for concise description of
r nch nd-
ound methods for integer progr ming.14 The
ooks
y Schrijver,15 Nemh user
nd Wolsey,16 nd P rker nd R rdin,17 cont in det iled expositions on integer p
rogr ming nd rel ted comput tion l issues. Br nch- nd
ound lgorithms designed
for determining optim l
B sic nd dv nced techniques in prost te
r chyther py
152
seed loc tions for prost te impl nts c n
e found in G ll gher nd Lee,10 Lee n
d Z ider,12 nd Lee et l.18 Genetic lgorithm This w s first proposed in connec
tion with the optim l lloc tion of tri ls in 1973.19,20 A genetic lgorithm is
heuristic optimiz tion method modeled on the
iologic l mech nisms of evolutio
n nd n tur l selection.21,22 In n ture, the ch r cteristics of n org nism re
encoded in stre ms of DNA known s chromosomes. Likewise, in genetic lgorithm
potenti l solution to pro
lem is encoded s stre m of sym
ols over give
n lph
et. Given n initi l popul tion of individu ls (i.e. potenti l solutions
encoded s sym
ol stre ms), su
set of the popul tion is selected to p rent of
fspring for the next gener tion. The p rent selection process is stoch stic,
ut

i sed tow rds selecting those individu ls th t re most fit, s me sured
y
preselected fitness function (e.g. the o
jective function th t one is trying to
optimize). After the p rents re selected, they re p ired off nd m ted. Th t i
s, su
sections of two-p rent sym
ol stre ms re interch nged, forming two new me
m
ers for the next gener tion. This is n logous to crossover in
iologic l repr
oduction, where childs genetic composition is com
in tion of its p rents. Mut
tions re lso possi
le. This is typic lly implemented
y r ndomly selecting
child sym
ol stre m nd r ndomly ltering one of its sym
ols. In order to ensure
th t the current
est solution is not lost, the str tegy of elitism c n
e empl
oyed. Th t is, the d t stre m with the highest fitness v lue is p ssed on unch
nged to the next gener tion. This is implemented
y simply overwriting one of th
e newly cre ted children. The lgorithm c n
e termin ted fter specified num

er of gener tions h ve
een cre ted (usu lly sever l thous nds), or
y ex mining
when the difference
etween the m ximum nd minimum fitness v lues
etween cons
ecutive gener tions rem ins less th n specified threshold for num
er of gene
r tions. On termin tion, the individu l in the fin l gener tion with the l rgest
fitness v lue is selected s the oper tive solution to the pro
lem t h nd. Sev
er l uthors discuss implement tion of genetic lgorithms for prost te impl nts.
11,2325 Simul ted nne ling This is lso referred to s Monte C rlo nne ling, pr
o

ilistic hill clim
ing, st tistic l cooling, nd stoch stic rel x tion,26 nd
w s first descri
ed s heuristic for solving computer design pro
lems,27 nd
the tr veling s lesm n pro
lem.28 Simul ted nne ling is the pplic tion of st t
istic l mech nics principles to com
in tori l optimiz tion. It h s proven effect
ive in gener ting ne r-optim l solutions for cert in l rge pro
lems. Anne ling i
s process in which solid is he ted
eyond its melting point nd then cooled
slowly nd c refully into perfect l ttice. The cryst lline structure of the pe
rfect l ttice represents minimiz tion of free energy for the solid. The coolin
g process determines if the ground st te is chieved or if the solid ret ins l
oc lly optim l l ttice structure with cryst l imperfections. The Metropolis lgo
rithm w s developed to ch r cterize cooling schedules th t would produce f vor

le results.29 The centr l fe ture of the lgorithm is the Metropolis condition:

 s the solid is cooled, the current configur tion of the toms is ccepted with
cert in pro

ility nd rejected otherwise. At non-zero temper tures, tr nsiti
ons out of loc l optim re lw ys possi
le. Thus, the free energy is not monoto
nic lly decre sed.
Tre tment pl nning for low nd high dose
153
Simul ted nne ling pplies these concepts to com
in tori l optimiz tion pro
l
em. The cost function, or o
jective, ssumes the role of the free energy functio
n. The set of fe si
le solutions is n logous to the st tes of solid. Let f(i)

e the v lue of the cost function for solution i. Suppose the o
jective is to m
inimize f. A tr nsition from st te i to st te j is ccepted ccording to the fol
lowing distri
ution: (13) The p r meter c is n rtifici l temper ture th t is usu
lly reduced s the num
er of iter tions incre ses. At l rge v lues of c, l rge
incre ses in the o
jective re ccepted while for sm ller v lues only sm ll incr
e ses re ccepted. Note th t decre ses in the o
jective re lw ys ccepted. Th
e cooling schedule is the method
y which c is decre sed. A simple cooling sched
ule specifies co s the initi l temper ture nd p r meter such th t ck=k co.27
Other cooling schedules h ve
een proposed.26 The implement tion of simul ted n
ne ling requires the following: (1) concise represent tion of the st te sp ce;
(2) method for r ndomly gener ting st te tr nsitions; (3) n o
jective functi
on me suring the cost/
enefit of tr nsitions; nd (4) cooling schedule p r meter
s nd stop criterion.27 Asymptotic convergence of the lgorithm under v rious co
nditions on the gener tion nd ccept nce distri
utions h s
een proven.30 The s
equence of st te tr nsitions produces discrete time M rkov ch in. The method c
n
e gener lized to pro
lems producing continuous time nd continuous st te sp
ce M rkov ch ins.30,31 Solutions r
itr rily close to optim l usu lly require ex
ponenti l run times,
ut the symptotic
eh vior c n
e pproxim ted in polynomi
l time.26 The neigh
orhood structure of pro
lem determines which solutions r
e ccessi
le in one tr nsition from the current solution. For sm ll pro
lems, th
e neigh
orhood structure c n h ve l rge imp ct on the time to find good soluti
ons. For pro
lems with l rge num
er of solutions nd rel tively uniform dist
ri
ution of v lues of the cost function, structure pl ys lesser role.9,26,32,3
3 Ex mples In this section, two-v ri
le integer progr m is solved using
r nc
h- nd-
ound.14 The most infe si
le integer v ri
le is used s the
r nching v r
i
le, nd
est-
ound is used for node selection. Consider the pro
lem: (14)
Initi lly, the set of ctive pro
lems, L, consists of just this pro
lem IP0. The
solution to the LP rel x tion is x01=2.5, x02=3.75, with v lue zR0=62.5. The mo
st infe si
le integer v ri
le is x1, so two new su
pro
lems re cre ted, IP1 wh
ere x13 nd IP2 where x12, nd L={IP1, IP2}.
B sic nd dv nced techniques in prost te
r chyther py
154
Both pro
lems in L h ve the s me
ound 62.5, so ssume the lgorithm r
itr rily
selects IP1. The optim l solution to the LP rel x tion of IP1 is x11=3, x12=2.5
, with v lue zR1=59. The most infe si
le integer v ri
le is x2, so two new su
p
ro
lems of IP1 re cre ted, IP3 where x23 nd IP4 where x22, nd now L={IP2, IP3,
IP4}. The lgorithm next ex mines IP2, since this is the pro
lem with the
est

ound. The optim l solution to the LP-rel x tion is x21=2, x22=4, with v lue zR2=
58. Since x2 is integr l fe si
le, zIP, the incum
ent lower
ound, is then upd t
ed to 58 nd IP2 is f thomed. Both of the two su
pro
lems rem ining in L h ve th
e
est
ound gre ter th n 58, so neither c n yet
e f thomed. Since these two su

pro
lems h ve the s me
ound 59, ssume the lgorithm r
itr rily selects IP3 t
o ex mine next. LP rel x tion to this pro
lem is infe si
le, since it requires t
h t x s tisfy x13, x23 nd 5x1+2x22 simult neously. Therefore, zR3=, an this noe ca

n be fathome by bouns since zR3zIP. That leaves the single problem IP4 in L. Th
e solution to the LP relaxation of this problem is x41=3.2, x42=2, with value zR
4=57.6. Since zR4zIP, this subproblem can also be fathome by
Figure 12.9 A branchanboun example.
bouns. The set L is now empty, hence x2 is an optimal solution for the integerp
rograming problem IP0. The progress of the algorithm is inicate in Figure 12.9
. Each box contains the name of the subproblem, the solution to the LP relaxatio
n, an its associate objective value.
Treatment planning for low an high ose
155
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13 Planning an implant: preoperative versus intraoperative planning
Ronal D Ennis Preoperative planning The prostate brachytherapy proceure evelo
pe by the Seattle group inclue a volume stuy as an integral part of the proc
ess. During this preoperative proceure, images of the prostate were obtaine at
5 mm intervals from base to apex.1 These images were then use to perform preop
erative treatment planning. The require positions of the sees an neeles were
etermine uring this process. Neeles were preloae prior to the implant pro
ceure itself as etermine by the preplan. During the implant proceure, the pa
tient was positione in similar manner to the position uring the preoperative v
olume stuy an the prostate position was reprouce. Then, the preloae neele
s were place into the prostate in the preetermine positions uner ultrasoun
guiance. This preoperative volume stuy session was novel an hel out several
avantages. First, a osimetryriven implant coul be performe. The alternativ
e brachytherapy technique was pioneere by Stock an Stone at Mt Sinai Hospital
(New York) an avocate also by rao.2,3 In this technique, the number of see
s place was etermine base on timehonore tables of activity per volume an
rules of thumb for the ratio of sees place in the central versus peripheral re
gions of the implante volume. The Seattle approach allowe one to prescribe a p
articular ose an then etermine the optimal position of the sees an neeles
to achieve this ose. Since ose is the biologically important parameter, it was
logical to attempt to eliver a particular ose to the prostate an to plan acc
oringly. Performing the volume stuy preoperatively allowe the physicist/osim
etrist the time to optimize the plan, since the volume stuy was typically perfo
rme a few weeks prior to the proceure itself. Asie from the prescription ose
, several other parameters can be varie, such as the egree of inhomogeneity wi
thin the prostate, an the ose to the ajacent normal tissues. The preplanning
process allowe ifferent plans to be evaluate to achieve these multiple goals.
An aitional avantage of preoperative planning was that the en result of the
preplanning process was to etermine the locations of each see. Since several
sees will typically be place at ifferent epths along a particular x, y posit
ion, a single neele coul be loae for each of these positions an unloae in
a single train thereby simplifying the implant proceure itself an obviating t
he nee to place each see iniviually. The preliminary results of prostate bra
chytherapy performe by the Seattle group were impressive an inspire many othe
rs to aopt their technique.1 However, several problems with the preoperative vo
lume stuy were appreciate. First, matching the prostate position, size, an sh
ape of the preplan to that which existe at the time of the

Basic an avance techniques in prostate brachytherapy

158
proceure itself was not always easily achievable. Differences in patient positi
oning an ifferences inuce by anesthesia, which is use only uring the brach
ytherapy proceure itself, are the presume causes of the iscrepancy. Data supp
orting these concerns are provie by a report from Messing et al. in which they
analyze the change in prostate volume between the preplan an the intraoperati
ve evaluation.4 There were significant changes in volume (greater than 5%) in 4
out of 5 patients. In aition, these investigators evaluate the osimetric res
ults of an implant if it precisely followe the preplan without any intraoperati
ve ajustments. The mean fraction of volume unerose was 22.6% with a range of
6.464.9%. In aition, a secon problem associate with the preplanning volume s
tuy is the nee for two sessions, which is cumbersome for patients an physicia
ns an expensive if both sessions are performe in an operating room setting. Be
cause of these concerns, several institutions began to perform the volume stuy
an the treatment planning in the operating room at the time of the proceure. T
he most important component necessary to perform this was computer software that
coul help perform a treatment plan quickly. In aition, those performing the
treatment plan ha to be knowlegeable an experience enough to know how many s
ees to orer for a particular size prostate an be able to optimize the plan qu
ickly in the operating room. Some groups evelope their own software, while oth
ers aapte commercially available software to this task. More rapi methos for
loaing sees into neeles were also neee to make this process feasible an s
everal manufacturers evelope ifferent loaing evices to spee this part of t
he process. Alternatively, some institutions use the Mick applicator (Mick Raio
Nuclear Instruments, Bronx, NY) in which iniviual sees are implante, obviat
ing the nee to loa neeles in a specific pattern as etermine by the treatmen
t plan. Successful implementation of this new approach woul require that simila
r or superior plans coul be evelope in the operating room in a reasonable amo
unt of time. In aition, the quality of the implant as assesse by postimplant
osimetry shoul be at least as goo, but ieally better, than that achieve wit
h a separate volume stuy. Finally, clinical outcome regaring isease control a
n toxicity shoul be at least as goo, but preferably better, than that achieve
 previously. Intraoperative preplanning Time requirements an osimetric result
s Investigators from the University of Rochester, NY were the first to escribe
the evelopment an implementation of an intraoperative preplanning metho.4 The
y evelope their own software known as Prostate Implant Planning Engine for Ra
iotherapy (PIPER). The software employe a genetic algorithm approach. The progr
am is given certain require osimetric parameters an then solves the problem.
Aitionally, it has a feature that inclues in the optimizations the uncertaint
y associate with actual see placement (i.e. it can provie an optimal plan giv
en that sees will be place with an error in location). In this stuy Messing e
t al. compare the plans of 10 patients. Each patient unerwent a preoperative v
olume stuy an a preplan was calculate using the PIPER. When the patient was b
rought to the operating room, an
Planning an implant
159
intraoperative preplan was evelope. The time for each step of the process was
quantifie. Then meian time to capture the transverse ultrasoun images on to t
he planning computer was 4 minutes, the meian time for image segmentation was 1
0 minutes, the meian time to run the PIPER was 2.2 minutes an the time to eval
uate the plan was 2 minutes. Thus, the overall time for the intraoperative plann
ing process was approximately 18 minutes. The plan erive in the stanar preop
erative fashion an in the intraoperative fashion were similar as assesse by th
e peripheral uniformity number, the uniformity number, an the fractional sprea

of the ifferential osevolume histogram aroun the mean tumor ose. Formal co
mparison by more traitional osimetric parameters was not performe. A visual 
isplay of the pure osevolume histograms, (i.e. ose to number of cc of PTV, ra
ther than the more common fraction of PTV receiving at least the ose) is provi
e in isplay form an o suggest that there are some ifferences between the tw
o plans. No comparison of postimplant osimetry was performe. At Columbia Unive
rsity College of Physicians an Surgeons, ewanter et al. implemente an intraop
erative preplanning proceure using commercially available software.5 We analyze
 the time require to perform the proceure an compare the osimetric paramet
ers of the first 10 patients treate in this manner with the previous 10 patient
s treate with the stanar preoperative volume stuy. Performance of the intrao
perative planning was performe using the commercially available software known
then as MMS (Multimeia Systems Inc, Charlottesville, VA). Varian Inc (Palo Alto
) has subsequently bought this company an the software is currently markete un
er the name of Varisee. The steps involve in the proceure an the mean time i
t took for each step were as follows: (1) positioning the patient an the ultras
oun probe in the proper position, 26 min; (2) capturing the ultrasoun images o
n the treatment planning computer at 5 mm intervals, 4 min; (3) image segmentati
on, 8 min; (4) performing the treatment plan, 18 min; (5) loaing the sees into
the neeles as etermine by the plan, 17 min; an (6) implanting the sees in
the prostate uner ultrasoun guiance, 57 min. Overall, the intraoperative prep
lanning implants took aitional time. However, incluing the time from the preo
perative volume stuy session negate this ifference. A key component of the Co
lumbia technique is the planning optimization function in the Varisee program. T
his allows one to enter the osimetric requirements an then it attempts to solv
e the problem. One can then a some human intelligence to moify the computere
rive plan to achieve ones goals. This optimization algorithm makes its calculati
ons quite rapily. In our stuy the mean time for treatment planning was 18 minu
tes. The preimplant plan osimetry was equivalent between the two plans as asses
se by the important parameters of %D80 an %D90 suggesting that it is possible
to create plans in the operating room that are similar to those performe after
a preoperative volume stuy has been performe. The %V100 was 100% in the stana
r approach an 99% in the intraoperative planning group. This ifference was st
atistically significant, but obviously not clinically significant. In aition,
the %D100 was 100% for the preplan
Basic an avance techniques in prostate brachytherapy
160
versus 93% for intraoperative planning. However, it is well known that the D100
is very sensitive to small ifferences an not a clinically useful parameter.6 A
more important measure of the acceptability of this approach is the postimplant
osimetric analysis. In our ata, the osimetric parameters were equivalent in
those plans evelope in the stanar manner versus those planne in the operati
ng room as assesse by the %D80, %D90, %D100, an %V100. Thus, we showe that co
mparable preoperative an postoperative osimetry was achieve, an that the tim
e to perform the proceure was acceptable. At the Clevelan Clinic Founation th
is proceure was also implemente although the planning was one manually rather
than by an optimization algorithm.7 In this retrospective report the results of
113 patients implante uring a time perio in which both intraoperative an pr
eoperative planning were performe on a similar number of patients were analyze
. The time require for the intraoperative an preoperative planning techniques
was not reporte. In their investigation, the intraoperative patients were impla
nte with a higher activity/volume an ha a higher preoperative V100 an V80. T
he postoperative osimetry was not surprisingly also superior as assesse by V80
, V90, V100, V150, V200, an D90. Thus, they emonstrate that intraoperative pl
anning coul be performe, even with manual planning, an achieve excellent qual
ity implants. However, these results shoul not be taken to emonstrate that int
raoperative preplanning results in superior postimplant osimetry since the prep

lans of those cases were also superior. Beyer et al. at Arizona Oncology Service
s evelope a timeefficient intraoperative technique.8 Their technique is a hyb
ri of the Mt Sinai approach an the osimetricriven approach of the Seattle gr
oup. (In fact, the Mt Sinai group now performs their implants in a similar hybri
 fashion.9) In this technique, stanar neele positions are use for all patie
nts. Neeles are place into these positions while the treatment plan using thes
e positions is evelope. Moifications in the plan are then manually performe
to optimize the plan an then carrie out. Doing planning an neele placement a
t the same time markely shortens the proceure time. In the eight patients anal
yze, the time neee to place the neeles, perform the plan, an implant the se
es was a mean of only 25 minutes compare with 100 minutes in the Columbia stu
y. Although no statistical analysis of the osimetric ata was performe, the po
stimplant osimetry was excellent an comparable among those implante with thei
r preoperative planning technique they ha use previously or the new intraopera
tive planning technique. The isavantages of this approach are that it is not a
truly ose optimize approach as most neele positions are fixe from the outse
t. In aition, in the interest of time, normal structures are not contoure an
their osevolume histograms not calculate nor examine. Kaplan et al. from th
e Joint Center for Raiation Therapy at Harvar University escribe performing
intraoperative planning in 107 patients.10 The mean time from transrectal ultras
oun placement until the patient left the room was a reasonable 104.2 minutes. T
he exact metho of treatment planning was not specifie. They performe the tra
itional preoperative planning an intraoperative planning in five patients. The
DVHs are isplaye graphically but no formal comparisons were mae. The rectal 
oses appeare consistently lower in the intraoperative plans, but no other clear
ifferences were emonstrate.
Planning an implant
161
DAmico et al, also from Harvar University, have evelope a novel prostate brach
ytherapy protocol.11 At their institution, magnetic resonance imaging (MRI) in t
he operating room is use to image the prostate an guie implantation of the pe
ripheral zone only. Intraoperative planning, using software they evelope, is p
erforme in this protocol. The time each step takes in their protocol has not be
en escribe, but the intraoperative DVH analysis of the first nine patients rev
eale excellent coverage of the peripheral zone. In conclusion, intraoperative p
replanning can be performe an results in similar osimetric outcome as the tra
itional preoperative preplanning. Taking into account the time require for the
preoperative volume stuy, there is no increase in overall time. In an effort t
o shorten the proceure time, some have evelope a hybri approach in which a l
imite form of intraoperative osimetry is performe with certain parameters of
the implant (e.g. neele positions) set from the outset. Whether an unrestricte
intraoperative preplanning approach or a hybri approach is superior remains to
be seen. Intraoperative planning on the basis of neele position The next step
in intraoperative planning is to plan on the basis of actual neele location ins
tea of base on a planne neele position in a preplan. Zelefsky et al. from Me
morial SloanKettering Cancer Center have reporte on such an approach.12 In the
ir approach, images of the prostate are capture onto the treatment planning com
puter only after neeles have been place in the prostate. A software program e
velope by the investigators then attempts to optimize the see placement while
satisfying the goals of ose elivery to the prostate, urethra, an rectum. The
postimplant osimetric analysis of 30 patients treate with this approach was co
mpare with that of 30 patients they ha treate previously with the Mt. Sinai a
pproach an 30 patients treate with a compute tomographic (CT)base preoperat
ive planning approach. The time to perform their proceure was not presente. Ho
wever, the osimetric results of the intraoperative plan were superior to the co
mbine results of the other two groups as assesse by multiple osimetric parame
ters. For example, the %D90 was 116% for the intraoperative planning versus 94%

for the Mt. Sinai technique an 88% for the CTbase preoperative planning techn
ique. Urethral an rectal oses were also lower. The limitation of this an this
limits the ability of the planning process to approach is that the neele posit
ions are preetermine optimize the see istribution. A more ieal approach wou
l be to allow the planning process full freeom to optimize all parameters an
then ajust for inaccurate neele placement as it occurs. Cormack et al, from Ha
rvar University, have evelope such an approach in their MRIriven program is
cusse above.13,14 The initial plan is not constraine to any particular neele
position. However, after each neele is place an MRI scan is performe an the
neele track ientifie. If the neele placement is inaccurate by more than a fe
w millimeters, it is repositione. If not, the sees are eposite along the tra
ck. The plan is then recalculate accounting for the actual neele position. At
the en of the proceure, aitional sees are place in areas neeing these on
the basis of the intraoperative analysis. In their report escribing this techni
que, 14 of 15 patients require a meian of two aitional neeles containing fi
ve aitional sees to optimize the coverage.
Basic an avance techniques in prostate brachytherapy
162
Unfortunately, intraoperative MRI is not wiely available. Therefore, the challe
nge is for other investigators to evelop conceptually similar approaches that c
an be more wiely aopte. Future irections The approaches iscusse above, how
ever, are still not base on actual see position. Ieally, each sees position s
houl be etermine at the time of the proceure an osimetry calculate on tha
t basis. Several techniques are being pursue to achieve this goal. Investigator
s from Riversie Research Institute are eveloping ultrasounbase techniques to
ientify each see within the prostate.15 Resonance, signature, an elastograph
ic methos are being pursue. Although still uner evelopment, these approaches
hol promise to perform see positionbase intraoperative postimplant osimetr
y. In aition, since they are performe using ultrasoun, they woul be wiely
applicable since most institutions perform ultrasounbase implants. An alternat
ive approach woul be to obtain igital raiographs from multiple angles to obta
in images of the sees. These images coul be capture by a software program, wh
ich woul then etermine each sees position in three imensions an then calcula
te the resultant ose istribution. Registering the raiographerive ose is
tribution to the ultrasoun prostate images woul allow the ose istribution to
be overlai on the ultrasoun images. Then, areas of insufficient see placemen
t coul be ientifie an fixe. Such a program is uner evelopment at Memorial
SloanKettering Cancer Center.16 References
1. Blasko JC, rimm PD, Rage H. Brachytherapy an organ preservation in the man
agement of carcinoma of the prostate. Semin Raiat Oncol 1993; 3:240249. 2. Stock
R, Stone NN, DeWyngaert JK, et al. Prostate specific antigen finings an biop
sy results following interactive ultrasoun guie transperineal brachytherapy f
or early stage prostate carcinoma. Cancer 1996; 77:23862392. 3. rao L, Larson
TR, Balch CS, et al. Actuarial iseasefree survival after prostate cancer brach
ytherapy using interactive techniques with biplane ultrasoun an fluoroscopic g
uiance. Int J Raiat Oncol Biol Phys 1998; 42:289298. 4. Messing EM, Zhang JBY,
Rubens DJ, et al. Intraoperative optimize inverse planning for prostate brachyt
herapy: Early experience. Int J Raiat Oncol Biol Phys 1999; 44:801808. 5. ewant
er RM, Wuu CS, Laguna JL, et al. Intraoperative preplanning for transperineal u
ltrasounguie permanent prostate brachytherapy. Int J Raiat Oncol Biol Phys
2000; 48:377380. 6. Yu Y, Waterman FM, Suntharalingam N, Schulsinger A. Limitatio
ns of the minimum peripheral ose as a parameter for ose specification in perma
nent I125 prostate implants. Int J Raiat Oncol Biol Phys 1996; 34:717725. 7. Wi
lkinson DA, Lee EJ, Ciezki JP, et al. Dosimetric comparison of preplanne an OR
planne prostate see brachytherapy. Int J Raiat Oncol Biol Phys 2000; 48:12411
244. 8. Beyer DC, Shapiro RH, Puente F. Realtime optimize intraoperative osim
etry for prostate brachytherapy: A pilot stuy. Int J Raiat Oncol Biol Phys 200

0; 48:15831589.
Planning an implant
163
9. Stock R, Stone NN, Lo YC. Intraoperative osimetric representation of the re
altime ultrasounguie prostate implant. Tech Urol 2000; 6:9598. 10. Kaplan ID
, Meskell EJ, Soon SJ, et al. Intraoperative treatment planning for raioactive
see implant therapy for prostate cancer. Urology 2000; 56:492495. 11. DAmico AV,
Cormack R, Tempany CM, et al. Realtime magnetic resonance imageguie intersti
tial brachytherapy in the treatment of select patients with clinically localize
prostate cancer. Int J Raiat Oncol Biol Phys 1998; 42:507515. 12. Zelefsky MJ,
Yamaa Y, Cohen , et al. Postimplantation osimetric analysis of permanent tran
sperineal prostate implantation: Improve ose istributions with an intraoperat
ive computeroptimize conformal planning technique. Int J Raiat Oncol Biol Phy
s 2000; 48:601 608. 13. Cormack RA, Tempany CM, DAmico AV. Optimizing target cover
age by osimetric feeback uring prostate brachytherapy. Int J Raiat Oncol Bio
l Phys 2000; 48:12451249. 14. Cormack RA, Kooy H, Tempany CM, DAmico AV. A clinica
l metho for realtime osimetric guiance of transperineal I125 prostate impla
nts using interventional magnetic resonance imaging. Int J Raiat Oncol Biol Phy
s 2000; 46:207214. 15. Feleppa EJ, Ramachanran S, Alam SK, et al. Novel methos
of analyzing raiofrequency echo signals for the purpose of imaging brachythera
py sees use to treat prostate cancer. Meical Imaging 2002; Ultrason Imaging S
ignal Processing 2002; 4687:127138. 16. Toor DA, Cohen N, Amols HI, et al. Film
base 3D see reconstruction in brachytherapy. Phys Me Biol 2002; 47:20312048.
14 The Wheeling approach to treatment planning for prostate brachytherapy
Wayne M Butler an regory S Merrick Introuction Favorable brachytherapy result
s have been obtaine with a variety of planning an intraoperative techniques, o
f which no metho has proven superior.1,2 Although quality is easy to conceptual
ize, it is more ifficult to quantitate. It is universally accepte that an aeq
uate implant shoul encompass the target volume, but no consensus exists as to w
hat represents the target. In aition, tolerance urethral an rectal oses have
not been wellefine an the significance an egree of ose homogeneity throug
hout the implante region remains unclear. Initially, the Seattle group utilize
a uniform seeloaing philosophy.3 However, because a purely uniform loaing sc
heme prouces a high central ose which may aversely affect the urethra,4 this
loaing philosophy has evolve into an approach using fewer central neeles an
more peripheral sees. The American Association of Physicists in Meicine (AAPM)
Task roup No. 56 (T56) recommene that treatment plans be esigne to place s
ees peripherally to improve ose homogeneity an to reuce unnecessary raiatio
n amage to the urethra.5 The American Brachytherapy Society (ABS) also recommen
e the aoption of moifie peripheral loaing techniques to minimize the length
of the urethra receiving >200% of the prescription ose.6 In general, four see
loaing philosophies (uniform loaing, peripheral loaing, moifie uniform loa
ing, an moifie peripheral loaing) have been utilize in prostate brachyther
apy.7 In a survey by the ABS, a moifie peripheral loaing approach was use by
75% of brachytherapists, while 25% use a moifie uniform approach.8 Although
most brachytherapy programs utilize a spectrum of loaing approaches, our approa
ch, base on a moifie uniform philosophy, has been use almost exclusively. Ev
en in patients with a pretreatment prostatespecific antigen (PSA) <10 ng/mL, ap
proximately 50% manifest extracapsular extension of malignant isease at the tim
e of raical prostatectomy.9 Thus, treatment of the periprostatic region remains
paramount. Our approach has the benefit to maximally respect urethral an recta
l tolerance with the ability to aggressively irraiate sites of extracapsular i
sease. At the periphery of the implant target volume, the raiation ose ecreas
es by up to 20 y per mm.10 Eema can also affect the actual ose of raiation 
elivere to the prostate glan an extracapsular regions.11 The utilization of t
reatment margins, however, significantly ecreases the effect of eema on postop

erative osimetry.11,12 Merrick an colleagues have previously reporte that imp
lant prescription oses of raiation measure via ay 0 osimetry can
The Wheeling approach to treatment
165
consistently be elivere to both the prostate an periprostatic region with the
utilization of extracapsular see placement.1316 In this chapter, we escribe ou
r planning approach utilizing a manual algorithm for a moifie uniform/ periphe
ral see loaing approach an provie a rationale for such an approach along wit
h recommenations for implant esign an evaluation. Preplanning philosophy Our
preplanning techniques an intraoperative proceures have previously been escri
be.1,2 In our preplanne approach, a transrectal ultrasoun volumetric stuy of
the prostate glan is obtaine in the ultrasoun suite prior to the operative p
roceure. The stuy is obtaine at 5 mm intervals extening from the proximal se
minal vesicles/ blaer neck/base of the prostate glan to the apex. Prior to ob
taining the ultrasoun, a urinary catheter is place so that the urethra can be
ientifie on each ultrasoun slice. A planning target volume (PTV, prostate plu
s periprostatic margin) is etermine by a 38 mm enlargement in all imensions (w
ith limitations on posterior extension) with a resultant PTV approximately twice
the ultrasoun volume. The margin extent an hence the enlargement of the
Figure 14.1 Transverse ultrasoun images illustrating enlargement of the prostat
e volume (white line) to the planning target volume (PTV, black line). The ureth
ra, ientifie by the presence of a catheter, is also marke by a small, central
white circle, (a) Section near miglan, 25 mm inferior to the base, (b) Secti
on 35 mm
Basic an avance techniques in prostate brachytherapy
166
inferior to the base an 5 mm superior to the apex.
ultrasoun volume to the PTV is patientspecific an consiers such factors as r
egions of likely extracapsular extension an the presence of a transurethral res
ection of the prostate (TURP). The prescription ose is then prescribe to the P
TV with margin. The rationale for this extracapsular margin is base on patholog
ic measures of the probability of microscopic extracapsular isease,9 an estima
tes that see uncertainty is approximately 5 mm longituinally an 3 mm in trans
verse imensions.17 As such, the crosssectional area of the PTV on any slice is
at least as large as the area of the prostate with margin on the next larger a
joining slice. The two ultrasoun images of a 31 cm3 prostate in Figure 14.1 ill
ustrate a typical relationship between the urethra, prostate, an PTV on transve
rse slices taken near miglan an near the apex. enerous periprostatic margin
s are of great utility in patients with any risk of extracapsular extension an
a low risk of pelvic lymph noe involvement/istant metastases. Our prostate imp
lant philosophy results in a mean periprostatic margin of 6.51.8 mm. These treatm
ent margins may potentially result in prostate cancer cures with a monotherapeut
ic approach for patients with low, intermeiate, an potentially even selecte h
igh risk isease.18 This volume ose escalation has been obtaine without any in
crease risk of urinary, rectal, or sexual ysfunction compare to other seelo
aing approaches.19,20 To eliver the prescribe ose to the PTV, it is necessar
y to irraiate a volume larger than the PTV given the constraints of using a sin
gle see strength throughout the implant an fixe gri coorinates for see pla
cement. If necessary, sees may be place outsie the PTV to pull the ose out to
the efine margin rather than use sees of greater strength place entirely wit
hin the PTV to push the ose out. Preplanning osimetry In our program, a lower cas
e plan is generate to eliver the minimal peripheral ose (mPD) which is the min
imum ose covering 100% of the efine volume (D100) to the PTV with margin. For

pallaium103 (103P), mean strengths of 2.8 U/see an 2.2 U/see are use for
125 y monotherapy an 90 y boost techniques, respectively, while for ioine1
25 (125I) monotherapy, average see strengths of 0.54 U/see are use. Table 14.
1 summarizes mean treatment planning parameters use in the implementation of mo
ifie uniform loaing uring calenar years 2001 an 2002. Because 610 extra see
s are orere an routinely implante to exten ose into the proximal seminal
vesicles an increase ose in the peripheral zone of the prostate, the actual nu
mber of sees implante is liste in Table 14.2 rather than the number planne.
Although most volume stuies an preplans are complete a week before the implan
t, the plans for some long istance referrals, particularly men requiring neoa
juvant hormones, cannot be complete in time for an accurate
The Wheeling approach to treatment
167
Table 14.1 Mean treatment planning parameters use for moifie uniform loaing
over the past 2 years
Parameter Overall 145 y
125
I 90 y 115 y
103
P 125 y

Ultrasoun volume (cm3) 33.69.4 37.59.0 31.610.8 35.47.3 35.08.9 Planning volume (cm3
) 66.413.7 72.413.4 63.415.7 68.49.9 69.012.9 Planning margin (mm) 5.20.6 5.10.5 5.20
5.10.6 5.20.5 Enlargement factor 2.020.22 1.960.14 2.070.24 1.960.19 2.010.21 Number o
sees* 12916 12714.0 12216 13512 13515 Number of neeles 273 284 263 28 3 283 See
th (U) 0.5420.0 10 2.160.09 2.570.07 2.800.10 * This is the number of sees actually
implante, which exceee the number of sees in the preplan by 610 extra sees.
These extra sees require an aitional 46 neeles.
Table 14.2 Preimplant osimetric evaluation criteria using appropriate see stre
ngths from Table 14.1
Evaluate quantity Parameter*
125
Value I
103
P
Patient specific nees TX volume, URP, etc. primary importance >99.8% volume Cov
erage of the planning volume V100 125%140% mPD D90 40%55% volume 55%70% volume Dose
homogeneity V150 15% volume 15%20% volume High ose volume V200 UV125 80%100% vol
ume 50%100% volume <15% volume <25% volume Urethra volume coverage UV150 UD50 130
%145% mPD 120%140% mPD 140%150% mPD 130%160% mPD Urethra ose UD10 * V100, V150 an
V200 are the percentage of the planning target volume (PTV) covere by 100, 150
an 200% of the prescribe ose (mPD), respectively, D90 is the minimum ose cov
ering 90% of the PTV. UV125 an UV150 are percent volume of the urethra at the 
efine mPD. UD50 an UD10 are the minimum oses covering 50% an. 10%, respectiv
ely, of the urethra volume.
see orer to be place with the see venor. An estimate for the number of see
s is mae using a regression equation base on the ultrasoun prostate volume, a
n the volume is ajuste as neee for the use of hormones by analytical equati
ons. For example, the initial prostate volume is iminishe by weeks of a lutein

izinghormonereleasing hormone (LHRH) agonist approximately as: (1)

Basic an avance techniques in prostate brachytherapy
168
The number of sees implante follows a power law equation because the enlargeme
nt ratio, PTV/ultrasoun volume, is nonlinear when a consistent margin is appli
e to each prostate. The equation for the number of 103P sees neee is given
by: (2) A similar equation is use for 125I where the coefficient is 3.83 an th
e power is 0.381. Figure 14.2 is a graph of Eq (2) preicting the number of 103P
 sees from the prostate volume for a monotherapy implant of 125 y an using a
see strength of 2.80 U. Moifie uniform loaing places sees on a 10 mm unifo
rm gri followe by moifications that place aitional sees on the lateral an
anterior periphery an reuce the linear ensity of sees in the central neele
s, but leave two thirs of the total sees on the original uniform gri points.1
The moification of uniform loaing improves ose homogeneity an urethral spar
ing (which is efine
Figure 14.2 raph of the power law Eq(2) preicting the number of implante see
s from the prostate volume for a 125 y monotheraphy 103 P implant using sees
of strength 2.80 U. (U=cY/cm2h).
The Wheeling approach to treatment
169
as a ose to the urethra of 100140% mPD) because in any uniform istribution of s
ees, the ose woul be greatest centrally ue to the cumulative effects. Two pl
anes efining the bounaries of the uniform gri are the base plane of the prost
ate an the posterior borer. In the posterior implant plane, a neele may occup
y the intersection of the sagittal mi plane with this plane or a position 0.5 c
m lateral to the intersection. This choice ientifies the main uniform loaing g
ri as upper case or lower case, where the sagittal miline column on the template i
s labele D an the columns 0.5 mm lateral to it c an . In our program, only lower c
ase plans are use. Neeles pass through all vali lattice points on a cm2 gri
at miglan that are within the PTV plus any vali gripoints that are within a
2 mm margin of the PTV rawn on the miglan slice. These neeles exten to the
base plane that efines the starting plane for vali see positions in 10 mm in
crements along each neele. All neeles are planne to eposit sees at the base
plane unless the see woul be more than 10 mm outsie the PTV at the base. A s
imilar margin applies to sees eposite on the apical plane, but a 4 mm margin
outsie the PTV limits see eposition from other planes. If the apex oes not f
all at an integer number of cm from the base, the uniformly space sees o not
en at the apical plane, an the see trains are extene 0.5 cm inferior to the a
pex with the PTV of the apical plane use as a guie for vali gripoints occupi
e by these sees. The first moification to uniform loaing places neeles on t
he lateral an anterior periphery. These neeles will eposit sees with a 1 cm
spacing, but the starting points are offset from the base by 5, 15, or 25 mm an
are also offset 5 mm in the y irection an usually 5 mm in the x irection fro
m the uniform loaing gri. Vali neele paths are no more than 5 mm insie the
PTV or 3 mm outsie at miglan, an vali see eposition points are no more th
an 5 mm insie or outsie the PTV on the relevant halfinteger offset transverse
image. At this point in the planning process, all neele paths, both uniform an
peripheral, are checke to ensure that they contain at least two sees with a u
niform 10 mm spacing throughout the length of each neele. The secon moificati
on reuces the linear see ensity in 46 central main gri neeles. In all cases,
these neeles retain the first an last see in the source train while one or t
wo of the remaining sees are iscare an replace with spacers or Mick applic
ator offsets. All plans are evaluate for each implant base on etaile osimet

ry, which inclues osevolume histograms (DVHs) of the PTV an urethra as well
as the ose profile points in the center of the urethra. Utilizing this algorith
m, a V100 (volume of the target area receiving 100% of the prescribe ose) typi
cally equals 100% an V105 excees 99.5%. Although there is no consensus regari
ng the optimum egree of ose homogeneity or its measure, particular care is tak
en to limit the volume of the target area receiving 150% of the prescribe ose
(V150) to approximately 4055% for 125I implants1,2 an 5570% for 103P implants. I
n aition, particular attention is taken to limit the volume of the target area
receiving 200% (V200) to <15% of the PTV for 125I an <25% for 103P. By reuci
ng the linear ensity of sees in the central neeles, the volume of the urethra
(UV) receiving a ose greater than 150% of the mPD (UV150) is maintaine <15% o
f the urethral volume for 125I an <25% for 103P. In terms of centroi ose poi
nts, the urethral ose is maintaine between 110% an 140% of the prescribe os
e (average ose 115120%). Because all uniform loaing neele paths within the bo
y of the prostate are use, some neele paths may transect or pass very near the
Basic an avance techniques in prostate brachytherapy
170
urethra, an some point oses may be very high. However, in executing the prepla
n, the path of the urethra is confirme in the operating room an the intene n
earby neele paths are isplace several mm to avoi the urethra. We o not rout
inely calculate a rectal ose via the preplan. The prescribe ose, however, enc
ompasses the posterior borer of the PTV with a 4 mm margin. The significance an
 utility of ose uniformity parameters such as V150 in etermining the quality
of an implant an urethral ose parameters in minimizing urethral morbiity rema
ins unknown. Highly inhomogeneous implants (V150>90% volume) make the concept of
prescribe ose less meaningful, may increase the likelihoo of raiation morbi
ity, an probably o little to increase tumor control so long as V100 an D90 a
re aequate. Although many loaing approaches can achieve an aequately low ose
near the urethra, the moifie uniform loaing approach results in greater ose
homogeneity measure in terms of DVH slope an orthogonal ose profiles without
creating a high ose annulus aroun the urethra. These guielines are utilize
for all implante patients except those who have previously unergone a transure
thral resection of the prostate (TURP). These patients are implante with a peri
pheral technique which explicitly places no neeles within 5 mm of the urethra a
n limits the urethral ose to approximately 110% mPD. Preimplant evaluation Mul
tiple postoperative osimetric analyses have evaluate the actual ose istribut
ion following prostate brachyther apy an have mae suggestions/recommenations
regaring quality.1216,2129 The ABS recommens that postoperative osimetry be ef
ine in terms of a V100 an D90.6 We have previously efine recommenations reg
aring preimplant evaluation.1,2 Our preimplant evaluation aheres to the criter
ia in Table 14.2. It is ensure that each ultrasoun slice has been appropriatel
y enlarge an that the ose has been prescribe to completely cover the PTV. Th
e V100 for the PTV shoul be >99.8% an D90 125140% of mPD. Urethral osimetry is
efine in terms of the average ose, UD50, an the UV150. These parameters sho
ul be <140% of mPD, an <15% of the urethral volume for 125I an <25% for 103P
. With this approach, 99% of our implants have utilize 2133 neeles per preplan.
In aition, at least two sees are place in each implant neele because of se
e uncertainty.17 Figure 14.3 is a typical osevolume histogram of the PTV, ult
rasoun prostate, an urethra illustrating the osimetric guielines iscusse a
bove. The urethra is clearly cooler than the PTV or prostate with a V150 of 12% vo
lume, but the entire urethra receives 125% of mPD. The PTV, overall, lies at hig
her ose than the prostate because the first moification to uniform loaing a
e sees to the anterior an lateral periphery of the PTV an the secon moific
ation reuce the ensity of sees in the mile of the prostate to reuce an h
omogenize the urethral ose. Figure 14.4 illustrates the homogeneity of ose thr
oughout the volume by tracing ose profiles along the urethra in one plot an al
ong a vertical line coinciing with the D implant column at the miglan. Although

peripheral loaing can achieve similar homogeneity along the urethra, profiles
perpenicular to the urethra are far from uniform in ose. The vertical profile
of Figure 14.4b, which is relatively flat from the posterior to anterior borer
of the PTV
The Wheeling approach to treatment
171
using moifie uniform loaing, woul have pronounce peaks at the anterior an
posterior borers with a eep valley between them to spare the urethra. Translat
ion of preimplant quality to postimplant osimetry Using the above treatmentpla
nning approach, ay 0 computer tomography (CT)base osimetry ha resulte in e
xtracapsular treatment margins of 6.5 an 9.6 mm at the 100% an 75% isoose lin
es, respectively.30 With the exception of the blaer neck an posterior prostat
e borer, the 100% isoose margin was 5 mm for all evaluate slices. In the cour
se of treatment planning an implantation, approximately 35% of sees are place
in extracapsular locations, an the overall see fixity was >98%.16 Investigato
rs at Thomas Jefferson University have also reporte the ability to aggressively
treat the extracapsular region.31 In aition, it
Figure 14.3 Preplan osevolume histograms (DVH) of the planning target volume (
PTV, soli line), ultrasoun prostate (ashe line), an urethra (otte line) f
or the patient whose prostate is illustrate in Figure 14.1. To eliver a boost
ose of 90 y with 103P sees of strength 2.14 U, 117 sees in 26 neeles were
planne. The steep slope of the curves at 50%
Basic an avance techniques in prostate brachytherapy
172
volume coverage inicates the homogeneity of the implant. The DVH of the PTV typ
ically lies at higher ose than the prostate because of sees ae to the anter
ior an lateral periphery of the PTV while the prostate has a reuce ensity of
sees in the mile to reuce the urethral ose.
has been reporte that ay 0 brachytherapy treatment margins outperform other o
simetric parameters, such as V100 an D90, in preicting 2 year prostatespecifi
c antigen (PSA) response following brachytherapy.32 In terms of ay 0 postimplan
t osimetric parameters, the crucial coverage values, V100 an D90, average 97.0
% 2.7% volume an 120%11% of mPD, respectively, with no ifference between isotope
s. Mean urethral average an maximum oses are 113%13% of mPD an 126%17% of mPD,
respectively, but there is a significant ifference between 125I an 103P oses
with the latter isotope about 6% cooler. A similar isotopic ose ivergence app
lies to the rectum where the mean anterior rectal average an maximum point ose
s are 38%14% of mPD an 58%23% of mPD, respectively. The ability to obtain generou
s extracapsular margins such as those we have reporte,30 calls into question th
e nee for supplemental external beam raiation therapy (EBRT) in selecte inter
meiate an high risk patients. Extensive pathologic evaluation of raical prost
atectomy specimens at the Mayo Clinic an Clevelan Clinic have reporte the mea
n extent of extracapsular extension to be 0.5 mm an 1.1 mm, respectively.33,34
The maximum extracapsular extension at the Mayo an Clevelan Clinics was 4.4 mm
an 10.0 mm, respectively.30,31 Both stuies conclue that brachytherapy margi
ns of 5 mm shoul encompass all extracapsular isease in approximately 99% of ca
ses eeme suitable for raical prostatectomy. Discussion No generally accepte
seeloaing philosophies have been aopte by the brachytherapy community, alth
ough the AAPM an the ABS have recommene peripheral loaing techniques.5,6 Des
pite marke ifferences in planning an intraoperative techniques, biochemical o
utcomes an complication rates have been comparable. Vicini an coworkers report
e the frequency of various prostate brachytherapy preplanning approaches with 2
7% of brachytherapists using a nomogram, 11% a least squares optimization techni

que, 35% ose specification criteria not state, an 11% uniform, 9% ifferentia
lly positione, an 6% peripherally positione.35 In our program, a moifie uni
form loaing approach has been use because of greater ose homogeneity in
The Wheeling approach to treatment
173
Figure 14.4 Profiles illustrating ose homogeneity using moifie uniform loain
g in the patient whose prostate is illustrate in Figure 14.1. (a) Urethral ose
moving inferiorly from the planning target volume (PTV) base at 0 mm to the PTV
apex at 45 mm. (b) Posterior to anterior ose profile at miglan. The posteri
or borer of the PTV is at 0 mm an the interior borer
Basic an avance techniques in prostate brachytherapy
174
at 32.5 mm. The urethra is at +17 an the rectum is below 5 mm.
terms of the DVH slope (Figure 14.3) an orthogonal ose profiles (Figure 14.4).
1,2 In a moifie uniform approach, misplacement of a single see results in lit
tle change to the ose istribution in comparison to peripherally loae techniq
ues.36 Despite the increase number of sees neee to cover our significantly e
nlarge PTV, there is no actual point ose escalation within the prostate glan.
Our approach, however, oes result in a greater integral ose seconary to the
therapeutic oses elivere to the extracapsular region. Yu an coworkers note
the uniformity of the ose within the target volume is poor if all of the sees
are place within the prostate glan, but ose uniformity is improve if peripro
static sees are utilize.37 In contrast, they reporte that the use of extracap
sular sees i not improve the ose coverage to the prostate. Ling an coworker
s reporte a raiobiologic moel of ose inhomogeneity in permanent 125I implant
s with the conclusion that oses 2030% greater than the ose that provies 99% co
verage of the target volume increase tumor cell kill an local control probabil
ity, but that higher oses i not further improve the results.38 One of the mos
t obvious ifferences in implant philosophies is the presence or absence an the
extent of periprostatic margin. In an ABS survey, Prete an coworkers reporte
that approximately 60% of brachytherapists ae margins aroun the prostate gla
n an use periprostatic sees with an average margin of 5 mm.8 Waterman an co
workers reporte that eema ha a minimal effect on ay 0 osimetry when the imp
lant was planne with margin versus prostate only volumes.11,12 Butler an cowor
kers also reporte that small prostates were more aversely affecte by eema th
an large prostates.11 The ientification of the urethra on the preplan further i
mproves the ability to generate a plan that respects urethral tolerance. Volume
parameters such as UV200 an UV150 shoul be kept small as shoul the similar o
simetric parameters UD10 an UD25, which are the minimal oses receive by 10 an
 25% of the urethra, respectively. In the ABS survey, Prete et al reporte that
twothirs of responents ientifie the urethra on the preplan.8 Previously, W
allner et al reporte that urinary morbiity was associate with a maximum ureth
ral ose >400 y.20 The ABS has recommene a moifie peripheral loaing techni
que to minimize the length of the urethra receiving >200% of the prescribe ose
.6 With our technique, we have never encountere a ose of such magnitue (excee
ing either 200% of mPD or 400 y) on the postimplant evaluation. We o not expl
icitly efine rectal oses on the preplan. The posterior borer of the target vo
lume, however, is implante with a osimetric margin of 4 mm. Our group has prev
iously reporte etaile rectal osimetry with the conclusion that the anterior
rectal mucosa can on average receive 100% an 120% of the prescribe ose to len
gths of 10 mm an 5 mm, respectively, without significant rectal toxicity.13,18
The ABS guielines i not aress rectal tolerance or formulate recommenations
.6 Faithful execution of the preplan oes not guarantee equivalent quality param
eters on the postplan. Utilizing our guielines, we have publishe ay 0 osimet

ric results with a V100, V150, an D90 of approximately 94%, 46%, an 108% with
the caveat that these implants use approximately 67% extra sees beyon the prep
lan.13 Resolution of eema is expecte to increase these osimetric values by 510
%.39 Dose is paramount to
The Wheeling approach to treatment
175
securing longterm biochemical control.24 With ay 30 osimetry, Stock an colle
agues reporte a D90 threshol of 140 y for optimal biochemical iseasefree ou
tcome following 125I monotherapy.27 Recently reporte biochemical results, howev
er, have suggeste that the ose threshol in terms of percent mPD may be less f
or 103P than for 125 40,41 I. Conclusions Despite a multitue of preplanning an
 intraoperative techniques an seeloaing philosophies, multiple groups have
reporte comparable results in terms of biochemical iseasefree survival an co
mplication rates. None of the seeloaing philosophies or ifferences in intrao
perative techniques have proven superior. We, however, are strong proponents of
a moifie uniform seeloaing approach because of the ability to eliver a rel
atively homogeneic ose to the prostate glan with a periprostatic margin, the a
bility to routinely maintain the average urethral ose at approximately 115% of
the prescription ose, an the fact that a moifie uniform approach is more forg
iving of local an systemic errors in see placement. References
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in prostate brachytherapy. Me Phys 2000; 27:381392. 2. Merrick S, Butler WM. Mo
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ce strength an source istribution for a transperineal prostate implant. Enocu
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of practice for brachytherapy physics: Report of the AAPM Raiation Therapy Task
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44:789799. 7. Prestige BR. Raioisotopic implantation for carcinoma of the pros
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13. Merrick S, Butler WM, Dorsey AT, et al. Potential role of various osimetri
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V, McShan DL, et al. Source placement error for permanent implant of the prostat
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hytherapy. Int J Raiat Oncol Biol Phys 2003; 55:342353. 30. Merrick S, Butler W
M, Wallner KE, et al. Extracapsular raiation ose istribution following perman
ent prostate brachytherapy. Am J Clin Oncol 2003; 26:E178E189. 31. Butzbach DA,
Waterman FM, Dicker AP. Can extraprostatic extension be treate by permanent pr
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l. The raial istance of extraprostatic extension of prostate carcinoma. Cancer
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rostatic extension in localize prostate cancer. Urology 2000; 55:382386. 35. Vic
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177
36. Roy JN, Ling CC, Wallner KE, Anerson LL. Determining source strength an so
urce istribution for a transperineal prostate implant. Enocurie/Hypertherm Onc
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ions of the minimum peripheral ose as a parameter for ose specification in per
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Ling CC, Roy J, Sahoo N, et al. Quantifying the effect of ose inhomogeneity in
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Raiat Oncol Biol Phys 1994; 28:971978. 39. Yue N, Dicker AP, Nath R, Waterman F
M. The impact of eema on planning 125I an 103P implants. Me Phys 1999; 26:76
3767. 40. Kollmeier MA, Stock R, Stone NN. Biochemical outcomes following prosta
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1. Wallner K, Merrick , True L, et al. I125 versus P103 for low risk prostat

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15 The Seattle Prostate Institute approach to treatment planning for permanent i
mplants
John Sylvester A brief history of prostate raioactive implants In 1903 Alexane
r raham Bell wrote there is no reason why a tiny fragment of raium seale in a f
ine glass tube shoul not be inserte into the very heart of the cancer, thus ac
ting irectly upon the isease material. Woul it not be worthwhile making exper
iments along this line? In 1910, Hugh Hampton Young (eveloper of the raical pro
statectomy) use intraurethral raium for the treatment of prostate cancer, with
encouraging results. He performe approximately 500 prostate brachytherapy proc
eures an 25 raical prostatectomies from 1915 to 1927. In 1930, Flocks first i
njecte raioactive gol into the prostate for the treatment of cancer. In the e
arly 1970s, Willet Whitmore an Basil Hilaris at Memorial SloanKettering Cancer
Center, New York, were the first physicians to perform I125 prostate see impla
nts. An abominal incision was use to implant the sees irectly into the expos
e glan. In 1983, Hans Holm, University of Copenhagen, Denmark, was the first p
hysician to perform the close or nonsurgical implant metho, which utilize transr
ectal ultrasoun (TRUS). In 1985, Haakon Rage, John Blasko, an Peter rimm, fu
rther moifie Holms approach in Seattle, Washington. They began using this treat
ment in November 1985. In 1987 Tim Mate an James ottesman begin a temporary se
e implant program (HDR) at the Sweish Meical Center, Seattle In the 1990s, a
ramatic increase in permanent see implantation occurs in the Unite States. Th
ere are avances in osimetry, patient selection, an implant technique. See im
plantation is linke with osimetry an patient selection. The past 18 years hav
e le to a continual refinement in patient selection, osimetry, an technique.
Backgroun Brachytherapy has been use in the efinitive treatment of prostate c
ancer since the early 1900s. One of the earliest reporte experiences was the se
ries of 100 patients treate by Denning publishe in 1922.1 However, they were n
ot able to accurately measure raiation oses in that era, complication rates we
re significant an control rates poor.
The Seattle Prostate Institute approach to treatment
179
Brachytherapy lost groun to surgery as surgical an anesthetic techniques avan
ce. During the 1960s to 1990s megavoltage external beam raiotherapy (EBRT) bec
ame more popular as it ha relatively fewer sie effects than surgery an simila
r survival rates. In the late 1960s Carlton an Scarino use permanent intersti
tial gol198 (198Au) (raioactive gol) sees combine with EBRT.2 At Memorial
SloanKettering Cancer Center (MSKCC) the use of raioactive ioine125 (125I) s
ees was pioneere.3 Using an open laporatomy approach, the sees were place i
rectly into the surgically expose prostate. The goal was to achieve a uniform 
istribution of sees using a nomogram table to calculate the appropriate number
of sees for a given see activity. In those patients in whom orthogonal xrays
reveale a high quality see istribution (matche peripheral ose of >140 y) a
chieve a local control rate of 60%. In those with a matche peripheral ose of
<120 y the local control was only 20%. Hilaris an colleagues reporte a 70% 15
year survival in B1 patients treate with high quality 125I see implantation.41
0 These results were at least as goo as the best contemporary surgical an EBRT
series in that era. However, the limite technology in the 1970s prevente this
retropubic technique from consistently achieving high quality implants. This in
consistency contribute to brachytherapy, once again, falling out of favor. Tech
nical breakthroughs The 1980s saw the introuction of multiple technologic avan
ces that le to the rebirth of prostate brachytherapy.11 Puthawala et al, at Lon
g Beach Memorial Hospital in southern California, pioneere transperineal low o
se rate temporary interstitial brachytherapy (performe at time of open laporato
my) combine with EBRT.12 Martinez et al use a transperineal applicator to gui

e the placement of the raioactive implant.13 In Denmark, Holm et al were the fi

rst to perform 125I see implantation via a transperineal approach using transre
ctal ultrasoun guiance for the placement of the sources.14 This technique prom
ise to allow consistently accurate placement of raioactive sees. This major t
echnologic avancement combine with improve patient selection ue to prostate
specific antigen (PSA) screening an improve raiation treatment planning syste
ms allowe the Seattle team to consistently eliver high quality implants to app
ropriately stage patients with appropriate oses of raiation base on the MSKC
C experience. Thus, in November 1985 Blasko an Rage preforme the first prepla
nne transrectal ultrasoun guie template guie transperineal permanent 125I
see implant in the Unite States. Dosimetry/treatment planning The primary ava
ntage to permanent see implantation is the ability to eliver significantly hig
her oses of raiation to the prostate an several millimeters (mm) margin in a
single outpatient setting than any other form of raiation therapy. However, os
e inhomogeneity is unavoiable ue to the fact that iniviual raioactive sourc
es (sees) are emitting raiation separately. Thus, hot spots (areas of higher 
oses of raiation) occur ajacent to the sees an coler areas (lower oses of
raiation) further away from the sees will inevitably occur. Despite this inhom
ogeneity the raiobiologic
Basic an avance techniques in prostate brachytherapy
180
effective ose of permanent see implantation has been shown to be higher than m
ore homogeneous raiation therapy approaches, such as threeimensional conforma
l raiotherapy (3DCRT) an intensity moulate raiotherapy (IMRT).15 With inho
mogeneous osimetry, stanarization of the prescription ose is necessary. In t
he ays of the retropubic approach a matche peripheral ose was use to escribe
the ose elivere to a volume equal to an ellipsoi volume with the same averag
e volume of the prostate being treate. This escription can be confusing. Treat
ment planning software an prostate volume evaluations have improve so that mos
t raiation oncologists use minimal peripheral ose (MPD) to escribe their pres
cription ose now. The MPD escribes the minimum ose elivere to the periphery
of the target volume. The target volume may vary from one center to the next. A
t some centers, the target volume equals the prostate with little to no margin,
at others (Seattle Prostate Institute, Schiffler Cancer Center, Seattle Veterans
Aministration, etc.) it equals the prostate plus several millimeters of margin
.11 Most centers prescribe 145 y MPD for 125I monotherapy implants an 120130 y
for pallaium103 (103P) monotherapy implants.16 In Seattle, we currently use
145 y an 110 y for 125I monotherapy an boost implants respectively. We presc
ribe 125 y an 100 y for 103P monotherapy an boost implants. Due to the inho
mogeneity issue two ifferent centers may prescribe the same MPD but have signif
icantly ifferent internal isoose curve osimetry. These variations are common
because of philosophic ifferences from center to center in seeloaing patterns
, see activity, an osimetry (uniform vs peripheral vs moifie approaches). F
rom 1985 to 1991, the Seattle team use a pure uniform osimetry approach. A rel
atively high number of low activity sees were evenly istribute through the pr
ostate. Urethral visualization techniques were not employe. As a result, the ce
ntral prostate oses were in excess of 150300% of the prescription ose (epenin
g on glan size). See spacing was planne to be 1 centimeter (cm) from see cen
ter to see center. This form of osimetry has the avantage of requiring less p
recision in see placement, as each see contributes less than 1% of the total p
rescription ose. However, there is a scallopingin effect at the eges of the i
mplant, which coul result in unerosing the eges of the glan, an there is a
n overosage of the central (urethral) portion of the glan (Figure 15.1). These
high central oses resulte in increase urinary morbiity in patients who prev
iously, or subsequently, unerwent a transurethral resection of the prostate (TU
RP).17 A pure peripheral implant is one in which the sees are place aroun the
periphery of the glan, usually just insie the capsule. These implants typical

ly require a lower number of higher activity sees. One woul expect these patie
nts (especially TURP patients) to experience less urinary toxicity ue to lower
urethral oses (Figure 15.2).18 Unfortunately, even slight misplacement of a few
sees can result in unerosage of the periphery of the prostate an overosage
of the urethra (if sees migrate centrally) or rectum (if sees migrate posteri
orly). The lower number of sees in these implants ecreases the cost of the imp
lant, but these are technically more challenging to perform. The initial biochem
ical relapsefree survival (BRFS) reports from centers using this technique were
not as high as those achieve with the Seattle approach.19,20
The Seattle Prostate Institute approach to treatment
181
Figure 15.1 Uniform loaing.
Figure 15.2 Peripheral loaing.
The moifie uniform peripheral loaing osimetric approach merges the best of b
oth philosophies. This planning approach (like the uniform approach) still uses
a relatively high number of low activity sees, thus ecreasing the osimetric e
rrors that woul occur if a see is misplace or migrates after it is place. Ho
wever, aitional sees are place in the periphery of the glan, the base, an
apex, an fewer sees are place centrally ajacent to the urethra. This reuces
the likelihoo of a scallopingin effect of the isoose curves at the glan per
iphery an reuces the ose to the urethra. This woul be expecte to result in
improve BRFS an less urethral toxicity than the pure uniform or pure periphera
l approaches. As a result, the vast majority of centers in the Unite States (in
cluing the Seattle Prostate Institute) currently use this osimetric approach (
Figure 15.3). The neele loaing pattern use in the Seattle Prostate Institute
is simple, symmetrical, an utilizes a minimum number of neeles. No plans with
only one see in a neele are ever accepte, an the num
Basic an avance techniques in prostate brachytherapy
182
Figure 15.3 Moifie uniform loaing.
ber of neeles with only two sees is kept to a minimum (Figure 15.4). Dose The
Memorial SloanKettering Cancer Center (MSKCC) note better local control rates
(in the retropubic era) when postoperative (postop) osimetry reveale a matche
peripheral ose of >140 y. In 1985, Dr Blasko of the Seattle team chose to pre
scribe a ose of 160 y (144 T43) minimal peripheral ose (MPD) in orer to achi
eve 140 y on postop osimetry. Fortunately, this chosen ose seems to have subs
equently been supporte by outcome analyses. rimm et al publishe local control
rates of 97%, base on igital rectal examination (DRE) an postimplant biopsie
s in patients treate with 125I monotherapy treate with a prescription ose on
160 y (144y, T43).21 Further support of this general ose level comes from r
eporte experiences in New York City from Stock et al at Mt Sinai Meical Center
an also from Potters an colleagues at MSKCC. Stock reporte improve BRFS in
patients treate with 125I achieving an MPD of 140 y on postop osimetry (comput
er tomographicbase).19 Potters et al note better BRFS in patients achieving a
D90 of > 90% of the prescription ose treate with 103 P or 125I monotherapy.2
2 These stuies were single institution retrospective reviews, involving a relat
ively small number of patients. However, base on the ata from Seattle, Mt Sina
i, an the ol retropubic an newer moern transrectal ultrasoun (TRUS) guie
ata from MSKCC, a ose of approximately 140 y for 125I monotherapy or a D90 of
90% in monotherapy patients (prescribe to approximately 145 y MPD for 125I an
 125130 y 103P, T43) seems reasonable. Using the moifie uniform approach a
significant portion of the peripheral zone of the prostate falls within the 150
%

The Seattle Prostate Institute approach to treatment

183
Figure 15.4 Neele loaing report.
isoose curves, this translates into a ose of 215 y (in 125I monotherapy cases
). Isotope selection Pallaium103 an ioine125 have ifferent photon energies
an halflives, thus ifferent prescription oses are use in orer to achieve
virtually ientical BRFS an long term toxicity outcomes. Much is written about
isotope selection because of these ifferences, but both 103P an 125I isotopes
are very low energy level sources (21 KeV an 28 KeV, respectively). Both are p
rescribe to very high oses compare to 3DCRT an IMRT. The big picture is tha
t the similarities outweigh the ifferences. Not surprisingly, there is no convi
ncing clinical evience that one isotope is superior to the other in terms of BR
FS or toxicity. In general, the Seattle team usually uses 125I for leason score
s 27 an 103P for leason scores 610. Since the majority of the patients treate
have leason scores 6 an 7, the isotope use epens more on physician an
Basic an avance techniques in prostate brachytherapy
184
patient preference than clinical evience. For example, if one prefers to use a
see with a low chance of migration 125I RAPID Stran woul be use. If one wante
 the raiation exposure to relatives to be a shorter uration 103P woul be us
e. The see activity orere by the Seattle Prostate Institute physicians is us
ually 0.27 mCi for 125I boosts an 0.297 or 0.326 for larger an smaller glans
treate with 125I monotherapy. For 103P we use 1.1 mCi for boosts an 1.51.7 mCi
for monotherapy implants. Limiting the activity to a selecte number of activit
ies per isotope helps to improve the consistency of the implants an leas to le
ss chance of errors. The Seattle Prostate Institute approach Treatment planning
New patients usually unergo a transrectal ultrasoun volume stuy (TRUVS) imme
iately prior to their initial consultation. This stuy, one in the clinic witho
ut anesthesia or antibiotics, is use to map out precisely the size an shape of
the prostate, rule out pubic arch blockage problems, an evience of prior surg
ery (transurethral resection of the prostate; TURP). Thus, it is use for treatm
ent planning an to ai in patient selection. This allows for a more etaile an
 thorough initial consultation. Some centers prefer to carry out this investiga
tion in the operating room (OR) at the time of the implant proceure itself. The
patient unergoes a bowel prep prior to entering the clinic, incluing an enema
. He is positione in the treatment position (extene lithotomy). The probe is
place into the rectum an the prostate ientifie on the ultrasoun monitor. Th
e miglan is ientifie an centere on the brachytherapy treatmentplanning g
ri. The posterior ege of the glan is positione approximately 1 mm below the
posterior row. We use row number 2 (instea of 1.5 for Siemens an row 1.5 instea
 of 1.0 for B&K ultrasoun equipment) as the posterior row because this places l
ess pressure on the prostate itself. This results in an image that is not as cle
ar an sharp (vs that achieve if row 1.0 is the posterior row), but the image q
uality is still acceptable. Our philosophy is that avoiance of glan shape ist
ortion is more important. Thus, the patient is more comfortable (an still) an
the glan is not as istorte as it woul be if we use row number 1. Iniviual
transverse images of the prostate are capture by the ultrasoun technologist i
n 5 mm increments, from the base an to the apex. On each image, the prostate ci
rcumference is outline an printe. Sagittal images are taken to verify the len
gth of the prostate. The pubic arch is then ientifie an overlai on the large
st prostate image. The frequency use is 5.0 megahertz (MHz), this frequency pen
etrates the glan better than higher frequencies, allowing better visualization
of the anterior aspect of the prostate. The base image is ientifie as the 0.0
retraction plane, the slice ajacent to the base is the 0.5 retraction plane, an

 so on until the final apical slice is ientifie an printe. The number of tr
ansverse images shoul be equal to two times the length of the glan plus 1. A 3
.5 cm long prostate shoul have eight transverse images printe out. If the pati
ent is known to have a history of a TURP, a urethrogram is performe at the time
of the volume stuy to better ientify the TURP efect. Every effort is use to
avoi prostate istortion an the probe angle is measure an note for future
reference.
The Seattle Prostate Institute approach to treatment
185
The TRUVS images are evaluate by the raiation oncologist, usually at the time
of the initial consultation. Target volumes will then be outline if the patient
ecies to procee with permanent interstitial raioactive see implantation. T
arget volumes an implant osimetry Target volumes can be outline by the raiat
ion oncologist irectly on the printe TRUVS images, or on the treatmentplannin
g computer. In Seattle, we outline irectly on the printe image, as it is faste
r for the physician. The osimetrist then transfers the prostate outlines an th
e target volume outlines into the treatmentplanning system. The goal is to aeq
uately cover, with our 100% isoose curve, the prostate an several millimeters
of margin outsie the prostate. This aitional margin is not circumferential.
There is more margin given laterally than posteriorly or anteriorly. The anter
iormost extent of our planning target volume (PTV) is the anterior most extent
of the prostate itself. The posteriormost extent is our posterior row (row 2.0
for Siemens an 1.5 for B&K) (Figure 15.5). In general the PTV outline for any on
e particular transverse image approximately equals the prostate volume of which
ever ajacent transverse prostate volume is largest. The margins are particularl
y large at the base an the apex. The base an apex have no ajacent planes (cra
nial or caual) to place sees, so to achieve an aequate ose at these extreme
ens of the implant we a extra sees laterally an anteriorly at the apex an
laterally an posteriorly (in the seminal vesicles) at the base. This preplan is
run by the osimetrist. Then it is reviewe an tweake by the raiation oncolo
gist. Then it is reviewe by the physicist, an finally approve by the raiatio
n oncologist. This preplan can be thought of as a roa map that the raiation on
cologist an urologist use in the OR. The preimplant osevolume histograms (DVH
) for 125I implants are V100 of 99100%, V150 of 3040%, an V200 of less than 20%.
The DVHs for 103P are similar except that V150 is planne for approximately 50%
. When using strane sources the central neeles ajacent to the urethra are al
ways loae with loose sees, not strane sees, even when a special loa (fewer
sees in the mile of the neele an more spacers) is not neee. Typically, th
e central 4 neeles are special loas to keep the urethral ose less than 120150% o
f the prescription ose. The integration of a high number of raioactive sees 
istribute in a symmetrical moifie uniform loaing
Basic an avance techniques in prostate brachytherapy
186
Figure 15.5 The Trus volume stuy.
osimetry plan that inclues a margin beyon the prostate, results in a robust a
n relatively bulletproof treatment plan. The Seattle Prostate Institute uses thi
s approach because one can safely moify the neele positions or the ultrasoun
probe angle in the OR if the prostate shape or size is somewhat ifferent than n
ote uring the preplan, without sacrificing the ultimate osimetric outcome. Mo
reover, it is reproucible from one raiation oncologist/ urologist team to anot
her an requires minimal OR time an personnel. The symmetry of the implant an
basic pattern reuces the possibility of placing a neele into the wrong positio
n. Implant technique The proceure itself is performe in the ambulatory surgica
l center at Sweish Hospital. A team approach combining the expertise of the ra

iation oncologists an regional urologists has always been utilize. The OR team
typically consists of the raiation oncologist, the urologist, the anesthesiolo
gist, the nuclear meicine technologist (seeorerer, seeloaer, an room rai
ation surveyor), an two OR nurses. We typically implant 57 patients a ay three
ays a week. The proceure can be performe uner general or spinal anesthesia,
95% of our cases are one uner the latter. The time it takes from the start of
one case to the start of the next (in the same OR room, incluing room turnover,
anesthesia, patient positioning, equipment setup, see implantation, fluorosco
py, cystoscopy, postimplant surgery notes, ischarge instructions an orers, an
 iscussion with family members) is typically 1 hour. The see implantation por
tion itself takes about 1525 minutes. The nurses retrieve the neele box containi
ng the neeles in their proper gri coorinates alreay preloae by the nuclear
meicine technologist (in the ajacent room) with the selecte isotope an acti
vity (Figure 15.6). The activity an loaing pattern an isotope is ouble check
e by the raiation oncologist. After spinal anesthesia is accomplishe the pati
ent is precisely positione squarely an symmetrically on the OR table, in an ex
tene lithotomy position (Figure 15.7). Patients with larger prostate
The Seattle Prostate Institute approach to treatment
187
volumes typically are a bit more extene than those with smaller prostate volum
es. The ultrasoun probe is inserte visualizing the prostate. The miglan is
centere on the gri as per the volume stuy (Figure 15.8). The glan height, th
ickness, an with are oublechecke. The urethra is visualize with aerate KY
Jelly (Figure 15.9). This is use instea of a catheter because it will not ist
ort the shape of the urethra or prostate as can a catheter, an it allows clear
visualization of the sulci on either sie of the veru montanum. After correct po
sitioning of the patient, ultrasoun probe, an connection of probe to the stabi
lization apparatus (which hols the ultrasoun probe, stepper unit, an neele g
uiance template), the neele insertion begins. The base is reverifie; the base
image shoul show a small amount of prostate anterior to the seminal vesicles (a
hea an bowtie appearance) (Figure 15.10). Iniviual preloae neeles are ins
erte one at a time, one row at a time in the transverse image moe with the ima
ge 1.0 cm (2 clicks on the stepper unit) from the base. The neeles are inserte
at the 1.0 retraction plane (instea of at the 0.0 retraction plane) for three
reasons: (1) to better visualize each neele; (2) to avoi blaer trauma; an (
3) to more reaily visualize prostate rift (Figure 15.11). We work one row at a
time moving from the anterior most row (placing the neeles an then epositing
those neeles sees) to the secon anteriormost row epositing their sees, an
so on
Figure 15.6 Preloae neeles with sees.
Basic an avance techniques in prostate brachytherapy
188
Figure 15.7 The extene lithotomy position.
until the final posteriormost row of neeles an their sees are eposite. Eac
h neele place has its position calle off by the OR nurse, an ouble checke
by the raiation oncologist, on the secon copy of the plan attache to the ultr
asoun unit. After the neele is place by the raiation oncologist or urologist
the final positioning is ouble checke (in the x an y imensions) by both ra
iation oncologist an urologist. After a typical row of neeles is inserte, un
er transverse imaging, to a epth that usually is at the 1.0
Figure 15.8 The ultrasoun gri: miglan.

The Seattle Prostate Institute approach to treatment

189
Figure 15.9 Urethra visualize clearly by using aerate KY JellyTM
retraction plane the spacing is ajuste so that each neele is approximately 1.
0 cm apart from each other an the central neeles are a minimum of 5 mm away fr
om the urethra (from base to apex) (Figure 15.12). Then the neeles are avance
to their final preplanne retraction plane (e.g. 0.0 or 0.5) an any final x a
n ycoorinate ajustments are mae. Then the epth of the neele is ouble che
cke by measuring with a ruler (Figure 15.13). Finally, the probe is line up on
the lateral most neele an switche to the sagittal image moe. It is in the s
agittal image moe that final epth ajustments are mae, by the raiation oncol
ogist, an verifie by the urologist (Figure 15.14). The stylet istance from th
e neele hub is measure to ensure proper see positioning within the neele an
the neele is then slowly withrawn, while holing the stylet in place, sowing
a row of raioactive 103P or 125I sees (Figures 15.15 an 15.16). This process
continues one row at a time from the anteriormost row to the posteriormost row
. During the proceure the prostate position within the ultrasoun gri is conti
nually ajuste. The base plane is also closely tracke an movement of the pros
tate in the cranial irection ajuste for. The combination of transverse imagin
g at the 1.0 retraction plane an see epositing at the correct pre
Basic an avance techniques in prostate brachytherapy
190
Figure 15.10 The base shoul be ientifie accurately. The prostate an seminal
vesicles shoul be seen (a hea an bowtie appearance).
planne epth uner the sagittal image plane makes it easy to make these frequen
t minor ajustments throughout the implant proceure, thus eliminating the chanc
e of unerosing the base or overosing the rectum or urethra. When working on t
he posterior row the neeles are inserte so that 23 mm of prostate tissue is vis
ible poste rior to the neeles in the mi plane (Figure 15.17). The neeles are
tracke on transverse an/or sagittal imaging from base to apex to ensure proper
istance exists between the neele an the rectum. Ajusting the angle of the p
robe can create more space between the neele an the rectum at the apex of the
prostate (Figure 15.18). Sometimes, the posterior row neeles are inserte in th
e template at a higher row number (i.e. row 2.5 instea of the usual 2.0 level)
so that the angle conforms to the rectum in a more optimal orientation, thus avo
iing having the neele an sees too close to the rectum at the apex of the pro
state yet still posterior enough at the base of the prostate. A final fluoroscop
ic an ultrasoun qualitative evaluation is carrie out at the en of the proce
ure. Postoperative compute tomography (CT) quantitative evaluation is one the
next ay as an outpatient. The postop plan is evaluate not only for DVH values
but also for isoose curve shapes an coverage. The goal being aequate coverage
of the prostate plus a lateral margin an avoiance of oses over 150% of the p
rescription ose to any portion of the urethra an the rectum. Urethral oses us
ually fall in the 95120% isoose ranges (Figure 15.19). Results The goal of see
implantation is elimination of prostate cancer, thus patients can avoi the ebi
litating effects of
The Seattle Prostate Institute approach to treatment
191
Figure 15.11 Insert neeles while 1 cm from base.
Figure 15.12 A row of neeles in position.

Basic an avance techniques in prostate brachytherapy

192
Figure 15.13 Establish a reference epth.
Figure 15.14 Final epth ajustments in the sagittal image moe.
The Seattle Prostate Institute approach to treatment
193
Figure 15.15 See number verification.
Figure 15.16 Withraw neele over stylet.
Basic an avance techniques in prostate brachytherapy
194
Figure 15.17 In the posterior row of neeles, 23 mm of prostate tissue is visible
.
metastatic isease an eath, while experiencing a reasonably goo quality of li
fe an minimal inconvenience. Surrogate enpoints are use, such as biochemical
relapsefree survival (BRFS). As note above, ata closely link BRFS with postimp
lant CT osimetry. The Seattle Prostate Institute has recently evaluate the ay
1 postop osimetry of over 2000 consecutively treate patients. The patients we
re treate with the TRUVS, osimetric planning philosophy, an the OR technique
note above. Day 1 postop osimetry reveals consistent achievement of high quali
ty implants. The average D90 is 102% of the prescription ose (Table 15.1). A sm
all number of iniviual patients were uner a D90 of 90%, but virtually all of
these patients ha unergone prior TURP an were purposely planne
The Seattle Prostate Institute approach to treatment
195
Figure 15.18 The angle position of the ultrasoun probe allows more space betwe
en the neele an the rectum at the apex of the prostate.
Figure 15.19 Postimplant CTbase osimetry.
Basic an avance techniques in prostate brachytherapy
196
Table 15.1 All patients (2115): ay I osevolume histogram (DVH) analysis: 4/2003
. (Reprouce with the permission of the Seattle Prostate Institute.)
Vol. No. sees V100 V150 D90
Mean 37 113 91% 55% 102%
to receive a slightly lower ose to the TURP efect region. Currently, the shape
of the isoose curves within the implant volume is analyze to ensure aequate
margin beyon the ege of the prostate is obtaine an the urethra achieves less
than 150% of the minimal peripheral ose (MPD) on every transverse image. Bioch
emical relapsefree survival Biochemical relapsefree survival (BRFS) is use as
the enpoint for isease control ue to the long natural history of prostate ca
ncer, which results in a large portion of patients (even biochemical failure pat
ients) ying of other iseases other than prostate cancer. The BRFS reports from
the Seattle Prostate Institute concern patients iagnose an treate in the la

te 1980s an early 1990s. These patients are from a ifferent era than those cur
rently treate with 3DCRT, IMRT, an HDR brachytherapy. Multiple stuies from t
he surgical an raiotherapy literature show that patients treate a ecae ago
o more poorly in terms of BRFS than patients toay. This is probably ue to a v
ariety of factors, such as improve leason scoring, stage migration, improve i
maging, improve treatment techniques, PSA screening, etc. Thus, it is impressiv
e that the results of see implantation in these patients from 1987 to 1994 (man
y of whom were leason unerscore, ha no postop CT osimetry, were treate wit
hout sagittal imaging, an with reusable neeles) compare so favorably to more r
ecent series. rimm reporte the Seattle experience with 125I monotherapy. From
1988 to 1990 a total of 125 patients were consecutively treate with 125I.21 The
se were low an intermeiate risk patients, base on the risk cohort system use
by MSKCC, Seattle Prostate Institute, an others. The average followup of the
nonecease patients was 94.5 months. The local control rate by igital rectal
examination (DRE) an biopsy was 97%. The metastatic isease rate was 3%. The lo
w an intermeiate risk patients experience a 10 year BRFS of 87% an 76%, resp
ectively. A moification of the American Society of Therapeutic Raiology an On
cology (ASTRO) efinition of BRFS is use in all the Seattle Prostate Institute
reports where only two rises in prostatespecific antigen (PSA) are neee to e
fine a patient as a failure instea of the ASTRO criteria of three consecutive r
ises. This increases the sensitivity of etecting a biochemical failure by appro
ximately 20%. There were a significant proportion of patients in this an all th
e Seattle reports that were classifie by the community hospital general patholo
gists as having leason score 24 isease. These patients were in all likelihoo u
nergrae as they were iagnose by ultrasounguie neele biopsies, the pati
ents assigne leason score 24 cancers BRFS were slightly (not statistically sign
ificant) worse than the leason score 56 patients, an when ranomly reviewe lat
er were upgrae by an
The Seattle Prostate Institute approach to treatment
197
average of two leason scores. This unergraing phenomenon has also been report
e elsewhere in the urologic literature.2326 Blasko et al reporte the Seattle ex
perience with 103P monotherapy.27 These patients ha higher leason scores than
the patients treate with 125I monotherapy rimm et al reporte.21 A total of 2
32 patients were treate with 103P monotherapy from 1/89 to 12/95, the average
followup was 49 months. The local control rate was also 97%. The metastatic is
ease rate was 6%. The 5 year BRFS for the low, intermeiate, an high risk patie
nts was 94%, 82%, an 65% respectively (but only a small number of high risk pat
ients were treate with 103P monotherapy). Those patients with leason score 710
isease an a PSA 10 ng/mL experience a 5 year BRFS of 80%. None of the patient
s in the above reports by Blasko an rimm receive any anrogen ablation therap
y. Sylvester et al reporte on the patients treate with anrogen ablation plus
either 125I or 103P monotherapy. These patients receive an average of 5 months
of anrogen ablation for a variety of reasons incluing ownsizing, high risk f
eatures, an/or for patients to have time to think about their options (the latt
er was usually by outsie nonSeattle physicians). The 5 year BRFS for the low, i
ntermeiate, an high risk patients was 90%, 80%, an 60%, respectively, both fo
r those patients treate with see implantation alone an also for those treate
with anrogen ablation plus see implantation. Thus, the aition of short cour
se anrogen ablation to see implantation i not improve (or worsen) the BRFS i
n any risk group. The mean followup was similar in each group.28 The Seattle ex
perience with short course anrogen ablation combine with EBRT plus 125 I or 10
3P (combination raiation therapy) in high risk patients reveale no significan
t improvement in the 5 year BRFS.29 The BRFS of patients treate with anrogen a
blation plus combination raiation therapy was no better than that experience b
y patients treate with combination raiation therapy alone. Blasko et al report
e the 5 year BRFS of patients treate with 125I or 103P with or without extern

al beam raiotherapy (EBRT).30 Again there was no significant improvement in BRF

S in those that receive the supplemental EBRT. However, this was a retrospectiv
e review an the patients typically receive the supplemental EBRT for a reason,
they were felt to be higher risk (within each risk group) than the patients tre
ate without the supplemental beam. They often receive the supplemental beam be
cause of a higher number of positive biopsy cores involve with isease on sexta
nt biopsies or perineural invasion. Thus, this paper was not able to answer the
question whether or not the aition of supplemental EBRT will improve BRFS. Syl
vester et al recently reporte the 10 year BRFS of the Seattle patients treate
with EBRT plus 125I/103P. The 10 year BRFS for the low, intermeiate, an high
risk patients treate with combination therapy were 85%, 77%, an 47%, respectiv
ely using the simple risk grouping metho (SR) typically use by us, MSKCC, etc
.31 If the DAmico risk grouping (DR) formula is use the BRFS for the low, inter
meiate, an high risk groups were 86%, 90%, an 48%, respectively. When the Mt
Sinai risk grouping system (MR) favore by Stock an Stone19 is use, the 10 ye
ar BRFS were 84%, 93%, an 57% for the low, intermeiate, an high risk groups,
respectively. The 10 year BRFS of intermeiate risk patients treate with 125I/1
03P monotherapy uring that time perio was 73% an 79% using the SR an MR m
ethos, respectively. The 5 year BRFS
Basic an avance techniques in prostate brachytherapy
198
outcomes of raical prostatectomy at The Hospital of the University of Pennsylva
nia an at Brigham an Womens Hospital, an of 3DCRT raiotherapy at MSKCC an o
f 103P see monotherapy in Seattle are shown in Table 15.2. Also in Table 15.2
are the 10 year BRFS of 125I monotherapy an of combination see implantation pl
us 45 y EBRT (using three ifferent risk grouping systems). Sie effects Tempor
ary acute sie effects of see implantation are expecte to inclue frequent uri
nation, urgency to urinate, an a slow urinary stream. These sie effects occur
in virtually all implante patients to some egree. The risk of urinary incontin
ence in the Seattle experience in patients without TURPs is less than 1%. Those
patients with a history of prior TURP who were treate in the late 1980s with un
iform loaing osimetry experience a greater than 30% rate of urinary incontine
nce (usually stress incontinence).32 Treatment with alpha blockers usually relie
ves acute obstructive symptoms significantly, until prostate swelling has subsi
e.33 These acute obstructive urinary symptoms resolve in the majority of patien
ts by 612 months status postimplantation.34 In orer to reuce the ose to the ur
ethra, sees are place more than 5 mm from the urethra at time of implantation.
The urethra is visualize with aerate KY Jelly.35 The risk of rectal complicatio
ns with see implantation monotherapy is 3% in the Seattle experience an 7% if
EBRT is combine with see implantation. The vast majority of patients who exper
ience raiation proctitis have grae 1 or grae 2, an respon well to supposito
ries, stool softeners, an a low roughage iet, if any treatment is neee at al
l.32,36 We recommen that biopsies an/or electrocauterization of the anterior r
ectal wall ajacent to the prostate are avoie. These invasive maneuvers may le
a to ulcer an/or fistula formation. Summary Moern transperineal permanent 125
I/103P see implantation of the prostate has increase in popularity over the p
ast 20 years because of technical avances, such as transrectal ultrasoun an t
ransperineal guiance, an computerize treatment planning, as well as early ia
gnosis ue to PSA screening, an improve information flow aie in part by the
internet an meia. The current Seattle approach attempts to minimize potential
for treatment errors, postoperative osimetry variance an complications by comb
ining preplanning with a symmetrical moifie
Table 15.2 Biochemical relapsefree survival (BRFS) outcomes at 5 years an 10 y
ears
Risk group 5 yr BRFS 10 yr BRFS Surgery 3DCRT Sees Sees Sees+EBRT 38 37 27 2
1 DAmko Zelefsky Bksko rimm Sylvester31 (HUP) (B&W) (MSKCC) (Seattle) (Seattle)

(Seattle) (Seattle) (Seattle)

The Seattle Prostate Institute approach to treatment
199
(DR) (DR)
(SR)
(SR) (SR) (SR)
(MR)
(DR)
Low 85% 83% 90% 94% 87% 85% 84% 86% Intermeiate 65% 50% 70% 82% 76% 77% 93% 90%
High 22% 28% 47% 65% 47% 57% 48% 3DCRT, threeimensioual conforms! raiothera
py; DR, DAmico risk grouping; SR, simple risk grouping; MR, Mt Sinai risk grou
ping; HUP, Hospital University of Pennsylvania; B&W, Brighouse an Williams,
uniformperipheral loaing philosophy (which treats the prostate plus a 48 mm mar
gin), meticulous intraoperative technique, an careful patient selection. The 5
an 10 year BRFS with this one ay outpatient treatment matches or excees the b
est reports in the surgical an EBRT literature. The Seattle results with prosta
te brachytherapy are currently being reprouce at several other centers. Refere
nces
1. Denning CL. Carcinoma of the prostate seminal vesicles treate with raium. S
urg ynecol Obstet 1922; 34:99118. 2. Scarino P, Carlton C. Combine interstitia
l an external irraiation for prostatic cancer. In: Javapour N, e. Principles
an management of urologic cancer. Baltimore, Williams & Wilkins, 1983:392408. 3
. Whitmore WF Jr, Hilaris B, rabstal H. Retropubic implantation to ioine 125
in the treatment of prostatic cancer. J Urol 1972; 108:918920. 4. DeLaney TF, Shi
pley WU, OLeary MP, et al. Preoperative irraiation, lymphaenectomy, an 125ioi
ne implantation for patients with localize carcinoma of the prostate. Int J Ra
iat Oncol Biol Phys 1986; 12:17791785. 5. Fuks Z, Leibel SA, Wallner KE, et al. T
he effect of local control on metastatic issemination in carcinoma of the prost
ate: longterm results in patients treate with 125I implantation. Int J Raiat
Oncol Biol Phys 1991; 21:537547. 6. iles M, Bray LW. 125Ioine implantation a
fter lymphaenectomy in early carcinoma of the prostate. Int J Raiat Oncol Biol
Phys 1986; 12:21172125. 7. Kuban DA, elMahi AM, Schellhammer PF. I125 interst
itial implantation for prostate cancer. What have we learne 10 years later? Can
cer 1989; 63:24152420. 8. Schellhammer PF, Whitmore WF, Kuban DA, et al. Morbiit
y an mortality of local failure after efinitive therapy for prostate cancer. J
Urol 1989; 141:567571. 9. Kovacs , alalae R, Loch T, et al. Prostate preservat
ion by combine external beam an HDR brachytherapy in noal negative prostate c
ancer. Strahlenther Onkol 1999; 175(suppl 2):8788. 10. Hilaris B, Fuks Z, Nori D,
et al. Interstitial irraiation in prostatic cancer: Report of 10year results.
In: Rolf, e. Interventional raiation therapy techniques/brachytherapy. Berlin
: Springer, 1991:235. 11. Sylvester J, Blasko JC, rimm P, et al. Interstitial i
mplantation techniques in prostate cancer. Surg Oncol 1997; 66:6575. 12. Puthawal
a A, Sye A, Tansey L. Temporary iriium implant in the management of carcinoma
of the prostate. Enocurie Hypertherm Oncol 1985; 1:2533. 13. Martinez A, Emuns
on K, Cox RS, et al. Combination of external beam irraiation an multiplesite
perineal applicator (MUPIT) for treatment of locally avance or recurrent pros
tatic, anorectal, an gynecologic malignancies. Int J Raiat Oncol Biol Phys 198
5; 11:391 398.
Basic an avance techniques in prostate brachytherapy

200
14. Holm HH, Juul N, Peersen JF, et al. Transperineal 125ioine see implantat
ion in prostatic cancer guie by transrectal ultrasonography. J Urol 1983; 130:
283286. 15. Ling CC. Permanent implants using Au198, P103 an I125: raiobiol
ogical consierations base on the linear quaratic moel. Int J Raiat Oncol Bi
ol Phys 1992; 23:8187. 16. Nag S, Beyer D, Frielan J, et al. American Brachythe
rapy Society (ABS) recommenations for transperineal permanent brachytherapy of
prostate cancer. Int J Raiat Oncol Biol Phys 1999; 44:789799. 17. Talcott JA, Cl
ark JA, Stark PC, Mitchell SP. Longterm treatment relate complications of brac
hytherapy for early prostate cancer: a survey of patients previously treate. J
Urol 2001; 166:494499. 18. Wallner K, Lee H, Wasserman S, Dattoli M. Low risk of
urinary incontinence following prostate brachytherapy in patients with a prior t
ransurethral prostate resection. Int J Raiat Oncol Biol Phys 1997; 37:565569. 19
. Stock R, Stone NN, Tabert A, et al. A oseresponse stuy for I125 prostate
implants. Int J Raiat Oncol Biol Phys 1998; 41:101108. 20. Wallner K, Roy J, Zel
efsky M, et al. Shortterm freeom from isease progression after I125 prostate
implantation. Int J Raiat Oncol Biol Phys 1994; 30:405409. 21. rimm P, Blasko
J, Sylvester JE, et al. 10year biochemical (prostatespecific antigen) control o
f prostate cancer with 125I brachytherapy. Int J Raiat Oncol Biol Phys 2001; 5
1:3140. 22. Potters L, Cao Y, Calugaru E, et al. A comprehensive review of CTbase
 osimetry parameters an biochemical control in patients treate with permanen
t prostate brachytherapy. Int J Raiat Oncol Biol Phys 2001; 50:605614. 23. Stein
berg D, Sauvageot J, Piantaosi S, Epstein J. Correlation of prostate neele bio
psy an raical prostatectomy leason grae in acaemic an community settings.
Am J Surg Pathol 1997; 21:566576. 24. Epstein J. leason score 24 aenocarcinoma o
f the prostate on neele biospy. Am J Surg Pathol 2000; 24:477478. 25. Iczkowski
KA, Bostwick D. The pathologist as optimist: cancer grae eflation in prostati
c neele biopsies [Eitorial]. Am J Surg Pathol 1998; 22:11691170. 26. Allsbrook
WC Jr, Mangol KA, Johnson MH, et al. Interobserver reproucibility of leason g
raing of prostatic carcinoma: urologic pathologists. Hum Pathol 2001; 32:7480. 2
7. Blasko JC, rimm PD, Sylvester JE, et al. Pallaium103 brachytherapy for pro
state carcinoma. Int J Raiat Oncol Biol Phys 2000; 46:839850. 28. Sylvester JE,
Blasko JC, rimm P, Cavanagh W. 125Ioine/ 103Pallaium brachytherapy with or
without neoajuvant brachytherapy for early stage prostate cancer. Int J Raiat
Oncol Biol Phys [Abstract] 2000; 48:310. 29. Sylvester JE, Blasko JC, rimm P,
et al. Impact of short course anrogen ablation on the biochemical progression
free survival of high risk prostate cancer patients manage with permanent brach
ytherapy. Int J Brachyther 2001; JulySept: 173180. 30. Blasko JC, rimm PD, Sylv
ester JE, Cavanagh W. The role of external beam raiotherapy with I125/P103 b
rachytherapy for prostate carcinoma. Raiother Oncol 2000; 57:273278. 31. Sylvest
er JE, Blasko JC, rimm P, et al. 10 year biochemical relapse free survival fun
ctions following brachytherapy with external beam raiotherapy for patients with
localize prostate cancer: the Seattle experience. Int J Raiat Oncol Biol Phys
2003; 57:944952. 32. Blasko JC, rimm PD, Rage H. 6 an 7 year results of perma
nent see implantation. In: Transperineal brachytherapy: Into the mainstream. Se
attle, WA: Pacific NW Cancer Founation, 1995. 33. rier D. Complications of per
manent see implantation. J Brachyther Int 2001; 17:205210. 34. Lee WR, Hall MC,
McQuellon RP, et al. A prospective qualityoflife stuy in men with clinically
localize prostate carcinoma treate with raical prostatectomy, external beam r
aiotherapy, or interstitial brachytherapy. Int J Raiat Oncol Biol Phys 2001; 5
1:614623.
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201
35. Sylvester JE, rimm PD, Blasko JC. Urethral visualization uring transrectal
ultrasoun guie interstitial implantation for early stage prostate cancer. In
: Annual Meeting of the Raiological Society of North America, Chicago, IL, 1998

. 36. Snyer K, Stock R, Hong S, et al. Defining the risk of eveloping grae 2
proctitis following 125 I prostate brachytherapy using a rectal osevolume hist
ogram analysis. Int J Raiat Oncol Biol Phys 2001; 50:335341. 37. Zelefsky MJ, Fu
ks Z, Hunt M, et al. High ose raiation elivere by intensity moulate confor
mal raiotherapy improves the outcome of localize prostate cancer. J Urol 2001;
166:876881. 38. DAmico AV, Whittington R, Malkowicz SB, et al. Clinical utility o
f the percentage of positive prostate biopsies in efining biochemical outcome a
fter raical prostatectomy for patients with clinically localize prostate cance
r [See comments]. J Clin Oncol 2000; 18:11641172.
103
16 P brachytherapy: rationale, esign, an evaluation
Michael J Dattoli The controversy: pallaium103 vs ioine125
Pallaium103 (103P) an ioine125 (125I) are the most commonly use raioisot
opes for permanent prostate brachytherapy. The choice of pallaium versus ioine
is typically base on physician preference, although it is sometimes riven by
the patient. While the controversy continues regaring which is the superior sou
rce, pallaium has long been my isotope of choice, a preference that ates back
to my experience with both 103P an 125 I at New York University Meical Center
an Memorial SloanKettering in the mi 1980s. My research an clinical practice
in Tampa uring the following ecae an my more recent Sarasota experience hav
e confirme the avantage of 103P even for low grae prostate malignancies. Whi
le there have been no efinitive (prospective, ranomize) human clinical trials
to ate comparing tumorcontrol rates with 103P an 125I, stuies have reporte
a lower complication rate for 103P,1 an a faster recovery rate from raiation
inuce prostatitis.2 Peschel an colleagues also note that the raiobiology mo
el preicts the log 10 cell kill for 103P implant will be greater than that of
an 125I implant for all tumor oubling times (high grae tumors an low grae t
umors). Both pallaium an ioine are effective implant sources, an both have th
e avantage over other therapies of continuous rather than fractionate elivery
(Table 16.1). But
Table 16.1 A comparison of raiotherapy moalities for treatment of localize (l
ocoregional) prostate cancer
Moality
3DCRT IMRT Protons/Neutrons 103 P/125I Brachytherapy (Monotherapy) HDR 192Ir (
Monotherapy) 3DCRT/IMRT an 103P/ 125I EBRT an HDR 192Ir
Typical ose (cy)
Delivery
70008000 Fractionate 70009000 Fractionate 70008000 Fractionate 12 500/14 400 Con
tinuous ? Fractionate 40005000 Fractionate/ 800012 000 Continuous 40005000 Fracti
onate 15002500 Fractionate 3DCRT, threeimensional conformal external beam ra
iotherapy; IMRT, intensity moulate raiotherapy; HDR, high ose rate, 192Ir,
iriium192; EBRT, external beam raiotherapy.
103P brachytherapy
203
I have foun the shortlive, preictable sie effects associate with 103P to
be especially attractive in the context of a large brachytherapybase practice.
The following iscussion is not intene to settle the ebate, but rather to ex
plain my rationale for choosing 103P. Raiobiology an the ose rate phenomenon
The physics of 103P versus 125I has been rigorously stuie (Figures 16.216.4).
A 103P implant is usually planne to eliver an 11 50012 500 cy to full ecay

at an initial ose rate of approximately 20 cy per hour. 125I implants eliver
raiation at a ose rate of approximately 510 cy per hour. The halflife of 103P
 is 17 ays compare to 60 ays with 125I. With pallaium, most of the raiatio
n osage is elivere in 3 months compare to up to one year with ioine. While
ose elivery is as state, sie effects with each isotope may be longer ue to
clinical lag time. Results favoring pallaium might be expecte given the raiob
iological consierations. Most raiobiological ata
Figure 16.1 Prescription oses EBRT vs 125I an 103P. EBRT, external beam raio
therapy.
Figure 16.2 Halflife of 103P vs 125|.
Basic an avance techniques in prostate brachytherapy
204
Figure 16.3 Average photon energy of 103 P an 125|.
Figure 16.4 Initial ose rate to prostate periphery: 103P vs 125|.
are erive from theory or base on in vitro stuies. It is known that raiobiol
ogical effect (RBE) ecreases with ecreasing ose rate primarily as a result of
the tumors ability to repair potentially lethal an sublethal amage, but also b
ecause of recruitment of a relatively quiescent subpopulation of cells an repop
ulation of initial target cell populations. If the ose rate is too low, tumors
associate with rapi cell cycles (e.g. 25 ays) may not be effectively kille. A
lthough no human clinical ata with longterm followup are available, the higher
ose rate of 103P woul theoretically be more successful in eraicating aggres
sive an rapily proliferating tumors. In this regar, it shoul be note that l
ow energy photons have a higher linear energy transfer (LET), associate with hi
gher RBE. The greater raiobiological effect is presumably the outcome of greate
r energy elivere per cell that the photon traverses. The average energy of 103
P photons is 21 keV (photons of 20, 23, 40, an 357 keV are emitte) as compare
 to 28 keV with 125I (photons of 27, 31, an 36keV are emitte). Thus, pallaiu
m woul be expecte to have a slightly higher LET an RBE compare to ioine. In
vivo an in vitro stuies In vivo animal moels (e.g. stuies of rat prostate t
umors) an also in vitro stuies o in fact emonstrate a significant benefit wi
th 103P for higher grae tumors, but also an
103P brachytherapy
205
avantage in low grae tumors. A stuy by Nag an colleagues emonstrate the tu
moricial effect of 103P was greater than 125I by a factor of two or more.3 The
RBE of 103 P versus 125I was compare by Ling an colleagues using rat embryo
cells transfecte with Haras oncogene. While the applicability of results from
such experiments to the clinic is limite, this stuy reporte an RBE of 1.9 for
pallaium versus 1.4 for ioine.4 These favorable 103P results may be base at
least in part on the oserate phenomenon. Only one piece of ata,5 suggeste t
hat 125I might be more effective for low grae tumors while 103P woul be super
ior for high grae carcinomas. This was a highly theoretical moel base on the
biologic effective ose (BED) formula, with questionable alpha/beta ratio assump
tions. The stuy has virtually no clinical applicability, as the central mathema
tical equation use to calculate the cell survival level relie on variables abo
ut which little or nothing is known when applie to human prostate cancer. Chang
es in source calibration Both 103P an 125I were affecte by source calibration
changes that were implemente in the late 1990s. Several alterations of apparen
t activity were applie to 103P, as summarize by an American Brachytherapy Soc
iety (ABS) article.6 The recommene prescription ose for monotherapy is now 12

500 cy, with the aoption of the National Institute of Stanars an Technolog
y (NIST)99 airkerma strength stanar for 103P using the new oserate constan
ts, which are unique to each manufacturer. In the case of 125I, the American Ass
ociation of Physicists in Meicine (AAPM) Raiation Therapy Committee Task roup
No. 43 (T43) recommene changing over to airkerma parameters establishe by
the Interstitial Collaborative Working roup (ICW).7,8 The 125I prescription 
ose change from 16 000 cy to 14 400 cy.9,10 Overall, these changes in nominal
ose represent approximately a 10% ecrease for 125I an an 8% increase for 103
P (Figures 16.5 an 16.6). While the alterations in state oses may not be of
major clinical significance, they shoul not be ignore, especially when publish
e ata spans the time frame in which these osimetry changes were implemente.
Because of the lower energy of photons emitte by 103P compare to 125I (21 keV
ave. vs 28 keV ave.), raial ose falloff is more steep at any istance from 1
03P sources, especially since attenuation coefficients (e.g. tis
Figure 16.5 Nominal 103P oses before an after aoption of NIST wie angle fre
eair chamber (WAFAC) calibration.
Basic an avance techniques in prostate brachytherapy
206
Figure 16.6 Nominal prescription ose changes for 125I before an after aoption
of T43 calculations.
sue, scatter, other sees) increase rapily with ecreasing photon energy an ar
e in fact exponential. Therefore, at greater istance from a 103P implant, the
ose is significantly reuce when compare to 125I. At a istance of 10 cm in t
issue, the ose of 103P is approximately one tenth that of 125I.11 This is not
insignificant clinically an shows that tissue penetration is istinctly issimi
lar between the two isotopes espite their energies being relatively similar. Th
e same phenomenon, however, may len itself to col spots if pallaium sees are n
ot accurately place. Further source comparisons Prouce in a cyclotron by bomb
aring rhoinium with protons, 103P ecays via electron capture an conversion
to rhoinium103. 125I is prouce from xenon in a nuclear reactor, an ecays v
ia electron capture an conversion to tellurium. The Therasee 103 P source is m
anufacture by Theragenics, the largest supplier of pallaium. Alternative moel
s of the 125I source are available from a number of manufacturers incluing Nyco
meAmersham, Best Inustries, an CR Bar. The marketplace for sees is increas
ingly competitive, an the costs of 125I an 103P are roughly similar, regarle
ss of activity. 125I offers a greater range of source strengths, an is availabl
e either as loose sees or suturemounte 125I (RAPIDStran). 103 P sources have
the same imensions as 125I sources (4.50.8 mm), but the two sources appear iff
erently on raiographs epening on the manufacturer.12,13 The two isotopes empl
oy ifferent raioense markers. 125I is asorbe on a silver filament, which is
encase in a titanium capsule. The inner wire is visible raiographically, but
the outer capsule is not. 103P is plate on raiolucent graphite ros, with a l
ea wire at the center of the source to make it raiographically visible. Disav
antages associate with 103P are primarily associate with the short halflife.
They require replacement an/or osimetric corrections if not use at the initi
al planning ate. This rarely allows for reutilization of the isotope. Also, as
iscusse below, technical accuracy of source placement with 103P is more ema
ning. As such, I always avise newcomers to use 125I first, since ioine is far
more forgiving of geographical misses than is 103P. Anisotrophy is a minor con
sieration for both 103P an 125I. Both sources have more ose attenuation at t
he ens than at the central plane because of absorption at the ens of the metal
capsule an also absorption by the silver or aluminum carrier. If the source
103P brachytherapy
207

istribution were uniformly aligne, the ecrease ose at the ens of the sourc
e might affect ose istribution. In current practice, however, source orientati
on is more ranom, an allows for a uniform correction factor, assuming ranom s
ource rotation from the longituinal prostatic axis. There woul be little clini
cal benefit erive from calculating the actual anistrophic effect. 103 P sees
are generally use at a source strength of 1.01.8 mCi (NIST99). The lower activ
ity sources are typically use for boost implants combine with supplemental ext
ernal raiation, although I have most commonly use 1.4 mCi for both mono therap
y an combine treatment. The source strength for 125I runs from 0.3 mCi to 0.9
mCi. Dicker an colleagues reporte that the ifference in average energy betwee
n 103P an 125 I has only minor effects on most planning parameters.14 While no
ting that both isotopes were similarly effective with long potential tumor oubl
ing time (Tpot), the Dicker stuy also emonstrate that 103P was more effectiv
e with short Tpot. Ling ha previously argue that the shorter acting isotope sh
oul be use to treat tumors with more rapi proliferation rates (shorter Tpot)
to allow less time for cell repopulation.5 Source insertion There are two princi
pal means for inserting 103P an 125I sources: the Mick applicator an preloae
neeles. In my practice, I primarily employ the Mick applicator because it allow
s for a hoc see placement an greater flexibility with intraoperative planning
. There are other avantages as well. The evice causes less raiation exposure
to personnel, an less preparation is require compare to the teious process o
f preloaing sources into neeles. The Mick applicator is also more cost effectiv
e in a large brachytherapy practice, as a limite number of neeles are use per
patient. Imaging techniques The resurgence of interest in prostate brachytherap
y over the past two ecaes was primarily riven by the technological innovation
of transrectal ultrasoun (TRUS), which allows for realtime imaging uring tre
atment planning an is also use for monitoring intraoperative neele placement.
TRUS imaging is supplemente by computerize tomography (CT) an enorectal mag
netic resonance imaging (MRI). Each imaging moality has its avantages an isa
vantages. TRUS, MRI, an CT images all reveal pretreatment prostate contours an
 are use in tanem to etermine the number an placement of implant sources. W
hile the appearance of the prostatic an periprostatic regions varies qualitativ
ely between the imaging moalities, the size an shape of the prostate are fairl
y consistent between techniques if interprete correctly. The visualization of p
rostate margins with these complementary moalities ultimately etermines the ra
ioactivity require an where it is place. TRUS an colorflow Doppler ultraso
un At our institution, colorflow Doppler ultrasoun is utilize since it provi
es enhance visualization an greater efinition compare to the conventional g
rayscale technique.15
Basic an avance techniques in prostate brachytherapy
18
208
While there is an art to interpreting both spectral an power colorflow Doppler
images, tumors ten to emonstrate increase perfusion or hypervascularity as f
inings consistent with malignancy. The stanar TRUS shows hypoechogenic areas
typically as arker shaes of gray. A colorflow Doppler ultrasoun may show the
same image, but it provies aitional insight into how much perfusion of bloo
is going into the region an can reveal whether just one prostate noule is inv
olve or if there is more cancer isperse throughout the glan (Figures 16.7 an
 16.8). In aition, biopsies guie by colorflow Doppler ultrasoun have the
avantage of showing the optimal sites from which to secure tissue samples.16,17
Once the initial iagnosis has been establishe, I typically request that the s
pecimen slies be reviewe by a pathologist who specializes in prostate patholog
y.
Figure 16.7 Colorflow Doppler ultrasoun imaging for tumor elineation, (a) CFD

, right mi cancer, axial image, (b) CFD, base cancer, longituinal image. (Repr
ouce with permission from Feleppa EJ, et al. Ultrasonic Spectrum analysis an
neuralnetwork classification as a basis for ultrasonic imaging to target brachy
therapy of prostate cancer. Brachytherapy 2002; 1(1):4853.)
103P brachytherapy
209
Figure 16.8 Prostate brachytherapy using colorflow Doppler optimization. (Repro
uce with permission from Feleppa EJ, et al. Ultrasonic spectrum analysis an n
euralnetwork classification as a basis for ultrasonic imaging to target brachyt
herapy of prostate cancer. Brachytherapy 2002; 1(1):4853.)
The sharp efinition of TRUS images can potentially help to minimize interobserv
er an intraobserver variability, although ifferences of 5% to 25% have been re
porte when patients are scanne in succession by the same observer.19,20 For sc
ans performe in succession by two ifferent observers, an approximate 10% varia
bility has been reporte.19 There are conflicting reports in the literature as t
o whether or not TRUS images accurately measure prostate volume when compare to
that etermine by the prostatectomy specimen. Rahmouni an colleagues foun th
at TRUS volume was 70% of that etermine by the resecte glan.21 Other stuies
have reporte less of a iscrepancy between TRUS an resecte prostate volumes.
22 The if ferences reporte in these stuies may reflect iscrepancies in TRUS
interpretations, isparate formulas use to measure volume (elliptical volume fo
rmula vs stepsection planimetry), an ifferences in equipment an in methos u
se to process specimens. Operator an team experience are probably inversely pr
oportional to such iscrepancies as reporte elsewhere. It shoul also be note
that prostate volume an shape can change when patients are anesthetize. Volume
tric inconsistencies can also stem from physiologic changes. Measure by TRUS or
MRI, prostate volume can change from aytoay by approximately 10%. This vari
ation is consistent with both moalities an may reflect actual changes in volum
e rather than inconsistent technique.23 The impact of such reproucibility probl
ems on clinical results is unknown. It is possible that miscalculate low volume
s coul lea to inaequate ose coverage of the prostatic periphery, an miscalc
ulate high volumes coul lea to complications ue to
Basic an avance techniques in prostate brachytherapy
210
overraiation of normal tissue.24 Such potential problems in treatment planning
an evaluation can be avoie by registration an crossreferencing the TRUS, MR
, an CT imaging techniques. Even with the wie variation in how the moalities
are implemente, clinical results with transperineal brachytherapy appear highly
favorable. Effective planning an operator skill appear to be the most signific
ant factors of practical consequence. CT an MRI The main avantages of the CT s
can over the other moalities are that CT offers a finer elineation of sources
an more accurate imaging of the pubic bones. In aition, the CT requires less
patient preparation an is less operator epenent than TRUS. The CT scan is wi
ely use for postimplant osimetry an quality control. The lack of realtime im
aging is the primary isavantage of CTbase implants. While CT scans provie l
ess efine images of the outer prostatic contour an internal architecture, CT
images o accurately elineate the spatial relationship between the prostate, re
ctum an pubic bones. As with TRUS, there are conflicting stuies in the literat
ure with regar to the volumetric accuracy of CT scans. Roach an colleagues fou
n CT volumes were approximately 30% larger than MRI volumes.21 Wallner has sugg
este that the volumes in that stuy (an those in a similar stuy by the Fox Ch
ase Cancer Center,25) were excessive because the levator ani musculature was incl
ue within the target volume (Wallner et al).26 A University of Washington stuy
compare TRUS an CT volumes rawn inepenently by three observers (Baiozaman

i, Wallner, an Blasko). They reporte the imaging moalities were consistent in
measuring anteriorposterior, lateral, an cranialcaual imensions.27,28 The si
gnificance of this fining is that CT an TRUS images are actually in close corr
esponence in etermining preimplant volumes. When interprete correctly, the CT
an TRUS volumes are interchangeable. Enorectal MR images (obtaine in the gla
n eforme by the enorectal balloon coil) show markely sharper prostatic marg
ins than either TRUS or CT. Image interpretation with MRI is therefore less oper
atorepenent an more reproucible. CT scans lack the attenuation patterns an
highresolution etails associate with MRI. Magnetic resonance spectroscopic i
maging (MRSI) is the most iscriminating test in terms of both the internal arch
itecture of the prostate glan an etermining whether or not there is extracaps
ular extension (EPE). With its high egree of etail, the enorectal MRI can sho
w whether or not there is rectal or seminal vesicle involvement. Blaer invasio
n can also be etecte by an enorectal MRI, while its not commonly seen with a C
T scan or an ultrasoun stuy. Zaier an colleagues reporte a biologicbase o
ptimization technique that registers MRSI images to intraoperative ultrasoun im
ages in orer to achieve ose escalation to intraprostatic tumor eposits.29 Sim
ilarly, Mizowaki an colleagues reporte on integrating functional imaging moal
ities with the registration of MRSI to TRUS an CT images.30 At our institution,
most patients unergo an enorectal MRI (preferably MRSI) in aition to TRUS a
n CT prior to prostate brachytherapy. The only reason a patient woul not have
an MRI is if his insurance company oes not cover it an the patient
103P brachytherapy
211
cannot affor the test, as it is expensive. The cost of a iagnostic pelvic MRI
is at least two to three times higher than the cost for a TRUS or CT. Setting as
ie cost consierations, the art of brachytherapy planning an esign epens in
large part on optimal integration of these complementary imaging moalities. Pr
eimplant osimetry an planning The purpose of preimplant osimetry is to eterm
ine the number of sources that will be use an where they will be place. At my
institution, a pretreatment stuy utilizing colorflow Doppler ultrasoun gener
ally takes place 23 ays prior to treatment. Preplanning obviates the nee for pr
olonge intraoperative planning. Inee, the most appealing aspect of preplannin
g is the increase time available to evaluate an refine the plan. Without use o
f preplanning images, an aitional 3060 minutes of operation room (OR) time may
be ae to the proceure. Another avantage affore by pretreatment planning i
s the ability to know in avance whether pubic arch interference (PAI) will pose
a problem, so that appropriate measures (e.g. hormonal ownsizing or intraopera
tive positioning) may be taken in anticipation of the problem. Planning images a
n target efinitions Both pallaium an ioine implants are planne using image
s of the prostate glan that are taken at 5 mm intervals from the base through t
he apex. The patient is place in the orsolithotomy position, as close as poss
ible to the position that he will be in uring the proceure. Care must be taken
that the glan is not eforme by excessive probe pressure. The prostate is to
remain centere on the image gri, while the posterior margin is aligne along a
gri row. The most proximal image is the zero plane, or 0.0 plane. This plane i
s locate by visualization of the most proximal image or base, which typically i
nclues a portion of the seminal vesicles. Source positions an images are efin
e by their istance from the zero plane. Measure caually, transverse planes a
re numbere by their istance in cm from the zero plane. The 1.0 plane, for exam
ple, is 1.0 cm caual to the zero plane. The most caual image, the apex is usua
lly less istinct with transverse imaging but I fin this to be easily confirme
with sagittal imaging. The sagittal view helps verify the base an apical plane
s, with the continuous contour of the glan visually extrapolate from the mise
ction of the prostate (Figure 16.9). The crucial importance of the zero plane an
 zero retraction point must be emphasize. The zero plane is etermine by the
preplan. Typically, we o not go back as much as 45 mmit is generally less, but ev

ery case is ifferent. We use 23 mm as a rough guieline. I will shoot a see in

an then etermine if it really went into the reference place by visualizing tha
t on ultrasoun. Again, throughout the proceure, we constantly check to make su
re that reference point has not change, that the sees
Basic an avance techniques in prostate brachytherapy
212
Figure 16.9 The aero plane can be etermine by moving the TRUS probe through th
e prostatic base. The 0.0 plane typically shows seminal vesicles blening into t
he prostate (top mile panel). The 0.5 plane shows seminal vesicles an blaer
but not the prostate. The tissue plane between the seminal vesicle base an post
erior blaer (*) can be useful lanmark to ientify the plane above the prostat
ic base (0.5). the sagittal view (left) can be use to verify that you are at the
103P brachytherapy
213
base. (Aapte with permission from Wallner K, Brachytheraphy mae complicate,
2n e. Seattle, WA: Smart Meicine Press, 201:8.15.)
are being place accurately using that reference guie. Typically, I o not o t
he proceure while resting my ultrasoun at the reference point, but rather go b
ack approximately 1.0 centimeter, a couple of clicks, or one click an a half, o
r possibly a half click, sometimes where there is a lanmark like a stone or a c
yst or some part of the glan contour that is easily recognizable. Treatmentpla
nning efinitions for brachytherapy were erive from ICRU50 planning efinitio
ns for external beam raiation therapy (ICRU, Han31). The gross target volume (
TV) is visualize on TRUS images as the prostate margin itself. The clinical tar
get volume (CTV) covers the glan an periprostatic regions of potential microsc
opic invasion. With external beam treatment planning, the planning target volume
(PTV) elineates a margin aroun the CTV to allow for patient setup changes an
prostatic motion, as well as for targeting periprostatic tissue. With the brach
ytherapy proceure, because the position of the prostate on the template oes no
t change, the PTV is not applicable whereas the TV an CTV parameters apply equ
ally well to brachytherapy an to EBRT. The treatment margin (TM) refers to the
perpenicular istance between the TV an the prescription isoose. The treate
volume (TV) refers to the area encompasse by the prescription ose. These trea
tmentplanning parameters are crucial for etermining the amount of tissue treat
e an the ose elivere to the prostate, rectum, urethra, an proximal penile
tissues. It shoul be kept in min that both 103P an 125I brachytherapy elive
r oses that are much greater than the prescription ose, because of the use of
treatment margins an the high ose regions in close proximity to the sources. T
reatment planning an target volumes At the start of the treatment planning stu
y, the TV is ientifie using TRUS images an incorporate (igitize) into a t
reatmentplanning program. Ientifying the prostate margins can pose a challenge
because they can be blurre, especially in the region of the apex, where the gl
anular tissue an pelvic floor musculature are ifficult to istinguish. When t
here are oubts, it is wise to aopt a policy of being slightly more generous wi
th margins at the apex. A retropubic urethrogram may be helpful since the prosta
te apex is 1.01.2 cm above the bulbar urethra. The margins can usually be visuali
ze more clearly posteriorly near the rectum, where care must be taken to limit
the ose. Aitionally, the proximal penile tissues (PPT) which are thought to b
e important for preserving erectile function are within 1.0 cm of the apex.32 Th
e steep raial ose falloff of 103P shoul allow more generous coverage of the
apex while sparing the PPT to a preictive egree when compare to 125I. On a c
asebycase basis, our treatment planning stuies utilize all available preimpla
nt ata, for example, stage, prostatespecific antigen (PSA), prostatic aci pho
sphatase (PAP), leason score, perineural invasion as well as iagnostic stuies

(colorflow Doppler ultrasoun, enorectal MRI, etc.). Depening on those preim

plant parameters, I
Basic an avance techniques in prostate brachytherapy
214
typically use 35 mm of margin an upwars of 1.0 cm as inicate. It shoul be no
te that the margin may vary throughout the glan: L versus R, base versus apex,
an the anterior margin. No aitional margin is ae posteriorly. Smaller gla
n volumes typically allow for an require larger margins, an extracapsular pla
cement is utilize in all but the largest glans (50 cm3). The proximal portion o
f volume, especially when there is clinical or raiographic the seminal vesicles
is commonly inclue in the target evience of seminal vesicle involvement or w
hen the tumor is at the junction of the base an the seminal vesicles. The most
proximal portion of the seminal vesicles (between the blaer an prostatic base
) can certainly receive a cancerocial ose with brachytherapy.33 However, exten
sive seminal vesicle invasion into the istal aspect oes not typically len its
elf to treatment with brachytherapy. Using osevolume histogram (DVH) analysis,
Stock an colleagues reporte that vesicle tissue above the top of the prostate
receives less than 50% of the prescription ose.34 Aitionally, extensive semi
nal vesicular invasion may be a harbinger of metastatic isease. As expecte, r
awing more generous target volumes will increase the volume irraiate an total
103P activity require per implant. Because of the steep raial ose falloff a
ssociate with 103P, sources are routinely place in extracapsular locations as
per TRUS planning to achieve satisfactory ose coverage as evaluate by postimp
lant CT base osimetry.35 Tumors occur most frequently in the posteriorlateral
regions, which is where extraprostatic extension (EPE) is most likely to occur.
36,37 Consequently, larger treatment margins shoul be use where the likelihoo
of EPE is higher.38 McNeal an colleagues reporte that prostatic carcinoma is
multifocal in 50% to 80% of cases, which woul suggest that the entire prostate
be treate with the prescription ose.37 Ieally, an implant will eliver a canc
erocial ose not only to the entire glan, but with a target margin large enoug
h to cover extracapsular extension. This extraprostatic coverage is especially a
ppealing since a substantial number of patients may harbor unforeseen microscopi
c extracapsular isease extension. Prostatectomy specimen stuies have shown tha
t EPE is usually limite to a istance of 3 mm measure raially from the prosta
tic ege, an therefore a 3 mm target margin is a reasonable guieline.38,39 Set
ting asie source placement errors an implantrelate eema, a preplanning marg
in of 35 mm will typically achieve a postimplant margin of 35 mm. In my practice,
with enhance visualization affore by colorflow Doppler ultrasonography, I ro
utinely place aitional sees intraprostatically, or extracapsular to a visuali
ze lesion abutting or breaching the capsule (Figure 16.10). While it may not be
practical to prescribe a higher ose to one portion of the glan, the prostatic
margin closest to the tumorbearing region can be rawn more generously uring
preplanning. These extra a hoc sources aress high risk areas within the prostat
e, though care must be taken to keep sees away from the rectum an urethra. Ext
ras are typically place laterally to minimize rectal an urethral oses. Periop
erative steroi to control swelling A great eal of literature has been evote
to the inconsistencies between preimplant an postimplant target margins cause
by implantrelate swelling an prostatic volume
103P brachytherapy
215
changes.31,40,41 Han an colleagues reporte that postimplant target margins (TM
) correlate only loosely with preplanne TMs ue to implantrelate prostatic v
olume changes. Baiozamani an colleagues compare implantrelate imensional c
hanges with preimplant clinical parameters, incluing volume, hormonal eprivati

on, an supplemental EBRT, concluing that no single factor can accurately prei
ct the egree of implantrelate swelling. The stuy reporte that postimplant t
arget volumes increase by an average factor of 1.7.27,28 In my practice, periop
erative an postoperative sterois an nonsteroial antiinflammatory agents (cel
ecoxib or ibuprofen) are use to control swelling an to reuce urinary retentio
n. Perioperative examethasone has been shown to reuce swelling up to four week
s after the implant.4246
Figure 16.10 Tumor, as visualize on colorflow Doppler ultrasoun, can be easil
y elineate an utilize for ose optimization/escalation. (a) Brachytheraphy a
n extracapsular see placement for ose optimization. (b)
Basic an avance techniques in prostate brachytherapy
216
Brachytheraphy ose optimization using extracapsular sees.
Speight an colleagues also reporte an improve osevolume histogram associate
with perioperative examethasone. In aition, celecoxib (Celebrex) has been sh
own to significantly reuce eema following brachytherapy an also appears to e
crease urinary retention.47 Dosimetry analysis an moifie peripheral loaing I
n a sizable patient population, I have evaluate glan size an see motion from
the ay of implant, then every two weeks up to 3 to 4 months. There has been no
substantial change in glan size when using perioperative an postoperative ste
rois an nonsteroial antiinflammatory agents. Therefore, CT scanning an posti
mplant osimetry analysis nee not be elaye, an many patients even unergo CT
scanning for postimplant osimetry on postop ay 1. Immeiate postimplant analy
sis allows for any mistakes to be reveale as soon after the fact as possible, m
aking for easier correction. Narayana an colleagues reporte that limiting sour
ces to the periphery allows for optimal treatment of prostatic margins without e
xcessive central oses.48 Moifie peripheral source placement patterns avoi th
e urethral ose extremes of homogeneous loaing that places sees in a uniform p
attern throughout the prostate. Wallner an colleagues emonstrate that excessi
ve urethral oses are correlate with urethral morbiity.49 Urethral oses shoul
 be kept within approximately 150% to 250% of the prescription ose. While in p
ractice, it is often assume that the urethra is locate miline, at the time of
the implant the urethra may turn out to be quite circuitous in its course throu
gh the prostate. Intraoperative ajustments can be mae simply by moving neeles
several millimeters away from the urethra. The prescription isoose is generall
y 45 mm outsie the most peripheral sees, an therefore, achieving a 35 mm treatm
ent margin aroun the prostatic ege requires that sources be place very near t
he ege. I generally place sources aroun the periphery at 1.0 cm spacing an wi
thin 25 mm from the margin. Because sees at the periphery o not provie aequat
e coverage of the entire volume, aitional sources are place more centrally an
/or peripherally to achieve optimal target coverage, with acceptable central o
ses. With sees limite to the periphery, the margins can receive aequate oses
without central overosing.48 Comprehensive peripheral loaing is not practical
because placing all the sources at the periphery may result in unerosing the
central portion of the glan. Merrick an colleagues have reporte on the ration
ale for moifie peripheral loaing.43 46 With this technique, the central ose i
s generally kept below 200% of the prescription ose, thus minimizing the likeli
hoo of urinary complications. Extraprostatic source placement I am an avocate
of extraprostatic source placement, an typically place sees 15 mm outsie the p
rostatic capsule. It shoul be note that the clinical target volume (CTV)
103P brachytherapy
217
extens anteriorly an laterally beyon the prostate margin (TV), with the ae

 target margin most marke at the apex an base. Extraprostatic see placement,
as assesse by CTbase postimplant osimetry, achieves broaer an more unifor
m coverage without escalating urethral or rectal oses (Figures 16.11 an 16.12)
. Placing sources outsie the prostate has not resulte in increase clinical pr
oblems (e.g. RTO grae 3 an 4 toxicity or increase erectile ys function). Th
is consieration is especially appealing since urethral morbiity is the most se
vere sie effect associate with 103P brachytherapy. It is also an important co
nsieration for prior TURP patients since increase central oses may result in
increase incontinence. In our experience, the immeiate periprostatic tissues (
1 mm to 1 cm beyon the capsule) allow for source placement without substantial
concern for migration.35 The concave ens of 103P Therasee may contribute to so
urce fixity. In contrast to the more convexshape sees, Therasee sources ten
to anchor themselves within the immeiate periprostatic tissue. Placement of a
itional sees peripheral to the ultrasounefine TV is necessary to achieve b
oth optimal uniform coverage an the prescribe minimum peripheral ose. The ste
ep raial ose falloff with 103P tens to reuce the minimum peripheral ose e
ven when using a moifie peripheral see loaing pattern. When using our prosta
te osimetry, an increase number of 103P sources place outsie the prostate v
olume bring us closer to our osimetry objectives (minimum peripheral ose cover
age of 80%), without increase toxicity. To achieve this egree of ose coverage
on postimplant CT analysis, substantial increases in preimplant ultrasoun targ
et volumes have been necessary over the years when using a moifie nonuniform
peripheral see loaing pattern. While recognizing that the evaluation of implan
t quality is multifacete an epens to a large extent on target ientification
an postimplant osimetric methos, it nonetheless seems logical that improving
uniformity throughout the glan an targeting extracapsular regions woul not o
nly result in iminishe morbiity, but shoul also increase the likelihoo of t
umor eraication. Total activity an source strength The total activity require
for 103P an 125I implants increases as the prostate volume increases. However
, there is only a rough corresponence between activity an volume because the a
ctual activity epens on a number of
Basic an avance techniques in prostate brachytherapy
218
Figure 16.11 Preimplant TRUS an postimplant CT showing extracapsular see place
ment. (Reprouce with permission from Wallner K, Brachytherapy mae complicate
, 2n e. Seattle, WA: Smart Meicine Press, 2001:6.25.)
Figure 16.12 Tumor elineation on postimplant CT for osimetric analysis. Brachy
therapy ose optimization.
other factors incluing the shape of the glan, how large the treatment margins
are an how many targeting ajustments have been mae to limit the ose to the r
ectum an urethra. Prostate brachytherapy has utilize a wie variety of source
strengths, with no evience of any effect on clinical outcomes. 103P sources ty
pically vary in strength from 1.4 mCi to 2.0 mCi, while 125I sources range from
0.2 mCi to 0.9 mCi (NIST99). Moifie peripheral loaing with higher strength s
ees shoul lower costs by reucing the number of sources require.50 Some inves
tigators have argue that the use of higher activity sources coul lea to incre
ase complications an/or inaequate target coverage, but that argument is not s
upporte by any clinical ata or by common sense. Raiationinuce morbiity is
relate to the ose elivere rather than iniviual source strength. Newcomers
to brachytherapy might be avise to use lower strength sources because there i
s greater margin for error an less risk of overosing. The fact that a few see
s are misplace is less of a concern when those errors are sprea out among a gr
eater number of lower strength sources.
103P brachytherapy

219
Hormonal ownsizing Anrogen eprivation therapy (ADT) ecreases prostate volume
by approximately 25% to 50%.5153 When ADT is use prior to 103P brachytherapy,
treatment planning is base on the reuce prostatic volume. Planning images are
obtaine after at least 12 weeks of ADT when maximal shrinkage has been achieve
. Hormonal treatment is typically optional for patients having intermeiate ris
k features but encourage for patients having high risk features. High risk fact
ors woul inclue two or more of the following: PSA >10, leason 7, cancer stage
>T2b, an an elevate PAP. With the low risk or milly aggressive cancers, I o
not usually prescribe conventional ADT, that is, combine hormonal blockae usin
g an antianrogen an a LHRH (luteinizinghormonereleasing hormone) agonist, si
nce this may result in unwante sie effects, such as impotence, hot flashes, my
algia, an anthralgia. I often prescribe a miler or moifie version of hormone
s (antianrogen  Proscar/Avoart), a protocol that is just enough to arrest the c
ancer an to allow the patient to make a ecision about treatment without the r
ush an urgency often associate with it. It shoul be note that my own ata o
not emonstrate a strong avantage to utilizing hormones in patients having hig
h risk features, but I cannot ignore the fact that numerous multiinstitutional
stuies both in the Unite States an abroa have emonstrate a benefit with th
e utilization of hormonal therapy. In aition, within my own ata, the patients
who receive hormones ha far more aggressive tumors an yet fare similarly to
the other patients uner stuy.54 Implant technique an evaluation Positioning
the patient After inuction of anesthesia (spinal, general, or local), the patie
nt shoul be place in the orsolithotomy position, making every effort to repl
icate the planning position. The patients legs are rotate symmetrically with ext
ension at the hips an knees to avoi pelvis rotation on the longituinal axis.
Care is taken to make sure the patient is line up at the mile of the table wi
th the perineum near the table ege, allowing sufficient space to facilitate man
euvering the TRUS probe. Pubic arch interference (PAI) can usually be rectifie
by placing the patient in the extene lithotomy position, tilting back the pelv
is in orer to reorient the pubic bones away from the anterior of the prostate.
The pelvis can be tippe back by ajusting the stirrups or by manually maneuveri
ng the patients pelvis towar the en of the table. Care shoul also be taken to
make sure the scrotum is kept away from the perineum. This can be accomplishe b
y using tape, a wet cloth or towel to hol the scrotum to the abominal wall.
Basic an avance techniques in prostate brachytherapy
220
Urethral imaging The urethra is visualize uring the proceure to avoi misplac
ement of sees in those patients with asymmetrical urethras. Misplace intrauret
hral sources will be expelle, lowering the planne ose. A catheter can be use
to visualize the urethra an blaer neck; however, this technique creates an a
rtificial enlargement of the glan (e.g. an 18French catheter is sizable when co
mpare to a 2030 cc glan). In my practice, I routinely utilize the catheter prio
r to starting the implant, aministering 50 cc of 100% Renografin into the blae
r an obtaining a lateral fluoroscopic cystogram, while aitional ye is instil
le into the catheter. Keeping the image on a secon fluoroscopic screen uring
the proceure provies a reliable urethral reference. Information regaring the
blaer can be obtaine (e.g. a smooth blaer wall vs a heavy trabeculate wall
), while an enlarge prostatic meian lobe may also be appreciate (both may pre
ict voiing problems, in which case postimplant meical management can be inten
sifie). The catheter is remove after obtaining the reference image, but only a
fter carefully ientifying an recoring the location of the urethra as it cours
es through the glan from base to apex. A small amount of ye (1015cc) remains in
the blaer throughout the proceure in orer to visualize the blaerprostate
interface. Too much ye may istort the glan, making it ifficult to replicate
the preimplant glan contour. TRUS probe positioning Before placing neeles, th

e imaging planes shoul closely match those of the planning images. The posterio
r margin of the miprostate shoul line up on the gri as it i on the plannin
g images. Ajustments can be mae by angling the probe slightly in various irec
tions. Probe pressure shoul be gentle, as excessive pressure can cause image i
stortion an artifactual eformation of the glan. As iscusse, the 0.0 plane i
s visualize by moving the TRUS probe through the confluence of seminal vesicles
, blaer, an prostatic base. Once the TRUS images correspon to those of the p
replan, seeloaing epth is etermine (Figure 16.12). The neeleguie templa
te is mounte on the stepping unit close to the perineum, with a 12 cm margin bet
ween the skin an template so that the neele irection can be ajuste manually
if necessary. Accoring to the preplan, each neele will typically carry 510 see
s. It shoul be note that the Mick applicator eposits sees beyon the tip of
the neele, an therefore, the neele tip shoul be retracte 25 mm before placin
g a see at the zero plane. A common operator loaing error occurs when the TRUS
probe is retracte too far from the reference (> 1.0 cm). When this happens, th
e most posterior superior aspect of the base of the glan may be unerloae bec
ause the operator is uner the mistaken impression that he is loaing the glan
as it appeare before the TRUS probe was pulle back. This problem can be avoie
 by constantly monitoring the position of the reference plane.
103P brachytherapy
221
A simple solution to prostatic motion Even without the pressures of neele inser
tion an the ultrasoun probe, the prostate can be very mobile, as external beam
stuies have emonstrate.55 The egree of prostatic motion varies from patient
to patient, with most occurring in the anteriorposterior irection. During imp
lant neele insertion, there may be lateral an cephala isplacement up to 1 cm
. In orer to minimize prostatic motion an the potential for misplacement, I ro
utinely insert two crossing neeles through the posterior aspect of the prostate
, thereby anchoring the glan in place.56 The neeles are fixe in the pelvic fl
oor musculature at an angle (Figure 16.13). Two 18gauge stainlesssteel neeles
(provie by Mick Nuclear Instruments, Inc) are inserte transperineally, obliq
uely into the prostate, using manual an fluoroscopic guiance. These neeles ar
e left in place uring the proceure. In my experience, maximum lateral isplace
ment is ecrease from approximately 1 cm to 2.0 mm, while craniocaual motion i
s virtually eliminate. Neele insertion Neeles are inserte through the templa
te holes to penetrate the perineal skin. After the skin is pierce, rapi avanc
ement minimizes neele eviations. When neele eflection is etecte, the neel
e may be withrawn several centimeters an then reinserte, making sure the nee
le is exactly parallel to the TRUS probe. A twisting motion may enable the neel
e to penetrate obstructing tissue planes (e.g. ue to intraprostatic calculi) wi
th minimal eflection. Monitoring the neeles fluoroscopically uring insertion
helps guar against misplacement (Figure 16.14). In aition, fluoroscopic monit
oring minimizes urethral piercing. Sources can migrate into the retroperitoneum,
the lungs or heart, but espite concerns often raise in this regar, loss is t
ypically minimal an osimetrically inconsequential. Merrick an colleagues repo
rte a low incience of source migration even with a large percentage of extrapr
ostatic sources.4346 Stuies have reporte that sources migrate to the pulmonary
vasculature in 11% to 25% of patients.57,58 No morbiity has been escribe in c
ases of pulmonary migration. A goo implant epens on accurately etermining th
e epth of the neeles an maintaining correct positioning in the transverse TRU
S images. Inserting the neele while viewing the appropriate plane helps to ensu
re the proper epth is achieve. The insertion can also be viewe fluoroscopical
ly. Lanmarks, such as intraprostatic calculi, cysts, etc., may be valuable to a
ssess current epth of neele placement throughout the proceure. It may help to
measure the istance in centimeters that the neele shoul be
Basic an avance techniques in prostate brachytherapy

222
Figure 16.13 Insertion of two crisscrossing neeles to minimize prostatic motion
, with two fluoroscopic view. (Reprouce with permission from Wallner K, brachy
theraphy mae complicate, 2 e. seattle, WA: Smart Meicine Pres, 2001:8.25.)
103P brachytherapy
223
Figure 16.14 Neele epth checke on sagittal TRUS view (Reprouce with permiss
ion from Wallner K, Brachytherapy mae complicate, 2n e. Seattle, WA: Smart M
eicine Press, 2001:8.19.)
istal to the template. Sagittal TRUS imaging can also be use to check neele 
epth. Because of the vacuum create when a neele is withrawn, sees may slie
along the track as the neele is retracte. This type of source isplacement can
be avoie by removing the neeles in a slow, steay motion, an with 103P The
rasees, utilizing a rapi clockwise rotation of the neele after it releases th
e sees from their concave ens. Newcomers to brachytherapy are avise to take
special care to avoi the pubic bones, as a rapi thrust of the neele tip again
st the pubic arch can ben or even break a neele. After multiple sources have b
een inserte, there is typically some egree of image egraation. Visual obfusc
ation can be reuce by inserting the anterior neeles before the more posterior
neeles. This anteriorposterior progression reuces the likelihoo that the po
sterior neeles will obscure the more anterior neeles. Proper neele positionin
g can be verifie on the TRUS images proximal to the template (caual to the pro
static apex), where the image is less likely to be obscure by sources. Even in
experience hans, sees can sometimes be misplace or migrate into the perirect
al region, the blaer or the lungs. While these errant sees are not known to c
ause any clinically significant problems, excessive see loss can lea to uner
osing part of the prostatic target.39 Errant sees in the urethra or blaer are
unusual, an when they occur, they typically pass spontaneously. Such errors ca
n be minimize by juicious use of intraoperative fluoroscopy an TRUS, an by c
ontinually checking that the reference has not change. Regaring intraoperative
planning, I am currently unsatisfie with the state of ynamic osimetry, fining
it to be unreliable while aing little value. Until this technique avances, I
believe that experience with both TRUS an fluoroscopy is more than satisfactor
y, proviing ample operator feeback as to the exact placement of sees. I consi
er intraoperative moifications with physics support for osimetric analysis to
satisfy the efinition of ynamic osimetry. Between the TRUS an fluoroscopy
Basic an avance techniques in prostate brachytherapy
224
moalities (an consierable experience an intraoperative osimetry), I am conf
ient that each patient has or approaches having the perfect implant. After all, y
ou cannot teach experience. Pubic arch interference Pubic arch interference (PAI
) can usually be circumvente by shifting the neele path in relation to the pub
ic arch. Tincher an colleagues have evaluate the effects of pelvic rotation an
 neele angle on PAL.59 Extening the lithotomy position rotates the pubic arch
superiorly, allowing more space for maneuvering the neeles unerneath the arch
(Figure 16.15). After changing the patients position, the template gri shoul b
e checke to be sure the prostate is aligne properly. Altering TRUS probe angul
ation or moving the probe tip away from the prostate may enable the neele to pa
ss more reaily uner the prostate; however, care must be taken when altering th
e probes position not to shift the gri alignment. Another technique for circumve
nting PAI is to ben the tip of the neele slightly, at about 34 cm from the tip.
The neele is then inserte approximately 0.51.0 cm meial an posterior to the

intene gri position, with the neele tip angling away from the arch. As the i
nsertion procees, the neele is rotate 180 egrees, thereby forcing the tip aw
ay from the probe after it passes the arch. This metho shoul be use as a last
resort, as it may increase tissue trauma an the likelihoo of urinary retentio
n.60 The various methos use to circumvent PAI can lea to unerosing, but for
tunately, when this occurs, the unerose portion of the glan is usually the m
ost anterolateral, where cancer is less likely to be present an where minor ev
iations from the target plan are not likely to be of clinical significance.36,37
Postimplant evaluation Postimplant osimetry an evaluation provie for essenti
al quality assurance through precise an objective analysis of prostate brachyth
erapy results. Postimplant evaluation tells us whether an implant was goo or su
boptimal, an this fining can have potential legal as well as clinical ramifica
tions. If a patient has serious complications an takes legal action, postimplan
t osimetry will be central to the case on both sies. With more favorable outco
mes, the osimetry provies assurance for the patient, an many are anxious to h
ear the specifics of the report even in highly technical terms. Postimplant imag
ing an osimetric analysis routinely compare isoose volumes with the prostate
margin. The sources an margins are ientifie using TRUS, CT, an/or MRI, each
with its avantages an isavantages. While CT scans allow for easy source ien
tification, the prostatic margins are frequently obscure, especially near the ap
ex. MRI provies better images of the margins, but is less effective at source i
entification. TRUS allows for realtime osimetry, though there is typically so
me egree of postproceural image egraation cause by the placement of sources
(not to mention the iscomfort!). In my experience, CT is practical an effecti
ve, taking into account the tenency to erive slightly larger volumes from CT s
cans. It shoul also be note that espite problems with interobserver variabili
ty, CTbase osimetric stuies have been fairly
103P brachytherapy
225
consistent between experience brachytherapy teams, an CTbase osimetry oes
correlate with biochemical control rates.61,62 Casual inspection of postimplant
images may show whether or not sources have been place too close to the urethra
or rectum. Postimplant osimetric results are most
Figure 16.15 Extening the lithotomy position can reuce pelvic arch interferenc
e. Note how in this patient the pubic arch (arrow) obstructe the neele at the
C5 template position (*). The extene lithotomy position allows the right sie
of the pubic arch to move away from the TRUS probe, allowing insertion to the C5
position. (Reprouce with permission from Wallner K. Brachytherapy mae compli
cate, 2n e. Seattle, WA: Smart Meicine Press, 2001:8.29.)
effectively analyze by visual evaluation of the isooses overlai on cross sect
ions of the prostate (Figure 16.16). Dose istribution obtaine from the overlay
s may reveal col spots an misplacements. In cases of suboptimal prescription 
ose coverage, aitional sources can be ae to the unerose portion of the g
lan, or in some cases supplemental external raiation (preferably IMRT) may be
ae. When aing sources after the original proceure, care must again be take
n to avoi excessive rectal an urethral oses.
Basic an avance techniques in prostate brachytherapy
226
Evaluating target coverage Current practice is to assess ose coverage of the en
tire glan, assuming it to be uniformly at risk for cancerous involvement. Isoo
se overlays are use to evaluate implants for iniviual patients, while summary
osimetric inices are use to analyze oserelate clinical effects across gro
ups of patients. The osevolume histogram (DVH) graphs the percent of target co

vere versus increasing oses an can be use to erive an array of osimetric p
arameters (Figure 16.16 an Table 16.2). The V100 inex measures the percent of
target volume covere by the prescription ose. A stuy by Roy an colleagues re
porte V100 values of 80% using postimplant CT scans.63 This inaequate coverage
was later attribute to implantrelate swelling.38,39 The effect of swelling i
s not as great as might be suggeste by reporte volume changes. Willins an Wal
lner emonstrate that with an average volume change of 25%, target coverage fel
l only by an average of 5%, ue to preplan treatment margins that allow for swel
ling, keeping most of the volume increase insie the prescription ose. I have a
voie problems with postimplant swelling by using aggressive perioperative an
postoperative steroi therapy, so that postimplant CT can be utilize at postimp
lant ay 1 or even at 3 months. V100 an D90 correlate best with biochemical con
trol, an both are commonly use. The minimum prostatic ose (Dmin) is not a use
ful inex because it has proven too sensitive to minor source placement variatio
ns.62 It shoul be note that inclusion of the levator ani musculature or the pu
borectalis in the target volume or rawing the apex too generously leas to fals
ely large prostate volumes an falsely low V100 values. Our current quality cont
rol criteria for an aequate implant require that at least 80% of the CTefine p
rostate is covere by the prescribe isoose surface (12 500 cy minimum periphe
ral ose for pallaium alone; 80009000 cy minimum peripheral ose when combine
with supplemental external raiation; IMRT). Most patients o in fact receive at
least 90% minimum peripheral ose. Failure to obtain 80% ose coverage manates
either reimplantation or supplemental external raiation IMRT.
103P brachytherapy
227
Figure 16.16 (a) CTbase evaluation of 11 500 cy 103P implant an (b) osevolu
me histogram. (Reprouce with permission from Wallner K. Brachytheraphy mae co
mplicate, 2n e. Seattle, WA: Smart Meicine Press, 2001:9.10; 9.11.)
Hot spots Hot spots occur because tissue ajacent to the sees receives extremely
high oses, an therefore, a significant amount of the prostate receives far gre
ater than the prescription ose. High ose regions near the urethra or rectum ma
y increase the likelihoo of morbiity, although serious complications (incontin
ence, rectal ulcers, or parenchymal necrosis) are uncommon. The lack of correlat
ion between the magnitue of high ose regions an clinical outcomes is probably
ue to the fact that current moifie peripheral loaing patterns minimize vari
ations in magnitue of high ose regions.6367
Basic an avance techniques in prostate brachytherapy
228
Urethral an rectal oses The maximum acceptable urethral ose for 103P is appr
oximately 300 y. With a moifie peripheral loaing pattern, the central urethr
al ose is easily kept well below the guieline of 2.5 times the prescription o
se. My implants commonly reuce the urethral ose to unity or less.35 Since the
avent of peripheral loaing patterns in the 1990s, no relationship between uret
hral oses an morbiity has been reporte, probably ue to the limite range of
urethral oses seen in current practice.6466 Serious rectal complications, such
as ulcerations an fistulas, are uncommon with brachytherapy. There are
Table 16.2 Various inices for quantifying implant quality
Percent of prostate volume receiving prescription ose or higher V100 Dose cover
ing 100% of prostate volume D100 Dose covering 90% of prostate volume D90 Percen
t of prostate volume receiving twice the prescription ose or higher V200 V200/V
100 The fraction of prostate volume receiving more than twice the prescription 
ose Minimum ose elivere to any part of the prostate Dmin

little clinical ata with regar to rectal tolerance an how to minimize rectal
morbiity. Han an colleagues reporte that rectal surface an volume oses are
correlate with postimplant proctitis an rectal bleeing.68 However, they also
suggeste that some patients may be preispose to higher rectal oses because l
ow levels of perirectal fat cause the rectum to be in closer proximity to the pr
ostate. Wallner an colleagues reporte only a loose correlation between rectal
bleeing or ulceration with oses in excess of 100 y.49 Howar an colleagues r
eporte no correlation between high ose regions an the likelihoo of serious r
ectal complications.69 Bice an colleagues evaluate osimetry from five experie
nce brachytherapy teams, with each reporting a low incience of rectal morbiit
y. A rough guieline may be suggeste by their maximum rectal wall oses, which
were approximately twice the prescription ose.70 Newcomers to brachytherapy are
avise to inclue urethral an rectal oses in their postimplant osimetric an
alysis to be certain that implants fall within acceptable guielines. With exper
ience, osimetric analysis can be limite to assessing the extent of prostate co
verage, with urethral an rectal ose calculations restricte to those patients
who later evelop serious complications. In those cases, oses are calculate at
the time the complication is iscovere, usually long after the proceure. Comp
lications Urinary morbiity Following both 103P an 125I brachytherapy, most pa
tients temporarily experience some egree of implantrelate prostatitis. Voiin
g symptoms may inclue frequency an
103P brachytherapy
229
urgency, a weakene stream, an occasionally, urinary burning. These symptoms ar
e not ebilitating, but rather a nuisance an patients are encourage to continu
e their normal level of activity. While patients unergoing 103P brachytherapy
o experience increase shortterm sie effects, the more longterm urinary morb
iities have been emonstrate to be extremely favorable (Figure 16.17).7174 In f
act, Stone emonstrate that patients with marke symptoms (high American Urolog
ical Association (AUA) scores) prior to treatment erive statistically signific
ant improvement in symptoms an quality of life (QOL) after brachytherapy. Merri
ck showe no significant ifference (not even a tren) when comparing brachyther
apy patients to an untreate control group at a meian of 69 months. Because the
halflife of pallaium is 17 ays, urinary symptoms typically last 10 to 12 wee
ks. By contrast, the halflife of ioine is 60 ays, an symptoms may persist for
10 to 12 months. Other investigators using either 125I or both isotopes reporte
 temporal resolution of urinal morbiity within 6 to 12 months.4346,75 Most pati
ents return to baseline AUA scores. Preimplant parameters, such as prostate volu
me, age, preimplant AUA scores, an urinary flow parameters o not appear to inf
luence long term AUA scores. Postimplant urinary retention is the most common ac
ute morbiity. Retention may occur in 510% of patients, but usually lasts only a
few ays.76,77 A very small percentage of patients may evelop refractory retent
ion. In my experience urinary incontinence is virtually nil. Superficial urethra
l necrosis (SUN) is uncommon since the wiesprea aoption of peripheral loaing
patterns.78 Urethral stricture is also uncommon, an may be relate to excessiv
e apical oses.7981 I have never ha a patient who require permanent urinary iv
ersion because of amage to the urethra. It appears that while the urethra tens
to play an important role with brachytherapy in terms of the sie effect profil
e, the urethra generally is able to withstan the ose, an once the sources ec
ay, the sie effects resolve spontaneously. Transurethral resection of the prost
ate Patients who have previously unergone TURP are at higher risk for evelopin
g incontinence. Although incontinence is a very rare complication associate wit
h brachytherapy in generalit is less than 1% in virtually all the series. However
, patients who have unergone TURP may be at higher risk for incontinence, up to
50% in some early series (Blasko82). More recent series with peripheral loaing
patterns emonstrate a 3% or less risk of incontinence.78,83 The reason that in
continence risks may be higher in these patients is because the TURP typically r

emoves the superior (or internal or proximal) sphincter, leaving only the istal
(or external) sphincter. The high oses of raiation elivere by the implant m
ay impair that remaining sphincter. Another factor to consier is how large the
TURP is compare to the size of the prostate. There must be enough prostate tiss
ue aroun the TURP to hol the sees; in cases where the TURP is excessive, ther
e may not be enough tissue to anchor the sees, an that may be a contrainicati
on.
Basic an avance techniques in prostate brachytherapy
230
Figure 16.17 Late urinary function after permanent prostate brachytherapy. EBRT,
external beam raiotherapy; IPSS, International Prostate Symptom Score.73 It is
rare that I woul issuae a patient from unergoing brachytherapy because of a
TURP, but if a patient has ha a TURP, the implant proceure has to be mappe o
ut very carefully an the sees nee to be arrange ifferently than with those
patients with no prior TURP. Sources are positione in a way that avois the TUR
P itself (Figure 16.18); otherwise they will be eposite into the empty cavity
where they will be urinate out, or potentially o amage. With prior TURP patie
nts, there shoul be a rim of tissue at least 1.0 cm remaining aroun the efect
, to ensure sufficient tissue to anchor the sees. I use a
103P brachytherapy
231
highly peripheral seeing loaing pattern in these patients, an take care to av
oi the remaining sphincter.
Figure 16.18 Transurethral resection of the prostate (TURP). Brachytherapy an e
xtracapsular see placement.
Rectal complications In my experience with the implant proceure, I o not have
one patient who has ha to have a colostomy or who has a persistent rectal ulcer
ation. Rectal ulceration is much less common than proctitis.68 Rectal bleeing f
rom raiation proctitis occurs in 2% to 10% of patients an usually manifests be
tween 6 an 18 months of the implant.6468 Rectal bleeing is typically painless,
with minimal bloo loss, an only rarely requires transfusion. Most patients who
experience rectal bleeing o not progress to rectal fistula, an most appear t
o heal spontaneously over time.8488 Erectile ysfunction Define in practical ter
ms as the inability to maintain an erection sufficient for intercourse, impotenc
e is a potential sie effect from any therapy use to treat the prostate. The e
finition has been criticize for being too imprecise an allowing too much latit
ue for interpretation by patients an investigators.8990 Within the limitations
of the efinition, in regar to potency preservation, brachytherapy appears to o
ffer an appealing avantage over both raical prostatectomy an conventional ext
ernal raiation IMRT. Any large stuy of patients with a long term followup sho
ul take into account normal, agerelate impotence, but all too often we forget
that there is a linear curve showing a 1.5% spontaneous ecrease in potency wit
h each year after the age of 40, an this is without any type of prostate treatm
ent.91 Evaluating the effect of treatment on potency is mae all the more proble
matical by the fact that prostate cancer affects an age group for which there is
a high incience of sexual ysfunction prior to treatment.
Basic an avance techniques in prostate brachytherapy
232
Approx imately 50% of prostate cancer patients are alreay impotent at the time
they are iagnose.89 My own ata at six years show that 70% of those patients w

ho are potent at the time of 103 P brachytherapy will maintain their potency. T
he numbers vary consierably even among experience teams, an I am aware of oth
er institutions reporting 59%,92 an 39%,73,74,7981 without pharmacologic support
(Table 16.3). I believe that the risk of impotence may be ecrease with palla
ium since the raial ose falloff an the amount of raiation actually elivere
 at any istance from the see to the neurovascular bunles (NVB) or proximal p
enile tissues is less with 103P than with any other isotope. Therefore, a sligh
tly higher potency rate might be expecte with pallaium than with other isotope
s. The importance of the NVD in preserving potency was probably overstate in ea
rly stuies at Johns Hopkins University Hospital.93 After Walsh an colleagues94
attribute postsurgical impotence to NVB trauma, it was assume by many investi
gators that implantinuce impotence might be relate to excessive raiation o
ses to the NVBs. Merrick an colleagues95 i not fin a correlation between NVB
ose an impotence with patients followe up to four years. More recent stuies
suggest that a more likely cause for raiationrelate impotence may be venous
insufficiency ue to overoses to the penile bulb
Table 16.3 Prostate brachytherapy an potency preservation
Stuy Potency preservation at 6 years
Dattoli (unpublishe) 70% Stock62 59% 39% Merrick79
Box 16.1 Brachytherapy ose to neurovascular bunle (NVB) an erectile ysfuncti
on (ED) Is it important? No relationship between ose to NVB (mean 215% 55% of pre
scription ose) an the evelopment of brachytherapyinuce ED Merrick S, et al
Int J Ra One 2000:4895 [Meian f/u 37 months] Wallner K, et al Int J Cancer 20
01:96122 [Meian f/u 49 months] f/u=follow up an proximal penile tissues, espec
ially the corpus cavernosum, which harbors the most erectile tissue (see Box 16.
1 an Figure 16.19). The penile bulb typically has a volume of 5.07.0 cc an is s
ituate approximately 0.51.0 cm below the prostatic apex. With brachytherapy, the
bulb usually receives a maximum ose of about 50% of the prescription ose, ep
ening on the treatment margins an the istance between the prostatic apex an
bulb. Merrick an colleagues emonstrate a correlation between excessive penile
bulb oses an posttreatment impotence.7981 External beam stuies have also show
n a strong correlation between bulb oses an impotence.9698 Revise NVB an peni
le bulb ose parameters are now
103P brachytherapy
233
typically incorporate into brachytherapy planning to maximize tumor eraication
an minimize the likelihoo of impotence. Brachytherapy oes not appear to pro
uce the steay ecline of potency that we have seen with full course external be
am raiation through the years. Rather, there appears to be a leveling off of th
e potency rate over time after implantation. Again, the patient is getting oler
an he may be taking hypertensive meications, iabetic meications, or have ot
her comorbi meical problems which coul interfere with his potency. Stock an
colleagues have
Figure 16.19 Prostate an periprostatic anatomy. Coronal crosssection of prosta
te an periprostatic structures.
shown that patients with substantial erectile ysfunction prior to treatment are
at an increase risk for impotency than patients who are fully potent prior to
treatment.92 For those men who lose potency, intracavernosal paparavine, prostag
lanin 1 (PE1) injection, an Viagra (silenafil) are very effective.7381,99,100
Viagra has altere the clinical situation consierably. For those men who are pot
ent at the time of treatment, 92% of patients having brachytherapy ( supplemental
external raiation) will maintain their potency (Figure 16.20).78 As of the tim
e of writing, two aitional oral erectile ais, Levitra (varenafil) an Cialis
(taalafil) are now available. Like Viagra, both are P1 inhibitors. With the m

ajority of patients who retain potency, the major change reporte is iminution
in the volume of the ejaculate. Shortly after the implant there may also be a i
scoloring or a ifferent consistency to the ejaculate.
Basic an avance techniques in prostate brachytherapy
234
Figure 16.20 Potency preservation/recovery with silenafil (brachytherapy EBRT).8
0
Typically, its escribe as being clearer an thinner, but patients have no probl
em with that as long as they are able to maintain an erection an achieve orgasm
. Dry ejaculate occurs in as many as 25% of patients. Between one thir to one h
alf of patients report painful ejaculations for 3 months or longer postimplant.7
3 Due to inflammation of the ejaculatory ucts an urethra, many patients experi
ence burning with ejaculation (orgalmasia) 612 months after the proceure. Blooy
ejaculate is common up to several weeks after the implant. Hematospermia appear
s to have little clinical significance. A patient can have normal sperm after th
e proceure. While there may be a short perio of oligospermia, a patient may ha
ve a return of sperm. However, because of the iminishe ejaculate an change in
milieu that the sperm will encounter, the likelihoo of successful impregnation
shoul be greatly reuce. Nonetheless, we counsel our patients to be careful,
because they cannot consier themselves to be sterile because of the proceure.
Supplemental external raiation: a cautionary note When external raiation is co
mbine with brachytherapy, the sequence is typically EBRT (preferably IMRT) foll
owe by an implant boost, with the oses of each moality moerate to achieve o
ptimal coverage while limiting rectal an urethral oses. The history of the com
bine approach suggests there may be consierable reason for concern that revers
ing the sequence (implant first followe by external raiation) may increase the
risk of rectal complications, in part because there is a significant interval w
hen patients are receiving simultaneous implant an external raiation IMRT. The
true pioneer of moern transperineal implants for prostate cancer, Dr Hans Henr
ik Holm of the University of Copenhagen, utilize 125I see implants first, foll
owe by a higher ose per fraction of EBRT than is generally use in the Unite
States. His complication rate of 44% require subsequent colostomies or surgical
urethral repairs. This result, couple with a high local recurrence rate of 40%
, le him to abanon the proceure altogether, with great isappointment.101
103P brachytherapy
235
Dr Holm has since been faulte for using full ose 125I followe by higher ose
per fraction EBRT. While this might account for many of the complications, it o
es not explain why 40% of his patients still ha cancer of the prostate. Another
group, Patel an colleagues followe a similar protocol,102 but use lower ose
s similar to those commonly use in the Unite States. This group encountere si
milar complications an recurrence rates. In both series, severe complications i
n most patients i not occur until four to seven years following the protocol.
Even with shorter followup (mean: 33 months), Zeithin an colleagues reporte e
arly complication rates (incontinence, 2.8%; rectoprostate fistula, 2.4%; rectal
wall breakown, 0.5%; urethral stricture, 0.5%) with the simultaneous approach
utilizing either 125I or 103P combine with EBRT.103 With sees place prior to
external raiation, there is also concern for raiation interactions (e.g. the
Photo Electric effect, the Compton effect, Bremsstrahlung) that occur with high
energy photons striking metallic objects, such as stainlesssteel, titanium, or
gol sees. These interactions may cause ose escalation an raiation scatterin
g to ajacent structures, such as the blaer, rectum, an urethra. Moreover, th
ese interactions make it virtually impossible to accurately calculate the ose l
evels elivere to relevant tissues an organs in the treatment fiel. Another i

ssue of concern with the sees first metho (with the exception of very early stag
e prostate cancer) is the potential for active cancer cells to be release into
the bloostream as a result of the implant proceure itself. This potential was
first ientifie when prostate cancer patients who unerwent TURP were iscovere
 to have a high risk of eveloping metastatic isease.104 More recent ata have
emonstrate that patients unergoing raical prostatectomy are also at high ri
sk for spreaing active cancer cells into the normal circulation.105 This potent
ial threat is eliminate with the sequential implantboost approach, because the
external raiation has sterilize the peripheral fiel prior to the insertion o
f implant neeles. Our combine approach (namely, IMRT followe by brachytherapy
), in aition to eactivating cancer cells prior to neele insertion, has resul
te in minimal complications compare to the use of sees followe by external r
aiation in the stuies cite. Cure rates Summary of 10 year results During the
last ten years, brachytherapy has progresse to the point that it has become the
treatment of choice for the majority of patients with clinically localize pros
tate cancer. Meicare ata inicate the number of brachytherapy proceures perfo
rme annually now excees the number of prostatectomies, which peake in the ear
ly 1990s (Figure 16.21). The tren in favor of seeing has been encourage by th
e growing boy of literature regaring tumor control rates. The majority of the
publishe series, incluing my own, have reporte brachytherapy results equal to
or superior to both raical surgery an conventional external raiation therapy
, with significantly lower complication rates.
Basic an avance techniques in prostate brachytherapy
236
As summarize here, my personal series ates from 1991 with 103P an supplement
al external raiation, utilizing threeimensional conformal raiation therapy (
3DCRT).54 A total of 175 consecutive patients with stage T2aT3 prostate cancer
were treate from 1991 through 1995. Each patient ha at least one of the follo
wing averse
Figure 16.21 CMS Meicare ata: sees vs raical prostatectomy (1996 2001). (Repr
ouce courtesy of Theragenics Corporation.)
risk factors: stageT2b (27 patients T2b, 83 patients T2c, 60 patients T3 as per t
he fourth eition of the American Joint Committee on Cancer Manual for Staging o
f Cancer); PSA15 ng/mL (74 patients); leason score 710 (68 patients); an/or elev
ate PAP (53 patients): 77% of patients ha two or more high risk features. Enzy
matic aci phosphatase was etermine using the metho of Roy an colleagues.106
Because interobserver variability in leason graing is a matter of concern, sl
ies for the majority of patients were reviewe by a single, highly regare pat
hologist (Dr Lawrence True at the University of Washington). Patients whose biop
sy slies coul not be reviewe were not inclue in the statistical analysis of
leason score. Patients receive meian 4140 cy 3DCRT to a limite pelvic fie
l followe by 103 P boost (minimum peripheral ose 80009000 cy, meian see ac
tivity 1.4 mCi). The clinical target volume was rawn approximately 0.51.0cm ante
rolaterally to the TRUS prostate margin. Patients receive neoajuvant or ajun
ctive hormones, meian uration 4 months (maximum 6 months). Biochemical failure
was efine as a serum PSA level greater than 0.2 ng/mL. Biochemical success wa
s analyze using a posttreatment PSA of 0.2 ng/mL. Patients whose PSA plateaue a
t greater than 0.2 were score as failures. Freeom from failure was calculate
by the metho of KaplanMeier. The overall actuarial freeom from biochemical fa
ilure at 10 years was 79%. Meian followup was 7.3 years. Using a log rank mult
ivariate analysis, the strongest preictor of failure was elevate PAP (p=0.003)
, followe by PSA (p=0.06), leason score (p=0.08), an stage (p=0.14). Morbiit
y was limite to temporary RTO grae 12 urinary symptoms. One patient who ha bo
th a TUIP an TURP posttreatment evelope low volume stress incontinence. No pa
tient evelope rectal ulceration. No patient was ocumente to have local failu
re by prostatic biopsy at the time of PSA rise. Hormonal therapy conferre no

103P brachytherapy
237
significant avantage although those patients receiving hormones ha the most a
verse features.
Table 16.4 Long term outcomes after treatment with EBRT an 103P for patients w
ith higher risk prostate carcinoma107
Material an methos; 19911995 161 consecutive patients: clinical stage was not i
nclue in risk stratification to avoi clinician subjectivity, Only one patient
ha a staging pelvic lymphaenectomy. Patients were followe at 3, 6, an 12 mo
nths, an every 612 months thereafter, Definition of biochemical success: PSA0.2 m
g/mL Followup prostatic biopsies were performe only on failing patients. All 
ata inepenently rereviewe, incluing slies (single pathologist: Dr Lawrence
True) at the University of Washington, Seattle. Patient characteristics Mean PS
A: 18.3. Meian PSA: 14.1. 51 patients ha elevate enzymatic PAPs (>2.5 U). Me
ian age: 67 (range: 4580). Other Followup: 7.3 years meian (minimum 5 years). 1
18 patients followe beyon 5 years. 48 patients receive a meian of 4 months (
range: 28 months) hormonal ablation. Results 79% overall actuarial freeom from b
iochemical progression at 10 years using strict PSA nair of 0.2 mg/mL (Freeom f
rom failure calculate by metho of KaplanMeier. Differences between groups were
etermine by the log rank metho.) Treatment relate morbiity was limite to
temporary RTO grae 12 symptoms. No patient experience RTO grae 34 toxicity. A
ll failing patients unerwent prostatic biopsies: No pathologically ocumente l
ocal failure, nor any clinical evience of local failure. Anrogen ablation i
not affect the failure rate (p=0.48), although these patients ha at least 2 out
of 3 averse features (excluing stage). PAP was the strongest preictor of lon
g term biochemical failure. (p=0.0001), followe by PSA (p=0,04) an leason sco
re. (p=0.13) using a log rank multivariate regression metho. EBRT, external bea
m raiotherapy; PSA, prostatespecific antigen; PAP, prostate phosphate aci pho
sphatase; RTO, Raiation Therapy Oncology roup.
Biochemical freeom from failure using combine 3DCRT or IMRT an 103P boost f
or clinically localize high risk prostate cancer is quite high even when using
strict PSA nairs. Morbiity has been very acceptable. Despite the aggressive na
ture of the stuy group, with followup prostate biopsies performe on all faili
ng patients, there was no
Basic an avance techniques in prostate brachytherapy
238
pathological ocumentation of local failure; nor was there clinical evience of
local failure. These results were consistent with those in my most recent publis
he series (2003) (Table 16.4, Figures 16.2216.24).107 Consiering the increase
likelihoo of biochemical failure in patients with an elevate pretreatment PAP
an other high risk features (eg leason 810, PSA>20), with these patients we hav
e begun aing an aitional 540 to 1440 cy to the periprostatic tissue, blocki
ng the implant to the 1000 cy isoose line. At the time of writing, we have a
ta out to 12.5 years, with 8 year meian followup an results are actually impr
oving to a point in excess of 80% biochemical iseasefree survival. Comparing m
oalities No moern ranomize stuies comparing brachytherapy, raical surgery
, an external raiation have been performe. Instea, we rely on uncontrolle r
etrospective comparisons using PSA categories to stratify patient groups an com
pare the effectiveness of the various moalities. This represents a fairly reaso
nable form of analysis, given the overall prognostic consistency between instit
utions since the avent of PSA (Zietman,108 Zagars109). It shoul be emphasize
that we are evaluating reporte ata an not mere opinions. When comparing brach

ytherapy with raical surgery in the PSA era, finings have been consistent when
grouping patients in low, intermeiate, an high risk categories. With a follow
up of 10 years or longer, both prostatectomy an seeing appear to be effective
in 80% to 90% of patients with low tumor burens (low PSAs), as reporte by tea
ms from the leaing specialty centers. It is probably fair to say that with low
risk favorable tumors (PSA<10, leason score6, clinical stage T2a or less) the tw
o moalities are essentially comparable. But with intermeiate an high risk pat
ients, the ata now appear to favor brachytherapy markely over both raical sur
gery an conformal raiation therapy (3DCRT). Patients with greater tumor buren
(PSA>10) have a high risk for biochemical failure with either prostatectomy or
3DCRT. Inee, it is with the higher risk groups that the results obtaine wit
h surgery an conventional external raiation have eteriorate to the point of
being woefully unacceptable. The lack of any plateau in the iseasefree surviv
al curves of surgery patients with a pretreatment PSA above 10 is especially str
iking coming from leaing institutions like Johns Hopkins,110 the University of
Pennsylvania,111 an Brigham (Figures 16.2516.26).112,113 Conclusions With cure r
ates at 80% an higher, brachytherapy is proving to be the only reasonable choic
e for intermeiate an high risk patients at whatever age they are treate. A ab
le plateaux of iseasefree survival with implants alone number of investigators
have emonstrate highly favoror when combining brachytherapy with supplementa
l EBRT.7981,107,114117 In aition, a prospective ranomize multicenter trial com
paring pallaium103 brachytherapy with or without supplemental beam raiation i
s reporting no increase blaerurethralrectal toxicities with the combine tr
eatment group.118
103P brachytherapy
239
With the emergence of brachytherapy as the new gol stanar treatment for clini
cally localize prostate cancer, there will continue to be naysayers in the surg
ical community who will want longer term followup before accepting the proceur
e as meeting that stanar of care. As we are now arriving at the benchmark of 1
5 year followup, the argument appears to be on the verge of resolution, with a g
rowing number of brachytherapy teams reporting remarkably effective tumor contro
l rates an favorable morbiity. It is safe to say that as far as the foreseeabl
e future, the proceure is here to stay an its quality assurance is estine to
become even more reliable as more proficient operators join the fiel.
Basic an avance techniques in prostate brachytherapy
240
Figure 16.22 (a, b) Freeom from biochemical progression for 161 patients with P
SA>10 or leason
103P brachytherapy
241
score7 treate with 103P plus 41 y beam raiation.107
Figure 16.22 (c) Freeom from biochemical progression for 161 patients with PSA>
10 or leason score7 treate with 103P plus 41 y beam raiation.107
Figure 16.23 Likelihoo of biochemical failure (rising prostatespecific antigen)
by preoperative serum PSA.110 (Data erive from the
Basic an avance techniques in prostate brachytherapy

242
series by Dr Patrick Walsh, Johns Hopkins Hospital, 19822001.)
Figure 16.24 The likelihoo of biochemical failure (rising prostatespecific anti
gen) by preoperative biopsy leason score (a) an by pathologic leason score (b
). (Data erive from the series by Dr Patrick Walsh, Johns Hopkins Hospital, 19
82 2001.)
Figure 16.25 Likelihoo of biochemical failure (rising prostatespecific antigen)
by postoperative pathologic stage an margin status. EPE, extraprostatic extens
ion. OC,
103P brachytherapy
243
organ confine; SM, surgical margins; SV, seminal vesicles; LN, lymph noes. (Da
ta erive from the series by Dr Patrick Walsh, Johns Hopkins Hospital, 19822001.
)
Figure 16.26 Permanent prostate brachytherapy compare to prostatectomy.119 (a)
Biochemical iseasefree survival (bNED) for
Basic an avance techniques in prostate brachytherapy
244
selecte prostatectomy an brachytherapy series,120,121 stratifie by pretreatme
nt prostatespecific antigen level, (b) Biochemical iseasefree survival (bNED)
for selecte prostatectomy,119 an brachytherapy series,120,121 stratifie by 
leason score of at least 5.
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calize prostate cancer: the Seattle experience. Int J Raiat Oncol Biol Phys 20
03; 57(4):944952. 116. Martinez A, onzalez J, Spencer W, et al. Conformal high 
ose rate brachytherapy improves biochemical control an cause specific survival
in patients with prostate cancer an poor prognostic factors. J Urol 2003; 169(3
):974979. 117. Rage H, rao L, Nair BS. Brachytherapy for clinically localize
 prostate cancer: thirteenyear iseasefree survival of 769 consecutive prosta
te cancer patients treate with permanent implants alone. Arch Esp Urol 2001; 54
(7):739747. 118. haly M, Wallner K, Merrick , et al. The effect of supplemental
beam raiation on prostate brachytherapyrelate morbiity: morbiity outcomes
from two prospective ranomize multicenter trials. Int J Raiat Oncol Biol Phys
2003; 55:12881293.
Basic an avance techniques in prostate brachytherapy
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119. Poun CR, Partin AW, Epstein JI, et al. Prostate specific antigen after ana
tomic raical retropubic prostatectomy: patterns of recurrence an cancer contro
l. Urol Clin north Am 1997; 24:395406. 120. Blasko JC, rimm PD, Sylvester JE. Pa
llaium103 brachytherapy for prostate carcinoma. Int J Raiat Oncol Biol Phys 2
000; 46:839850. 121. Merrick S, Butler WM, Lief JH, albreath RW. Permanent pros
tate brachytherapy: Do prostatectomy an external beam measure up? J Brachyther
Int 2001; 17:189204
17 Ultrasounguie 103P prostate brachytherapy
Jerrol Sharkey, Zucel Solc, William Huff, Raymon J Behar, Stanley D Chovnick,
Ramon Perez, Juan N Otheguy, an Richar I Rabinowitz Introuction In this chapt
er, we escribe our improve an evolving techniques in pallaium103 (Therasee)
brachytherapy for patients with stage T1 an T2 aenocarcinoma of the prostate.
We began this system in 1991.1 With our technique, brachytherapy is an effectiv
e, low morbiity, cost effective treatment for aenocarcinoma of the prostate. W
hile both ioine125 (125I) an pallaium103 (103P) have been use extensively
over the last 14 years,2 our opinion is that 103P (Therasee) provies a more ra
pi initial ose an therefore may give better control of higher leason grae t
umors. We have assesse an highly refine our methoology for 103P brachythera
py in patients with prostate cancer. Stanar brachytherapy techniques3 were mo
ifie to inclue a combination of preplanning with realtime ajustment, placing

all neeles at the same time to minimize prostate movement, using monitore ane
sthesia control (MAC), seation uring preoperative volume stuy an cystoscopy,
an several other small but important changes. At two an six years posttreatme
nt, 9198% of patients ha not experience prostatespecific antigen (PSA) failure
, by the American Society of Therapeutic Raiology an Oncology (ASTRO) efiniti
on. Biopsies were negative in 91% at two years. In our practice we use 103Pa (T
herasee) exclusively for stage T1 an T2 prostate cancer. Retrospective reviews
of pathology graes have reveale unergraing of original leason scores, which
makes the theoretical avantages of 103Ps higher initial ose even more signific
ant. Blasko has reporte that 103P is as effective as 125I in patients with low
er leason scores, an no ifference in patient outcomes (pers comm 1999). Using
103 P, a calculate ose of 13 500 cy can be elivere to the periphery of th
e glan, while conventional external beam raiotherapy (EBRT), an even the newe
r intensity moulate raiotherapy (IMRT) technology elivers only 66007800 cy t
o the prostate an surrouning tissues. The short range of pallaium raiation r
euces the serious blaer an bowel complications that can occur after EBRT, an
 the risks of incontinence an impotence are less than that of raical prostate
ctomy.4,5 Also, postoperative irritative symptoms appear to have a shorter urat
ion when compare to 125I because of the shorter halflife of raioactivity.6
Basic an avance techniques in prostate brachytherapy
252
Methos As part of our continuing efforts to improve results of 103P brachyther
apy over the last several years, we have moifie our techniques for see implan
tation, see activity, an total activity implante. Our current proceure is a
significant moification of techniques an treatment escribe by Blasko,3 an S
tone et al 710 in that we combine preplanning with realtime ajustment, place al
l the neeles at once to minimize prostate movement, an use monitore anesthesi
a control (MAC) seation for the preoperative volume stuy an cytoscopy.11 Spec
ifically, our revise methos inclue: 1. The use of anesthesia for the preplann
ing ultrasoun volume stuy with cystoscopy at the same sitting 2 weeks prior to
the implant. The patient is place in precisely the same extene lithotomy pos
ition he will be in at the time of the actual implant. 2. Use of a biplanar prob
e for both the preoperative volume stuy an the implantation proceure itself.
3. Increase see activity from 2.0 to 2.25 U/see (NIST 2000 equivalent). 4. In
crease number of sees use by approximately 2530% since 1992 (i.e. for a typica
l 35 cc glan, the number of sees has increase from approximately 75 to 95 see
s). 5. Placement of all neeles first, rather than one row or neele at a time
before see loaing. 6. Use of aitional sees to the tumor area or if intraope
rative volume changes occur uring the implant proceure. 7. The presence of a t
eam comprising: raiation physicist, raiation oncologist, specially traine pro
state ultrasoun technician an urologist in the operating room (OR) uring the
implant proceure. 8. Careful consieration of pubic arch interference (PAI) usi
ng a preoperative compute tomography (CT) scan of the pelvis if inicate. 9. U
se of neoajuvant Lupron with or without antianrogen to ownsize the glan when
the volume is over 50 cc or PAI is emonstrate. 10. More extensive an careful
preoperative evaluation of blaer outlet obstruction an treating it preoperat
ively rather than postoperatively is critical. 11. During the planning process p
lacement of a foley catheter allows for better recognition of the urethra an tr
ansurethral resection of the prostate (TURP) efects to allow for urethral sparin
g. 12. Use of sharper, isposable neeles for better visualization of neeles an
with less trauma to the prostate. 13. Use of a fixe stan to stabilize the ult
rasoun probe an gri uring the proceure as well as for the preoperative volu
me stuy. From 1991 through March 2004 we have treate 1442 patients with 103P
(Therasee) brachytherapy exclusively. The mean age of the patients was 72.0 year
s, an about one quarter (25.1%) ha unergone TURP before receiving brachythera
py. This TURP might have been one years earlier for relief of blaer outlet ob
struction before a iagnosis of cancer was mae. The other reasons for preoperat

ive TURP was if ownsizing was not effective in getting the glan below 50 cc af
ter 69 months or because of severe blaer
Ultrasounguie 103P prostrate brachytherapy
253
outlet obstructive symptoms associate with resiual urine. We o a moifie TUR
P, carefully leaving tissue near the veru/sphincter area an not carrying resect
ion own to the capsule. This leaves enough retaine tissue to hol sees withou
t the obstruction remaining. We then wait three months for complete healing (con
firme visually by cystoscopy) an regrowth of a healthy lining in the prostatic
urethra. The average preoperative PSA was 7.46 ng/mL. leason scores range fro
m 2 to 10, although more than two thirs (71%) of the patients ha scores <7. St
anar pretreatment assessments Stanar staging stuies, igital rectal examina
tions, an PSA tests were use to confirm that the isease is organconfine. Pro
state ultrasoun was use to etermine prostate size. Each patient ha 1216 prost
ate mapping biopsies an 24 seminal vesical biopsies (12 per sie). Mapping means
that each core is place in a separate test tube an labele for location in the
prostate (i.e. left lateral base, left apex, etc.). Aitionally, a bone scan a
n pelvic CT scan are one. Where prostate glans are larger than 50 cc, preoper
ative neoajuvant hormonal therapy (NHT) is aministere until the prostate size
ecreases to <45 cc, as etermine by transrectal ultrasoun (TRUs). In patient
s with any egree of blaer outlet obstruction, we consier it critical that th
is be aresse an resolve before, rather than after, see implantation to avo
i postoperative urinary retention, increase symptomatic irritation from raiat
ion, an the nee to o a TURP after see implantation. The incience of inconti
nence is much higher when a TURP is one postimplantation.12 Therefore, before s
ee implantation, we ientify an correct any blaer outlet obstruction. Americ
an Urological Association (AUA) symptom score, resiual urine, an uroflow are a
ssesse, as are complex uroynamics when necessary. When significant obstruction
is iscovere, we perform a transurethral incision of the prostate (TUIP) or TU
RP (25.1% of the patients) an elay implantation for three months to allow time
for healing. At the time of the preoperative volume stuy a cystoscopy is carri
e out, an mucosal healing must be complete (visually by cystoscopy) or the imp
lant is elaye. If healing is not complete superficial urethral necrosis can oc
cur several months later after raiation from the effects of the sees on the ne
wly forme an usually ischemic mucosa that follows TURP. We generally treat mil
er blaer outlet obstruction with alphablockers for several weeks before an
after brachytherapy to avoi the nee for TURP an to prevent postoperative voi
ing problems. Preoperative volume stuy An accurate preoperative volume stuy (b
y the urology ultrasoun technician), is essential because it etermines the geo
metric volume to be targete. The see pattern, which etermines how successfull
y the glan is covere by the actual implantation, is base on this stuy. Patie
nts have the preoperative prostate volume stuy in the operating room in the lit
hotomy position.
Basic an avance techniques in prostate brachytherapy
254
Patients receive moifie anesthesia control (MAC) with Diprivan (propofol), Subl
imaze (fentanyl citrate), an Verse (miazolam hyrochlorie). We feel seation i
s necessary because variations in apparent prostate shape an length have been o
bserve between the ay of the preoperative stuy an the intraoperative volume
stuy in patients who have not been seate. Such changes can be cause by varyi
ng egrees of pelvic muscle relaxation an a ifferent lithotomy position; both
of which occur when a patient is uncomfortable without seation. A simultaneous
cystoscopy to rule out blaer pathology an urethral strictures, an to etermi
ne the egree of blaer outlet obstruction is also performe. It is not uncommo

n for us to iscover unsuspecte blaer tumors or urethral strictures at this p

reimplant cystoscopy. A stepping an stabilizing evice is use with the ultraso
un probe in both the preoperative volume stuy an the actual implantation proc
eure to improve the stability an accuracy an ecrease setup time. Allen stir
rups are calibrate for position setting of the patients lithotomy position an t
his is recore so that the patient is place in this exact position setting ur
ing both volume stuy an implantation proceure. Fleet enemas are given the even
ing before an the morning of the stuy. The patient is place in the lithotomy
position with buttocks flat, horizontal, an parallel to the floor. A foley cath
eter is place to efine the urethra, an inicates the location of the blaer
neck. A transrectal biplanar ultrasoun probe is place parallel to the anterior
rectal wall an the longituinal moe is use to check this alignment. This is
important to prevent neeles from passing through the rectal wall in orer to re
ach the base of the glan. A longituinal ultrasoun image is obtaine at the lo
ngest cephalacaual axis to etermine the number of transverse slices of the p
rostate. Then transverse ultrasoun images are recore from the base to the ape
x of the glan at 5 mm intervals. We label each image so that maximum informatio
n is available to the physicist an raiation oncologist. The location of the po
sitive biopsies, leason score, an PSA are also provie to both physicist an
raiation oncologist. The prostate glan, the urethra, the blaer neck an any
TURP efects are outline in each slice, which enables the physicist to evise a
see pattern that avois excessively high oses to these areas. Because pubic a
rch interference can prevent neeles being place anteriorly or laterally in men
with large glans an narrow pelvic openings, we obtain pelvic CTs to evaluate
this interference when the glan is >50 cc, or in any situation where we suspect
a problem might occur. If the interference involves a major part of the glan,
then implantation is not one an external raiation is offere to the patient.
If this is a minor interference we can change the angle of the probe at the en
of the proceure to implant the problem areas an avoi the obstruction to neel
e passage by the pelvic bones. Because of improve ultrasoun techniques (i.e. t
he use of the longituinal moe an a foley catheter), we can localize the bla
er neck, the base of the glan an the urethra, without the nee for fluoroscopy
. As a result of accurate visualization, sees are rarely retrieve in the posto
perative cystoscopy, an, because of the isposable neeles an rapi placement
of all neeles, there is minimal swelling ue to hematoma in or aroun the prost
ate.
Ultrasounguie 103P prostrate brachytherapy
255
Planning proceures base on the volume stuy 1. The planning process begins wit
h the raiation physicist an the raiation oncologist reviewing both the transv
erse an the longituinal images of the volume stuy, an the total number of cu
ts to be use are agree on. 2. The prostate an the urethra are entere as stru
ctures in the planning program. 3. A threeimensional (3D) moifie peripheral
loaing philosophy is use to etermine the see pattern. It is recognize that
the base an apex of the glan are part of the periphery, an therefore these pl
anes are usually fully loae. In aition, any neele, or portion of a neele,
which is at the periphery of the glan is uniformly loae with sees. For 125I,
the spacing is always 1 cm in these neeles. However, because of the shorter ra
nge of the 103P photons, it is sometimes necessary to place the 103P sees cm
apart in orer to maintain the esire margin aroun the glan. In general, the
interior neeles have a see at base an apex, an just enough sees within the
glan to maintain the esire ose throughout the volume of the glan. The ureth
ra as well as any TURP efect is wellefine an clearly marke in each cut. The
refore, it is a simple matter to avoi placing sees near these raiationsensiti
ve structures. 4. Williamson et al have publishe a etaile review of the NIST
1999 calibration stanars as they apply to 103P osimetric calculations.13 Bas
e on that analysis, they conclue that prior to 1997,103P monotherapy patient

s were receiving a ose of 120 y, an after 1997 the ose was 135 y. When we c
onverte all calculation parameters to conform to the new stanar, we continue
to calculate the ose to the glan at 135 y for monotherapy, an 105 y for pa
tients who receive external beam raiation. In recent years we have reimplante
42 see failures. The ose for these resees is calculate at 110 y. From 1991
to 1993, the activity per see was 2.0 U (NIST 2000 equivalent), with total imp
lante activity as etermine by the original SloanKettering nomograms an a fo
rmula presente by Anerson et al.14 In October 1993, we increase the target se
e count by 15% after a review of postoperative CTs reveale ifficulty achievin
g consistently aequate coverage with the number of sees preicte by this nomo
gram. In October 1996, we again increase the total target activity, by increasi
ng the number of sees an by using sees of 2.25 U. For a typical glan of 35 c
c, with an average imension of 3.8 cm, this woul correspon to an increase in
an implante activity from 150 U (NIST 1999 equivalent) in 1992, to 215 U in 199
9. 5. The see pattern for a particular patient is essentially inepenent of th
e prescribe ose. The esire ose is obtaine by varying the see activity fro
m 2.25 U for monotherapy to 1.75 U for combine therapy, an 1.85 U for resees.
6. The evaluation of the final ose istribution is base first on a visual ins
pection of both the planebyplane isoose istribution, an the 3D renering of
the ose clou to insure that the glan is covere with a suitable margin (35 mm
) aroun the lateral lobes, an somewhat less on the rectal sie. The final eval
uation is base on the osevolume histogram (DVH) with the following criteria:
Basic an avance techniques in prostate brachytherapy
256
(a) The percentage of the glan receiving more than 150% of the prescribe ose
(V150) shoul be between 60% an 70%. This high ose region shoul be confine m
ainly to the lateral lobes where the higher probability of cancer exists. (b) Th
e V150 for the urethra an/or the TUR efect shoul be less than 0.5%. (c) For r
esee patients, the V150 for the urethra shoul be less than 1%, if possible. Th
is planning process coul be one in realtime in the OR with little or no chang
e in philosophy. Doing it approximately two weeks prior to the actual implant, h
owever, allows the physicist to etermine in avance the exact number of sees t
hat will be neee. As mentione previously, three to five aitional sees are
orere for each patient, so that realtime ajustments to the plan can be mae
in any case where the geometry of the prostate encountere at the time of the im
plant is slightly ifferent from that marke on the volume stuy. Implantation p
roceure 1. See implantation is performe in our outpatient surgery center usin
g general or spinal anesthesia. 2. Once the entire team is present the ultrasoun
 images are recreate an matche as closely as possible to the preoperative vo
lume stuy ultrasoun. 3. The urologist then places 23 neeles in the preplan is
tribution nearest to the center of the glan. This helps to prevent the glan fr
om rotating an istorting the implant volume. 4. Then all the neeles are place
 by the urologist in the pattern preetermine by the raiation physicists plan
from the posterior (bottom) row to the anterior (top) row, rather than inserting
one neele at a time. The previous fixation makes it possible to place the rema
ining neeles more accurately than if they were inserte one neele or row at a
time.
Figure 17.1 Diagram showing transverse (T) versus longituinal (L) ultrasoun im
ages.
Ultrasounguie 103P prostrate brachytherapy
257
5. Once all the neeles are in place, we procee to check the prostate in each t
ransverse slice, making sure that the eges or periphery of the prostate align w

ith the initial setup, then rotating to the longituinal ultrasoun, we count on
ce more, the number of slices, an the position of the apex, the balloon an con
sequently the base of the prostate. We then iscuss potential or real changes th
at will have to be mae using the realtime ultrasonography. If we encounter pub
ic arch interference (PAI) causing an inability to place some of the neeles in
the periphery of the prostate we place these neeles at the completion of the im
plant (Figure 17.1). 6. Ultrasoun imaging is switche from the transverse to th
e longituinal view by the raiation oncologist in orer to visualize the prosta
teblaer neck interface an to compensate for prostate motion as a result of t
he initial positioning of the neeles. We start by checking the neeles on a pla
nebyplane basis, starting with the most anterior plane. This allows us to ensu
re that the neele is inserte in the same coorinate throughout the cephalaca
ual axis. It also ensures that it is locate in the proper plane using the long
ituinal view. 7. Careful attention is also given to the peripheral neeles to e
nsure that they are locate within the prostate (as oppose to within the extrac
apsular tissue) unless otherwise specifie in the preplan. The see placement is
one neelebyneele, from the base going caually to the apex, following the
preplan, keeping the spacing as planne. Likewise, if a TURP is present, we make
sure that the tip of the neele approaches, but oes not exten into the TURP c
avity. 8. We ensure that the spacing between the sees remains accurate as the n
eele is move from base to apex. Aitionally, even though the number of sees
to be place in each neele an the spacing between sees were etermine by the
preoperative plan, the plan can be moifie intraoperatively when the size or
Basic an avance techniques in prostate brachytherapy
258
shape of the glan is not exactly the same as it ha been in the volume stuy. T
hese changes can be cause by pressure from the foley balloon, the position of t
he balloon on the ultrasoun probe in the rectum, the amount of flui in the bla
er, or even slight isplacement of the neeles from their intene position (F
igure 17.2). 9. As the raiation oncologist places the sees in each planne coo
rinate, using the MICK applicator, it is very important to ensure that the posi
tion of the sees is within the coorinate plan. Occasionally, as the neele is
retracte (from base to apex) the neele pulls the prostate caually. If no corr
ection is mae for this, then the two sees will be less than the intene ista
nce apart. In certain cases, it is necessary to pull the neele caually up to 1
cm more than the original plan, let the prostate go back to its initial, origin
al position an then, push the neele cephala back to its intene position, an
 thereafter place the see. Likewise, as the neele is retracte caually, it c
oul create a vacuum, which will move or isplace the see caually, sometimes u
p
Ultrasounguie 103P prostrate brachytherapy
259
to 1 cm away from an intene placement (that vacuum also
reate by the action of retracting the plunger within the
As the sees are place from the most anterior plane own
have notice that as we get to the posteriormost planes

has been note to be c

MICK applicator). 10.
towars the rectum, we
some ebris has

Figure 17.3 (a) Longituinal ultrasoun image of the prostate showing foley cath
eter (F), which ais in sees being place away from
Basic an avance techniques in prostate brachytherapy
260

urethra, an neeles (N). (b) iagram of (a).

accumulate in the MICK applicator. It is seen coming out of the neele as the p
lunger moves forwar, to push the see in place. That ebris is hyperechoic, an
it coul be mistaken for a see. Careful observation ensures this oes not inte
rfere with the proper placement of each see. Furthermore, in the lower most pla
nes, occasionally there has been some bleeing note between the rectum an the
posterior wall of
Figure 17.4 Diagram showing how the pubic arch can interfere with irect placeme
nt of neeles into anterior an lateral prostate. Note also that the movement of
the probe can be up or own an sieways to reach those areas.
the prostate, especially towars the apex, after the placement of the initial ne
eles an as we continue to place the sees. Consequently, realtime ajustments
of the neeles to ensure placement within the prostate an not outsie of it ha
s to be one in these instances.
Ultrasounguie 103P prostrate brachytherapy
261
11. Occurrences, such as viewing a neele in its longituinal plane going throug
h the catheter, the presence of an unetecte subcervical lobe, realization that
there is more prostate tissue (initially not visualize in the volumetric ultra
soun) lateral to the most lateral neeles, will make the realtime changes in t
he placement of the sees necessary to assure goo peripheral coverage. As menti
one above, we avoi placement of sees too close to the urethra, an a extra
sees in prostate tissue that was unrecognize initially. These fine points are
crucial to success (Figure 17.3). 12. There are occasional cases in which some o
f the peripheral neeles cannot be place, ue to a PAI. Once all the sees have
been place in the neeles alreay place in their proper coorinates, we proce
e to move the probe slightly to the contralateral sie an then angle the entir
e stan back towar the sie we nee to implant. The image on the TV monitor is
set to the wiest transverse prostate image. Next, a neele is place at the par
t of the glan in the esire plane at an angle. The gri lettering is of no val
ue in this istorte view, so the placement may take several trials before the n
eele hits the esire location in the glan (Figure 17.4). Once complete, the
image is change to the longituinal moe, an the neele is assesse to make su
re that it is locate within the proper plane. Sees are then place starting at
the base an moving caually towars the apex, in that previously unreachable a
rea of the prostate. 13. An extra 35 sees are orere for each case; they are us
e to ajust for ifferences between the volume stuy an the actual treatment g
eometry, or to a an extra ose to the areas of positive biopsy. This is one i
n the longituinal moe by changing the number of sees place in each neele to
cover the area of the prostate being treate. 14. This has the avantage of not
using preloae neeles an using realtime control in two planes. The ability
to visualize the catheter as well as the entire length of the glan in the longi
tuinal moe makes it possible to avoi implanting close to the urethra or to an
y TURP efects. It also ensures that the posterior plane of neeles is entirely
within the prostate an not in the anterior rectal wall. 15. When implantation i
s finishe, the ultrasoun probe is remove an a cystoscopy is performe on all
patients to retrieve any sees that may have migrate into the blaer or ureth
ral lumen. This happens rarely since the newer ultrasoun probes have improve v
isualization. Bloo clots, if any, are evacuate before placing the foley cathet
er, which is left in place for one ay after surgery. 16. A healthcare nurse vis
its the patient on the ay of ischarge from the surgery center, an again the f
ollowing morning to remove the foley catheter.
Results PSA, osimetry, biopsy Patients are evaluate by both the urologist an
the raiation oncologist 2 weeks after the proceure an every 6 months thereaft
er. Multiple sextant biopsies (approximately seven per sie) an mapping (labeli

ng the location in the prostate each biopsy was taken from an placing each biop
sy core in a separate tube) are performe at 1,2, an 5 years. For patients with
negative biopsies, we evaluate PSA levels at 6 month intervals an perform
Basic an avance techniques in prostate brachytherapy
262
another biopsy only when a change in PSA is observe. PSAs are recore every 6
months for 5 years then yearly from the ate of implantation. Over the last 12 y
ears all patients ha postimplant CT scans of the pelvis with Verisee 5 mm step
s through the prostate, 10 ays postoperatively. These were use for postimplant
osimetry using the MMS planning system. Our current proceure is yieling a po
stoperative mean V100 of more than 90%, which means that 90% of the prostate vol
ume is receiving the prescribe ose of 135 y. A separate review is planne on
our pre an postimplant osimetry. Figure 17.5 shows a typical example of our p
ostoperative DVH taken from postoperative CT scans. Evaluation of treatment outc
omes was base on ASTRO efinitions applie to Freeom from PSA recurrence, as w
ell as biopsy ata. In our latest paper1 we analyze our results not only using
initial PSA groups, but also low risk, intermeiate risk an high risk groups, b
ase on both PSA an leason scores. These results confirm 12 years of success i
llustrate in current tables. We also show improvement in results using suppleme
ntal Ext.Raiation Therapy an Hormonal Therapy in Intermeiate an High Risk gr
oups. We further
Figure 17.5 Dosevolume histogram taken from postoperative CT scans. Tissue volu
me (V) istribution irraiate by the implant, with respect to ose (D). The pro
state (re line) gets 100% of prescribe ose throughout the glan (V100); 68% o
f prostate gets 150% of prescribe ose. Urethra (green line): 90% of urethra ge
ts 100%
Ultrasounguie 103P prostrate brachytherapy
263
of precribe ose; 0.4% of the urethra gets 150% of prescribe ose.
Figure 17.6 Postoperative compute tomographic (CT) scan. (a) Peripheral see i
stribution. (b) Typical CT scan 2 weeks postimplant. (c) Xray simulation of pos
toperative see count an istribution.
show outstaning results compare to Raical Prostatectomy in all risk groups us
ing the techniques outline in this chapter.1 Figure 17.6 shows an example of a
postoperative CT scan (see also Tables 17.1 an 17.2). Complications All patient
s experience some egree of shortterm blaer an bowel irritation, which requi
res only symptomatic treatment. Most patients became symptomfree by 36 weeks or
less, as assesse by urinalysis, postvoi resiual ultrasoun reaings, an AUA
symptom scores, an careful history an physical examination. No patient ha rec
tal ulceration, fistula, chronic proctitis, raiation cystitis, or reamission f
or sepsis or urinary
Basic an avance techniques in prostate brachytherapy
264
retention. It is notable that the patients who ha unergone TURP experience no
postoperative irritative problems in the immeiate postoperative perio. During
our followup, fewer than 5% of the patients experience incontinence, an this
sie effect was limite to only those patients who ha unergone previous TURP a
n ha evelope calcification in the prostatic fossa (superficial urethral necr
osis), usually 12 years postimplant. Impotence occurre in fewer than 15% of pati

ents by retrospective history, which we know is not precise. These numbers are a
pproximate an we currently are carrying out a prospective stuy on all our brac
hytherapy an raical prostatectomy patients, using sexual questionnaires an pe
nile Doppler stuies, to more precisely etermine the exact incience of these c
omplications. One patient ie from isease relate causesthis man ha a high ini
tial PSA (>20) an ha probably been unerstage espite negative preoperative C
T an bone scans. There are several other possible complications of brachytherap
y, which occur rarely if at all in our practice. To some extent this may reflect
the meticulous attention to each etail of the proceure by every member of our
team. Possible complications an suggeste means for prevention an/or treatmen
t are presente below.
Table 17.1 Proportion of patients (%) free of PSA failure (ASTRO efinition) aft
er see implantation
Initial PSA (ng/ML) 1 yr 2 yrs 3 yrs 4 yrs 5 yrs 6 yrs
Brachytherapy (103P) monotherapy 04.0 100% 100% 98% 98% 98% 92% 4.110.0 100% 98%
96% 94% 92% 88% 10.120.0 100% 93% 86% 86% 72% 72% >20.0 100% 100% 100% 100% 100%
100% Total 100% 98% 96% 95% 93% 89% Brachytherapy (103p) plus hormone therapy 04
.0 100% 99% 96% 96% 96% 96% 4.110.0 100% 98% 96% 94% 93% 93% 10.120.0 100% 96% 93%
93% 93% 87% >20.0 100% 91% 91% 91% 91% 91% Total 100% 98% 97% 96% 95% 93% All p
atients 04.0 100% 100% 98% 97% 97% 94% 4.110.0 100% 98% 97% 95% 93% 91% 10.120.0 10
0% 95% 91% 89% 89% 84% >20.0 100% 94% 94% 94% 94% 94% Total 100% 98% 96% 95% 94%
91% ASTRO, American Society of Therapeutic Raiology an Oncology.
Perioperative bleeing in the perineum or blaer of significance is rare an pr
eventable by leaving a foley catheter in place overnight an making certain anti
coagulants (aspirin/NSAIDs) are stoppe one week before. Urinary retention can b
e prevente by careful preoperative evaluation of outlet obstructive symptoms (A
UA symptom scores), postvoi resiual urine etermination, an flexible cystosco
py at the time of volume stuy. If significant blaer outlet
Ultrasounguie 103P prostrate brachytherapy
265
obstruction is foun it shoul be taken care of preoperatively, as shoul unsusp
ecte blaer tumors or urethral strictures. Urinary frequency is common an tre
ate with Aleve (which ecreases prostate swelling an inflammation) twice aily
for 721 ays. Pyriium, Urise, an sterois can also be use. Blaer outlet obs
tructive symptoms, if they evelop early, shoul be treate with alphablockers,
or intermittent catheterization if large resiuals evelop. Blaer ultrasoun
for resiual urine shoul be one at the first postoperative visit if symptoms a
re present. Proctitis may be treate with cortisone suppositories or enemas, sit
z baths, stool softeners, an Metamucil. Persistent colorectal complications shou
l be referre for consultation. Superficial urethral necrosis in postTURP pati
ents can be treate with careful TUR ebriement, electrohyrostatic lithotripsy
(if neee), an care to avoi the sphincter area.
Discussion We have escribe a true collaboration an attention to small etails
by a urologist, raiation oncologist, an raiation physicist. We have perioic
ally reviewe our results an complications with the goal of making our proceur
e more accurate an therefore more effective for our patients. Brachytherapy, wi
th a seeosing emphasis on the peripheral zone an sparing of the periurethral
area, may leave more viable (PSAproucing) prostate than external raiation to
account for PSAs not falling to zero. Our urethral ose is maintaine at less t
han 150% of the prescribe ose of the preplan an confirme by postoperative o
simetry. This is especially true when sees are use as monotherapy without exte
rnal raiation ae. In cases of enlarge prostate (>50 cc), neoajuvant thera
py can reuce the volume of prostate tissue expose to the raiation oses in mo
st cases, thereby requiring the placement

Table 17.2 Patients with negative biopsy after see implantation

Initial PSA (ng/mL) 1 yr 2 yrs
Brachytherapy (103P) monotherapy 04.0 95/107 (92%) 50/55 (91%) 4.110.0 120/128(87
%) 48/56 (86%) 10.120.0 13/18 (72%) 11/11 (100%) >20.0 5/6 (83%) 3/4 (75%) Total
238/271 (88%) 112/126(89%) Brachytherapy (103P) an hormone therapy 04.0 109/112
(97%) 57/59(97%) 4.110.0 204/214 (.95%) 104/112 (93%) 10.120.0 53/57(93%) 26/30(87
%)
Basic an avance techniques in prostate brachytherapy
>20.0 Total All patients 04.0 4.110.0 10,120.0 >20.0 Total 21/24 (88%) 6/8 (75%) 38
8/408 (95%) 193/209 (92%) 208/220 (95%) 107/114(94%) 325/353 (92%) 152/168 (90%)
67/76 (89%) 37/41 (90%) 26/30 (88%) 9/12 (75%) 626/679 (92%) 305/335 (91%)
266
of fewer neeles an permitting fewer sees to be implante in the brachytherapy
proceure.7 Early perioic biopsies uring the first two years after implantati
on of 103P are critical to etermine the efficacy of this treatment an to allo
w early salvage treatments such as reseeing any col areas to be institute in pa
tients who fail therapy before the isease becomes metastatic. We o mapping biops
ies of the prostate (1416 iniviually labele specimens) to tell us the exact ar
ea of the prostate sample. We have reseee 41 patients primarily from our first
two years of experience in whom CT scans showe col spots that correspone to th
e area of positive biopsy. The response rate to reseeing has been about 91% in
terms of PSA falling to less than 1.5 ng/mL at the six year point, with 62% an
69% of biopsies negative at one an two years, respectively. It is too early to
comment on its routine use in patients that fail. With greater experience an im
prove osimetry reseeing has rarely been neee. For optimal efficacy of brach
ytherapy, experience with the technique is critical. We believe that our learnin
g curve has been shortene by carefully reviewing our failures, we now know that
our initial ultrasoun equipment was inferior compare with toays improve equi
pment. Also, in terms of visualization as well as the precision of see placemen
t using transverse an longituinal views, as compare to only the transverse vi
ew initially use ramatically improve our results. As mentione earlier, we ha
ve also increase our see activity to correspon with stanars commonly use t
oay an have not neee to use fluoroscopy, rapi strans (only available for 1
25I sees), or fixation neeles in our technique. As a consequence of meticulous
care uring the planning proceure to keep the ose of raiation to the urethra
within acceptable limits (1% of the urethral volume receiving 150% of the presc
ribe ose), we have manage to avoi the high incience of incontinence an sup
erficial urethral necrosis reporte by clinicians who use uniform loaing techn
iques (e.g. 5% in our series compare with 12% reporte by Blasko).3 enerally,
an incontinence rate of 510% occurs with raical prostatectomy (RP) an about 510%
with external beam raiotherapy (EBRT).3 In aition, sexual function has been
maintaine in approximately 85% of our patients; our 15% rate of impotence compa
res with an impotence rate of 2050% for RP an 1030 % for EBRT.3 We believe that t
echnical improvements in brachytherapy will continue to avance its role in the
treatment of localize prostate cancer. This is a relatively noninvasive, onetim
e outpatient proceure, especially valuable in the oler patient who may be unco
mfortable with watchful waiting. Brachytherapy eliminates technical raiation pr
oblems, such as target motion, aily setup variations, an localization errors
that are
Ultrasounguie 103P prostrate brachytherapy
267
problems with 7 weeks of external raiation. We believe this represents the ultim
ate in conformal raiation treatment of the prostate. Biopsy ata have been an w

ill continue to be inclue in our analyses, as this information is crucial to t

he unerstaning of PSA ata an to initiate salvage therapy promptly. A negativ
e biopsy with a rising PSA is inicative of excellent local control by the impla
nt, but extraprostatic sprea of the cancer. A positive biopsy at 1824 months in
icates the nee for aitional local treatment if possible. Very few stuies are
available with both biopsy an PSA results in the raiation literature.3 Aiti
onally, we feel it is important to the longterm outcome of prostate cancer for
the urologist to control all aspects of treatment of this isease. If these resu
lts stan the test of time then brachytherapy, which is a less morbi treatment,
will be an important aition to the urologists armamentarium. Our patient selec
tion, exclusions, an use of postoperative osimetry over the years are in agree
ment with the recently publishe American Brachytherapy Society (ABS) recommena
tions for transperineal permanent brachytherapy of prostate cancer.16 Intraopera
tive osimetry (with immeiate aition of sees to col areas will be the next im
portant avance in our proceure. When conucte with the care an attention to
etail employe in our clinic, see implantation takes only one hour of the phys
icians time an one halfay of the patients an outpatient facilitys time. Despite
the fact that a consierably higher raiation ose is elivere to the prostate
, brachytherapy is associate with less morbiity than stanar EBRT. We have e
scribe several changes in our implantation techniques, which we offer as a mean
s to improve the precision of see placement. Acknowlegments The authors acknow
lege the outstaning help of Sany Winham, RN, Susan Ranall, RN an Marissa Y
oung Aministrative Assistant, for their organizing the proceure to be one in
a costeffective manner in our outpatient surgical center an for careful patien
t ata collection an followup. We also recognize the expert prostate ultrasoun
 volumes pre an intraoperatively by James Steele an Chuck Webster, physician
assistants. References
1. Sharkey J, Cantor, A, et al. Brachytherapy versus raical prostatectomy in pa
tients with clinically localize prostate cancer. Current Urology Reports 2002;
3:250257. 2. Porter AT, Blasko JC, rimm PD, et al. Brachytherapy for prostate ca
ncer. CA Cancer J Clin 1995; 45:165178. 3. Blasko JC, rimm PD, Rage H. Brachyth
erapy an organ preservation in the management of carcinoma of the prostate. Sem
in Raiat Oncol 1993; 3:240249. 4. Henricks J, Kaplan SA. What the literature r
eveals about the complications of raical retropublic prostatectomy. Contemp Uro
logy 1997; 9:1322. 5. Wallner K. Raiation safety parameters following prostate b
rachytherapy. Int J Raiat Oncol Biol Phys 1999; 45:397399. 6. Peschel RE, Chen Z
, Robert K, Nath R. Raiation oncology investigations: Clinical an basic resear
ch. 1999; 7:278288.
Basic an avance techniques in prostate brachytherapy
268
7. Stone NN, Forman JD, Sogani PC. Transrectal ultrasonography an 125I implanta
tion in patients with prostate cancer. J Urol 1988; 139:604A. 8. Stock R, Stone
NN, Wesson MP, DeWyngaert JK. A moifie technique allowing interactive ultraso
un guie threeimensional transperineal prostate implantation. Int J Raiat O
ncol Biol Phys 1995; 32:219225. 9. Stone NN, Ramin SA, Wesson MP, et al. Laparosc
opic pelvic lymph noe issection combine with realtime interactive transrecta
l ultrasoun guie transperineal raioactive see implantation of the prostate.
J Urol 1995; 153:15551560. 10. Stone NN, Stock R, DeWyngaert JK, Tabert A. Pros
tate brachytherapy: improvements in prostate volume measurements an ose istri
bution using interactive ultrasoun guie implantation an threeimensional o
simetry. Raiat Oncol Investig 1995; 3:185195. 11. Sharkey, J, Chovnick SD, Behar
RJ, et al. A minimally invasive treatment for localize aenocarcinoma of the p
rostate: a review of 950 patients treate with ultrasounguie pallaium 103 b
rachytherapy. J Enourol 2000; 14:4. 12. Hu K, Wallner K. Urinary incontinence i
n patients who have a TURP/TUIP following prostate brachytherapy. Int J Raiat O
ncol Biol Phys 1998; 40:4783786. 13. Williamson et al. Recommenations of the Amer
ican Association of Physicists in Meicine on 103 P interstitial source calibra

tion an osimetry: Implications for ose specification an prescription. Me Ph
ys 2000; 27:634642. 14. Anerson LL, Moni JV, Harrison LB. A nomograph for perman
ent implants of pallaium103 sees. Int J Raiat Oncol Biol Phys 1993; 27:129135
. 15. Wallner KE, Roy J, Harrison L. Dosimetry guielines to minimize urethral a
n rectal morbiity following transperineal 125I prostate brachytherapy. Int J R
aiat Oncol Biol Phys 1995; 32:465 471. 16. Nag S, Beyer D, Frielan J, et al. A
merican Brachytherapy Society (ABS) recommenations for transperineal permanent
brachytherapy of prostate cancer. Int J Raiat Oncol Biol Phys 1999; 44:789799.
18 Optimizing realtime, interactive, ultrasounguie prostate brachytherapy
lenn A Healey Introuction For treatment of localize prostate cancer, brachyth
erapy has come of age as the alternative to external beam raiation therapy an
raical prostatectomy.15 Although improvements in ultrasoun inspire the renaiss
ance in brachytherapy,6 a major impeiment to ultrasounguie implant excellen
ce continues to be ifficulty visualizing conventional sees in realtime.7 Hist
orically, ultrasounguie implant techniques took avantage of the stateofth
eart equipment at the time. Early ultrasoun units ha poor sagittal imaging (or
none at all). This prompte early users to evelop the preloae neele techniq
ue;8 there was reliance on hubstylet measurement, hubtemplate measurement, an
base plane ientification, rather than realtime sagittal imaging.9 In the past
five years, enhancements in ultrasoun technology, in particular the evelopmen
t of eicate probes, have ramatically improve the sagittal ultrasoun image.
The harware an software avances are an impetus to optimize ultrasounguie
techniques. However, intraoperative etection of a conventional see is critica
lly epenent on the see location relative to the plane of the ultrasoun trans
ucer. A conventional see is apparent when it is in the transucer plane, but i
sappears if it lies only slightly offaxis. Therefore, it has been rare to visuali
ze every see in a line of implante sees when viewing in sagittal section, bec
ause it is rare for all of the sees to be precisely coincient with a single sa
gittal ultrasoun axis angle. The recently approve EchoSee (Amersham Health, Pr
inceton, NJ) was the first see specifically esigne to be easier to visualize
in sagittal section because it continues to be visible offaxis. The innovative
groove esign of the external surface of the EchoSee makes the see more echog
enic. This allows visualization even when the ultrasoun axis plane is rotate o
ff the axis of the see. A line of implante EchoSees is more likely to be visu
alize in its entirety because a Compromise sagittal axis can be ientifie that
captures all of the sees. The enhance ability to visualize the implant has at
least three obvious benefits that set apart EchoSee implants from conventional
see implants: 1. As each iniviual see is place, the user can immeiately ev
aluate the see in place. The next see in the line can then be place in relati
on to the actual location of other sees in the line, allowing ongoing ajustmen
t for prostate glan motion, neele eflection, an see rift.
Basic an avance techniques in prostate brachytherapy
270
2. Once the entire line of sees is in place, the user can evaluate the relation
ship of the entire line of sees with respect to the base an apex of the prosta
te glan to ensure that coverage is accoring to plan. Unintene col spots can
be assesse an taken care of, if eeme necessary, before moving on. 3. Rotati
ng the ultrasoun probe crale (rocking the crale) very slightly in sagittal sect
ion can capture the threeimensional location of an iniviual see. A see bec
omes highlighte (exhibits greatest signal intensity) when the axis plane of the u
ltrasoun probe is aligne with the axis of the see. The enhance ability to i
scriminate the precise location of an iniviual see will improve the performan
ce of computerassiste, realtime, seecapture/osebuiling programs. Intraope
rative evaluation of the eveloping oseclou base on improve see localizati
on gives the user greater implant quality assurance.

Techniques to optimize EchoSee visualization The benefit of improve visualizat

ion of the EchoSee is realtime, interactive implant optimization to more close
ly align an implant with its intene plan. To fully realize the benefits, howev
er, techniques to optimize the ultrasoun image are neee that take avantage o
f the EchoSees uniquely echogenic esign: Dim the operating room (OR) lights to
improve ultrasoun monitor performance. Select the ultrasoun frequency (usually
6.57.5 megahertz; MHz) that will optimize the prostate image. Dial own the ultr
asoun gain to ecrease extraneous echoes in orer to bring out the EchoSee sig
nal. Rock the crale to realign the sagittal axis an recapture the see signal.
Dim the operating room lights During the proceure, the operating room shoul b
e as ark as possible to optimize the use of ultrasoun. In a arkene operating
room (OR), the users eyes become arkaapte, improving the ability to see the g
rayscale contrasts of ultrasoun imagery. The arkene OR also minimizes extrane
ous light reflecting off the ultrasoun monitor screen, which can egrae the im
age. However, a arkene OR poses proceural an safety challenges that must be
reviewe before proceeing. Key locations for task lighting shoul be ientifie
, incluing the surgical scrub table, the anesthesia station, the physics workst
ation, an the template itself. Lighte clipboars are helpful for critical pape
rwork. Select the ultrasoun frequency that optimizes the prostate image Each br
an an moel of an ultrasoun unit has an optimal working frequency for prostat
e glan visualization. In general, 6.57.5 MHz is the optimal range. The selection
of frequency can vary from case to case base on the inherent echogenicity of t
he
Optimizing realtime
271
prostate. It is useful to scroll through the frequency options at the beginning
of the case an chose a frequency that iminishes internal echoes within the pro
state. This is an especially useful exercise in the highly calcifie prostate gl
an. Once a frequency is selecte, it is recommene that the setting be use th
roughout the case. Manipulation of the gain is a much more sensitive way to reo
ptimize the image uring the proceure. Dial own the ultrasoun gain to bring o
ut the see signal The ultrasoun gain shoul be turne own to accentuate the 
ifference between the ark prostate glan an the highly echogenic EchoSee. The
trick is to set the gain as low as possible, preserving see visibility without
sacrificing prostate glan imagery. ain settings of 20% to 40% are usually opt
imal. As the user moves from see to see an from neele to neele, the inheren
t echogenicity of the ultrasoun image will change. It is useful to manipulate t
he gain to appreciate the setting that reoptimizes the image. During the case,
the prostate glan image can become increasingly echogenic ue to the presence o
f sees an introuce air. As the prostate image egraes, the gain can be turn
e own in orer to bring out the see signal. Rock the crale to recapture the
see signal Implant neeles are typically place in axial section, using institu
tionspecific stanar proceures. Once the neeles are place, the ultrasoun i
s switche to the sagittal view to visualize the neele along its entire length
an to appreciate the relationship of the neele tip to the base of the prostate
. Sees are then place uner irect vision in sagittal section. This allows the
operator to assess in realtime the location of the sees in the glan as they
are being place (Figure 18.1). Avancing an retracting iniviual neeles with
in the glan will cause unesirable yet somewhat preictable movement of the pro
state glan.9 Insertion of the neele tens to cause rotation of the glan (Figu
re 18.2a). Despite a vigilant technique to relax any tension on the glan cause
by neele insertion (Figure 18.2b), a see can ultimately lie in a position awa
y from the axis of the neele (Figure 18.2c). To recapture the see signal, it i
s necessary to rock the crale (rotate the ultrasoun probe) very slightly (Figu
res 18.2 an 18.3). The echogenic tip of the neele is still easily seen even t
hough the operator has now move off of the axis of the neele tip an on to the
axis of the see. As the next see is release along the neele track (Figure 1

8.2e), the process of rocking the crale is repeate to maintain visualization o

f the sees (Figure 18.2f). The technique is to rop a see, rock the crale to
optimize the see signal, retract the neele an rock the crale to reoptimize t
he image, then rop the next see. Once the sees in a given line of sees have
been place an the neele is out of the glan, the crale is rocke to select t
he sagittal axis that lies between the axes of the first an subsequent sees in
orer to see the entire line of EchoSees (Figure 18.4). The apex an base of t
he prostate are seen in relation to the sees, which allows the physician to imm
eiately etermine if the sees are positione as planne before moving on to th
e next neele. If there is a
Basic an avance techniques in prostate brachytherapy
272
Figure 18.1 Establishing anatomical reference from the sagittal view of prostate
brachytherapy raioactive see implant placement proceure.
loss of see signal at any time uring the case, the signal can be recapture by
rocking the crale an further optimize by turning the gain own. Conclusions
Prostate brachytherapy has become an acceptable treatment option for the treatme
nt of localize prostate cancer. However, espite recent improvements in ultraso
unguie raioactive see placement technique an ultrasoun technology, the ab
ility to visualize sees in realtime uring implantation is limite by the phys
ical properties of conventional sees. Whereas intraoperative visualization of c
onventional sees is critically epenent on the see location in relation to th
e ultrasoun transucer plane, the unique groove structure of the newly esigne
 an recently approve EchoSee permits visualization even when the ultrasoun
plane is rotate offaxis. The unique esign of the EchoSee is capable of takin
g full avantage of the new stateoftheart in ultrasoun equipment. Minor moi
fications in intraoperative technique will optimize the echogenic avantages of
the see. Improve visualization allows the user to make intraoperative ajustme
nts to more closely align the eveloping implant with the intene plan. The enh
ance visualization is an asset for computerassiste, realtime, intraoperative
, seecapture/osebuiling programs. Acknowlegment
Optimizing realtime
273
The author thanks Amersham Health, Princeton, NJ for their help with preparation
of the manuscript.
Figure 18.2 Principal steps for placing raioactive sees, incluing key points
of technique for sequential see placement that allow for glan istortion an o
ptimize see signal capture an image visualization. US, ultrasoun probe.
Basic an avance techniques in prostate brachytherapy
274
Figure 18.3 Rocking the crale, a technique for manipulating the ultrasoun tran
sucer to optimize the image an further guie see placement.
Optimizing realtime
275
Figure 18.4 On an offaxis ultrasoun visualization of see implant
References
1. Stock R, Stone NN, Tabert A, et al. A oseresponse stuy for 125I prostate

implants. Int J Raiat Oncol Biol Phys 1998; 41:101108. 2. Zelefsky MJ, Wallner K
E, Ling CC, et al. Comparison of the 5year outcome an morbiity of threeimen
sional conformal raiotherapy versus transperineal permanent ioine125 implanta
tion for earlystage prostatic cancer. J Clin Oncol 1999; 17:517522. 3. Brachman D
, Thomas T, Hilbe J, et al. Failurefree survival following brachytherapy alone
or external beam irraiation alone for T12 prostate tumors in 2222 patients: res
ults from a single practice. Int J Raiat Oncol Biol Phys 2000; 48:111117. 4. ri
mm PD, Blasko JC, Sylvester JE, et al. 10year biochemical (prostate specific an
tigen) control of prostate cancer with 125I brachytherapy. Int J Raiat Oncol Bi
ol Phys 2001; 51:3140.
Basic an avance techniques in prostate brachytherapy
276
5. Merrick S, Butler WM, albreath RW, et al. Relationship between percent posi
tive biopsies an biochemical outcome after permanent interstitial brachytherapy
for clinically organconfine carcinoma of the prostate glan. Int J Raiat Onco
l Biol Phys 2002; 52:664673. 6. Holm HH, Juul N, Peersen JF, et al. Transperinea
l ioine125 see implantation in prostatic cancer guie by transrectal ultraso
nography. J Urol 1983; 130:283286. 7. Nag S, Ciezki JP, Cormack R, et al., for th
e Clinical Research Committee, American Brachytherapy Society. Intraoperative pl
anning an evaluation of permanent prostate brachytherapy: Report of the America
n Brachytherapy Society. Int J Raiat Oncol Biol Phys 2001; 51(5):14221430. 8. r
imm PD, Blasko JC, Rage H. Ultrasoun guie transperineal implantation of ioi
ne125 an pallaium103 for the treatment of early stage prostate cancer. Techn
ical concepts in planning, operative technique an evaluation. Atlas of the Urol
ogic Clinics of North America 1994; 2(2):113125. 9. Stock R, Stone NN, Wesson MF
, et al. A moifie technique allowing interactive ultrasounguie threeimens
ional transperineal prostate implantation. Int J Raiat Oncol Biol Phys 1995; 32
:219225.
19 Realtime prostate brachytherapy: transition from intraoperative nomogram pla
nning to virtual planning
Nelson N Stone, Jeffrey H Chircus, an Richar  Stock Introuction The realtim
e implantation of permanent sources into the prostate was introuce at Mount Si
nai Meical Center in New York in 1990. The esire to evelop a realtime implan
t technique came from the concern that the preplan metho woul not aequately a
ress glan motion an changes in patient setup. While the concept that the us
e of low energy raioactive sources coul prove attractive in the treatment of l
ocalize prostate cancer, attaining uniform goo results has been more elusive.
The transition from the open, retropubic technique to the transperineal approach
with ultrasoun guiance was a necessary step in ensuring osimetric consistenc
y.14 The initial transrectal ultrasoun probes use in prostate brachytherapy wer
e crue compare to toays equipment. Problems with image quality an proper plan
ning le to many false starts an even cause the initiator of this approach, Ho
lm, to abanon it altogether.2 Rage ha visite Holm an ecie to take the ul
trasoun approach back to America.5 Working in Seattle with Blasko an rimm, he
evelope the preplanne implant in the mi 1980s. The two most significant con
tributions in prostate cancer for urologists in the last 15 years have been the
introuction of prostatespecific antigen (PSA) an the biplanar transrectal ult
rasoun probe. Perhaps it was a coincience that they were both introuce towar
s the en of the 1980s, but nonetheless, the iagnosis an treatment of prostat
e cancer raically change once they became part of the typical urologic practic
e. Ultrasounguie biopsy using the B&K moel 8551 biplanar probe (Bruel & Kjae
r; B&K, Wilmington, MA) along with the springloae neele mae this proceure
a common event.6 It was the initial experience with this probe in 1988 that le
to the concept of creating a realtime metho for placing the raioactive source
s in the prostate.7 The impetus to evelop a realtime technique was base on th
e concern that one coul preplan an implant an at some later ate place the see

s accoring to this plan an expect the final result to be the same. The intro
uction of the biplanar ultrasoun probe for prostate biopsy with its sagittal im
age of the glan provie a completely ifferent perspective than the monoplanar
axial imaging probe (B&K moel 1850, B&K Meical Systems, Inc, Wilmington, MA)
initially use for the preplan implant. Axial imaging provie important informa
tion in etermining prostate size through step section planimetry, but sagittal
imaging gave strategic information for sampling the prostate glan.8 The sagitta
l image allowe visualization from the base to apex of the glan, view
Basic an avance techniques in prostate brachytherapy
278
of the blaer an seminal vesicles, an rectum all in one view. The prostate bi
opsy experience also emonstrate how mobile the prostate was. The small spring
loae biopsy neeles cause movement of the glan in several irections creatin
g the potential for major glan movement with the use of many larger implant nee
les. Such movement has recently been emonstrate in a stuy that measure glan
 motion an position uring the neele placement process.9 The introuction of
the B&K biplanar probe generate the initial interest for the creation of the re
altime implant. The implant neeles coul be place using axial imaging an the
sees eposite with the ai of a sagittal transucer. The next step was to ec
ie on how to place the sees. Two methos were possible. The Seattle group favo
re preloae neeles. An applicator metho was selecte for the realtime appro
ach. In keeping with the philosophy of the realtime metho, control of the posi
tion of each iniviual see as it was place was critical. Preloae neeles un
necessarily compromise the inherent flexibility in iniviual see insertion wi
th an applicator (Mick TP 200, Mick RaioNuclear Instruments, Inc, Mount Vernon
, New York). The same can be sai for strane sees. Any inherent avantage to
these proucts may be lost by the brachytherapists inability to control the place
ment of ownstream sources. The next issue to be solve was the planning of the
implant. In keeping with the philosophy of intraoperative planning a set of rule
s was evelope to accomplish this. The total activity neee for the case woul
be etermine prior to the implant by comparing the prostate size to a lookup
table (nomogram). The prostate size was measure by the urologist who etermine
the three imensions of the glan an multiplie them by 0.52.7 This, of course
, require the prostate volume measurement to be accurate otherwise not enough s
ees woul be available for the implant in the operating room (OR).10 The nomogr
am was initially evelope from the Anerson tieline use for the open retropubic
implant at Memorial SloanKettering Cancer Center.11 The tieline was converte t
o a cc/mCi table. It was also ecie to place the sees equally throughout the
glan following the pattern suggeste by Quimby.12 The activity per see was sel
ecte to allow equal spacing of sees throughout the glan. Thus, a 35 cc glan
woul require 17 mCi of ioine125 to eliver a ose of 160 y an sees of 0.5
mCi woul be use. In the OR the prostate volume was remeasure by step section
planimetry an the amount of activity to place in the glan was recalculate. Th
e peripheral neeles were place using a spacing of 1 cm between neeles an arr
anging them just insie the capsule. The number of interior neeles was etermin
e by evaluating the istance from urethra to capsule an maintaining a istance
of at least 5 mm from the urethra. The interior neeles were not place at this
time. The total number of neeles was summe an this number was ivie into t
he total number of sees to be place accoring to the nomogram. After periphera
l neele placement, the sees were place at the base, just uner the capsule, t
he secon miway, an the remaining see at the apex. All sees were iniviuall
y place, taking care to etermine the position of the tip of the neele prior t
o see insertion. Once the periphery was finishe, the interior neeles an see
s were inserte. The see placement looke ieal on the anteriorposterior scout
film. The sees conforme to the shape of the glan with consistent spacing betw
een themselves. One of the most significant avances that were mae early on in
this work was the evelopment

Realtime prostate brachytherapy

279
of a 3D, compute tomographic (CT)base software program that allowe the posti
mplant etermination of osevolume histograms of the prostate, urethra, an rec
tum. Experience in the first year resulte in significant moifications of this
early technique. In orer to achieve the goal of 9095% coverage of the glan by t
he prescription ose, the total activity place in the prostate ha to be increa
se several times an the istribution of the sources slowly evolve from 50/50
to 75/25 periphery/interior.10 This process took almost 5 years, but resulte in
a more consistent target D90. The changes in postimplant osimetry results from
1990 to 1995 reflect these changes. The nomogram changes from 1990 to 1995 also
attest to the magnitue of the increase in activity require to achieve these 
osimetry results.10 The next significant change occurre with the aition of th
e biplanar electronic (linear array) probe (B&K moel 8558). This probe uses two
nonoverlapping transucers, the first, a curve pa occupying the istal anter
ior surface of the probe, an a 5 cm rectangular array just proximal to it. The
ifference in imaging between the new electronic probe an the oler mechanical
one was remarkable. While the axial image was only moestly improve, the linear
or sagittal image was ramatically ifferent. The probe was introuce in 1996
for cryoablation, because of its superior imaging of the prostaticrectal interf
ace. It was aapte for the realtime implant when its superior sagittal imaging
qualities were recognize. After a trial year at Mount Sinai, B&K agree to cre
ate harware to accompany the probe for see implantation. The avantage over th
e oler probes was ocumente in a recent report on postimplant osimetry result
s.13 In concert with the avances in the realtime metho, preplan brachytherapi
sts were reware with the introuction of computer planning software, which imp
rove the efficiency an accuracy of the plan. Several companies release their
versions of software that allowe archiving of the ultrasoun images into a trea
tmentplanning system permitting the physicist to plan the neele an see arran
gements in avance of the implant. Towars the en of 1995, Multimeia Meical S
ystems (MMS) (Varian Meical Systems, Inc, Varian BrachyTherapy, Charlottesville
, VA) introuce their treatmentplanning software (TherpacPlus 6.6 B3DTUI an T
herpacPlus B3DTUI 6.7). While the realtime brachytherapist ha no interest in p
replan software, the technicians of MMS were intereste in coming up with a solu
tion that woul be usable in the OR with this technique. They introuce a versi
on of 6.7, which permitte movement of the planning neele off the gri points,
allowing realtime neele placement with subsequent matching of treatmentplanni
ng neele positions. The moifie software was introuce into the realtime tech
nique in 1998.14 It is still wiely use throughout the worl in those centers t
hat have been traine in this methoology. TherpacPlus 6.7 Technique The prostat
e volume is etermine in the urologists office by measuring the height, with an
 length (in sagittal) of the glan an multiplying by 0.52. This volume is give
n to the raiation oncologist, who using the lookup table specific for the manu
facturers
Basic an avance techniques in prostate brachytherapy
280
source, orers the appropriate see strength an total activity. The prostate vo
lume etermination is repeate in the OR using 5 mm stepsection planimetry. Impl
antation begins by insertion of neeles into the periphery of the glan using th
e largest ultrasoun (US) transverse iameter cut as a guie. Neeles are insert
e just interior to the capsule an place no more than 1 centimeter from the su
rrouning neeles. Determination of glan circumference at the largest transvers
e section yiels the total number of neeles require in the periphery. After al
l peripheral neeles are place the prostate is contoure using US, the urethra

ientifie by an arrow, an each image is store by the treatmentplanning system

. On each acquire prostate slice, the prostate contour was copie an the ureth
ral marker circle. In aition, the anterior rectum was segmente (perirectal f
at an mucosa). The planning system create a 3D gri matrix with x, y, an z ax
es. The sees are then implante with a Mick applicator using sagittal US imagin
g as a guie to neele an see location. The implant is starte at the most lat
eral posterior (7 oclock) neele. The neele is avance to the base of the glan
an the first see place while observing that the neele is just proximal to t
he prostate capsule at the base. The goal for each row is to place the first see
 at the base, the last one at the apex, an intervening sees (usually 24) evenl
y space between the two ens of the glan. The number of sees place through t
he peripheral neeles is etermine by taking 75% of the total number of sees r
equire for the implant an iviing by the number of peripheral neeles. Longer
length rows require more sees, while the shorter rows fewer. The istance betw
een sees in the periphery is always less than 1 cm. After finishing the first n
eele, the probe is rotate a few egrees clockwise an the next lateralanterio
r neele is locate an implante. The entire peripheral implant takes about 20
minutes. During this time, the physicist works to complete the osimetric repres
entation of the implant.14 The position of the neeles in the treatment planning
system is etermine base on the acquire US images with the actual implant ne
eles in place. Neele positions are ientifie by the echobright flash present
on the acquire transverse images. The neele position is first ientifie by l
ocating the nearest gri position to the neele. This point is then ragge to t
he spot corresponing to the image of the neele on the acquire ultrasoun imag
e. The location of the sees in the planning matrix is etermine manually by ex
amining the path of the neele through the transverse capture prostate images.
Sees can be place on any of the 5 mm slices or 2.5 mm above or below any slice
. After placement of all of the peripheral sees, the corresponing isoose line
s are visualize. The next step involves placing the internal neeles. The remai
ning 25% of the sees are inserte via these neeles. Typically, between 6 an 9
neeles are inserte into the interior such that they encompasse the periphery
of the base an apical slices an are 0.51 cm from the urethra. Three to 4 sees
are inserte into these neeles with one see at the base an apex of the glan
. Once these neeles are inserte, the best imaging transverse cut visualizing t
hese neeles is acquire by the planning system. The neele positions are locate
 on the planning matrix. If the prostate has not move or change shape, then t
he new interior neele positions are marke using the newly acquire US image. I
f the prostate ha shifte position, then the neele positions from the US image
are shifte to match the position of the interior neeles in relation to the pr
ostate from the actual implant. The positions of the sees eposite are then e
termine on the planning software in a similar fashion to those implante by the
peripheral neeles. Once the
Realtime prostate brachytherapy
281
interior sees have been place the final isoose istributions are visualize a
n osevolume histograms (DVH) generate. The next avance require a change fro
m working on intraoperative archive images to an interactive system with live sag
ittal imaging. There was also more of a nee to move in the irection of a prepl
an methoology, not because it was necessary to have a plan prior to neele inse
rtion, but to be able to generate enough information for the team as quickly as
possible. Once the plan ha been generate, it neee to be instantaneously upa
te with every change occurring uring neele an see placement. Finally, the p
rogram ha to generate a live sagittal image from which the physicist coul trac
k iniviual see placement an avise the raiation oncologist about the number
an location of each see along with the corresponing isoose contours. The en
gineers an technicians at Varian built the software in parts, which were inivi
ually teste. The new version, VariSee 7.0 (recent upate 7.1) with implant vi

ew took over two years to evelop. The program was release in the fall of 2001
an, after 34 months of testing was reay to be wiely introuce into the brachy
therapy community. The VariSee 7.0 was not just introuce for the realtime tre
atment methoology. Many brachytherapists, whether using a preplan or realtime
methoology will fin its features an enhancement over the oler version. In a
ition, several other companies have avance their brachytherapy planning softwa
re to the point where the user may fin greater avantages for their style of im
plantation over the Varian prouct. It is not the purpose of this stuy to compa
re or recommen the ifferent treatmentplanning systems, but rather to escribe
how the realtime metho evolve, which invariably inclue the use of the Varia
n software. VariSee 7.1 Technique The prostate volume is etermine in the offi
ce using a biplanar transucer by calculating the height, with, an
Basic an avance techniques in prostate brachytherapy
282
Figure 19.1 Use of the Bar/ProSee planning moule to rapily generate the nee
le an see placement locations. Selecting this planning moule within the image
view moule creates an instantaneous plan. The 3 longituinal measurements, onc
e entere, give the number of peripheral an interior neeles along with the cor
responing number of sees.
length of the glan on the largest transverse an sagittal images an multiplyin
g by 0.52. The total activity neee is calculate from a nomogram an elivere
alreay loae in isposable cartriges. The patient is positione an the prob
e is place in the rectum. The prostate glan is contoure at 5 mm intervals fro
m base to apex. The images are store in the treatmentplanning program. The phy
sicist uses several new tools in the VariSee 7.1 program to rapily autocontou
r the prostate an ientify the urethra an rectum. Containe within special ver
sions of the 7.1 program is a Bar/ProSee planning moule which allows the physic
ist to create a full plan, following the rules establishe for realtime plannin
g in a matter of a few minutes. In essence, once the urologist completes the pla
nimetry stuy, leaves the room to scrub, returns to prep an rape the patient,
the planning is complete (Figure 19.1). The urologist places the peripheral nee
les accoring to the plan by observing the computer monitor. The urologist looks
at a live transverse image of the glan with the virtual image of the prostate,
urethra, an rectum superimpose over the ultrasoun
Realtime prostate brachytherapy
283
image. Virtual neeles also appear an the urologist places the applicator neel
es through the template (gri) so they en up at or near the intene positions.
It is not necessary to get an exact match, because the physicist can rag the v
irtual neeles to the flash positions of the inserte neele (Figure 19.2). After
all of the peripheral neeles have been place the prostate is recapture into t
he planning system (Figure 19.3). This is a crucial step because the neele plac
ement has significantly altere the original plan. The neeles cause the prostat
e to move cranial, isplace it off the rectum an istort its eges.15 The neel
es also o not en up in the exact same position as the plan calle for. The phy
sicist upates the plan by ajusting the contours an aing or eleting sees f
or each neele (Figure 19.4). Once the plan has been upate, the raiotherapist
can start placing the sees with the Mick applicator. Starting at the 7 oclock p
osition, the first neele is ientifie an avance to the base of the glan. T
he physicist is looking at the corresponing live image on the planning system w
ith the virtual prostate image, neele, an sees in front of him (Figure 19.5).
He can irect the number of sees to be implante in each row after checking th
e isoose contours overlying that sagittal image as well as the osing parameter
s that were previously set. Once the raiation oncologist starts placing the see

s, the physicists can track there positions an overlay the virtual sees on to
p of the actual sees in the glan (Figure 19.6). The isoose contour at each su
cceeing neele is representative of the composite osimetry of all of the sees
alreay place an
Figure 19.2 Live transverse image with 5 posterior neeles place. Arrow points
to next intene neele position. The posterior neele placement has move the p
rostate anterior,
Basic an avance techniques in prostate brachytherapy
284
necessitating the recontouring of the prostate after all peripheral neeles have
been place (see text).
Figure 19.3 Image recapture with all of the peripheral neeles place. The newer
images are capture on top of the initial planning images.
Realtime prostate brachytherapy
285
Figure 19.4 The physicist upates the plan once all of the peripheral neeles ha
ve been place by aing or eleting sees for these neeles or by ajusting the
neele an sees for the interior neeles (which have not been place at this p
oint). The 140,160, an 240 y isoose lines are isplaye.
Basic an avance techniques in prostate brachytherapy
286
Figure 19.5 Implantation of posterior neele in miline or D position. The plan in
icates 4 sees from base to apex with the corresponing isoose contour. The 16
0 y line covers the entire posterior aspect of the prostate with little ose to
the rectum.
Realtime prostate brachytherapy
287
Figure 19.6 See placement of a posterior neele. The physicist has aligne the
virtual sees to the corresponing implante ones. In this way the plan is conti
nually upate after the sees are place.
the sees yet to be place. In this way the physicist is continually upating an
 moifying the plan as the physicians work to complete it. After finishing the
periphery, the probe is returne to axial an the interior neeles are place in
a similar fashion to the peripheral ones. The finetuning of the interior neel
e positions an number of sees to be place in these neeles is epenent on th
e number an positions of the peripheral sees alreay place. In many cases, it
is not unusual to en with fewer sees than originally recommene by the plann
ing moule. The osing criteria ahere to are: ose to 90% of prostate (D90) 16
0180 y, 30% of the urethral volume (UD30) <150% of prescription (160 y) an vol
ume of rectum (rectal V100) covere by the prescription ose < 1.3 cc. Results T
en consecutive patients unerwent ioine125 (125I) see implant between April a
n September 2003 using the VariSee 7.1 with implant view an the Bar/ProSee
planning moule. Mean intraoperative prostate volume was 49.8 cc (range: 32.698.1
) compare to 54.9 cc (range: 33.4113.5, p=0.019) as etermine by the 30 ay pos
timplant CT stuy. The mean intraoperative prostate D90 was 193 y (range: 184 20
7) compare to 197 y (range: 177229, p=0.417) from the postoperative stuy.

Basic an avance techniques in prostate brachytherapy

288
Results for the prostate V100, V150, rectal V100 (in cc) an urethral ose (to 3
0% of volume) are shown in Table 19.1. The intraoperative an postplan values we
re compare by taking a ratio of the two. The mean D90 ratio was 0.98 (95% CI 0.
921.03) an the mean V100 was 1.06 (0.98 1.15). The other variables are shown in T
able 19.2. The mean ifference in D90 values (intraoperative minus postoperative
results) was 411 cy (range: 30411696 cy). Discussion The ieal implant techniqu
e shoul yiel results for target tissue osimetry that closely match the planni
ng stuy, regarless of the metho use. Unfortunately, most stuies, even from
centers of excellence, have yiele less favorable results.16 We have avocate
the realtime technique over the preplan metho for the reasons state above. Ma
tzkin has compare the twostep preplant metho (n=142) to the Varian 6.7 intrao
perative metho (n=214) escribe above an foun only 58.4% achieve their targ
et ose with the preplan metho, compare to 95.2% with realtime.17 Newer techn
ology in meicine oes not always result in improve clinical outcomes. This is
our first attempt to
Table 19.1 Mean values for intraoperative versus postplan (30 ay CTerive) fo
r prostate D90 (ose to 90% of glan), V100 (% of volume of prostate covere by
160 y), V150 (% of volume of prostate covere by 240 y), rectal V100 (volume o
f rectum covere by 160 y) an urethra D30 (ose to 30% of urethral volume)
Intraoperative Postimplant pvalue Prostate volume (cc) 48.7 54.9 0.019 D90 (y)
193 197 0.417 V100 (%) 103 97.4 0.96 V150 (%) 52% 36% 0.015 Rectal V100(cc) 0.8
1.2 0.244
Urethra D30 (y) 194 250 <0.001
Realtime prostate brachytherapy
289
Table 19.2 Mean ratio with 95% confience interval (CI) of intraoperative to pos
tplan stuies
Ratio 95% CI
D90 0.98 V100 1.06 V150 1.67 Rectal V100 0.81 Urethra D30 0.77 Prostate volume 0
.91 0.921.03 0.981.15 1.052.30 0.261.36 0.710.84 0.821.01
quantitate our osimetry results in a small cohort of patients implante with Va
rian 7.1 with implant view. While the initial results appear to be very goo, th
e intraoperative to postimplant D90 ratio was 0.98, is it substantially better t
han what can be achieve with Varian 6.7 using the realtime metho? In a stuy
of 77 patients comparing the intraoperative D90 to the postimplant ose using 6.
7, Stone also foun a ratio of 0.98.18 These ata suggest that there may be litt
le avantage to switching to the newer metho. However, a closer analysis of the
6.7 ata in the 125I patients reveale a mean OR D90 of 178 y (range: 126214) v
ersus the postimplant D90 of 188 y (142225). The current stuy (Varian 7.1) reve
ale a much tighter an closer match between the OR an postimplant ata: 193 y
versus 197 y, with a much smaller range of oses. These ata also substantiate
that the use of an intraoperative planning system, combine with the realtime
implant yiels very favorable postimplant osimetry results. Attaining a postimp
lant prostate D90 of at least 140 y in patients receiving an 125I implant is ne
cessary to insure a favorable iseasefree (PSA) outcome.19 For local control, 
oses of 160 y may be more appropriate. Stock has shown that patients receiving
a ose of at least 160 y ha a 4% likelihoo of local recurrence (as etermine
by biopsy) 2 years after implantation.20 Thus, consistency of implant results,
above a certain threshol (either 140 y or 160 y) has significant implications
on cancerspecific outcomes. Selecting the ieal implant metho as a means of ass

uring this consistency is controversial. Many believe that the preplan metho is
superior, while others woul argue for some form of intraoperative or realtime t
echnique. Most brachytherapists have starte to move towar more of an intraoper
ative methoology, either running the preplan in the OR or by performing the int
eractive technique escribe here. Intraoperative planning has the avantage of
minimizing the iscrepancies in repositioning the patient an saves time by elim
inating the preplanning carrie out in a ifferent setting. However, pubic arch
interference, prostate glan movement, an ege istortion from neele insertion
can still cause plan eviation, which may aversely affect implant quality. ew
anter foun a ecrease in overall planning time with the intraoperative preplann
ing approach. There was no ifference in osimetry from their stanar preplanni
ng an their intraoperative technique (V145:80% vs 83%).21 The ratio of mean %D9
0 comparing the preimplant to postimplant osimetry results was also quite high
(1.34 an 1.54, respectively). The intraoperative planning ratio was much higher
because of significant overplanning in the operating room (mean D90:131%) compa
re to the postimplant osimetry results (mean D90:85%). Wilkinson performe a s
imilar
Basic an avance techniques in prostate brachytherapy
290
stuy an foun comparable results.22 Messing et al reporte on an optimize inv
erse planning system.23 Patients first ha a computerize preplan performe >1 m
onth prior to implantation. Planning was repeate in the operating room with thr
ee stabilizing neeles in place. An average of 23% unerosing was emonstrate
when the preplan osimetry results were compare to the intraoperative replannin
g results. No postimplant osimetry ata were reporte, so making any jugment a
s to whether this system is an improvement over stanar preplanning is not poss
ible. Beyer has also evaluate the application of intraoperative osimetry. In a
pilot stuy of 17 patients, 9 control patients (stanar preplan) ha a meian
V100 (145 y) of 97%, while 8 intraoperative planne patients ha a meian V100
of 94%.24 Beyers approach is similar to that reporte by ewanter with some moif
ications. A greater attempt was mae to incorporate intraoperative eviations, s
uch as pubic arch interference, within the planning system. This stuy, like the
other two reports on intraoperative preplanning, i not fin a osimetric ava
ntage over the stanar preplan technique. In fact, Beyer foun the postimplant
osimetry was somewhat inferior. Of the 9 patients, 3 (33%) ha V100 < 100% of w
hich 2 < 90%. No comparison was mae between the intraoperative an the postoper
ative plans. Zelefsky treate 248 patients with an intraoperative computeroptim
ize technique.25 He use an inhouse program to optimize see positions after p
eripheral neele placement along with their own osing constraints. Postimplant
osimetry was obtaine 4 hours after see insertion. Comparisons were mae to 24
7 patients treate with the stanar preplan technique before the institution of
the computerize metho. Meian V100 improve from 88% to 96% (p<0.05) an D90
from 95% to 120% (prescription 145 y, p<0.05). While a ecrease was also note
in urethral ose, both the V150 an rectal oses were higher in the intraoperati
ve computeroptimize treate patients. This is in contrast to the current stuy
, where postimplant urethral oses were note to be higher than the oses genera
te in the OR (194 vs 250 y to 30% of the urethra). However, a catheter was not
place for the CT stuy an the urethral position was just estimate. The recta
l oses were not increase an the V150 was substantially less than what was rep
orte in the Zelefsky stuy (36% vs 74%) suggesting that the 7.1 metho may give
a more homogeneous implant. It is clear from this stuy an the one reporte by
Zelefsky that an intraoperative approach combine with an interactive planning
system coul improve the osimetry outcomes of prostate brachytherapy. These tec
hniques yiel consistent prostate oses that are sufficient for eraicating the
tumor an low enough at critical structures, urethra, an rectum to protect the
patient from longterm morbiity. More centers shoul consier aopting similar
methoologies in orer to enhance their prostate brachytherapy program. Referenc

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t al. Intraoperative optimize inverse planning for prostate brachytherapy: earl
y experience. Int J Raiat Oncol Biol Phys 1999; 44:801808. 24. Beyer DC, Shapiro
RH, Puente E Realtime optimize intraoperative osimetry for prostate brachyth
erapy: a pilot stuy. Int J Raiat Oncol Biol Phys 2000; 48:15831589.

Basic an avance techniques in prostate brachytherapy

292
25. Zelefsky MJ, Yomaa Y, Marion C, et al. Improve conformality an ecrease
toxicity with intraoperative computeroptimize transperineal ultrasounguie
prostate brachytherapy. Int J Ra Oncol Biol Phys 2003; 55:956963.
20 The ProSee approach: a multicenter stuy of the results of brachytherapy tra
ining
Nelson N Stone, Jeffrey H Chircus, Richar  Stock, Joseph Presser, an the ProS
ee team Introuction Prostate brachytherapy has become an accepte means of tre
ating localize prostate cancer. The new brachytherapy is istinguishe from prost
ate see implantation performe prior to 1985 by the use of ultrasoun guiance
to plan an place the raioactive sources.13 The oler brachytherapy techniques u
se a freehan metho of see placement, which resulte in inaequate coverage of
the prostate glan by the intene raiation an a higher clinical failure rate
than ha been anticipate.45 While the ultrasounguie implant technique is co
nsiere to be far superior to the oler implant metho, little ata have emerge
 that ocuments that quality implants consistently result with its use. Implant
quality irectly relates to clinical outcome, yet significant ata are lacking
on postimplant osimetry from most centers.6 Two issues are emerging that coul
be crucially important to patients as they search for what they consier to be t
heir best choice for treating their localize prostate cancer: can a quality imp
lant always be performe an has the physician been traine aequately to get co
nsistent high quality results? Prostate brachytherapy is a highly technical proc
eure requiring extensive training an experience in orer to gain proficiency.
We set up a training program that offere classroomstyle instruction along with
onsite proctorship of the treating physicians until a sufficient level of prof
iciency was achieve. After completing training, the physicians were allowe to
treat patients on their own. The results of the training were assesse by eterm
ining the quality of the implant while the proctor was present an after the phy
sician ha complete training. This is the first report that analyses this type
of postgrauate (resiency) training an the impact that it has on assuring pati
ents that they can receive a new type of treatment without having to worry that
their physician will have to go through a learning curve by gaining sufficient exp
erience on them. Materials an methos Technique The realtime technique of see
implantation was the metho for prostate brachytherapy taught to the physicians
. Prior to implantation, the prostate volume measurement was
Basic an avance techniques in prostate brachytherapy
294
obtaine using: heightwithlength multiplie by 0.52. This ellipsoi volume was give
n to the raiation oncologist, who use an activity nomogram orere the appropr
iate amount of activity to bring to the opearting room (OR). Details of this tec
hnique for prostate brachytherapy have been previously reporte.2,7 Patients wit
h biopsy proven aenocarcinoma of the prostate who ha a leason score 6 were imp
lante with 125ioine (125I) to a minimum ose of 140 y (T43). Patients were
brought to the OR where the ultrasoun probe [Bruel & Kjaer; B&K, moel 8551 (pr
ior to 1998) or moel 8558] was place into a special crale an stepping evice
an attache to the base unit. The probe was next positione in the rectum an
using transverse imaging, serial contours of the prostate were measure at 5 mm
intervals from prostate base to apex. This planimetry volume was use when calcu
lating the amount of activity to implant. The total activity, as etermine by a
nomogram, was ivie by the activity per see (usually 0.30.5 mCi/see for 125I
) gives the total number of sees to implant. After the volume measurements were
complete, imaging was switche to longituinal an measurements were mae ante
rior an posterior to the urethra an posteriorly from base to apex. These longi

tuinal measurements were use to etermine the spacing between the sees. The i
mplant was begun by first fining the largest transverse image of the prostate a
n by placing Mick neeles (Mick Raionuclear Instruments) in the periphery of t
he glan. Starting posteriorly, an keeping at least 7 mm above the anterior rec
tal wall, iniviual applicator neeles were place approximately 1 cm apart. Wo
rking in a clockwise fashion, the entire periphery of the glan was implante wi
th the neeles. The number of neeles was summe an ivie into the number of
sees to be implante in the periphery. For ioine125 an pallaium103, 75% of
the total activity was place in the periphery. Spacing between sees in each ro
w was etermine by calculating the average length of the prostate. The average
length was etermine from the three length measurements mae in longituinal im
aging. The spacing between sees was then calculate by: sees per row/n1, wher
e n=average height of the prostate. The maximum space allowe between sees in t
he periphery was no greater than 10 mm. Implantation of the sees was accomplish
e with a Mick applicator (Mick TP200, Mick Raionuclear Instruments). Imaging
was switche to sagittal an the probe was avance in a cephala irection unti
l the entire prostate was overlying the transucer hea. The probe was then rota
te to the left (counter clockwise) an the most lateral neele was image. The
tip of the neele was ientifie an brought to the base of the glan. The rai
ation oncologist then place the raioactive sources using the Mick TP 200 appli
cator. Working from base to apex an keeping the tip of the neele uner constan
t ultrasoun monitoring, each iniviual see was place until the apex of the g
lan was reache. The interior neeles were then place using ultrasoun to spac
e the neeles 10 mm from the periphery, at least 5 mm from the urethra an appro
ximately 10 mm apart. The implant proceure was enhance with the aition of th
e Varian 6.7 software (Varian, Palo Alto, CA) in 1999, Varian 7.0 in 2002 an 7.
1 in 2003. The aition of the planning software allowe for intraoperative ose
ajustment.89 The planning rules, as etaile above, remaine consistent, even w
ith the aition of the software.
The ProSee approach
295
Training The training consiste of two phases, a iactic session an proctoring
. All personnel who participate in the proceure were require to atten the i
actic session, which laste about four hours. This inclue urologists, raiati
on oncologists, meical physicists, osimetrists, nurses, an all OR personnel i
nvolve in the proceure. A urologist, raiation oncologist, an nurse gave lect
ures. Materials hane out inclue a etaile proceure manual, a iactic book
an vieotape emonstrating the proceure. At the en of the iactic session t
he participants were traine on the use of the equipment using a phantom moel.
After completion of the iactic session, physicians scheule cases in their ow
n OR. A physician proctor (either raiation oncologist or urologist), with signi
ficant experience in this technique, attene all cases until both the provier
raiation oncologist an urologist were proficient in the metho. This usually r
equire a minimum of five cases, but was not specifically limite to a case numb
er. It was up to the jugment of the last physician proctor to etermine if an i
niviual provier coul perform cases inepenently (postproctor). Yearly upa
tes were recommene an provie both a clinical review an proceure enhanceme
nt with aitional OR training. Implant quality evaluation One month after impla
ntation the patient returne to have the implant evaluate. Compute tomography
(CT) images of the prostate were taken at 3 mm intervals from the base of the bl
aer to the sphincter to assess the quality of the implant. Initially, CT image
s were sent to a central site where all the osimetry was performe. Transverse
CT images were analyze by one physician (RS) who trace out the prostate, rect
um, an blaer on each slice. The images an see positions were then igitize
by a commercially available brachytherapy software package (MMS TherpacPLUS, Mu
ltimeia Meical Systems, 700 Harris Street, Suite 109, Charlottesville, VA, 229
03). The software moels ose istributions with the new osimetry formalisms a

opte by the American Association of Physicists in Meicine (AAPM) Task roup 43

. The output is a graphical epiction of the isoose istribution of each transv
erse slice. A osevolume histogram (DVH) was also generate to provie volume in
formation of the implante ose. The osimetry parameters Dn, Rectn, an Blan,
which are the oses encompassing n% of the prostate, rectal wall, an blaer w
all, respectively, were interpolate from the DVH. Results were reporte as ose
volume histograms an ose surface histograms of the prostate, rectal wall, an
base of blaer, respectively. The ose to 90% of the prostate volume (as eter
mine by the CT) was calculate as was the percent of the glan covere by the t
arget ose (140 y for 125I, 115 y for full 103P, an 83 y for partial 103P)
. The oses to 10% an 30% of the rectal an blaer volumes were also calculate
. After 1999, hospitals that obtaine the Varian planning software through ProS
ee performe their own postimplant osimetry an sent the ata in for analysis.
All ata was entere into a centralize atabase. Comparisons were mae for the
entire group as well as for the proctore an postproctore cases using stuen
t t test.
Basic an avance techniques in prostate brachytherapy
296
Results The initial four ProSee hospitals were starte in 1996 an were: Northw
est Hospital Center, Baltimore, MD,
Figure 20.1 Number of implants per hospital. There was a mean of 26 cases per ho
spital (range: 1108).
Holy Cross Hospital, Silver Spring, MD, Baptist Hospital, Nashville, TN, an Res
ton Hospital Center, Reston, VA. By the en of 1997 an 1998, 19 hospitals an 4
4 hospitals were performing the ProSee implants. Over 120 hospitals throughout
the Unite States, Europe, the Mile East, an Africa have performe ProSee im
plants to ate. From 1996 to February 2001, a total of 990 patients receive a p
rostate implant at 35 centers (mean 26 cases/ center) of which 289 (29.1%) were
proctore (group 1) an 701 were one by physicians who ha been certifie to pe
rform the implants inepenent of a proctor (group 2) (Figure 20.1). There were
665 125I (target ose 140 y), 135 full ose 103 P (target ose 115 y) an 19
partial 103P (target ose 77 y) implants (Figure 20.2). Mean D90 for
Figure 20.2 Distribution of cases by teaching (proctor) an implant type (p103P
is partial 103P).
The ProSee approach
125
297
I for group 1 vs 2 were 164 y vs 162 y (p=0.4). Mean D90 for full 103P for gr
oup 1 (n=45) vs 2 (n=89) were 133 y vs 125 y (p=0.02). Mean D90 for partial 10
3P for group 1 (n=47) vs 2 (n=144) were 88 y vs 92 y (p=0.1). Aitional pros
tate, rectal, blaer, an urethral ose comparisons are shown in Tables 20.120.3
. Of the 665 125I implants, 9.1% (18/197) of group 1 an 8.3% (39/468) of group
2 ha a D90 < 140 y an 1.5% vs 1.9% ha a D90 < 120 y (Figure 20.3). In the f
ull 103P patients, 13.3% of the proctore cases ha a D90 < 115 y vs 23.6% of
the traine physicians, while 0% of group 1 vs 2% of group 2 resulte in D90 < 1
00 y (Figure 20.4). For the partial 103P cases, 23% vs 13.2% were foun with a
D90 < 77 y, while 0% of each group ha a D90 < 60 y (Figure 20.5).
Table 20.1 Comparison of proctor (n=197) to certifie (n=468) 125I postimplant 
osimetry oses. D50100: ose covering percent of prostate volume; rectal 10 an 3
0: ose to 10% an 30% of rectal volume; same for blaer an urethra. Values ar
e in gray an represent means

Proctor Certifie pvalue

D100 D95 D90 D80 D50 Rectal 10 Rectal 30 Blaer 10 Blaer 30 Urethra 10 Urethr
a 30 81 143 164 190 242 125 64 102 59 201 186 77 139 162 186 240 122 60 98 59 19
8 184 0.03 0.02 0.4 0.2 0.5 0.4 0.1 0.2 0.9 0.8 0.8
Table 20.2 Comparison of proctor (n=45) to certifie (n=89) 103P postimplant o
simetry oses. D50100: ose covering percent of prostate volume rectal 10 an 30:
ose to 10% an 30% of rectal volume; same for blaer an urethra. Values are
in y an represent means
Proctor Certifie pvalue
D100 D95 D90 D80 D50 Rectal 10 51 111 133 163 228 87 50 0.6 105 0.01 125 0.02 15
2 0.02 221 0.4 86 0.7
Basic an avance techniques in prostate brachytherapy
Rectal 30 Blaer 10 Blaer 30 Urethra 10 Urethra 30 37 87 45 184 170 34 0.3 75
0.06 39 0.12 183 0.9 167 0.6
298
Table 20.3 Comparison of proctor (n=47) to certifie (n=144) partial 103P posti
mplant osimetry oses. D50100: ose covering percent of prostate volume; rectal
10 an 30: ose to 10% an 30% of rectal volume; same for blaer an urethra. V
alues are in gray an represent means
Proctor Certifie pvalue
D100 D95 D90 D80 D50 Rectal 10 Rectal 30 Blaer 10 Blaer 30 Urethra 10 Urethr
a 30 32 73 88 110 161 63 28 46 21 140 127 36 0.04 76 0.06 92 0.1 110 0.1 158 0.7
61 0.7 26 0.4 54 0.05 28 0.006 135 0.6 123 0.6
Discussion Prostate brachytherapy was first escribe almost 90 years ago an ha
s evolve into a highly technical proceure.8,10 Ten year ata suggest that pros
tate brachytherapy is equivalent to raical prostatectomy in biochemical cure.11
Other stuies with shorter followup also suggest equivalent results to externa
l beam irraiation an raical prostatectomy.122020 With these ata now reaily a
vailable to clini cians an their patients, brachytherapy is rapily becoming a
popular metho of treating localize prostate cancer. Physician training in pros
tate brachytherapy has typically been limite to one or two ay courses offering
at most iactic laboratory sessions. In aition, newly grauate urologists a
n raiation oncologists are emerging from their resiency programs with little
to no training because most acaemic programs are just beginning their own brach
ytherapy programs. There are no formal
The ProSee approach
299
Figure 20.3 Percent of patients with D90 (125I) values by ose cutoff point.
Figure 20.4 Percent of patients with D90 (103P) values by ose cutoff point.
programs that also train the hospital staff. Because of the highly technical nat
ure of prostate brachytherapy, aequate training woul appear to be critically i
mportant in assuring patients of a successful an safe outcome. While clinical o
utcome (e.g. biochemical freeom from failure base on PSA), can be one measure
of a successfully performe implant, it can take at least 5 years in a prostate
cancer patient to know if the treatment has been successful. In aition, late r
aiation complications, such as proctitis, urethritis, an cystitis, can take 2
to 3 years before they manifest themselves. It is not reasonable to wait for suc
h outcomes to know whether or not the treating physician is elivering proper th
erapy. Quality assessment of an implant can be etermine shortly after the proc
eure by a osimetric evaluation. Oler systems relying on plain or orthogonal r
aiographs i not accurately ientify the relationship between the raioactive

sources an the soft tissue structures (prostate, rectum, an blaer). Stone e
scribe the use of CT scan transverse images, which were igitize with a softwa
re program that accurately reconstructe the position of the sees an the prost
ate, urethra, rectum, an blaer. The ose covering 90% of the prostate was es
cribe as well as the ose to the surface area of the rectum.21 Willis an
Basic an avance techniques in prostate brachytherapy
300
Figure 20.5 Percent of patients with D90 (partial 103P) values by ose cutoff p
oint.
Wallner reporte on their experience with CTbase osimetry in 20 unselecte pa
tients who ha receive an 125I implant.22 They reporte an average of 84% of th
e target volume (prostate) receiving a ose of 140 y. They consiere an implan
t to be aequate if 140 y covere 80% of the prostate. Stock, in a oserespons
e stuy of 125I, was the first to report that a D90 ose of 140 y was necessary
in orer to achieve a substantial PSA biochemical freeom from failure (93% vs
48% in those patients who receive less than 140 y).6 Biopsy stuies have emon
strate 98% local control with a D90 of at least 100 y (preNIST 1999) in patie
nts who ha receive a 103P implant.12 Aequate osing for permanent implants c
ombine with external beam irraiation (EBRT) has been harer to efine. Dattoli
use 103P at a ose of 80 y with external beam irraiation in high risk patie
nts an foun a favorable outcome.23 Likewise, Blasko reporte on a group of hig
h risk patients who receive the combination treatment an receive a ose of 86
y of pallaium.24 Finally, the American Brachytherapy Society recommens that
the brachytherapy ose be reuce to 5075% of the full ose (for pallaium: 5886 
y) when combine with EBRT.25 The ata from this stuy emonstrate two important
points. One, that community hospitals, performing the ProSee implant can achie
ve high quality results in the majority of their patients. An secon, with a co
mprehensive training program, centers can quickly learn the realtime implant tec
hnique. The ProSee training program was esigne as a turnkey approach where th
e entire team, incluing nursing staff, physicists, urologists, raiation oncolo
gist, an hospital aministration were traine in their respective responsibilit
ies. The training took place over several months until the physician team was co
nfient enough to perform the cases inepenent of the proctor. The mean D90 for
the 125I patients when the proctor was supervising the case was 164 y compare
to 162 y when the case was performe after the physicians ha receive their c
ertification. Raiation oses to the rectum, urethra an blaer were also no i
fferent for the two groups. In aition, a similar number of cases (9.1% vs 8.3%
) fell below the recommene therapeutic ose of 140 y. Lee et al escribe a b
rachytherapy learning curve in reporting the osimetry results of the first 63 pat
ients treate at their center.26 They reporte a C90 (90% of the prostate receiv
ing a percentage of the prescription ose: 144 y). The mean C90 for the first 3
0 patients was 79.9% vs 89.9% for the next 33 (p=0.00009). Conversion of these v
alues to D90 in gray woul yiel 115
The ProSee approach
301

y vs 127 y for the two groups. When compare to the D90s reporte for the ProS
ee implants, these values are substantially inferior. While a irect comparison
of the ata from Lees center to the ProSee ata is not appropriate, it is clear
from the large ose iscrepancy that some explanations are warrante. Lee chose
to perform a preplanne technique, while all of the implants performe in the P
roSee centers were one by the realtime metho. While there are no ranomize
trials evaluating a possible avantage of one metho of see implantation over a
nother, stuies are starting to emerge inicating a preference of the realtime
metho. Matzkin reporte the osimetric outcomes of 142 consecutive patients tre

ate by the preplan metho an compare these to 214 who were implante using th
e realtime metho (Sourasky, Tel Aviv, Israel is a ProSee center). The V90 (pr
escription ose 160 y) for the preplanne metho was 67.5% vs 97.9% (p<0.01) fo
r the realtime metho.27 The authors of this stuy iscount any learning curve eff
ect an attribute the osimetry ifferences solely to the ifferences in techniq
ue. Technique asie, it is clear from the ata presente in this stuy, that an
intensive formal training program in prostate brachytherapy is highly effective
in proviing physicians with enough skills to inepenently perform goo quality
implants. The ata from the Lee stuy also suggest that without such a program,
even a year of practice may also not be enough to bring a center up to the level
of expertise necessary to perform the implants uniformly well. References
1. Holm HH, Peersen JF, Hansen H, Stroyer I. Transperineal I125 ioine see im
plantation in prostatic cancer guie by transrectal ultrasonography. J Urol 198
3; 130:283286. 2. Stock R, Stone NN, Wesson MF, De Wyngaert JK. A moifie techn
ique allowing interactive ultrasounguie threeimensional transperineal pros
tate implantation. Int J Raiat Oncol Biol Phys 1995; 32:219225. 3. Blasko JC, Wa
llner K, rimm PD, Rage H. PSA base isease control following ultrasoun guie
 I125 implantation for stage T1/T2 prostatic carcinoma. J Urol 1995; 154:109610
99. 4. Kuban DA, ElMahi AM, Schellhammer PF. I125 interstitial implantation f
or prostate cancer: What have we learne 10 years later? Cancer 1989; 63:24152420
. 5. Fuks Z, Leibel SA, Wallner KE, et al. The effect of local control on metast
atic issemination in carcinoma of the prostate: Longterm results in patients t
reate with I125 implantation. Int J Raiat Oncol Biol Phys 1991; 21:537547. 6.
Stock R, Stone NN, Tabert A, et al. A ose response stuy for I125 prostate im
plants. Int J Raiat Oncol Biol Phys 1998; 41:101. 7. Stone NN, Stock R. Brachy
therapy for prostate cancer: realtime threeimensional interactive see implan
tation. Tech Urol 1995; 1:7280. 8. Stock R, Stone NN, Lo YC. Intraoperative osi
metric representation of the realtime ultrasoun guie prostate implant. Tech
Urol 2000; 6:9598. 9. Stone NN, Hong S, Lo YC, Howar V, Stock R. Comparison of
intraoperative osimetric implant representation to postimplant osimetry in pa
tients receiving prostate brachytherapy. Brachytherapy 2003; 2(1):1725. 10. Paste
au O, Degrais P. The raium treatment of cancer of the prostate. Journal Urol (P
aris) 1913; 4:341366. 11. Rage H, AbelAziz AE, Snow PB, et al. Tenyear iseas
e free survival after transperineal sonographyguie Ioine125 brachytherapy w
ith or without 45ray external beam irraiation
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in the treatment of patients with clinically localize, low to high leason gra
e prostate carcinoma. Cancer 1998; 83:9891001. 12. Stock R, Stone NN, DeWyngaert
JK, et al. Prostate specific antigen finings an biopsy results following inte
ractive ultrasoun guie transperineal brachytherapy for early stage prostate c
arcinoma. Cancer 1996; 77:23862392. 13. Stock R, Stone NN. The effect of prognos
tic factors on therapeutic outcome following transperineal prostrate brachythera
py. Semin Surg Oncol 1997; 13:454460. 14. Wallner K, Roy J, Harrison L. Tumor con
trol an morbiity following transperineal ioine 125 implantation for stage T1/
T2 prostatic carcinoma. J Clin Oncol 1996; 14:449453. 15. Beyer DC, Priestley Jr
JB. Biochemical iseasefree survival following 125I prostate implantation. Int
J Raiat Oncol Biol Phys 1997; 37:559563. 16. Blasko JC, Rage H, Luse RW, et al.
Shoul brachytherapy be consiere a therapeutic option in localize prostate c
ancer? Urol Clin North Am 1996; 23:633650. 17. rao L, Larson TR, Balch CS, et
al. Actuarial iseasefree survival after prostate cancer brachytherapy using in
teractive techniques with biplane ultrasoun an fluoroscopic guiance. Int J Ra
iat Oncol Biol Phys 1998; 42:289298. 18. Kaye KW, Olson DJ, Payne JT. Detaile p
reliminary analysis of 125Ioine implantation for localize prostate cancer usin
g percutaneous approach. J Urol 1995; 153:10201025. 19. Rage H, Blasko JC, rimm
PD, et al. Interstitial Ioine125 raiation without ajuvant therapy in the tr
eatment of clinically localize prostate carcinoma. Cancer 1997; 80:442453. 20. S

harkey J, Chovnick SD, Behar RJ, et al. Outpatient ultrasounguie pallaium 10

3 brachytherapy for localize aenocarcinoma of the prostate: A preliminary repo
rt of 434 patients. Urology 1998; 51:796803. 21. Stone NN, Stock R, DeWyngaert J
K, Tabert A. Prostate brachytherapy: Improvements in prostate volume measurement
s an ose istribution using interactive ultrasoun guie implantation an thr
eeimensional osimetry. Raiat Oncol Investig 1995; 3:185195. 22. Willins J, Wa
llner K. CTbase osimetry for transperineal I125 prostate brachytherapy. Int
J Raiat Oncol Biol Phys 1997; 39:347353. 23. Dattoli M, Wallner K, Sorace R, et
al. 103P brachytherapy an external beam irraiation for clinically localize,
highrisk prostatic carcinoma. Int J Raiat Oncol Biol Phys 1996; 35:875. 24. Bl
asko JC, Rage H, Cavanagh W, Sylvester J, et al. Longterm outcomes of external
beam irraiation an I125/P103 brachytherapy boost for prostate cancer. Int
J Raiat Oncol Biol Phys 1996; 36:198. 25. Nag S, Bair M, Blasko J, et al. Amer
ican Brachytherapy Society survey of current clinical practice for permanent bra
chytherapy of prostate cancer. J Brachyther Int 1997; 13:243251. 26. Lee WR, De 
uzman AF, Bare RL, et al. Postimplant analysis of transperineal interstitial per
manent prostate brachytherapy: evi ence of a learning curve in the first year a
t a single institution. Int J Ra Oncol Biol Phys 2000; 46:8388. 27. Matzkin H, K
aver I, BrmanteSchreiber L, et al. Comparison between two ioine125 implant te
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rs. Raiother Oncol 2003; 68:289294.
Contributing hospitals in brachytherapy training Avera McKennan Hospital, Sioux
Falls, SD; Baptist Memorial East, Nashville, TN; Baptist Meical Center, Birming
ham, Memphis, Alabama; Bouler Community, Bouler, CO; Columbia reenview Meica
l Center, Bowling reen, KY, Columbia; Danbury Hospital, Danbury, CT; DCH Region
al Meical Center, Tuscaloosa, AL; DePaul, Norfolk, VA; eisinger Wyoming Valley
Meical Center, Wilkes Barre, PA lens Falls
The ProSee approach
303
Hospital, lens Falls, NY; Kalispell Regional Hospital, Kalispell, MT; Lewisal
e Meical Center, Salem, VA; Los atos Hospital, Los atos, CA; Lucy Lee Health
Care System, Poplar Bluff, MO; Mather Memorial, Port Jefferson, NY; Me Center O
ne Hospital, Bismark, ND; Mills Peninsula Health Services, San Mateo, CA; Mount
Carmel Regional Meical Center, Pittsburg, KS; Maryview Meical Center, Portsmou
th, VA; Niagra Falls Memorial Meical Center, Niagra Falls, NY; Phelps County Re
gional Meical Center, Rolla, MO; Pinnacle Health System, Harrisburg, PA; Reain
g Hospital an Meical Center, West Reaing, PA; Pulaski Community Hospital, Pul
aski, VA; Rex Health Care, Raleigh, NC; River Parishes Hospital, LaPlace, LA; Ri
versie Meical Center, Kankakee, IL; Shaysie Hospital, Pittsburgh, PA; Silver
cross Hospital, Joliet, IL; Saint Mary Mercy Hospital, Livonia, MI; Saint Ritas,
Lima, OH; Saint Tammany Parish Hospital, Covington, LA; Tulare District Health
Care System, Tulare, CA; WCA, Jamestown, NY; Virtua Health Memorial Hospital, Mo
unt Holly, NJ; Nelson N.Stone MD, Jeffrey H.Chircus MD an Richar .Stock MD we
re the original founers of ProSee, LLC an have a financial interest in ProSee
, Inc, a subsiiary company of C.R.Bar. Joseph Presser is a consultant physici
st, The ProSee Team: Laura Tweett, Mike Krachon, Julie Darity, Russ Dean an Er
ic Brown work in the ProSee Office at Bar Urologic Division, Covington, A.
21 Functional image registration in brachytherapy
Takashi Mizowaki an Marco Zaier Backgroun Imageguie raiation therapy has
become clinically important as a result of technical avances in both iagnostic
imaging an raiation therapy. To conuct tumortargete raiation therapy, the
ability to etect an treat regions with aggressive tumor eposits that inclue
raioresistant or hypermetabolic cells is inispensable. Ling et al propose the
term biological target volume (BTV) for the escription of these regions.1 Mali
gnant cells within the BTV are consiere clinically significant cancer because,

at least in raiobiological terms, it is these cells that quite likely etermin

e the ultimate outcome of raiation therapy. Therefore, an ieal imaging tool fo
r raiotherapy treatment planning (RTTP) shoul provie information on the locat
ion an the number of these cells.1 A number of new techniques have been evelop
e to overcome the rawbacks of conventional imaging moalities. In comparison w
ith conventional (socalle anatomical) imaging, they are classifie as biologic
al imaging because of the ability to provie information on the metabolic, funct
ional, physiological, or biochemical status of the tumor.1 Essentially, they can
help to improve the etectability of the BTV within the gross tumor volume. Int
erest in these new imaging moalities has increase in irect proportion to impr
ovements in RTTP an in ose elivery, such as externalbeam intensitymoulate
raiation therapy, or imageguie intraoperative planning in brachytherapy,13 w
ith the result that one has the ability to esign an implement treatment plans
that safely escalate the ose to the BTV within the target volume. As a function
alimaging tool for the treatment of localize prostate cancer, magnetic resonan
ce spectroscopy (MRS) is consiere to be most promising.4 It is base on the ob
servation that a significant increase in choline an ecrease in citrate levels
are observe in regions harboring cancer.59 On biochemical grouns, choline/citra
te ratio is expecte to reflect an increase rate of cell proliferation. In ai
tion, there is limite empirical evience of a correlation between choline level
s an histological grae inicate by leasons scores.6 Our preliminary ata furt
her confirm that MRS can etect most of the regions containing cells with high 
leason score (8 or higher). MRS is currently in clinical use at our institution
for prostate implant brachytherapy. Treatment planning performe intraoperativel
y, makes use of this information to escalate the ose at the MRS voxels ientifi
e as having increase choline/citrate ratios.2,10 The prostate as a whole is tr
eate to 100% of the prescription ose (144 y for 125I sees); however, MRS pos
itive voxels (tumor burens) are prescribe 200% of the prescription
Functional image registration
305

ose with no upper limit. For every treatment (with or without MRS ose escalati
on) the urethral ose is kept uner 120% of the prescription ose (Figure 21.1).
A key element in the implementation of the above technique is the ability to ma
p the location of MRSpositive voxels to the corresponing ultrasoun (US) image
s for RTTP. The reason for this is the fact that an enorectal coil is necessary
to conuct magnetic resonance imaging (MRI) an MRS examinations, an this resu
lts in a eformation of the prostate volume. MRI an MRS of the prostate are obt
aine with the combination of an enorectal an a pelvic phase array receiver c
oil.5,6 The enorectal balloon probe (which supports the enorectal coil, see Fi
gure 21.2) is inflate to a total volume of 100 cm3 of air an uner these coni
tions the prostate glan is pushe anteriorly against the pubic bone, which caus
es a slight flattening of its shape (Figure 21.3). Because treatment planning in
prostate implants is performe base on US images, where the eformation of the
prostate by the US probe is minimal, it was necessary to evise a proceure for
mapping points of interest from MRS to US images. In the present chapter, our m
etho for image registration of the prostate from the MRI/MRS (MR) to the US spa
ce is escribe. Image registration algorithm The mapping algorithm escribe be
low is base on the assumption that points within the glan maintain the
Figure 21.1 An example of the oseistribution for prostate implants that incorp
orates MRS information. This
Basic an avance techniques in prostate brachytherapy
306
plan is optimize to give at least 200% of the prescription ose (144 y for 125

I sees) to the registere MRSpositive area on a US image (inclue in an area

shae in yellow).
Figure 21.2 An enorectal balloon probe for MRI/MRS stuy.
Figure 21.3 Magnetic resonance spectroscopy (MRS) information superimpose on th
e corresponing T2weighte fast spinecho image (TR,
Functional image registration
307
5000 ms; effective TE, 102 ms) of a 54yearol patient with leason grae 7, pr
etreatment prostatespecific antigen (PSA) level of 4.5 ng/mL A leftposterior v
oxel labele P is juge as possible cancer by MRS. R_B inicates the rectum inflate
with the balloon probe. Deformation of the prostate by the inflate enorectal b
alloon probe is apparent (arrows).
same relative position with respect to the axial contours of the prostate an ar
e isplace along the zaxis (craniocaual irection) in irect proportion to th
e craniocaual imension of the glan with reference to the centerofmass (COM)
of the prostate. The algorithm is as follows (Figures 21.4 an 21.5): (a) Calcu
late the COM of the prostate in the MRI/MRS (MR) (C1) an US (C2) space. (b) For
a particular point [A1:(x1, y1, z1)] in the MR space, obtain the zcoorinate o
f the corresponing point [A2:(x2y2, z2)] in the US space from:
(1)
Here, zT1 an zT2 refer to the zcoorinate of the superior aspects of the prost
ate in the MR an US space, respectively, an zB1 an zB2 refer to the zcoorin
ate of the inferior aspects of the prostate in the MR an US space, respectively
. zC1 an zC2 are the zcoorinate of the prostate COM in the MR space an US sp
ace, respectively. (c) With the z position thus calculate, map the (x1, y1) in
the MR space on to the (x2, y2) in the US space as follows:
(2)
Basic an avance techniques in prostate brachytherapy
308
Figure 21.4 Schematic rawing of the mapping metho in the sagittal plane: the 
iagram inicates the registration of a point (A1) in the MRI/MRS space to the co
rresponing point (A2) in the US space. The zaxis is along the superiorinferio
r irection.
Figure 21.5 Schematic rawing of the mapping metho in the axial plane: the iag
ram inicates the registration of a point (A1) in the MRI/MRS space to the corre
sponing point (A2) in the US space. The xaxis inicates rightleft
Functional image registration
309

irection an the yaxis represents the anteriorposterior irection.

Here, yA1 an yA2 are the ycoorinate of the anterior aspects of the prostate i
n the MR an US space, respectively; an yP1 an yP2 represent the ycoorinate o
f the posterior aspects of the prostate, respectively. It is unerstoo that (x2
, y2) are calculate in the axial slice that correspons to the z2 position obta
ine from Eq. (1). Valiation of the algorithm Phantom stuy The valiity of the
algorithm (Eqs 1,2) was verifie with the ai of an anthropomorphic pelvic phan
tom (Figure 21.6) built to our specifications by Computerize Imaging Reference

Systems, Inc (CIRS, Norfolk, VA). The phantom is mae mainly of a proprietary ma
terial (Zerine, a waterbase polymer evelope by CIRS to be CT, US, an MRI/MR
S compatible), which accurately mimics human tissues for MR, US, an CT imaging.
The phantom contains simulate prostate, blaer, urethra, seminal vesicles, an
 rectum, all mae of Zerine, an pelvic bones mae of epoxy. Backgroun materi
al, which occupies all regions outsie the structures efine above, is also ma
e of Zerine. Accoring to the manufacturer, Zerine was mae to simulate the ul
trasoun characteristics of human liver tissue. The spee of soun, acoustic att
enuation, an backscatter properties of this material can be ajuste to simulat
e ifferent tissues. Thus, the contrast for ifferent structures (built by moli
ng Zerine into the respective shapes) in this phantom is such that they are vis
ible using both ultrasoun an CT (the prostate is arker than backgroun materi
al, urethra material is arker than prostate, etc.). Seventyfive ummy sees (phys
ically ientical to moel 6711 125I sees) were place in the prostate in a quas
iregular pattern. The coorinates of these sees coul be exactly etermine wi
th a CT stuy, an thus they serve as reference markers for stuying prostate ef
ormation when a rectal probe was inflate insie the rectum of the phantom (Figu
re 21.7). Two series of CT scans of the phantom were obtaine, one without the e
norectal probe (CTSeries_A) an the other with the enorectal balloon probe in
flate with 100 ml of air (CTSeries_B). Therefore, the CTSeries_A represents t
he status of US image acquisition, while the CTSeries_B represents the status o
f MRI/MRS acquisition (Figure 21.7). The slice thickness of the CT images was 3
mm. The actual coorinates of the center of each see were etermine by using a
ppropriate computer software (Interplant Postimplant Analysis System, Version 1.
0: Burette Meical Systems, Inc, Champaign, IL) for each CT series. This softwa
re can reconstruct axial CT images of 1 mm thickness for positional eterminatio
n of the sees an plan evaluations. Then, the previously escribe mapping meth
o was applie for the registration of each see position from the CTSeries_B t
o the CTSeries_A, which simulate the registration from MRI/MRS to US images. T
hereafter, coorinates of each
Basic an avance techniques in prostate brachytherapy
310
see in CTSeries_A calculate by the registration algorithm were compare with
the actual see position irectly efine with the CT ata (see
Figure 21.6 A custommae phantom that simulates the male pelvis was use to eva
luate the mappin accuracy of our algorithm. This phantom can simulate eformatio
ns an shifts in structures cause by the enorectal balloon probe inflate with
in the rectum (R).
Figure 21.7 A compute tomography (CT) image of the phantom: its original config
uration (without the enorectal
Functional image registration
311
balloon probe: left) an with the inflate enorectal probe (right). R in the left
image inicates the empty rectum an R_B in the right represents the rectum with infl
ate balloon probe. Insie the prostate of the phantom, 75 ummy sees are implant
e. The eformation of the prostate an the positional isplacement of the implant
e sees by the enorectal probe are evient.
Figure 21.8 A flowchart of the steps neee to evaluate the accuracy of the imag
e registration algorithm. CT, compute tomography; US, ultrasoun.
Basic an avance techniques in prostate brachytherapy

312
Figure 21.9 A schematic representation of the location of sees with a positiona
l error of 4 mm or larger in 3D space (inicate by+).
positions etermine by the computer software as escribe above), an the magni
tue of the positional isplacement was evaluate for each see (Figure 21.8). I
n this way, the accuracy of the mapping algorithm was irectly verifie. In the
phantom stuy, the absolute value of the threeimensional (3D) positional ispla
cements between the registere an actual see positions was 2.2 mm 1.2 mm (avera
ge  stanar eviation; SD). Only 6 of the 75 implante sees ha 3D positional 
isplacement larger than 4 mm (Figure 21.9). The maximum value of the 3D isplace
ments was 4.9 mm. The absolute value of the 3D positional error in this metho w
as significantly smaller than that of our earlier metho (p<0.0001 by both paire
 ttest an Wilcoxon signe rank test) (Figure 21.10).11 Patient stuy To vali
ate this algorithm in vivo we have evaluate changes in see positions ue to th
e eema resolution of
Functional image registration
313
Figure 21.10 The absolute value of the 3D registration errors resulting from the
phantom stuy as obtaine with the current metho (with corrections in zcoorin
ates) or with an earlier metho (without corrections in zcoorinates).2 Positio
nal accuracy for mapping in the current metho was significantly smaller than th
at in the earlier approach (p<0.0001).
Figure 21.11 The absolute value of the 3D errors in the registration metho as e
stimate for 20 sees in a patient who unerwent permanent prostate implant.
Basic an avance techniques in prostate brachytherapy
314
the prostate in a patient who receive permanent see implant for prostate cance
r. Two series of CT scans were obtaine on this particular patient: one immeiat
ely after the implant an the other 6 weeks after the proceure. On the CT image
s obtaine immeiately after the see implant, the eema of the prostate cause
by the invasive proceure was observe. Throughout the perio of 6 weeks that se
parates the two CT stuies, there was eema shrinkage of about 30% in volume of
the prostate that cause changes in the relative position of the sees. Using th
e same methoology as in the phantom stuy (Figure 21.8), the accuracy of the ma
pping algorithm was evaluate on 20 sees within the prostate of this particular
patient. The result was similar to the outcome from the phantom stuy. The abso
lute value of the 3D positional error between preicte an actual see position
s was 2.4 mm 1.3 mm (averageSD) (Figure 21.11). Current problems an future irect
ions At this time, magnetic resonance spectroscopy (MRS) appears to be the most
promising biological imaging moality for localize prostate cancer.4 In recent
stuies it was reporte that proton MRS can well istinguish cancer ominant reg
ions within the prostate glan from the normal prostate tissue base on the fin
ing of significant reuction in citrate levels an increase in choline levels re
lative to the normal peripheral zone.59 In aition, cancerpositive voxels etec
te by 3D MRS can be precisely mappe on the corresponing MR images, which thus
provies positional information on the location of the lesions within the prost
ate.5,12 Although MRS has relatively poor sensitivity an specificity (0.76 an
0.57, respectively,12 an argument can be mae base on tumorcontrol probability
moels that a clinical avantage in brachytherapy may be expecte when osimetr
ic hot spots, which are unavoiable, are place at MRSpositive voxels rather th
an at ranom locations in the glan.2 A major limitation in the use of MRS in ra

iotherapy treatment planning (RTTP) is the nee to account for changes in the s
hape of the prostate uring the MRI/MRS stuy.2 Therefore, a metho for performi
ng image registration for a eformable object (prostate) is inispensable. We ha
ve evelope an empirical registration metho using proportional corrections bas
e on the external con tour of the prostate. Despite its simplicity, this metho
is sufficienty accurate (2.2 mm 1.2 mm) when compare to both the MRS voxel size
(6.25 mm6.25 mm3.0 mm) an the typical slice thickness (5 mm) of the ultrasoun (
US) stuy for prostate implant. Because the spectroscopic signal obtaine from a
voxel (6.25 mm6.25 mm 3.0 mm) reflects the average response of tissue in it, wit
h 2.2 mm uncertainty, there is no reason to be concerne about misplacement of t
he voxel. When an MRSpositive voxel is locate laterally an close to the poste
rior surface of the prostate one may encounter the situation where the preicte
position of the voxel (usually a part of the voxel) in the US space locates out
sie the prostate. This is ue to the fact that, in contrast to the normal prostat
e, the MRS prostate is a istinctly nonconvex object. The a hoc solution we have
aopte is to move the voxel anteriorly insie the prostate; this is quite accep
table in view of the relatively moest spatial
Functional image registration
315
resolution of the MRS stuy an the fact that low ose rate brachytherapy patien
ts o not have extracapsular extension of the isease. Another possibility is to
reverse the orer of the calculation in Eq (2) of the algorithm, that is, calcu
late the xcoorinate first an then the ycoorinate, as shown in Figure 21.12 (
originally, ycoorinates are calculate first followe by the calculation of x
coorinates as inicate in Figure 21.5). The mapping accuracy of this reverse
metho was also examine in the phantom stuy: the
Figure 21.12 Schematic explanation of the reverse mapping metho. If the voxel is
locate close to the posterior surface of the concaveshape prostate, the origi
nal metho may result in a registere point outsie the prostate. One can avoi
this by simply conucting the same proportional calculation in Eq (2) in reverse
 orer (obtain the x2coorinate first followe by the calculation of the y2coo
rinate).
Basic an avance techniques in prostate brachytherapy
316
Figure 21.13 Absolute values of 3D registration errors in the original an rever
se methos.
Figure 21.14 An example of automate registration of magnetic resonance spectros
copy (MRS) positive voxels in our treatmentplanning system. The table contains
Functional image registration
317
the proportions of Eqs (1,2), as evaluate in the MRS space. The coorinates of
the positive MRS voxels are automatically mappe to the US images.
absolute value of the 3D registration error was 2.2 mm  0.9 mm (average  SD), whic
h is almost the same as in the original metho (Figure 21.13). Some of the probl
ems examine here may be ameliorate with the imminent availability for clinical
use of a new 3.0 tesla MR unit, which offers better resolution in both spatial
an MRS spectral characteristics compare with the current 1.5 tesla unit, an t
hus allows the utilization of either a smaller enorectal coil that will cause l
ess eformation of the prostate or pantsstyle coil that oes not require any en

orectal probe. A ifferent kin of limitation of this stuy comes from possible
ifferences in the response to eformations between pelvic organs an the phant
om material. For instance, the cancer burens may have elasticity properties if
ferent from the healthy prostate tissue, while the phantom is mae of a uniform
eformable material. Also, for in vivo valiation eema shrinkage may not provi
e a realistic simulation of the actual treatment situation. A stuy to valiate
this metho with CT scans (with/without enorectal balloon coil) of patients who
unerwent permanent prostate implant is uner consieration. The mapping algori
thm has been implemente in our RTTP system for USbase intraoperative optimiza
tion of prostate see implants (Figure 21.14). Specifically, after the prostate
is outline on both MRS an US axial images, software specially evelope for th
is purpose automatically fins the centerofmass of the glan, calculates the p
ositions of the MRSpositive voxels in the US frame of reference with the ai of
Eqs (1,2), an inserts the coorinates of these points in the list of constrain
ts use in treatment plan optimization. As alreay mentione, these points are g
iven at least 200% of the prescription ose with no upper limit. Conclusions In
conclusion, the algorithm for image registration reporte here has acceptable ac
curacy an is sufficiently practical for clinical implementation. It shoul be u
seful for patientspecific tumortargete see implants for prostate cancer with
the potential expectation of improve treatment outcome as well as reuction in
treatmentassociate morbiity. In aition, this registration scheme shoul be
helpful for guiing the pathologist to those regions of the glan where MRS posi
tive signals were etecte. References
1. Ling CC, Humm J, Larson S, et al. Towars multiimensional raiotherapy (MDC
RT): biological imaging an biological conformality. Int J Raiat Oncol Biol Phy
s 2000; 47:551560. 2. Zaier M, Zelefsky MJ, Lee EK, et al. Treatment planning fo
r prostate implants using magneticresonance spectroscopy imaging. Int J Raiat O
ncol Biol Phys 2000; 47:10851096.
Basic an avance techniques in prostate brachytherapy
318
3. Rosenman J. Incorporating functional imaging information into raiation treat
ment. Semin Raiat Oncol 2001; 11:8392. 4. Thornbury JR, Ornstein DK, Choyke PL,
et al. Prostate cancer: what is the future role for imaging? Am J Roentgenol 200
1; 176:1722. 5. Kurhanewicz J, Vigneron DB, Hricak H, et al. Threeimensional H1
MR spectroscopic imaging of the in situ human prostate with high (0.240.7cm3) s
patial resolution. Raiology 1996; 198:795805. 6. Kurhanewicz J, Vigneron DB, Mal
es R, et al. The prostate: MR imaging an spectroscopy. Present an future. Ra
iol Clin North Am 2000; 38:115138, viiiix. 7. Kurhanewicz J, Vigneron DB, Nelson S
J, et al. Citrate as an in vivo marker to iscriminate prostate cancer from beni
gn prostatic hyperplasia an normal prostate peripheral zone: etection via loca
lize proton spectroscopy. Urology 1995; 45:459466. 8. Kurhanewicz J, Dahiya R, M
aconal JM, et al. Citrate alterations in primary an metastatic human prostati
c aenocarcinomas: 1H magnetic resonance spectroscopy an biochemical stuy. Mag
n Reson Me 1993; 29:149157. 9. Costello LC, Franklin RB, Narayan P. Citrate in t
he iagnosis of prostate cancer. Prostate 1999; 38:237245. 10. Zelefsky MJ, Cohen
, Zakian KL, et al. Intraoperative conformal optimization for transperineal pr
ostate implantation using magnetic resonance spectroscopic imaging. Cancer J 200
0; 6:249255. 11. Mizowaki T, Cohen N, Fung AY, Zaier M. Towars integrating fun
ctional imaging in the treatment of prostate cancer with raiation: the registra
tion of the MR spectroscopy imaging to ultrasoun/CT images an its implementati
on in treatment planning. Int J Raiat Oncol Biol Phys 2002; 54(5):15581564. 12.
Scheiler J, Hricak H, Vigneron DB, et al. Prostate cancer: localization with th
reeimensional proton MR spectroscopic imagingclinicopathologic stuy. Raiology
1999; 213:473480.
22 A novel prostate brachytherapy technique: use of preloae neeles without sp
acers. The Frankfor Hospital experience

Eric L ressen, Jinyu Xue, Frank M Waterman, an Jay Hanler Introuction Multip
le techniques have been utilize to perform permanent prostate brachytherapy. Th
ere are marke ifferences in pretreatment planning in terms of the istribution
of raioactive sees an the treatment margin, efine in ICRU50 as the istan
ce from the prostatic ege an the prescription isoose.1 This chapter will esc
ribe in etail the process at our institution in evaluating an managing patient
s with prostate cancer treate with raioactive see implantation either alone o
r combine with another treatment moality. Special attention will be given to t
he technique use for planning an executing the see implantation. Patient eval
uation All patients unergo a complete history an physical examination. Key ele
ments of the history inclue urinary, bowel, an sexual function. Patients who h
ave unergone a transurethral resection of the prostate (TURP) for blaer outle
t obstruction may not be a suitable implantation caniate ue to the potential
for increase urinary incontinence after the proceure compare to patients who
have not receive a TURP. Rage et al reporte a 12% incontinence rate for see
implantation patients with history of a prior TURP.2 Blaer outlet obstruction
an pyuria remain urologic concerns for selection for see implantation since po
stoperative urinary retention presents a troublesome an challenging ilemma. An
y hip/pelvic injury or limitations are taken into account to ensure tolerance of
the lithotomy position for implantation. A significant cariac an/or respirato
ry history may preclue one from performing implantation uner any form of anest
hesia. Physical examination always inclues a igital rectal exam ination (DRE)
for staging purposes an for etermining an approximate size of the prostate gla
n. A heme occult test is performe on the stool specimen after DRE with a gastr
ointestinal consultation requeste for an unexplaine hemepositive stool prior
to see implantation to rule out colorectal malignancy. The pathology slies are
always reviewe at our institution. The leason score, the number an percentag
e of involvement of each specimen, an the presence of perineural invasion are a
ll taken into account to assess the risk of extracapsular extension, seminal ves
icle an lymph noe involvement. Compute tomography (CT) of the abomen an
Basic an avance techniques in prostate brachytherapy
320
pelvis is performe on all patients to further efine the prostate size an to a
ssess for lymphaenopathy with the rare aitional benefit of ientifying a seco
n malignancy in the genitourinary or gastrointestinal tract. Patients who are s
ee caniates with a glan size of greater than 50 grams as measure by CT or t
ransrectal ultrasoun (TRUS) at the time of their initial biopsy are offere an
rogen ablation to minimize pubic arch interference an the number of sees neces
sary for implantation. Patients are eeme optimal caniates for see implantat
ion alone if they satisfy the following criteria: leason score<7, prostatespec
ific antigen (PSA)<10, T1c/T2a isease, prostate glan size 50 grams, absence of
perineural invasion, cancer note in 3 specimens, no history of TURP. The prescri
ption ose for monotherapy with ioine125 (I125) sees is 145 y calculate bas
e on the American Association of Physics an Meicine (AAPM) Task roup No. 43
(T43) recommenations.3 Combination treatment consisting of external beam raiot
herapy (EBRT) followe by see implantation is offere to patients with an incre
ase risk of extracapsular extension but a low risk of pelvic lymph noe involve
ment. Patients fitting these criteria are those with a PSA in the range of 1020 a
n/or a leason score of 7. Also, perineural invasion signifies an increase ris
k of extracapsular invasion an is taken into account for combination treatment.
Combination treatment consists of 45 y EBRT to the prostate, seminal vesicles
an surrouning tissue followe by 108 y elivere by 125I see implantation 36
weeks later. Ultrasoun volume stuy All patients, once eeme meically to be c
aniates for see implantation an preliminarily anatomical caniates for see
implantation, unergo a TRUS for treatment planning an to assess pubic arch in
terference. CT is no longer use to assess for interference since TRUS has been
proven to be at least as effective in visualizing the pubic arch as CT.4,5 If an

rogen ablation has been institute, TRUS is performe 3 months after institutin
g the hormonal agents to account for most of the prostate glan reuction. For p
atients treate with combination therapy consisting of EBRT an see implantatio
n, TRUS is performe prior to commencing the external beam treatment to insure t
hat there is no anatomic limitation to performing see implantation an to minim
ize the rectal irritation from the proceure. All patients take two preparations
of Fleet Phospha Soa 45 mL orally separate by 3 hours the ay prior to the pr
oceure. In rare cases, iazepam is offere to patients to ecrease anxiety attr
ibute to unergoing the TRUS proceure. There is an overall high tolerance leve
l for this proceure. A eicate Hitachi EUB6500 ultrasoun unit equippe with
a 7.5 MHz enorectal ultrasoun probe (Hitachi Meical Corporation of America,
Tarrytown, NY) combine with a stepping an stabilizing apparatus table mount Su
rePoint II (Amertek Meical, Inc, Singer Islan, FL) is use for the TRUS stuy
. The prostate, rectal wall, an urethra are well visualize. The eicate pros
tate brachytherapy ultrasoun technologist outlines the prostate an the pubic a
rch for treatment planning an to assess for pubic arch interference. If necessa
ry, a urethrogram using aerate gel is performe to ientify the urethra. A rai
ologist, specializing in TRUS, verifies the lack of or the amount of pubic arch
interference. If minimal to no pubic arch interference is appreciate on TRUS,
A novel prostate brachytherapy technique
321
patients are place on alpha1 blockers at least 2 weeks prior to unergoing see
 implantation to further minimize the risk of urinary retention. Treatment plan
ning Treatment planning is one using VariSee planning system (Varian Oncology S
ystems, Milpitas, CA). The TRUS images from the volume stuy are igitally impor
te into the planning system. The contours of prostate, urethra, an rectum are
rawn an a planning tumor volume (PTV), which inclues the prostate with the pr
escribe treatment margin, is generate. We utilize a treatment margin that is v
arie from 35 mm epenent on a series of risk factors etermining the probabilit
y of extracapsular extension. In a stuy of 396 patients status postraical retr
opubic prostatectomy at the Mayo Clinic, a 35 mm treatment margin by brachytherap
y woul encompass all known tumor in approximately 99% of the prostatectomy spec
imens.6 Sohaya an colleagues observe similar results at the Clevelan Clinic
in 255 raical prostatectomy specimens with a 4 mm tumor margin encompassing all
isease in 90% of the cases.7 Of the three types of see istributions escribe
 by Task roup 64,8 uniform loaing, moifie peripheral loaing, an periphera
l loaing, none has proven to have superior clinical outcomes. Technically, peri
pheral loaing can reuce the ose to the urethra compare to the other types, s
ince the sees are further isplace from the urethra. In aition, the peripher
al technique requires the least number of neeles, reucing prostate trauma from
neele placement an shortening the time of the proceure. There are two seein
g patterns for the peripheral loaing technique, linear source peripheral loain
g where sees are abutting without a spacer between two ajacent sees, an poin
t source peripheral loaing where sees an spacers are alternate along each ne
ele track. The latter approach requires approximately half the number of sees
per neele compare to linear source peripheral loaing, but woul require appro
ximately oubling the see strength to maintain the same total source strength.
The use of fewer sees of high strength was cite as a isavantage to periphera
l loaing by Butler et al because it requires more precise placement of each see
 to avoi inavertently elivering a high ose to a critical structure.9 Our pr
imary seeing approach is peripheral loaing with a specific pattern of see an
spacer sequence for each neele track tailore to optimize ose coverage of the
PTV. Figure 22.1 emonstrates the ifference between our primary seeing approa
ch compare to pure linear or point source seeing patterns. This moifie loai
ng pattern allows the use of meium strength sees (~0.3750.450 mCi). Compare to
our seeing approach, the point source peripheral loaing typically requires a
higher activity see an the linear source peripheral loaing requires a large n

umber of sees. Our variation of peripheral loaing effectively minimizes the o

se to the ajacent critical structures without compromising ose to the PTV. The
preloae neele technique has proven to be an effective approach to optimize s
ee implantation. The 100% isoose line is planne to cover the PTV taking into
account subtle variations in contouring the prostate seconary to patient moveme
nt an internal organ motion with a wie treatment margin at the prostatic apex
ue to
Basic an avance techniques in prostate brachytherapy
322
Figure 22.1 Three peripheral see loaing patterns escribe in the context: poi
nt, linear, an hybri (from top to bottom). Our variation of peripheral loaing
is a hybri of point an linear source peripheral loaing tailore to optimize
ose coverage of the planning tumor volume (PTV).
the inherent uncertainty of contouring the apical region. The objectives of trea
tment planning inclue the relative sparing of the urethra by maintaining the ma
jority of the urethra outsie the 150% isoose line. The planne D90 for the ure
thra is approximately 100% of prescribe ose to the prostate. Once a plan is op
timize, an isoose plan is printe to review with the raiation oncologist, em
arcating the prostate contour with the 100% an 150% isoose lines, as emonstra
te in Figure 22.2.
A novel prostate brachytherapy technique
323
Preamission testing All patients unergo a complete bloo count, basic metaboli
c profile, international normalize ratio (INR), protime (PT), prothrombin time
(PTT), urinalysis, an electrocariogram prior to the see implantation. If ther
e is an unexplaine electrocariogram fining, the patient is sent to a cariolo
gist for cariac clearance. All plateletinhibiting agents are stoppe at least o
ne week prior to implantation. Warfarin, an anticoagulant, is iscontinue 5 ay
s prior to the proceure with an INR, PT, an PTT taken the morning of the impla
nt to ensure a return to normal values. Preparation of the neeles an sees for
implantation The loaing sequence of sees an spacers for each neele is print
e on a iagram for visualization of the see pattern, an to facilitate the loa
ing process. The neele tip is seale with surgical bone wax of approximate 5 m
m length. The length of the wax is accounte for when the sees are eposite in
to the prostate. A rapi seeloaing evice, QuickLoa (Prostate Services of A
merica, Inc, Singer Islan, FL) is use to significantly reuce the time for pre
loaing the neeles an to provie quick verification of the loaing pattern. An
extra spacer is ae to the en of the last see in each neele to minimize mi
gration of the sees uring the epositing process. The preloae neeles for an
iniviual patient are store in a shiele box, an each neele is place into
a hole having an alphanumeric coorinate corresponing to the template hole for
neele insertion into the prostate. See implantation Prior to arrival to the c
ystoscopy suite, patients are given intravenous antibiotics an are place in th
ighhigh Te stockings. The vast majority of patients are given general anesthes
ia. Spinal anesthesia is given at the iscretion of the anesthesiologist base o
n the patients meical conition an meications. Patients are set up in the lith
otomy position with careful attention to symmetry of the pelvis an the position
ing level of the stirrups to maximize reproucibility with the preplan TRUS volu
me stuy. The implant proceure is guie by TRUS image. Before insertion of the
neeles, each image slice is carefully checke relative to the template gri an
 aligne against the position of the preoperatively planne contours for the pr
ostate an the urethra. Agreement between two TRUS volume stuies is expecte wi
thin a couple of millimeters

Basic an avance techniques in prostate brachytherapy

324
Figure 22.2 Source placement as planne on each axial slice of transrectal ultra
soun (TRUS) image (green spot: 125I see). The re line is the contour of prost
ate an the ark green line in the center of the prostate emarcates the urethra
. The gray line is the planning tumor volume (PTV) contour generate by the expa
nsion of prostate with an appropriate margin. Cyan an pink lines are the 100% a
n 150% prescription isoose lines, respectively.
for the imension of the prostate an the location of the urethra. Variations in
volume an shape are common with subtle changes in neele placement performe 
uring implantation to account for these ifferences. Beaulieu et al reporte 63%
of the volumes taken in the operating room iffere from the pretreatmentplann
ing volume in the 35 cases evaluate at his institution.10 If the alignment cann
ot uplicate the imension of the prostate within the acceptable eviation for m
any image slices, an appropriate ajustment of the neele positions is necessary
to ensure goo ose coverage. Offsetting a few neeles usually fulfills the aj
ustment, but aing extra neeles or eliminating planne neeles may be require
to optimize the implant.
A novel prostate brachytherapy technique
325
Two stabilizing neeles (MDTech, ainesville, FL) with a hooktype mechanism are
place into the glan symmetrically along the same gri row. 125I sees (BrachyS
ource, Covington, A) (0.3750.450 mCi/ source, T43) are use for implantation. T
he preloae implant neeles are inserte through the template an perineum, an
into the prostate, uner irect visualization with the TRUS. All neeles are gu
ie by the preplan. Anterior lateral neeles are place first to assess the nee
 for further elevation of the lower extremities in the lithotomy position to av
oi pubic arch interference an to avoi poor visualization of the anterior port
ion of the prostate from ultrasoun transmission interference from posterior pla
ce sees. The physicist instructs the raiation oncologist which neele to plac
e base on the plan an any moifications necessary base on subtle ifferences
between the preplanne ultrasoun volume stuy an the ultrasoun volume stuy i
n the cystoscopy suite. Prior to eposition, an urethrogram with aerate gel is
performe to insure at least a 5 mm istance between the sees an the urethra.
Before inserting the posterior row of neeles, the shift of the rectal wall is v
erifie through all slices from the base to apex an the neeles are place at l
east 5 mm from the anterior rectal wall. When all the planne sees have been pl
ace, an anteriorposterior raiograph is taken to assess the general location o
f the sees noting see migration into the blaer or periprostatic tissue. In r
are circumstances when a see eficit is apparent in a portion of the prostate g
lan ue to see migration, aitional sees may be place taking into account t
he pretreatment plan. Cystourethroscopy When the see implantation is complete,
the urologist performs a cystourethroscopy to evaluate the blaer an urethra p
ostimplant. It is not uncommon for the urologist to retrieve raioactive sees,
inavertently place into the urethra or blaer. An inwelling urinary catheter
is not routinely place after this proceure. Patients are given prescriptions
for oral antibiotics, analgesics an alpha1 blockers upon ischarge from the ho
spital. Urinary retention If patients are unable to voi 4 to 6 hours after the
implant, an inwelling urinary catheter is place for acute urinary retention an
 remains in for several ays to allow for reuction in prostatic eema. If urin
ary retention persists, multiple trials of either successively longer uration c
ontinuous inwelling catheters or intermittent selfcatheterization via a clean
technique is performe until patients are able to uri nate freely without interv
ention. In troublesome cases of urinary retention 8 to 10 months post see impla
ntation, further intervention is ecie by our urologic consultants. Patients a

re given alpha1 blockers as a preparative regimen at least two weeks prior to i

mplantation to lessen urinary irritative symptoms postimplant, an maintaine fo
r 612 months.
Basic an avance techniques in prostate brachytherapy
326
Raiation precautions All patients are instructe to utilize a conom uring sex
ual intercourse for the first two weeks postoperatively in case a see is releas
e uring ejaculation. Pregnant women or potentially pregnant women shoul avoi
prolonge personal contact with the patient for the first 2 months after see i
mplantation with no limit to the length of time in the same room if she maintain
s a istance of at least 6 feet (2 meters) from the patient. Chilren are not al
lowe to sit on the lap of the patient uring the first two months following the
implant. Postimplantation evaluation Postimplant osimetry an evaluation is e
sire on all patients to assess the quality of the implant. Axial CT
Figure 22.3 Postimplant osimetry performe in approximately 30 ays after impla
ntation. Source is ientifie as green spots on the axial slice of compute tomo
graphy (CT) scan at 5 mm spacing. Re line is the contour of prostate. Cyan an
pink lines are the
A novel prostate brachytherapy technique
327
100% an 150% isoose line of prescription, respectively.
images at 5 mm intervals are acquire 46 weeks after implantation since most of t
he prostatic eema inuce by brachytherapy resolves in one month.11,12 The rai
oactive sources an contoure images are entere into our treatment planning sys
tem using VariSee 7.0 Software (Varian Meical Systems, Inc, Charlotteville, VA
). An example of the resultant images is isplaye in Figure 22.3. A reunancy
check is performe on see localization to prevent uplication of sees. Base o
n the recommenations of the American Brachytherapy Society (ABS), the ose give
n to 90% of the postimplant prostate volume (D90) an the percent of postimplant
prostate volume covere by the prescription ose (V100) are obtaine from the 
osevolume histograms (DVH) an reporte.13 The D90 typically falls between 160 
y an 180 y with the V100 in the range of 90% to 100%. Conclusions The process
of see implantation an evaluation postimplant are evolving. Improvements in ul
trasonic technology has allowe for better visualization of the prostatic anatom
y an surrouning structures to enhance treatment planning an see placement. R
eserving catheter placement for the minority of patients that evelop urinary re
tention improves patient satisfaction by avoiing the common symptoms of pelvic
an penile iscomfort attribute to foley catheters. Peripheral loaing with var
iations in the istribution of sees an spacers optimizes ose coverage of the
planning tumor volume while reucing the maximal urethral ose to below 150% of
the prescribe ose. Careful attention to the entire see implantation process h
as resulte in goo patient satisfaction an high quality implants base on post
implant compute tomography (CT) osimetry in the majority of cases. References
1. International Commission on Raiation Units an Meicine (ICRU). Prescribing,
recoring, an reporting photon beam therapy. ICRU Report 1993;18. 2. Rage H, B
lasko JC, rimm PD, et al. Interstitial ioine125 raiation without ajuvant th
erapy in the treatment of clinically localize prostate carcinoma. Cancer 1997;
80:442453. 3. Nath R, Anerson LL, Luxton , et al. Dosimetry of interstitial bra
chytherapy sources: recommenations of the AAPM Raiation Therapy Committee Task
roup No. 43. American Association of Physicists in Meicine. Me Phys 1995; 22
:209234. 4. Strang J, Rubens DJ, Brasacchio RA, et al. Realtime US versus CT e
termination of pubic arch interference for brachytherapy. Raiology 2001; 219:38
7393. 5. Wallner K, Ellis W, Russell K, et al. Use of TRUS to preict pubic arch

interference of prostate brachytherapy. Int J Raiat Oncol Biol Phys 1999; 43:58
3585. 6. Davis BJ, Pisansky TM, Wilson TM, et al. The raial istance of extrapro
static extension of prostate carcinoma: implications for prostate brachytherapy.
Cancer 1999; 85:26302637.
Basic an avance techniques in prostate brachytherapy
328
7. Sohaya C, Kupelian PA, Levin HS, et al. Extent of extracapsular extension in
localize prostate cancer. Urology 2000; 55:382386. 8. Yu Y, Anerson LL, Li Z,
et al. Permanent prostate see implant brachytherapy: report of the American Ass
ociation of Physicists in Meicine Task roup No. 64. Me Phys 1999; 26:2054 2076
. 9. Butler WM, Merrick S, Lief JH, et al. Comparison of see loaing approache
s in prostate brachytherapy. Me Phys 2000; 27:381392. 10. Beaulieu L, Aubin S, T
aschereau R, et al. Dosimetric impact of the variation of the prostate volume an
 shape between pretreatment planning an treatment proceure. Int J Raiat Onco
l Biol Phys 2002; 53:215221. 11. Prestige BR, Bice WS, Kiefer EJ, et al. Timing
of compute tomographybase postimplant assessment following permanent transper
ineal prostate brachytherapy. Int J Raiat Oncol Biol Phys 1998; 40:11111115. 12.
Waterman FM, Yue N, Corn BW, et al. Eema associate with I125 or P103 prost
ate brachytherapy an its impact on postimplant osimetry: an analysis base on
serial CT acquisition. Int J Raiat Oncol Biol Phys 1998; 41:10691077. 13. Nag S
, Beyer D, Frielan J, et al. American Brachytherapy Society (ABS) recommenati
ons for transperineal permanent brachytherapy of prostate cancer. Int J Raiat O
ncol Biol Phys 1999; 44:789799.
23 Raioimmunoguie prostate brachytherapy
Roney J Ellis Introuction Prostate brachytherapy has gaine wiesprea accepta
nce as an alternative to external beam raiotherapy or surgery for patients with
localize prostate aenocarcinoma. As the technique has evolve, significant a
vances have refine the proceure. Early attempts at brachytherapy with permanen
t raioisotopes in the 1970s were marke by unsatisfactory results ue to inaeq
uate technology for proper treatment planning. The wiesprea availability of im
prove imaging moalities an planning software in the 1980s le to the evelopm
ent of prostate brachytherapy as it is wiely practice toay, with a lowmorbii
ty transperineal approach that can be performe on an outpatient basis.1 Despite
these avances, there is still no accepte technique to plan an implant proceu
re base on the istribution of tumor within the glan, rather than on the size
an shape of the prostate itself. Theoretically, therapy esigne to target spec
ific foci of isease within the prostate woul yiel better local isease contro
l with ecrease morbiity. Various imaging techniques incluing ultrasoun (US)
, compute tomography (CT), an magnetic resonance imaging (MRI), give informati
on about the size an shape of the glan but lack the anatomical imaging sensiti
vity an specificity require to ifferentiate normal prostate tissue from malig
nancy. Of the three moalities available, MRI appears to elineate tumor from no
rmal prostate tissue most effectively. Most of the reporte literature on MRI ev
aluation of the prostate relies on either enorectal or whole boy coil MRI tech
niques to etermine capsular penetration or seminal vesicle invasion. This helps
in selecting patients for prostatectomy, but oes not help in planning brachyth
erapy. Whereas stanar MRI may be able to help etect macroscopic isease, its
ability to etect lesions less than 5 mm is limite.25 Magnetic resonance spectro
scopy (MRS) relies on the metabolic activity of cancers an appears to increase
the specificity of enorectal coil MRI.6 Its use in localizing intraprostatic i
sease for specific treatment approaches is currently being explore at other ins
titutions.710 It may help ientify regions of isease within the prostate but its
utility in evaluating the pelvis or abomen woul be limite by the amount of t
ime require to scan such large areas by spectroscopy. Although some groups have
been using MRS to attempt to etect occult tumor foci, this moality is not wi
ely available. The use of a raiolable antiboy, inium111 (111In) capromab pe

netie (ProstaScint), specific for prostatespecific membrane antigen (PSMA) allo

ws the use of immunoscintigraphy to etect metastatic isease beyon the prostat
e.11 When we began our prostate brachytherapy program in February 1997, we eci
e to use a ProstaScint scan on all patients as part of the routine workup in or
er to help us select appropriate caniates for the proceure. The concept of r
aioimmunoguie brachytherapy for
Basic an avance techniques in prostate brachytherapy
330
colorectal carcinoma ha alreay been explore,12,13 an we attempte to transla
te this technique to prostate brachytherapy. Although ProstaScint imaging has bee
n criticize for having falsepositives beyon the prostate glan when use for
evaluation of metastatic isease, coregistration to CT, or MRI has greatly impro
ve the ability to interpret regions of increase antiboy uptake in the pelvis,
abomen, or chest by etermining the unerlying anatomical structure. Once the
coregistration is complete, the contrast is increase on the singlephoton emi
ssion compute tomography (SPECT) images to remove any resiual uptake seen outs
ie of the prostate within the surrouning musculature. Regions within the prost
ate that persist to have visualize antiboy concentration are selecte as targe
t lesions for raiotherapy either by brachytherapy see placement within the reg
ion, or possibly by intensity moulate raiotherapy (IMRT)targete ose escalat
ion. PSMA is upregulate an overexpresse by both cancerous tissue an prostati
c intraepithelial neoplasia (PIN), but not atypical aenomatous hyperplastic les
ions (AAH) or benign prostatic hypertrophy (BPH).14 While the expression of PSMA
in PIN may result in a falsepositive reaing for carcinoma within the prostate
glan with the image fusion, we believe that PIN shoul be targete an treate
as cancer uring brachytherapy or IMRT, as it has been shown to be a likely pre
cursor of invasive carcinoma.15 At the beginning of our series, we explore the
possibility of using the ProstaScint scan to etermine where the areas of highest
tumor buren were locate within the prostate glan. We theorize that by obtai
ning SPECT images through the region of the prostate glan, we woul be able to
improve our istribution of raioactive sources at the time of implantation. We
planne to eliver a higher ose of raiation to regions with a higher tumor bur
en while sparing sensitive normal structures, such as the urethra, rectum, an
blaer if there was low antiboy uptake in the prostate tissue ajacent to thes
e structures. Thus, immunoscintigraphy with ProstaScint woul allow us to target
the tumor within the prostate using ose escalation with the overall goals of r
eucing biochemical failure an toxicity. We present in this chapter the accurac
y of this metho an the acute an chronic toxicity an outcome ata from the fi
rst 124 consecutive patients with prostate cancer treate with raioimmunoguie
brachytherapy either alone or in combination with electron beam raiotherapy (E
BRT). Methos an results Image fusion In orer to evaluate the ability of the g
amma camera to etect regions of inium 111 (111In) capromab concentration withi
n the prostate, regions of interest (ROIs) were place within the anterior an p
osterior portions of the glan. Counts were etermine from each region uring t
he patients secon scan four ays post injection with the raiolabele antiboy.
The total counts for each ROI were ivie by the backgroun activity, as efine
 by the number of counts within a similarly place control region within the ex
ternal obturator muscle. We have emonstrate previously that these ROI prostate
tomuscle ratios (P/M ratios) appear to have a strong correlation with the prost
ate biopsy results using a ratio of 3.0 or greater as a cutoff to efine regions
suspicious for
Raioimmunoguie prostate brachytherapy
331
containing carcinoma.16 Image fusion was complete through manual alignment of a

natomic structures common to both the CT scan an ProstaScint image, such as bon
e marrow an vessels. The contrast of the ProstaScint stuy was increase to rem
ove backgroun activity from all nonvascular structures. The remaining regions
of high antiboy concentration were ientifie as sites suspicious for aenocarc
inoma. We were able to see clearly anatomical relationships between the areas of
antiboy concentration an the surrouning prostate glan. In our current progr
am at University Hospitals of Clevelan, we utilize a preplanne ultrasoun volu
me stuy an postoperative CTbase osimetry using a Rosses treatmentplanning
computer (Rosses Meical, Columbia, MD). We target the regions containing high a
ntiboy concentrations within the V150 isoose line, or 150% of the prescribe 
ose. A moifie peripheral loaing pattern is utilize in all of our implants; h
owever, the central loaing is altere base on clinical, pathological, an rai
ographic finings. Aitionally, we are now eveloping a technique to import the
coregistere images into the treatment planning to allow 3D reconstruction an
intraoperative osimetry. This woul then allow comparison of the preoperative
plan with the postoperative CTbase osimetry, improving our ability to report p
artial organ osimetry within the prostate. Correlation of prostate cancer foci
an fusion images Between June 1998 an March 1999, we evaluate the biopsy ata
from 7 patients to assess the accuracy of this imaging technique. Each patient
unerwent prostatic biopsies at 12 sites etermine inepenently of the imaging
stuies prior to a prostate brachytherapy proceure. We use 18 gauge trucut n
eeles to obtain samples of prostate tissue transperineally. The biopsies were o
btaine uner ultrasoun guiance through the stanar template use for the tra
nsperineal implant prior to performing the proceure (Bruel & Kjaer; B&K Leopar
ultrasoun unit an template, Copenhagen, Denmark). Anterior an posterior sext
ant samples were taken from the base, mi, an apex of the glan 1 cm lateral to
the miline of the glan an 1 cm above an below the transverse miplane. Thes
e sites were labele as left/right, anterior/posterior, base/ mi/apex, an sent
in separate containers to a pathologist bline to the results of the imaging s
tuies. The samples were each rea by a pathologist an reporte to have either
aenocarcinoma of the prostate or benign tissue incluing: benign prostatic hype
rtrophy, prostatic parenchymal tissue, prostatic intraepithelial neoplasia (PIN)
, or atypical acinar proliferation. The image fusion stuies were rea by two cl
inical investigators to be either positive or negative for high antiboy concent
ration within each anterior an posterior sextant region prior to reviewing the
pathologic results. Truepositives (TP) were efine as regions where both the i
mage an pathology were rea as having antiboy concentration an positive patho
logy for aenocarcinoma. Truenegatives (TN) were efine as regions where both
the image an pathology were rea as having low or no antiboy concentration an
negative pathology for aenocarcinoma. Falsepositives (FP) were efine as reg
ions where the image an pathology were rea as having high antiboy concentrati
on but negative pathology for aenocarcinoma. Falsenegatives (FN) were efine
as regions where the image an pathology were rea as having low or no antiboy
concentration
Basic an avance techniques in prostate brachytherapy
332
Table 23.1 A comparison of biopsy an imaging results by anatomic site
Patient
1
RAB RPB RAM RPM RAA RPA LAB LPB LAM LPM LAA LPA
Pathology (+) () () () () () () (+) () () () () Image (+) () (+) (+) () ()
Pathology (+) (+) (+) (+) (+) (+) () (+) () () () (+) Image () (+) (+) (+) (+) (+)
) (+) (+) (+) (+) (+) 3 Pathology () () () (+) () (+) () (+) (+) (+) (+) (+) Image
() () (+) () () () (+) (+) (+) () (+) 4 Pathology (+) () (+) () (+) () () ()
() () (+) () (+) () () () (+) () () () 5 Pathology (+) () (+) (+) (+) (+) ()

() (
(+
()
() (
() (

e (+) () (+) (+) (+) (+) () () (+) () () () 6 Pathology () () () () () () () ()

() () () () () () () () () () () (+) 7 Pathology (+) (+) () () () (+) () () (
(+) () () () () () (+) () () () () RAB, right anterior base; RPB, right posterior ba
AM, right anterior mi; RPM, right posterior mi; RAA, right anterior apex; RPA,
right posterior apex. LAB, left anterior base; LPB, left posterior base; LAM, l
eft anterior mi; LPM, left posterior mi; LAA, left anterior apex; LPA, left po
sterior apex.
Table 23.2 Truepositivenegatives (TP/TN) an falsepositivesnegatives (FT/TN) by
location within the prostate
RAB RPB RAM RPM RAA RPA LAB LPB LAM LPM LAA LPA
TP 3 TN 2 FP 0 FN 2 2 5 0 0 3 3 1 0 3 3 1 0 3 4 0 0 2 3 0 2 0 6 1 0 3 3 1 0 1 2
3 1 1 5 1 0 0 5 1 1 2 3 2 0
but positive pathology for aenocarcinoma (Tables 23.1 an 23.2). Accuracy of Pr
ostaScint For the 84 biopsies obtaine from these seven patients, we ha 23 TP,
44 TN, 11 FP, an 6 FN reaings. There i not appear to be a strong correlation
between anatomic site an TP/TN/FP/FN rate. Of note, the left anterior base, le
ft posterior mi, an left anterior apex ha a high TN rate an a low TP rate. W
hen the anterior portion of the glan was compare with the posterior, the TP/TN
/FP/FN rates were very similar. The anterior glan ha 10 TP, 22 TN, 6 FP, an 4
FN values. The posterior glan ha 13 TP, 22 TN, 5 FP, an 2 FN values (see Tab
les 23.1 an 23.2). After comparison of the scans with the pathologic results, o
ur metho yiele an overall accuracy of 80% (TP+TN/total number of samples). We
ha a sensitivity of 79% (TP/TP+FN) an a specificity of 80%
Raioimmunoguie prostate brachytherapy
333
(TN/TN+FP). The positive preictive value (PPV) for the stuy was 68% (TP/TP+FP)
with a negative preictive value (NPV) of 88% (TN/TN+FN).19 These results were
encouraging, but it is important to note that the multifocal nature of prostate
cancer can create significant sampling error when evaluating prostatic biopsies.
Stanar anatomical imaging of the glan with ultrasoun at the time of TRUS bi
opsy oes not always visualize tumor foci well. These biopsies were obtaine in
a routine fashion, but even repeat sextant biopsies in patients with a known ia
gnosis of cancer may fail to etect isease in 20% of patients.17,18 A positive
image for which pathology was negative coul therefore represent a falsenegativ
e biopsy or a falsepositive scan reaing. Although these preliminary results we
re encouraging, a
Figure 23.1 (a) Wholemounte prostate specimen. The lesion is 7 mm in maximal 
imension in the left posterior mi region of the prostate glan, (b) The fusion
image. The region of antiboy concentration matching the location of the lesion.
Uptake in the right anterior near miline matches a 3 mm foci of aenocarcinoma
ientifie 4 mm superior to the isplaye slice on serial wholemounte histopa
thology.
current stuy is unerway to obtain a more etaile comparison utilizing wholem
ounte specimens obtaine after prostatectomy for correlation with preoperative
iagnostic stuies (Figure 23.1). Patients Between February 1997 an December 20
00, 124 consecutive patients with prostate cancer unerwent ultrasounguie tra
nsperineal implantation with pallaium103
Basic an avance techniques in prostate brachytherapy
334
(103P) or ioine125 (125I) sees either alone or in combination with external
beam raiation therapy (EBRT) at MetroHealth Meical Center or University Hospit

als of Clevelan, both in Clevelan, Ohio. Preoperative evaluation inclue a hi

story an physical examination, a ProstaScint scan, a thin slice pelvic CT scan
with intravenous contrast, a prostate volume stuy, a pubic arch stuy if inica
te, an measurement of prostatespecific antigen (PSA), prostatic alkaline phos
phatase (PAP), an alkaline phosphatase. Lymph noe sampling was performe in ap
propriate patients at the iscretion of their urologist. Patients range in age
from 45 to 79 years (meian: 67 years). The pretreatment PSA values range from
1.5 to 42 ng/mL (meian: 8.1 ng/mL). TNM staging was base on the 1993 American
Joint Committee on Cancer staging an the patients were stage T1c to T3b (meian
: T1C). leason scores range from 3 to 9 (meian 6). Risk factors (RF) were ef
ine by: PSA greater than 10 (38 patients, 30.6%), Stage T2b or greater (26 pati
ents, 21%), an leason score 7 to 10 (39 patients, 31.5%). enerally, patients
with lower risk factors unerwent brachytherapy alone (see implant; SI alone);
while patients were treate with EBRT an a brachytherapy boost (EBRT plus SI) i
f they exhibite at least one of the risk factors. For the 80 patients that une
rwent SI alone, 60 patients were low RF (0 RF); 17 patients were intermeiate RF
(1 RF) an 3 were high RF (2 or 3 RF). For the 44 patients having EBRT plus SI,
1 patient was low RF (0 RF); 15 patients were intermeiate RF (1 RF) an 28 wer
e high RF (2 or 3 RF). Thirtythree patients receive neoajuvant hormone epriv
ation (Lupron or Casoex) prior to or uring treatment at the iscretion of thei
r urologist, in conjunction either with SI alone (13 patients) or with EBRT plus
SI (20 patients). For the 91 patients who i not receive hormonal therapy, 67
patients were SI alone an 24 patients were EBRT plus SI. If the patient receive
 hormone therapy prior to implant, the pretreatment PSA was that recore prior
to the initiation of hormone therapy. Tables 23.3 an 23.4 summarize the patien
t characteristics of these 124 patients. Treatment For the 80 SI alone patients,
53 patients receive 103P at a minimal peripheral ose of 80 to 125 y (meian
: 115 y) an 27 patients receive 125I at a minimal peripheral ose of 140160 y
(meian: 144 y). For the 44 patients treate with EBRT plus a brachytherapy bo
ost, all patients were treate with 45 y of EBRT elivere to the prostate with
a 1.5 cm margin aroun the prostate an seminal vesicles. The EBRT was elivere
 with four fiels in a
Table 23.3 Patient characteristics
Characteristic n
Stage T1c T2a T2b T2c T3a T3b 46 27 6 0 1 0
SI alone Percent (%)
57.5 33.8 7.5 0 1.25 0
n
19 6 15 1 2 1
EBRT+SI Percent (%)
43.2 13.6 34.1 2.3 4.5 2.3
Raioimmunoguie prostate brachytherapy
335

rae leason 5 18 22.5 3 6,8 leason 6 56 70 8 18.2 leason 7 3 3.75 28 63.6 l

eason 8 3 3.75 4 9.1 leason 9 0 0 1 2.3 PSA 24 13 16.3 2 4.5 4.110 56 70 15 34.1
10.115 7 8.75 10 22.7 15.120 3 3.75 11 25 2040 1 1.25 3 6.8 >40 0 0 3 6.8 Hormone t
herapy Yes 13 16.3 20 45.5 No 67 83.7 24 54.5 SI, see implant; EBRT, external b
eam raiotherapy; PSA, prostatespecific antigen.
1.8 y fraction per ay over five weeks. Approximately one month after the compl
etion of EBRT, an ultrasounguie brachytherapy boost was performe. Fortyone
patients receive 103P at a minimal peripheral ose of 70100 y (meian: 90 y)
an 3 patients receive 125I at a minimal peripheral ose of 108 y (Table 23.4)

. The ose was escalate to 150% of the prescribe ose for regions with increas
e ProstaScint uptake. Toxicity stuy For the first 43 patients who unerwent mo
notherapy from February 1997 to August 1998, we followe PSA outcomes an the ac
ute an late complications of treatment. The meian PSA value at 10 months posti
mplant was 0.7 ng/mL, with a meian PSA in the patients with an without ajuvan
t hormonal therapy of 0.33 ng/mL an 0.7 ng/mL, respectively. Raiation Therapy
Oncology roup (RTO) graing criteria were applie to rank the patients symptoms
. Acute complications within the first month following implantation inclue gra
e 1 urinary symptoms in 13 patients (30%) an grae 2 urinary symptoms in 24 pa
tients (56%) requiring an alphablocker. Acute high grae complications inclue
grae 3 complications in six patients (14% total, incluing one patient with rec
tal bleeing an five patients who require a foley catheter beyon the first we
ek following the implant), an no patients with grae 4 complications. Beyon 4
months, late complications inclue grae 1 urinary symptoms in 12 patients (28%
) an grae 2 urinary symptoms requiring an alphablocker in 17 patients (40%).
High grae complications were limite to one grae 3 patient who require interm
ittent selfcatheterization which resolve at one year (2%), an one grae 4 pati
ent who unerwent a TURP to relieve urinary obstruction (2%). All acute symptoms
were within expecte tolerance. Minimal late complications were note; there we
re no complaints of urinary incontinence.20
Basic an avance techniques in prostate brachytherapy
336
Statistics Clinical evaluation, incluing igital rectal examination (DRE) an P
SA etermination, was performe one month following implant, every three months
for the first two years after implant, biannually for two years, an yearly ther
eafter. PSA values were followe in all 124 patients to evaluate isease outcome
s. If the patient i not continue to follow with one of the investigators, PSA
values were obtaine from the patients current physician. Followup was also obta
ine with selfaministere questionnaires, phone calls, an chart review. Follo
wup was calculate from the ay of implantation. Chisquare (x2) or Fishers exac
t tests were use to examine the association between two categorical factors. Th
e Wilcoxon rank sum test was use to examine the mean or meian ifference of co
ntinuous measures between two groups. Three biochemical iseasefree survivals (
bDFS) were estimate. One was efine by American Society of Therapeutic Raiolo
gy an Oncology (ASTRO), the other two were efine by serum PSA level. The ise
asefree survival time was measure from the ate of implantation to the time of
biochemical failure or last ate of followup. The institution of any hormonal
intervention posttherapy was efine as a biochemical failure event for all thes
e three efinitions. Patients were censore at the ate of last followup, or th
e ate of eath by other isease, if biochemical failure ha not occurre. The o
verall survival rate was measure from the ate of implantation to the ate of 
eath an censore at the ate of last followup for survivors. Survival istribut
ion was estimate using KaplanMeier methos an ifference between/ among group
s was teste using logrank test. All tests were twosie.
Table 23.4 Aitional patient characteristics
Characteristic
Age (yrs) SI alone EBRT+SI Pretreatment PSA (ng/mL) SI alone EBRT +SI leason sc
ore SI alone EBRT +SI Followup (mths) SI alone EBRT+SI Stage SI alone EBRT+SI S
I alone osage (y) 125 I implant 103 P implant EBRT+SI osage (y)
n
124 80 44 124 80 44 124 80 44 124 80 44 124 80 44 27 53
Mean
66 67 64 9.6 7.5 13.6 6.1 5.8 6.8 36 37 36

SD
8 8 8 7 4 9.3 1 0.9 0.8 15 15 15
Meian
67 68 45 8,1 7.3 12 6 6 7 33 36 33 T1c T1c T2a 144 115
Range
4579 4579 4977 1.542 1.527.3 242 39 38 59 1161 1161 1159 T1cT3b T1cT3a T1cT3b
pvalue*
0.04
<0.00 1
<0.001
0.678
145.5 110.6
4.6 10.0
Raioimmunoguie prostate brachytherapy
337
EBRT ose 44 45 0 45 45 125 I implant 3 108 0 108 108 103 P implant 41 87.8 6.9
90 70100 * The pvalue was between two treatment groups. SD, stanar evotion,
See Table 23.3 for other abbreviations,
Survival analysis Table 23.5 shows a yearly overall an biochemical iseasefree
survival (bDFS) rate for the whole cohort. The 4 year overall survival was 89.3%
. The 4 year (bDFS) rate for the whole group was 93.1% by ASTRO consensus efini
tion (Figure 23.2a), an it was 82.2% an 72.5% efine by PSA<1.0 an PSA<0.5,
respectively (Figure 23.2b).
Table 23.5 Yearly survival rate (%) for the entire patient population
Survival rate n 1 year 3 year 4 year 5 year pvalue*
124 992 91.1 89.3 89.3 Overall survival SI alone 80 100 90.9 88.2 88.2 0.947 EBR
T +SI 44 97.7 91.3 91.3 bDFS ASTRO 124 96.8 91.1 93.1 93.1 SI alone 80 100 97.4
97.4 97.4 0.015 EBRT+SI 44 90.9 85.5 85.5 PSA<1.0 124 94.5 82.2 82.2 82,2 SI alo
ne 80 97.5 85.2 85.2 85.2 0.203 EBRT+SI 44 88.6 76.6 76.6 PSA<0.5 124 94.4 74.1
72.5 67.4 SI alone 80 97.5 78.1 75.9 75.9 0.18 EBRT+SI 44 88,6 66.2 66.2 * ASTRO
; American Society of Therapeutic Raiology an Oncology; bDFS, biochemical ise
asefree survival. See Table 23.3 for other abbreviations,
Basic an avance techniques in prostate brachytherapy
338
Figure 23.2 (a) KaplanMeier biochemical iseasefree survival (bDFS) efine by
ASTRO consensus for all the 124 patients with a 95% confience interval, (b) Ka
planMeier
Raioimmunoguie prostate brachytherapy
339
bDFS efine by PSA<1.0 or PSA<0.5 for all the 124 patients. Table 23.6 Yearly s
urvival rate (%) by American Society of Therapeutic Raiology an Oncology (ASTR

O) graing
bDPS rate n 1 year 3 year 4 year 5 year pvalue
Risk factor Low risk 61 100 100 100 100 Intermeiate risk
0.002 High risk 31 87.1 79.6 79.6 Baseline PSA 10 86 100
38 89.5 86.6 86.6 86.6 Hormonal status HT 33 97 89,6 89.6
.4 94.4 94.4 bDFS, biochemical iseasefree survival; HT,

32 100 93.3 93.3 93.3

95.9 95.9 95.9 0.04 >10
0.483 No HT 91 96.7 94
hormonal therapy.

When stratifie by treatment group, for the 80 patients treate with SI alone, t
he 4 year bDFS rate was 97.4% by ASTRO consensus efinition, bDFS was 85.2% an
75.9% efine by PSA<1.0 an PSA<0.5. For the 44 patients treate with EBRT plus
see implant (SI), bDFS was 85.5% by ASTRO, 76.6% an 66.2% by PSA<1.0 an PSA<
0.5 efinition, respectively. Table 23.6 shows a yearly survival rate by ASTRO c
onsensus efinition stratifie by risk factor (RF), baseline PSA, or hormonal th
erapy (HT). The 4year actuarial bDFS rate for the low risk group (0 RF) was 100
%, for the intermeiate risk group (1 RF) it was 93.3%, an for the high risk gr
oup it was 79.6% (2 or 3 RF) (Figure 23.3a). For the patients with baseline PSA10
, bDFS was 95.9%, however, for those with PSA>10, bDFS was 86.6% (Figure 23.3b).
For the patients with neoajuvant hormones, bDFS was 83.3%, an for those witho
ut neoajuvant hormones, bDFS was 94.4% (Figure 23.3c). A plateau on the bDFS cur
ve for all the groups by any of the efinitions occurre by 3 years. For all the
patients without HT, the meian PSA nair was 0.2 ng/mL. The meian time to na
ir was 19 months. Discussion The favorable results observe in the present stuy
are likely attributable to the raioimmunoguie prostate brachytherapy techniq
ue. In a previous publication, we emonstrate a semiquantitative correlation w
ith prostate biopsy results utilizing prostate to muscle ROI ratios for specific
regions of interest (ROIs) within the prostate glan compare to backgroun mus
cle regions of interest place over the external obturator muscle.16 A sensitivi
ty of 91% an a specificity of 92% were emonstrate in the stuy. Although the
stuy emonstrate a strong correla tion between transrectal
Basic an avance techniques in prostate brachytherapy
340
ultrasoun guie biopsy results an antiboy concentration within the prostate
glan, it has at least two methoologic weaknesses. First, it is ifficult to e
termine the exact site of biopsy within the prostate glan by transrectal ultras
oun upon review of a written pathology report. While the majority of the patien
ts biopsies were reporte in sextant fashion, several of the reports merely refer
ence right or left prostate glan. Secon, the use of regions of interest withi
n the prostate glan to etermine counts rather than the use of the actual fuse
image between the compute tomography (CT) scan an a ProstaScint scan mae it 
ifficult to translate this information to the operating room at the time of the
interstitial brachytherapy proceure. We are now in the process of obtaining fur
ther ata to valiate the accuracy of the fusion stuy by correlating histopatho
logic finings with the computer images. Initially in our current series, a yna
mic implant technique, epenent solely on prostate volume, was chosen using the
Mick applicator. As we have become more comfortable with the image fusion techni
que, we are now relying more heavily on preplanning. However, we still prefer to
use free sees place with the Mick applicator to allow for aaptation from the
preplan at the time of implant. The role of raiolabele antiboies in the iagn
osis an treatment of cancer continues to expan. Inium111 capromab penetie
(ProstaScint) scanning is becoming wiely available for staging patients with pro
state cancer. We hope to emonstrate that it is useful not only to stage patient
s with prostate cancer an to help select appropriate patients for prostate brac
hytherapy, but also to tailor the implant itself base on histopathologic change
s within the glan not reaily apparent by anatomic imaging proceures. By oing
so, we hope to increase control rates while reucing the toxicity of treatment.
Fusion of the pelvic CT an ProstaScint scans ientifies regions within the pros
tate felt to be at high risk of local failure, which can then be targete with a

itional sees at the time of implantation. Peripheral loaing is applie to av

oi an excessive urethral ose. While the initial patients in this stuy were al
l implante using a nomogram approach an 2D osimetry, current patients are bei
ng treate using a 3D ultrasounbase preplanning technique to help improve targ
eting of the high risk regions. The concept of targeting intraprostatic isease
with higher concentrations of raiation is not a raical or novel one. We have p
reviously presente ata epicting the feasibility of raioimmunoguie prostate
brachytherapy.20 Other groups have ientifie localize isease with enorectal
MRI/MRS an esigne external beam therapy to provie an enhance ose to these
particular areas.21 The avantage inherent in such an approach is the ability t
o provie an increase ose to areas of isease concentration while sparing ise
asefree areas aroun the raiosensitive tissues of the urethra an rectum. Our
approach focuses on applications with brachytherapy, but our imaging technique m
ay also be useful in other treatment moalities, such as 3D conformal raiothera
py with intensity moulate raiotherapy (IMRT) for localize ose escalation.
Raioimmunoguie prostate brachytherapy
341
Figure 23.3 KaplanMeier biochemical iseasefree survival (bDFS) efine by AST
RO consensus, (a) Patients were stratifie by risk factor (RF). There were 61 pa
tients with 0 RF (low risk), 32 patients with
Basic an avance techniques in prostate brachytherapy
342
1 RF (intermeiate risk), an 31 patients with 2 or 3 RF (high risk). The pvalu
e is 0.002 among the three groups, (b) Patients were stratifie by pretreatment
PSA. There were 86 patients with PSA10; 38 patients PSA >10. The pvalue is 0.04.
(c) Patients were stratifie by hormonal therapy (HT): 33 patients with HT; 91 p
atients without HT. The pvalue is 0.483.
References
1. Blasko J, Rage H, Luse R, et al. Shoul brachytherapy be consiere a therap
eutic option in localize prostate cancer? Urol Clin North Am 1996; 23(4):633650.
2. Bates TS, illatt TA, Cavanagh PM, et al. A comparison of enorectal magneti
c resonance imaging an transrectal ultrasonography in the local staging of pros
tate cancer with histopathological correlation. Br J Urol 1997; 79(6):927932. 3.
Ikonen S, Karkkainen P, Kivisaari L, et al. Magnetic resonance imaging of clinic
ally localize prostatic cancer. J Urol 1998; 159(3):915919. 4. Deasy NP, Conry B
, Lewis JL, et al. Local staging of prostate cancer with 0.2 boy coil MRI. Cli
n Raiol 1997; 52(12):933937.
Raioimmunoguie prostate brachytherapy
343
5. Maio A, Rifkin MD. Magnetic resonance imaging of prostate cancer: upate. Top
Magn Reson Imaging 1995; 7(1):5468. 6. Scheiler J, Hricak H, Vigneron D, et al.
Prostate cancer: localization with threeimensional proton MR spectroscopic im
agingclinicopathologic stuy. Raiology 1999; 213:473480. 7. Pickett B, Vigneault
E, Kurhanewicz J, et al. Static fiel intensity moulation to treat a ominant
intraprostatic lesion to 90 y compare to seven fiel 3imensional raiother
apy. Int J Raiat Oncol Biol Phys 1999; 43:921929. 8. Ling CC, Humm J, Larson S,
et al. Towars multiimensional raiotherapy (MDCRT): biological imaging an b
iological conformality. Int J Raiat Oncol Biol Phys 2000; 47(3):551 560. 9. Zai
er M, Zelefsky MJ, Lee EK, et al. Treatment planning for prostate implants using
magneticresonance spectroscopy imaging. Int J Raiat Oncol Biol Phys 2000; 47(4

):10851096. 10. Kurhanewicz J, Vigneron D, Males R. The prostate: MR imaging an

spectroscopy. Raiol Clin North Am 2000; 38:115138, viiiix. Review. 11. Kahn D, Wi
lliams RD, Haseman MJ, et al. 111 Iniumcapromab penetie in the evaluation of
patients with resiual or recurrent prostate cancer after raical prostatectomy
, The ProstaScint Stuy roup. J Urol 1998; 159(6):20412046. 12. Nag S, Hinkle ,
Mojzisik C, et al. Raioimmunoguie brachytherapy (Rigby): A new technique fo
r implantation of occult tumors. Antiboy Immunoconj Raiopharmaceut 1993; 6(1):
2937. 13. Nag S, Ellis RJ, Martin EW, et al. Feasibility stuy of raioimmunogui
e ioine125 brachytherapy for metastatic colorectal cancer. Raiat Oncol Inves
tig 1995; 2:230236. 14. Chang SS, Reuter VE, Heston WD, et al. Short term noeaju
vant anrogen eprovation therapy oes not affect prostate specific membrane ant
igen expression in prostate tissues. Cancer 2000; 88(2):407415. 15. Qian J, Wolla
n P, Bostwick D. The extent an multicentricity of highgrae prostatic intraep
ithelial neoplasia in clinically localize prostatic aenocarcinoma. Hum Pathol
1997; 28(2): 143148. 16. Soee DB, Ellis RJ, Samuels MA, et al. Prostate cancer a
n prostate be SPECT imaging with ProstaScint: Semiquantitative correlation wit
h prostatic biopsy results. Prostate 1998; 37:140148. 17. Donohue RE, Millere J.
Aenocarcinoma of the prostate: biopsy to whole mount. Denver VA experience. Ur
ol Clin North Am 1991; 18(3):449452. 18. Stroumbakis N, Cookson MS, Reuter VE, Fa
ir WR. Clinical significance of repeat sextant biopsies in prostate cancer patie
nts. Urology 1997; 49(3 A suppl): 113118. * 19. Ellis RJ, Kim EY, Conant R, et al
. Raioimmunoguie imaging of prostate cancer foci with histopathological corre
lation. Int J Raiat Oncol Biol Phys 2001; 49(5):12811286. * 20. Ellis RJ, Soee
DB, Spirnak JP, et al. Feasibility an acute toxicities of raioimmunoguie pro
state brachytherapy. Int J Raiat Oncol Biol Phys 2000; 48(3):683687. * Reprinte
from these two papers with permission from Elsevier. 21. Pickett B, Vigneault E
, Kurhanewicz J, et al. Static fiel intensity moulation to treat a ominant in
traprostatic lesion to 90 y compare to seven fiel 3imensional raiotherapy
. Int J Raiat Oncol Biol Phys 1999; 43:921929.
24 Prostate brachytherapy uner local anesthesia
Sanra Arthurs an Kent Wallner Introuction Since its reintrouction, transperi
neal prostate brachytherapy has been performe uner spinal (epiural) or genera
l anesthesia, similar to what is stanar for more invasive proceures, such as
transurethral resection of the prostate (TURP) an prostatectomy. In fact, perfo
rming an implant is less invasive than a prostate biopsy, since no tissue is tak
en with the former, an shoul not require more than the little or no anesthesia
, as is customary for biopsies.1 Spinal or general anesthesia entails a substant
ial cost, some egree of health risk, an burensome scheuling requirements. Sw
itching to local anesthesia provies substantial practical avantages, incluing
less cost, less physician time coorination (bypassing anesthesiologist), an a
voiance of epenence on operating room time. Prostate brachytherapy uner loca
l anesthesia was first escribe by the late Dr Jean Roy, who performe over 100
proceures in a fully equippe operating room using intraprostatic liocaine (l
ignocaine) injections, along with intravenous (IV) seation.2 Local anesthesia w
as institute at Puget Soun Health Care System, Seattle in 1999, performing the
proceure in the simulator suite of the raiation oncology epartment, without
anesthesia personnel in attenance. Technique3 An intravenous (IV) line is start
e in a clinic room, just prior to the proceure. At that time, a brief preopera
tive interview is conucte by the brachytherapist, noting meications an previ
ous meical history. The patient is then brought into the simulator suite an po
sitions himself on the simulator table. The patient has a cariac monitor attach
e an a urinary catheter is inserte. He is then place in the lithotomy positi
on, using stirrups attache to the simulator table (Figure 24.1). A 68 cm patch o
f perineal skin an subcutaneous tissue is anesthetize by local infiltration of
1% liocaine (lignocaine), using a 25 gauge 1.5 inch neele (Figure
Prostate brachytherapy uner local anesthesia
345

Figure 24.1 The patient is fully awake for an implant proceure in the simulator
suite in the Veterans Affairs Puget Soun Health Care System.
24.2a). All parenteral liocaine (lignocaine) is given in a 0.5% solution with e
pinephrine (arenaline) (1:100000). The transrectal ultrasoun (TRUS) probe is t
hen inserte an positione to reprouce the planning images. A 3.0 inch 22 gaug
e spinal neele is use to inject liocaine (lignocaine) up to the prostatic ape
x, in a pattern aroun the periphery of the prostate (Figure 24.2b). Once the pe
lvic floor an prostatic apex are anesthetize, a 7.0 inch, 22 gauge spinal nee
le is inserte through an 18 gauge 3 inch spinal neele into the peripheral plan
ne neele tracks, monitore by TRUS (Figure 24.2c, an Figure 24.3). As the nee
les are avance to the prostatic base, about 1.0 cc of liocaine (lignocaine)
solution is injecte in the intraprostatic track. At this point in the proceure
, a total of approximately 200400 mg of liocaine (lignocaine) is injecte (Figur
e 24.4). Not all tracks are necessarily injecte, epening on the total number
an how patients tolerate the injections. A limite number of central tracks are
also injecte. The total ose is limite to 500 mg (approx. 7 mg/kg).
Figure 24.2 (a) Injection of liocaine (lignocaine) into perineal skin an subcu
taneous tissue, (b) periapical
Basic an avance techniques in prostate brachytherapy
346
region, an (c) eep prostatic tissue. The eeper injections are mae with 22 ga
uge spinal neeles inserte through the transrectal ultrasoun (TRUS) template.
Figure 24.3 (a) Typical liocaine (lignocaine) injection points (white ots) for
periapical an prostatic sites. 22 gauge spinal neeles visualize well on trans
rectal ultrasoun (TRUS) (b).
When using a Mick applicator, a maximum of two neeles are in the patient at any
one time. During the implant proceure, aitional liocaine (lignocaine) is inj
ecte into one or more neele tracks if the patient experiences excessive iscom
fort. Neither an anesthetist nor a nurse is present for the implant proceure. L
iocaine (lignocaine) injections are generally carrie out by a physician, but c
oul easily be one by a traine nurse, physician assistant, or raiation therap
ist. The total time neee to position the patient, perform the liocaine (ligno
caine) injections, an implant the sees is typically less than two hours.3 At t
he completion of the source placement, the TRUS probe is remove an plain ortho
gonal pelvic raiographs taken with the catheter in place. The catheter is remov
e, an the patient then has his postimplant CT taken. About a total of two hour
s after completing the implant, the patient is ischarge. Patients are allowe
to rive themselves home, proviing no seative was given.
Prostate brachytherapy uner local anesthesia
347
Figure 24.4 Approximate amounts of liocaine (lignocaine) injecte into four reg
ions. The precise amount varies from patient to patient, accoring to iniviual
tolerance. The pelvic floor is typically the least sensitive site to neele ins
ertion an liocaine (lignocaine) injections.4
Patient tolerance Patients tolerate brachytherapy uner local anesthesia surpris
ingly well. Their heart rate an iastolic bloo pressure usually show minimal c
hanges, consistent with mil iscomfort.5 Serum liocaine (lignocaine) levels ar
e typically below or at the low range of therapeutic.5 Postimplant CTefine tar
get coverage has range from 80% to 95%; well within publishe criteria for tech
nical aequacy.3 Large prostate glans have not pose a particular problem in co
mpleting the proceure.3 Anesthesia, as escribe here, still allows for maneuve

ring in orer to avoi pubic arch interference. The arch itself is not particula
rly painsensitive, an can be anesthesize if necessary. To evaluate comfort le
vel, patients were interviewe by telephone an aske to rate their pain with th
eir prostate biopsy versus their prostate implant on a scale of 0 to 10. Of 58 p
atients interviewe at a meian of 6 months since implantation, the meian biops
y pain score was 4.5 an the meian implant pain score was 3.0 (Figure 24.5).6 I
n general, there has been little correlation of patients biopsy pain scores an t
heir implant pain scores (Figure 24.6).3 Five of the 58 patients interviewe by
Smathers an colleagues (9%) state that they woul have preferre to have the p
roceure uner general anesthesia.6 Currently, however, we suspect that fewer th
an 9% woul prefer general anesthesia if aske, now that we have gaine experien
ce with the use of local anesthesia.
Basic an avance techniques in prostate brachytherapy
348
Figure 24.5 Meian patientreporte pain scores relate to their prostate biopsy
versus implant proceure.6
Figure 24.6 Patientreporte pain scores from the aministration of liocaine (l
ignocaine) an see placement, versus their biopsyrelate pain scores. Due to ov
erlap of scores, some points represent more than one patient3
Prostate brachytherapy uner local anesthesia
349
As of October 2004, more than 1150 patients have receive implants uner local a
nesthesia at Puget Soun. Only one patient has been switche to general or spina
l anesthesia since June 1999after lying own on the simulator table he ecie no
t to procee, before letting us begin the local anesthesia process. Other than e
xtreme patient apprehension, we have encountere no contrainication to local an
esthesia, proviing patients are meically stable. Nonetheless, there was a lear
ning curve, an the proceure has become easier with increasing staff experience
. Juging from the experience of visiting physicians, we estimate that performin
g local anesthesia for 10 patients woul allow most practitioners to feel comfor
table with the technique. Seation In aition to local liocaine (lignocaine) i
nfiltration, we have use a variety of seation techniques over the last three y
ears, incluing oral agents (benzoiazepines), conscious seation with miazolam
/fentanyl, an nitrous oxie (laughing gas). After a series of patient acceptanc
e quality stuies, we have abanone the routine use of seation, an currently
rely on local liocaine (lignocaine) infiltration alone.6,7 Our reasoning has be
en that the local liocaine (lignocaine) is the primary moe of pain control, an
 that patient anxiety is best allaye with personal contact/communication urin
g the proceure. In cases of severe anxiety, IV miazolam is given. Problems Per
forming implants with local anesthesia can entail some problems. Patients are mo
re likely to move, requiring some ajustments in positioning of the TRUS probe.
But with an easily ajustable TRUS stan, this has not been a major problem. The
proceure is facilitate by a lighthearte rapport between the patient an sta
ffour personalities (patients or staff) make the experience less pleasant. Concl
usions The fact that patients typically rate their implant proceurerelate pai
n as being somewhat less unpleasant than their prostate biopsy (a very common pr
oceure that is rarely one uner anesthesia), shoul allay any ethical concerns
about using local anesthesia for implants. While a small percentage of patients
state in retrospect that they woul have preferre general over local anesthesi
a, it is far more common for patients to express relief about not neeing genera
l or spinal anesthesia.6 We have no plans to substantially alter the proceure f
rom that escribe here.

Basic an avance techniques in prostate brachytherapy

350
References
1. Nash PA, Bruce JE, Inuhara R, et al. Transrectal ultrasoun guie prostati
c nerve blockae eases systematic neele biopsy of the prostate. J Urol 1996; 15
5:607609. 2. Roy J, Pouliot J, Taschereau R. Permanent transperineal prostate imp
lants performe uner local anesthesia. Int J Ra Oncol Biol Phys 1998; 42(suppl
):296. 3. Mueller A, Wallner K, Corriveau J, et al. A reappraisal of local anest
hesia for prostate brachytherapy. Raiother Oncol 2003; 37(5):309312. 4. Wallner
K, Blasko J, Dattoli M. Patient preparation. In: Dattoli M, e. Prostate brachyt
herapy mae complicate, 2n en. Seattle: Smart Meicine Press, 2001; 7.17.18. 5
. Wallner KE, Simpson C, Roof J, et al. Local anesthesia for prostate brachyther
apy. Int J Ra Oncol Biol Phys 1999; 45:401406. 6. Smathers S, Wallner K, Simpson
C, et al. Patient perception of local anesthesia for prostate brachytherapy. Se
min Urol Oncol 2000; 18:142146. 7. Simpson C, Wallner K, Sanchez D, et al. Patien
t anxiety an tolerance of prostate brachytherapy uner local anesthesia. J Brac
hyther Int 2001; 17:7781. 8. Wallner K. Prostate brachytherapy uner local anesth
esia: lessons from the first 600 patients. Brachytherapy 2002; 1:145148.
25 The impact of hormonal therapy on pubic arch interference
Aam P Dicker, Christopher T Chen, JD Liu, Richar C Valicenti, an Frank M Wate
rman Introuction Prostate cancer is the most frequent cancer an the secon mos
t frequent cause of eath for men in North America. Much attention has been focu
se on the early etection of isease an recent reports appear to inicate that
a greater percentage of patients are now etecte with early stage isease. Ra
ical prostatectomy an raiotherapy are reporte as having goo five year surviv
al rates in men with early stage isease (stageT2b, PSA<10 ng/mL, an leason sco
re6), although the avantages an isavantages are the subject of active ebate.
There has been a recent resurgence in using permanent interstitial raioactive
see implants for prostate cancer because of avances in transrectal ultrasoun
(TRUS) an in compute tomography (CT).15 Transperineal interstitial permanent pr
ostate brachytherapy (TIPPB) requires an unobstructe access to the prostate. Pe
lvic bones efining the pubic arch can block access to the anterior aspect of th
e glan; commonly referre to as pubic arch interference/ overlap (PAI/PAO) (Figur
e 25.1). Assessment of potential PAI is critical when eciing whether or not a
patient can be a caniate for a transperineal prostate implant,6 an can be per
forme with either CT,79 or TRUS.10,11 An example of a TRUS image of the pubic ar
ch is shown in Figure 25.2. Anrogen suppression treatment can be use to reuce
both the size of the glan an the egree of PAI in those patients with signifi
cant PAI, although no benefit to reuction in biochemical control has been note
.12 Most literature reports to ate have evaluate anrogen suppression therapy
an its ability to ownsize the overall prostate volume.13 To ate, there is lim
ite information that evaluates the egree of PAI/PAO prior to an after anroge
n suppression therapy in the context of TIPPB. To ai in this evaluation of PAI
in the planning of permanent prostate implants, we aapte technology primarily
evelope for the treatment planning of external beam raiotherapy (EBRT). The p
otential for PAI/PAO was evaluate by isplaying the prostate contours superimpo
se on the pubic arch from the perspective of the neele template (neeleseyevi
ew). We use this methoology to evaluate patients prior to an after anrogen su
ppression therapy to assess pubic arch interference an other prostate imension
s. The software an harware methoology escribe here shoul be applicable wit
h most external beam treatmentplanning software packages.
Basic an avance techniques in prostate brachytherapy
352
Treatment Men with biopsy proven aenocarcinoma of the prostate were referre to

the multiisciplinary prostate program at the Kimmel Cancer Center at Thomas Je

fferson University. Patients believe to be appropriate caniates for brachythe
rapy were counsele. Patients at high risk for pubic arch interference (volume>6
0 cm3), or whose initial raiographic evaluation suggeste pubic arch interferen
ce, receive a three month course of either luteinizinghormonereleasinghormone
(LHRH) agonist alone (Zolaex, Zeneca Pharmaceuticals, 3.6 mg subcutaneous epot
, monthly or Lupron, TAP Pharmaceuticals, 7.5 mg intramuscular, monthly) or total
anrogen suppression (LHRH agonist an Flutamie, ScheringPlough, 250 mg three
times a ay) or antianrogen alone (Casoex, Zeneca Pharmaceuticals, 50 mg, ail
y). The form of anrogen suppression was by preference of the referring physicia
n. No attempt was mae to ranomize patients to any form of hormonal therapy. Pr
ior to the aministration of anrogen suppression therapy a CT scan was performe
 to evaluate PAO/PAI. Limite bowel prep was performe prior to the scan (eithe
r 5 ounces of magnesium citrate or a Fleets enema), to clear the lower colon an
rectum. A pelvic CT scan was performe on a flatbe helical CT scanner (Picker P
Q5000, Clevelan, OH) with the patient in the supine position, legs straight to
evaluate pubic arch interference. The urethra was lubricate an anesthetize wi
th 10 mL 2% liocaine (lignocaine) hyrochlorie jelly
The impact of hormonal therapy
353
Figure 25.1 Threeimensional renering of the pelvic bones (white) an prostate
(re). The AQuSim software allows for a cubecut view for greater appreciation of
the relationship between the prostate an pubic arch, (a) Represents a view from
the perineum an (b) an oblique view.
(International Meication Systems, South El Monte, CA). A foley catheter was ins
erte an the balloon inflate with 710 mL of ilute (50% original concentration
) iatrizoate meglumine (Renografen60, Mallinckrot Corp, St Louis, MO). The bla
er was instille with 30 mL ilute iatrizoate meglumine (2% in saline). The 
ye was ilute to minimize artifact on the scan. The prostate was image using 2
.0 mm slice thickness an 2.5 mm inexing from base to apex. Using the Picker AQ
uSim software program, the periphery of the prostate glan was contoure either b
y author APD or author CC, but all contours were reviewe by APD. After completi
on of the contours an the computation of the prostate volume, the virtual simul
ation moule was activate. The image setting use was esigne to
Basic an avance techniques in prostate brachytherapy
354
emphasize the igital reconstructe raiograph (DRR) for smooth bone. To obtain
a perspective from the neele template (neeleseyeview) the gantry an table ang
le parameters were ajuste an the following settings were use in the virtual
simulation moule: antry 90, Table 90, Winow 425, Level 225. For the purposes of
statistical analysis, the ifference between pre an postanrogen suppression
therapy for each patient was use with respect to a measurement, an was teste
against a ifference of 0 mm change. Meian, range, an quartiles were calculate
 (25% an 75%). Means an stanar eviations were not use because those measu
rements may not be istribute normally an the population sample was limite. T
he signe rank test was applie to test the hypothesis that the change was posit
ive. The signe rank test was selecte because it oes not require the assumptio
n of normal istribution for the change.
Figure 25.2 Neeleseyeview. Patient 5 (a) prior to an (b) after anrogen suppre
ssion therapy. Potential for pubic arch interference is assesse by isplaying t
he prostate contours superimpose on the pubic arch from the perspective of the
template. Note the significant overlap of the prostate contours, with the anteri
orlateral aspect of the pubic arch extening to the outer cortex. The calculate

 prostate volumes an measurements prior to an after anrogen suppression ther
apy are
The impact of hormonal therapy
355

etaile in Tables 25.125.4. (Reprouce courtesy of Dr Peter rimm, Seattle Pros

tate Institute.)
Results All men were compliant with their anrogen suppression therapy as measur
e by followup prostatespecific antigen (PSA) an serum testosterone levels. Po
tential for pubic arch interference was evaluate by isplaying the prostate con
tours superimpose on the pubic arch from the perspective of the neele template
(Figure 25.3). This perspective was obtaine by ajusting the AQuSim software pa
rameters to isplay the gantry an table, an rotate 90 for a template/neelesey
eview. The egree of pubic arch overlap can be ientifie an measure. In our e
xperience, pubic arch overlap extening to the outer cortex represents a serious
impeiment to a transperineal
Figure 25.3 Transrectal ultrasoun (TRUS) image of pubic arch interference. The
yellow line enotes the outline of the pubic arch, (a) PreLHRH monotherapy. (b)
Post LHRH monotherapy. (Reprouce courtesy of Dr Peter rimm, Seattle Prostate
Institute.)
implant, an anrogen suppression therapy is one possible solution. In aition,
prostate volume, length, anterioposterior an longituinal measurements can be
calculate easily from the treatmentplanning software an can be use to verify
information erive from a volume stuy from a ifferent source (ultrasoun, ma
gnetic resonance imaging). Anrogen suppression therapy can have a ramatic effe
ct on the imensions of the prostate (Tables 25.125.4). Of the three measure im
ensions of the prostate,
Basic an avance techniques in prostate brachytherapy
356
neoajuvant hormonal therapy appears to have the greatest effect on the longitu
inal measurement (meian change: 10.0 mm, range: 032.4 mm, p<0.0001) as compare
with the AP (meian change: 8.1mm, range: 0.619.2 mm, p<0.0001) an with the late
ral (meian change: 5.2 mm, range: 028.2 mm, p<0.0001). Although this can be epe
nent on the interpretation of the base an apex as well as the CT slice thickne
ss. Prostatic volume change significantly after hormonal therapy, ranging from
1.4% to 61.8% (meian change: 23.4 cm3, range: 7.847.0 cm3, p<0.0001). The small
sample size of this pilot stuy preclues any conclusions about the metho of an
rogen suppression an the egree of volume reuction. The most striking part of
this stuy was the measurable change in PAI/PAO after three months of anrogen
suppression therapy. All patients emonstrate a ecrease in PAI/PAO an more th
an half of the patients showe a 50% or greater change. The meian change for th
e right an left pubic arches was 4.6 mm (range: 2.36.2 mm, p<0.0001) an 3.8mm (
range: 1.611.6 mm, p<0.0001), respectively. The ecrease in PAO usually was not s
ymmetrical, with one sie having the greater change. Large changes in prostatic
volume i not always correspon with similar changes in PAI/PAO an vice versa.
Discussion The role of hormonal therapy (HT) in the management of prostate canc
er has evolve over the past ecaes. The initial use of HT was for the primary
management of metastatic isease. During the past ten years its use for patients
with localize isease in the neoajuvant setting for both surgery an raiatio
n therapy has increase. The role for neoajuvant HT for patients being consier
e for interstitial permanent prostate brachytherapy (TIPPB) is limite to owns
izing the glan to reucing the amount of pubic arch overlap, an to facilitatin
g the technical placement of raioactive sees. Although the concept of ownstag
ing is attractive, an surgical ata now emonstrate that it is possible to shif

t clinical stage T3c isease into a lower stage, there has been no associate pa
tient benefit note in freeom from biochemical recurrence.1417 Whether hormonal
ownsizing will improve local tumor control with TIPPB by reucing tumor volume
before raiation is elivere, can be answere only by properly esigne stuies
. Until ranomize prospective stuies properly evaluate this effect for TIPPB,
we suggest that neoajuvant HT be use only for patients with early stage iseas
e. Stuies of the use of neoajuvant HT with threeimensional conformal external
beam raiotherapy (3DCRT) emonstrate that reuction in size of the prostate c
an be accompaine by a concomitant reuction in raiation ose to normal surrou
ning organs.4,18,19 Downsizing of the prostate can result in ecrease acute co
mplication rates.2023 This concept, however, is not reaily transferable to TIPPB
. The steep ose graient achieve by brachytherapy limits the amount of rectum
an blaer receiving a significant raiation ose. This stuy was esigne to
investigate the effect of HT on the egree of pubic arch overlap. It uses a uniq
ue approach to quantitating PAI/PAO by utilizing beameyeview technology to superi
mpose the contours of the prostate on the pubic arch. A similar approach was pub
lishe by Tincher et al.24 Different methos of neoajuvant HT were
The impact of hormonal therapy
357
use, accoring to referring physician preference. A reuction of 3355% in glan
size an volume has been reporte by others,2533 an a similar reuction was obse
rve in this stuy. Neoajuvant hormonal therapy ha a ramatic effect by ecrea
sing the proportion of PAI/PAO. Although this stuy was not esigne to evaluate
how ifferent methos of anrogen ablation affect PAI/PAO, there may be a sugge
stion that total anrogen ablation carries no significant benefit in reucing PA
I/PAO when compare with an LHRH agonist alone. One concern arising from this st
uy woul be interobserver variation of contouring the prostate.21,22,34 User va
riation can be minimize by letting only two iniviuals perform contouring an
by having all contours reviewe by one iniviual. Transrectal ultrasoun (TRUS)
also has been use for evaluating pubic arch interference (Figure 25.3). Usuall
y, this is performe by imaging the apex region an by moving the probe caually
until the bright echoes of the pubic arch are note an a har copy is printe.
The prostate then is image at its wiest anterior imension an a har copy pr
inte. The prostate outline is trace on paper noting the position of the coori
nates. This tracing is superimpose on the pubic arch image an evaluate for ov
erlapping regions. A CT scan performe for iagnostic purposes also can be use
for this purpose. It has been suggeste that if greater than onethir of the gl
an is blocke by the pubic arch, the patient shoul either select a ifferent f
orm of treatment or consier hormonal ownsizing.6 Large changes in prostate vol
ume are not always associate with corresponing changes in PAI/PAO. One explana
tion for this is that PAI/PAO reflects a combination of
Table 25.1 Effect of anrogen suppression therpay on Ap (anteroposterior), later
al, an longituinal measurements of the prostate, as measure using CT
Patient Hormone Status
1 Pre Post 2 Pre Post 3 Pre Post 4 Pre Post 5 Pre Post 6 Pre Post 7 Pre Post 8 P
re Post
Anrogen AP Lateral Longituinal Volume Right Left suppression (mm) (mm) (mm) (c
m3) arch arch (mm) (mm)
Total LHRH LHRH Total Total LHRH LHRH Total 61.6 51.8 46.5 38.7 44.9 34.8 45.7 4
2.9 42.0 34.3 48.8 38.4 54.2 46.1 50.2 45.4 62.6 60.5 58.2 46.7 48.3 42.4 47.1 4
4.2 50.3 43.6 48.3 43.1 58.5 53.9 59.6 54.5 82.5 69,9 44.9 34.9 40.0 40.1 40.0 3
0.3 37.6 30.1 45.0 30.0 45.0 32.4 47.4 35.1 134.9 87.9 72.6 40.1 44.6 36.8 64.6
41.2 42.0 31.5 62.7 31.8 87.0 51 80.7 59 22.0 18,9 7.2 3.9 8.1 4.6 12.0 9.7 14.9
10.7 8.5 3.1 8.5 2.2 11.1 5.0 17.4 13.7 8.0 2.1 12.3 5.8 12.3 8.7 16.2 12.4 10.
4 8.4 6.7 0.8 9.6 5.9

Basic an avance techniques in prostate brachytherapy

358
9 Pre LHRH 41.6 50.2 33.1 31.2 6.22 2.18 Post 30.6 42.6 24.9 18.6 0 0 10 Pre Tot
al 47.6 65.6 57.5 48.9 9.4 9.9 Post 28.4 37.4 25.1 18.7 4.8 6.03 11 Pre LHRH 38.
4 47.8 44.9 51.1 7.4 8.5 Post 37.8 44.0 44.9 41.8 2.2 7.0 12 Pre LHRH 46.5 47.8
44.9 55.4 12.0 17.2 Post 31.1 47.8 29.9 31.1 7.6 5.6 13 Pre LHRH 43.2 58.9 37.6
48.4 13.1 13.7 Post 39.8 49.5 35.0 33.9 7.2 8.5 14 Pre Antianrogen 41.0 51.4 47
.5 47.2 8.2 12,4 Post 40.2 47.0 40.0 46.5 5.8 11.8 15 Pre Antianrogen 46.6 63.4
35.1 54.2 7.5 9.7 Post 43.8 61.1 35.1 46.6 3.5 9.5 Change in prostate volume wa
s calculate by the AQuSim software. Pubic arch interference was measure by isp
laying the prostate contours superimpose on the pubic arch from the perspective
of the template an is liste as right arch an left arch, respectively. LHRH, lute
inizinghormonereleasing hormone.
Table 25.2 Calculate ifference between pre an postanrogen suppression thera
py measurements
Patient Suppression metho
1 Total 2 LHRH 3 LHRH 4 Total 5 Total 6 LHRH 7 LHRH 8 Total 9 LHRH 10 Total 11 L
HRH 12 LHRH 13 LHRH 14 Antianrogen 15 Antianrogen
AP Lateral Longitudinal Volume Rarch Larch (mm) (mm) (mm). (mm) (mm) (cm3)
9.8 7.8 10.1 2.8 7.7 10.4 8.1 4.8 11 19.2 0.6 15.4 3.4 0.8 2.8 2.1 11.5 5.9 2.9
6.7 5.2 4.6 5.1 7.6 28.2 3.8 0 9.4 4.4 2.3 12.6 10.0 0.1 9.7 7.5 15.0 12.6 12.3 8
.2 32.4 0 15.0 2.6 7.5 0 47.0 32.5 7.8 23.4 10.5 30.9 36.0 21.7 12.6 30.2 9.3 24
.3 14.5 0.7 7.6 3.1 3.3 3.5 2.3 4.2 5.4 6.2 6.1 6.2 4.6 5.2 36.5 5.9 2.4 4.0 3.7
5.9 6.5 3.6 3.7 2.0 6.0 3.8 2.2 3.9 1.6 11.6 5.3 0.6 0.2
glanular size, position within the pelvis an pelvic bone architecture. For exa
mple, two patients can have ientical prostate volumes, yet one may have signifi
cant PAI/PAO an the other minimal PAI/PAO. This may be because the first patien
ts prostate has a more anterior location within the pubic arch. The anterioposter
ior location of the prostate is not well unerstoo but is believe to be a func
tion of the muscle tone of the levator ani an puborectalis muscles, plus the vo
lume of air in the rectum.
The impact of hormonal therapy
359
This stuy was unertaken to evaluate the effect of anrogen suppression on the
imensions of the prostate from the perspective of a physician consiering a tra
nsperineal interstitial permanent implant for a patient with prostate cancer. No
attempt was mae to compare ifferent methos of anrogen suppression. Although
ultrasoun is frequently use to compute prostate volumes for raioactive see
implantation, this stuy i not attempt to aress the ifferences in measuring
prostate imensions when comparing ultrasoun with CT,3537 but focuse strictly
on the subject of PAI/PAO. Software to ai the evaluation of pubic arch interfer
ence has been reporte by Pathak et al.11 The importance of patient positioning
has been aresse by Strang et al.9 Their initial observation was that at time
of TRUS volume scanning, the pubic arch was reaily visible in realtime. In a
ition, virtually every patient appeare to have some PAI at CT but not at subseq
uent TRUS. They chose to stuy this iscrepancy an hypothesize that this was 
ue to CT being performe with the patient in the supine position, whereas TRUS w
as performe with the patient in the orsal lithotomy position. Nine patients un
erwent preplanning CT an TRUS within 21 ays of each other an were not treate
 with hormonal therapy before or uring that interval. Every patient appeare t
o have PAI at CT (overall range: 820 mm, mean: 12.2 mm3.4). Four of the nine patie

nts ha overlap at TRUS; one ha at least a 5 mm overlap. The range of overlap a
t TRUS was 5 mm clearance (the istance between the anterior prostate an poster
ior pubic arch when there is no overlap) to 7 mm overlap (mean: 0.4 mm3.6). The s
ensitivity of CT for any PAI was 100% (95% CI: 40%, 100%), an the specificity w
as 0 (95% CI: 0%, 52%). CT thus resulte in overestimation of PAI by 11.8 mm, in
comparison with TRUS performe with the patient in a orsal lithotomy position
as the reference stanar. However, this is without any software manipulation of
the image (alteration of gantry angle) to take into account the lack of orsal
lithotomy leg position. Our clinical practice has incorporate aspects of this s
tuy. We acknowlege that the patient position in the CT scanner oes not mimic
that of the operating room. We i try to construct a evice that woul place th
e patients legs in a limite lithotomy position, but foun that this evice i n
ot place the legs in a satisfactory position, although Tincher et al have solve
this technical problem.24
Table 25.3 Percent change pre an postanrogen suppression therapy measurements
Patient Suppression % AP
1 Total 2 LHRH 3 LHRH 4 Total 5 Total 6 LHRH 7 LHRH 8 Total 9 LHRH 10 Total 11 L
HRH 15.9 16.8 22.5 6.1 18.3 21.3 14.9 9.6 26.4 40.3 1.6
% Lateral
3.4 19.8 12.2 6.2 13.3 10.8 7.9 8.6 15.1 43.0 7.9
% Longitudinal
15.3 22.3 0.3 24.3 19.9 33.3 28.0 25.9 24.8 56.3 0.0
% Volume
34.8 44.8 17.5 36.2 25.0 49.3 41.4 26.9 40.4 61.8 18.2
%R arch
14.2 45.4 43.7 19.1 28,4 63.8 73.5 55.5 100.0 48.9 69.9
% Larch
21.5 73.9 52.8 29.1 23.1 19.4 88.8 39.0 100.0 39.0 18.2
Basic and advanced techniques in prostate
rachytherapy
12 LHRH 13 LHRH 14 Antlandrogen 15 Antiandrogen 33.1 7.9 2.0 6.0 0 16.0 8.7 16 3
3.4 6.9 5.5 0 43.9 30.0 1.4 14.0
360
36.5 45.0 29.2 53.3 67.5 38.6 4.8 2.0
Ta
le 25.4 Statistical analysis of the difference
etween pre- and postandrogen
suppression therapy on prostatic dimensions (anteroposterior, lateral, longitudi
nal), volume, and right/left pu
lic arch overlap

AP (mm) Lateral (mm) longitudinal (mm) Volume (cm3) Eight arch (mm) Left arch (m
m)
Median Min Max 25% Q 75% Q Signed rank test p<
8.1 5.2 10.0 23.4 4.6 3.8 0.6 0.0 0.0 7.8 2.3 1.6 19.2 28.2 32.4 47.0 6.2 11.6 4
.8 3.8 7.5 12.6 3.5 3.6 10.4 7.6 12.6 30.9 5.9 5.9 0.001 0.001 0.001 0.001 0.001
0.001
Because the patient in the CT scanner is not in the dorsal lithotomy position we
have learned to make adjustments in the gantry angle to simulate the lithotomy po
sition. Our clinical operating experience has
een that no PAI has
een experien
ced when gantry angles ranging from 90 to 115 are used to demonstrate that no over
lap with the prostate and pu
ic arch exists. When gantry angles greater than 25 a
re necessary, we find that extended lithotomy positions are necessary to avoid P

AI, as has
een suggested
y Stone and Stock.38 Conclusion Androgen suppression
therapy can have a significant effect on overall prostate dimensions and can red
uce pu
ic arch interference. Software designed for external
eam radiation thera
py (EBRT) can
e used in the evaluation of pu
ic arch interference for patients
who are
eing considered for permanent transperineal prostate
rachytherapy. The
needles-eye-view can
e used as
oth a research and clinical tool for the evaluat
ion of pu
ic arch interference prior to and after androgen suppression therapy.
This does not require specialized software and the information o
tained can
e u
sed to corro
orate data o
tained from other volume studies to aid in the plannin
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80. 31. Weiner KX, Ciesla J, Jaffe AB, et al. Chromosomal location and structura
l organization of the human deoxycytidylate deaminase gene. J Biol Chem 1995; 27
0(32):1872718729. 32. Pinault S, Tetu B, Gagnon J, et al. Transrectal ultrasound
evaluation of local prostate cancer in patients treated with LHRH agonist and in
com
ination with flutamide. Urology 1992; 39(3):254261. 33. Kojima M, Ohe H, Wat
ana
e H. Kinetic analysis of prostatic volume in patients with stage D prostatic
cancer treated with LHRH analogues in relation to prognosis. Br J Urol 1995; 75
(4):492497. 34. Du
ois DF, Prestidge BR, Hotchkiss LA, et al. Intrao
server and i
ntero
server varia
ility of MR imaging- and CT-derived prostate volumes after tr
ansperineal interstitial permanent prostate
rachytherapy [See comments]. Radiol
ogy 1998; 207(3):785789. 35. Roach M, Faillace-Akazawa P, Malfatti, et al. Prosta
te volumes defined
y magnetic resonance imaging and computerized tomographic sc
ans for three-dimensional conformal radiotherapy. Int J Radiat Oncol Biol Phys 1
996; 35(5):10111018. 36. Narayana V, Ro
erson PL, Pu AT, et al. Impact of differe
nces in ultrasound and computed tomography volumes on treatment planning of perm
anent prostate implants. Int J Radiat Oncol Biol Phys 1997; 37(5):11811185. 37. N
arayana V, Ro
erson PL, Winfield RJ, et al. Impact of ultrasound and computed to
mography prostate volume registration on evalua tion of permanent prostate impla
nts. Int J Radiat Oncol Biol Phys 1997; 39(2):341346. 38. Stone NN, Stock RG. Pro
state
rachytherapy in patients with prostate volumes >/=50 cm3: dosimetic analy
sis of implant quality. Int J Radiat Oncol Biol Phys 2000; 46(5): 11991204.
26 Using the needle manipulation ruler
Brian J Moran Introduction Prostate
rachytherapy was initially performed using
radium. However, there were many pro
lems associated with its use, one of which
was radiation exposure to the hands of the
rachytherapist. In the 1970s, the us

e of iodine-125 (125I) was popularized at Memorial Sloan-Kettering Hospital usin

g an open suprapu
ic approach. This technique was a
andoned in the early 1980s,
not
ecause of radiation exposure risks,
ut rather,
ecause of flaws of seed pl
acement in the technique itself. With the development of transrectal ultrasound
(TRUS) and the transperineal interstitial implant technique of the prostate, see
d placement was dramatically improved. Currently, iodine-125 and palladium-103 a
re the radioactive isotopes of choice for this procedure. Both of these sources
have low energies with su
sequent dramatic fall-off of dose as distance increase
s (inverse square law E 1/d2). These sources therefore pose a very low exposure
risk to the healthcare professional working with them. Most institutions utilize
preloaded 18 gauge 20 cm needles to perform interstitial implant of the prostat
e. The radioactive sources (seeds) are placed into the needles prior to depositi
on in the patients prostate. The procedure is very well-tolerated and descri
ed i
n detail elsewhere in this
ook. It should
e noted that use of a Mick applicato
r has dramatically lower exposure risk, at least initially, since the needles us
ed with this technique do not contain seeds at the time of needle placement. It
is only later that the actual seeds are deposited from the Mick applicator into
the needles that are already within the prostate. Technique Using the preloaded
technique, needles are inserted through a perineal template that is attached to
the ultrasound device. The template has (x and y) coordinates to assist in accur
ate placement of the needles. Ideally, the needle tip will meet the designated c
oordinate on the ultrasound image. However, this is not always the case and the
needle tip image may
e off target. The implant needle at this time can
e redir
ected intracorporeally using the
evel at the tip of the needle. Then again, the

evel may not
e successful in redirecting the needle to the desired coordinate
, and additional maneuvers must
e undertaken. It is not infrequent that extraco
rporeal needle manipulation is required to direct the needle to its desired x- a
nd y-axis coordinates. Frequently, the
rachytherapist will redirect the needle

y use of the index finger placed
etween the perineum and the template. Pressur
e exerted on the needle
y the index finger in the
Basic and advanced techniques in prostate
rachytherapy
364
desired direction will usually result in proper placement of the preloaded needl
e resulting in su
sequent seed deposition. Although overall radiation exposure d
uring this procedure is minimal, the radiation exposure on the surface of the pr
eloaded needle is not negligi
le (Ta
le 26.1). However, sporadic exposure to rad
ioactive 125I and 103Pd in the setting of prostate
rachytherapy is pro
a
ly
Ta
le 26.1 Exposure rates for 103Pd and 125I at the needle surface
Pd (1.4 mCi/seed) (U/seed) Needle surface (mR/h)
103
I (0.333 mCi/seed) Needle surface (mR/h)
125
No. seeds/needle
3 22 14.1 4 29 21 5 36 23 6 40 27 Exposure rates of the needles containing 36 see
ds per needle were measured on the needle sjurface at the center of the active l
ength using a Victoreen 450B Dosimeter Survey Meter.
negligi
le. As this procedure gains acceptance and the volume increases, one sho
uld practice good radiation safety in accordance with ALARA (as low as reasona
l
y achieva
le). At our institution, with the large volume of prostate
rachythera
py
eing performed using the preloaded needle technique, we developed the needle
manipulation ruler. As one can see from Ta
le 26.1, the needles themselves, whe

n containing radioactive isotopes, exhi
it significant radiation exposure at the

surface of the needle and up to a distance of 6 cm. Therefore, if the
rachythe
rapists hands or fingers come within a 6 cm radius of the needle, he or she will

e exposed to radiation; o
viously, such exposure is undesira
le. In the past, a
1520 cm stainless steel ruler was used to assess the depth of needle insertion i
nto the prostate gland. This was a useful aid in exact implantation of the prost
ate. To control the direction of insertion of the needle once the needle is insi
de the patient, surgeons used their fingers to guide and manipulate the needle (
Figure 26.1). However, significant exposure to radiation can result to the finge
rs and hands of the
rachytherapist while touching the needle, which houses the
radioactive seeds prior to deposition in the prostate gland. The needle manipula
tion ruler descri
ed here is a medical device for use in
rachytherapy. It minim
izes the
rachytherapists exposure to radiation while still allowing the surgeon
to properly guide and manipulate the needle inside the patient (Figure 26.2). Wi
th a series of needle engagers contained on the end of the ruler, it is possi
le
to manipulate the needle into its proper location without physically touching t
he needle with fingers or hands. The needle engagers are shaped and positioned t
o allow the needle to
e adjusted in any direction from any position relative to
the manipulation ruler. The advantage of using the needle manipulation ruler is
that radiation exposure is
Using the needle manipulation ruler
365
Figure 26.1 Finger used to guide the needle.
Figure 26.2 The needle manipulation ruler. Ta
le 26.2 Exposure rates for 103P an
d 125I at a distance of 12cm
103
No. of seeds/needle
Pd (1.4 mCi/seed) (U/seed) 12 cm from needle surface (mR/h)
I (0.333 mCi/seed) 1 2 cm from needle surface (mR/h)
125
3 2.3 0.71 4 2.6 1.3 5 3.4 2.0 6 4.0 2.2 Exposure rates of the needles containin
g 36 seeds per needle were measured on the needle surface at the center of the ac
tive length using a Victoreen 450B Dosimeter Survey Meter.
greatly reduced when compared to using the finger to adjust the ruler (Ta
le 26.
2). By integrating a ruler into the needle manipulator, the surgeon has a single
device that can
e held in one hand and used to safely manipulate the needle an
d measure the depth of insertion of the needle into the prostate gland rather th
an having to work with two separate devices. The needle manipulation ruler is 15
cm in length and is composed entirely of stainless steel. There are three direc
tional notches which act as needle engagers located at the distal end. These eng
agers allow the
rachytherapist to direct the needle within the
Basic and advanced techniques in prostate
rachytherapy
366
patient. This occurs as the result of pressure exerted on the needle in a specif
ic direction to the desired coordinate. The contact point
etween the surface of
the needle and the edge of the stainless steel notch is quite small, measuring
less than 1 mm. This is a dramatic difference compared to the surface contact wh

en the index finger directs an implant needle. We have found this device to
e i
nvalua
le
ecause it allows a 10-fold reduction in radiation exposure, while all
owing very accurate placement of the needles used during the implant.
Figure 26.3 Perineal spacing.
Figure 26.4 Incorrect use of the needle manipulation ruler.
Since the needle manipulation ruler is very thin, it can
e utilized within a mi
nimal space a few millimeters
etween the perineum and the template. This is in
stark contrast to the amount of space required, usually a minimum of 25 mm (1 in
ch), to use the index finger for extracorporeal redirection of needles (Figure 2
6.3). While the manipulation component of this device is very practical and effe
ctive, there is a proper way to use it. One of the common errors is just placing
the device on the needle and applying pressure (Figure 26.4). This will result
in a
ent needle as
oth the template and the perineum are securing each end of
the needle. The ruler should never
e
Using the needle manipulation ruler
367
applied to the needle unless it is firmly placed against the perineum. The corre
ct amount of presFigure 26.5 The correct use of the needle manipulation ruler.
Figure 26.6 (a and
) Measuring with the needle manipulation ruler.
Basic and advanced techniques in prostate
rachytherapy
368
sure to direct the needle within the patient is surprisingly small (Figure 26.5)
. The ruler component of this device is utilized to ensure that the appropriate
depth of needle insertion has occurred into the patients prostate and equally imp
ortant, that the trocar has
een properly positioned and secured prior to needle
withdrawal and su
sequent seed deposition (Figure 26.6). Conclusion The needle
manipulation ruler allows one to have minimal exposure during prostate
rachythe
rapy using a preloaded needle technique while assisting real-time quality assura
nce that the z-axis orientation of seed deposition is occurring as planned in th
e preimplant evaluation.
27 Using the perineal pressure applicator device
Brian J Moran Introduction Hematomas are not uncommon sequelae of skin needle pu
ncture sites. Not infrequently, hematomas are noted at intravenous sites or just
after
lood specimen draws. The prostate is surrounded
y venous plexi and the
perineum has a rich
lood supply. These anatomic areas are necessarily violated
for the purpose of prostate
rachytherapy. Unfortunately to date, techniques of
prostate
rachytherapy have not evolved to avoid this side effect and most likel
y never will. Patients with perineal hematoma frequently complain of pain while
sitting on hard surfaces. Bruising and discoloration may accompany a perineal he
matoma, particularly on the perineum, scrotum, and medial aspects of the thighs.
While
ruising may take many weeks to resolve, a true perineal hematoma can tak
e many months to resolve. Therefore, developing a perineal hematoma should
e re
garded as a real possi
ility when counseling patients prior to the actual proced
ure. While hematoma is a possi
ility, it may
e limited or even preventa
le if t
opical pressure is applied to the needle puncture sites immediately after the pr
ostate
rachytherapy procedure. This is usually accomplished
y applying pressur
e on a sterile towel to the perineum. However, radiation exposure is not negligi

le to the operators hand when placing the hand in such close proximity to the ra

diation emitted from the iodine-125/palladium-103 prostate implant. Naturally, i

f a high dose rate remote afterloader technique is utilized, the perineum is not
radioactive and therefore there is no hazard
y applying direct pressure to the
needle sites on the needle removal. At our institution, it is our policy to imp
lant the prostate with the perineal template snug to the patients perineum. This
minimizes su
cutaneous
leeding and su
sequent perineal hematoma during the actu
al procedure. It has
een our experience that patients who exhi
it
right red
l
ood draining through the perineal template needle holes are usually at increased
risk for developing perineal hematoma (Figures 27.1 and 27.2). As a policy, we
apply direct pressure to the perineum upon removal of the template. Initially, t
his was done
y hand on a sterile towel (Figure 27.3)
ut now we use a modified
device to apply pressure.
Basic and advanced techniques in prostate
rachytherapy
370
Figure 27.1 Blood draining through the perineal template.
Figure 27.2 Blood draining through the perineum.
This modified device has
een named the perineal pressure applicator device (PPAD)
(Figure 27.4). The PPAD has a soft, flat ru

er surface, 12.5 cm in diameter at
tached
Using the perineal pressure applicator device
371
Figure 27.3 Perineal pressure using hand and sterile towel.
Figure 27.4 The perineal pressure applicator device (PPAD).
to a 50 cm acrylic
roomstick handle. The head portion of the plunger has
een f
illed with a soft foam material that is mallea
le. Furthermore, the flat end of
the head has a 3 mm lead surface. The flat, ru

er surface of the PPAD is placed
against a folded, sterile towel over the needle puncture sites immediately on c
ompletion of the implant prior to cystoscopy (Figure 27.5). This design takes in
to account the three principles of minimizing radiation exposure: time, distance
, and shielding. This philosophy is consistent with ALARA (as low as reasona
ly
achieva
le) principles. The study To
etter understand the actual impact this de
vice has on minimizing radiation exposure to the operators hand, we designed a st
udy. Ten consecutive 125I and ten consecutive
Basic and advanced techniques in prostate
rachytherapy
372
Figure 27.5 The perineal pressure applicator device (PPAD) in use following impl
ant procedure.
Figure 27.6 Resolution of hematoma following use of the perineal pressure applic
ator device (PPAD).
103
Pd prostate implant patients were evaluated in order to discover radiation expos
ure rates at the perineal surface and at a perpendicular distance of 50 cm. The
heights and weights of all patients were recorded as well as the total num
er of
seeds/implant, activity/seed, and total activity of each implant. On completion
of the implant, pressure was applied to the perineum using the handle of the PP
AD for 5 minutes. In each case, exposure rate measurements were o
tained with a
Victoreen 450B Dosimeter Survey Meter. The exposure rate at the center of the ne

edle puncture field on the perineum was measured. Exposure rate was then o
taine
d at the handle of the PPAD, 50 cm from the perineal surface. A right angle was
employed to ensure a perpendicular orientation of the PPAD handle to the perinea
l exposure rate site of measurement. All rate
Using the perineal pressure applicator device
373
Ta
le 27.1 Exposure rates with and without the use of the PPAD for
oth 125I and
103Pd
Isotope
I125 Pd103
No. Av. patients height (in)
10 10 70.3 68.7
Av. weight (I
s)
202.9 199.8
Av. no. seeds/ implant
104.5 97
A/seed A total Perineum PPAD (50 (mCi) (mCi) (mR/h) cm) mR/h
0.333 1.28 34.8 123.66 16.57 9.72 0.52 0.015
measurements were o
tained for a minimum duration of 1 minute. Our results showe
d that of the 20 patients evaluated in this analysis, none developed perineal he
matoma within 10 days after implant (Figure 27.6). There was a profound reductio
n in exposure rates o
tained at the handle of the PPAD compared to those at the
perineal surface (Ta
le 27.1). Conclusion The perineal pressure applicator devic
e (PPAD) is a simple way to reduce the incidence of perineal hematoma after tran
sperineal prostate
rachytherapy and greatly reduces radiation exposure to the o
perators hand
y a factor of 32 for 125I and 648 for 103Pd.
28 Permanent prostate
rachytherapy using sources em
edded in a
sor
a
le vicryl
suture and a preplanned, preloaded needle technique
W Ro
ert Lee and Brian J Davis Introduction The history of prostate
rachytherap
y extends nearly 100 years,
eginning in the early years of the 20th century in
Paris. Enthusiasm for the procedure waxed and waned over the years. A num
er of
renowned urologists contri
uted to the history of prostate
rachytherapy includi
ng Hugh Hampton Young, Benjamin Barringer, and Willett Whitmore.13 The technique
has evolved from an intraurethral approach using radium capsules to an interstit
ial approach relying on radon capsules to an open procedure returning
ack to a
closed transperineal approach. The present widespread acceptance of permanent pr
ostate
rachytherapy is the result of prostate screening and improved technology
that currently allows for an outpatient procedure that generally can
e accompl
ished in one to two hours.4 Coincident with the evolution of various techniques,
the sources used in the procedure have evolved. In the first application of
ra
chytherapy to prostate cancer, radium capsules were placed intraurethrally.2 Hug
h Hampton Young would develop his own intracavitary technique
ut it was Benjami
n Barringer at Memorial Hospital in New York that pioneered the interstitial app
roach.1 Barringers technique relied on the use of radon capsules. These capsules
were far smaller than the radium capsules and allowed for easier placement. The
next advance in radioactive source evolution was the a
ility to produce encapsul
ated iodine-125 (125I) sources on a large scale. Early 125I source designs posse
ssed a single radiopaque marker and the 125I was adsor
ed on two
eads with ion
exchange resin.5 Su
sequent source modification adsor
ed the radioactive 125I to
the surface of a silver wire which resulted in increased radiopacity and more c
onsistent cali
ration. Palladium-103 (103Pd) sources were introduced into clinic

al practice in the late 1980s. In the mid 1970s investigators
egan to experimen
t with placing radioactive sources into suture material.68 The early reports desc
ri
e a num
er of different techniques to insert sources in suture material. Thes
e early investigators
elieved that sources em
edded in suture would result in:
(1) even distri
ution of sources; (2) easy and rapid placement; and (3) reductio
n of source migration. Sources in suture were used to treat a variety of neoplas
ms including prostate, head and neck, lung, and gynecologic cancers.68 In January
1995, the US Food and Drug Administration (FDA) approved the use of a new medic
al device, the RAPIDStrand (Amersham Health). This device consisted of
Permanent prostate
rachytherapy
125
375
I sources em
edded in stiffened, a
sor
a
le suture. The primary use for this dev
ice is the treatment of prostate cancer
ut other uses have
een descri
ed.9 At
the time of pu
lication a num
er of stranded source devices have
een approved

y the FDA, each with different polymer material and as yet unreported rates of a

sorption. RAPIDStrand is the only stranded product with years of clinical experi
ence and results that are descri
ed in the peer-reviewed pu
lished literature. F
or that reason, this chapter will focus on RAPIDStrand, in particular, its use wi
th a preplanned, preloaded needle technique. For this chapter, RAPIDStrand will a
lso
e referred to as stranded sources or sources em
edded in suture (SES). Descript
ion of RAPIDStrand RAPIDStrand (Amersham Health) consists of 10 Model 6711 OncoSee
ds (Amersham Health) spaced at a fixed distance of 1 cm em
edded within polyglact
in 910 a
sor
a
le suture (Medi-Physics RAPIDStrand product information sheet). T
he sources are spaced 1 cm from center to center as shown in Fig 28.1. The usual
activity ranges
Figure 28.1 Photograph of RAPIDStrand showing three seeds each 1 cm apart.
from 0.191 mCi to 0.673 mCi/seed. All sources within each strand are within the
same activity range. The suture material is stiffened and su
sequently sterilize
d
y ethylene oxide. The stiffened suture material holds the sources in place to
minimize source movement and optimize the intended radiation dose distri
ution.
Studies of intramuscular implantation in rats show that non-stiffened suture ma
terial containing 125I sources is minimally a
sor
ed until a
out postoperative d
ay 40
ut a
sorption is essentially complete
etween 60 and 90 days postoperativ
ely. Potential advantages of stranded sources Advantages in source preparation R
APIDStrand is shipped as sequences of 10 sources in a single suture. This arrange
ment allows the potential for a decrease in needle loading time and reduced erro
rs in source and spacer count. To the authors knowledge, no pu
lished papers have
Basic and advanced techniques in prostate
rachytherapy
376
explicitly examined needle loading time with loose sources versus sources em
edd
ed in vicryl suture. Practitioners with clinical experience using
oth loose sou
rces and sources in vicryl suture confirm the o
servation that needle loading ti
me is less with sources in vicryl suture as compared to loose sources. Loading o
f loose sources requires loading each source and spacer individually, whereas lo
ading of sources in vicryl suture only requires cutting of the strands to the re
quired lengths. The strands themselves contain the desired num
er of sources and
spacers and thus only one contiguous o
ject needs to
e loaded in the needle. R
educed source migration Early investigators using the transperineal technique fo
und that the closed approach resulted in
etter dosimetry
ut source migration c
ontinued to
e a pro
lem.10 In particular, Kumar noted that loose sources tended
to migrate
y way of three mechanisms: (1) sources could
e pulled down along t
he needle track owing to suction created
y the needle; (2) peripheral sources o

ccasionally migrated cephalad via the venous system into the pelvic veins; and (
3) in patients treated with transurethral resection of the prostate (TURP), sour
ces would often
e lost
y way of the urethra. He developed a method to fix iodi
ne sources in vicryl carriers. In the first 14 patients he treated he o
served no
source migration. The pu
lished rates of loose source migration to the pelvis or
lungs range from 6% to 55%, with most series reporting migration rates of appro
ximately 20%.1115 The largest series examining the incidence of source migration
comes from investigators at the Seattle Prostate Institute.16 In this report pre
sented at the Sixth Annual Advanced Brachytherapy Conference (Seattle, April 200
3), the rates of source migration for loose sources 103Pd and RAPIDStrand sources
were compared in more than 1000 patients. The rate of source migration to the l
ungs was significantly less with stranded sources compared to loose sources (23%
vs 2%, p<0.0001). The only other report examining the rate of migration accordi
ng to source type (loose vs stranded) also found that migration was significantl
y less for sources em
edded in suture compared to loose sources (0.7% vs 11%, p=
0.0002).14 Although these patients were not randomized, the unique source delive
ry system makes it likely that this o
servation is consistent with clinical real
ity. To date, no clinical consequences of source migration to the lungs has
een
reported, although at least one case of pro
a
le radiationinduced rectal cancer
has
een reported.17 Furthermore, source migration to the myocardium and corona
ry arteries has
een o
served.18,19 Improved dosimetry with sources em
edded in
suture There is at least a theoretical argument that the fixed relationship of t
he sources in vicryl suture will result in a reduction in spacing errors leading
to improved postimplantation dosimetry.20 In a large report examining the spati
al distri
ution of dose within the prostate gland, the region of the prostate mo
st likely to receive a dose
elow the prescription dose is the anterior
ase.21
Anecdotal experience indicates that the sources most likely to migrate are those
placed on the periphery of the prostate or those placed anteriorly (near the do
rsal venous plexus). Given that the use of stranded sources results
Permanent prostate
rachytherapy
377
in: (1) lower rates of source migration; and (2) perhaps more consistent source
placement
y decreasing spacing errors, it is plausi
le that the use of stranded
sources may
e associated with improved postimplant dosimetry.
Ta
le 28.1 Dosimetric quantifiers for the entire study population stratified
y
source type
Varia
le Entire group (n=40) LS(n=20) SES (n=20) p-value*
Mean (SD)V100 90.3 (5.1) 86.5 (3.7) 94.1 (2.9) <0.0001 Mean (SD)V90 93.5 (4.2) 9
0.4 (3.2) 96.6 (2.2.) <0.0001 Mean (SD)V80 96.3(3) 94.1 (2.6) 98.5 (1.3) <0.0001
Mean (SO) D90 148 (22) Gy 132 (11) Gy 164 (17) Gy <0.0001 D90> 140 Gy 27/40 (67
%) 7/20 (35%) 20/20 (100%) <0.0001 LS, loose sources; SES, sources em
edded in s
uture; SD, standard deviation. *p-value is two-sided; LS vs SES. Modified with p
ermission from Lee et al Radiother Oncol 2002; 65(2):123127.22
Lee and others at Wake Forest University have examined a small group of patients
to determine if the use of sources em
edded in suture (SES) is associated with
improved postimplant dosimetry when compared to the use of loose sources.22 Thes
e clinicians had
een performing prostate implants for approximately three years
using exclusively loose sources. In May 2000, sources em
edded in suture were i
ncorporated into the treatment procedure. In this paper the investigators compar
ed postimplant dosimetric quantifiers of the first 20 patients treated with SES
to the last 20 patients treated with loose sources. The patient and treatment ch
aracteristics of the two groups were not different, with the exception that the
mean prostate volume of the patients treated with SES was slightly smaller (33.7
4 vs 39.55, p=0.0474). The dosimetric quantifiers for each group are listed in T
a
le 28.1. It is clear that the V100 and D90 are superior in the group treated w

ith SES. Fagundes has reported a similar improvement in postimplant dosimetry wi

th the use of SES. In work presented at the American Brachytherapy Society (ABS)
meeting in 2003, Fagundes reported on 473 patients treated with loose sources a
nd Mick applicator (n=337) or a novel afterloading approach using sources em
edd
ed in suture. The calculated V100 was superior for the patients treated with SES
(92.5% vs 88.4%, p<0.005). This improvement was achieved without an increase in
acute urinary mor
idity. Given the retrospective nature of the comparisons, the
result can only
e considered hypothesis generating and definitive statements r
equire a randomized trial. Such trials are ongoing.23 Improved ultrasound imagin
g with stranded sources Recent advances in treatment planning systems have led m
any investigators to develop techniques that incorporate some type of dosimetric
feed
ack during the procedure in the hope of improving postimplant dosimetric o
utcome and reducing mor
idity.2426 To date, most intraoperative treatment plannin
g has relied on the location of the needles rather than the individual sources.
Dosimetrically accurate intraoperative treatment planning will require accurate
source localization using ultrasound.
Basic and advanced techniques in prostate
rachytherapy
378
In a study
y Davis et al, comparing detecta
ility rates of loose sources in viv
o
y computed tomography (CT), magnetic resonance imaging (MRI), or transrectal
ultrasound (TRUS), it was determined that interpretation of TRUS images leads to
detection of only an average of 50% (95% CI: 4057.7%) of loose sources after the
y are deposited.27 In another study, investigating TRUS as a means of source det
ection, TRUS source detection was increased to 62% with advanced image-processin
g techniques.28 Although a num
er of investigators are examining methods of ultr
asound (US)-
ased source identification for intraoperative applications, the sho
rtcomings of conventional TRUS in identifying loose sources remains an impedimen
t towards achieving convenient US
ased intraoperative dosimetry. In this regard,
sources in vicryl suture may offer improved US imaging in vivo for two reasons.
In the first place, the vicryl suture material itself provides a surface from w
hich ultrasound reflects and produces an image. Second, the vicryl strands are m
ore likely to keep sources oriented in a parallel manner such that acoustic
ack
scatter from the sources and suture to the TRUS pro
e is more pronounced than ot
herwise. As such, it can reasona
ly
e expected that increased acoustic
ackscat
ter yields a more readily detected signal and, ultimately, a higher source detec
ta
ility rate as compared to loose sources. Quantitative data on imaging of sour
ces in vicryl suture material relative to standard loose sources are provided
e
low. Quantitative data regarding source and strand orientation as compared to lo
ose sources are not availa
le, although it is clear from clinical o
servations t
hat sources in strand appear more parallel under fluoroscopic imaging than do ty
pical loose source implants. Examination of fluoroscopic images of the seed dist
ri
ution following
rachytherapy reveals that seeds do not
Ta
le 28.2 Comparison of ultrasound
ackscatter signal strength
etween seeds in
vicryl suture material and standard loose seeds in deci
els (dB) as a function
of angle of seed orientation. Data for two standard TRUS
rachytherapy operating
frequencies, 5 MHz and 7.5 MHz, are provided
Seed orientation (degrees)
5 MHz
0
5
10
20

30
40
50
60
70

2.30 1.94 3.35 4.13 3.63 0.63 1.13 9.39 11.18 1.09 3.59 7.52 7.36 5.58 2.86 1.27 3.12
.14 7.5 MHz 0.48 1.93 6.71 5.91 4.76 1.29 2.73 7.83 12.6 0.38 3.59 10.13 7.72 7.27 3
.12 0.99 5.04 10.05 See orientation is given relative to the transucer; zero egr
ee orientation correspons to see position that is absolutely perpenicular to
the incient US beam. The range of values is the upper an lower stanar eviat
ions. The shae areas represent the see orientations where sees in suture mat
erial prouce greater acoustic backscatter than loose sees. (Aapte from Davis
et al.30)
necessarily line up completely parallel, but rather may assume various nonparal
lel orientations. In a stuy by Leif et al,29 it was emonstrate that the orien
tation of loose sees varies with respect to the TRUS probe an to one another f
requently up to 40 an
Permanent prostate brachytherapy
379
that a 10 variation in orientation is routine. Consequently, the USimaging prope
rties of sources as a function of their orientation is relevant. The acousticim
aging properties of sources in vicryl suture as compare to loose stanar sourc
es have been measure as a function of angle of orientation of the US beam relat
ive to source alignment (Table 28.2).30 Ultrasoun backscatter from loose source
s an sources in vicryl suture was measure from two parallel rows of 10 sources
with ultrasoun transucers operating at 5 MHz an 7.5 MHz. The angle of incie
nce of the scanning US beam was varie from perpenicular to 70 away from the per
penicular with 5 MHz an 7.5 MHz transucers. Ultrasoun images were igitize
with 12bit pixel resolution with pixel imension of 0.2 mm2. The signal was ana
lyze using the metho of integrate optical ensity (IOD) as a measure of the o
verall magnitue of the signal an, consequently, relates to source etectabilit
y.31 The results, given as a ecibel ratio of signal from sources in vicryl sutu
re to stanar sources, are that the sources in vicryl suture provie an increas
e backscatter signal from 4 to 45 incience away from the perpenicular. A mean o
f 3.6 B an maximum of 7.9 B was foun at 5.0 MHz, an a mean of 4.5 an maxim
um of 10.1 B was foun at 7.5 MHz over this range of angular orientations. To p
ut this in perspective, a typical TRUSimaging system has a ynamic range of 50
B at an imaging plane of epth, often referre to as the local ynamic range. The
refore, the improvement in ultrasoun backscattere signal strength of 3.6 B to
10.1 B over this clinically relevant range of see orientations represents a m
eaningful fraction of the physical capacity of the imaging system. Differences i
n the backscattere signal between the sources in the absorbable suture material
an loose sources are expecte because the suture material reflects an absorbs
ultra soun. The acoustic interaction between the sources embee in vicryl su
ture an the suture material itself are complex an involve a combination of ref
lection, refraction, scatter, an absorption. Measurements of relative acoustic
backscatter beyon 45 incience emonstrate that the sources in vicryl suture ma
terial prouce less backscattere signal than stanar sources. In those uncommo
n circumstances where the long axis of the sources in vicryl suture are oriente
at greater than 45 away from an incient ultrasoun beam, the vicryl suture mate

rial may serve to iminish the reflecte signal in a manner greater than if the
sources were loose. Representative images of five stanar sees an 5 SES at 5
MHz imaging frequency an angles of incience from 0 (perpenicular) to 50 away fr
om the perpenicular are shown in Figure 28.2. Of further note, ultrasoun scatt
er an absorption are physical interactions which are generally more frequency
epenent than reflection or refraction, so the small ifferences in backscatter
signal strength as a function of angle between 5 MHz an 7.5 MHz are not unexpec
te. Nevertheless, these ata strongly suggest that sees in vicryl suture will
provie improve ultrasoun imaging in vivo because of the ultrasoun imaging pr
operties of the strans an sees acting in concert an the more parallel an un
iform see orientation exhibite by strane sees as compare to loose sees. S
ummary Raioactive sources embee in suture have been use for nearly three e
caes in the treatment of prostate, hea an neck cancer, an gynecologic cancer
. Avantages for the
Basic an avance techniques in prostate brachytherapy
380
use of embee sources inclue: ecrease source migration, improve osimetric
outcome, an improve ultrasoun visualization. Refinements in source esign ov
er the past century have le to significant improvements in prostate brachythera
py.
Figure 28.2 Ultrasoun images of stanar sees compare to sees embee in su
ture material as a function of angle of orientation. The six panels show 5 stan
ar sees in the top row an 5 sees embee in suture material in the bottom r
ow of each panel. Zero egree orientation correspons to perpenicular incience
of the ultrasoun beam. Images taken at 5 MHz.
References
1. Aronowitz JN. Benjamin Barringer: originator of the transperineal prostate im
plant. Urology 2002; 60(4):731734. 2. Aronowitz JN. Dawn of prostate brachytherap
y: 19151930. Int J Raiat Oncol Biol Phys 2002; 54(3):712718. 3. Whitmore WF Jr, H
ilaris B, rabstal H. Retropubic implantation of ioine 125 in the treatment of
prostatic cancer. 1972. J Urol 2002; 167(2 Pt 2):981983. 4. Blasko JC, Mate T, S
ylvester JE, et al. Brachytherapy for carcinoma of the prostate: techniques, pat
ient selection, an clinical outcomes. Semin Raiat Oncol 2002; 12(1):8194. 5. Li
ng CC, Yorke ED, Spiro IJ, et al. Physical osimetry of 125I sees of a new esi
gn for interstitial implant. Int J Raiat Oncol Biol Phys 1983;9(11):17471752.
Permanent prostate brachytherapy
381
6. Martinez A, offinet DR, Palos B, et al. Sterilization of 125 I sees encase
in vicryl sutures for permanent interstitial implantation. Int J Raiat Oncol B
iol Phys 1979; 5(3):411413. 7. Beach JL, Meniono OA. A simple evice for loain
g raioactive sees into absorbable sutures. Raiology 1983; 146(3):842843. 8. Pa
los BB, Pooler D, offinet DR, Martinez A. A metho for inserting I125 sees in
to absorbable sutures for permanent implantation in tissue. Int J Raiat Oncol B
iol Phys 1980; 6(3):381386. 9. Rogers CL, Theoore N, Dickman CA, et al. Surgery
an permanent 125I see paraspinal brachytherapy for malignant tumors with spina
l cor compression. Int J Raiat Oncol Biol Phys 2002; 54(2):505513. 10. Kumar PP
, oo RR. Vicryl carrier for I125 sees: percutaneous transperineal insertion.
Raiology 1986; 159(1):276. 11. Nag S, Vivekananam S, MartinezMonge R. Pulmon
ary embolization of permanently implante raioactive pallaium103 sees for ca
rcinoma of the prostate. Int J Raiat Oncol Biol Phys 1997; 39(3):667670. 12. Ank
em MK, DeCarvalho VS, Harangozo AM, et al. Implications of raioactive see migr
ation to the lungs after prostate brachytherapy. Urology 2002; 59(4):555559. 13.
Merrick S, Butler WM, Dorsey AT, et al. See fixity in the prostate/periprostat

ic region following brachytherapy. Int J Raiat Oncol Biol Phys 2000; 46(1):21522
0. 14. Tapen EM, Blasko JC, rimm PD, et al. Reuction of raioactive see embol
ization to the lung following prostate brachytherapy. Int J Raiat Oncol Biol Ph
ys 1998; 42(5): 10631067. 15. Oler RA, Syner B, Krupski TL, et al. Raioactive
implant migration in patients treate for localize prostate cancer with interst
itial brachytherapy. J Urol 2001; 165(5):15901592. 16. rimm PD, Blasko JC, Sylve
ster JE. See migration: Linke versus loose sees. Sixth Annual Prostate Brachy
therapy Conference, Seattle, WA, April 2003. 17. Yurakul , e Reijke TM, Blank
LE, Rauws EA. Rectal squamous cell carcinoma 11 years after brachytherapy for c
arcinoma of the prostate. J Urol 2003; 169(1):280. 18. Davis BJ, Bresnahan JF, S
taffor SL, et al. Prostate brachytherapy see migration to a coronary artery fo
un uring angiography. J Urol 2002; 168(3):1103. 19. Davis BJ, Pfeifer EA, Wils
on TM, et al. Prostate brachytherapy see migration to the right ventricle foun
at autopsy following acute cariac ysrhythmia. J Urol 2000; 164(5):1661. 20. R
oberson PL, Narayana V, McShan DL, et al. Source placement error for permanent i
mplant of the prostate. Me Phys 1997; 24(2):251257. 21. Sihu S, Morris WJ, Spa
inger I, et al. Prostate brachytherapy postimplant osimetry: a comparison of pr
ostate quarants. Int J Raiat Oncol Biol Phys 2002; 52(2):544552. 22. Lee WR, e
uzman AF, Tomlinson SK, McCullough DL. Raioactive sources embee in suture
are associate with improve postimplant osimetry in men treate with prostate
brachytherapy. Raiother Oncol 2002; 65(2): 123127. 23. Fagunes HM, Keys RJ, Woj
ick MF, et al. Searching for a better prostate see implant: a new rapi stran
afterloaing technique. Presente at American Brachytherapy Society Meeting, Ma
y 2003. 24. Nag S, Ciezki JP, Cormack R, et al. Intraoperative planning an eval
uation of permanent prostate brachytherapy: report of the American Brachytherapy
Society. Int J Raiat Oncol Biol Phys 2001; 51(5):14221430. 25. Stone NN, Stock
R. Brachytherapy for prostate cancer: realtime threeimensional interactive s
ee implantation. Tech Urol 1995; 1(2):7280. 26. Zelefsky MJ, Yamaa Y, Marion C,
et al. Improve conformality an ecrease toxicity with intraoperative compute
roptimize transperineal ultrasounguie prostate brachytherapy. Int J Raiat
Oncol Biol Phys 2003; 55(4):956963. 27. Davis BJ, LaJoie WN, Mcee KP, et al. Se
e ientification rates as a function of imaging moality in permanent prostate
brachytherapy using CTMRTRUS image fusion. Proceeings of the American Brachyt
herapy Society, Orlano, FL, May 2002:42.
Basic an avance techniques in prostate brachytherapy
382
28. Holmes DR, Davis BJ, Robb RA. 3D localization of implante raioactive sourc
es in the prostate using transurethral ultrasoun. Stu Health Technol Inform 2
001; 81:199205. 29. Leif EP, Davis BJ, Kline RW, et al. Optimal CT slice spacing
for postoperation see localization in permanent interstitial implants. Optimal
CT slice spacing vs. raioactive source orientation for postimplant osimetry an
 quality control in permanent interstitial implants. Presente at the 9th Inter
national Brachytherapy Conference, Boston, MA, 1997:22. 30. Davis BJ, Kinnick RR
, Fatemi M, reenleaf JF. Experimental evaluation of ultrasoun imaging an back
scatter signal amplitue of stanar sees, echogenic sees an sees in suture
material as a function of angle of incience of the ultrasoun beam: application
to permanent prostate brachytherapy. Proceeings of the American Brachytherapy
Society. Orlano, FL, May 2002:60. 31. Oberholzer M, Ostreicher M, Christen H, B
ruhlmann M. Methos in quantitative image analysis. Histochem Cell Biol 1996; 10
5(5):333355.
29 The Utrecht technique in RAPIDStran TM afterloaing
Jan J Battermann, Ina M Schulz, Marinus A Moerlan, an Marijke van Deursen Intr
ouction The RAPIDStran evice (Amersham Health) comprising ioine125 sees emb
ee in a stiff polyglactin suture, has been available since June 1996 in Europ
e, an 1995 in the Unite States. Since 1996, we have use RAPIDStran (stran) f
or our permanent prostate brachytherapy (PPB) proceures, except for patients tr

eate with the Fully Integrate Raiotherapy See Treatment (FIRST) system, whic
h we introuce in January 2002. Our previous technique was using a Mick applica
tor to insert single sees into the prostate. We first positione all the neele
s an then loae each iniviual neele. With RAPIDStran, the neeles can eithe
r be preloae or manually afterloae. At our center in Utrecht, we prefer to p
lace all the neeles first, an use customize stran holers to facilitate the in
sertion of the strans into the prostate. In this chapter we escribe our techni
que. (See also Chapter 32 on the FIRST system, for general information on our in
traoperative treatment proceures. These are similar for strans an single see
s using the automate afterloaing system.) Implantation technique Pretreatment
evaluation an preparation are similar to the FIRST technique. The implant proce
ure begins with the introuction of two stabilization neeles into the prostate
an subsequent threeimensional (3D) imaging acquisition. During contouring, t
he neeles are introuce into the prostate prior to the autoplan. Using osevo
lume histograms (DVH), the team ecies whether extra neeles have to be place
or remove. In aition, the number of sees per neele is establishe, using i
fferential loaing of the more centrally locate neeles to lower the ose to th
e urethra. When the plan is approve, the strans can be prepare an place int
o the stran holers for insertion into the
Basic an avance techniques in prostate brachytherapy
384
Figure 29.1 The prepare stran is put into the stran holer.
prostate. In the preparation room ajacent to our brachytherapy operating room (
OR), strans are cut to lengths of 1, 2, 3, or more sees. This can be one with
a surgical knife or electrocoagulation. Any jamming of the stran in a neele c
an be avoie by electrocoagulating the stran. However, uring the past two yea
rs we have use jamming. The strans are then put into a stran holer both meth
os of cutting an have rarely experience any (Figure 29.1) an place in conta
iners that inicate the length on the strans. The containers are then taken to
the OR. Typically, the loaing is from periphery to central, so the shorter stra
ns are loae into the prostate first. The stran holer is place on the hub o
f the neele, after retraction of the obturator (Figure 29.2). The same obturato
r is use to introuce the stran in the neele. The holer is remove an with
the obturator the stran is place at the tip of the neele. This can be ientif
ie by the marking on the obturator, but is also sense when the stran is gentl
y pushe to the tip of the neele. While retracting the neele over the obturato
r with the stopper (Figure 29.3), the placing of the stran can be
Figure 29.2 The stran holer is place on the hub of the inserte
The Utrecht technique in RAPIDStran
385
neele to facilitate the introuction of the stran.
Figure 29.3 The stopper is place at the en of the obturator an the neele ret
racte.
viewe using sagittal transrectal ultrasoun (TRUS). TRUS can also be use to fi
ne tune the position of each iniviual neele just before loaing with the stra
n. This takes more time, but improves the quality of the implant. Although we u
se peripheral loaing for many years, only with the intraoperative planning sys
tem, we realize the better sparing of urethra an rectum when Using ifferentia
l neele loaing.2 With strans, this is possible by inserting one or two sees
at the prostate base an then retracting the neele for the require istance of
24 cm before placing the remaining see(s). Currently, we usually cut the stran
s in parts with a maximum of 2 or 3 sees. The raiographer can prepare an inven

tory of strans of ifferent lengths beforehan, especially when more than one p
atient is implante that ay. To obtain a stran of 4 or 5 sees, a a stran o
f 2 or 3 to another stran of 2. Jamming is no more of a problem than with a com
plete stran. Amersham Health recently introuce a new stran with thicker space
rs between the sees to further reuce the risk of jamming. Another reason for ha
ving longer strans in two parts is that with swelling of the prostate because o
f the implant proceure, a complete stran is not move in one irection. See m
igration rarely occurs in complete strans an the use of parts of a stran, eve
n with one see, oes not increase the migration rate. Postimplant care an post
planning are the same as the FIRST technique (see Chapter 32). Discussion We hav
e use RAPID Strans for more than seven years an foun our technique (with stra
n holers) very reliable. During this perio, we move from using a nomogram fo
r the calculation of the require number of sees, via preplanning to intraopera
tive
Basic an avance techniques in prostate brachytherapy
386
planning. In using intraoperative planning we have realize that in previous yea
rs the ose to urethra, an especially rectum, might have been too high in a sub
stantial proportion of patients. Inee, we i encounter some patients with rec
tal problems, bleeing, an also some ulceration. Although fewer than 10% of pat
ients experience problems, with intraoperative planning we expect to lower this
figure substantially. Late urethral complications are rare, but we feel that an
y early reaction is less severe with intraoperative planning than in previous ye
ars, incluing the acute urine retention.
Figure 29.4 Xray of sees in strans. Note the neater position of the sees compa
re to those in Figure 33.12.
On plain xrays, the istribution of sees over the prostate area looks neater wit
h strans than with single sees (Figure 29.4).3 This also applies to loose part
s as well as complete strans. However, the irection of each iniviual see ha
s little influence on the quality of the implant. Dosimetry parameters for RAPID
Stran an selectSee implants were similar. Conclusion In our experience, the i
mplant technique is similar for strans an single sees, using the Mick applica
tor or automate afterloaing with FIRST; however, we o not have any experience
with preloae neeles. The total time require for the ifferent techniques se
ems to be more or less the same, but OR time is substantially longer with intrao
perative planning, as is escribe in Chapter 32 on the FIRST system. On the oth
er han, accuracy is certainly improve with intraoperative planning an shoul
lea to better results with respect to postimplant osimetry an biochemical con
trol.
The Utrecht technique in RAPIDStran
387
References
1. Battermann JJ. Ioine125 see implantation for localize prostate cancer. J
Brachyther Int 1998; 14:2127. 2. Zelefsky MJ, Yamaa Y, Marion C, et al. Improve
conformality an ecrease toxicity with intraoperative computeroptimize tran
sperineal ultrasounguie prostate brachymerapy. Int J Raiat Oncol Biol Phys
2003; 55:956963. 3. Lee WR, euzman AF, Tomlinson SK, et al. Raioactive sources
embee in suture are associate with improve postimplant osimetry in men tr
eate with prostate brachytherapy. Raiother Oncol 2002; 65:123127.
30 The PIPER prostate brachytherapy planning system
Yan Yu Introuction PIPER (RTek Meical Systems, Pittsfor, NY) is a commercial
software system for preplanning, intraoperative planning, an postimplant osime

try of permanent prostate brachytherapy. Its esign goals were clearly aime at
realtime implant planning, guiance, an osimetry, incluing: simple, work flo
woriente graphical user interface that can be run by operating room personnel;
encapsulation of avance technologies in image processing, feature recognition
, an practical inverse planning. Figure 30.1 shows the Microsoft Tab layout of th
e PIPER user interface. Each tab activates a unique winow layout in which all o
f the functionalities require uner that step of the proceure can be accesse
without leaving the screen. TRUS/Contouring Tab This screen contains tools for a
cquiring ultrasoun images an contouring various anatomical structures on the i
mages. Image import may be via live vieo (e.g. irect connection to the ultraso
un vieo output), in DICOM or a number of common image formats, such as JPE, B
MP. Autocontouring is a unique technology available in PIPER to assist the user
to rapily efine the prostate an anterior rectal wall in a series of ultrasou
n images. This completely automatic tool is base on a synergistic combination
of sophisticate imageprocessing techniques an a traine knowlege set of the p
rostate morphology using avance mathematical moeling.1 In aition to autocon
touring, rawing an eiting tools are provie in this screen to efine the ure
thra, blaer, pubic arch, neurovascular bunles, an tumor focus. Figure 30.2 i
llustrates the TRUS/Contouring screen. The prostate an anterior rectal wall wer
e rawn automatically using the AutoContour technology, while the urethra, pubi
c arch, neurovascular bunles (estimate), an tumor focus (for illustration onl
y) were rawn manually.
The PIPER prostate brachytherapy planning system
389
Treatment Plan Tab Uner the Treatment Plan Tab, neele/see placement plans an
osimetry can be optimize via PIPERs unique Inverse Planning Engine, or by one
of several geometrically base automatic planning options, such as uniform, moi
fie uniform, an peripheral loaing. Of course, complete manual planning or man
ual eiting of plans is always available to the user. The PIPER enetic Algorith
m Inverse Planning Engine emboies unique esigns to prouce clinically relevant
, optimize neele placement plans in 12 minutes. Successive
Figure 30.1 Tab layout of the PIPERuser interface. Each tab leas to a main scre
en in which all of the functionalities uner a given proceural step can be acce
sse.
Figure 30.2 The TRUS/Contouring screen. The prostate an anterior rectal
Basic an avance techniques in prostate brachytherapy
390
wall were rawn automatically using AutoContour technology. A crosshatch was ac
tivate to illustrate the effect of the pubic arch on elimiting the planning sp
ace.
generations of the Inverse Planning Engine have evolve along with clinically or
iente research an investigation to inclue: specifying neele loaing rules, s
uch as backtoback see loaing at the apex or base; requiring a minimum number
of sees per neele (e.g. to avoi planning with 1seeers); multiobjective optimi
zation taking into account the prescribe ose, ose uniformity, minimizing ose
to the urethra, rectum, an/or neurovascular bunles (if efine); oseescalat
ing to the tumor focus (if efine); onthefly sensitivity analysis to assess t
he osimetric impact of a given mean isplacement of the sees uring optimizati
on; specifying a esire range of neeles use. The en result is an automate 
osimetryplanning system that generates practical, clinically acceptable implant
plans in a 12 minute running time. Figure 30.3 shows isoose istribution an a
osevolume histogram (DVH) of a sample osimetry plan generate uner the Treatm

ent Plan Tab. In aition to the typical ose conformity to the target volume an
 sparing of the critical structures, the Inverse Planning Engine also prouces
an islan of escalate ose aroun the tumor focus (posterolateral aspect) autom
atically. OR Support Tab The preplan or intraoperative plan, electronically sign
e by the treating physician from the previous step, is use as the starting poi
nt in the OR Support Tab, from which the neele/see placement positions can be
refine, ajuste, reoptimize, or built up incrementally as the implant procee
s, that is, the OR Support Tab is an environment for ynamic implant tracking an
 realtime osimetry (see Figure 30.4). The OR Support Tab consists of a main 
isplay winow, which can be toggle between live vieo, isoose isplay, 3D, an
DVH views. In aition, isoose isplay is always available in thumbnail images
on the screen. All of these isplays are upate in realtime uring the brachy
therapy proceure. The neele placement plan is tabulate in the Interactive Pla
nning Worksheet, which can be sorte into one of several customary orers. This
worksheet contains the neele coorinates, retraction istance, the number of se
es in each neele, an the see patterns. The neele an/or see patterns can b
e moifie, ae or elete, upon which the osimetry will be upate.
The PIPER prostate brachytherapy planning system
391
Figure 30.3 The Treatment Plan Tab. (a) Isoose isplay moe, (b) Dosevolume his
togram (DVH) moe. Note the relative high ose region aroun the tumor focus (fo
r illustration only),
Basic an avance techniques in prostate brachytherapy
392
which was automatically generate by PIPERs Inverse Planning Engine, without sacr
ificing ose an sparing of the urethra an rectum.
Figure 30.4 OR Support Tabs live vieo view.
Neele Tracking is a unique metho available in PIPER to achieve realtime osim
etry verification. It allows the user to use a single click on the live ultrasou
n view to efine the trajectory an eflection of the neele as it is being ins
erte. As the neele tip appears on live transverse ultrasoun as a hyperechoic
spot, the user ientifies its resting location by a single mouse click. PIPER wi
ll then report the lateral an anteroposterior eviations, x and y, and recalculat
e the dosimetry using a deflected needle track
y 3D
ackprojection to the templ
ate coordinates. This practical and clinically relevant design was
ased on actu
al intraoperative experience and requirements. It uses the same ultrasound view
as that during needle insertion, and requires the same needle tip identification
as when the clinician performs visually, therefore there is no extra time added
to the procedure. At the same time, this method of Needle Tracking delivers tru
e image-guided therapy with quantitative feed
ack and dosimetry in real-time. No
te that the a
ove methodology can
e used to examine the dosimetric consequence
of any needle placement error
efore the seeds are actually deposited into tissu
e. If the needle deflection results in unaccepta
le dosimetry, the needle may
e
reinserted, and the new needle tip reidentified,
efore seeds are actually impl
anted. This is another quality assurance tool in PIPER designed to assist the ph
ysician to deliver optimal prostate
rachytherapy.
The PIPER prostate
rachytherapy planning system
393
A running total seed count is automatically updated in the Interactive Planning
Worksheet as each needle is placed. Figure 30.5 is a screen shot of an actual
r

achytherapy implant at the end of the planned needle/seed placements. The prescr
i
ed treatment was a palladium-103
oost of 100 Gy to the prostate. If the treat
ing physician determines that supplemental seed insertions are necessary (e.g. i
n the posterior leftlateral aspect of the prostate in Figure 30.5), they can
e
added to the needle placement plan and the worksheet,
efore the real-time dosim
etry is electronically signed
y the physician. CT Contouring Ta
and a Validati
on Ta
The CT Contouring Ta
contains tools to import DICOM images, manually con
tour the prostate and critical structures, automatically localize all the seeds,
and display isodose distri
utions. The CT Validation Ta
analyses the dose-volu
me histogram (DVH) data, and permits the ultrasound-
ased dosimetry to
e compar
ed to computed tomographic (CT)-
ased postimplant dosimetry. The Find Seeds func
tion is a unique algorithm for automatically localizing all the implanted seeds
in a postimplant CT series. The methodology involves statistical and feature ana
lysis of CT intensities in
oth 2D and 3D. This ensures that redundancies (one s
eed exposed on two or more CT slices) are automatically recognized and assigned
their true
Figure 30.5 OR Support Ta
at the completion of
rachytherapy seed placement.
Basic and advanced techniques in prostate
rachytherapy
394
positions in 3D, that is, in contrast to other common methods of seed eliminatio
n, this algorithm does not require the user to assume that a certain num
er of s
eeds exists in the volume imaged.2 Furthermore, higher seed localization accurac
y can
e achieved with more closely spaced and thinner CT slices without requiri
ng any additional user time. Calcification will
e automatically eliminated rath
er than included as seeds, further ensuring dosimetric accuracy. The postimplant
CT-
ased dosimetry can
e compared with the ultrasound-
ased dosimetry (i.e. OR
real-time dosimetry) in the CT Validation Ta
. This tool provides another oppor
tunity for the
rachytherapy team to achieve Quality Assurance and Improvements.
Summary PIPER is a full-featured
rachytherapy planning system for interstitial
seed implantation of the prostate, designed to support intraoperative planning
and real-time dosimetry as well as conventional preplanning techniques. It conta
ins a num
er of unique, state-of-theart technologies designed
y practitioners f
or practitioners. These technologies make intraoperative planning, guidance, and
dosimetry feasi
le and clinically practical, as well as eliminate much of the t
ime spent
y the planning personnel in the preplanning setting. It will also ser
ve as a mature platform for other exciting technologies in the near future. Refe
rences
1. Liu H, Cheng G, Ru
ens D, et al. Automatic segmentation of prostate
oundarie
s in transrectal ultrasound (TRUS) imaging. Proc SPIE; 4684:412423. 2. Liu H, Che
ng G, Yu Y, et al. Automatic localization of implanted seeds from postimplant CT
images. Phys Med Biol 2003; 48:11911203.
31 Ro
ot-aided and 3D TRUS-guided intraoperative prostate
rachytherapy
Aaron Fenster, Lori Gardi, Zhouping Wei, Gang Wan, Chandima Edirisinghe, and Don
al B Downey Introduction Although current prostate
rachytherapy is widely accep
t-ed, it still suffers from limitations and varia
ility that have limited its fu
ll potential. Progress in overcoming limitations is
eing made in a num
er of la

oratories and companies
y addressing factors such as: The patients anatomy. Pu

ic arch interference (PAI) with the implant path occurs in many patients with la
rge pro-states (>60 cm3) and even in some patients with prostates <40 cm3. These
patients cannot
e treated with conven-tional
rachytherapy with parallel needl
e trajectories guided
y the conventional
rachytherapy template, as the anterio
r and/or the anterolateral parts of the prostate are
locked
y the pu
ic
one.13
In this chapter, we descri
e a ro
ot-aided approach, which removes the paral-le
l needle trajectory constraint of the template, allowing patients with PAI to
e
treated with
rachytherapy without undergoing additional lengthy hormonal thera

py to shrink the prostate. Needle placement accuracy using two-dimensional trans

rectal ultrasound (2D TRUS). Although needles are inserted with real-time 2D TRU
S guidance, lateral deflection is not detected easily due to the poor elevationa
l resolution of conventional 2D ultrasound (i.e. thick TRUS
eam). However, 3D T
RUS imaging provides views of the prostate not availa
le using 2D TRUS, allowing
the trajectory of the needle to
e detected in a coronal section.4,5 In this ch
apter, we descri
e an automated technique to track the needle in 3D and guide it
to the planned location in the prostate. Delay
etween preplan and treatment. T
ypically, a preplan is performed a
out 2 weeks prior to treatment. For a preplan
, 2D transverse TRUS images are o
tained at 5 mm intervals, the prostate contour
is manually traced and the dose plan and seed locations are calculated
ased on
prostate volume and location at that particular time. However, reports have sho
wn that the prostate volume can change during the delay
y as much as 50%.6 Belo
w, we descri
e a technique for rapid acquisition of a 3D TRUS image of the prost
ate, followed immediately
y prostate segmentation and dose planning, allowing p
lanning and treatment in the same session to avoid the pro
lem of volume change.
Anatomical changes during the procedure. The implantation process itself induce
s trauma and causes the prostate to swell due to edema.7,8 Variation of prostate
shape and volume during the procedure will result in misplaced seeds and loss of
proper dose coverage.9 Rapid 3D TRUS imaging, prostate segmentation, and dose pl
anning will allow
Basic and advanced techniques in prostate
rachytherapy
396
dynamic reoptimization of the procedure, intraoperatively, to take into account
changes in the prostate during the procedure.10 Delay
etween treatment and post
plan. Factors, such as prostate motion,
leeding and swelling during implantatio
n,7,8 TRUS imaging artifacts, migration of the seeds in the needle tracks,11 and
needle deflection, contri
ute to errors
etween the preplan and the actual pros
tate dose distri
ution.12 Thus, verification of the actual locations of the seed
s relative to the prostate margin, rectal wall, and
ladder is needed intraopera
tively to allow adjustments to the plan to compensate for potential cold spots. Cu
rrently, TRUS is not used to localize seeds
ecause they are too difficult to id
entify in 2D TRUS images, even with the use of the new echo-seeds. For this reason
, seed location verification is typically done with computed tomography (CT) or
fluoroscopy, which show the seeds well
ut the prostate margins poorly. Since th
is is performed after implantation, intraoperative corrections are not possi
le.
The availa
ility of high quality 3D TRUS images may provide the a
ility to iden
tify the seed locations intraoperatively. Advances in 3D TRUS imaging, ro
otics,
and segmentation approaches allows us to consider performing the procedure intr
aoperatively with dynamic reoptimization and without the constraints of the rect
ilinear template.1317 This type of approach would permit planning and implantatio
n at the same session there
y avoiding pro
lems of repositioning, prostate motio
n, and prostate size/contour changes
etween the preplan and the implantation. T
his will also partially overcome the pro
lem of pu
ic arch interference and perm
it the development of intraoperative postimplant verification, allowing immediat
e corrections of implantation errors. In the following, we descri
e our developm
ents of the tools for use in this type of procedure. Key to these developments i
s the use of 3D TRUS imaging and ro
otic aids. Ro
otic aids in
rachytherapy Red
ucing the effects of pu
ic arch interference (PAI) requires freeing needle inser
tions from parallel trajectory constraints, that is needle trajectories should

e positioned with considera
le flexi
ility, allowing o
lique trajectories. Medic
al ro
otic systems are playing an increasing role in different image-guided surg
ical procedures,18,19 including urology,2022 and CT-guided prostate
rachytherapy
.14 Although they introduce more complex instrumentation and increasing hardware
costs, ro
otic approaches provide significant advantages and cost saving in som
e cases. The key potential advantages of ro
otic aids in
rachytherapy relate to
their a
ility to position, orient, and manipulate needles in 3D space accuratel

y and consistently. These characteristics are important
ecause they help to fre
e parallel needle insertion constraints.23 In addition, they do not get tired, c
an
e dynamically programed and controlled, and can
e effectively integrated wi
th real-time imaging systems. 3D TRUS in
rachytherapy Our introduction of 3D TR
US has alleviated some technical limitations of 2D TRUS
y allowing interactive
viewing of the prostate in three dimensions for treatment planning, providing a
more efficient and reproduci
le delineation of the prostate
oundary in 3D
Ro
ot-aided and 3D TRUS-guided intraoperative
397
for accurate treatment planning, and allowing the development of 3D monitoring o
f needle placement during implantation.5,15,2427 In addition, recent advances in
computer and visualization techniques have allowed real-time reconstruction and
visualization of 3D ultrasound images and their manipulation on inexpensive desk
top computers. These developments, coupled with efficient software tools, have t
he potential to allow the development of a system capa
le of dynamic reoptimizat
ion and intraoperative postplan verification. In the following sections, we desc
ri
e the integration of ro
otic aids and 3D TRUS imaging in the development of a
system for prostate
rachytherapy. In our approach, a one-hole needle guide is
attached to the arm of a ro
ot so that the position and orientation of the needl
e targeting can
e changed as the ro
ot moves. At this phase of the project, we
are using the ro
ot as a dynamically mova
le needle guide, that is, the ro
ot po
sitions the needle,
ut the physician inserts the needle into the patients prosta
te. In a later phase of our work, we will include ro
ot insertion. By integratin
g the coordinate systems of the ro
ot, TRUS transducer, and 3D TRUS image, we ca
n determine the position of the guidance hole in the 3D TRUS image so that the n
eedle can
e guided to target any point identified in the 3D TRUS image. In addi
tion, we also developed a near real-time method for automatic segmentation and t
racking of
rachytherapy needles during o
lique insertion when the needle exits
the 2D TRUS image plane. System description A schematic diagram of our ro
ot-aid
ed and 3D TRUSguided system is shown in Figure 31.1 and a photograph in Figure.
31.2. The system consists of an A465 industrial ro
ot system (Thermo-CRS, Burlin
gton, Ontario, Canada) with 6 degrees of freedom and a 3D TRUS imaging system de
veloped in our la
oratory.15,24 A needle-guide is attached to the ro
ot arm, and
has only one hole to guide the
rachytherapy needle. Since the needleguide is a
ttached to
Figure 31.1 Schematic diagram of the ro
ot-aided and 3D TRUS-guided
rachytherap
y procedure. The use of
Basic and advanced techniques in prostate
rachytherapy
398
the ro
ot will remove the constraints of the rectilinear template, allowing angu
lated needle insertion. The transducer is mounted in a rotational mechanism allo
wing rotation along its long axis for 3D TRUS imaging.
Figure 31.2 A photograph of the ro
ot-aided and 3D TRUS-guided prostate
rachyth
erapy system showing the ultrasound transducer inserted into a phantom and ready
for 3D TRUS imaging.
the ro
ot arm, the position of the hole in the ro
ot coordinate system is known.
A software module transforms the ro
ot coordinate system to the 3D TRUS coordin
ate system allowing the needle-guide hole, and hence the needle trajectory, to

e displayed and coordinated with the 3D TRUS images. In the su
sequent sections,
we descri
e the 3D TRUS acquisition system, prostate segmentation approach, dos
imetry, cali
ration procedure, and evaluation of the errors with the cali
ration
, needle tracking and seed implantation. 3D TRUS imaging system Although our 3D

TRUS system can
e coupled to any ultrasound machine with a sidefiring transduce
r, our results descri
ed
elow were made using a B&K Medical 2102 Hawk ultrasoun
d machine (B&K, Denmark) with a sidefiring 7.5 MHz transducer. In our 3D TRUS sy
stem, the side-firing linear array transducer is coupled to a rotational mover d
eveloped in our la
oratory.15 The mover rotates the transducer around its long a
xis to generate a 3D image volume in the shape of a fan scan, with a rotation an
gle of a
out
Ro
ot-aided and 3D TRUS-guided intraoperative
399
100.24 As the transducer is rotated, 2D US images from the ultrasound machine are
digitized at 0.7 intervals at 30 Hz for a
out 9 seconds
y a frame gra

er and s
tored in the computer. The 2D images are reconstructed into a 3D image (as the 2
D images are acquired), which is immediately availa
le for viewing using 3D visu
alization software.15 Figure 31.3 shows an example of the quality of 3D TRUS ima
ges that can
e achieved. Prostate segmentation Outlining the prostate margins m
anually is time consuming and tedious. Thus, a semi- or fully automated prostate
segmentation technique is required that is accurate, reproduci
le, and fast. Be
cause 3D US images suffer from shadowing, speckle and poor contrast, fully autom
ated segmentation procedures result, at times, in unaccepta
le errors. Our appro
ach has
een to develop a semiautomated prostate segmentation that allows the us
er to correct errors.28,29 In our approach, the prostate is segmented in a serie
s of cross-sectional 2D image slices o
tained from the 3D TRUS image, and the re
sulting set of
oundaries is assem
led into a single 3D prostate
oundary. Our 3
D prostate segmentation algorithm has
een descri
ed in detail in previous pu
li
cations,28,29 and consists of the following three-step procedure as shown in Fig
ure 31.4. (1) The operator manually initializes the algorithm
y selecting four
or more points on the prostate
oundary in one central prostate 2D slice. A curv
e passing through these points is then calculated and is used as the initial est
imate of the prostate
oundary (Figure 31.4a). (2) The curve is converted to a p
olygon with equally spaced points, which are then deformed using a Discrete Dyna
mic Contour algorithm until it reaches equili
rium (Figure 31.4
). If required,
the polygon can
e edited
y manually repositioning selected vertices. (3) The 2
D segmented prostate
oundary in one slice is extended to 3D (Figure 31.4c)
y p
ropagating the contour to an adjacent slice and repeating the deformation proces
s. This is accomplished
y slicing the prostate in radial slices separated
y a
constant angle (e.g. 3) intersecting along an axis approximately in the center of
the prostate.29 The accuracy of the prostate segmentation algorithm was tested

y comparing its results with manual planimetry. Using the prostate volume o
tai
ned
y manual planimetry as a reference, the errors in the semi-automated approa
ch ranged from an underestimate of 3.5% to an overestimate of 4.1%. The mean err
or was 1.7% with a standard deviation of 3.1%.29 Segmentation of the prostate req
uires a
out 5 seconds when implemented on a 1 GHz PC. Dosimetry We use the AAPM
TG-43 formalism, which uses predetermined dosimetry data in dose rate evaluation
.30 The dose can
e calculated
y either considering the sources oriented in a l
ine in any trajectory, or as point sources where source orientation is ignored.
After delineating the organs,
Basic and advanced techniques in prostate
rachytherapy
400
Figure 31.3 Example of 3D TRUS image of the prostate. The 3D image has
een slic
ed to show: (a) a transverse view; (
) a sagittal view; and (c) a coronal view.
The coronal view cannot
e o
tained usuing conventional 2D ultrasound imaging.
the user selects the type of source to
e used and enters its cali
ration data.
Considering pu
ic arch interference (PAI), the possi
le needle insertion area is
outlined and the preplan is produced. The preplan consists of a
out 20 needles,

which can
e oriented in o
lique trajectories to avoid PAI. The isodose curves
are displayed in real-time on the 3D TRUS image as well as on a surface-rendered
view with the needles and the seeds. Each needle can also
e activated and deac
tivated individually and the modified isodose curves can
e o
served instantly.
The user can evaluate the plan using dose-volume histograms (DVH) for each organ
and make necessary modifications. Figure 31.5 shows an example of the use of th
e preplan software for o
lique trajectory needle planning. During the live plann
ing procedure in the operating room (OR), after inserting each needle, the actua
l needle location is determined and the isodose curves are modified and displaye
d in real-time. This helps the user to decide whether the needle position is
Ro
ot-aided and 3D TRUS-guided intraoperative
401
satisfactory. After retracting the needle, the actual seed locations are determi
ned (currently we use assumed positions) and the new isodose curves are displaye
d. At this time the user has the option to modify the rest of the plan according
to the real seed locations after the needle retraction. System cali
ration The ai
m of the cali
ration procedure is to determine the transformation
etween the 3D
TRUS image-
ased coordinate system and the ro
ot coordinate system. Our
Figure 31.4 Images showing the steps of the 3D prostate segmentation algorithm.
The 3D TRUS image is first resliced into 2D slices, (a) The user initializes the
algorithm
y placing 4 or more points on the
oundary as shown. A model-
ased i
nterpolation approach is used to generate an initial contour, (
) A deforma
le d
ynamic contour (DDC) approach is used to refine the initial contour until it mat
ches the prostate
oundary, (c) The contour is propagated to adjacent 2D slices
of the 3D TRUS image and refined using the DDC. The process is repeated until th
e complete prostate is segmented as shown.
Basic and advanced techniques in prostate
rachytherapy
402
Figure 31.5 Display of a typical dose plan with o
lique needle trajectories for
use with 3D TRUS guidance and ro
otic aids. Our 3D visualization approach allows
display of a texturemapped 3D view of the prostate, extracted planes, and graph
ical overlays of surfaces and contours, (a) Coronal view with delineated organs,
needles, seeds, and isodose curves, (
) Sagittal view, (c) Transverse view, (d)
Surface rendered view showing the organs and needles with seeds.
approach involves two cali
ration steps: (a) 3D TRUS image to the transducer coo
rdinate system cali
ration (image cali
ration), and (
) the transducer to the ro

ot coordinate system cali
ration (ro
ot cali
ration). The transformation
etwee
n any two different coordinate systems is found
y solving the orthogonal Procru
stes pro
lem as follows. Given two 3D point sets K={kj}, L={1j} for j=1,2,,N, fin
ding a rigid-
ody transformation F: lj=F (kj)=Rkj+T, where R is a 33 rotation mat
rix, T is a 31 translation vector, so that we minimize the cost function:
Ro
ot-aided and 3D TRUS-guided intraoperative
403
(1) A unique solution to Eq (1) exists if, and only if, the point sets K and L c
ontain at least four non-coplanar points.31 For image cali
ration, we designed a
phantom comprised of 1 mm diameter nylon strings positioned in a Plexiglas
ox
(Figure 31.6a). The nylon strings were immersed in agar and were placed in three
layers 1 cm apart. The strings were arranged with known separations and forming
non-coplanar intersections. The coordinates of these intersections in the trans
ducer coordinate system were known from the phantom design, whereas their coordi

nates in the 3D TRUS coordinate system were determined
y scanning these interse
ctions (Figure 31.6
). The transformation linking the two coordinate systems was
determined
y solving Eq (1) using the coordinates of the string intersections
in
oth coordinate systems. For ro
ot cali
ration, we used two orthogonal plates
mounted on the transducer holder and drilled with five hemispherical divots on
each plate (Figure 31.7). Homologous points in the transducer and ro
ot coordina
te systems are provided
y the centers of the hemispherical divots on these two
plates. The coordinates of these divot centers in the transducer coordinate syst
em are known from the plate design. The coordinates of these divot centers in th
e ro
ot coordinate system were determined
y moving the ro
ot to sequentially to
uch the divots with a stylus tip that was attached to the ro
ot arm. The transfo
rmation linking the two coordinate systems was determined
y solving Eq (1) usin
g the coordinates of the divots in the two coordinate systems. Needle segmentati
on Ro
ot-assisted
rachytherapy procedure allows needle implantation in a non-pa
rallel approach. Thus, the needle may
e inserted in an o
lique trajectory, whic
h will result in its image passing out of the real-time 2D US image (Figure 31.8
). Visual tracking of the needle tip in an US image while
eing inserted into th
e prostate is necessary to ensure proper placement and to avoid implanting seeds
outside the prostate. Thus, we developed a technique to track the needle as it
is
eing inserted o
liquely. In our approach, we use near real-time 3D US imagin
g to segment the needle in 3D and then display o
lique
Basic and advanced techniques in prostate
rachytherapy
404
Figure 31.6 (a) Photograph of the image cali
ration phantom with the transducer
inserted into the simulated rectum, (
) A 3D TRUS image of the image cali
ration
phantom showing the nylon strings as white lines. The string intersections were
used as fiducials to determine the image transformation used to integrate the 3
D TRUS and ro
ot coordinate systems.
Figure 31.7 Photograph of the plates used for the ro
ot cali
ration. The positio
ns of the divots in the transducer coordinate system are all known through the p
hantom design. The positions of the divots in the ro
ot
Ro
ot-aided and 3D TRUS-guided intraoperative
405
coordinate system were determined
y moving the ro
ot to touch these divots as s
hown in this figure.
Figure 31.8 Schematic diagram showing the ultrasound transducer mounted on the r
otational mover used to produce 3D TRUS images of the prostate. The diagram show
s that when the needle is inserted at an o
lique angle relative to the transduce
rs axis, multiple 2D US images are needed to track its insertion trajectory.
sagittal, coronal and transverse image views with the needle trajectory highligh
ted. Since the needle may
e angled, at most, approximately 20 from the orientati
on of the 2D US plane, and 2D images may
e acquired at 30 images per second, a
new 3D image may
e formed in less than 1 second. From these 3D images, the need
le may
e segmented automatically,32,33 and the three planes needed to visualize
the needle insertion may
e displayed. Figure 31.9 shows the results of needle i
nsertion tracking in a prostate using our near real-time o
lique needle segmenta
tion and viewing approach. System evaluation We evaluated the performance of the
ro
ot-aided and 3D TRUS-guided
rachytherapy system in a controlled la
oratory
setting using precisely
uilt test phantoms. This is descri
ed
elow.
Basic and advanced techniques in prostate
rachytherapy

406
3D TRUS image and ro
ot coordinate system integration Cali
ration methods Accura
cy analysis of the image and ro
ot cali
ration was performed via the method used
for analyzing accuracy of point-
ased rigid-
ody registration.34 This method in
volves the analysis of three errors: (1) fiducial localization error (FLE); (2)
fiducial registration error (FRE); and (3) target registration error (TRE). FLE.
The FLE is defined as the error in locating the fiducial points used in the reg
istration procedure.35 We assumed that the mean value of the error in locating t
he fiducial points is zero and calculated the root-mean-square (rms) distance
e
tween the exact and calculated fiducial positions, thus:36 (2) where and are the
variances of the errors in locating the fiducial points along the three orthogo
nal axes. The terms in Eq (2) are calculated as: (3) where, j=1,2,3 represents t
he components (i.e. x, y, or z), xijk is the kth measurement for ith fiducial po
int (for image cali
ration, i=1, 2, 3, 4, and for ro
ot cali
ration, i=1, 2, , 6)
, k is the num
er of measurements for each fiducial point. For
oth image and ro

ot cali
rations, n=10, and, (4) is the mean measurement for the jth component o
f the ith fiducial point.
Figure 31.9 Example of the views displayed during o
lique needle tracking in a p
atients prostate, (a)
Ro
ot-aided and 3D TRUS-guided intraoperative
407
O
lique sagittal view showing the o
lique plane with the needle, (
) O
lique cor
onal view showing the plane with the needle (this view cannot
e o
tained with a
conventional US system), (c) The 3D TRUS image has
een sliced in a transverse
direction showing the segmented needle trajectory, (d) The 3D TRUS image has
ee
n sliced in an o
lique sagittal direction showing the needle.
FRE. We know the exact positions of N fiducials P={pj; j=1,,N} in the transducer
coordinate system for either the image cali
ration phantom (Figure 31.6) or the
ro
ot cali
ration plates (Figure 31.7). For image cali
ration, we measured the p
ositions of N fiducials (intersection of nylon strings) in the 3D image coordina
te system Q={qj; j=1,,N}. For ro
ot cali
ration, we measured the positions of N f
iducials (small divots) Q={qj; j=1,,N} in the ro
ot coordinate system
y moving t
he ro
ot to touch those small divots. FRE is calculated as the rms distance
etw
een corresponding fiducial positions
efore and after registration: (5)
where F is the rigid
ody transformation that registers the exact fiducial posit
ions P with the measured fiducial positions Q. TRE. TRE is defined as the distan
ce
etween corresponding points other than fiducial points
efore and after regi
stration and calculated using Eq (5). We used four targets in the image cali
rat
ion phantom to determine the TRE for image cali
ration, and four other markers o
n the plates to determine the TRE for ro
ot cali
ration. Cali
ration results Our
a
ility to localize the intersections of the nylon strings in the 3D TRUS image
for the image cali
ration was analyzed along the X, Y, and Z directions and ta

ulated in Ta
le 31.1 as the average fiducial localization error (FLE). From Ta
l
e 31.1 it can
e seen that the FLE for localizing the intersection of the string
s was similar in the X or Y
Ta
le 31.1 Fiducial localization error (FLE) (mm) for
oth image and ro
ot cali

ration
Image cali
ration Ro
ot cali
ration
Basic and advanced techniques in prostate
rachytherapy
408

X
Y
Z
X
Y
Z
Mean 0.05 0.06 0.11 0.17 0.21 0.20 SD (0.01) (0.02) (0.01) (0.07) (0.11) (0.10)
SD, standard deviation.
Ta
le 31.2 Fiducial registration error (FRE) and target registration error (TRE)
(in mm) for image and ro
ot cali
ration. These values were o
tained after the c
oordinate systems of the ro
ot and 3D TRUS image had
een integrated
Image Ro
ot cali
ration cali
ration FRE TRE FRE THE
Mean 0.12 0.23 0.52 0.68 SD (0.07) (0.11) (0.18) (0.29) SD, standard deviation
directions and larger in the Z direction. This is attri
uted to the fact that th
e measurement error in the Z direction corresponded to the elevation (i.e. out-o
f-plane) direction of the acquired 2D images, which has the poorest resolution i
n 3D TRUS images.15 Ta
le 31.1 also shows the FLE for localizing the divots on t
he two orthogonal plates used for ro
ot cali
ration. From Ta
le 31.1, it can
e
seen that the FLE for the divot localization was approximately the same in the t
hree directions. This error, caused
y the flexi
ility of the ro
ot arm and
ack
lash in the ro
ot arm joints, is greater than that for the string intersection l
ocalization, and will dominate the cali
ration errors, affecting the accuracy of
the system. The FRE and TRE values for the ro
ot cali
ration are shown in Ta
le
31.2. The mean FRE for the ro
ot cali
ration was 0.52mm0.18mm, and the mean TRE
was 0.68 mm0.29 mm, greater than those for image cali
ration. This results from t
he greater FLE for ro
ot cali
ration as discussed a
ove. Since system errors wil
l result from
oth the image and ro
ot cali
ration, the errors in the ro
ot cali

ration dominate the accuracy of integration of the two coordinate systems. Need
le placement accuracy The first phase of our testing involved measuring the accu
racy of needle placement and angulation
y the ro
ot at the patients skin. Needle p
lacement accuracy was determined
y using the ro
ot to move the needle tip to ni
ne locations on a 5 cm5 cm grid that represented the patients skin, (i.e. 33 grid of
targeting points). A threeaxis stage (Parker Hannifin Co, Irwin, PA) with a meas
uring accuracy of 2 m was then used to locate the needle tip. The displacement d b

tw

n th
m
asur
d and targ
t
d position of th
n

dl
tip was found as follows
: (6)
Robot-aid
d and 3D TRUS-guid
d intraop
rativ

409
wh
r
(x, y, z) ar
th
coordinat
s for th
targ
t
d point, (xi, yi, zi) ar
th

coordinat
s for th
ith m
asur
d point. Th
m
an n

dl
plac
m
nt
rror, and th

standard d
viation (SD) from 10 m
asur
m
nts at
ach position was found to b

0.15 mm0.06 mm. N

dl
angulation accuracy To m
asur
th
accuracy of n

dl
angu
lation using th
robot, w
attach
d a small plat
to th
n

dl
hold
r and us
d
th
robot to tilt th
plat
in four angl
s v
rtically and lat
rally (0, 5, 10, 15).
Aft
r
ach tilt, w
m
asur
d th
ori
ntation of th
plat
using th
thr

-axis s
tag
and d
t
rmin
d th
angulation
rror by comparing th
m
asur
d and plann
d p
lat
angl
. Tabl
31.3 shows th
m
an angl
diff
r
nc
s b
tw

n th
m
asur
d and
plann
d angulation by th
robot and th
ir standard d
viations. As s

n in Tabl

31.3, all m
an angl
diff
r
nc
s w
r
l
ss than 0.12 with a m
an of 0.07. Sinc
t

h
angulation
rror will caus
an incr
asing displac
m
nt
rror with incr
asing
n

dl
ins
rtion distanc
s, w

stimat
d th
displac
m
nt
rror aft
r an ins
rti
on of 10 cm from th
n

dl
guid
. Using th
m
an and maximum angulation
rrors,
th
m
an and maximum displac
m
nt
rrors for a 10 cm ins
rtion will b
0.13 mm a
nd 0.50 mm, r
sp
ctiv
ly. N

dl
targ
ting accuracy W
us
d tissu
-mimicking phan
toms mad
from agar37 and contain
d in a Pl
xiglas box to d
t
rmin
th
n

dl
i
ns
rtion accuracy. On
sid
of th
box was r
movabl
, allowing th
ins
rtion of
th
n

dl
(Figur
31.2). Each of two phantoms contain
d two rows of 0.8 mm diam

t
r stainl
ss b
ads as shown in Figur
31.10.38 This approach provid
d four dif
f
r
nt b
ad targ
ting configurations: two diff
r
nt n

dl
ins
rtion d
pths and
two diff
r
nt distanc
s from th
ultrasound transduc
r. Th
s
b
ad configuration
s form
d a 444 cm3 cub
to simulat
th
approximat
siz
of a prostat
. Th
targ
t
ing
xp
rim
nts involv
d first producing a 3D TRUS imag
, id
ntifying a b
ad to
b
targ
t
d, choosing a traj
ctory, positioning th
robot to allow ins
rtion of
th
n

dl
to th
targ
t, and th
n ins
rtion of th
n

dl
to th
targ
t. Th
s

dl
ins
rtion
xp
rim
nts w
r
carri
d out with a rigid rod to avoid n

dl
d

fl
ction. Th
accuracy was calculat
d by d
t
rmining th
d
viations of th
n

d
l
tip from th
pr
ins
rtion b
ad locations in th
3D TRUS imag
. By av
raging t
h
r
sults from all targ
ting
xp
rim
nts, th
m
an
rror was found to b
0.74 m
m 0.24 mm. In addition, w
also plott
d th
r
sults of th
n

dl
targ
ting accur
acy as a 3D scatt
r plot of th
n

dl
tips r
lativ
to
Tabl
31.3 R
sults of accuracy
valuation of n

dl
angulation by th
robot for
v
rtical and lat
ral tilts
V
rtical angulation
Targ
t
d angl
s 5 0.02 M
an angl
diff
r
nc
Standard d
viation (0.02) Maximum angl

diff
r
nc
0.08 10 0.04 (0.06) 0.22 15 5 0.06 0.07 (0.05) (0.05) a 18 0.12
Lat
ral angulation
10 0.10 (0.03) 0.15 15 0.12 (0.08) 0.28
Basic and advanc
d t
chniqu
s in prostat
brachyth
rapy
410
Figur
31.10 Sch
matic diagram of th
prostat
phantom us
d for
valuation of n

dl
targ
ting accuracy. Th
four rows of circl
s r
pr
s
nt th
four diff
r
nt b

ad configurations. Th
n

dl

nt
r
d th
phantom from th
front, parall
l to t
h
x-axis. TRIP, top row, long p
n
tration; TRSP, top row, short p
n
tration; BR
LP, bottom row, long p
n
tration, BRSP, bottom row, short p
n
tration.
th
targ
t. In Figur
31.11, w
show th
s
r
sults as w
ll as th

llipsoid
nco
mpassing 95% of th
n

dl
tip locations (r
lativ
to th
targ
ts). Accuracy of
n

dl
tracking W
compar
d th
obliqu
n

dl
tracking r
sults to thos
obtain

d from th
parall
l traj
ctory sinc
th
traj
ctory of n

dl
ins
rtion parall
l
to th
US transduc
r axis can b
v
rifi
d by obs
rving th
n

dl
ins
rtion in
th
r
altim
2D TRUS imag
s. W
us
d th
robot to guid
th
ins
rtion of a n

dl

at diff
r
nt angl
s (5, 10, 15) with r
sp
ct to th
parall
l ins
rtion traj
ctory. F
or
ach obliqu
n

dl
traj
ctory, th
n

dl
was track
d automatically using th

algorithm d
scrib
d abov
, its ins
rtion
Robot-aid
d and 3D TRUS-guid
d intraop
rativ

411
Figur
31.11 N

dl
targ
ting accuracy is display
d as th
95% confid
nc

llips
oid. Th
origin of th
coordinat
syst
m r
pr
s
nts th
targ
t and th
n

dl
ti
p positions aft
r ins
rtion (r
lativ
to th
targ
ts) ar
pr
s
nt
d by th
squar

s. Th
proj
ctions of th
n

dl
tip positions and th

llipsoid (on th
thr

orthogonal plan
s) ar
also shown. Th
s
r
sults ar
for th
targ
ts n
ar th
tr
ansduc
r and for a short p
n
tration, as shown in Figur
31.10.

angl
d
t
rmin
d automatically and compar
d with th
parall
l ins
rtion angl
. T
his t
st was carri
d out with agar and a chick
n tissu
phantom,39 and r
p
at
d
fiv
tim
s for
ach angl
to d
t
rmin
th
m
an tracking
rror and its standard
d
viation. Tabl
31.4 shows th
r
sults of th
n

dl
tracking algorithm
valuat
ion. In th
chick
n tissu
phantom, th
av
rag

x
cution tim
was 0.130.01 s
con
ds, and th
av
rag
angulation
rror was 0.540.16. In agar phantoms, th
av
rag

x

cution tim
was 0.120.01 s
conds, and th
av
rag
angulation
rror was 0.580.36. Th

r
sults shown in Tabl
31.4 d
monstrat
that n

dl
ins
rtion can b
track
d i
n n
ar r
al-tim
and that th
tracking
rror do
s not significantly d
p
nd on in
s
rtion angl
. Figur
31.9 shows th
vi
ws provid
d to th
physician during n

d
l
tracking.
Tabl
31.4 N

dl
tracking r
sults for chick
n tissu
(a) and agar (b) phantoms
for n

dl
ins
rtions at diff
r
nt angl
s. Th
valu
s ar
th
s
gm
ntation tim
s
(s
conds) and th
angulation
rrors (d
gr

s)
Basic and advanc
d t
chniqu
s in prostat
brachyth
rapy
412
Angl
15 10 5 5 10 15
(a) Time (s) 0.13 0.11 0.12 0.12 0.12 0.14 Error 0.50 0.51 0.43 0.37 0.74 0.74 (b) Tim
e (s) 0.12 0.12 0.12 0.11 0.12 0.13 Error 0.31 0.71 0.48 0.68 0.42 0.86

See implantation accuracy We use a brachytherapy neele to implant 0.8 mm sphe

rical steel beas instea of brachytherapy sees to test the accuracy of see im
plantation using robot ais an 3D TRUS guiance. These beas were implante int
o agar phantoms at ifferent preefine positions, an using ifferent neele tr
ajectories (0, 5, 10, 15 with respect to the TRUS probe axis). These tests were carrie
out by first choosing a trajectory an a targete position in the 3D TRUS image
. Then, the robot was use to guie the neele an a bea was implante. By obta
ining a 3D TRUS image after the implantation, ientifying the bea in the 3D TRU
S image, an comparing the actual position of the bea to the planne position,
the error in implantation coul be etermine. The bea implantation accuracy is
tabulate in Table 31.5, showing that the mean error was 2.59 mm  0.76 mm. The g
reatest error was observe in the Y irection (in vertical plane), corresponing
to the bevel irection of the neele. Discussion an conclusions We have emons
trate the technical feasibility of a robotaie prostate brachytherapy proceur
e using 3D TRUS guiance. The experimental results inicate that, with robotic a
ssistance, the brachytherapy neele can be guie accurately an consistently to
target points in the 3D TRUS image along various trajectories, incluing obliqu
e, as specifie by a preplan. We expect that with the introuction of a robotic
system an 3D TRUS tools for automatic neele etection for oblique insertion, l
ocalization of the implante sees, an monitoring of prostate changes, an effec
tive intraoperative prostate brachytherapy proceure will be possible. While mos
t of the tools to accomplish this goal have been evelope, see segmentation fr
om 3D TRUS images, such as the one shown in Figure 31.12, has not yet been accom
plishe an is require for a complete intraoperative system. However, base on
the work to ate, we can raw the following conclusions. In registering the coor
inate systems of the robot an the imaging system, fiucial points in the 3D TR
US image must be etermine. Errors in locating these fiucial points occur ue
to the poorer resolution in the 3D scanning irection, an will be greater for t
he points further away from the TRUS transucer.15 These errors will propagate,
an result in targeting errors, causing systematic errors. Calibrating the robot
coorinate system an integrating it with the 3D TRUS image requires that fiuc
ial points relate to the robot be ientifie (see Figure 31.7). Comparing Table
31.2 with Table 31.1, it is apparent that the fiucial localization error (FLE)
for the robot fiucials is greater
Robotaie an 3D TRUSguie intraoperative

413
Table 31.5 Results of the see implantation accuracy evaluation. The values are
the error (mm) components along the coorinate axis between the planne an actu
al implante bea locations. The errors have been measure for a range of neele
trajectories
20 15 10 5 0 5 10 15 20
X 0.62 0.33 Y 2.79 2.01 Z 0.89 0.61 Total error 2.99 2.13 MeanSD SB, stanar ev
iation 0.34 0.78 0.41 0.54 0.65 0.40 0.51 1.02 2.97 1.58 2.97 3.27 3.03 1.99 0.7
9 0.75 0.53 0.75 0.98 0.63 0.87 1.33 3.16 1.72 3.11 3.48 3.12 2.23 2.59 mm 0.76 m
m
Figure 31.12 (a) A 3D TRUS prostate image postimplantation slices to reveal a tr
ansverse plane, (b) 3D TRUS image of the same patient as in (a), but slice in t
he transverse an longituinal irections showing the sees, (c) The same image
slice in the coronal plane, showing the sees arrange along the neele path. T
his view cannot be obtaine using
Basic an avance techniques in prostate brachytherapy
414
conventional ultrasoun (US) techniques.
than that for the 3D TRUS fiucials. Therefore, errors in calibrating the robot
coorinate system will ominate an will eteriorate the overall accuracy of the
system. Comparing the results obtaine in the evaluation of the neele targetin
g accuracy using a rigi ro with those obtaine for the bea implantation accur
acy with a brachytherapy neele, it is apparent that the errors observe in the
latter are larger an ominate the overall performance of the system. The large
implantation error was primarily cause by neele eflection from the planne tr
ajectory ue to the bevel at the neele tip. Solving this source of error will g
reatly improve the performance of the system. In our prototype robotassiste an
 3D TRUSguie system, the robot an 3D TRUS image coorinate systems are regi
stere through specially esigne calibration phantoms so that the position of t
he neele guiance hole coul be relate to the 3D TRUS image. This approach has
allowe us to remove the parallel neele trajectory constraints present in curr
ent prostate brachytherapy proceures. Mounting the neele guie on a robot arm,
rather than having it fixe to the ultrasoun transucer holer, provies great
er flexibility over the neeles trajectory reucing the effects of pubic arch int
erference. While neele eflection ue to the neele bevel contributes significa
ntly to the overall error, robotic ais an 3D visualization may allow us to com
pensate for this eflection. Acknowlegments The authors gratefully acknowlege
the financial support provie by the Canaian Institute of Health Research an
the Ontario R&D Challenge Fun. AF hols a Canaa Research Chair in Biomeical E
ngineering, an acknowleges the support of the Canaa Research Chair Program. Z
W acknowleges partial funing provie by Ontario rauate Scholarship in Scien
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32 Initial experience with the FIRST system in Utrecht
Jan J Battermann, Ina Schulz, Marinus A Moerlan, an Marijke van Deursen Intro
uction Permanent prostate brachytherapy is now performe in many centers worlw
ie an is well accepte in North America an Western Europe. Different techniqu
es are employe with either single sees or strans, using preloaing as well as
afterloaing, an preplanning or intraoperative planning. A new evice was rec
ently introuce by Nucletron Inc, the FIRST system (Fully Integrate Raiothera
py See Treatment). In this system, both an intraoperative planning system (SPOT
; Sonographic Planning of Oncology Treatment) an an automate see afterloaer
(seeSelectron) are combine. This system is in routine use at the raiation onc
ology epartment at University Hospital Utrecht since January 2002. The FIRST sy
stem The FIRST system combines intraoperative planning (SPOT) an automate see
elivery (seeSelectron). The configuration of the sees, calculate with the S
POT system, is automatically transferre to the seeSelectron steering software.
Increasingly, treatment centers are using intraoperative planning with the ava
ntages of exact patient position an realtime ajustments.16 SPOT is a threeim
ensional (3D) planning system, using 3D imaging of the prostate by ultrasoun wi
th a special electronic ultrasoun probe with a transversal an longituinal arr
ay. With 3D imaging an contouring of the prostate, a first plan is mae base o
n geometric rules, the socalle autoplan. At the iscretion of the raiation onco
logist an physicist, aing or eleting neeles an sees can moify this plan.
The autoplan can be evaluate by osevolume histograms (DVH)both the cumulative
an the natural DVH. The cumulative DVH assesses the placement of the ose ist
ribution over the target volume an provies prostate volume an coverage. The n
atural DVH assesses the homogeneity of the implant accoring to the with of the
peak. The natural prescription ose (NPD) for a given ose istribution is loca
te at the base of the peak at the low ose sie an is the optimal prescription
ose for the implant. The ratio between the NPD an the prescribe ose (PD) (u
sually 144 y) is calle the natural ose ratio (NDR) an shoul be aroun 1 for
an optimal plan. The higher the
Basic an avance techniques in prostate brachytherapy
418
value, the hotter the implant. Without the use of the natural DVH, overose easily
escapes etection.7,8 With SPOT, a neele upate can be mae after insertion of
the neeles to their actual position with 3D reconstruction. Hence, a live plan c
an be mae before the sees are place an evaluate for further moification of
see istribution. The calculate neele an see configuration is transferre

to the seeSelectron user interface. Since the seeSelectron has separate cartri
ges with raioactive sees an spacers, any combination of active sees an spa
cers in one neele can be mae. After a train of sees an spacers is built up i
n the compose element, a rive wire pushes the complete train into the prostate
to the tip of the neele. The neele is retracte automatically 7 mm beyon the
proximal see in the train with the rive wire still in position. The wire is th
en again retracte 7 mm to avoi any sticking of the proximal see to the rive
wire. Subsequently, the wire is completely retracte an the neele is remove m
anually. The cartriges can hol up to 100 active sees. However, cartriges wit
h 90, 80, 70, 60, 50, or 10 sees are also available. Cartriges with spacers al
ways contain 100 spacers. However, cartriges can only be use for one patient,
since bloo can be introuce into the cartrige an cause contamination in the
next patient unergoing this proceure. Implantation technique Pretreatment eval
uation All patients referre for permanent see implantation are screene for th
eir eligibility by ultrasoun imaging at the urology epartment in our hospital.
This proceure is performe as a combine examination by the urologist an rai
ation oncologist. The prostate is screene for extracapsular tumor extension, wh
ich is an exclusion criterion for brachytherapy, an the prostate volume is meas
ure. Since intraoperative planning is performe, no complete volume stuy is ma
e on this occasion. Prostate volumes with a maximum of 50 cc are accepte, know
ing that, in general, the volume will be approximately 1020% larger with 3D ultra
soun imaging. Questionnaires for lower urinary tract symptoms (International Pr
ostate Symptom Score; IPSS) an sexual activity will have been complete before
this first visit. The urine flow is measure before the ultrasoun proceure. Pa
tients are seen by the anesthesiologist, who will estimate the patients eligibili
ty for anesthesia an iscuss the type of anesthesia. Most patients are treate
uner epiural anesthesia, although it is avisable to use general anesthesia in
a center just starting this proceure. The patients receive instructions for bo
wel cleansing an a prescription for a laxative to be use for five ays before
treatment. Pretreatment preparation Patients are hospitalize some hours prior t
o the proceure to allow ample time for the nursing staff to prepare him for tre
atment. The perineum is shave an a rectal enema is given about one hour before
implantation.
Initial experience with the FIRST system in Utrecht
419
The actual implant is carrie out in a eicate operating room (OR), which is a
lso use for other brachytherapy proceures. Prophylactic antibiotics are amini
stere as an intravenous bolus. With the patient anesthetize, his legs are plac
e in the stirrups in the lithotomy position with the femurs in a vertical plane
. A foley catheter is introuce to rain the blaer an visualize the urethra
on ultrasoun. The catheter balloon is fille with a solution of contrast meium
to ientify the blaer outlet uring fluoroscopy. The scrotum is isplace upw
ars with ahesive ressing an the perineum is washe with an antiseptic soluti
on. In orer to perform fluoroscopy uring the proceure, see Figure 32.1 for se
tup of patient an equipment. After the transrectal ultrasoun (TRUS) probe is
inserte into the rectum an locke in the stepping unit the template is mounte
on to the stepping unit. The stepping unit an support evice are aligne in su
ch a way that the orsal contour of the prostate is at the lowest line of the te
mplate (row 1). The prostate contour shoul be symmetric
Figure 32.1 Patient setup with legs in stirrups, stepping unit in place, Carm,
TRUS system an planning system.
in the template, usually with the urethra at the Dline of the gri. Implantatio
n proceure Stabilization neeles are manatory in orer to reuce movement of t
he prostate uring insertion of the neeles an 3D imaging acquisition. After in
sertion of these locking neeles, routinely in the center of the prostate (Figur
e 33.3), a first 3D scan is taken using the sagittal moe of the probe. The pros

tate is contoure, first using a sagittal view to ientify the base, apex, an t
hickness of the prostate in the misagittal plane (Figure 32.2). Then, on trans
verse imaging at increments of 2.5 or 5 mm, the contour is epicte from base to
apex (Figure 32.5).
Basic an avance techniques in prostate brachytherapy
420
Following contouring of the prostate, the urethra is contoure, again using both
sagittal an transverse imaging. The process of contouring takes about 15 minut
es. From this contour a treatment plan is mae in the autoplan moe. Neeles an
sees are place insie the prostate volume accoring to geometric rules, (e.g.
see placing in a line at 10 mm, between lines at 5 mm, an see staggering). N
ote that an autoplan only places sees homogeneously in the prostate volume an
this woul result in an overose to the central part of the prostate, incluing
the urethra (Figure 32.4). The DVH, as escribe above, is essential for evaluat
ing an optimizing the plan. At the iscretion of the raiation oncologist an p
hysicist, neeles can be ae or remove, an the see composition per neele c
an be ajuste. Centrally place neeles, in general, only nee one see at the
base an one at the apex, resulting in a substantial reuction of the ose to th
e urethra. We aim at a prostate coverage of over
Figure 32.2 Contouring of prostate an urethra in transversal, sagittal an coro
nal planes, an 3D composition of these images. Note the position of the two loc
king neeles as echos on the transversal an coronal planes.
Initial experience with the FIRST system in Utrecht
421
Figure 32.3 Contouring in transverse plane.
95%, a margin of 23 mm aroun the prostate volume, a urethral ose lower than 150
%, an the volume receiving 150% an 200% of the prescribe ose less than two t
hirs an one thir of the prostate volume, respectively. The natural ose ratio
(NDR) is a simple an helpful tool for evaluating over or unerosing of the t
arget volume (Figure 32.4). In centers with experience in this technique, the in
sertion of neeles is routinely starte after 3D scanning, without waiting for t
he treatment plan. All neeles are place (Figure 32.5), using both transverse a
n sagittal imaging to etermine the position of each neele in relation to the
base an apex of the prostate, starting at the most ventral row to reuce image
interference from other neeles. The neele is navigate using the bevel irecti
on to fine tune the final position of the neele. The special neeles, necessary
for the FIRST system, have an obturator 5 mm shorter than the neele length; he
nce the obturator oes not
Figure 32.4 Autoplan base on geometric rules (left). Re line inicates the con
toure outline of the prostate. The blue line is the 150% isoose line, covering
the whole prostate. The purple line is the
Basic an avance techniques in prostate brachytherapy
422
prescription ose of 145 y. Natural ose volume histogram (right) shows a narro
w peak, inicating a very homogenous ose istribution ue to the high ose that
is receive by the whole glan. This is also reflecte by the high natural ose
ratio of 1.70.
Figure 32.5 Situation with all neeles, incluing two locking neeles, in place.

The neele position is ajuste for the correct position in relation to the bas
e plan.
have to be retracte while inserting the neele. After insertion an proper plac
ement of all neeles, fluoroscopy is performe to verify the relation of the nee
le tips with the catheter balloon in the blaer. On the xray, shown in Figure
32.6, note that the tip of the obturator is at a ifferent level to the tips of
the neeles. A secon 3D ultrasoun scan is then mae with all the neeles in p
osition. With SPOT, there is the option of a 3D reconstruction of the neele pos
ition to its actual position in the prostate an hence more accurate treatment p
lan
Initial experience with the FIRST system in Utrecht
423
Figure 32.6 Definitive neele an see positions, accoring to final treatment p
lan in schematic view (upper part). Loaing pattern per iniviual neele (lower
part).
Figure 32.7 Upate treatment plan, showing reuce ose to urethral an rectum
in transversal plane (left) an exact neele position on sagittal plane (right).
Basic an avance techniques in prostate brachytherapy
424
(Figure 32.7). This secon scan is helpful in eciing the final see configurat
ion. When the team has agree on the plan, it is save an printe out (Figure 3
2.8). Intraoperative planning parameters, such as V100 (percent
Figure 32.8 Definitive neele an see positions, accoring to final treatment p
lan in schematic view (upper part). Loaing pattern per iniviual neele (lower
part).
prostate volume covere by the prescription ose) an D90 (minimal ose in 90% o
f the prostate) can be obtaine, as well as other ata. The seeSelectron is mou
nte on the stepping unit an connecte to the SPOT system. After a trial run, t
he compose element, two cartriges containing active sees an spacers, an the
rive wire element are inserte into the seeSelectron (Figures 32.9 an 32.10).
The transfer tube of the seeSelectron is attache to a small container to rele
ase one see for valiation of the see activity. A particular quality assessmen
t (QA) feature of the seeSelectron is iniviual measurement of raioactivity p
er see uring see composition. The transfer tube is first connecte to one of
the neeles with the tip at the
Initial experience with the FIRST system in Utrecht
425
base plane, to ensure that all the subsequent neeles are within the elivery ra
nge. If necessary, the position of the seeSelectron on the stepper can be ajus
te. The hook of the seeSelectron has to be positione at the flange on the tra
nsfer tube to ensure
Figure 32.9 Disposables use for the FIRST implant. Not shown are the locking an
 insertion neeles. The lea pot will contain the ispose see for verificatio
n of the see activity to calibrate the system.
Basic an avance techniques in prostate brachytherapy
426

Figure 32.10 Schematic view of seeSelectron with cartriges containing active s

ees an spacers.
that the see train reaches the tip of the neele. The first train of sees an
spacers is compose, the activity of each see checke as well as the compositio
n of the train, an then the see train is automatically transporte to the tip
of the neele. See elivery can be performe either in the automatic or manual
moe. In the automatic moe, the tip
Figure 32.11 See istribution over prostate glan, irectly after implant proce
ure. See also Figure 29.4.
positions are automatically erive from the calibration of the first neele at
the base plane an the retraction values of the respective neeles in the treatm
ent plan. In the manual moe, the hook has to be positione for each neele. The
manual moe has an avantage in that each neele position can be checke by sag
ittal ultrasoun an, if necessary, ajuste before actual see elivery. Usuall
y, neeles have to be pushe a few mm eeper to obtain optimal placement of see
s. With sagittal ultrasoun in place, eposition of sees can also be checke. T
he eposition of the see trains begins, as escribe above, with a eeply posit
ione neele. After this first neele, all the remaining are elivere similarly
, starting with the highest row an going from left to right.
Initial experience with the FIRST system in Utrecht
427
When all the neeles have been remove, incluing the locking neeles, a final 3
D scan is mae as well as fluoroscopy (Figure 32.11) to assess the number of imp
lante sees. The perineum is rape with sterile gauze. Postimplant care In Utr
echt, we hospitalize the patient for one night after implantation. The urinary c
atheter is left in situ overnight an remove the next ay, after performing a c
ompute tomography (CT) scan for postplanning an fluoroscopy for a secon check
on the number of sees. Postoperative care is usually routine postanesthesia ca
re. Patients receive naproxen 250 mg three times a ay to reuce swelling of the
prostate. Alphablockers are not routinely prescribe: only for those patients w
ith large volumes or a high IPSS. The patient is ischarge after being checke
that he can voi normally. In our total practice of almost 1000 patients we only
once ha to postpone hospital ischarge for acute retention after removal of th
e catheter. Postplanning Postimplant osimetry is either one after CT or magnet
ic resonance imaging (MRI) one month after treatment when swelling shoul have g
one own.911 Although we prefer MRI, because of better contouring of the prostate
, we o not use MRI for all patients ue to costs an facility availability. MRI
, CT, an Xray ata are matche to optimize prostate contouring an see locali
zation. DVH parameters are very useful inices in assessing the quality of the i
mplant. The emergency tool An emergency kit (Figure 32.12) is inclue in the FI
RST system so that the implant proceure can continue shoul the system break o
wn. The implant neeles, alreay in situ, can be use with this tool. The two ca
rtriges with active sees an spacers can be place in the tool, an a see tra
in can be compose manually. This train is then manually inserte by placing the
tool on the neele an pushing the see train into the prostate. The neele is
then retracte manually. However, at the time of writing, we have implante more
than 150 patients with the FIRST system an have ha to use the emergency kit o
nly twice. Moifications in the FIRST system uring 2003 have mae it very relia
ble. Discussion Different techniques are use in ifferent centers aroun the wo
rl. Intraoperative planning is becoming increasingly popular an has the avant
ages of accurate patient position an realtime osimetry, resulting in a better
ose istribution.16 Although the total OR time is substantially longer than aft
er preplanning, especially with preloae neeles, the total time is reuce bec
ause neither a complete volume stuy nor a preplan

Basic an avance techniques in prostate brachytherapy

428
have to be mae. Table 32.1 gives a summary of the treatment times for ifferent
implantation techniques. From our own experience,
Figure 32.12 Emergency kit to finish the implant proceure manually in case a of
breakown of the FIRST system. The two cartriges are mounte together an wit
h the knob, a composition of sees an spacers can be mae accoring the origina
l plan. The small tube is connecte to the iniviual neele to insert the train
of sees an spacers manually with the neele obturator
we foun that for a proper volume stuy approximately 20 minutes is require. Fo
r patients treate with intraoperative planning, an ultrasoun examination will
be repeate after referral from another urology epartment to confirm the eligib
ility of the particular patient an assess the prostate volume. One shoul take
into account that the volume might be up to 20% larger with a complete volume st
uy or 3D volume assessment. Preplanning with current available 2Dplanning syst
ems will take 15 to 20 minutes. The FIRST system combines intraoperative plannin
g with automate see afterloaing. The avantages of this system inclue optima
l raiation protection an a straightforwar composition of sees an spacers in
any combination, especially when central neeles can be loae with just one ac
tive see at the beginning an at the en. Checking raioactivity of each see,
automatic train composition, an check on composition, improve quality control a
n save time, compare to measuring the activity at ranom of loose sees or som
e strans. Furthermore, it saves time (an personnel) in
Initial experience with the FIRST system in Utrecht
429
preparing see trains, an a preparation station in a separate source room is un
necessary. The length of time neee to insert the neeles is similar for all te
chniques. We prefer to insert all the neeles first, as this has the avantage o
f a better fixation in the prostate, an optimal control of the position of the
neeles, using both sagittal ultrasoun scanning an fluoroscopy. When iniviua
l
Table 32.1 Estimate treatment times for ifferent implant techniques
Technique Preplan an preparation OR time Total time
45 min 1 h 30 min 45 min 2h 1 h 45 min 1 h 45 min 1 h 45 min 1 h 45 min Mick app
licator 45 min Str Strans preloae 45 min 4+30 rain Strans afterloae Live p
lan FIRST Live plan
neeles are introuce, their position checke, an sees place into the prosta
te separately, the treatment time is longer. This is because the control of the
neele positions is at the en of the proceure. The loaing of sees in the pro
state takes a little longer than the placing of sees from preloae neeles an
about the same time as the loaing of sees in sutures (RAPID Stran). A isav
antage of the FIRST system coul be cartriges, although these are available wit
h 10, 50, 60, 70, 80, 90, an 100 sees. Unuse sees from a cartrige cannot be
use in another patient, because the cartrige may be contaminate with bloo.
If insufficient sees are present in a cartrige, extra sees can be use (e.g.
from a 10 see cartrige) or, if available, loose sees or RAPIDStran, but, in
general, the neeles in the FIRST system are not compatible to other elivery sy
stems, such as the Mick applicator or the Utrecht stran holers for RAPID Stran
s. (See Chapter 29.). Another problem is see loss an see migration. With per
ipheral loaing of the sees over the prostate volume, see loss is quite rare (
Table 32.2), an see migration harly ever encountere. With selectSees an pe
ripheral loaing, we see a higher migration rate, mainly resulting in one or mor

e sees entering the lungs. Although no etrimental effect of this migration has
been escribe so far, we shoul try to reuce any ose at locations not to be
irraiate to the minimum. Postplanning Since SPOT offers the possibility of 3D
reconstruction of the actual neele position, the moifie plan can be regare
as a postplan. In general, sees are not as neatly line up as with RAPID Stran
s (see Figures 32.11 an 29.4), which may reuce the homogeneity of the implant,
but in our experience this has little influence on prostate coverage. We teste
the FIRST system by the insertion of half of the prostate with Strans, an hal
f with selectSees. We controlle the position of selectSees an strans with f
luoroscopy an sagittal ultrasoun uring the actual insertion of the see in th
e glan. We foun that selectSees came approximately 5 mm closer to the prostat
e base than sees
Basic an avance techniques in prostate brachytherapy
430
Table 32.2 See loss an migration after ifferent implant techniques use at UM
C Utrecht
Single see Strans of sees FIRST
No. patients 101 536 70 No. sees 4227 38 903 5095 Lost at ischarge 129 16 6 Lo
st at 1 month 26 233 7 Lost at 1 year* 46 211 1 Total lost 216 (5.1%) 460 (1.2%)
14 (0.3%) Migration 11 (0.3%) 25 (<0.1%) 41 (0.8%) Exclue are patients with p
re or post (<1 year) implant transurethral resection of the prostate (TURP); p
atients with combine strans an Selectsees (total 19 patients). UMC, Universi
ty Meical Hospital. *34 patients with Selectsees have not yet complete 1 year
followup, an 83 patients with strans.
in strans. This is ue to a 2.5 mm vicryl coating on the first see in a stran
, but espite this, the FIRST system places sees exactly at the en of the nee
le tip. These ifferences resulte in slightly better coverage with selectSees
compare to strans.1214 Nowaays, we introuce the neeles for loaing with stra
ns a few millimeters eeper than we i originally. When ifferent parameters f
or osimetry are compare, we foun harly any ifferences between stran implan
ts an selectSee implants. Although the moifie plan after the 3D neele posit
ion upate shows a coverage of almost 100% in all patients, postplanning one ay
later with CT imaging often shows some reuce coverage to 85 95%. This can be a
ttribute to postimplant swelling of the glan. The V200 an V150, in general, a
re below one an two thirs of the total prostate volume, respectively. D90 valu
es obtaine 4 weeks postimplant are normally between 100 y an 160 y an impro
ve with intraoperative planning.14 Conclusions The FIRST system is a reliable t
echnique that offers intraoperative planning an automate see afterloaing. Th
e total treatment time for an iniviual patient oes not substantially iffer f
rom other techniques, but OR time is longer, mainly because of intraoperative pl
anning. This requires more OR time, inepenent of the implantation technique. O
n the other han, intraoperative planning is more accurate an results in better
osimetry parameters. Avantages of FIRST are the quality control of see stren
gth an seespacer combination, the straightforwar composition of a ifferentia
l loaing pattern per neele, an optimal raiation safety. Automatic compositio
n of seespacer combinations saves time, personnel, an time in the preparation
room. Disavantages inclue the isposal of unuse sees from a cartrige an th
e higher percent of see migration. The followup time since the introuction of
FIRST is too short to give any reliable information on biochemical control so f
ar. However, as osimetry parameters are similar, or even better than in other t
echniques, we expect similar biochemical control results.
Initial experience with the FIRST system in Utrecht
431

References
1. Cormack RA, Tempany CM, DAmico AV. Optimizing target coverage by osimetry fee
back uring prostate brachytherapy. Int J Raiat Oncol Biol Phys 2000; 48:124512
49. 2. Beyer DC, Shapiro RH, Puente E Realtime optimize intraoperative osimet
ry for prostate brachytherapy: a pilot stuy. Int J Raiat Oncol Biol Phys 2000;
48:15831589. 3. Nag S, Ciezki JP, Cormack R, et al. Intraoperative planning an
evaluation of permanent prostate brachytherapy: report of the American Brachythe
rapy Society. Int J Raiat Oncol Biol Phys 2001; 51:14221430. 4. Thompson SA, Fun
g AY, Zaier M. Optimal neele arrangement for intraoperative planning in perman
ent I125 prostate implants. Phys Me Biol 2002; 47:N209215. 5. Zelefsky MJ, Yama
a Y, Marion C, et al. Improve conformality an ecrease toxicity with intraop
erative computeroptimize transperineal ultrasounguie prostate brachytherap
y. Int J Raiat Oncol Biol Phys 2003; 55:956963. 6. Lee EK, Zaier M. Intraoperat
ive ynamic ose optimization in permanent prostate implants. Int J Raiat Oncol
Biol Phys 2003; 56:854861. 7. Riet A vant, Loo HJ te, Ypma AF, et al. Ultrasonica
lly guie transperineal see implantation of the prostate: moification of the
technique an qualitative assessment of implants. Int J Raiat Oncol Biol Phys 1
992; 24:555558. 8. Moerlan MA, Laarse R van er, Luthman RW, et al. The combine
use of the natural an the cumulative osevolume histograms in planning an ev
aluation of permanent prostatic see implants. Raiother Oncol 2000; 57:279284. 9
. Moerlan MA. The effect of eema on postimplant osimetry of permanent Ioine
125 prostate implants: a simulation stuy. J Brachyther Int 1998; 14:225231. 10.
Yue N, Chen Z, Peschel R, et al. Optimum timing for imagebase ose evaluation
of 125I an 103PD prostate see implants. Int J Raiat Oncol Biol Phys 1999; 45:
10631072. 11. ellekom MP van, Moerlan MA, Kal HB, Battermann JJ. Biologically e
ffective ose for permanent prostate brachytherapy taking into account postimpla
nt eema. Int J Raiat Oncol Biol Phys 2002; 53:422433. 12. Moerlan MA, Wijrema
n HK, Beersma R, et al. Evaluation of permanent I125 prostate implants using ra
iography an magnetic resonance imaging. Int J Raiat Oncol Biol Phys 1997; 37:
927933. 13. Nag S, Bice W, DeWyngaert K, et al. The American Brachytherapy Societ
y recommenations for permanent prostate brachytherapy postimplant osimetric an
alysis. Int J Raiat Oncol Biol Phys 2000; 46:221230. 14. ellekom MP Van, Moerla
n MA, Wrjreman HK, Battermann JJ. Quality of permanent prostate implants using
automate elivery with seeSelectron versus manual insertion of RAPID Strans.
Raiother Oncol 2004; 7:4956.
Part IV Treatment planning an techniques for high ose rate prostate brachyther
apy
High ose rate
192
33 Ir prostate brachytherapy
Kas R Baiozamani, Timothy P Mate, an James E ottesman Introuction High ose
rate (HDR) brachytherapy is an establishe an rapily avancing technique use
to eliver highly conformal oses of raiation in the treatment of prostate canc
er. Remote afterloaing evices using high activity iriium192 (192Ir) sources
are commonly use in a variety of centers throughout the worl, making HDR prost
ate brachytherapy increasingly available to many patients. This chapter is inten
e to provie the reaer with a basic unerstaning of HDR implants, incluing
appropriate patient selection, implant techniques, an outcomes. The rationale f
or the use of HDR brachytherapy in prostate cancer is iscusse, as are areas fo
r future research an evelopment. Backgroun an rationale HDR brachytherapy wa
s first use for prostate cancer in the mi 1980s in Kiel, ermany,1 an others
soon evelope pilot programs in the Unite States, but the relatively limite a
vailability of afterloaers preclue early, wiesprea aoption of the techniqu
e. Nonetheless, several factors make HDR an attractive an promising approach to
the treatment of prostate cancer. Conformal ose escalation has been the major

theme in prostate irraiation for more than a ecae. Avances in computer techn
ology have allowe for the evelopment of complex, threeimensional (3D) treatm
ent plans that allow greater precision in ose elivery to the prostate to spare
the rectum an blaer. It is hope that intensity moulate raiotherapy (IMRT
) will further this cause. However, organ motion an aily variations in setup
have continue to pose challenges to external irraiation techniques. Although p
ermanent see implants avoi this problem, accurate placement of sees within th
e glan is technically challenging, an significant eviations from the preopera
tive plan may occur.2 These ifficulties potentially compromise accurate an rel
iable ose escalation with sparing of normal structures. Remote afterloaing wit
h high ose rate iriium192 (192Ir) theoretically remeies some of the osimetr
ic uncertainties of see implants an conformal external beam irraiation. This
technology can precisely place a single, movable high activity 192Ir source anyw
here insie an afterloaing neele, an then vary the time spent at a particular
location (well time) to control ose eposition. When use in the context of a
prostate implant with multiple neeles, one may eliver a highly conformal ose
to the periphery of the prostate while minimizing blaer an rectal oses. Fur
thermore, oses maybe ifferentially elivere within the prostate glan as esi
re while sparing the centrally locate urethra. Because well times may be aju
ste after the neeles have
Basic an avance techniques in prostate brachytherapy
434
been place, small inaccuracies in neele placement can be overcome. For these r
easons, HDR brachytherapy is currently the most accurate metho for elivery of
conformal irraiation to the prostate.3 Stuies of the raiobiology of prostate
cancer provie further support for the use of HDR. Recently, there have been rep
orts suggesting that prostate cancers may be more susceptible to large fraction
sizes compare with stanar fractionation.4,5 Base on the linearquaratic mo
el of clonogenic cell eath in response to irraiation, Brenner an Hall have su
ggeste that prostate cancers have an alphabeta ratio of approximately 1.5,4 an
there have been clinical ata to support this fining.5 Although a etaile is
cussion of the linearquaratic moel an its application to raiation osing is
outsie the scope of this chapter, such finings have prompte raiation oncolo
gists to consier hypofractionate regimens for prostate cancer.6 The potential
for increase late morbiity with large fraction sizes remains a concern, howeve
r, an for this reason the precise ose control affore by HDR makes it particu
larly useful for regimens incorporating hypofractionation. Patient selection an
pretreatment evaluation All patients with clinically localize isease may be a
ppropriate caniates for HDR therapy. At the Sweish Cancer Institute in Seattl
e, Washington, we have tene to treat those patients with a high tumor buren,
base on the following criteria: sextant biopsies with two or more zones positiv
e, leason scores of 7 or higher, an pretreatment prostatespecific antigen (PS
A) values greater than 10 ng/mL. This relatively averse selection has resulte
from the local presence of a permanent see program emonstrating successful mon
otherapy in patients with low risk isease, but there is little to suggest that
HDR woul be less effective in these patients. Martinez is currently investigati
ng HDR monotherapy in patients with favorable characteristics, specifically thos
e with stage T1T2a isease, leason scores of 7 or less, an PSA values of 10 n
g/mL or less. Preliminary results have been encouraging thus far.7 As more ata
regaring the efficacy an morbiity of this treatment become available, it is a
nticipate that more low risk patients will pursue HDR therapy. Patients with a
prior history of transurethral resection of the prostate (TURP) shoul be cautio
ne about the potential risk of incontinence, but in our experience even these p
atients have faire well, likely owing to the moest oses that may be aministe
re to the urethra without compromising peripheral zone coverage. Likewise, larg
e glan size (e.g. >60 cc) is not a contrainication. Although pubic arch interf
erence (PAI) may preclue neele placement in the anterior portions of large pro

states, this efect can be overcome by moifying source well times. We have impla
nte prostates over 100 cc with little ifficulty. All patients who may be consi
ering an HDR implant are require to unergo a complete pretreatment evaluation
, incluing appropriate biopsy with leason scoring, serum PSA evaluation, an 
igital rectal examination (DRE). Bone scans are recommene for patients with PS
A levels greater than 20 ng/mL, or leason scores >8. No pretreatment compute t
omographic (CT) scan or transrectal ultrasoun (TRUS) is require for planning,
although exceptions are mae if the brachytherapist iscovers an
High ose rate 192Ir prostate brachytherapy
435
exceptionally large glan on DRE, in which 23 months of neoajuvant anrogen abl
ation therapy may be use for ownsizing. Magnetic resonance imaging (MRI) has t
he potential to elineate areas of extracapsular extension or gross isease with
in the prostate, but this has not been stanar in our practice. Implant techniq
ue Our technique at the Sweish Cancer Institute is the one escribe below. The
re are, however, variations on this metho incluing the use of realtime osime
try, as escribe by Martinez et al, an the intereste reaer is referre to th
e citations liste. (1,812) However, the various techniques all have in common se
veral salient features, incluing transrectal ultrasoun guiance to place after
loaing neeles into the prostate, with threeimensional ose (3D) optimizatio
n base on the resulting neele istribution. Dose scheules have been highly va
riable across institutions, an our own practice patterns have also unergone si
gnificant evolution since our first report (Table 33.1). Preoperative bowel prep
inclues a low fiber iet for 23 ays prior to the implant, with only clear liqu
is
Table 33.1 High ose rate (HDR) brachytherapy: variations in prescribe oses
Institution
interisciplinaryBrachytherapy Center, Kiel, ermany
Pelvic EBRT* HDR boost
50 y pelvis 40 y prostate
Timing of No, operative implant proceures boost
9 y2 entire After 20 y 2 glan;15 y2 an40 y of to peripheral EBRT zone Sweis
h Cancer Institute, Seattle, Initial report: Initial: 3.04.0 2 weeks 1 WA 50.4 y
prostate y4 Current 8 before Current 4550 y2 EBRT y prostate William Beaumont H
ospital, Royal 46 y pelvis 5.56.6 y3 or Weeks 1 2 Oak, MI 8.251 0.5 y2 an 3 of E
BRT * EBRT, External beam raiotherapy.
allowe after lunch until minight on the evening before the proceure, followe
by one Fleets enema in the morning. Occasionally, an intraoperative rectal lava
ge may be neee if TRUS visualization is compromise by persistent stool in the
rectum. In the operating room, patients are preppe an rape in the lithotomy
position uner general or spinal anesthesia, but no foley catheter is inserte
at this point. TRUS is performe to assess glan size an align the prostate wit
h respect to the mounte template on the ultrasoun probe. enerally, we have al
igne the glan such that the 3D position of the template correspons to the pos
ition of the urethra; aerate gel can be inserte into the urethra to assist wit
h visualization but this is usually not necessary. Three raiopaque sees are
Basic an avance techniques in prostate brachytherapy
436
inserte at the apex uner TRUS guiance to later assist with target localizatio

n. Afterloaing neeles, usually 1520, are inserte into the prostate in a patter
n that is esigne to preferentially treat the peripheral zone. Relatively more
neeles are place in the peripheral zone of the prostate compare to the anteri
or glan, since tight control of osing is esire in this region, ue to the pr
oximity of the rectum an the propensity of cancers to arise there. Coorinate 3
D of the template, corresponing to the path of the urethra, is typically not lo
ae. We continue to use metal neeles to create tracks in the prostate an peri
neum through which flexible plastic neeles are substitute to allow for CT plan
ning as escribe later. After the plastic neeles are completely inserte, the
TRUS probe is remove an the plastic template with the neeles is suture to th
e patients perineum. A rectal examination is one to assess posterior placement o
f the neeles an also to place a bellaonna an opiate (B an O) suppository to m
inimize blaer spasms. The patient is then prepare for a cystoscopy. This allo
ws for visualization of the blaer neck while the transperineal neeles are av
ance. We attempt to place the superior portion of each neele such that some bu
lging of the blaer neck is seen on cystoscopy, without any sharp tenting of the
mucosa. This ensures that aequate coverage of the prostatic base can be obtaine
 while minimizing blaer trauma. This precision in neele placement is one of
the avantages of the HDR technique an has contribute to low rates of urinary
retention in the acute setting. Once the cystoscope is remove, a foley catheter
is place an connecte to blaer irrigation of normal saline, an the patient
is transferre from the operating room. After ischarge from the postanesthesia
care unit, the patient unergoes a treatmentplanning CT scan with the plastic a
fterloaing neeles in place. First, a lateral scout view is obtaine to verify
that the neele tips are just beneath the blaer. Next, images are obtaine fro
m the base to the apex at 5 mm intervals perpenicular to the neele array to re
cor neele position an ientify the prostate borers, blaer, urethra, an re
ctum. The target volume is efine as the prostates peripheral margin as rawn on
the postimplant CT scan, an 100% of the prescribe ose is elivere to this v
olume. Maximum urethral oses are kept to 120% of the prescription. In orer to
keep the urethral oses at this level, however, a small anterior portion of the
prostate may be omitte from the high ose region. iven the low probability of
isease in this location, we have continue with this compromise in osing, alth
ough typically the ose receive by this region is not lower than 80% of the pla
nne prescription. (See Figures 33.133.9.) Once the plan is reviewe an approve
, the patient is transferre to a shiele room for treatment. The afterloaing
evice is then connecte to the extraperineal portion of the interstitial cathet
ers, which are colorcoe at the time of CT scan to help ensure that they are m
atche with the appropriate source channels. The first treatment of 8 y to the
prostate periphery is elivere on the afternoon of the operative proceure an
typically lasts approximately 15 minutes, after which time the patient is transf
erre back to the hospital floor for overnight observation. We have maintaine p
atients on a morphine patientcontrolle analgesia (PCA), for comfort in aitio
n to continuous blaer irrigation with saline via a foley catheter. The next mo
rning, a secon application of 8 y is elivere an the catheters an template
are remove in unison. Before each ose application, plain raiographs are use
to etermine the position of the catheters with respect to the marker sees plac
e at the prostatic apex intraoperatively. Occasionally,
High ose rate 192Ir prostate brachytherapy
437
minor shifts in position are neee, but this is rare. Previously, we aminister
e four applications of 3.04.0 y each, but the majority of patients have tolerat
e the more hypofractionate regimen of two applications, which offers patients
the avantage of a shorter inpatient stay. Martinez an others have emonstrate
success with fewer applications, an as iscusse above this may be relatively
avantageous from a raiobiologic stanpoint.5 Perioperative morbiity has been
limite to perineal bruising an mil iscomfort that typically resolve in a few

ays. Acute urinary retention has been a rare event, occurring in 2% of patient
s. Patients are ischarge from the hospital shortly after the secon fraction a
fter they have emonstrate the ability to urinate without the use of the foley
catheter. Outcomes Outcomes with this technique have compare favorably with tho
se from prostatectomy, external irraiation, an permanent see implants perform
e in patients with similar stages of isease. At William Beaumont Hospital, Roy
al Oak, Michigan, patients with any of the following tumor characteristics were
treate with external beam an HDR boost; leason score 7, T2b or T3 isease, or
PSA levels 10.0 ng/mL. In this relatively high risk population, biochemical contr
ol was achieve in 67% of patients at five years using the American Society for
Therapeutic Raiology an Oncology (ASTRO) efinition of biochemical failure.13
Using the same enpoint, results from our institution have been comparable. In a
n early pilot report from our institution, patients with pretreatment PSA levels
<20 ng/mL were foun to have an 84% freeom from PSA progression at 5 years.14
Eulau recently upate this series an stratifie patients accoring to observe
risk factors: pretreatment PSA level >15 ng/mL; leason score > 6; an tumor st
age >T2b. Outcomes were reporte using the ASTRO criteria. At 10 years, patients
with 01 risk factor showe biochemical control rates of 97% an 69%, respectivel
y. Patients with more avance isease i not fare as well, with significantly
lower biochemical relapsefree rates. However, it is not known how many of these
failures were ue to isolate local progression, as only three patients ha a b
iopsyproven local recurrence.15 Biopsy proven local control at 18 months has be
en at least 90% in two stuies that prospectively performe such evaluations.8,1
2 Interpretation of postirraiation biopsies is complicate,16 but these local c
ontrol results have been encouraging an consistent with the favorable biochemic
al enpoints reporte for earlytointermeiate stage patients thus far. Table 3
3.2 shows reporte results from various institutions using HDR therapy. Acute an
 long term morbiity rates have been very low in the available reports on HDR b
oost techniques, although prospective quality of life assessments are still lack
ing. In our experience, acute urinary retention has evelope in only 23% of pati
ents an late urethral strictures were seen in only 6.7%.15 Investigators at Wil
liam Beaumont Hospital foun only 5% grae 3 acute toxicity, with no patients ex
periencing grae 45 complications. Late urinary toxicity was corresponingly low,
an no patient evelope any late grae 3 gastrointestinal morbiity.13 At 5 ye
ars, the actuarial rate of grae 3 complications was 9%. Of patients, 29% evelo
pe impotence at a meian of nearly one
Basic an avance techniques in prostate brachytherapy
438
year posttreatment. In the publishe series from Sween, most patients experienc
e some mil iarrhea within 6 months of therapy, but only four patients reporte
 persistent rectal symptoms severe enough to require treatment after that time.
8 Urinary morbiity beyon 6 months was seen in only six of 50 patients, with on
ly 8% suffering grae 3 uropathy. The Kiel group has the longest experience an
has reporte similarly low rates of severe late toxicity. In their experience, a
TURP performe within 6 months of HDR implant le to markely higher rates of u
rinary symptoms,1 but overall rates of grae 23 complications were less than 10%
(Table 33.3). In general, the low complication rates reporte in HDR series have
been one of the more encouraging aspects of this form of local treatment. Acute
an long term morbi ity have compare favorably with those reporte from other
forms of therapy. Most likely, this is ue to the precise osecontrol that HDR
allows compare to external beam an permanent see implants. The physical ose
istributions prouce with HDR strongly suggest that this treatment shoul off
er the wiest therapeutic winow currently available with efinitive raiation.
However, as mentione above, prospective quality of life ata are still lacking
an protocols are currently being evelope to etermine whether these seemingly
low complication rates will withstan further scrutiny. Future irections Curre
nt ata have establishe HDR brachytherapy as an effective form of local treatme

nt for prostate cancer. However, several areas remain for further investigation.
The optimal osing regimen for HDR remains unclear. Initially, we use four app
lications over a 40 hour perio, elivering 3.04.0 y with each fraction, followe
 by 50.4 y of external beam irraiation elivere with a four fiel technique.
Martinezs initial series was treate using three fractions of 5.56.0 y each in c
ombination with 46 y of EBRT. Results from both series were encouraging an sin
ce those initial reports, both of our institutions have move to using two fract
ions of HDR boost. We are currently using 8 y with each fraction, while the Wil
liam Beaumont group has reporte early results with ose escalation up to 21 y
elivere in two implants.13 Dose inhomogeneities that occur with brachytherapy
complicate assessment of optimal osing. While prescriptions to the periphery of
the glan can be stanarize, the number of catheters place an the variation
s in well times can prouce highly variable oses within the target volume. Fur
thermore, efinitions of the prostate periphery can be variable across experienc
e brachytherapists, often a result of personal preference.2 Although target ef
inition for osimetric analysis of permanent see implants has been geare to mi
nimizing inclusion of extraprostatic tissue,17 we have continue to use a more g
enerous target volume in our HDR practice, an these ifferences may complicate
irect comparisons of oseepenent outcome. As more ata are gathere, osevo
lume histogram (DVH) analysis may help eluciate some of these issues. One of th
e limitations of current HDR techniques is that supplemental EBRT is generally r
ecommene for all patients. However, there may exist a subgroup of favorable st
age patients for whom monotherapy with HDR alone may be aequate, an Martinez i
s actively enrolling patients on such a protocol.7 Preliminary results have been
High ose rate 192Ir prostate brachytherapy
439
publishe using four fractions of 9.5 y each, an as for boost techniques, opti
mal osing strategies also remain uncertain for monotherapy. Publications have t
hus far suggeste that morbiity from the proceure is fairly low, but etaile
an prospective quality of life analysis with actuarial assessment an valiate
instruments is still lacking. As these ata are generate, DVH analysis may all
ow for specific osing guielines to be mae for optimal HDR techniques. iven t
he raiobiologic consierations escribe previously, couple with the precision
of ose application from HDR,
Figure 33.1 Intraoperative TRUS setup. Note how the mounte blue guiance templa
te is attache to the perineal applicator (top showing) in the picture. Steel ne
eles will be passe through the template an applicator in the esire loaing
pattern.
Basic an avance techniques in prostate brachytherapy
440
Figure 33.2 Placement of flexible catheters. After metal neeles are place in t
he esire pattern, flexible catheters are inserte into the tracks create by t
he metal neeles. Each of the flexible catheters contains a metal wire to mainta
in rigiity uring placement. Since these catheters will hol the iriium source
, their integrity must be ensure.
it appears that this technique woul provie a higher therapeutic ratio than oth
er forms of raiation treatment. If HDR methos are able to convincingly emonst
rate a significantly improve quality of life for the same egree of tumor contr
ol (or better) as other forms of therapy, the proceure is likely to be offere
to increasing numbers of patients in the years to come.
Figure 33.3 The perineal applicator an flexible catheters are in place, with th
e ultrasounmounte template remove. The applicator is stitche in position. A

small screw beneath the surgeons inex finger is use to tighten the applicator
apertures aroun each catheter, but not before final ajustment of the neele po
sition uring cytoscopy.
High ose rate 192Ir prostate brachytherapy
441
Figure 33.4 Cytoscopy. The patient is remove from lithotomy position, an a cyt
oscopy is performe. Irrigation is use to clear seminal flui from the blaer
for improve visualization of the blaer neck.
Figure 33.5 Cytoscopy image. The blaer neck with the cytoscope in the urethra
is visualize while the neeles are avance. Some bulging of the blaer neck is
esire, but any sharp tenting of the mucosa is to be avoie. In this patient,
meian lobe hypertrophy obscures the urethral opening.
Basic an avance techniques in prostate brachytherapy
442
Figure 33.6 Neeles may be avance up to 4 cm at the time of cytoscopy epenin
g on initial placement.
Figure 33.7 Lateral CT scout view of the neeles in place. Note the position of
the neele tips in relation to the blaer (fille with contrast material). Prec
ise placement of the source carriers at the prostatic base is one of the attract
ive features of the high ose rate (HDR) brachtherapy technique.
High ose rate 192Ir prostate brachytherapy
443
Figure 33.8 Compute tomography (CT) image of prostate with catheters in place.
A peripheral loaing pattern is use, with some sparing of the anterior zone.
Figure 33.9 Example of treatment isoose plan. The urethra in the center of the
glan is kept to less than 120% of the prescribe ose.
Basic an avance techniques in prostate brachytherapy
444
Table 33.2 Reporte biochemical outcomes from institutions performing high ose
rate (HDR) boost
Stuy
Mate 199814
No. patients Meian PSA (ng/mL)
104 12.9
Meian follow 5 year bNEDa up
45 mths
iPSA<20;
161 9.9
12.15 8
with no
logy an
ecifie,

84%b iPSA>20:50% 104 12,9 6.3 yrs Overall: 83% 5 yrs Euiau15 77% 10 yrs
2.5 yrs 83% Kestin 200013 Borghee 19978 50 NR 45 mths 84%:18 mthsc 144
yrs 74%:5 yrs alalae 20021 (mean: 25.6) 69%:8 yrs a bNED, biochemical
evience of isease. Base on the American Society of Therapeutic Raio
Oncology (ASTRO) efinition of biochemical failure, unless otherwise sp
b Results stratifie by pretreatment prostatespecific antigen level (i

PSA), in ng/mL. c Define as patients with PSA level of <1.0 ng/mL,  Three cons
ecutive rises in PSA, greater than 1.0 ng/mL.
Table 33.3 Longterm morbiity*
Stuy
Mate 199814 Kestin 200013 alalae 20021

astrointestinal
enitourinary
2% grae 2 proctitis 6.7% urethral stricture No grae 3 morbiity 4% stricture 4
% grae 3 2% grae 3 (cystitis) 7% grae 2 4% grae 2 10% grae 1 12% grae 1 Borg
hee 19978 8% grae 2 proctitis 12% grae 13 No grae 3 morbiity 0 urethral stri
ctures * Base on the Raiation Therapy Oncology roup (RTO) graing scale, occ
urring beyon 6 months after therapy.
References
1. alalae RM, Kovacs , Schultze J, et al. Longterm outcome after elective irr
aiation of the pelvic lymphatics an local ose escalation using highoserate
brachytherapy for locally avance prostate cancer. Int J Raiat Oncol Biol Phy
s 2002; 52(1):8190. 2. Wallner K, Blasko J, Dattoli MJ. Prostate brachytherapy ma
e complicate, 2n en. Smart Meicine Press, 2001:6.16.42; 9.19.44. 3. Hsu IC, P
ickett B, Shinohara K, et al. Normal tissue osimetric comparison between HDR pr
ostate implant boost an conformal external beam raiotherapy boost: potential f
or ose escalation. Int J Raiat Oncol Biol Phys 2000; 46(4):851858. 4. Brenner D
J, Hall EJ. Fractionation an protraction for raiotherapy of prostate carcinoma
. Int J Raiat Oncol Biol Phys 1999; 43:10951101. 5. Brenner DJ, Martinez AA, Em
unson K, et al. Direct evience that prostate tumors show high sensitivity to
fractionation (low / r tio), simil r to l te-responding norm l tissue. Int J R di
t Oncol Biol Phys 2002; 52(1):613.
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r chyther py
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6. M dsen B, Jones D, His RA, et l. Development of pp r tus nd ph se I tri l
for stereot ctic hypofr ction ted ccur te r diother py of the prost te (SHARP).
Proceedings of the 43rd Annu l Meeting of the Associ tion of Ther peutic R diol
ogy nd Oncology (ASTRO), 2001. 7. M rtinez AA, P t ki I, Edmundson G, et l. Ph
se II study of the use of conform l high-doser te
r chyther py s monother py
for the tre tment of f vor
le st ge prost te c ncer: fe si
ility report. Int
J R di t Oncol Biol Phys 2001; 49(1):6169. 8. Borghede G, Hedelin H, Holm ng S, e
t l. Com
ined tre tment with tempor ry short-term high dose r te Iridium-192
r
chyther py nd extern l
e m r diother py for irr di tion of loc lized prost ti
c c rcinom . R diother Oncol 1997; 44:237244. 9. Kini VR, Edmundson GK, Vicini FA
, et l. Use of three-dimension l r di tion ther py pl nning tools nd intr oper
tive ultr sound to ev lu te high-dose-r te prost te
r chyther py impl nts. Int
J R di t Oncol Biol Phys 1999; 43(3):571578. 10. Dem nes DJ, Rodriguez RR, Altie
ri GA. High dose r te prost te
r chyther py: the C liforni Endocuriether py (C
ET) method. R diother Oncol 2000; 57(3):289296. 11. M rtinez AA, Kestin LL, Strom

erg JS, et l. Interim report of im ge-guided conform l highdose-r te
r chythe
r py for p tients with unf vor
le prost te c ncer: the Willi m Be umont ph se I
I doseesc l ting tri l. Int J R di t Oncol Biol Phys 2000; 47(2):343352. 12. Stro
m
erg J, M rtinez A, Gonz lez J, et l. Ultr sound-guided high dose r te conform
l
r chyther py
oost in prost te c ncer: tre tment description nd prelimin ry
results of ph se I/II clinic l tri l. Int J R di t Oncol Biol Phys 1995; 33(1
):161171. 13. Kestin LL, M rtinez AA, Strom
erg JS, et l. M tched-p ir n lysis
of conform l high-doser te
r chyther py
oost versus extern l-
e m r di tion th
er py lone for loc lly dv nced prost te c ncer. J Clin Oncol 2000; 18(15):28692

880. 14. M te TP, Gottesm n JE, H tton J, et l. High dose-r te fterlo ding Iri
dium-192 prost te
r chyther py: fe si
ility report. Int J R di t Oncol Biol Phy
s 1997; 41(3):525533. 15. Eul u SM, M te TP, V nHolle
eke L, et l. High dose r t
e Iridium192
r chyther py in loc lized prost te c ncer: results nd toxicity wi
th m ximum follow-up of 10 ye rs. Proceedings of the 42nd Annu l Meeting of the
Associ tion of Ther peutic R diology nd Oncology (ASTRO), 2000. 16. Prestige B,
K pl n I, Cox RS, et l. Predictors of surviv l fter positive post-irr di ti
on prost te
iopsy. Int J R di t Oncol Biol Phys 1993; 28:1722. 17. B dioz m ni K
R, W llner K, C v n gh W, Bl sko J. Comp r
ility of CT-
sed nd TRUS
sed pros
t te volumes. Int J R di t Oncol Biol Phys 1999; 43(2):375378.
34 High dose r te prost te
r chyther py. Tre tment pl nning nd results from Me
mori l Slo n-Kettering C ncer Center
Yoshiy Y m d Introduction Br chyther py represents the e rliest use of r dioth
er py in the tre tment of prost te c ncer. B rringer descri
ed using r dium need
les to tre t prost te c ncer t Memori l Hospit l s e rly s 1917.1 Tod y, the
use of high dose r te (HDR)
r chyther py for the definitive tre tment of prost
te c ncer rem ins st nd rd tre tment option t Memori l Slo n-Kettering C ncer
Center (MSKCC). There re
oth physic l nd r dio
iologic r tion le for the use
of HDR
r chyther py which m ke it n excellent technique to tre t prost te c n
cer. High dose r te
r chyther py c theters securely hold the prost te in reli

le tre tment position. There is very little likelihood of t rget org n motion.
Simil rly, the position of critic l norm l tissue structures, such s urethr
nd rectum, c n
e ccur tely loc ted with high degree of confidence. Therefore
, there is very little uncert inty reg rding doses received
y the t rget or nor
m l tissue. The high degree of dosimetric cert inty which HDR provides en
les c
linici ns to prescri
e l rge fr ctions of r diother py with ssur nce th t the d
ose is delivered s prescri
ed. Elimin tion of such uncert inties gives HDR
r c
hyther py distinct dv nt ge over extern l
e m r diother py (EBRT), which mus
t ccount for org n motion
y exp nding the volume of irr di ted tissue
eyond t
he prost te to ensure th t the intended dose is delivered to the t rget. Therefo
re, surrounding norm l tissues dj cent to the prost te, such s the rectum, wil
l receive r di tion to l rger volume rel tive to HDR tre tment. Tre tment pl n
ning Tre tment pl nning t MSKCC utilizes computeroptimized postimpl nt computed
tomogr phy (CT)
sed dosimetry. E ch HDR c theter s well s the urethr , rectu
m, nd
l dder re re dily visu lized, m king ex ct dosimetry possi
le without t
he worry of org n motion. With str tegic c theter pl cement, postimpl nt dosimet
ry c n re dily
oost re s of concern, including re s suspicious for extr c psu
l r extension of dise se s well s intr prost tic re s suspicious either
y
i
opsy or MRI/MRI spectroscopy. Although HDR delivers inhomogeneous dosimetry, thi
s is dv nt geous in the setting of prost te c ncer. With computer optimiz tion,
structures, such s the urethr , c n
e m de to
e rel tively cold while re s of
concern c n
e m de hot rel tive to the prescri
ed dose
High dose r te prost te
r chyther py
447
(see Figure 34.1). Thus, with proper c theter pl cement, the peripher l zone of
the prost te or other tumor-
e ring re s c n e sily receive over 150% of the pr
escri
ed dose while urethr l doses re m int ined t less th n 120% of the presc
ri
ed dose. This phenomenon is due to the inverse squ re l w of
r chyther py ph
ysics. Since r di tion doses f ll off in n inverse squ re rel tionship rel tive
to dist nce, high doses of r di tion c n
e given to the prost te, while the re
ctum receives signific ntly lower dose of r di tion. CT-
sed tre tment pl nni
ng w s performed using softw re developed in-house. The t rget volume, urethr ,
rectum, s well s e ch
r chyther py c theter w s contoured on e ch relev nt CT
slice. Computer-optimized inverse tre tment pl nning w s performed using softw
re developed in-house. The m in dose-constr int v ri
les governing the optimiz
tion engine include 100% t rget volume cover ge, urethr Dm x120%, dose uniformit

y points
etween 100% nd 175%, nd dose conformity index
etween 1 nd 1.1. In

c ses of extr c psul r extension, the re s identified on MRI sc ns were include
d in the t rget volume. Doses were prescri
ed to the isodose line which encomp s
sed the t rget volume, except t the
l dder neck, where lower dose cover ge w s
ccepted. In some c ses, due to geometric nd n tomic consider tions, full dos
e w s not given to the prost te dj cent to the
Figure 34.1 Computed tomogr phy (CT)-
sed tre tment pl nning. E ch c theter is
identified s n ir pocket on CT. Different colored lines represent levels of r
di tion dose intensity (isodose lines). Extr c psul r
B sic nd dv nced techniques in prost te
r chyther py
448
extension identified on m gnetic reson nce im ging (MRI) nd impl nted with high
dose r te (HDR) c theters (circled in yellow).

l dder neck nd the superior nterior
se of the prost te, where no gl ndul r
tissue exists nd the likelihood of c rcinom is very low (see T
le 34.1). Repo
rted m ximum doses were c lcul ted voxel-
y-voxel, nd often represent single
voxel m ximum. Thus, the extreme m ximum doses c lcul ted to the urethr m y pp
e r to
e high,
ut typic lly represent doses to very sm ll volumes. Over ll, th
e me n urethr dose w s less th n 120% of the prescri
ed dose (see T
le 34.2).
T
le 34.1 T rget dosimetry
Medi n R nge
V150 (%} 41.3 V100 (%) 97.8 D90 (Gy) 19.2 2370 94100 1722
At MSKCC, we undertook det iled n lysis of rect l doses given
y HDR techniqu
es comp red to three-dimension l conform l extern l
e m r di tion (3D-CRT). A s
ignific ntly lower dose of r di tion w s received
y the rectum with the use of
HDR rel tive to 3D-CRT. The HDR nd 3D-CRT rect l volumes for e ch p tient were
not found to
e signific ntly different. The me n rect l V50, V90, nd V100 for
HDR vs 3DCRT w s found to
e 6.3 cc
T
le 34.2 Urethr l dosimetry
Medi n R nge
Urethr l Dm x 138% Urethr l v. dose 118% Urethr l V100 (cc) 2,2 Urettel V150 (c
c) 0 142331% 50145% 14 02
vs 40.6 cc, 0.6 cc vs 14.8 cc, nd 0.3 cc vs 11.7 cc, respectively. The me n D50
, D90, D100, nd Dm x were found to
e 5.1 Gy vs 35 Gy, 2.7 Gy vs 23.4 Gy, 2.4 G
y vs 15.1 Gy, nd 20.7 Gy vs 53.4 Gy for HDR doses nd 3D-CRT doses, respectivel
y. Rect l doses rel tive to t rget prescription doses (50.4 Gy for 3D-CRT nd 16
.5 Gy for HDR) were found to
e 11% vs 30%, 15% vs 47%, nd 27% vs 71% (HDR vs 3
D-CRT) for D100, D90, nd D50, respectively. The p-v lues for ll n lyzed f cto
rs were found to
e highly signific nt (p<0.00001). Simil r findings h ve
een r
eported
y others.2 Currently, t MSKCC, CT-
sed computeroptimized postimpl n
t tre tment-pl nning lgorithm is utilized to provide HDR
oost of 19.5 Gy to th
e periphery of the prost te given in three fr ctions (6.5 Gy e ch) t le st 6 ho
urs p rt, with n ddition l 50.4 Gy given to the prost te nd semin l vesicles
in 28 fr ctions using 3D-CRT techniques. We re continuing progr m of dose es
c l tion with the HDR portion of tre tment.
High dose r te prost te
r chyther py
449
R dio
iology Bec use of less tre tment uncert inty nd computeroptimized tre tme
nt pl nning providing lower doses to critic l norm l structures, HDR is ide lly

suited for hypofr ction ted ther py. Hypofr ction refers to fr ction tion sche
dule which prescri
es l rger doses of r di tion (i.e. >22.5 Gy per fr ction), whi
le reducing the num
er of fr ctions given. Although the tot l cumul tive dose of
r di tion is typic lly lower th n with st nd rd fr ction tion schedules,
ec us
e of the gre ter imp ct of l rger doses of r di tion per fr ction, the
iologic
lly effective dose chieved c n
e equ l to or gre ter th n th t of st nd rd fr
ction tion schemes. One method of expressing the re ction of cells to r di tion
is expressed s n / r tio
sed on line r-qu dr tic form lism. Prost te c ncers
re thought to
e on the extreme low end of the spectrum of / r tios (1.23.5 in vitr
o) of hum n m lign ncies.36 Furthermore, prost te c ncer cells lso exhi
it low
l
eling index (0.62.5%), nd Tpot estim tes r nge from 16 d ys to 64 d ys, ll
suggesting th t prost te c ncers prolifer te slowly.5 Bec use sm ll / r tio impl
ies l rger sensitivity to ch nges in fr ction size, hypofr ction ted tre tment
schedules re more likely to
e effective g inst prost te c ncer cells. Also,
tumor repopul tion is n unlikely signific nt r dio
iologic p r meter.6 T
le 34
.3 illustr tes c lcul ted
iologic effective doses (BED) for v rious HDR schedul
es
sed on n / of 10 comp red to 1.5. The most extreme ex mple of hypofr ction t
ion included in T
le 34.3 is 9.5 Gy4 (no EBRT), which lso permits the most sp r
ing of cute re cting tissues,
ut delivers the highest effect on l te respondin
g tissue, such s prost te c ncer. As n ex mple
sed on ctu l p tients, n n
lysis of p tients tre ted with HDR prost te
r chyther py s
oost found impr
oved prost te-specific
T
le 34.3 B iologic effective doses (BED) comp red
BED10 BED1.5
8640 cGy/48 102.0 190 5040 cGy/28+: 59.5 111 10 Gy2 100.0 264 6 Gy3 88.5 201 5 Gy4
89.5 198 8.5 Gy2 91.0 224 9.5 Gy4 74.0 279 Note: time corrections re not included
in the c lcul tions.

ntigen (PSA) rel pse-free surviv l when the num
er of fr ctions w s reduced fro
m 3 to 2. This clinic l correl tion suggests th t prost te c ncers do h ve low / r
tios th t re more sensitive to ch nges in fr ction size.7 T
le 34.3 lso sugg
ests th t hypofr ction tion should confer sp ring of e rly re ctions rel tive to
l te re ctions. Thus, there is signific nt r dio
iologic rgument for the use
of hypofr ction ted tre tment schedules in the m n gement of prost te c ncer.
B sic nd dv nced techniques in prost te
r chyther py
450
Technique P tients underwent HDR impl nt prior to extern l
e m r diother py. Th
e
r chyther py is typic lly performed under gener l nesthetic. E ch c theter
is pl ced tr nsperine lly under tr nsrect l ultr sound (TRUS) guid nce through
templ te which is tt ched to the perineum. A cystoscopy is performed fter c
theter pl cement to ensure th t no c theters tr nsgress the
l dder mucos or ur
ethr . The p tients re pl ced on strict
edrest while c theters re in pl ce. A
three-w y c theter is pl ced t the end of the procedure, nd
l dder irrig tio
n is performed on PRN ( s required)
sis. P tients re given intr venous nti

iotics. CT-
sed computer-optimized tre tment pl nning is performed on p tients
fter recovery from the nesthesi . C theters re m rked t the templ te with p
erm nent ink s qu lity control me sure g inst c theter migr tion. Antiem
oli
sm infl ting
oots re prescri
ed. P tients re pl ced on strict
edrest while t
he c theters re in pl ce. All p tients re tre ted with 192Ir source. The imp
l nt is removed t the
edside fter the l st fr ction h s
een dministered. P
tients re usu lly disch rged following sever l hours of o
serv tion. Outcomes A
det iled n lysis of 54 p tients with loc lized prost te c ncer who were tre te
d with HDR
oost nd 3D-CRT to
T
le 34.4 Clinic l ch r cteristics of high dose r te (HDR) p tients
Medi n R nge

Num
er of p tients 54 Age (yrs) 65 4575 Gle son score 7 69 Clinic l st ge T1c T1
T3

Pretre tment PSA 9.2 1.260 Pretre tment IPSS 4 018 Pretre tment potency 74% Foll
ow-up (mths) 18 1238 PSA, prost te-specific ntigen; IPSS, intern tion l prost te
symptom
the prost te (3D-CRT)
etween 1998 nd 2000 t MSKCC w s undert ken. Of the p ti
ents, 50 were prescri
ed 550 cGy3, nd 4 p tients were prescri
ed 600 cGy3; 7 p ti
ents received 4500 cGy in 25 fr ctions nd 47 p tients received 5040 cGy in 28 f
r ctions using six
e m 3D-CRT technique. All c ses tre ted during this time i
nterv l were included in this n lysis. All p thology w s reviewed t our instit
ution prior to initi ting tre tment. Clinic l ch r cteristics of the p tients r
e summ rized in T
le 34.4. Typic lly, p tients considered for HDR were not
High dose r te prost te
r chyther py
451
f vor
le p tients, with either Gle son score 6, PSA >10, or >T2
. HDR
r chyther
py w s not encour ged s tre tment option if the p tient presented with signi
fic nt urin ry symptoms (intern tion l prost te symptom score; IPSS >12), if the
p tient could not undergo CT-
sed tre tment pl nning (i.e.
il ter l hip repl
cements), could not toler te nesthesi , or otherwise could not toler te the pro
cedure. If the p tient w s thought to h ve prost te l rger th n 60 cc, hormon
l ther py (HT) consisting of com
ined ndrogen
lock ge w s dministered for pp
roxim tely 3 months prior to the impl nt. P tients with p rticul rly high risk f
e tures (Gle son >8, PSA>10, T3 dise se) were considered for HT prior to nd in
conjunction with r di tion ther py. Follow-up w s performed regul rly t 36 month
interv ls, including the use of IPSS/qu lity of life questionn ires nd R di ti
on Ther py Oncology Group (RTOG) toxicity gr ding. No p tients were lost to foll
ow-up. K pl nMeier st tistics were used for n lysis. The medi n follow up for t
he entire group w s 24 months (1644 months). Of the p tients, 74% reported full p
otency (sufficient for v gin l intercourse) prior to initi ting tre tment; 41% o
f p tients were given neo- djuv nt HT prior to tre tment. The medi n pretre tmen
t IPSS w s 4 (018). The clinic l ch r cteristics of this cohort re summ rized in
T
le 34.4. No gr de 3 RTOG urin ry toxicity h s
een reported. Three p tients
experienced urin ry retention requiring c theter t some time during their pos
ttre tment course. In ll c ses, the use of c theters for urin ry retention w s
tempor ry. Two of the three p tients h d tr um tic c theteriz tions during the i
mpl nt procedure ( fter cystoscopy). As illustr ted in Figures 34.2 nd 34.3, si
gnific nt urin ry toxicity fter tre tment w s unusu l. The me n pretre tment IP
SS w s 5.3. The IPSS score pe ked t the time
Figure 34.2 Urin ry toxicity following high dose r te (HDR)
r chyther py. RTOG,
R di tion Ther py Oncology Group.
B sic nd dv nced techniques in prost te
r chyther py
452
Figure 34.3 Urin ry toxicity following high dose r te (HDR)
r chyther py. Inter
n tion l prost te symptom scores (IPSS) nd qu lity of life (QOL) scores.
of first follow-up (3 months), nd su
sequently settled ne r to pretre tment lev
els. The s me p ttern w s noted for RTOG urin ry toxicity scores. Of p tients, 7
% reported gr de 2 toxicity prior to tre tment, nd pe ked t 15% t the time of
first follow-up; 9% of p tients continued to report gr de 2 toxicity in su
sequ
ent follow-up. These p tients were still using lph -
lockers to ssist urin tio
n. Potency (erections sufficient for v gin l penetr tion) w s lso rel tively we
llm int ined. Prior to tre tment, 76% of p tients reported full potency, 13% wer
e
le to chieve erections with the use of silden fil citr te (Vi gr ) nd 11%
were not
le to chieve erections with the use of silden fil citr te. At the ti

me of l st follow-up, 58% were still potent without the use of medic tions, nd
28% of p tients reported erections sufficient for v gin l penetr tion with the u
se of silden fil citr te. This represents 15% incre se in the use of silden fi
l citr te. Over ll, only one p tient who w s potent prior to tre tment experienc
ed loss of sufficient erectile function nd did not respond to silden fil citr t
e (see T
les 34.5 nd 34.6). P tients were s tisfied with their urin ry qu lity
of life (QOL). Urin ry QOL w s r ted
y e ch p tient t e ch follow-up, using
sc le of 06 (0, delighted; 1, ple sed; 2, mostly s tisfied; 3, mixed feelings; 4
, mostly diss tisfied; 5, unh ppy; 6, terri
le). The me n pretre tment QOL w s 1
.5, pe ked t 3 months with me n v lue of 2, nd t 12 months, the me n report
ed v lue w s 1.2 (see Figure 34.2). Signific nt rect l toxicity w s not seen in
this cohort. No p tients experienced RTOG cute or l te rect l RTOG toxicity gr
de 2 or higher. No PSA f ilures (ASTRO definition8) h ve yet
een o
served in th
is cohort, lthough medi n follow-up is still
High dose r te prost te
r chyther py
453
T
le 34.5 Potency r tes of high dose r te (HDR) p tients
MSKCC HDR
Full potency (%) Silden fil citr te (%) Impotent (%) Pre tx, pretoxicity.
Pre tx L st follow-up % Ch nge
76 13 13 58 28 14 24% 115% 7%
Table 34.6 Potency rates at two years postreatment compare to hormonenanve pati
ents
Hormone nave HDR 3D CRT
Full potency (%) 83 78 Potent incluing silenafil citrate (%) 93 84 HDR, high 
ose rate; 3DCRT, threeimensional conformal external beam raiotherapy.
short. One of the longest HDRbase outcomes in the literature comes from alala
e et al.9 HDR was utilize to eliver two fractions of 15 y an 9 y to the pro
state, while giving 50 y to the pelvis to 144 patients. The meian followup fo
r this group was 8.2 years (range: 514). Of the patients, 60% ha a PSA>10 (mean
PSA: 25.6) an
Table 34.7 Long term outcomes for patients treate with high ose rate (HDR) bra
chytherapy9
Prognostic groups Mesian PSA (ng/mL) Meian grae (WHO 5 year bNED 8 year bNED
Mostofi) (%) (%)
93 81 64 64 75 32
PSA<10 ng/mL 1 46 2 94.7 2 PSA 1020 ng/mL 12.8 2 83.3 12 PSA>20 ng/mL 1 36 2 68 2
PSA<10 ng/mL 3 3.8 3 72 PSA 1020 ng/mL 15.7 3 77 3 PSA>20 ng/mL 3 40.4 3 38 
, RTO (Raiation Therapy Oncology roup) grae. PSA, prostatespecific antigen;
bNED, biochemical with no evience of isease.
nearly one thir of the patients were stage T3. The majority of patients were n
ot low risk patients. The reporte outcomes base on risk stratification are pre
sente in Table 34.7. In this relatively high risk cohort with mature followup,
HDR in conjunction with EBRT offers a high rate of isease control.
Basic an avance techniques in prostate brachytherapy
454
Summary There is mounting evience that prostate cancers ten to be relentless,
but very slow in the progression through their clinical natural history, in comp

arison with most other human malignancies. Using linearquaratic moeling, rai
obiologists suggest that localize prostate cancers may have / r tios in the neigh

orhood of 1.21.5the extreme low end of hum n m lign ncies, which re typic lly s
signed / v lues 8. Such d t would suggest th t hypofr ction tioned schedules, for
which high dose r te (HDR)
r chyther py is ide lly suited, is the optim l ppro
ch for the tre tment of prost te c ncer. A low / would lso suggest th t t equiv
lent dose levels, the cute toxicity should
e lower with hypofr ction tion. Ou
r experience indic tes th t signific nt cute toxicity is infrequent. This is li
kely due to superior dosimetry provided
y computer-optimized tre tment pl nning
nd lso the inherent rel tive sp ring of e rly responding tissues fforded
y
hypofr ction tion. Gr de 3 l te rect l toxicity ssoci ted with HDR
r chyther p
y h s
een reported
etween 04%.9,10 The p tients with signific nt l te toxicity
were tre ted when sophistic ted computer optimized CT-
sed tre tment pl nning w
s not v il
le, nd this complic tion is now extremely r re in current tre tme
nt pl nning. Our d t indic te th t computer optimiz tion c n successfully provi
de excellent t rget volume cover ge while sp ring rectum nd urethr . Although t
he follow-up of our cohort is not long, the cute toxicity h s
een thus f r ver
y ccept
le. Reports with long term follow-up utilizing HDR
r chyther py re n
ow emerging. Although most of the p pers descri
e rel tively high risk p tients,
the
iochemic l with no evidence of dise se (
NED) r te is surprisingly high.912
Thus the HDR hypofr ction tion p r digm ppe rs to
e well suited for the tre t
ment of prost te c ncer from ll spects of m n gementtre tment pl nning, r dio
i
ology, toxicity, s well s dise se control. References
1. B rringer BS. R dium in the tre tment of c rcinom of the
l dder nd prost t
e. JAMA 1917; 58:12271230. 2. Hsu IJ, Pickett B, Shinoh r K, et l. Int J R di t
Oncol Biol Phys 2000; 46(4):851858. 3. King CR, Fowler IF. A simple n lytic der
iv tion suggests th t prost te c ncer lph /
et r tio is low. Int J R di t Onco
l Biol Phys 2001; 51(1):213214. 4. Fowler J, Ch ppell R, Ritter M. Is lph /
et
for prost te tumors re lly low? Int J R di t Oncol Biol Phys 2001; 50(4): 1021103
1. 5. Duschesne GM, Peters LI. Wh t is the / r tio for prost te c ncer? R tion le
for hypofr ction ted high-dose r te
r chyther py. Int J R di t Oncol Biol Phys
1999; 44(4):747 748. 6. Brenner DJ, H ll EJ. Fr ction tion nd protr ction for r
diother py of prost te c rcinom . Int J R di t Oncol Biol Phys 1999; 43(5):109511
01.
High dose r te prost te
r chyther py
455
7. Brenner DJ, M rtinez AA, Edmundson GK, et l. Direct evidence th t prost te t
umors show high sensitivity to fr ction tion (low / Biol Phys 52(1):613. r tio), si
mil r to l te-responding norm l tissue. Int J R di t Oncol 8. Americ n Society f
or Ther peutic R diology nd Oncology Consensus P nel. Consensus st tement: guid
elines for PSA following r di tion ther py. Int J R di t Oncol Biol Phys 1997; 3
7(5):10351041. 9. G l l e RM, Kov cs G, Schultze J, et l. Long-term outcome fte
r elective irr di tion of the pelvic lymph tics nd loc l dose esc l tion using
high-dose-r te
r chyther py for loc lly dv nced prost te c ncer. Int J R di t
Oncol Biol Phys 2002; 52(1):8190. 10. M te TP, Gottesm n JE, H tton J, et l. Hig
h dose-r te fterlo ding 192 Iridium prost te
r chyther py: fe si
ility report.
Int J R di t Oncol Biol Phys 1998; 41(3):525533. 11. Deger S, Boehmer D, Turk I,
et l. High dose r te
r chyther py of loc lized prost te c ncer. Eur Urol 2002
; 41(4):420426. 12. M rtinez AA, Gust fson G, Gonz lez J, et l. Dose esc l tion
using conform l high-dose-r te
r chyther py improves outcome in unf vor
le pro
st te c ncer. Int J R di t Oncol Biol Phys 2002; 53(2):316327.
35 High dose r te fterlo ding 192Ir prost te
r chyther py
Alv ro M rtinez, Jeffrey Dem nes, R zv n G l l e, How rd J Korm n, Hgen Berterm n
n, C rlos V rg s, Jose Gonz lez, nd G ry Gust fson Introduction During the l st
two dec des, surgery nd r diother py h ve
een the tre tment mod lities most f
requently used to tre t p tients with prost te c ncer. However, the specific ind

ic tions for the different surgic l procedures nd/or r diother py techniques v

il
le rem in under de
te. Although thorough discussion of this gener l topi
c is outside the scope of this ch pter, it is import nt to recognize the promine
nce
r chyther py tre tments h ve chieved. To this respect, The Americ n Urolog
ic Society,1 nd the Americ n College of R diology,2 p tterns of c re of utiliz
tion h ve reported the signific nt incre se of utiliz tion of
r chyther py duri
ng the l st dec de. We will limit the extent of our d t n lysis nd comments t
o type of r diother py c lled high dose r te (HDR)
r chyther py whether used
s monother py or s
oost ther py com
ined with pelvic extern l
e m with or
without ndrogen depriv tion ther py (ADT). Prost te
r chyther py h s
een perf
ormed using one of two tre tment techniques, low dose r te (LDR) or HDR. LDR
r
chyther py involves the perm nent impl nt tion of multiple r dio ctive seeds; ty
pic lly iodine-125 (125I) or p ll dium-103 (103Pd) seeds, into the prost te in o
rder to deliver ther peutic dose of r di tion to the prost te gl nd over sever
l weeks to months. This signifies th t the p tient rem ins r dio ctive for wh
ile nd must comply with the st te rules nd regul tions for p tients h r
oring
r dio ctive m teri l. HDR
r chyther py, on the other h nd, uses single high i
ntensity r dio ctive source stored in ro
otlike m chine c lled remote fterlo
der. The fterlo der sends nd retr cts this single source sequenti lly into e c
h impl nted needle, nd is
le to deliver ther peutic dose of r di tion in
very short period of time, typic lly 1015 minutes. Hence, the p tients re no lon
ger r dio ctive fter the completion of tre tment. Adv nt ges of high dose r te

r chyther py HDR h s num
er of dv nt ges over LDR, th t re p tient- nd t r
get-specific. They re summ rized
elow: 1. The over ll tre tment time is reduce
d from m ny weeks with LDR to sever l minutes with HDR. This elimin tes the unce
rt inties rel ted to volume ch nges occurring over
High dose r te fterlo ding
457
weeks (typic l with LDR) due to tr um nd swelling or su
sequent shrink ge due
to postr di tion fi
rosis. 2. HDR signific ntly improves the r di tion dose dist
ri
ution second ry to the
ility to modul te nd ccur tely control the sp ti l
source position nd v ry the source dwell time during tre tment. 3. The intr op
er tive (or re l-time) optimiz tion used with HDR llows ide l selection of need
le pl cement on re l-time nd
etter source position t rgeting, hence modul ting
the intensity with the potenti l for limiting tre tment toxicity. 4. HDR c n si
gnific ntly reduce the cost of tre tment,
ec use the r dio ctive sources re no
t purch sed per c se tre ted, s is done with LDR. 5. Adv nt ges in r di tion s
fety nd protection since the p tient is not r dio ctive when he returns home. T
he HDR single r di tion source is retr cted into the ro
ot t the completion of
tre tment. 6. There re multiple r dio
iologic l consider tions f voring HDR sin
ce the tre tment is given in sever l minutes for which repopul tion, cell cycle,
nd recovery of su
leth l d m ge c nnot occur. For p tients with e rly st ge pr
ost te c ncer, r dic l prost tectomy, extern l
e m r diother py, nd
r chyther
py re commonly used tre tment mod lities. These tre tment options h ve
een su
pported
y the excellent
iochemic l outcomes reported during the l st few ye rs
for p tients with simil r st ge dise se nd f vor
le prognostic v ri
les. Alt
hough the most ppropri te definition of
iochemic l f ilure m y
e controversi
l, the Clevel nd Clinic Found tion,3 nd Willi m Be umont Hospit l, Michig n,4 h
ve reported simil r outcomes for p tients with f vor
le prognostic fe tures un
dergoing either r dic l prost tectomy or extern l
e m r diother py t single
institution. The multi-institution l report from DAmico et l5 indic ted simil r

iochemic l outcomes were chieved for f vor
le p tients fter ny of three tre
tment mod lities, r dic l prost tectomy, extern l
e m r diother py, or interst
iti l
r chyther py. In ddition, sever l other investig tors h ve reported exce
llent results with interstiti l prost te
r chyther py,611 with
iochemic l contr
ol r tes comp r
le to those of p tients tre ted with either r dic l prost tecto
my or extern l
e m irr di tion. The
ove series h ve supported the r tion le f

or the use of interstiti l
r chyther py in the tre tment of e rly st ge prost t

e c ncer. While most monother py series h ve utilized LDR technique with 125I se
eds or 103Pd seeds, HDR
r chyther py with iridium-192 (192Ir) is g ining popul
rity.1012 A wide v riety of tre tment ppro ches h s
een used for p tients with
unf vor
le prost te c ncer. However, tre tment rem ins ch llenge. Surviv l r
tes from series in which r dic l prost tectomy is performed for st ge C or T3 di
se se,13,14 nd/or low dose extern l
e m r di tion ther py re given rem in su

optim l.15 To improve on these results three r diother peutic str tegies h ve
e
en tested, including the ddition of hormon l
l tion
efore st nd rd r diother
py,16,18 the ddition of p rticle
e m s
oost to extern l
e m r diother py
,19,20 nd dose esc l ting conform l r diother py. Dose esc l tion h s
een cco
mplished
y using 3-dimension l (3D) conform l extern l
e m r diother py (EBRT)
,21,22 or
r chyther py using conform l high dose r te
oost.23 Tumor dose esc
l tion should hypothetic lly overcome r dioresist nce of tumor clonogens seen
t convention l dose levels. The question rem ins s to which of these two str te
gies
est esc l tes the dose sufficiently to o
t in gre ter ther peutic g in.
B sic nd dv nced techniques in prost te
r chyther py
458
The 3D conform l r diother py EBRT tri ls incorpo r te cert in dr w
cks, such
s system tic nd r ndom setup errors, intern l org n motion nd deform tion, whi
ch h ve
een well documented nd m y limit effic cy.2426 Bec use of these dr w
c
ks, we
eg n dose esc l ting prospective clinic l tri l in 1991 using tr nsrec
t l ultr sound-guided 3D conform l modul ted high dose r te
r chyther py to del
iver the
oost dose.23 A key component for 3D conform l high dose r te prost te

r chyther py w s our development of n inter ctive online dose optimiz tion pro
gr m,27,28 which we c lled the hypofr ction ted high dose r te sm rt seed techniq
ue. This new ppro ch llowed direct visu liz tion of isodose curves with rect l
nd urethr l doses (Figure 35.1 ,
) in rel tion to re l-time prost te im ges n
d computerized selection of ide l needle loc tion with online identific tion of
ctu l needle position (Figure 35.1c). In 1991, our prospective Hum n Investig t
ion Committee (HIC) pproved clinic l tri l, which w s designed to test the hy
pothesis th t for p tients with poor prognostic f ctors loc l f ilure is rel ted
to the l rge volume of cell m ss nd r dioresist nt cell clones,
oth of which
require
iologic lly higher r di tion dos ges, th n those convention lly used. T
hus, we selected hypofr ction ted 3D conform l high dose r te
r chyther py
o
ost s the method of dose esc l tion. In 2001, we reported the interim three ye
r results of 145 p tients enrolled in this tri l.23 We h ve exp nded the d t
s
e, incre sed the num
er of p tients to 207, nd upd ted the follow-up (5 ye rs)
nd d t n lysis, now focusing on p tients with poor prognostic f ctors.29 More
recently, we reported n upd te com
ining the d t from Kiel University, using
simil r tre tment progr m,30 nd lso T
le 35.1 with the d t from the C lifo
rni Endocurither py C ncer Center (CET) (T
le 35.1).31 Equ lly import nt to su
rviv l outcomes re the toxicity nd side effects ssoci ted with ny cur tive t
re tment mod lity, whether surgery, extern l
e m irr di tion, or
r chyther py.
Both the physici n recommending tre tment nd the p tient receiving it must con
sider the potenti l for tre tment-rel ted mor
idities nd the imp ct they m y h
ve on p tients qu lity of life. As gener l st tement, it ppe rs th t prost t
ic
r chyther py is the most convenient tre tment nd m y h ve the lowest r te o
f long term complic tions when comp red to r dic l prost tectomy or extern l
e
m r diother py.32 During the l st 5 ye rs, these ppe r to
e the prim ry re son
s for signific nt incre se in the utiliz tion of prost te
r chyther py s p
rim ry nd cur tive tre tment ppro ch for p tients with e rly st ge prost te c
ncer.1,2,32 M teri ls nd methods Hypofr ction ted tre tment progr m High dose r
te
r chyther py s
oost Our tre tment technique h s
een previously descri

ed.18,19 The pelvis w s tre ted with n isocentric dose of 46 Gy in 23 fr ctions
using 4-field technique. All p tients underwent pretre tment pelvic computed
tomogr phy (CT) with contr st to ssist in defining the prost te nd norm l tiss

ue volumes. Pelvic EBRT w s interdigit ted

High dose r te fterlo ding
459
Figure 35.1 ( ) Urethr l doses in per cent t 5 mm p rt with isodose lines t t
he reference pl ne. (
) Prost te
ound ries contour with overlying outline of th
e prost tic urethr in depicted green cricles. (c) Computer selected ide l templ
te needle coordin tes (
lue dots).
with tr nsrect l ultr sound (TRUS)-guided tr nsperine l conform l interstiti l 1
92Ir impl nts. The over ll tre tment time w s compressed to only 5 weeks. No EBR
T w s given on the d y of the outp tient impl nt. After we est
lished enough ex
perience with p tients toler nce to HDR prost te
r chyther py nd th t their tox
icity w s ccept
le, we decided to decre se the num
er of (conform l) C-HDR imp
l nts from three to two. From 1991 to 1995, ll p tients underwent three TRUS-gu
ided C-HDR impl nts, during the first, second nd third weeks of tre tment. Afte
r Octo
er 1995, ll p tients underwent two HDR impl nts during the first nd thi
rd weeks of pelvic EBRT. This ch nge w s implemented to elimin te dministr tion
of spin l nesthesi nd the surgic l tr um of third impl nt. P tients with
prost te gl nd volume >65 cm3 or length of 5.5 cm were initi lly ineligi
le fo
r the protocol. These p tients underwent downsizing with short course of hormo
n l ther py (<6 months) nd were the su
ject of sep r te n lysis.34 Br chythe
r py dosimetry w s never done using prepl nning TRUS performed
efore the ctu l
HDR impl nt. Inste d, using our HDR sm rt seed technique progr m the dosimetry w
s done in re l-time intr oper tively. The impl nt procedure w s performed under
spin l nesthesi with the p tient in the lithotomy position with extreme pelvic
B sic nd dv nced techniques in prost te
r chyther py
460
flexion. A 7.5 MHz
ipl n r TRUS pro
e w s fixed to the t
le to llow only long
itudin l motion. The pex nd
se of the prost te gl nd were identified online
using tr nsverse nd s gitt l TRUS im ges. The pro
e w s positioned s p r llel
s possi
le to the prost tic urethr (Figure 35.2). The length of the prost te
nd corresponding tre tment length w s considered the dist nce from the
se to t
he pex. No m rgins were dded. The prost te gl nd w s sc nned t 5 mm interv ls
from 1.0 cm
ove the
se to 1.0 cm
elow the pex of the prost te gl nd on th
e tr nsverse pl ne (only for intr oper tively pl nning purposes, not for tre tme
nt). The urethr w s m pped on e ch 5 mm tr nsverse im ge s well. The tr nsvers
e im ge with the l rgest cross-section l prost te re w s considered the refere
nce pl ne. This re w s contoured with no pl nning m rgins dded. Consequently,
the clinic l t rget volumes (CTV) nd pl nning t rget volumes (PTV) were the s
me (Figure 35.3). The optim l needle positions within the reference pl ne were d
etermined intr oper tively using our re l-time, inter ctive optimiz tion progr m
. The computer pl nning softw re g ve the physici n the needle coordin tes with
reference to the perine l templ te.2729 Under TRUS guid nce, the needles were pl
ced p r llel to the TRUS pro
e using templ te
T
le 35.1 P tients ch r cteristics for high dose r te (HDR)
r chyther py
oost

y institution
KM (n=160) WBH (n=315) CET (n=459)
57 yrs 4.1 yrs 4,2 yrs Medi n Age 69 yrs 69 yrs 68 yrs PSA 14.8 ng/mL 8.9 ng/mL 9
.6 ng/mL Tre ted volume 30.8 cc 35 cc 30 cc 33% 31% 55% Hormones Gle son score 26
14% 38% 40% 7 51% 42% 43% 810 35% 20% 17% Clinic l st ge T1cT2 19% 40% 46% T2
T2c
49% 50% 42% T3 31% 10% 12% KM, Kiel University Hospit l; Germ ny; WBH, Willi m
Be umont Hospit l, Michig n; CET, C liforni Esdocuriether py C ncer Center; PSA
, prost te-specific ntigen.

High dose r te fterlo ding

461
Figure 35.2 S gitt l tr nsrect l ultr sound (TRUS) view showing the foley c thet
er inside the urethr .
mounted nd fixed to the pro
e. After pl cement of ll needles, cystoscopy w s p
erformed to reconfirm the prost te tre tment length with dequ te depth
y virtu
e of
l dder mucos tenting (Figure 35.4). To reconfirm the gl nd pex during cy
stoscopy, the TRUS pro
e w s pl ced in the s gitt l pl ne t the pex. The veru
mont num (1 cm
ehind) w s used to correl te with the TRUS pro
e position in the
longitudin l pl ne. After cystoscopy, contr st m teri l w s instilled into the

l dder nd fluoroscopy (
y w y of C- rm) w s performed
efore nd fter conne
cting the tr nsfer tu
es to verify nd document the ppropri te needle tip posit
ions (Figure 35.5). Bec use the
Figure 35.3 Re l-time tr nsrect l ultr sound (TRUS) im ge showing no m rgins dd
ed to the prost te contour. Clinic l t rget-volume (CTV); pl nning t rget volume
(PTV).
B sic nd dv nced techniques in prost te
r chyther py
462
Figure 35.4 Cytoscopy performed
y the urologists with the flexi
le cytoscope to
minimize tr um nd m ximize exposure, especi lly with the retroverted view.
needle positions m y h ve shifted slightly during cystoscopy, the fin l TRUS nee
dle positions, s well s the fin l urethr l loc tions
efore tre tment, were re
c ptured to determine the ctu l tre tment dwell positions nd times. Intr oper
tively, ll dosimetric c lcul tions were performed using re l-time inter ctive o
ptimiz tion softw re (Figure 35.6).27,29 The tre tment w s optimized using st nd
rd geometric optimiz tion.27 On e ch tr nsverse TRUS im ge, the 100% isodose li
ne encomp ssed the contoured prost te volume (Figure 35.7). The urethr w s limi
ted to 125% of the tre tment dose in e ch tr nsverse pl ne, s seen in Figure 35.
8. The rect l dose w s c lcul ted t the nterior edge of the TRUS pro
e within
the reference pl ne nd w s limited to <75% of the tre tment dose. For those p t
ients who underwent three initi l impl nts, the dose to this tre tment volume w
s su
sequently esc l ted from
High dose r te fterlo ding
463
Figure 35.5 ( ) C- rm for fluoroscopy fter cytoscopy is performed. Contr st m t
eri l instilled in the
l dder, (
) Xr y film documenting the rel tionship of go
ld seeds pl ced t the prost tic
se with tip of the needles nd
l dder contr
st.
5.5 Gy for e ch initi l impl nt to 6.0 Gy, nd fin lly to 6.5 Gy. This group con
stituted the low dose group. P tients who received two impl nts initi lly receiv
ed 8.25 Gy during e ch impl nt, then 8.75 Gy, 9.50 Gy, 10.5 Gy, nd 11.5 Gy. Thi
s w s the high dose group (T
le 35.2). When the tri l
eg n in 1991, the line r
-qu dr tic formul w s used to c lcul te the
iologic effective dose (BED).35 Th
e / v lue r tio for tumor control pro

ility w s 10 nd 4 for norm l tissue compl
ic tions. The
sic ssumption w s th t EBRT of 46 Gy in 23 d ily fr ctions to t
he pelvis followed
y prost te EBRT
oost of 24 Gy in 12 fr ctions (tot l dose
70 Gy t 2 Gy increments) in 7 weeks will deliver BED to 5.5 Gy3 in weekly HDR
fr ctions interdigit ted with 23 pelvic EBRT doses in 5 weeks. The expected
io
logic effect w s decre se in tumor
B sic nd dv nced techniques in prost te
r chyther py

464
Figure 35.6 Immedi tely prior to tre tment nd fter cytoscopy, the entire prost
tic volume w s im ged nd fin l needle position rec ptured prior to definitive
dosimetric n lysis.
Figure 35.7 An lysis of multiple pl ne tr nsrect l ultr sound (TRUS) im ges demo
nstr ting the dosimetry cover ge with excellent conform lity t the reference pl
ne. C lcul ted rect l dose of 68% of the prost te dose.
control pro

ility of 5% for the EBRT+HDR regimen. Simil rly, when we elimin te
d one impl nt in 1995, the s me formul tion nd
iologic ssumptions (/ of 10) wer
e used. There is evidence now th t the / r tio for prost te c ncer is much lower.3
638 In 1998, we pu
lished the v lid tion of this low / v lue. The v lue 1.2 w s der
ived from this EBRT+HDR clinic l tri l.39 For comp rison, we h ve listed the BED
s for ll levels of our dose esc l tion tri l using / r tio of 10 nd 1.2 (T
le 3
5.2). For this n lysis, the LDR group receiving three impl nts using n / of 1.2
h d BED of <93 Gy, for the HDR group with two impl nts, the BED >93 Gy. With
n HDR dose of 11.5 Gy2, the
High dose r te fterlo ding
465
BED using n / r tio of 1.2 w s 136.3 Gy, dose unlikely to
e chieved with n E
BRT delivery system.
Figure 35.8 Urethr l nd prost tic dose-volume histogr m (DVH) using the Nucletro
n Swift guid nce system: 80% of the prost te is receiving 115% of the dose; <15%
of the prost te is receiving 150% nd 0% of the dose; the urethr is receiving 115
% of the prost te dose. T
le 35.2 High dose r te (HDR)
oost ch r cteristics fo
r p tients tre ted t Willi m Be umont Hospit l MI
Dose level No. p tients Medi n follow-up BED1.2 G
Low dose group (3 HDR fr ctions) High dose r te group (2 HDR fr ctions) 5.53 63 6.
53 8.252 8.752 9.52 10.52 19 15 27 26 26 39 103 10.3 yrs 9.8 yrs 8.4 yrs 5.5 yrs 6.1
yrs 5.8 yrs 3.4 yrs 80.2 86.1 92.5 94.2 99.9 108.9 122.0
B sic nd dv nced techniques in prost te
r chyther py
11.52 45 BED,
iologic effective dose; LDR, low dose r te. 2.7 yrs
466
136.3
High dose r te
r chyther py s monother py The BED cceler ted hypofr ction ted
regime w s selected
sed on HDR f vor
le r dio
iologic l consider tions descr
i
ed
ove nd physic l dose delivery dv nt ges of TRUS guid nce,10 with confor
m l intensity modul ted re l-time dosimetry of prost te HDR
r chyther py.11,22,
24 Figure 35.9 depicts n HDR intr oper tive impl nt using the Nucletron Swift g
uid nce system. All p tients h d
iopsy-proven denoc rcinom of the prost te. T
hey were st ged with the 2003 Americ n Joint Committee on C ncer (AJCC) clinic l
st ge II
Figure 35.9 ( ) Re l time intr oper tive ultr sound im ge from Nucletron Swift sho
wing the computer-selected ide l needle positions to guide urologists nd r di t
ion oncologists during needle pl cement, (
) Re l-time intr oper tive 3D renditi
on from executed needle pl cement depicting the prost te contour, urethr tr jec
tory, nd semin l vesicles.
(T1cT2 ) dise se, Gle son score 7, nd pretre tment PSA12 ng/mL. The m jority of p
tients presented with wh t would
e considered low risk or f vor
le prost te c
ncer. St nd rd pretre tment work-up w s performed for st ging. Prior to gl nd im

pl nt tion, nd with the p tient in the tre tment position, TRUS w s performed
with or without light sed tion to determine the n tomic l nd geometric l suit

ility of the gl nd for
r chyther py; the criteri were: prost te gl nd volume
1565 cc; length 5.5 cm; nd dequ te pu
ic rch sep r tion. Once it w s determine
d th t the p tient met ll clinic l
High dose r te fterlo ding
467

nd n tomic l criteri for monother py, the HDR nd LDR
r chyther py tre tment
options were discussed nd then the p tient selected the
r chyther py mod lity
. A short course of neo- djuv nt ndrogen depriv tion (6 months) w s utilized for
downsizing the gl nd volume in 31% of Willi m Be umont Hospit l p tients, in eq
u l proportions
etween perm nent seeds nd HDR nd in 30% of the C liforni End
ocurither py C ncer Center p tients (T
le 35.3). All procedures were done under
spin l nesthesi . Willi m Be umont Hospit l Experience All
iopsy specimens we
re reviewed t our institution. The volume nd length were
sed on TRUS perform
ed in the dep rtment of r di tion oncology, with the p tient under light sed tio
n in tre tment position. Between J nu ry 1996 nd Decem
er 2002, 253 p tients wi
th clinic lly loc lized prost te c ncer, were tre ted with cceler ted hypofr ct
ion ted
r chyther py s the sole tre tment mod lity. Of the p tients, 92 were t
re ted with high dose r te (HDR)
r chyther py lone using 92Ir, nd 161 p tient
s were tre ted with low dose r te (LDR)
r chyther py lone using 103Pd. The me
n p tient ge in the HDR nd LDR tre tment groups w s 64 nd 66, respectively. T
he me n preimpl nt gl nd volume w s 39 cc for
oth tre tment groups. For the imp
l nt procedure s well s for p in control during the entire tre tment time, spi
n l nesthesi w s dministered following pl cement of n epidur l c theter. The
p tient w s supine in the lithotomy position. A foley c theter w s pl ced with
the
lloon cont ining 7 cc of contr st m teri l, nd the
l dder filled with 15
0 cc of sterile w ter. A modific tion of our st nd rd rigid perine l templ te w
s ffixed to the
ipl n r 7.5 MHz ultr sound pro
e. The pro
e w s then introduce
d into the rectum, positioned s p r llel to the urethr s possi
le, nd fixed
to the t
le, llowing c ud d-ceph l d movement only. In the s me m nner s in t
he
oost technique, using
oth tr nsverse nd s gitt l views, the TRUS w s used
to visu lize nd identify the
se nd pex of the prost te, s well s norm l s
tructures, such s urethr , rectum, nd
l dder. Im ges were c ptured, nd the u
rethr w s m pped t e ch 5 mm tr nsverse slice from
se to pex. The tr nsvers
e im ge with the l rgest cross-section l re w s considered the Reference pl ne.
The tre tment volume w s cylindric l in sh pe, extending from
se to pex, wit
h the crosssection corresponding to the reference pl ne. No m rgin w s dded ro
und the prost te gl nd either t the reference pl ne or t the pex or prost tic

se. Optim l needle positions were then gener ted intr oper tively using n on
line, inter ctive inhouse softw re progr m.16,19 Flexi
le pl stic needles with m
et l stylets, either 20 cm or 23 cm long nd
T
le 35.3 P tients ch r cteristics for high dose r te (192Ir) or low dose r te

r chyther py (103Pd) monother py
y institution
HDR
r chyther py WBH (n=92)
Medi n follow- 25 mths up 65 yrs Me n ge 6.0 Me n
LDR
r chyther phy WBH (n=161)
40 mths 66 yrs 5.3
HDR
chyther py CET (n=77)
20 mths 63 yrs 7.9
B sic nd dv nced techniques in prost te
r chyther py
pretre tment Me n gl nd volume

468
41 cc (1693)
39 cc (15111)
38 cc (91 34)
No, p tients (%} No p tients (%) No, p tients (%) Clinic l st ge T1 T1c 55 (60) 1
00 (62) 47(61) T2 34 (37) 55 (34) 21 (27) T2
2 (2) 4 (2) 9 (12) T2c 1 (1) 2 (1
) Pretreatmeat PSA 3.9 11 (12) 36 (22) 14 (18) 4.09.9 75 (82) 123 (77) 61 (79) 10.0
6 (6) 2 (1) 13 (17) leason score 5 5 (6) 22 (14) 14 (18) 6 80 (87) 131 (82) 61
(79) 7 6 (6} 6 (4) 2 (3) 8 1 (1) NAD 28 (30) 50 (31) 20 (26) WBH, William Beaumo
nt Hospital, MI; CET, California Eaocuritherapy Cancer Center.
1.9 mm in iameter, were place through the template uner TRUS guiance (Figure
35.10). Location of the prostate base etermine the epth of neele insertion.
Dosimetry was continuously upate in realtime base on the actual location of
neeles to compensate for organ istortion an motion an to assure conformal c
overage of the glan.19 ol see markers were then place uner TRUS guiance a
t the base an at the apex of the prostate to assess an measure possible interf
raction neele isplacement (Figure 35.5). Flexible cystoscopy was performe aft
er placement of all neeles to verify that the blaer an urethral mucosa were
not perforate. Tenting of the blaer mucosa from the plastic neeles was a req
uirement. Before elivery of the raiation, the entire prostate was image again
, with final neele an urethral positions capture by TRUS an a final treatmen
t plan create. The blaer was fille with 150 cc of contrast material, an flu
oroscopy was performe to recor the relationship of the tip of the neeles to t
he bony anatomy an the blaer base. After removing the metal stylets, plastic
collars were tightene aroun the neeles to fix them to the template. This prev
ente any cauacephala movement of the neeles with respect to the template.
The template was then suture to the perineum (Figure 35.11). The treatment plan
was geometrically optimize to etermine well positions an times.19 The ose
prescription was to a point of minimum ose in the implante volume. The rectal
ose was calculate at the anterior ege of the TRUS probe an was limite to 75%
of the prescription ose. The ose to any segment of the urethra was limite to
125% of the prescription ose. The prescription ose was 950 cy elivere four
times for a total ose of 3800 cy. Two fractions were elivere aily over 2 a
ys, with at least 6 hours separating each fraction. The brachytherapy neeles wi
th the template were then remove as well as the epiural catheter. The patient
was ischarge home after voiing spontaneously, typically
High ose rate afterloaing
469
34 hours after completing treatment. Using the linearquaratic formula,22 this 
ose fractionation is the equivalent of an external beam regimen of 76.4 y (50.4
y in 28 fractions plus a boost of 26 y in 13 fractions). This can be erive
using an / v lue of 7 for estim ting tumor control, rel tively conserv tive ssu
mption.
Figure 35.10 Under tr nsrect l ultr sound (TRUS) guid nce, the flexi
le pl stic
c theters were pl ced tr nsperine lly following the computer-gener ted intr oper
tive pl n.
C liforni Endocurither py C ncer Center Experience Between J nu ry 1996 nd Dec
em
er 2002, 77 p tients with clinic lly loc lized prost te c ncer were tre ted w
ith interstiti l
r chyther py s the sole tre tment mod lity. P tient ch r cter
istics re presented in T
le 38.1. The me n p tient ge w s 69 ye rs nd medi n
prost te volume w s
etween 30 nd 35 cc. The C liforni Endocuriether py (CET)

method of high dose r te
r chyther py is descri
ed s
eing prost te-specific

cceler ted HDR
r chyther py in Dem nes et l.13 Either the Syed or CET type of
perine l templ te w s pl ced freeh nd on the perineum nd not fixed to the h nd
-held
ipl n r tr nsrect l ultr sound (TRVS) pro
e. A st nd rd p ttern consistin
g of 17 Flexiguide c theters with met l wire (Best Industries) w s inserted r
ound the prost te
B sic nd dv nced techniques in prost te
r chyther py
470

nterior nd l ter l to the c psule nd just within the posterior c psule t the
reference pl ne, nd into the prost te. C re w s lso t ken to void the non-line
r course of the prost te urethr . Cystoscopy w s lso performed to ensure th t
the c theters did not penetr te the urethr or
l dder while dequ tely covering
the prost te
se. The templ te w s sutured to the
Figure 35.11 Completed monother py impl nt with templ te sutured to the perineum
. Pl stic stifferners re used to prevent tu
e kinking, nd secured with red c p
s.
perineum, nd fter recovery the p tient underwent du l method of simul tion r
diogr phy consisting of pl in film loc liz tion for pplic tor djustment nd q
u lity control nd computed tomogr phy (CT) sc n w s performed. The im ges wer
e downlo ded to the tre tment-pl nning computer nd three-dimension l (3D) recon
struction w s c rried out. The dose w s prescri
ed to point 56 mm
eyond the pr
ost te c psule l ter lly nd 3 mm posteriorly. Multiple point doses to the
l dd
er neck, urethr , nd nterior edge of the TRUS pro
e were c lcul ted. A dose-vo
lume histogr m (DVH) nd virtu l im ges of the n tomy, clinic l t rget volume (
CTV), nd pl nning t rget volume (PTV) were o
t ined. A series of six HDR fr cti
ons w s dministered twice d y in two sep r te procedures one week p rt. E ch
fr ction delivered 7 Gy for tot l dose of 42 Gy. The dose to ny segment of t
he urethr w s limited to 103%, the
l dder neck 80%, nd the rectum 75% of the
prescription dose. Postoper tive m n gement w s simil r to th t descri
ed for th
e WBH experience. Results Tre tment results re presented in two p rts. First, t
he long term surviv l nd toxicity results with HDR s
oost with or without
djuv nt/concurrent hormon l ther py is discussed. Second, the monother py result
s re reviewed.
High dose r te fterlo ding
471
High dose r te
r chyther py s
oost The c ses were clinic lly st ged. Consid
ering the in ccur cies of clinic l st ging, if these c ses h d
een p thologic l
ly
Figure 35.12 Outcome for ll p tients tre ted with high dose r te (HDR)
oost t
Willi m Be umont Hospit l. CSS, c use specific surviv l; OS, over ll surviv l;
BC,
iochemic l control.
st ged ( fter prost tectomy) they would h ve
een up st ged
y more th n 60% in
st ge nd Gle son c tegories. There w s no difference in prost te gl nd volume

sed on different num
ers of prognostic f ctors or
r chyther py dose level. Me
n follow-up for the entire group (n=383) w s 4.8 ye rs (r nge: 0.412). Me n follo
w-up w s 4.3 ye rs (r nge: 0.410.3) for p tients with one poor prognostic f ctor,
5.6 ye rs (r nge: 1.512) for two, nd 6 ye rs (r nge: 111.3) for ll three. Me n
followup w s 8.1 ye rs (r nge: 2.012) for the low dose r te (LDR) group nd 4.1 (
r nge: 0.4 8.2) for the high dose r te (HDR) group. The 5 nd 10 ye r ctu ri l
n lysis of
iochemic l control, over ll surviv l, dise sefree, nd c use-specifi
c surviv l for ll WBH p tients is shown in Figure 35.12. B sed on the Americ n
Society of Ther peutic R diology nd Oncology (ASTRO) consensus p nel definition

of
iochemic l f ilure, the 5 ye r ctu ri l
iochemic l control r te w s 52% f

or the LDR group versus 87% for the HDR group (p<0.001). In ddition, the me n d
ose w s 83.2 Gy for
iochemic lly controlled c ses versus 75.6 Gy for
iochemic
l f ilures (t-test, p<0.001). The
iochemic l control in the coll
or tive group
for the three institutions (n=934) is shown in Figure 35.13. The difference in

iochemic l control (p<0.001) is second ry to the difference in risk f ctors mo
ng the three institutions. P tients t CECC h d more f vor
le clinic l st ge
(T) cl ssific tion nd Gle son score comp red to the rem ining two institutions.
Figure 35.14 depicts the 5 ye r nd 10 ye r
iochemic l control str tified
y t
he use of hormon l ther py (HT). No difference in
iochemic l control w s seen
t 10 ye rs. When
B sic nd dv nced techniques in prost te
r chyther py
472
p tients with ll three risk f ctors (n=177) were selected (T2
, PSA 10 ng/mL, n
d Gle son score 7), p tients receiving HT h d decre sed c use-specific surFigure 35.13 Biochemic l control
y institution (n=934). CET, C liforni Endocur
ither py C ncer Center; WBH, Willi m Be umont Hospit l, Michig n; Kiel, Kiel Uni
versity Hospit l, Germ ny.
Figure 35.14 Biochemic l control str tified
y the use of hormon l ther py (n=93
4). cmHDR, conform l modul ted high dose r te (HDR); ADT, ndrogen depriv tion t
her py.
viv l, s seen in Figure 35.15 (p=0.02). The inferior outcome for the p tients r
eceiving neo- djuv nt/concurrent hormon l suppressive ther py with high dose r d
iother py r ised the question
out the
enefit of HT when com
ined with optim l
r di tion doses. It is uncle r if the deleterious effect of HT w s due to its
dministr tion, dur tion, or timing.
High dose r te fterlo ding
473
There w s no difference in prost te gl nd volume
sed on the different prognost
ic f ctors or
r chyther py dose levels. The 5 ye r r tes of loc l tre tment f i
lure
y digit l rect l ex min tions (DRE) were 8% nd 6%, respectively. Medi n p
tient ge w s 69 ye rs (r nge: 4885), which is typic l of r diother py series. A
n lysis of
iochemic l control, over ll surviv l, dise se-free surviv l, nd c u
sespecific surviv l t ges less th n 65, 6575 nd gre ter th n 75 ye rs demonstr
ted no signific nt differences.
Figure 35.15 C use-specific surviv l for p tients with ll three poor prognostic
f ctors str tified
y the use of hormon l ther py (HT) (n=177). cmHDR, conform
l modul ted high dose r te (HDR); ADT, ndrogen depriv tion ther py.
Whether n lyzing
y these su
groups or s continuous v ri
le, ge w s not
signific nt predictor of outcome, s younger nd older p tients
enefited equ ll
y (
iochemic l control: p=0.633). The R di tion Ther py Oncology Group (RTOG) gl
oss ry w s used to ssess complic tions nd exp nded to include toxicity common
to
r chyther py. RTOG gr des 12 toxicity w s seen t the level expected from the
extern l
e m component of the tre tment progr m. No incre ses in percent ge of
gr de 1 or 2 complic tions were seen s consequence of the impl nts. Gr de 2
l te urin ry complic tions, mostly urin ry strictures, were seen in 7 of 58 p ti
ents tre ted with three sep r te impl nts or low dose, nd 3 of 149 tre ted with
two impl nts or high dose (Pe rson Chi-squ re: p=0.006). Multiv ri te n lysis
of tot l urethr l stricture, dose per fr ction, segment of urethr , highest dose
, length of follow-up, nd num
er of impl nts (2 vs 3) reve led th t only three
impl nts correl ted with incre sed risk of stricture. Medi n time w s 2.0 ye rs

for l te gr de 3 genitourin ry stricture, 2.7 for low dose, nd 1.8 for high dos
e. The 5 ye r ctu ri l r te of RTOG l te genitourin ry complic tions w s 8% for
gr de 3 nd 0% for gr de 4. Gr de 3 urin ry incontinence developed in only one
p tient following tr nsurethr l resection of the prost te performed 3.8 ye rs f
ter r diother py. The corresponding 5 ye r ctu ri l r te for RTOG g strointesti
n l complic tions w s 0.5% for gr de 3 nd 0.5% for gr de 4 ( n symptom tic rec
t l ulcer developed in one p tient).
B sic nd dv nced techniques in prost te
r chyther py
474
No p tients experienced gr de 5 cute or l te toxicity. For p tients cl iming to

e sexu lly potent the 5 ye r ctu ri l impotence r te w s 51%, with impotence
occurring t medi n interv l of 0.7 ye rs (r nge: 0.05.2). No difference in imp
otence r te w s o
served
sed on dose level (p=0.897). High dose r te
r chythe
r py s monother py A tot l of 330 prost te c ncer p tients were tre ted with ei
ther high dose r te (HDR) 192Ir or low dose r te (LDR) 103Pd
r chyther py lone
. The medi n follow-up for ll p tients w s 32 months (r nge: 382). D t re pres
ented for ll p tients, s well s for only those p tients who did not receive h
ormon l m nipul tion for gl nd downsizing. E ch toxicity event w s censored s
sep r te event. In other words, the s me p tient who presented in followup with
dysuri , hem turi nd incre se in urin ry frequency w s counted s 3 sep r te
events. For chronic complic tions, the s me p tient w s uncensored only once (T

les 35.435.6). Acute toxicity T
le 35.4 shows the results for cute toxicity c
cording to tre tment technique with nd without neo- djuv nt hormon l m nipul ti
on. HDR
r chyther py lone w s ssoci ted with st tistic lly signific nt reduct
ions in the cute r tes of dysuri , from 65% with 103Pd seeds to 38% with HDR mo
nother py (p<0.001), s well s urin ry frequency nd/or urgency, from 94% (103P
d) to 53% (HDR, p<0.001) nd urin ry retention from 43% (103Pd) to 29% (HDR, p=0
.012). In ddition to reduced cute genitourin ry symptoms, HDR w s lso ssoci
ted with lower r tes of rect l p in, 18% (LDR) versus 7% (HDR, p=0.025). Hormon
l ndrogen
l tion w s given to 31% of p tients in
oth groups. HDR monother py
nd perm nent seeds were ssoci ted with simil r r tes of cute urin ry inconti
nence, di rrhe , nd rect l
leeding. Acute gr de 1 urin ry retention consisting
of the use of c theter for sever l d ys in the immedi te postoper tive period
w s seen more often fter LDR perm nent seeds impl nt, where s the r tes of mor
e prolonged gr de 2 retention tre ted with lph
lockers w s the s me. The m jor
ity of cute toxicities in
oth groups were gr de 1. Over ll, however, there w s
gre ter percent ge of gr de 3 cute genitourin ry toxicities seen in LDR p ti
ents, 15%, comp red to only 4% with HDR. There were no gr de 4 toxicities in eit
her tre tment group. Chronic toxicity T
le 35.5 shows the results for chronic t
oxicity ccording to tre tment technique, with nd without hormon l m nipul tion
. Ag in, HDR
r chyther py lone w s ssoci ted with reduced urin ry frequency
nd urgency, 54% (103Pd) versus 32% (HDR) (p<0.001). As mentioned
ove, 31% of t
he p tients in
oth groups received hormon l ndrogen depriv tion. Ag in, the m
jority of toxicities were gr de 1. There
High dose r te fterlo ding
475
T
le 35.4 Acute urin ry toxicity for high dose r te (LDR) (192Ir) nd low dose
r te (HDR) (103Pd)
r chyther py s monother py
Acute toxicity* HDR lone with 192Ir (n=92) LDR lone with 103Pd (n=427) Gr de G
r de Gr de All n Gr de Gr de Gr de All n 1 2 3 toxicities 1 2 3 toxicities
0% 0% 2% 5% 1% 0% 0% 0% 38% 35 7% 6 32% 29 53% 49 4% 4 14% 13 7% 6 2% 2 48% 3% 2
4% 54% 1% 9% 15% 2% 14% 3% 12% 25% 0% 2% 3% 0% 3% 0% 1% 13% 0% 1% 0% 0% 65% 83 6
% 8 36% 46 94% 119 1% 1

Genitourin ry Dysuri 30% 8% Incontinence 5% 1% Urin ry retention 13% 16% Urin r

y frequency or 33% 15% urgency Hem turi 1% 2% G strointestin l Di rrhe 13% 1%
Rect l p in 7% 0% Rect l
leeding 2% 0% * No gr de 4 toxicities were o
served.
11% 14 18% 23 2% 2
T
le 35.5 Chronic urin ry toxicity for high dose r te (HDR) (192Ir) nd low dos
e r te (LDR) (103Pd)
r chyther py s monother py
Chronic toxicity* HDR lone with 192Ir (n=92) LDR lone with 103Pd (n=127) Gr de
Gr de Gr de All n Gr de Gr de Gr de All n 1 2 3 toxicities 1 2 3 toxicities
2% 0% 2% 0% 0% 0% 0% 0% 15% 14 7% 6 18% 17 32% 29 11% 10 4% 4 4% 4 9% 8 17% 8% 9
% 39% 6% 3% 5% 2% 2% 2% 10% 14% 0% 0% 1% 1% 0% 0% 3% 1% 0% 0% 0% 0% 20% 25 9% 12
28% 35 54% 69 6% 8 3% 4 6% 7 2% 3
Genitourin ry Dysuri 8% 6% Incontinence 7% 0% Urin ry retention 4% 13% Urin ry
frequency or 13% 8% urgency Hem turi 6% 6% G strointestin l Di rrhe 4% 0% Rect
l p in 4% 0% Rect l
leeding 9% 0% * No gr de 4 toxicities were o
served.
were no differences in the rem ining chronic toxicity r tes of urin ry incontine
nce or retention, hem turi , di rrhe , rect l p in, or rect l
leeding
etween t
he two tre tment groups. Considering the limit tions of the current definition f
or urin ry retention, ll chronic retention c ses were rel ted to the us ge of >
6 months of lph -
lockers (no p tient necessit ted prolonged use of c theter)
. The r te of urethr l stricture requiring dil t tion w s 3% with HDR comp red t
o 1% with 103Pd (p=0.3). The medi n time to
B sic nd dv nced techniques in prost te
r chyther py
476
development of urethr l stricture w s 17 months, with r nge of 437 months. Figu
re 35.16 shows, the cumul tive proportion of chronic gr de 1 genitourin ry toxic
ity
y tre tment mod lity nd over time. Ex mining the re under the curve cle
rly demonstr tes more toxicity for the 103P LDR group (p=0.028). No difference w
s noted
etween the two tre tment types.
T
le 35.6 Surviv l for high dose r te (HDR) (192Ir) nd low dose r te (LDR) (10
3Pd)
r chyther py s monother py
4 ye r results HDR
r chyther py LDR
r chyther py HDR
r chyther py pWBH (n=92)
WBH (n=161) CET (n=77) v lue*
0.345 0,602
Over ll surviv l 100% 93% 98% C use-specific 100% 100% 100% surviv l Biochemic
l 99% 98% 96% control * K pl n-Meier log r nk, WBH, Willi m Be umont Hospit l, M
I: CET, C liforni Endocuriether py C ncer Center,
Figure 35.16 Cumul tive incidence of chronic gr de 1 genitourin ry toxicity for
high dose r te (HDR) 192lr nd low dose r te (LDR) 103Pd
r chyther py s monoth
er py.
High dose r te fterlo ding
477
Potency Potency w s ev lu ted only on those p tients in whom the pretre tment po
tency st tus w s known, nd in whom posttre tment potency st tus w s lso ev lu
ted. Hormon l m nipul tion for up to 6 months for gl nd volume downsizing w s us
ed equ lly in 30% of the LDR nd HDR c ses. Reg rdless of the use of djuv nt ho
rmon l ther py ll c ses were included. This included 51 p tients tre ted with H
DR
r chyther py lone nd 71 p tients tre ted with perm nent 103Pd. The 4 ye r

pro

ility of impotency w s 38% for ll p tients with v il
le d t . As shown

in Figure 35.17, the pro

ility w s 49% for LDR p tients, nd 21% for HDR p tie
nts (p=0.006). The me n times to impotency for the HDR nd LDR tre tments were 3
.9 ye rs nd 3.2 ye rs, respectively.
Figure 35.17 Cumul tive incidence of impotency for high dose r te (HDR) (192lr)
nd low dose r te (LDR) (103Pd)
r chyther py s monother py.
Conclusions Biochemic l control Although this is n interim report, we h ve pres
ented the 4 ye r surviv l n lysis nd
iochemic l control r tes. Using the Amer
ic n Society of Ther peutic R diology nd Oncology (ASTRO) consensus p nel defin
ition of three consecutive rises
ove the n dir level, the 4 ye r
iochemic l c
ontrol r te w s the s me for
oth
r chyther py tre tment mod lities. It w s 98%
t WBH nd 96% t CET using HDR monother py, nd 98% using 103Pd seeds. T
le 3
5.6 depicts the surviv l outcome comp rison of the three
B sic nd dv nced techniques in prost te
r chyther py
478
tre tment groups. No st tistic l difference w s noted in over ll nd/or c use-sp
ecific surviv l ccording to tre tment technique or
etween centers. Cost n lys
is A cost n lysis for
oth types of
r chyther py procedure w s c rried out. Th
e technic l (f cility) cost n lysis of
oth procedures showed HDR to
e, on ve
r ge, 2329% lower in cost th n LDR. This differenti l is rel ted to the num
er of
103Pd seeds used. The profession l (pr ctitioner) tot l cost n lysis reve led
no signific nt difference in cost. References
1. Mettlin CJ, Murphy GP, McDon ld CJ, Menck HR. The n tion l C ncer D t B se R
eport on incre sed use of
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te c rcinom in the USA. C ncer 1999; 86:18771882. 2. Lee WR, Mough n J, Owen JB
, Zelefsky JM. The 1999 p tterns of c re study of r diother py in loc lized pros
t te c rcinom . A comprehensive survey of prost te
r chyther py in the United S
t tes. C ncer 2003; 98(9):19871994. 3. Kupeli n P, K tcher J, Levin H, et l. Ext
ern l
e m r diother py versus r dic l prost tectomy for clinic l St ge T1-T2 pr
ost te c ncer: Ther peutic implic tions of str tific tion
y pretre tment PSA le
vels nd
iopsy Gle son scores. C ncer J Sci Am 1997; 3:7887. 4. M rtinez AA, Gon
z lez JA, Chung AK, et l. A comp rison of extern l
e m r di tion ther py versu
s r dic l prost tectomy for p tients with low risk prost te c rcinom di gnosed,
st ged nd tre ted t single institution. C ncer 2000; 88(2):425432. 5. DAmico
AV, Whittington R, M lkowicz SB, et l. Biochemic l outcome fter r dic l prost
tectomy, extern l
e m r di tion ther py or interstiti l r di tion ther py for c
linic lly loc lized prost te c ncer. JAMA 1998; 280:969974. 6. Bl sko JC, Grimm P
D, Sylvester JE, et l. P ll dium-103
r chyther py for prost te c rcinom . Int
J R di t Oncol Biol Phys 2000; 46:839850. 7. Stock RG, Stone NN, T
ert A, et l.
A dose-response study for I-125 prost te impl nts. Int J R di t Oncol Biol Phys
1998; 41:101108. 8. W llner K, Merrick G, True L, et l. I-125 versus Pd-103 for
low risk prost te c ncer. Mor
idity outcomes from prospective r ndomized mult
icenter tri l. C ncer J Sci Am 2002; 8:6773. 9. Beyer DC, Priestley JB. Biochemic
l dise se-free surviv l following I125 prost te impl nt tion. Int J R di t Onco
l Biol Phys 1997; 37(3):559563. 10. M rtinez AA, P t ki I, Edmundson G, et l. Ph
se II prospective study of the use of conform l high-dose r te
r chyther py s
monother py for the tre tment of f vor
le st ge prost te c ncer: A fe si
ility
report. Int J R di t Oncol Biol Phys 2001; 49(1):6169. 11. Grills IS, M rtinez A
, Holl nder M, et l. High dose r te
r chyther py s prost te c ncer monother p
y reduces toxicity comp red to low dose r te P ll dium seeds. J Urol 2004; 171:1
0981104. 12. Yoshiok Y, Nose T, Yoshid K, et l. High-dose-r te interstiti l
r
chyther py s monother py for loc lized prost te c ncer: Tre tment description
nd prelimin ry results of ph se I/II clinic l tri l. Int J R di t Oncol Biol Ph
ys 2000; 48(1):675681. 13. Dem nes JD, Rodriguez RR, Altieri GA. High dose r te p
rost te
r chyther py: the C liforni Endocuriether py (CET) Method. R diother O
ncol 2000; 57:289296. 14. Morg n WR, Bergstr lh EJ, Zincke H. Long-term ev lu tio

n of r dic l prost tectomy s tre tment for clinic l st ge C (T3) prost te c nce
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15. Ger
er GS, Thisted RA, Chod k GW, et l. Results of r dic l prost tectomy in
men with loc lly dv nced prost te c ncer: Multiinstitution l pooled n lysis.
Eur Urol 1997; 32:385 390. 16. Oefelein MG, Smith ND, Gr yh ck JT, et l. Long-te
rm results of r dic l retropu
ic prost tectomy in men with high gr de c rcinom
of the prost te. J Urol 1997; 158:14601465. 17. H nks GE, Di mond JJ, Kr ll JM, e
t l. A ten-ye r follow-up of 682 p tients tre ted for prost te c ncer with r di
tion ther py in the United St tes. Int J R di t Oncol Biol Phys 1987; 13:499505.
18. Pilepich MV, C pl n R, Byh rdt CA, et l. Ph se III tri l of ndrogen suppr
ession using goserelin in unf vor
le-prognosis c rcinom of the prost te tre te
d with definitive r diother py: Report of R di tion Ther py Oncology Group Proto
col 8535. J Clin Oncol 1997; 15:10131021. 19. Boll M, Gonz lez D, W rde P, et l.
Improved surviv l in p tients with loc lly dv nced prost te c ncer tre ted wit
h r diother py nd goserelin. N Engl J Med 1997; 337:295300. 20. Ro ch M, Lu J, P
ilepich M, et l. Predicting surviv l nd the role of ndrogen suppressive ther
py (AST): R di tion Ther py Oncology Group (RTOG) ph se III r ndomized prost te
c ncer tri ls. Int J R di t Oncol Biol Phys 1998; 42:177. 21. Shipley WU, Verhey
JJ, Munzenrider JE, et l. Adv nced prost te c ncer: The results of r ndomize
d comp r tive tri l of high dose irr di tion
oosting with conform l protons com
p red with convention l dose irr di tion using photons lone. Int J R di t Oncol
Biol Phys 1995; 32:312. 22. Form n JD, Duclos M, Sh rm R, et l. Conform l mixe
d neutron nd photon irr di tion in loc lized nd loc lly dv nced prost te c nc
er: Prelimin ry estim tes of the ther peutic r tio. Int J R di t Oncol Biol Phys
1996; 35:259266. 23. Zelefsky MJ, Fuks Z, Wolfe T, et l. Loc lly dv nced prost
te c ncer: Long-term toxicity outcome fter three-dimension l conform l r di ti
on ther py dose esc l tion study. R diology 1998; 209:169174. 24. H nks GE, Lee W
R, H nlon AL, et l. Conform l technique dose esc l tion for prost te c ncer: Bi
ochemic l evidence of improved c ncer control with higher doses in p tients with
pretre tment prost te-specific ntigen >10ng/ml. Int J R di t Oncol Biol Phys 1
996; 35:861 868. 25. M rtinez AA, Kestin LL, Strom
erg J, et l. Interim report o
f im geguided conform l high dose r te
r chyther py for p tients with unf vor

le prost te c ncer: The Willi m Be umont ph se II doseesc l ting tri l. Int J R
di t Oncol Biol Phys 2000; 47:343352. 26. Meli n E, M g er s GS, Fuks Z, et l. V
ri tion in prost te position qu ntit tion nd implic tions for three-dimension
l conform l tre tment pl nning. Int J R di t Oncol Biol Phys 1997; 38:7381. 27. Y
n D, Zi j E, J ffr y D, et l. The use of d ptive r di tion ther py to reduce
setup error: A prospective clinic l study. Int J R di t Oncol Biol Phys 1998; 4
1:715720. 28. Y n D, J ffr y DA, Wong JW. A model to ccumul te fr ction ted dose
in deforming org n. Int J R di t Oncol Biol Phys 1999; 44(3):665675. 29. Edmun
dson GK, Rizzo N, Te h n M, et l. Concurrent tre tment pl nning for outp tient
high dose r te prost te templ te impl nts. Int J R di t Oncol Biol Phys 1993; 27
:12151223. 30. Edmundson GK, Y n D, M rtinez A. Intr oper tive optimiz tion of ne
edle pl cement nd dwell times for conform l prost te
r chyther py. Int J R di
t Oncol Biol Phys 1995; 33:1257 1263. 31. M rtinez AA, Gonz lez J, Spencer W, et
l. Conform l high dose r te
r chyther py improves
iochemic l control nd c us
e specific surviv l in p tients with prost tic c ncer nd poor prognostic f ctor
s. J Urol 2003; 169:974980. 32. M rtinez AA, G l l e R, Gonz lez J, et l. No pp
rent
enefit t 5 ye rs from course of neo djuv nt/concurrent ndrogen depriv
tion for prost te c ncer p tients tre ted to high tot l r di tion dose. J Uro
l 2003; 170(6):22962301.
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r chyther py
480

33. M rtinez AA, G l l e R, Mitchell C, et l. L ck of
enefit from short cour

se of ndrogen depriv tion for unf vor
le prost te c ncer p tients tre ted with
n cceler ted hypofr ction ted regime. Int J R di t Oncol Biol Phys 2003; 57:S
175. 34. Bl sko JC, Grim PD, Sylvester JE, et l. P ll dium-103
r chyther py fo
r prost te c rcinom . Int J R di t Oncol Biol Phys 2000; 46(4):839850. 35. Brenne
r DJ, M rtinez AA, Edmundson GK, et l. Direct evidence th t prost te tumors sho
w high sensitivity to fr ction (low / r tio) comp r
le to l te-responding norm l
tissues. Int J R di t Oncol Biol Phys 2002; 52(1):613. 36. Cox DR. Regression mod
els nd life-t
les. J Roy St t Soc 1972; B34:187220. 37. Fuks Z, Lei
el SA, W ll
ner KE, et l. The effect of loc l control on met st tic dissemin tion in c rcin
om of the prost te: Long-term results in p tients tre ted with 125-I impl nt ti
on. Int J R di t Oncol Biol Phys 1991; 21:537547. 38. DAmico AV, Whittington R, M
lkowicz SB, et l. Biochemic l outcome fter r dic l prost tectomy, extern l
e
m r di tion ther py or interstiti l r di tion ther py for clinic lly loc lized p
rost te c ncer. JAMA 1998; 280:969974. 39. M te TP, Gottesm n JE, H tton J, et l
. High dose-r te fterlo ding iridium-192 prost te
r chyther py: Fe si
ility re
port. Int J R di t Oncol Biol Phys 1998; 41:525533. 40. R dge H, Elg m l AA, Snow
PB, et l. Ten-ye r dise se free surviv l fter tr nsperine l sonogr phy-guided
iodine-125
r chyther py with or without 45-Gr y extern l
e m irr di tion in t
he tre tment of p tients with clinic lly loc lized, low to high Gle son gr de pr
ost te c rcinom . C ncer 1998; 83:9891001. 41. Edmundson GK, Rizzo NR, Te h n M,
et l. Concurrent tre tment pl nning for outp tient high dose r te prost te temp
l te impl nts. Int J R di t Oncol Biol Phys 1993; 27:1215. 42. Edmundson GK, Y n
D, M rtinez AA. Intr oper tive optimiz tion of needle pl cement nd dwell times
for conform l prost te
r chyther py. Int J R di t Oncol Biol Phys 1995; 33:125
7. 43. N g S, Ciezski JP, Corm ck R, et l. Intr oper tive pl nning nd ev lu ti
on of perm nent prost te
r chyther py: Report of the Americ n Br chyther py Soc
iety. Int J R di t Oncol Biol Phys 2001; 51(5):14221430. 44. M rtinez AA, Gonz le
z J, Spencer W, et l. Conform l high dose r te
r chyther py improves
iochemic
l control nd c use specific surviv l in p tients with prost te c ncer nd poor
prognostic f ctors. J Urol 2003; 169:974980. 45. Bl sko JC, H kon R, Grimm P. T
r nsperine l ultr sound-guided impl nt tion of the prost te: Mor
idity nd compl
ic tions. Sc nd J Urol Nephrol Suppl 1991; 137:113. 46. Zelefsky MJ, W llner KE,
Ling C, et l. Comp rison of the 5-ye r outcome nd mor
idity of three-dimensio
n l conform l r diother py versus tr nsperine l perm nent iodine-125 impl nt tio
n for e rlyst ge prost tic c ncer. Oncology 1999; 17(2):517522. 47. Zelefsky MJ,
Hollister T, R
en A, et l. Five-ye r
iochemic l outcome nd toxicity with tr
nsperine l CT-pl nned perm nent I-125 prost te impl nt tion for p tients with lo
c lized prost te c ncer. Int J R di t Oncol Biol Phys 2000; 47(5):12611266. 48. K
ng SK, Chou RH, Dodge RK, et l. G strointestin l toxicity of tr nsperine l int
erstiti l prost te
r chyther py. Int J R di t Oncol Biol Phys 2002; 55(1):99103.
49. Gel
um DY, Potters L. Rect l complic tions ssoci ted with tr nsperine l i
nterstiti l
r chyther py for prost te c ncer. Int J R di t Oncol Biol Phys 2000
; 48(1):119124. 50. Merrick GS, Butler WM, Dorsey AT, et l. Rect l dosimetric n
lysis following prost te
r chyther py. Int J R di t Oncol Biol Phys 1999; 43(5
):10211027. 51. W llner K, Roy J, H rrison L. Dosimetry guidelines to minimize ur
ethr l nd rect l mor
idity following tr nsperine l I-125 prost te
r chyther py
. Int J R di t Oncol Biol Phys 1995; 32(2):465471.
36 High dose r te
r chyther py in p tients with high IPSS, l rge gl nds, or wit
h prior TURP
Glen Gejerm n Introduction Prost te c ncer is the most common c ncer di gnosed
mong men in the United St tes, with n estim ted 230 110 new c ses nd 29 900 de
ths in 2004.1 In the prost tespecific ntigen (PSA) er , the m jority of men r
e di gnosed t n e rly st ge, nd tre tment options include surgery, extern l

e m r diother py (EBRT), nd interstiti l
r chyther py. Bec use of the risks of
impotence nd incontinence ssoci ted with r dic l prost tectomy, m ny men opt
for non-surgic l ppro ch. During the p st dec de prost te
r chyther py h s

een incre singly used s monother py or in com
in tion with EBRT.25 Exponenti l g

rowth h s
een forec sted so th t while only 4% of men di gnosed with prost te c
ncer in 1996 were tre ted with
r chyther py, it is estim ted th t pproxim tel
y h lf of those di gnosed in 2006 will
e impl nted.6 A 1999 medic re utiliz tio
n review demonstr ted th t
r chyther py m y suppl nt prost tectomy s the tre t
ment of choice for loc lized prost te c ncer.7 The m jority of prost te intersti
ti l
r chyther py is performed with perm nent seed impl nts with or without sup
plement l extern l
e m r diother py. Ten ye r PSA-free surviv l r tes of 8090% h
ve
een chieved with ccept
le r tes of mor
idity.25 The development of the hi
gh dose r te (HDR) iridium192 (192Ir) remote fterlo der nd its success in tre
ting gynecologic, pulmon ry, nd he d nd neck c ncers prompted interest in tre
ting prost te c ncer with tempor ry HDR interstiti l
r chyther py. The initi l
experience with multifr ction ted HDR
r chyther py in conjunction with EBRT not
ed excellent toler nce with superior conform lity nd minim l mor
idity (T
le 3
6.1).814 M te et l delivered four HDR tre tments with minimum peripher l dose
r nging from 3 Gy to 4 Gy.8 Dwell times in the periurethr l needles were reduced
limiting the m ximum urethr l dose to 120% while chieving 67 Gy per fr ction to
the posterol ter l prost te. Approxim tely 10% of p tients developed l te genit
ourin ry toxicity. However, s the impl nt technique evolved nd steel needles w
ere repl ced with pl stic c theters the incidence of urethr l strictures
ec me
uncommon. G l l e et l com
ined irr di tion of the prost te nd pelvic lymph ti
cs with HDR impl nts interdigit ted fter 20 Gy nd 40 Gy extern l
e m dose.9 T
he clinic l t rget volume (CTV) w s divided so th t 15 Gy w s delivered to CTV1,
defined s the peripher l zone nd 9 Gy to CTV2, which included the entire pros
t te gl nd. At medi n follow-up of 8 ye rs, the dise se-free surviv l w s 83%
with n ccept
le toxicity profile. Of p tients, 12% developed signific nt l te
genitourin ry toxicity consisting of incontinence, urethr l stricture, or
l dd
er sphincter sclerosis. These p tients h d undergone tr nsurethr l resection o
f the prost te (TURP) with medi n interv l to r diother py of less th n 5
B sic nd dv nced techniques in prost te
r chyther py
482
months. In contr st, none of 10 p tients who h d undergone TURP with medi n in
terv l to r diother py gre ter th n 6 months experienced incontinence. M rtinez
et l tre ted high risk prost te c ncer p tients with 46 Gy pelvic EBRT nd incr
e sing doses of HDR
r chyther py using from 5.5 Gy to 11.5 Gy per fr ction.10 A
n improved c use-specific surviv l w s noted in p tients tre ted with higher
io
logic l effective doses (>93 Gy). Since HDR
r chyther py c n mitig te the techn
ic l difficulties sometimes encountered during n impl nt procedure, it h s seve
r l potenti l dv nt ges over perm nent seed
r chyther py. The success of per
m nent seed impl nt is dependent on the correct deline tion of the t rget volume
nd optim l pl cement of needles nd seeds. Bec use tre tment pl nning nd dose
optimiz tion of HDR
r chyther py is performed fter the impl nt, the risk th t
prost te movement, needle deflection, nd seed migr tion will imp ir the intend
ed dosimetry is voided. N rrow pu
ic rch n tomy is less of n o
st cle in HDR

r chyther py
ec use the
ility to optimize dwell positions nd dwell times i
n the nterol ter l c theters will llow dequ te dosimetric cover ge even if pu

ic rch interference prevents ide l needle pl cement. HDR optimiz tion m kes it
possi
le to ccur tely cover the t rget
T
le 36.1HBR/EBRT studies
Author Ye rs P t St ge EERT HDR ient no.
1986 144 1992 T1
50Gy 15Gy T3 inter 2 ictate fract with ions HDR to to perip her
al pros tate zone T2 45y 910 T3 after y HDR 2 fx T1b 50.4y 34 T3 after y HDR 4 fx
T1c 36y 2 sep T3b before arate or proce alter ures HDR 6y/fx T1 50y 2 sep T3 s
plit arate course proce ures
Tx Follow DFS Acute Acute Chronic Chronic Plan up U I U U ning Toxi Toxi To
xi Toxi city>1 city>1 city>1 city>1

Trus Meian 83% NA base 96 months NA 6% 11%

alalae9
Pmges11 Mate8
1992 82 1994 1989 104 1995
Demanes13 1991 491 1998
Sim Meian 79% 11% base 24 months CT Meian 84% NA base 45 months Sim NA NA NA
base
8.50% 10%
4%
NA
9%
NA
NA
NA
NA
Borghee12 1998 50 1994
Trus Meian 96% 8% /Sim 45 base months
70%
2%
8%
High ose rate brachytherapy in patients
10y/fx 207 T1c 46y 23 sep T3c interig arate itate proce with ures HDR 5.51
483
Martinez10 1991 2000
Real Mean time 4.4 Trus years base
74% NA
NA
12%
NA
Sye14
1996 1999

200 T1c 39.6 T3b 45y before or after HDR

1.5y/fx 5.5 CT Mean 93% 10% 6.5 base 30 y months 4 fx
20%
2%
1.50%
volume while simultaneously setting tolerance constraints for the urethra an re
ctum. This ifferential osing is particularly useful for patients at higher ris
k for postimplant uropathy, such as those with high international prostate sympt
om scores (IPSS) an those with a prior TURP.1518 The ynamics of postimplant ee
ma must be consiere when evaluating prostate an urethral ose after permanent
see implantation.19,20 Since prostatic eema remains relatively stable uring
the immeiate postoperative perio, osimetric measurements of HDR brachytherapy
may be more reliable.21 HDR catheters can be place in the periprostatic tissue
s so that patients at risk for seminal vesicle or extraprostatic isease can be
aequately treate. The temporary uration of HDR implants an the use of remote
afterloaers obviate the nee for raiation precautions. This has been shown to
be a significant factor in a patients ecision when consiering the ifferent tr
eatment moalities.21 Finally, recent calculations of a low / r tio for prost te c
ncer suggests th t the use of hypofr ction ted HDR
r chyther py m y incre se t
he ther peutic r tio.22,23 Nonetheless,
ec use of the dur
le 10 ye r PSA-free
surviv l nd low mor
idity seen with perm nent seed impl nts, choosing
etween p
erm nent seeds nd HDR
r chyther py c n
e difficult. In the
sence of wellest

lished criteri , t H ckens ck University Medic l Center, New Jersey, we h ve
limited the use of HDR
r chyther py to those p tients who re not ide l c ndid
tes for perm nent seed
r chyther py, th t is, those with l rge gl nds, high IPS
S, nd those with prior TURP. An incre sed risk of urin ry mor
idity h s
een
found for p tients with l rge prost te gl nds. Prost te volumes gre ter th n 3540
cc h ve
een ssoci ted with gr de 2 urin ry toxicity nd cute urin ry retenti
on.2427 Gel
lum et l found th t p tients with prost te volumes gre ter th n 35 c
c h d 52.6% gr de 2 urin ry toxicity r te comp red with 35% in those whose gl
nds were sm ller th n 35 cc.24 Lee et l reported 25% risk of retention for p
tients whose pretre tment pl nning ultr sound t rget volume me sured more th n 4
5 cc.26 Crook et l demonstr ted th t the prost te volume w s signific nt pred
ictor of cute urin ry retention nd th t the risk incre sed for ny given size
when downsizing with ndrogen suppression w s used.27 IPSS is lso highly predic
tive of postimpl nt urop thy.15,16,24 Terk found n ssoci tion
etween the
se
line IPSS score nd the risk of urin ry retention fter seed impl nt tion.15 IPS
S scores <10, 1019, nd >20 correl ted with retention r tes of 2%, 11%, nd 29%,
respectively. Gel
lum et l reported th t men with
seline score gre ter th n
7 h d 59.2% r te of gr de 2 urin ry toxicity nd
B sic nd dv nced techniques in prost te
r chyther py
484
gre ter ch nce of h ving residu l symptoms fter one ye r.24 Bucci et l demonst
r ted th t high IPSS score w s predictive for the need for nd the dur tion of c
theteriz tion.16 The me n IPSS v lues for the no c theter nd c theter-requirin
g p tients were 6 nd 10 (p=0.004). P tients who h ve undergone TURP prior to
perm nent seed impl nt tion h ve higher risk for urin ry incontinence.28,29 Wh
ile the risk of urethr l injury is decre sed when peripher l lo ding technique
is employed,18 most
r chyther pists consider prior TURP to
e rel tive con
tr indic tion. The H ckens ck University experience Between 1998 nd 2003, 235 p
tients with st ge T1 T2
were tre ted with intensity modul ted r diother py (IMR
T) in com
in tion with HDR
r chyther py. P tients were st ged clinic lly
y his

tory, physic l ex m, digit l rect l ex m, nd serum PSA me surement. Addition l

studies including CT sc n,
one sc n, nd endorect l MRI were o
t ined s clinic
lly indic ted. The medi n ge w s 70 (r nge: 5280 ye rs); medi n PSA w s 7 (r ng
e: 1.2116); medi n ultr sound volume 25 cc (r nge: 11105); medi n erectile functio
n index 15 (r nge: 025), nd medi n IPSS 9 (r nge: 029). Of the p tients, 45% h d
prior TURP, 48% h d signific nt lower urin ry tr ct symptoms (IPSS>10), nd 24
% h d l rge prost te volumes (>45 cc); 13% of p tients h d pretre tment incontin
ence; 72% of p tients were ndrogen
l ted. All p tients received 50.4 Gy IMRT
prior to HDR
r chyther py. P tients underwent tre tment pl nning CT simul tion
in the supine position with v clock immo
iliz tion. The gross t rget volume (GTV
) included the prost te nd proxim l semin l vesicles with 3 mm m rgins round t
he prost te (except t the rect l interf ce). The pl nning t rget volume (PTV) w
s circumscri
ed
y the Corvus pl nning system (NOMOS Corp) nd w s determined

y com
ining the immo
iliz tion uncert inty with the loc liz tion uncert inty. Th
e result nt tot l uncert inty lloc ted the following m rgins: nterior, 10.2 mm
; posterior, 10.2 mm; right, 5.8 mm; left, 5.8 mm; superior, 10.2 mm; inferior,
10.2 mm1.8 Gy d ily fr ction w s prescri
ed for tot l of 50.4 Gy over 28 tre tm
ents. Inverse tre tment pl nning en
led dose optimiz tion so th t the prescript
ion go l to the GTV could
e met while constr ining the norm l tissue dose. The
norm l tissue dose w s restricted llowing no more th n 10% of the
l dder volum
e to receive gre ter th n 41.4 Gy nd no more th n 10% of the rect l volume to r
eceive 50 Gy. Dose-volume histogr ms (DVH) were reviewed nd only those pl ns wi
th 100% cover ge of the GTV nd t le st 90% of the PTV were pproved. The dose
w s prescri
ed to 7695% (medi n: 86%), the prost te m ximum dose w s 5266 Gy (medi
n: 60 Gy), the
l dder m ximum dose w s 5165 Gy (medi n: 58 Gy), nd the rect l
m ximum w s 4859 Gy (medi n: 55 Gy). After completion of IMRT, volumetric study
w s o
t ined to document the prost te volume nd the extent of the tissue defec
t in p tients with prior TURP. HDR
r chyther py w s performed within 6 weeks of
completing IMRT.
High dose r te
r chyther py in p tients
485
Oper ting room technique P tient positioning nd templ te P tients underwent

owel prep consisting of one d y full liquid diet followed
y Golytely on the e
vening
efore the impl nt. This regimen f cilit tes good ultr sound im ges nd
voids
domin l cr mping during the su
sequent constip tion from Lomotil nd mor
phine. After induction of nesthesi , p tients were pl ced in the dorsolithotomy
position with the femurs t 90 degree ngle to the OR t
le. Improper positio
ning will not llow the templ te to
e sutured to the perineum in flush positi
on (Figure 36.1). If pu
ic rch interference (PAI) required n extended lithotom
y position, the legs were repositioned once the nterior c theters were pl ced.
Prophyl ctic nti
iotics
eg n perioper tively nd continued for 5 d ys fter th
e impl nt. A 7.5 MHz
ipl n r ultr sound pro
e w s tt ched to floor-mounted s
tepping unit nd inserted in to the rectum. A needle guide/perine l templ te (Fi
gure 36.2) w s tt ched to the stepping device nd dv nced until flush g inst
the perineum. Over the ye rs, sever l ch nges h ve
een m de to the templ te
ut
the
sic design h s rem ined const nt. A needle guide templ te with det ch

le perine l portion is used. The needle guide h s holes with 0.5 cm sp cing nd
the perine l templ te cont ins silicone insert which prevents c theter movemen
t rel tive to the templ te. A foley c theter w s pl ced in the dist l urethr n
d 30 cc of n erosolized
Figure 36.1 Perine l templ te. Note th t the superior portion is not flush g in
st the perineum.
B sic nd dv nced techniques in prost te
r chyther py
486

Figure 36.2 Needle guide/perine l templ te in the stepping device.

Figure 36.3 Cystoscopy demonstr ting mucos l penetr tion.
surgilu
e mixture w s injected. The prost te w s im ged in 5 mm tr nsverse secti
ons nd the urethr l ( nd TURP defect) loc tion w s deline ted. Proper urethr l
visu liz tion is import nt
ec use the urethr m y
e more nterior or l ter l t
h n expected.30 TURP defect sizes nd loc tions c n v ry, therefore proper delin
e tion is critic l. While mucos l penetr tion would
e su
sequently detected wit
h flexi
le cystoscopy (Figure 36.3), identifying the offending c theter c n
e d
ifficult nd time consuming, nd voiding mucos l tr um is prudent. Flexiguide
c theter pl cement nd flexi
le cystoscopy Prior to flexiguide insertion, 18-g u
ge met l needles were pl ced through the templ te nd dv nced to the
High dose r te
r chyther py in p tients
487
Figure 36.4 ( ) Volumetric study with tr nsurethr l resection of the prost te (T
URP) defect (
) Corresponding im ge during impl nt.
mid-gl nd. The result nt needle tr ct prevents excessive pressure on the flexigu
ide tip while piercing the skin nd templ te. C theter devi tion is less likely
nd p r llel rr y of c theters is f cilit ted. The order in which the needles
were pl ced gener lly followed typic l sequence. The first two needles (1 nd
2) were pl ced l ter lly to ensure proper pl cement t the c psule edge nd to
st
ilize the gl nd. Needles 3 nd 4 were pl ced t the nterior surf ce of the
gl nd (11 nd 1 oclock positions) on either side of the urethr . In p tients with
TURP defects, these needles were moved posterior nd l ter lly s judged
y the
urethrogr m (Figure 36.4). Needle 5 w s pl ced t the prost te-rect l interf ce
. These needles were repl ced
y 6 French (inner di meter 1.65 mm) c theters wit
h stylets nd dv nced to the
se s seen on the tr nsverse im ge. Using the s
gitt l view, the c theters were dv nced 5 mm
eyond the
se (Figure 36.5). The
su
sequent needles nd
B sic nd dv nced techniques in prost te
r chyther py
488
Figure 36.5 ( ) Tr nsverse ultr sound im ge, (
) Corresponding s gitt l im ge.
c theters were sequenti lly pl ced (Figure 36.6) nd dv nced to the previously
determined depth. Typic lly, 1825 c theters were impl nted. The det ch
le perine
l templ te w s unscrewed from the needle guide (Figure 36.7) nd the stepping u
nit nd TRUS pro
e were removed. After the templ te w s sutured, the p tient w s
t ken out of the lithotomy position nd pl ced in frog-legged position for fl
exi
le cystoscopy. Cystoscopy confirms th t the c theters h ve
een dv nced to
the
se
y checking for tenting of the
l dder mucos t the
l dder neck (Figu
re 36.8). If ny of the c theters h d tr versed the prost tic urethr or pierced
the
l dder mucos , they were identified nd withdr wn to su
mucos l position
with cystoscopic visu liz tion. The c theters were covered during cystoscopy to
prevent them from getting wet. At completion of the procedure, three-w y fole
y c theter w s pl ced nd the p tient w s tr nsferred to recovery room.
High dose r te
r chyther py in p tients
489
Figure 36.6 Sequence of needle insertion.
Figure 36.7 Det ching the needle guide from the perine l templ te.

B sic nd dv nced techniques in prost te
r chyther py

490
Figure 36.8 ( ) Flexi
le cystoscopy without mucos l tenting; (
) with mucos l te
nting.
CT tre tment pl nning P tients were tr nsferred to custom
r chyther py m ttre
ss nd tre tment pl nning
eg n within 3 hours of the impl nt. A helic l CT w s
t ken with the g ntry ngle p r llel to the perpendicul r pl ne of the c theters
. The p tients position w s checked with lignment l sers nd tri ngul ting BBs w
ere pl ced. E ch c theter-templ te interf ce w s m rked, rect l m rker w s pl
ced, the foley c theter w s cl mped, nd 40 cc of contr st w s injected in to th
e
l dder. Anterior nd l ter l scout films confirmed th t the p tient h d
een
properly positioned nd th t the c theters h d
een dv nced superiorly enough i
n to the
se of the gl nd (Figure 36.9). Tr nsverse im ges of the impl nt volum
e (with 3 cm superior nd inferior m rgins) were collected in 5 mm
utting slic
es. The CT films were printed nd the clinic l t rget volume, urethr , TURP defe
ct (when pplic
le), nd rectum were outlined nd digitized into the Nucletron
Br chyther py Pl nning System (Figure 36.10). The c theters were digitized from
the visi
le tips in the
se of the gl nd until 5 mm
elow the pex. The first d
well position w s offset 5 mm from the tip nd ll dwell positions outside of th
e t rget were dis
led. The optimiz tion procedure w s performed with dwell ti
me gr dient f ctor of 2.5 in order to minimize the num
er of dwell positions wit
h neg tive times. Dose optimiz tion w s performed using full polynomi l fit to
dose points pl ced on the surf ce of the t rget volume with 10 mm dist nce
e
tween dose points. The rel tive weight for e ch dwell position w s m nu lly dju
sted to enh nce the t rget cover ge while m int ining the urethr l dose
elow 11
0% nd the rect l dose t 100% (Figure 36.10). Once the t rget dose of 5 Gy to t
he clinic l t rget volume (CTV) w s chieved, dose-volume histogr m n lysis nd
second ry h nd c lcul tions were performed.
High dose r te
r chyther py in p tients
491
P tients received 20 Gy in four fr ctions. The tre tments were delivered over 242
8 hours with minimum of 6 hours
etween e ch fr ction. Prior to e ch
r chythe
r py tre tment, the c theter-templ te interf ce w s checked to rule out c theter
displ cement from the templ te (Figure 36.11).
Figure 36.9 Anterior ( ) nd l ter l (
) scout films of c theters with stylets.
B sic nd dv nced techniques in prost te
r chyther py
492
Figure 36.10 ( ,
) Computed tomogr phy (CT) with corresponding dosimetry.
Repe t CT c theter movement In order to investig te the const ncy of the c thete
r position during the tre tment period, seri l CT sc ns were o
t ined for 30 p t
ients. During the initi l tre tment pl nning, fter the 5 mm slices were collect
ed, second set of CT im ges w s o
t ined in 2 mm slices. These were tr nsferre
d to the Voxelq workst tion (Philips Medic l Systems), the c theter tips were di
gitized, nd nterior nd l ter l digit lly reconstructed r diogr phs (DRR) were
gener ted with rendering of the c theter tips, ischi l tu
erosity, nd perine l
High dose r te
r chyther py in p tients
493
templ te (Figure 36.12). The me n dist nce
etween the ischi l tu
erosity nd th

e c theter tips w s me sured nd recorded. The dist nce
etween the ischi l tu
e
rosity nd fixed point on the templ te w s simil rly me sured. This 2 mm CT se
t w s repe ted
(c, d) P tient with prior tr nsurethr l resection of the prost te (TURP) in p
tient with prost te gl nd >100 cc.
nd n lyzed t different interv ls: one group h d the CT repe ted
efore the se
cond fr ction, second group
efore the third fr ction nd third group
efore
the fourth fr ction. After the c theters were digitized, DRR films were gener t
ed nd the me n dist nce
etween the c theter tips nd the ischi l tu
erosity w
s c lcul ted nd comp red to the first me surement to ssess c theter movement.
Using the me n dist nce voided the necessity to m tch corresponding c theters

etween the CT sets nd potenti l errors
B sic nd dv nced techniques in prost te
r chyther py
494

ssoci ted with this process. A simil r n lysis w s performed to ssess templ t
e movement. We found no movement of the c theters rel tive to the templ te. The
templ te nd c theters were lmost lw ys displ ced in c ud l direction. The
mount of displ cement w s time-dependent. The ver ge displ cement
efore the se
cond fr ction w s 2 mm,
efore the third 8 mm, nd
efore the fourth 10 mm (Figu
re 36.13). Without redress of this movement, signific nt dosimetric ch nges r
e encountered.31 When comp ring the first HDR tre tment with the third fr ction,
medi n decre ses in the following dosimetric p r meters were noted: dose to 90%
of the prost te volume, 35% (r nge: 060%); minim l dose to the
Figure 36.11 The c theter-templ te interf ce is checked
efore e ch tre tment.

se, 35% (r nge: 1765%); nd m xim l dose to 1 cc, 13% (r nge: 319%). The reducti
on in the D90, nd more signific ntly in the D
se, demonstr te the loss of dose
t the prost te
se th t results from the un v il
le dwell positions. Thus, t
he potenti l of HDR
r chyther py to provide ultim te conform lity m y
e limite
d
y the c theter displ cement th t occurs with multifr ction ted ther py. While
some centers resolve this pro
lem
y restricting the
r chyther py pplic tion
to one or two
High dose r te
r chyther py in p tients
495
Figure 36.12 Digit lly reconstructed r diogr phs (DRR) of templ te nd c theters
.
fr ctions per impl nt, their p tients re su
jected to multiple impl nt procedur
es. Others continue to deliver three or four HDR tre tments fter one impl nt pr
ocedure
ut compens te
y dv ncing the c theter under fluoroscopy prior to e ch
tre tment. Bec use of the potenti l to push the prost te superiorly s the c th
eters re djusted, this me sure m y compound the dosimetric error. We currently
o
t in repe t CT sc n prior to the third fr ction, ev lu te c theter position
t the
se, nd if displ cement is noted, dv nce the c theters. A repe t CT s
c n ccur tely documents the extent of displ cement nd when c theter dv ncemen
t is needed, repe t pl nning is necess ry to ensure ppropri te cover ge of the
t rget volume. P in m n gement Given the discomfort ssoci ted with interstiti l

r chyther py, urethr l c theteriz tion, nd
ed confinement, ggressive p in m
n gement is vit l to successful procedure. Bec use of logistic l del ys
etwe
en the time p tient reports p in nd the time n lgesic medic tions re dmini
stered, the nticip tion of p in dds su
jective response to physiologic event
s. Since studies h ve shown lower levels of p in-rel ted nxiety when p tients
re in control of their n lgesic regimen,32 we h ve prescri
ed morphine delivere
d
y p tient-controlled n lgesi (PCA) devices.32 With the use of PCA,
s l

infusion r te
B sic nd dv nced techniques in prost te
r chyther py
496
Figure 36.13 Time dependence of c theter displ cement.
m int ins non-zero
lood concentr tion so th t self dministr tion of sm ll opi
oid doses sufficiently
oosts drug concentr tion into the ther peutic r nge. P t
ients c n therefore self-titr te the mount of medic tion needed for the intensi
ty of the p in. In order to cl rify the n ture nd intensity of p in experienced
during
r chyther py, 102 p tients were ssessed with v lid ted visu l n log
ue sc le from 0 (no p in) to 10 (severe p in)
efore impl nt, 2 hours fter, nd

efore e ch of the four tre tments. P tients were sked to r te their
ck p in
nd p in from the foley nd perine l c theters s worst, ver ge, nd now. The type o
f PCA, num
er of perine l c theters, nd the presence of di
etes, o
esity, or
rthritis were n lyzed for ssoci tion with p in severity. Over ll, the reported
worst p in w s mild-to-moder te with the following me ns:
ck, 2.4; foley c th
eter, 3; perine l c theters, 2.7. On univ ri te n lysis,
ck p in incre sed ov
er time (p=0.001) while perine l c theter p in decre sed over time (p=0.005), n
d incre sed with the num
er of c theters (p=0.001). Foley c theter p in w s wors
e in those with rthritis (p=0.0025) nd o
esity (p=0.001). Multiv ri te n lysi
s with c theter num
er, di
etes, rthritis, nd o
esity s cov ri tes demonstr
ted th t only the type of PCA w s ssoci ted with p in intensity. P tients m n g
ed with
upivic ine-epidur l PCA experienced signific ntly less worst p in th n th
ose with morphinep renter l PCA (p<0.0001). In our experience, when PCA is use
d, prost te HDR c uses mild-to-moder te p in nd n epidur l dministr tion sign
ific ntly reduces the p in intensity (T
le 36.2). Toxicity All p tients underwe
nt HDR impl nt fter completion of extern l
e m irr di tion. The medi n interv
l
etween IMRT nd HDR w s 3 weeks (r nge: 0.56). Urethrogr phy w s used to delin
e te the urethr l course nd
High dose r te
r chyther py in p tients
497
T
le 36.2 Visu l n logue sc le p in intensity
y route of PCA dministr tion
Me n
ck p in Me n foley c theter p in Me n perine l c theter p in
Epidur l PCA 0.5 0.6 P renter l PCA 2.7 3.4 PCA, p tient-controlled n lgesi . 0
.7 3.0
medi n of 19 (r nge: 1228) flexiguide c theters were pl ced. Cystoscopy confirmed
mucos l penetr tion in 22% of p tientsmost of these were e rly in the series nd
in p tients who h d l rge TURP defects. HDR dosimetry w s optimized to deliver
20 Gy to the prost te t rget volume while keeping the medi n urethr l D50 to 91%
(r nge: 60 123%) nd urethr l Dm x to 112% (r nge: 90140%) of the prescri
ed dose
. Four fr ctions of 5 Gy were sep r ted
y t le st 6 hours. RTOG nd LENT/SOMA
scores were used to qu ntify toxicity (T
les 36.3 nd 36.4).33 Acute toxicity D
uring IMRT, 19% of p tients reported gr de 1 cute g strointestin l (GI) toxicit
y: 21% of p tients experienced gr de 1 cute genitourin ry (GU) toxicity nd 16%
of p tients gr de 2 toxicity. Symptoms resolved within 3 weeks of completing r
diother py in 66% of p tients nd ll returned to
seline
y 10 weeks. After re
mov l of the interstiti l impl nt gr de 1, cute GU toxicity w s noted in 26% n
d gr de 2 cute GU toxicity in 18% of p tients. These symptoms resolved within 4
weeks. No cute GI toxicity w s encountered. The following p r meters were test
ed for ssoci tion with toxicity: IPSS, ge, TURP, prost te size, ndrogen suppr
ession, num
er of interstiti l c theters, mucos l penetr tion, IMRT/HDR dose to
prost te rectum nd urethr , nd TURP defect dose. On univ ri te n lysis prio
r TURP w s ssoci ted with lower incidence of cute GU toxicity >1 with toxici
ty seen in 13% of TURP p tients versus 30% in those without (p=0.06). Continuous

signific nt predictors of gre ter th n gr de 1 cute GU toxicity included m xim

um prost te nd rect l IMRT dose (see T
le 36.5). L te toxicity The medi n foll
ow-up is 25 months (r nge: 660). Of the p tients, 23% developed gr de 1 chronic G
I toxicity
T
le 36.3 Modified RTOG gr ding criteri for cute r di tion toxicity
Gr de 1
G strointestin l symptoms Incre sed frequency or ch nge in
owel h
its not requ
iring medic tion. Rect l discomfort not
Gr de 2
Gr de 3
Gr de4
Di rrhe requiring Di rrhe requiring IV Acute or su
cute medic tion (eg, suppo
rt. Severe mucous o
struction, Fistul or Lomotil). Mucous disch rge requiring p
erfor tion. Rect l disch rge infrequently extended use of p ds.
leeding requiri
ng requiring p ds. Proctitis A
domin l distension. more th n 1 requiring n lges
ics or Proctitis requiring frequent tr nsfusion. A
domin l
B sic nd dv nced techniques in prost te
r chyther py
requiring n lgesics. occ sion l n rcotics. Mild rect l
leeding.
498
n rcotics. Rect l
leeding p in or tenesmus requiring 1 tr nsfusion, requiring

owel diversion. Genitourin ry Frequency or nocturi Frequency of nocturi Hem tu
ri requiring symptoms Frequency less frequent th n hourly or more, Dysuri more
th n 1 or nocturi twice the hourly. Dysuri or p in or sp sm requiring tr nsfu
sion.
seline. Dysuri not
l dder sp sm frequent n rcotics. Gross Hospit liz t
ion for requiring medic tion. requiring medic tion hem turi requiring 1 sepsis
due to (Pyridium). Infrequent tr nsfusion. Prolonged o
struction nd/or gross he
m turi . urin ry o
struction due to ulcer tion or necrosis Tempor ry prost te in
fl mm tion or of the
l dder. c theteriz tion. clots requiring c theteriz tion,
RTOG, R di tion Ther py Oncology Group; IV, intr venous.
T
le 36.4 RTOG nd LENT gr ding criteri for del yed r di tion toxicity
Gr de 1
G strointestin l symptoms Excess
owel movements twice the
seline. Slight rect
l disch rge or
lood.
Gr de 2
Gr de 3
More th n 2 ntidi rrhe ls/ d y At le st 1 tr nsfusion or more th n 2 co gul tio
ns for
leeding. Prolonged steroids per enem , Hyper
ric oxygen for
leeding/ul
cer tion. Regul r dil tion. Persistent use of protective p ds. Regul r n rcotic
use for p in.
Gr de 4
Dysfunction requiring surgery. Perfor tion. Lifethre tening
leeding.
More th n 2 ntidi rrhe ls/ week. 2 or fewer co gul tions for rect l
leeding. O
cc sion l steroids for ulcer tion. Occ sion l dil t tion. Intermittent use of pr
otective p ds. Regul r non-n rcotic or occ sion l n rcotic medic tion for p in.
Genitourin ry Moder te frequency. symptoms Nocturi Nocturi more th n twice the

seline. twice the
seline, Microscopic Gener lized hem turi . Light tel ngec
t si . mucos l trophy nd Intermittent minor tel ngect si . m croscopic hem tur

i . 2 or fewer co gul tions, Intermittent use of p ds. Regul r non-n rcotic or o

cc sion l n rcotic medic tion for p in. LENT
Severe frequency nd dysuri , Nocturi more frequent th n every hour. Reduction
in
l dder c p city (150 cc). Frequent hem turi requiring t le st 1 tr nsfusio
n. More th n 2 co gul tions for hem turi . Hyper
ric oxygen for
leeding/ ulcer
tion. n. Persistent use of protective p ds. Regul r n rcotic use for p in.
Severe hemorrh gic cystitis or ulcer tion requiring urin ry diversion nd/or cys
tectomy
T
le 36.5 Acute g strointestin l (GI) nd genitourin ry (GU) toxicity
Acute GI toxicity 01 Acute GU toxicity>1 p-v lue
Me n prost te m x Me n rect l m x 59.25 Gy (SD 304) 54.96 Gy (SD 248) 60.43 Gy (
SD 142) 55.83 Gy (SD 145) 0.02 0.06
High dose r te
r chyther py in p tients
SEX st nd rd devi tion,
499
T
le 36.6 Signific nt predictors of chronic genitourin ry (GU) toxicity
Chronic gr de 2 GU toxicity p-v lue
Me n prost te US volume 18 cc(SD 8) 0.04 Me n c theter num
er 18 (SD 3) 0.03 Me
n rect l HDR m x 311 cGy(SD 85) 0,04 US, ultr sound; HDR, high dose r te; SD, st
nd rd devi tion.
consisting of slight mucoid disch rge nd frequent
owel movements. No interve
ntion w s required nd resolution w s noted
y the 1 ye r follow-up: 9% of p tie
nts experienced gr de 2 chronic GU toxicity consisting of stress incontinence re
quiring intermittent use of protective p ds. All
ut two of these p tients h d p
reexisting incontinence th t resulted from prior TURP. The following p r meter
s were tested for ssoci tion with l te toxicity: IPSS, ge, TURP, prost te size
, ndrogen suppression, num
er of interstiti l c theters, mucos l penetr tion, I
MRT/HDR dose to prost te rectum nd urethr , nd TURP defect dose. On univ ri te
n lysis, postimpl nt incontinence w s ssoci ted with prior TURP (18% of p t
ients with TURP vs 0% without TURP, p=0.002) nd pretre tment incontinence (46%
of p tients with prior incontinence vs 2% without p=0.0001). Continuous signific
nt predictors of chronic GU toxicity included prost te volume, num
er of inters
titi l c theters, nd m ximum rect l HDR dose to 1 cc (T
le 36.6). The sm ll nu
m
er of p tients with l te gr de 2 GU toxicity nd the simil rity of pretre tmen
t v ri
les did not llow for st
le multiv ri te model. Conclusion High dose
r te (HDR)
r chyther py in com
in tion with intensity modul ted r diother py (I
MRT) is highly conform l tre tment chieving signific nt dose to the prost te
gl nd with minim l mor
idity. Excellent long term prost te-specific ntigen (PSA
)-free surviv l r tes h ve
een reported with 10 ye r
iochemic l cure r tes gre
ter th n 70%.34 The l ck of toxicity found in p tients considered t highest ri
sk for sequel e is encour ging nd r ises the possi
ility of further dose esc l
tion. Further study is needed to resolve the issue of interfr ction c theter mov
ement nd to determine the optim l
l nce of IMRT dose nd HDR fr ction tion. R
eferences
1. Jem l A, Tiw ri RC, Murr y T, et l. C ncer st tistics 2004. C ncer J Clin 20
04; 54:829. 2. R gde H, A
del-Aziz AE, Snow PB, et l. Ten ye r dise se free surv
iv l fter tr nsperine l sonogr phy guided Iodine-125
r chyther py with or with
out 45-Gr y extern l
e m irr di tion in the tre tment of p tients with clinic l
ly loc lized low to high Gle son gr de prost te c rcinom . C ncer 1998; 83:989100
1.
B sic nd dv nced techniques in prost te
r chyther py

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3. Sylvester JE, Bl sko JC, Grimm PD, et l. Ten ye r rel pse free surviv l fte
r extern l
e m r di tion nd
r chyther py tor loc lized prost te c ncer: the S
e ttle experience. Int J R di t Oncol Biol Phys 2003; 57:944952. 4. D ttoli M, W
llner K, True L, et l. Long term outcomes fter tre tment with extern l
e m r
di tion ther py nd p ll dium 103 for p tients with higher risk prost te c rcino
m . C ncer 2003; 97:979983. 5. Critz FA, Willi ms H, Levinson KA, et l. Prost te
specific ntigen
ounce fter simult neous irr di tion for prost te c ncer: the
rel tionship to p tient ge. J Urol 2003; 170:18641867. 6. N g S. Br chyther py
for prost te c ncer: summ ry of Americ n Br chyther py Society recommend tions.
Semin Urol Oncol 2000; 18(2):133136. 7. Hudson R. Br chyther py tre tments incre
sing mong Medic re Popul tion. He lth policy
rief of the Americ n Urologic Ass
oci tion, Inc. 1999; 9:18. 8. M te TP, Gottesm n JE, H tton J, et l. High dose r
te fterlo ding 192Iridium prost te
r chyther py: fe si
ility report. Int J R
di t Oncol Biol Phys 1998; 41:525533. 9. G l l e RM, Kov cs G, Schultze J, et l.
Long term outcome fter elective irr di tion of the pelvic lymph tics nd loc l
dose esc l tion using high dose r te
r chyther py for loc lly dv nced prost t
e c ncer. Int J R di t Oncol Biol Phys 2002; 52:8190. 10. M rtinez AA, Gust fson
G, Gonz lez J, et l. Dose esc l tion using conform l high dose r te
r chyther
py improves outcome in unf vor
le prost te c ncer. Int J R di t Oncol Biol Phys
2002; 53:316327. 11. Dinges S, Deger S, Koswig S, et l. High dose r te intersti
ti l with extern l
e m irr di tion for loc lized prost te c ncerresults of pro
spective tri l. R diother Oncol 1998; 48:197202. 12. Borghede G, Hedelin H, Holm
n S, et l. Com
ined tre tment with tempor ry short-term high dose r te Iridium192
r chyther py nd extern l
e m r diother py for irr di tion of loc lized pr
ost tic c rcinom . R diother Oncol 1991; 44:237244. 13. Dem nes DJ, Rodriguez RR,
Altieri GA. High dose r te prost te
r chyther py: the C liforni endocuriether
py (CET) method. R diother Oncol 2000; 57:289296. 14. Syed AMN, Puth w l A, Sh
rm A, et l. High dose r te prost te
r chyther py in the tre tment of c rcinom
of the prost te. C ncer Control 2001; 8:511521. 15. Terk MD, Stock RG, Stone NN
. Identific tion of p tients t incre sed risk for prolonged urin ry retention f
ollowing r dio ctive seed impl nt tion of the prost te. J Urol 1998; 160:13791382
. 16. Bucci J, Morris WJ, Keyes M, et l. Predictive f ctors of urin ry retentio
n following prost te
r chyther py. Int J R di t Oncol Biol Phys 2002; 53:9198. 1
7. Bl sko J, R gde H, Grimm PD. Tr nsperine l ultr sound guided impl nt tion of
the prost te: mor
idity nd complic tions. Sc nd J Urol Nephrol 1991; 137:113118.
18. W llner K, Lee H, W sserm n S, D ttoli M. Low risk of urin ry incontinence
following
r chyther py in p tients with prior tr nsurethr l prost te resectio
n. Int J R di t Oncol Biol Phys 1997; 37:565569. 19. W term n FM, Dicker AP. The
imp ct of post impl nt edem on the urethr l dose in prost te
r chyther py. Int
J R di t Oncol Biol Phys 2000; 47:661664. 20. Gejerm n G, Mullok ndov EA, S ini
AJ, et l. The effects of edem on urethr l dose fter p ll dium-103
r chyther
py. Med Dosim 2002; 27:221225. 21. M rtinez AA, P t ki I, Edmundson G, et l. Ph
se II prospective study of the use of conform l high dose r te
r chyther py s
monother py for the tre tment of unf vor
le st ge prost te c ncer: fe si
ilit
y report. Int J R di t Oncol Biol Phys 2001; 49:6169. 22. Duchesne G, Peters L. W
h t is the lph /
et r tio for prost te c ncer? R tion le for hypofr ction ted
high dose r te
r chyther py. Int J R di t Oncol Biol Phys 1999; 44:747748. 23. B
renner DJ, M rtinez AA, Edmundson GK, et l. Direct evidence th t prost te tumor
s show high sensitivity to fr ction (low / r tio) comp r
le to l te responding no
rm l tissue. Int J R di t Oncol Biol Phys 2002; 52:613.
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r chyther py in p tients
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24. Gel
lum DY, Potters L, Ashley R, et l. Urin ry mor
idity following ultr sou
nd guided tr nsperine l seed impl nt tion. Int J R di t Oncol Biol Phys 1999; 45
:5967. 25. H n BH, Demel KC, W llner K, et l. P tient reported short term compli

c tions fter prost te
r chyther py. J Urol 2001; 166(3):953957. 26. Lee N, Wuu
C, Brody R, et l. F ctors predicting for postimpl nt tion urin ry retention ft
er perm nent prost te
r chyther py. Int J R di t Oncol Biol Phys 2000; 48:145714
60. 27. Crook J, McLe n M, C tton C, et l. F ctors influencing the risk of cut
e urin ry retention fter trus-guided perm nent prost te seed impl nt tion. Int
J R di t Oncol Biol Phys 2002; 52:453 460. 28. R gde H, Bl sko JC, Grimm PD, et
l. Interstiti l I-125 r di tion without djuv nt ther py in the tre tment of cli
nic lly loc lized prost te c rcinom . C ncer 1997; 80:442453. 29. W llner K, Bl s
ko JC, C v n gh W. Br chyther py in the m n gement of prost te c ncer. In: DAmico
AV, H nks GE (eds) R diother peutic m n gement of prost te denoc rcinom . New
York: Oxford University Press, 1999, 135149. 30. Gejerm n G, Richter F, L nteri V
, et l. Imp ct of urethr l loc liz tion during tr nsrect l ultr sonogr phic lly
-guided tr nsperine l prost te
r chyther py. J Br chyther Int 2000; 16:249255. 3
1. Mullok ndov E, Gejerm n G. An lysis of seri l CT sc ns to ssess templ te nd
c theter movement in prost te HDR
r chyther py. Int J R di t Oncol Biol Phys 2
004; 58:10631071. 32. Upton RN, Semple TJ, M cintyre PE. Ph rm cokinetic optimis
tion of opioid tre tment in cute p in ther py. Clin Ph rm cokinet 1997; 33:22524
4. 33. Storey MR, Poll ck A, Z g rs G, et l. Complic tions from r diother py do
se esc l tion in prost te c ncer: prelimin ry results of r ndomized tri l. Int
J R di t Oncol Biol Phys 2000; 48:635642. 34. G l l e RM, M rtinez A, M te T, et
l. Long term outcome
y risk f ctors using conform l high dose r te
r chyther
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oost with or without neo djuv nt ndrogen suppression for loc lized prost t
e c ncer. Int J R di t Oncol Biol Phys 2004; 58:10481055.
P rt V Com
in tion of extern l
e m r diother py nd prost te
r chyther py
37 Com
ining extern l
e m r diother py with prost te
r chyther py: issues nd
r tion le
Cl riss Fe
les nd Rich rd K V licenti Introduction The optim l tre tment for c
linic lly loc lized prost te c ncer is controversi l. For low risk prost te c nc
er p tients (T1c/T2 , Gle son score <7, prost te-specific ntigen (PSA) 10.0 ng/m
L), monother py with either r dic l prost tectomy, three-dimension l conform l r
diother py (3D-CRT), intensity modul ted r diother py (IMRT), or prost te
r ch
yther py, result in simil r
iochemic l rel pse-free surviv l.13 P tient nd phys
ici n preference usu lly influence tre tment selection,
sed on critic l ssess
ment of rel tive side effect profiles, nd qu lity of life ev lu tions. While
r
chyther py lone in low risk p tients c n yield excellent dise se control nd
reported 93% 5 ye r freedom from
iochemic l f ilure,
r chyther py s monother
py in intermedi te nd high risk dise se (Gle son score >6, nd/or PSA >10 ng/m
L) is less th n optim l.3 Ku
n et l reported in the l te 1980s the higher loc
l f ilure r tes o
served in p tients with st ge B2 nd C, moder tely well, nd p
oorly differenti ted prost te c ncer tre ted with
r chyther py lone, using the
retropu
ic impl nt method.4 With the introduction nd routine use of PSA follow
-up, loc l control in intermedi te nd high risk p tients is much lower th n pre
viously reported. Even with modern
r chyther py techniques, intermedi te nd hi
gh risk p tients f ir poorly with
r chyther py lone. In recent report
y Kwo
k et l, 5 ye r results with iodine-125 (125I) prost te
r chyther py s monothe
r py w s dis ppointing 63% nd 24% in intermedi te nd high risk p tients, res
pectively.5 The com
in tion of extern l
e m r diother py (EBRT) nd prost te
r
chyther py h s
een used to improve outcomes in intermedi te nd high risk p ti
ents. This ch pter focuses on the use of extern l
e m r diother py with prost t
e seed impl nt tion nd provides review of the r tion le nd v il
le d t in
com
ining these mod lities. R tion le for com
ined mod lity ther py Improved di
se se-free surviv l, freedom from dist nt met st sis nd over ll surviv l h s
e
en shown with delivery of high r di tion doses to the prost te.58 Multiple ppro
ches h ve
een used to deliver dose esc l tion to the prost te: intensity modul
ted r diother py (IMRT), high energy neutrons, hyperfr ction ted r di tion, nd
prost te
r chyther py
oosts in conjunction with extern l
e m r diother py (EB
RT). The ddition of EBRT provides
ro der delivery of r diother py nd the
e
nefit of gre ter dose distri
ution nd cover ge of tumor th t h s extended
eyon

d the prost te c psule. Br chyther py lone, however, m y
e limited in its
il

ity to deliver dequ te doses to dise se extending
eyond the prost te. By com
i
ning
r chyther py nd threeB sic nd dv nced techniques in prost te
r chyther py
504
dimension l conform l r diother py (3D-CRT) one g ins the
enefit of higher dose
delivery provided
y
r chyther py long with cover ge of dise se th t m y exte
nd outside of the prost te gl nd proper with the use of EBRT. Furthermore, if c
ncer cells h ve spre d to the dr ining lymph nodes,
r chyther py will not ddre
ss these re s of dise se. An ddition l
enefit of com
ined
r chyther py nd E
BRT is in p tients who h ve received su
optim l impl nts. Com
ined mod lity m y

e justifi
le in those p tients who h ve received su
optim l impl nts nd requi
re supplement l doses of r di tion to compens te for underdosed re s of dise se
. Achieving higher doses The import nce of tre ting to higher r di tion doses h
s
een est
lished
y multiple dose-response studies.68 There re
oth 3D-CRT nd
prost te
r chyther py d t supporting dose-response in intermedi te nd high
risk p tients.7,9,10 The R di tion Ther py Oncology Group (RTOG) reported impro
ved dise se-specific nd over ll surviv l in p tients with high Gle son scores w
ho received higher doses of EBRT.9 Stone nd Stock reported their experience wit
h perm nent 125I impl nts nd found th t dose w s the most signific nt predictor
of
iochemic l control.10 The
enefit w s gre ter for those p tients presenting
with PSA levels >10 ng/mL. The 4 ye r freedom from
iochemic l f ilure (PSA <1.
0 ng/mL) w s 51% nd 100% in p tients with D90 (the dose covering 90% of the p
rost te) <140 Gy versus D90>40 Gy, respectively (p=0.009). Tre tment of extr pri
ost tic extension c ncer A second
enefit of com
ining EBRT with
r chyther py i
s the dded r di tion doses to dise se th t h s extended through the prost tic c
psule nd/or into the semin l vesicles. Both extr c psul r extension (ECE) nd
semin l vesicle inv sion (SVI) re dverse prognostic f ctors th t c n
e estim
ted
y PSA nd Gle son scoring, using the following equ tions derived from P rti
n nd descri
ed
y Ro ch. 1113 ECE=(3/2) PSA+[(GS3)10] SVI=PSA+[(S6)10] The extent t
o which raioactive sees place within the prostate can aequately treat extrac
apsular isease has been ebate. Patients at higher risk of ECE may benefit les
s from brachytherapy alone. Davis et al evaluate postprostatectomy patients an
foun that extraprostatic extension (EPE) measure on average 0.8 mm, with a ra
nge from 0.04 mm to 4.4 mm.14 Current brachytherapy techniques typically encompa
ss 35 mm beyon the glan. However, postprostatectomy measurements of ECE may not
represent in vivo istance of actual tumor sprea. As the risk of EPE increases
, the benefit of brachytherapy as monotherapy becomes less, an combine EBRT an
 brachytherapy or EBRT alone ought to be consiere.
Combining external beam raiotherapy
505
Treatment of pelvic lymph noes With increasing tumor (T) stage, PSA, an tumor
grae, men with prostate cancer are at higher risk of lymph noe sprea. The est
imate risk of lymph noe metastasis can be estimate from the formula evise b
y Roach:13 +LN=(2/3) PSA+[(S6)10] Often, men with >15% risk of lymph noe involve
ment are treate with whole pelvis external beam irraiation. Clearly, if patien
ts are at an increase risk of lymph noal metastases that can be eraicate wit
h raiotherapy, brachytherapy is not aequate treatment. Thus, the aition of E
BRT can aress both extracapsular isease as well as subclinical lymph noal me
tastases. Results from the RTO 9413 trial show a significant improvement in pro
gressionfree survival in men with prostate cancer treate with whole pelvis EBR
T in conjunction with hormonal therapy, compare with men treate with hormones
an raiation to the prostate only.15 These ata strongly suggest pelvic irraia
tion benefits patients with intermeiatetohigh risk isease. Supplementation of

prostate brachytherapy ose Aequate ose elivery with interstitial see impla
ntation is epenent on proper visualization of the prostate, osimetric plannin
g, correct placement of raioactive sees, an ultimately physician expertise. D
espite improvements in brachytherapy techniques an better visualization with tr
ansrectal ultrasoun (TRUS), often the preplanne osimetry oes not match the a
ctual oses elivere to the prostate at the time of postimplant evaluation. If
on postimplant osimetric analysis, subtherapeutic oses have been raiationel
ivere to the prostate, a patient may be consiere for see reimplantation or s
upplemental EBRT to provie aequate oses an avoi treatment failure. Postimpl
ant osimetric analysis The American Brachytherapy Society (ABS) recognizes the
nee for aequate postimplant osimetry in elivering optimal patient care an h
as establishe guielines for postimplant osimetric analysis base on an expert
panels review of the literature. The ABS recommens that compute tomography (CT
)base postimplant osimetry be performe on all patients unergoing permanent
prostate brachytherapy. The enlargement of the prostate ue to eema immeiately
postimplantation can result in a 10% mean ecrease in ose elivere to the pro
state compare to osimetry obtaine one month postimplantation.16 enerally, im
aging is obtaine one month post implant, however, the optimal timing of imaging
remains unclear. The ABS recommens that postimplant osimetry be performe at
a consistent interval with ocumentation of the 50, 80, 90, 100, 150, an 200% i
soose lines, osevolume histograms (DVH) an the minimal ose covering 90% of t
he prostate volume (D90). The percent volume of the prostate receiving at least
100% or 150% of the prescribe minimal peripheral ose or V100 an V150 also
Basic an avance techniques in prostate brachytherapy
506
are recommene as parameters to be measure. The rectal an urethral oses shou
l be reporte an correlate with clinical outcome. Ultimately, the earlier the
postimplant osimetry is performe, the earlier an unerose implant can be i
entifie an aitional treatment provie with either reimplantation or aitio
nal external beam raiotherapy.17 Caniates for combine moality therapy As ou
tline by the ABS, brachytherapy is an option for patients with a life expectanc
y of 5 years or more, who are without istant metastasis an without large trans
urethral resection of the prostate (TURP) efects.18 In patients with a signific
ant risk of isease outsie the implant volume, the aition of EBRT or hormonal
therapy is avise. The risk of lymph noe an seminal vesicle involvement as w
ell as the risk of extracapsular extension shoul be calculate for each patient
with the use of Partin Tables or other risk stratification moels. The recommen
e brachytherapy oses when use in combination with EBRT are shown in Table 37
.1. Treatment outcomes with combine permanent see implant an EBRT No ranomiz
e trials have been conucte to evaluate the benefit of combine brachytherapy
an external beam irraiation.
Table 37.1 ABS recommene see implant oses with combine EBRT
Isotope
103
Brachytheraphy ose (y)
EBRT ose (y)
P 8090 4050 125 I 100110 4050 ABS, American Brachytherapy Society EBRT, external be
am raiotherapy;
Dattoli et al, at the University Community Hospital in Tampa, provie the earli
est ata with combine EBRT an prostate brachytherapy. A total of 73 patients w
ith T2aT3 prostate cancer with one or more of the following risk factors: stage T2
b, leason score 710, PSA >15 ng/mL, or elevate prostatic aci phosphatase (PAP)

receive pallaium103 (103P) implant followe by EBRT; 10 patients i, howeve

r, receive 2 months of hormonal therapy prior to raiation. With a meian follow
up time of 2 years, actuarial freeom from biochemical failure (PSA<1.0 ng/mL)
was 79% at 3 years.19 PAP was the only significant preictor of biochemical fail
ure (p=0.04). After poor results with 125I interstitial implants alone, Frank Cr
itz, at the Raiotherapy Clinics of eorgia began treating patients with combine
 brachytherapy an EBRT in the late 1970s. Critzs experience with simultaneous int
erstitial see implant an EBRT is the largest to ate. Over 1000 men with T1T2 l
ymph noe negative NO low to high risk aenocarcinoma of the prostate receive 1
25I implant followe 21 ays later by EBRT, thus being expose simultaneously to b
oth the raioactive sees an external beam
Combining external beam raiotherapy
507
irraiation.20 Despite variable implant oses, which are consiere less than th
e current stanar, excellent iseasefree survival was achieve. With a meian
followup of 3 years, iseasefree survival, efine as achieving an maintainin
g a PSA nair of 0.5 ng/mL was 79% an 72% at 5 an 10 years, respectively. Meia
n time to recurrence was 3.5 years. Of note, patients who unerwent a retropubic
125I implant ha a 73% 5 year DFS while those patients receiving the ultrasoun
guie transperineal implant ha a higher 5 year iseasefree survival (DFS) of
92% (p=0.0001). Again, emphasizing the importance of technique an the benefit
of the more moern ultrasounguie transperineal approach. The secon largest
reporte experience with combine permanent interstitial implant an EBRT comes
from Seattle, Washington. Rage et al reporte their upate results of 229 pati
ents with T1T3 aenocarcinoma of the prostate treate with transrectal ultrasoun
(TRUS)guie 125I implant with or without EBRT:21 82 men consiere at high ris
k of extracapsular extension (leason score >6 an/or stage >T2b) receive a mea
n peripheral ose (MPD) of 120 y by implant following EBRT (see Table 37.2). Of
note, this prescribe implant ose is higher than that recommene by the ABS a
n American Association of Physicists in Meicine (APPM) Task roup 43. Using th
e upate American Society for Therapeutic Raiology an Oncology (ASTRO) efini
tion of biochemical failure (three consecutive rises in serum PSA level measure
6 months apart) the observe 10 year biochemical with no evience of isease (b
NED) for the monotherapy group was 66% an 79% in the combination therapy group.
Four patients ie from prostate cancer yieling a iseasespecific 10 year sur
vival rate of 98%. The higher
Table 37.2 Comparison of biochemical iseasefree survival (DFS) with combine b
rachytherapy an EBRT
Stuy No. EBRT Implant Meian % patients 10 yr Definition 5 yr patients ose os
e (f/u) receiving biochemical biochemical of PSA ts (y) (y), (mths) hormones D
FS (%) DFS (%), failure source
1029 82 73 72c 45 80, 125I 45 122 24 47 0 0 14 79 79b 72 88 79.9a 45 120, 125I 4
1 80, 103P 45 120, 125I 72 PSA 70.5 ng/ml 79a ASTROa PSA 1.0 ng/mll 2 successive
rises in PSA ASTROa
Critz20 Rage21 Dattoli19 rao24
100, P 66 45 90, 125I 54 0 Merrick26 110, 103 p 25 103 MSKCC 65 50.4 P 36 86
a three consecutive rises in PSA level measure 6 months apart
103
87b
ASTROa
Basic an avance techniques in prostate brachytherapy

b c
508
3 year freeom from biochemical failure, hormone nave group.  patients expose t
o anrogen eprivation.
biochemical control rate observe in the combine moality group, although not s
ignificant, is nonetheless encouraging an further supports the notion that exce
llent isease control can be obtaine in intermeiate an high risk patients wit
h combine moality therapy. The upate Rage et al results are comparable to t
he earlier publishe ata which use a PSA>0.5 ng/mL as the efinition of bioche
mical failure. In their earlier publishe ata, a benefit in tumor control in th
e combine group was note when a PSA enpoint of 0.4 ng/mL was use. The isease
free survival was 63% in the combine moality group versus 74.5% in the implan
t alone group (p=0.046).22 The authors suggest that combining EBRT with a brachy
therapy implant might be the best moality for patients with clinically localize
 prostate cancer, with results comparable to prostatectomy. While some physicia
ns, such as Critz, propose combine implant an EBRT for all locally confine pr
ostate cancer patients, others, incluing Rage et al, use the following criteri
a for implant alone therapy: PSA<10 ng/mL, clinical stage T2a, an leason score
<7.21 While the level of PSA nair necessary for optimal treatment outcome is e
bate (<1.0 ng/mL or <0.5 ng/mL), a low posttreatment PSA nair is a known prei
ctor of DPS.23 Critz et al showe that 77% of patients treate with combine EBR
T an prostate implant ha a PSA of 0.5 ng/mL or less at 60 months. Posttreatmen
t PSA values ecrease most rapily within the first 3 months postimplant, an m
ore graually thereafter, an by 24 months 52% of patients achieve a PSA nair
of 0.5 ng/mL or less.23 The rate of PSA ecrease, however, was not a prognostic
inicator. rao et al publishe on their actuarial DFS after prostate cancer br
achytherapy using interactive techniques with biplane ultrasoun an fluoroscopi
c guiance. A total of 553 T1T3c prostate cancer patients (with no PSA, leason s
core, or hormonal therapy restriction) were treate with 125I or 103P see impl
antation. 490 patients were analyze, of whom 70 were at risk of having capsular
involvement (etermine by igital rectal examination, TRUS, or biopsy) an rec
eive 45 y ajuvant EBRT. Two aitional patients receive EBRT after brachythe
rapy because of suboptimal ose elivery uring the brachytherapy. Twenty six pa
tients in the implant alone group an 10 patients in the combinationmoility gr
oup receive anrogen eprivation therapy. There was no significant ifference i
n iseasefree survival between the group treate by implant alone versus the gr
oup unergoing implant plus xray therapy. For the hormone nave group the 5year
isease free survival was 80% for the implant alone group an 72% for the combin
etreate group, while those patients receiving hormonal therapy ha a 5year D
FS of 83% vs 88%, respectively.24 The Memorial SloanKettering Cancer Center (MS
KCC) reporte their early but favorable results with 103P permanent implant an
3DCRT in intermeiate an unfavorable risk prostate cancer patients. PSA relapse
free survival at 3 years was 87%.25 More recently, Merrick et al reporte favor
able results with combine EBRT an permanent see implantation. Five year bioch
emical DFS in 66 hormone naive patients with high risk prostate cancer (leason
score 7, PSA10 ng/mL, clinical stage T2b) treate with supplemental EBRT an perman
ent see implant was 79.9%.26 A summary
Combining external beam raiotherapy
509
of institutional experiences with combination EBRT an brachytherapy boost is pr
ovie in Table 37.2. Treatmentrelate sie effects Rectal an urinary toxicity
Morbiity from the combine approach appears to be comparable to high ose 3DC
RT or surgery for similar risk patients. The most commonly observe toxicity wit
h combine 3DCRT an brachytherapy boost are RTO grae 12 rectal an urinary to

xicity. In the stuy by Singh et al, 13% of patients experience rectal bleeing
an 8% experience increase frequency of bowel movements, while no patients ex
perience grae 3 or 4 rectal toxicity.25 The most commonly reporte urinary si
e effects are symptoms of frequency, urgency, an nocturia, which are easily man
age with alphablockers.19,24 Less commonly observe are urinary obstructive sy
mptoms requiring intervention. A 6% rate of temporary foley catheterization was
observe by Singh et al,25 an less than 3% of men require a TURP postimplantat
ion for persistent urinary obstructive symptoms in the stuy by Datoli et al.19
Observe rates of stress incontinence range from 1% to 5%.19,20,25 One patient
in the stuy by Dattoli et al evelope complete urinary incontinence.19 Althoug
h complications of urinary incontinence, obstruction, an urethral stricture (le
ss than 3%) are rare with combine moality therapy, they are more likely to be
seen in men with a prior history of urinary obstructive symptoms an prior TURP.
19,20 In Critzs review of 1000 men treate with combine implant an EBRT, all ca
ses of urinary incontinence, urethral necrosis, an urethral occurre in men wit
h a prior history of TURP.20 Similar to the urinary toxicity profile, rectal tox
icity with combine moality therapy is most likely to be of grae 1 or 2 (RTO)
. In the MSKCC experience, 13% of patients experience rectal bleeing an 8% ex
perience increase frequency of bowel movements an no grae 3 or 4 rectal toxi
cities were observe.25 rao et al reporte a rare 1% occurrence of rectal fist
ula.24 A more recent report ranomizing low risk patients to implant alone (125I
or 103P) an intermeiate risk patients to 103P implants (90 y vs 115 y) wi
th supplemental EBRT (44 vs 20 y), reporte higher American Urological Associat
ion (AUA) scores at one month in the groups receiving higher prescription oses
of 103P (125 y 103P alone an 115 y 103P plus 20 y EBRT).36 Similarly, rec
tal morbiity was significantly higher in the combine moality group only at on
e month. Rectal morbiity consiste mostly of increase frequency an mucous pas
sage an no cases of rectal ulceration or fistula were ocumente. The ose of E
BRT receive (20 y vs 44 y) i not correlate with urinary or rectal morbiity
, suggesting that the aition of EBRT oes not impact on overall treatment morb
iity. Furthermore, at one year, little ifference in morbiity was observe bet
ween all treatment groups.36 Longer followup, an aitional ranomize stuies
will help to clarify the effect of supplemental EBRT on rectal an urinary toxi
city as well as eluciate its effect on other parameters such as potency preserv
ation an quality of life.
Basic an avance techniques in prostate brachytherapy
510
Potency preservation Rates of potency preservation greater than 80% have been re
porte with brachytherapy as monotherapy.28 Potency following EBRT, however, is
less preserve with reporte rates of only 50% at 6 years.3 From the limite at
a available on the effects of combine prostate brachytherapy an EBRT on sexual
function, the rates of sexual potency, not surprisingly, fall between those rep
orte for either moality alone. Dattoli et al reporte sexual potency rates of
82% an 77% at 1 an 3 years, respectively.19 Similar rates of potency were obse
rve by Singh et al, an a reporte 26% of sexually potent men evelope erectil
e ysfunction after combine moality treatment.25 No ifference in the rate of
erectile ysfunction was observe among those men receiving neoajuvant anroge
n eprivation an men who i not receive hormonal therapy.25 Age has been shown
to play a role in preservation of sexual function. Critz et al showe that men
younger than 65 ha an 85% rate of potency preservation, while men oler than 65
ha a 65% rate of potency (p=0.05).27 Future trials incluing an ongoing RTO p
hase II stuy for intermeiate risk prostate cancer patients receiving combine
EBRT an prostate will provie further ata on treatmentrelate toxicity an th
e overall safety an efficacy of the combine approach. High ose rate brachythe
rapy boost The largest experience with temporary high ose rate (HDR) implants h
as been in Europe. This remote afterloaing technique enables treatment planning
to take place after neeles have been securely place into the prostate. The o

se elivere to the prostate is then calculate an controlle by the well time
of the raiation source at specifie locations within each neele. Several inst
itutions have reviewe their experience with EBRT in combination with HDR brachy
therapy for locally avance prostate cancer. Mate et al reporte their prospect
ive results of 144 men with T1bT3 clinically noenegative prostate cancer treate
between 1986 an 1992 with whole pelvis EBRT combine with two fractions of HDR
brachytherapy consisting of 15 y/fraction to the peripheral prostate an 9 y
to the entire prostate. Overall survival an bNED survival at 5 years was 80.4%
an 74%, respectively.29 A match pair analysis was performe by Keston et al to
compare patients treate with external beam irraiation alone with combine EBRT
an interstitial HDR brachytherapy boost. Five year biochemical control rates f
or EBRT +HDR was significantly higher than for EBRT alone (67% vs 44%, p<0.001).
The combine moality group achieve a lower PSA nair (0.4 ng/mL vs 1.1 ng/mL)
an sustaine longer intervals of PSA nair (1.5 vs 1.0 years).30 On multivaria
te analysis, leason score, tumor (T) stage, an the use of EBRT were significan
tly associate with freeom from biochemical failure. A correlation between bioc
hemical control an causespecific survival was also emonstrate. Aition of h
ormonal therapy In aition to ose escalation, the use of hormonal therapy has
also been use to achieve better isease control in intermeiate an high risk p
rostate cancer patients. While the
Combining external beam raiotherapy
511
aition of hormonal therapy to EBRT or surgery has shown a benefit in iseasef
ree an overall survival, the use of ajuvant hormonal therapy with brachytherap
y has not been well establishe. Data supporting hormonal therapy (HT) in conjun
ction with brachytherapy are limite by retrospective esign an small sample si
ze. Multiple prospective ranomize trials have shown a benefit from the aitio
n of anrogen ablation to EBRT in patients with locally avance prostate cancer
. Both RTO 8610 an RTO 8531 showe a benefit in local control an iseasefree
survival.31,32 The EORTC stuy, publishe in The New Englan Journal of Meicine
, showe improvement in overall survival in aition to iseasefree survival an
 local control with the aition of ajuvant HT to EBRT.33 Neoajuvant HT is r
outinely use for ownsizing large prostate glans prior to brachytherapy with t
he goal of shrinking the prostate an allowing for greater ease of implant an a
better ose istribution. The ABS recognizes the use of HT with brachytherapy a
n EBRT an recommens its use only in the context of ownstaging large prostate
glans (>60 cc) prior to see implantation.17 Several institutions have reviewe
 their experiences with combine HT an brachytherapy, however, no prospective
ranomize trials exist to ate. Stone an Stock reporte their results with neo
ajuvant HT an brachytherapy in 115 patient with intermeiate risk prostate can
cer treate with leuprolie an flutamie for 3 months prior to implant an an a
itional 3 months post implant.34 A benefit in local control is suggeste in th
e group receiving neoajuvant HT with local control measure by routine prostat
e biopsy at 2 years (3.8% vs 7.7%, p=0.05). Two aitional retrospective stuies
, both limite by number of patients an mean followup time have evaluate the
aition of anrogen ablation to combine EBRT an brachytherapy see implant. S
ylvester et al performe a matche pair subset analysis of 98 patients with inte
rmeiatetohigh risk prostate cancer of which 21 patients receive combine imp
lant an EBRT plus anrogen ablation.35 The overall rate of freeom from biochem
ical failure at 5 years was 77% in the HT group an 58% in the nonHT group (p=0
.08). In the rao et al retrospective implant review: 36 of 490 patients with T
1T3c prostate cancer who unerwent brachytherapy see implantation ha receive p
rior anrogen eprivation therapy for prebrachytherapy reuction of prostate vol
ume or per patient request. Ten of the 36 patients at risk of extracapsular exte
nsion receive combine brachytherapy with EBRT. The 5 year iseasefree surviva
l for the implant alone group an implant combine with EBRT were 83% an 88% re
spectively.24 No strong evience exists for the aition of ajuvant or neoaju

vant HT to prostate brachytherapy. The stuies publishe thus far are limite bo
th in patient size an uration of followup. The role of HT in aition to pros
tate brachytherapy remains unanswere an we await future prospective trials. Th
e potential benefit of HT in combination with brachytherapy has not yet been tho
roughly investigate an no consensus exists for the routine use of hormonal the
rapy with brachytherapy in intermeiatetohigh risk patients. Conclusions an fu
ture irections Prostate cancer remains the most common noncutaneous cancer ia
gnose in men, an the secon leaing cancer cause of eath. With the wiesprea
use of prostatespecific
Basic an avance techniques in prostate brachytherapy
512
antigen (PSA) testing, more men are being etecte with localize prostate cance
r. Optimal therapy for organconfine prostate cancer remains an ongoing ilemma.
There is abunant evience showing the benefit of higher raiation oses in tre
atment outcomes in men with intermeiate an high risk features. While favorable
risk patients have excellent outcomes with monotherapy, combine external beam
an brachytherapy is an excellent treatment option for men with intermeiate to
high risk prostate cancer. Combining threeimensional conformal raiotherapy (3
DCRT) with brachytherapy boost is a safe an effective way of elivering high ra
iation oses to the prostate an can achieve results similar to favorable risk p
atients. Brachytherapy an combine EBRT has been shown to be both safe an effe
ctive, however, we await the results of recently complete an future prospectiv
e ranomize trials to verify these finings. While the aition of hormonal the
rapy to external beam irraiation has proven beneficial, the use of hormonal the
rapy with brachytherapy remains less clear. Future prospective clinical trials w
ill help better efine the role of combine external beam raiotherapy an brach
ytherapy an the aitional use of hormonal therapy. References
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al prostatectomy, external beam raiation therapy or interstitial raiation ther
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MJ, Wallner K, Ling CC, et al. Comparison of the 5year outcome an morbiity of
threeimensional conformal raiotherapy versus transperineal permanent ioine
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12. Partin AW, Mangol LA, et al. Contemporary upate of prostate cancer staging
nomograms (Partin tables) for the new millennium. Urology 2001; 58:843848. 13. R
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o preict pathological stage in men with localize prostate cancer. J Urol 1993;
150:1923. 14. Davis BJ, Pisansky TM, Wilson TM, et al. The raial istance of e
xtraprostatic extension of prostate carcinoma: implications for prostate brachyt
herapy. Cancer 1999; 85(12):26302637. 15. Roach M, Lu JD, Lawton C, et al. A Phas
e II trial comparing wholepelvic (WP) to prostate only (PO) raiotherapy an ne
oajuvant to ajuvant total anrogen suppression (TAS): Preliminary analysis of
RTO 9413. Proceeings of the 43r Annual ASTRO Meeting, 2001. 16. Waterman FM,
Yue N, Corn BW, et al. Eema associate with I125 or P103 prostate brachyther
apy an its impact on postimplant osimetry: An analysis base on serial CT acq
uisition. Int J Raiat Oncol Biol Phys 1998; 41:10691077. 17. Nag S, Bice W, DeWy
ngaert K, et al. The American Brachytherapy Society recommenations for permanen
t prostate brachytherapy postimplant osimetric analysis. Int J Raiat Oncol Bi
ol Phys 2000; 46:221230. 18. Nag S. Brachytherapy for prostate cancer: Summary of
American Brachytherapy Society recommenations. Semin Urol Oncol 2000; 18(2):13
3136. 19. Dattoli M, Wallner K, Sorace R, et al. 103PD brachytherapy an external
beam irraiation for clinically localize, highrisk prostatic carcinoma. Int J
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H, et al. Simultaneous raiotherapy for prostate cancer: 125I prostate implant f
ollowe by externalbeam raiation. Cancer J Sci Am 1998; 4(6):359 363. 21. Rage
H, Leroy K, AbelAziz E, et al. Prostate specific antigen results in 219 patien
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e H, Elgamal AA, Snow PB, et al. Tenyear isease free survival after transperin
eal sonographyguie ioine125 brachytherapy with or without 45ray external
beam irraiation in the treatment of patients with clinically localize, low to
high leason grae prostate carcinoma. Cancer 1998; 83(5):9891001. 23. Critz FA,
Levinson K, Williams WH, et al. Prostatespecific antigen nair of 0.5 ng/ml or
less efines isease freeom for surgically stage men irraiate for prostate c
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al iseasefree survival after prostate cancer brachytherapy using interactive t
echniques with biplane ultrasoun an fluoroscopic guiance. Int J Raiat Oncol
Biol Phys 1998; 42:289298. 25. Singh A, Zelefsky MJ, Raben A, et al. Combine 3
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iat Oncol Biol Phys 2000; 90:275280. 26. Merrick S, Butler WM, Lief JH, et al. B
iochemical outcome for hormonenave patients with highrisk prostate cancer manag
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tion. Cancer J 2002; 8(4):322327. 27. Critz FA, Tarlton RS, Hollaay DA, et al. P
rostate specific antigenmonitore combination raiotherapy for patients with pro
state cancer: I125 implant followe by external beam raiation. Cancer 1995; 75
:23832391. 28. Wallner KE, Roy J, Zelefsky M, et al. Dosimetry guielines to mini
mize urethral an rectal morbiity following transperineal 125I prostate brachyt
herapy. Int J Raiat Oncol Biol Phys 1995; 32:465471. 29. Mate TP, otttesman JE,
Hatton J, et al. High oserate afterloaing iriium192 prostate brachytherap
y: Feasibility report. Int J Raiat Oncol Biol Phys 1998; 41:525533. 30. Kestin L
L, Martinez AA, Stromberg JS, et al. Matchepair analysis of conformal highos
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31. Pilepich MV, Winter K, John W, et al. Phase III Raiation Therapy Oncology 
roup (RTO) trial 8610 of anrogen eprivation ajuvant to efinitive raiotherap

y in locally avance carcinoma of the prostate. Int J Raiat Oncol Biol Phys 20
01; 50(5):12431252. 32. Pilepch MV, Caplan RW, Byhart RW, et al. Phase III trial
of anrogen suppression using goserelin in unfavorableprognosis carcinoma of t
he prostate treate with efinitive raiotherapy: Report of Raiation Therapy On
cology roup Protocol 8531. J Clin Oncol 1997; 15(3):10131021. 33. Bolla M, onzal
ez D, Ware P, et al. Improve survival in patients with locally avance prosta
te cancer treate with raiotherapy an goserelin. N Engl J Me 1997; 337(5):2953
00. 34. Stone NN, Stock R, Unger P. Effects of neoajuvant hormonal therapy on
prostate biopsy results after 125I an 103P see implantation. Mol Urol 2000; 4
(3):163168. 35. Sylvester J, Blasko JC, rimm PD, et al. Shortcourse anrogen ab
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38 The role of external beam raiotherapy an permanent prostate brachytherapy i
n patients with localize prostate cancer
Louis Potters Introuction Permanent prostate brachytherapy (PPB) is an acceptab
le curative treatment moality for men with localize prostate cancer.16 However,
as currently performe, it is use either as monotherapy or in conjunction with
a short course of external beam raiotherapy (RTPPB). The reasons for combining
therapy are multifactorial (Table 38.1). Without prospective ata, it is iffic
ult to ascertain which is the best inication. The most frequently use inicati
on is base on the statistical risk of extracapsular isease associate with leas
on score, prostatespecific antigen (PSA), an stage.1 An implant only provies
ose to the prostate with a 35 mm margin an may exten to inclue just the base
of the seminal vesicles.2 The fiel effect of external raiation to encompass the
seminal vesicles an a larger prostatic margin potentially are at risk for extra
capsular isease not treate by the implant. This approach has continue without
much change since originally suggeste by Blasko et al uring the initiation of
the moern era of prostate brachytherapy. Doses of 45 y with 75% of the prescr
ibe implant ose are consiere stanar when using combine therapy. The Ameri
can Society of Brachytherapy (ABS) has publishe criteria for prostate brachythe
rapy an recommens
Table 38.1 Inications for combining external beam raiotherapy (EBRT) with perm
anent prostate brachytherapy (PPB)
1. Risk of extracapsular isease (a) LN risk (b) Seminal vesicle risk (c) Extrac
apsular penetration establishe capsular penetration focal capsular penetration
2. Dose escalation 3. Smooth out inhomogeneities
monotherapy in patients who have stage T1c an T2a, leason scores of 26, an a P
SA <10 ng/mL. Patients with stage T2b or leason score 810 or PSA >20 ng/mL are r
ecommene to unergo RTPPB.3
Basic an avance techniques in prostate brachytherapy
516
When using combination therapy, there is no consensus by the ABS for the sequenc
e of combine therapy. The avantages reporte for initiating treatment with ext
ernal beam raiotherapy (EBRT) inclue a ecrease in toxicity an better toleran
ce of the entire treatment course. Those sequencing with the implant first belie
ve that the see outline assists in locating the prostate at simulation an that
combine, or simultaneous treatments may be more beneficial.4 A review of the l
iterature is not helpful in assessing the inications or avantages of combining
external raiation an brachytherapy (Table 38.2). Therefore, we will review ou
r experience with RTPPB versus monotherapy PPB. Methos Potters et al recently
reporte on 1476 consecutive patients with clinically localize prostate cancer
treate with PPB between September 1992 an September 2000.5 The treatment crite
ria for monotherapy or RTPPB were base loosely on the ABS efinitions.7 Howeve

r, patient selfselection an preference allowe for an overlap of treatment meth

oologies an risk factors. Monotherapy use ioine125 (125I) or pallaium103
(103P) prescribe to 144 y (T43) or 140 y (NIST1999), respectively.6,7 RTPP
B consiste of raiation to 41.4 y or 45 y an a 125I or 103P implant to 108
y or 105 y, respectively. These elivere oses were kept constant, which mean
s that the written prescription oses have change to account for changes in the
airtowater kerma strength for 125I an a change in the calibration stanar f
or 103P. A total of 276 patients receive neoajuvant anrogen eprivation (NA
AD) to reuce the prostate volume or were
Table 38.2 Review of current brachytherapy literature for permanent prostate bra
chytherapy (PPB) performe as mono therapy or combine with external raiotherap
y (CMT)
Stuy
rao18 CMT Monotherapy Rage19 CMT Monotherapy Critz20 CMT Dattoli21 CMT Zekfsk
y22 Monotherapy 70 392 93 75 144 689 73 48 248 71% 48 36 79% 66% 88% 79% ASTRO N
air<0.2 PSA<1.0 All high risk patients CTbase 125I implants. Inclues patient
s with clinical
n Meian f/u Biochemical Definition (maths) control
46 81% 85%
Comments
PSA<1 No selection criteria between ng/mL groups no analysis performe comparing
groups ASTRO High risk patients treate with CMT: no analysis performe comparin
g groups
The role of external beam raiotherapy
Low risk Monotherapy Intermeiate risk Monotherapy High risk Monotherapy Leerma
n23 CMT Low risk CMT Intermeiate risk Monotherapy High risk Monotherapy Blasko1
3 CMT Monotherapy Low risk CMT Monotherapy Intermeiate risk CMT Monotherapy Hig
h risk. CMT Monotherapy Potters24
517
T3 isease
112 92 22 48 348 165 124 59 58 403 231 75 279 104 111 52 11 35
88% 77% 38% PSA>1.0 PSA cutoff for risk at 20 ng/mL ng/mL 77% 88% 75% 59% ASTRO
88% 79% 94% 79% 85% 84% 62% 54% ASTRO (Kattan)
p=0.06
p=0.86
p=0.53 p=0.54
CMT 314 81.5% Monotherapy 1162 82.1% Low risk (matche); CMT 38 87.7% Monotherap
y 40 93.4% Intermeiate risk (matche): CMT 174 84.8% Monotherapy 191 79.7% High
risk (matche): CMT 102 68.6% Monotherapy 84 60.5% f/u, followup; PSA, prostat
especific antigen; ASTRO, American Society of Therapeutic Raiology an Oncolog
y; CT, compute tomography.
p=0.54
p=0.64
p=0.49

Basic an avance techniques in prostate brachytherapy

518
treate as per the patients urologist. The implant was performe with peripheral
see loaing. Postimplant analysis consiste of stereo shift Xray films (n=468)
an as of late 1994, patients unerwent compute tomography (CT)base osimetr
y at 3 weeks postimplant (n=829). The D90% was use to assess implant quality in
this stuy an is efine as the D90 ose etermine by CT evaluation relative
to the prescribe ose.8 The American Society for Therapeutic Raiology an Onco
logy (ASTRO) efinition of PSA failure following EBRT was applie in this stuy.
9 This efinition marks failure at the mipoint in time between the posttreatmen
t nair an the first of three consecutive PSA rises with time zero the ate of
implant. However, two important conservative moifications were mae.10 First, t
he requirement that the three rises have to be consecutive was relaxe. If three
rises occurre with intervening stable PSA values, but the PSA never ecrease,
the patient was consiere a fail ure, at the mipoint in time between his firs
t rise an the PSA immeiately prior to the first rise. Secon, for patients who
se most recent PSA values were rising at their last followup, but in whom failu
re ha not occurre, followup time was truncate to the PSA immeiately prior t
o the first rise. Results From the initial 1476 patients, 174 patients were excl
ue having only a single posttreatment PSA value. The remaining cohort characte
ristics are presente in Table 38.3. The meian followup is 42.1 months with an
overall an iseasespecific survival at 72 months of 97% an 99%, respectively
. A total of 89% of patients obtaine a PSA value within the last year of follow
up, an the characteristics of
Table 38.3 Patient characteristics (from Potters et al)8
n a 1016 (%) Monotherapy n=1016 (%) RTPPB n=281(%) pvalue*
Isotope D90%a NAADa Stage T1c T2a T2b 2 3 4 5 6 7 598 (58.9) 384 (37.8) 34 (3.3)
76 (0.6) 16 (1.6) 42 (4.8) 105 (10.3) 587 (57.8) 225 (27.1) 144 (45.9) 131 (41.
7) 39 (12.1) 2 (0.7) 8 (2.8) 9 (3.2) 83 (29.5) 142 (50.5) 34 (12.1) I P Mean No
Yes
103 125
245 (24.1) 771 (75.9) 100 799 (79) 217 (21)
39 (13.9) 242 (86.1) 105 174 (61.9) 107 (38.1)
0.001 0.072 0.001 0.012

leason score
0.007
The role of external beam raiotherapy
8 Pretreatment PSA Mean Meian Range Age Mean Meian Range Followup Meian Rang
e * MannWhitney test. a D90 ose normalize to the prescription ose. b Neoaj
uvant anrogen ablation. 27 (2.7) 9.4 10.6 0.2112 69.6 70.2 4390 35.1 791
519
3 (1.1) 14.0 13.5 0.885 67.6 68.8 4884 33.0 685
0.014
0.082
Table 38.4 Evience of failure information from the entire cohort (Potters et al
)8
First evience of failure

Biochemical relapse Clinical relapse Hormonal therapy Salvage prostatectomy Deat

h from isease None (censore) Meian (maximum) months of followup for censore
patients Total number of PSA values obtaine at followup Percent of censore p
atients who i not have their PSA measure within 1 year of analysis
n=1297
131 4 14 2 0 1146 35 (91) 8783 11
Table 38.5 Cox regression analysis on all 1297 patients to preict PSA freeom f
rom recurrence base on leason sum, pretreatment PSA value, stage, NAAD, an RT
PPB
Characteristic pvalue Hazar ratio (95% CI)
Pretreatment PSA value 0.0001 * leason sum score 0.0001 * The aition of NAAD
0,0018 0.48 (0.31:0.76) Clinical stage 0.874 1.10 (0.78:1.55) T2a vs T1c 1.10 (0
.78:1.55) T2b vs T1c 1.05 (0.58:1.90) The aition of EBRT 0.058 0.67(0.46:1.01)
* Splines were use for continuous variables, NAAD, neoajuvant anrogen epri
vation: EBRT, external beam raiotherapy.
Basic an avance techniques in prostate brachytherapy
520
Table 38.6 Cox regression analysis to preict for PSA freeom from recurrence ba
se on pre an posttreatment variables
Characteristic pvalue Hazar ratio (95% CI)
Pretreatment PSA value 0.0001 * D90% <0.0001 * leason sum score 0.0008 * The a
ition of NAAD 0.086 0.55 (0.30:1.00) Clinical stage 0.366 T2a vs T1c 0.86 (0.49
:1.50) T2b vs T1c 1.46 (0.64:3.32) The aition of EBRT 0.561 1.00 (055:1.85) *
Splines were use for continuous variables EBRT, external beam raiotherapy.
recurrence are presente in Table 38.4. Of the patients, 35 (2.6%) are lost to f
ollowup an are censore base on their last PSA value. The PSA freeom from re
currence (FFR) for the entire cohort is 83.2%. The results of the Cox regression
presente in Table 38.5 emonstrate that the results of the following variables
an their impact on PSAFFR: pretreatment PSA (0.0001); leason sum (0.0001); t
he use of neoajuvant anrogen eprivation (NAAD) (0.0018); clinical stage (0.8
74); an the aition of EBRT (0.058). Table 38.6 presents the same analysis inc
luing the D90% variable as a continuous variable an change the results as fol
lows: pretreatment PSA (0.0001); D90% (<0.0001); leason sum (0.0008); the use o
f NAAD (0.086); clinical stage (0.366); an the aition of EBRT (0.561). Figure
38.1 illustrates the hazar ratio for the D90 ose that emonstrates a steep o
seresponse for a D90% of less than 100%, which then plateaus at a D90% above th
at. KaplanMeier PSAFFR was performe with stratification of the D90% cutoff po
int at 90% of the prescribe ose base on the use of RTPPB an is shown in Tab
le 38.7. Discussion This retrospective stuy represents a large cohort of patien
ts treate with moern prostate brachytherapy techniques an is the first such s
tuy that incorporates postimplant osimetry to better aress the role of RTPP
B.
The role of external beam raiotherapy
521
Figure 38.1 Hazar ratio plotte for a D90 ose. Table 38.7 PSA relapsefree sur
vival (PSAFFR) for patients stratifie by the D90% variable (Potters et al)8
Treatment metho (n) PSAFFR pvalue
D90<90%a Monotherapy (176) RTPPBb (45) D9090%a 72.1 % 85.6% 0.057
0.086 Monotherapy (437) 96.4% RTPPB(171) 83.9% a D90% is the D90 ose normalize

 to the prescription ose. b Combine moality therapy.

Base on Cox moeling, there appears to be no avantage for RTPPB over PPB as m
onotherapy. The most common rationale for combining external raiation an brach
ytherapy is base on the nee to expan the sphere of raiation ose beyon the
prostate capsule when patients exhibit a high risk for extracapsular isease. It
is clear from several stuies that pathologic analysis of prostate specimens ha
s establishe the risk of extracapsular extension of tumor being associate with
the pretreatment PSA value, leason sum, an clinical stage.1 Nonetheless, Davi
s et al were able to etermine that focal extracapsular extension of isease was
within 3.3 mm of the prostate capsule over 90% of the time an that the maximum
istance of isease was 1.2 cm.2 With moern transperineal
Basic an avance techniques in prostate brachytherapy
522
brachytherapy techniques, osimetric evaluation ientifies that this narrow extr
acapsular region is encompasse by a high raiation ose. In fact, moern planni
ng systems will automatically place a esire margin aroun the prostate to acco
unt for extracapsular sprea.11 Others have suggeste that the nee to combine e
xternal raiation an PPB is to account for suboptimal implants. This is the so
calle spackle effect where aitional raiation will account for col or unerose
areas within the target or prostate. There are many reasons why
Figure 38.2 Acturial potency in patients that i not receive neoajuvant anrog
en eprivation (NAD), comparing those treate with an implant as monotherapy or
in combination with external beam raiotherapy (EBRT).
the target ose may not be achieve an these inclue inexperience, ifficulty w
ith intraoperative visualization of the target, an see movement through the ne
ele tracks. Nonetheless, the Potters stuy appears to emonstrate that RTPPB i
s not an inepenent variable to preict PSAFFR, either with or without consie
ring implant osimetry (Tables 38.5 an 38.6). However, base on the hazar rati
o plot (Figure 38.2) that examines the D90 ose (Figure 38.1), there oes appear
to be a steep oseresponse that preicts for PSAFFR. This ose effect, on its
own, has a significant impact on clinical outcome for patients treate with mon
otherapy PPB, but not for patients treate with RT
The role of external beam raiotherapy
523
PPB.8 Since the elivere implant ose (D90 ose) reflects the quality of the im
plant, an since the aition of external raiation oes not appear to be an in
epenent variable to preict PSAFFR, it is appropriate to consier the nee to
maximize implant quality above the nee to offer combination therapy, regarless
of the inherent risk associate with the presenting PSA value an leason score
. Therefore, the more relevant question is to ensure an acceptable implant on a
consistent basis. One consieration for improving the skill of the surgical team
is to use intraoperative ynamic osimetry,12 which is beyon the iscussion of
this current subject. Nonetheless, even this may not guarantee an aequate impl
ant in all cases. Therefore, consieration for the use of combine external rai
ation an PPB may best be reserve for cases with inaequate osimetry. However,
this will require further stuy. The combination of aing EBRT to brachytherap
y remains a popular metho for treating high risk patients. Some authors even ha
ve suggeste that all patients unergoing brachytherapy shoul receive combine
therapy. Unfortunately, a review of the current literature oes not allow one to
generate any firm conclusions on this subject (Table 38.8). Blasko et al have c
ompare their patients treate with combination EBRT an brachytherapy verses br
achytherapy monotherapy an ientifie an improvement in the biochemical freeom

from failure for the monotherapy subset as compare to the combine therapy sub
set (88% vs 79% 8 year PSAFFR, respectively).13 The stratification of risk fact
ors in their series preicts for this outcome base on the pretreatment PSA valu
es an leason sum for each treatment group. Further, when these authors stratif
ie their patients into three basic risk categories, no ifference in outcome wa
s observe between the treatment groups within each category, respectively. Anot
her stuy from Rage et al that has been use to assert the nee for combine th
erapy in all patients, presents 12 year crue biochemical control rates of 76% i
n the
Table 38.8 PSA freeom from recurrence for patients stratifie by the aition o
f external beam raiotherapy (EBRT) to brachytherapy relative to postimplant os
imetric analysis
Treatment
Monotherapy Combine EBRT an brachytherapy
Stuy Rage14 Blasko13(65) Potters8(14) (090 ose<90%)
60% 76% 88% 79% 72.1% 85.6%
Potters8(14) (D90 ose90%)
96.4% 83.9%
combine therapy group as compare to 60% for those treate with monotherapy.14
Although multivariate analysis was not performe in the Rage stuy, it appears
that the combine therapy patients ha higher risk isease as compare with the
monotherapy group. This counterintuitive result may be explaine on the basis of
not achieving a esire osimetry in the monotherapy group of patients. When co
mpare with the escribe stuy from Potters et al, the use of patient stratific
ation base on a osimetry cutoff point makes clear a possible explanation of bo
th the intuitive an counterintuitive results of Blasko et al an Rage et al, r
espectively (Table 38.8). This comparison
Basic an avance techniques in prostate brachytherapy
524
suggests that implant quality may be a more important factor for preicting outc
ome than the actual nee for combine therapy, even in high risk patients. Other
s have suggeste that perhaps there is an aitive effect of combining external
raiation following an implant. While the results presente by Critz et al appea
r to inicate acceptable biochemical control rates in a large series of men trea
te with this approach,20 there is no clear evience that their ata are any bet
ter than the Potters et al or Blasko et al series. Further, comparison of their
results is tainte by their use of an unconventional efinition of fail ure usin
g a strict PSA nair, an their potential misuse of that efinition. Further, th
e Critz group uses smaller aily fractions of external raiation than is stanar
. Another confouning factor associate with the prescribing of combination ext
ernal raiation an brachytherapy is the type of external fiels that are being
use. Anecotal evience suggests that many centers that combine moalities are
using threeimensional (3D) or intensity moulate raiation techniques (IMRT),
encompassing the prostate an seminal vesicle only. If one was to consier the
fiel effect of combination therapy, a more likely approach woul be that of the
Raiation Therapy Oncology roup (RTO) experience where true pelvic raiation
fiels are place in orer to encompass the potential sprea of isease into lym
ph noes, since it has alreay been shown that the implant covers the areas of e
xtracapsular sprea. Combining therapies may increase treatment toxicities. In a
review of both urinary an rectal complications, elblum et al were unable to s
how that combine therapies are associate with higher treatmentrelate toxicit
y.15 However, a stuy from Zeitlin et al reporte a rectoprostatic fistula rate
of 2.3% when external raiation follows

Table 38.9 Data from 48 responers comparing quality of life (QOL) inices for p
atients treate with combine external beam raiotherapy an permanent prostate
brachytherapy (RTPPB) versus brachytherapy as monotherapy
RAND: 36item health survey UCLA prostate inex
Physical function n.s. Urinary function 0.01 Role limitations 0.02 Urinary bothe
r 0.02 Boily pain 0.03 Bowel function n.s. eneral health perceptions n.s. Bowe
l bother 0.02 Emotional wellbeing 0.02 Sexual function n.s. Role limitations n.
s. Sexual bother 0.04 Social function 0.01 Cancer interference with life 0.03 En
ergy/Fatigue 0.03 Cancer interference with family 0.002 UCLA, University of Cali
fornia, Los Angeles: n,s., not significant.
PPB.16 While this egree of rectal toxicity has not been reporte elsewhere, it
remains a concern that combine therapy may impact on rectal injury. In a stuy
on potency preservation following PPB, Potters et al ientifie that the aitio
n of external beam raiation with PPB contribute to a rop in potency preservat
ion of about 1520% (Figure 38.2). In a stuy using quality of life (QOL) paramete
rs that examine prostate
The role of external beam raiotherapy
525
cancer patients treate with either monotherapy or RTPPB, Braneis et al foun
that urinary function an bother, bowel bother, sexual function an bother, Amer
ican Urological Association (AUA) symptom score, an cancer interference with li
fe were all statistically worse in the RTPPB group even when ajusting for base
line ifferences between groups (Table 38.9).17 Further stuy on toxicity is req
uire an nees to be balance against outcome. Conclusions The use of combine
EBRT an permanent prostate brachytherapy (PPB) continues for efinitive treatme
nt of clinically localize prostate cancer. It is clear from the presente ata
that the role for combination therapy remains suspect, at best. Future stuies a
re require to aress the nee for combine treatments in lieu of toxicity an
expense. Until such time, efforts to maximize implant quality shoul assure the
treating physician that monotherapy PPB offers acceptable prostatespecific anti
gen freeom from recurrence (PSAFFR) an can be safely offere to all patients
eeme eligible for PPB. References
1. Partin AW, Kattan MW, Subong EN, et al. Combination of prostatespecific antig
en, clinical stage, an leason score to preict pathological stage of localize
prostate cancer. A multiinstitutional upate. JAMA 1997; 277(18):14451451. 2. Da
vis BJ, Pisansky TM, Meyers RP, et al. The raial istance of extraprostatic ext
ension of prostate aenocarcinoma: Implications for prostate brachytherapy. Int
J Raiat Oncol Biol Phys 1998; 42(suppl 1):132132. 3. Nag S, Beyer D, Frielan J
, et al. American Brachytherapy Society (ABS) recommenations for transperineal
permanent brachytherapy of prostate cancer. Int J Raiat Oncol Biol Phys 1999; 4
4(4):789799. 4. Critz FA, Levinson AK, Williams WH, et al. Simultaneous raiother
apy for prostate cancer: 125I prostate implant followe by externalbeam raiati
on [See comments]. Cancer J Sci Am 1998; 4(6):359363. 5. Fleming I, Cooper JS, He
nson DE, et al. AJCC Cancer staging manual, 5th en. Philaelphia: LippincottRa
ven, 1997. 6. Beyer D, Nath R, Butler W, et al. American Brachytherapy Society r
ecommenations for clinical implementation of NIST1999 stanars for (103)palla
ium brachytherapy. Int J Raiat Oncol Biol Phys 2000; 47(2):273275. 7. Nath R Ro
berts K, Ng M, et al. Correlation of meical osimetry quality inicators to the
local tumor control in patients with prostate cancer treate with ioine125 in
terstitial implants. Me Phys 1998; 25(12):22932307. 8. Potters L, Cao Y, Calugar
u E, et al. A comprehensive review of CTbase osimetry parameters an biochemi
cal control in patients treate with permanent prostate brachytherapy. Int J Ra
iat Oncol Biol Phys 2001; 50(3):605614. 9. Consensus statement: guielines for PS
A following raiation therapy. American Society for Therapeutic Raiology an On
cology Consensus Panel. Int J Raiat Oncol Biol Phys 1997; 37(5):10351041. 10. Ka

ttan MW, Fearn PA, Leibel S, Potters L. The efinition of biochemical failure in
patients treate with efinitive raiotherapy. Int J Raiat Oncol Biol Phys 200
0; 48(5):14691474.
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11. Merrick S, Butler WM. Moifie uniform see loaing for prostate brachyther
apy: rationale, esign, an evaluation. Tech Urol 2000; 6(2):7884. 12. Zelefsky M
J, Yamaa Y, Cohen , et al. Postimplantation osimetric analysis of permanent t
ransperineal prostate implantation: improve ose istributions with an intraope
rative computeroptimize conformal planning technique. Int J Raiat Oncol Biol
Phys 2000; 48(2):601608. 13. Blasko JC, rimm PD, Sylvester JE, Cavanagh W. The r
ole of external beam raiotherapy with I125/P103 brachytherapy for prostate c
arcinoma. Raiother Oncol 2000; 57(3):273278. 14. Rage H, Korb LJ, Elgamal AA, e
t al. Moern prostate brachytherapy. Prostate specific antigen results in 219 pa
tients with up to 12 years of observe followup. Cancer 2000; 89(1):135141. 15.
elblum DY, Potters L. Rectal complications associate with transperineal inters
titial brachytherapy for prostate cancer. Int J Raiat Oncol Biol Phys 2000; 48(
1):119124. 16. Zeitlin SI, Sherman J, Raboy A, et al. High ose combination raio
therapy for the treatment of localize prostate cancer [Discussion: 9596]. J Urol
1998; 160(1):9195. 17. Braneis JM, Litwin MS, Burnison CM, Reiter RE. Quality o
f life outcomes after brachytherapy for early stage prostate cancer. J Urol 2000
; 163(3):851857. 18. rao L, Larson TR, Balch CS, et al. Actuarial iseasefree
survival after prostate cancer brachytherapy using interactive techniques with
biplane ultrasoun an fluoroscopic guiance. Int J Raiat Oncol Biol Phys 1998;
42(2):289298. 19. Rage H, Elgamal AA, Snow PB, et al. Tenyear isease free sur
vival after transperineal sonographyguie ioine125 brachytherapy with or wit
hout 45gray external beam irraiation in the treatment of patients with clinica
lly localize, low to high leason grae prostate carcinoma. Cancer 1998; 83(5):
9891001. 20. Critz FA, Williams WH, Levinson AK, et al. Simultaneous irraiation
for prostate cancer: intermeiate results with moern techniques [Discussion: 74
1743]. J Urol 2000; 164(3 Pt 1):738741. 21. Dattoli M, Wallner K, Sorace R, et al.
103P brachytherapy an external beam irraiation for clinically localize, hig
hrisk prostatic carcinoma. Int J Raiat Oncol Biol Phys 1996; 35(5):875879. 22.
Zelefsky MJ, Hollister T, Raben A, et al. Fiveyear biochemical outcome an toxi
city with transperineal CTplanne permanent I125 prostate implantation for pat
ients with localize prostate cancer. Int J Raiat Oncol Biol Phys 2000; 47(5):1
2611266. 23. Leerman S, Cavanagh W, Albert PS, et al. Retrospective stratificat
ion of a consecutive cohort of prostate cancer patients treate with a combine
regimen of externalbeam raiotherapy an brachytherapy. Int J Raiat Oncol Biol
Phys 2001; 49(5):12971303. 24. Potters L, Cha C, Ashley R, et al. The role of ex
ternal beam irraiation in patients unergoing prostate brachytherapy. Urol Onco
l 2000; 5:112117.
39 Simultaneous irraiation for prostate cancer: iseasefree survival rates
Frank A Critz Introuction Simultaneous irraiation (SI) is efine by the initi
al performance of a raioactive ioine125 (125I) see implant, which has a 60 a
y halflife, followe by external beam irraiation (EBRT). Since the external be
am component is given 3 weeks postimplantation, intraprostatic malignant epithel
ium as well as benign prostate epithelium is irraiate simultaneously by two se
parate sources, which prouces ose intensification. In aition, potential extr
acapsular isease is also treate by the followup external beam irraiation. Th
us, integration of both methos of raiation shoul effectively treat clinically
localize prostate cancer. Base on this theory of SI, a formal stuy of this t
reatment process began in January 1984 with prostate implantation performe by t
he obsolete retropubic implant metho. In 1992, a transition was begun from the
retropubic implant metho to the ultrasounguie transperineal implant approach
. This stuy concerns only men implante with the transperineal technique as a p

art of SI an upates a prior report on this subject.1 Materials an methos Fro
m August 1992 through to December 2000, 3007 consecutive men with clinical stage
T1T2, Nx, Mo prostate cancer who i not receive neoajuvant anrogen epriva
tion (NAAD) were treate with SI employing the ultrasounguie transperineal i
mplant technique (meian 12 000 cy). The exclue men either receive NAAD befo
re consultation or it was initiate at this facility to ownsize an enlarge pro
state with severe obstructive symptoms. NAAD has not been given with SI to treat
prostate cancer. The clinical characteristics of all the men are escribe in T
able 39.1. leason score was taken from community pathology reports through 1998
without review. A review of prostate biopsies began in 1999. Staging was perfor
me by the 1992 American Joint Committee on Cancer (AJCC) criteria. External bea
m irraiation to the prostate seminal vesicles an periprostatic
Basic an avance techniques in prostate brachytherapy
528
Table 39.1 Clinical characteristics of all 3007 men; 14 of these i not have a
leason score
No %
Pre Treatment PSA groups 04 272 9 4.0110.0 2024 67 10.0120.00 555 19 >20.00 156 5 T
otal 3007 100 leason score 24 168 6 56 2059 68 7 650 22 840 116 4 Total 2993 100 S
tage T1a an T1b 25 1 T1c 1608 53 T2a 768 25 T2b 440 15 T2c 166 6 Total 3007 100
Risk groups Low 1577 52 Intermeiate 884 30 High 532 18 Total 2993 100 Min Me
Max 0.3 6.7 105.1 PrePSA 36.0 65.0 88.0 Age PSA, prostatespecific antigen.
tissue was begun 3 weeks postimplant at a aily ose of 150 cy, fives ays per
week for a total of 4500 cy. Men with averse prognostic factors efine as le
ason score 7, PSA 10.1 ng/mL or prostatic base involvement by either biopsy or pal
pation receive an aitional 750 cy to the seminal vesicles an prostatic base
. All men were treate three or more years ago. Followup was performe 3 months
postimplant (approximately 2 weeks after all treatment), 3 months later an then
every 6 months thereafter. There was no change to annual followup. Meian follo
wup was 4 years (range: 3 mths11 yrs). Disease freeom was efine as the achiev
ement an maintenance of a prostatespecific antigen (PSA) 0.2 ng/mL or less, an
 failure was efine as PSA nair >0.2 ng/mL or a subsequent PSA rise above thi
s level. For men with less than 5 year followup an who ha not yet achieve PS
A nair 0.2 ng/mL, isease freeom was efine by a falling PSA. Because of the
higher probability of PSA
Simultaneous irraiation for prostate cancer
529
bounce uring the first 5 years postimplant, men were efine iseasefree if th
ey ha not yet achieve PSA nair 0.2 ng/mL but ha a single PSA rise at last fo
llowup. Men were efine as having recurrence if there were two PSA rises. Dise
asefree survival rates were calculate by the KaplanMeier metho an compariso
n of curves by the log rank test. Results PSA nair an the efinition of iseas
e freeom A subset of the atabase, 1658 men treate five or more years ago (199
21998), was use to correlate PSA nair achieve with subsequent isease freeom
efine by the American Society of Therapeutic Raiology an Oncology (ASTRO) e
finition of recurrence (Figure 39.1). Men who achieve PSA nair 0.2 ng/mL ha a
98% 10 year iseasefree survival (DFS) rate compare with an 8 year DFS rate of
26% for those men who achieve PSA nair 0.30.5 ng/mL, a highly significant iffe
rence (p<0.0001). Men who achieve PSA nair 0.6 ng/mL ha an even worse outcome.
Diseasefree survival rates Figure 39.2 ocuments the overall 10 year iseasef
ree survival (DFS) rate of 85% for all 3007 men in this stuy. For the 283 men w
ho recurre, the meian time to recurrence was 30 months (range: 396 mths). Outco
me accoring to pretreatment PSA is ocumente in Figure 39.3. There was a highl

y significant ifference between each of the four PSA groups analyze. Disease f
reeom accoring to the leason score is ocumente in Figure 39.4. There was no
ifference in outcome between men with leason score 26, but there was a signifi
cant ifference between men who ha leason score 6 compare to 7, an between me
n who ha leason score 7 compare with leason score 810. Figure 39.5 is an anal
ysis of isease freeom by stage. Men were analyze accoring to risk group with
risk factors efine as PSA10.1 ng/mL, leason score 7 or stage T2b, T2c (Figure
39.6). Low risk was efine by none, intermeiate risk by one, an high risk by
two or more of
Basic an avance techniques in prostate brachytherapy
530
Figure 39.1 Disease freeom accoring to prostatespecific antigen (PSA) nair a
chieve for men treate 5 or more years ago with isease freeom efine by the
American Society of Therapeutic Raiology an Oncology (ASTRO) efinition of rec
urrence.
Figure 39.2 Overall iseasefree survival (DFS) rate for the 3007 men in this st
uy.
Simultaneous irraiation for prostate cancer
531
Figure 39.3 Diseasefree survival (DFS) rate accoring to pretreatment PSA group
. There was a highly significant ifference (p>/=<0.0001) between each of the gr
oups.
Figure 39.4 Diseasefree survival (DFS) accoring to the leason score. There wa
s no significant ifference between leason score 24 compare with 56, but there w
as a significant ifference between leason score 6
Basic an avance techniques in prostate brachytherapy
532
compare with 7 (p=0.0001), an between leason score 7 compare with 810 (p=0.03
).
Figure 39.5 Diseasefree survival (DFS) accoring to clinical stage: 25 men with
stage T1aT1b were exclue.
Figure 39.6 Diseasefree survival (DFS) accoring to risk group. There
Simultaneous irraiation for prostate cancer
533
was a significant ifference (p=0.0001) between each of these groups.
these factors. There was a highly significant 10 year DFS rate between each of t
hese groups. On multivariate analysis, pretreatment PSA an leason score were s
ignificantly associate with isease freeom but not stage. Discussion Prior to
evaluation of the outcomes of raiotherapy, isease freeom must be efine. Sin
ce 1987 when clinical use of the prostatespecific antigen (PSA) test began, a w
ie variety of efinitions of isease freeom after irraiation for prostate can
cer has been propose.2 In an attempt to arrive at a consensus on this issue, th
e American Society of Therapeutic Raiation Oncology (ASTRO) efinition of isea
se freeom was create in 1997.3 Since then, almost all reports on raiotherapy

for prostate cancer have use the ASTRO efinition, or some variant to evaluate
outcomes of irraiation. However, significant flaws in the ASTRO efinition of 
isease freeom have been escribe.35 Further, the ASTRO efinition of recurrence
was create for external beam irraiation of prostate cancer an not for brachy
therapy. More importantly, stuies after both raical prostatectomy an irraiat
ion have ocumente that calculations with the ASTRO efinition prouce signific
antly better iseasefree survival (DFS) rates than those calculate with an un
etectable PSA.68 Thus, comparisons of outcomes between calculations performe wit
h the ASTRO efinition an those with an unetectable PSA are inaccurate an mis
leaing. A stanar efinition of isease freeom from prostate cancer after all
treatment methos is neee. In the first research paper publishe on SI from t
his program using men implante by the obsolete retropubic technique, the PSA na
ir achieve correlate with isease freeom.9 In a later report, a PSA cutoff p
oint of 0.5 ng/mL was recommene as the efinition of isease freeom for irra
iation of prostate cancer.10 With better PSA assays an evaluation only of men t
reate with SI using the moern transperineal implant technique, a PSA cutoff po
int of 0.2 ng/mL was recommene as the efinition of isease freeom for irrai
ation of prostate cancer.8 Figure 39.1 upates the association of PSA nair an
isease freeom. This eviencebase efinition coincientally is the ientical
efinition etermine by Freelan et al who correlate various PSA nair levels
with outcome after raical prostatectomy.11 Consequently, a PSA cutoff point of
0.2 ng/mL has been recommene as the stanar efinition of isease freeom fr
om prostate cancer after both surgery an irraiation an is use to evaluate th
e results of SI in this report. The overall 10 year iseasefree survival rate o
f men treate with SI using the moern transperineal ultrasounguie implant t
echnique is 85% (Figure 39.2). Subset analysis accoring to pretreatment PSA em
onstrates a significant ifference between each of the four stanar PSA groups
(Figure 39.3). Analysis accoring to the leason score an clinical stage is als
o ocumente (Figures 39.4 an 39.5) an accoring to risk group in Figure 39.6.
The lower than usual percent of men with a leason score 710 in this report (Tab
le 39.1) maybe relate to a lack of biopsy review.12 A substantial number of men
with leason score 7 may have been misclassifie as leason score 6. This may hav
e
Basic an avance techniques in prostate brachytherapy
534
an averse effect on evaluation of men with lower leason scores an low risk gr
oup (Figures 39.4 an 39.6). On multivariate analysis, both the pretreatment PSA
an leason score are significantly relate to isease freeom. These outcomes
compare favorably with results reporte from raical prostatectomy for prostate
cancer performe in the PSA era. The 10 year isease free survival rate from Joh
ns Hopkins with raical prostatectomy, calculate with a PSA cutoff point of 0.2
ng/mL, is 80% which is approximately the same result achieve by simultaneous i
rraiation (SI) (Figure 39.2). Aitionally, the results of this report support
the principles of ose intensification for intraprostatic isease an treatment
of potential extraprostatic cancer by followup external beam irraiation. Furth
ermore, the same efinition of isease freeom, PSA cutoff point of 0.2 ng/mL, f
or both SI an raical prostatectomy suggests estruction of not only all malign
ant but also all benign prostate epithelium by SI which coul prevent the theore
tical evelopment of a secon new prostate cancer in the future. Conclusions Sim
ultaneous irraiation is a treatment process that logically integrates a raioac
tive 125I prostate implant with subsequent external beam irraiation. The theory
of SI is supporte by the overall 10 year iseasefree survival rate of 85% cal
culate with an unetectable prostatespecific antigen. References
1. Critz FA, Williams WH, Levinson AK, et al. Simultaneous irraiation for prost
ate cancer: Intermeiate results with moern techniques. J Urol 2000; 164:738743.
2. Vicini FA, Kestin LL, Martinez AA. The correlation of serial prostate specif
ic antigen measurements with clinical outcome after external beam raiation ther

apy of patients for prostate carcinoma. Cancer 2000; 88:23052318. 3. American Soc
iety For Therapeutic Raiology an Oncology Consensus Panel: Consensus Statement
. uielines for PSA Following Raiation Therapy. Int J Raiat Oncol Biol Phys 1
997; 37:10351041. 4. Horwitz EM, Uzzo R, Hanlon AL, et al. Moifying the America
n Society for Therapeutic Raiology an Oncology efinition of biochemical fail
ure to minimize the influence of backating in patients with prostate cancer tre
ate with 3imensional conformal raiation therapy alone. J Urol, 2003; 169:215
32159. 5. Pickles T, KimSing C, Morris WJ, et al. Evaluation of the Houston bioc
hemical relapse efinition in men treate with prolonge neoajuvant an ajuvan
t anrogen ablation an assessment of followup leatime Bias. Int J Ra Oncol
Biol Phys 2003; 57:1118. 6. retzer MB, Trock BJ, Han M, Walsh PC. A critical ana
lysis of the interpretation of biochemical failure in surgically treate patient
s using the American Society for Therapeutic Raiation an Oncology criteria. J
Urol 2002; 168:14191422. 7. Amling CL, Bergstralh EJ, Blute ML, et al. Defining p
rostate specific antigen progression after raical prostatectomy: What is the mo
st appropriate cut point? J Urol 2001; 165:11461151.
Simultaneous irraiation for prostate cancer
535
8. Critz FA. A stanar efinition of isease freeom is neee for prostate can
cer: Unetectable prostate specific antigen compare with the American Society o
f Therapeutic Raiology an Oncology consensus efinition. J Urol 2002; 167:13101
313. 9. Critz FA, Tarlton RS, Hollaay DA. Prostate specific antigenmonitore c
ombination raiotherapy for patients with prostate cancer. Cancer 1995; 75:238323
91. 10. Critz FA, Levinson AK, Williams WH, et al. Prostatespecific antigen na
ir: The optimum level after irraiation for prostate cancer. J Clin One 1996; 14
:28932900. 11. Freelan SJ, Sutter ME, Dorey F, Aronson WJ. Defining the Ieal c
utpoint for etermining PSA recurrence after raical prostatectomy. Urology 2003
; 61:365369. 12. Steinberg DM, Sauvageot J, Piantaosi S, Epstein JI. Correlation
of prostate neele biopsy an raical prostatectomy leason grae in acaemic a
n community settings. Am J Surg Pathol 1997; 21:566576. 13. Han M, Partin AW, Pi
antasoi S, et al. Era specific biochemical recurrencefree survival following r
aical prostatectomy for clinically localize prostate cancer. J Urol 2001; 166:
416 419.
Part VI Permanent raioactive sees: issues an features
40 Raioactive sources for insterstitial brachytherapy
Manny R Subramanian, Krishnan Suthanthiran, an Anatoly Dritschilo Introuction
Brachytherapy is an establishe moality for treating cancer of the hea an nec
k, breast, cervix, prostate, an soft tissue sarcomas.1,2 Although technically 
emaning, brachytherapy techniques offer substantial avantages in ose istribu
tions by proviing a conformal geometry to tumor volumes of interest. Recent int
erest in brachytherapy has been enhance by applications to prostate cancer trea
tment an partial breast irraiation. Both permanent an temporary high ose rat
e (HDR) interstitial brachytherapy treatments are currently employe in the trea
tment of malignant an nonmalignant iseases. Brachytherapy involves the placem
ent of raioactive elements within or near the target tissue. The first use of b
rachytherapy was for the treatment of lupus,3 shortly after the iscovery of ra
ioactivity an the isolation of raium.4 Raium applications prove to be useful
in the treatment of a variety of tumors an noncancerous pathologies an le t
o the evelopment an improvement of brachytherapy technology to its present for
m. Both temporary an permanent HDR interstitial brachytherapy treatments are cu
rrently use in the treatment of malignant an nonmalignant iseases. (The trea
tment of cancer or other tissue pathologies by placement of raioactive elements
within or near the target tissue is terme brachytherapy.) This form of raiati
on therapy was attempte soon after Becquerel iscovere raioactivity (1896) an
 Curie isolate raium from pitchblen (1898).1 The first isease treate with

brachytherapy was lupus.1 Following this there were an increasing number of repo
rts outlining successful application of brachytherapy to a variety of forms of c
ancer.2,3 Since then a variety of tumors an some normal tissue pathologies have
been successfully treate with brachytherapy.4 A normal tissue pathology is tha
t which is successfully treate with brachytherapy as a consequence of atheroscl
erosis, namely vascular restenosis. Brachytherapy has been prescribe since the
early 1900s. For example, Henchke et al reporte the successful use of iriium1
92 (192Ir) an ioine125 (125I) sources for the treatment of prostate cancer.5,
6 Recent evelopments an techniques evelope by Blasko et al7 have resulte in
the accurate placement of sees in the prostate base on preplanne osimetry a
n transrectal ultrasonography.7,8 Several groups have reporte encouraging long
term survival an biochemical outcomes comparable to raical prostatectomy.9 St
uies are currently unerway employing factors, such as quality of life, cost of
Basic an avance techniques in prostate brachytherapy
538
proceure, an biochemical/survival outcome comparing ifferent treatment moali
ties.10 In this chapter we will briefly look at ifferent raioisotopebase ev
ices that are available for brachytherapy applications. Iriium192 An iriium1
92 source has been available for brachytherapy applications since the early 1970
s. A iagram of the Best iriium192 source is shown in Figure 40.1. Iriium192 h
as an energy of 375 keV an a halflife of 74 ays. The commonly use isotopes i
n temporary interstitial brachytherapy are: iriium192, aurum (gol)198, ioin
e125, an cesium137. The energy of photons from 125I (average) is 28 keV an t
he energy from 192 Ir photons (average) is 375 keV.11 Isotopes, such as 125I an
103P, are use in permanent interstitial brachytherapy applications.12 In ai
tion to 192Ir, a few other isotopes are also use for high an low ose rate bra
chytherapy applications, as shown in Table 40.1. Iriium192 is normally provie
 in ribbons or as a platinum (Pt)cla wire. The sees are about 3 mm long an
0.5 mm in iameter. There are two types of 192Ir configurations currently availa
ble. One contains a 0.2 mm thick stainless steel wall an has a 0.1 mm iameter
core of 30% Ir70% Pt. The other esign has a 0.3 mm iameter core of 10% Ir90%
Pt cla in a 0.1 mm thick Pt wall.13 Best 192Ir sees are of the former type, an
a schematic iagram of the Best see is shown in Figure 40. 1b. Strans or ribbo
ns can be orere from Best Meical (Springfiel, VA) at a stanar spacing of 1
cm. Customize spacings are also available. Meiumtohigh activity 192Ir ribbo
ns
Figure 40.1 Diagram of the Best iriium192 source, (a) 192lr sees in nylon ribb
on, (b) 125Ir see imensions.
Raioactive sources for interstitial brachytherapy
539
Table 40.1 Common isotopes use in brachytherapy
Isotope
192
Energy
375 keV
Halflife
74.2 ays
Applications
Oncology Cariology 198 Au 420 keV 2.7 ays Oncology 125 I 28 keV 59.6 ays Onco

logy 103 P 21 keV 17 ays Oncology Cariology 32 p 690 keV 14 ays Cariology 9
0 Sr/90Y 970 keV 28 yrs Cariology 137 Cs 662 keV 30 yrs Oncology 32 P, phosphou
s32, 90Sr, strontium90; 90Y, yttrmm90;137Cs, cesium137.
Ir
(15 Ci) are use in intravascular applications using a manual afterloaing evice
. A iagram of the manual afterloaing evice use in low ose rate (LDR) oncolo
gy applications, evelope by Best Meical, is shown in Figure 40.2.14 Despite m
oerately goo clinical outcomes using 192Ir sees in a LDR form, it soon became
apparent that the raiation exposure to hospital staff an others, along with t
he nee for the patient to remain in hospital for 23 ays, necessitate the evel
opment of high ose rate (HDR) evices.15 High ose rate iriium192 evices Whe
reas in LDR, raioactive sees are place in ribbons with appropriate openings t
ailore to the preplanne ose estimates, in HDR, a single source with an activi
ty of approximately 10 Ci is place in a preetermine site for a perio of time
etermine by the esire ose. Several manufacturers (Nucletron, Varian, Best)
, have evelope raioactive source elivery systems for placement at the iseas
e site. The HDR systems evelope by Nucletron an Varian are use in the treatm
ent of oncologic iseases, an use an automate elivery evice. The manual evi
ce (Figure 40.2) has been use for the elivery of intermeiatetohigh oses fo
r nonmalignant iseases, such as cariovascular abnormalities an other oncolog
y applications. Detaile escriptions of automate HDR 192 Ir evices have been
provie in the literature.16 loine125 Laurence Soft XRay Corporation introu
ce 125I sees in the 1970s, with initial clinical stuies performe at the Memo
rial SloanKettering Cancer Centre in New York. Over the last 30 years, raioact
ive sees have been use in the treatment of localize cancers, such as prostate
cancer.17 Currently, there are over 10 manufacturers supplying 125I sources for
the brachytherapy community. Figure 40.3a shows the Best Moel 2301 125I source
as a typical
Basic an avance techniques in prostate brachytherapy
540
Figure 40.2 The 192Ir manual afterloaing evice.
example.18 Other sources available inclue those from Amersham, Oncura, Bar, Me
ntor, NASI, IsoAi, etc. The sources currently available are escribe by Heintz
et al,19 an in the American Association of Physicists in Meicine (AAPM)/Raio
biological Physics Centers file.20 Detaile escriptions of these sources are bey
on the scope of this review; however, most of the sources contain a portion con
sisting of a raiographic marker an a portion containing 125I. The configuratio
n is encapsulate in a metal cyliner of a similar evice to the seee source.
Ioine125 sees are mainly use in the treatment of localize prostate cancer.
High activity sees (>1 mCi/see) are use in the treatment of ocular cancer, ca
ncer of sarcomas, etc., in temporary implants. Pallaium103 Pallaium103 sourc
es have been available since the 1970s. Henschke was one of the earliest researc
hers to suggest that 103P coul be suitable for use in brachytherapy applicatio
ns. There are at least three ifferent 103P sees that are currently available
on the market. These inclue the sees manufacture by Best, NASI, an Therageni
cs. Although raiobiologically it has been shown in vitro that 103P may be a be
tter isotope than 125I for the treatment of prostate cancer, no conclusive multi
center clinical ata are available to ate.21 It has been suggeste that some of
the sie effects, such as rectal bleeing an incontinence, isappear much
Raioactive sources for interstitial brachytherapy
541
Figure 40.3 (a) The ouble wall Best Moel 2301 ioine125 source. (b) The ouble

wall Best Moel 2335 pallaium103 source.

earlier in patients treate with 103P than those treate with 125I sees. A pre
liminary report comparing 125I versus 103P in a clinical trial setup was recen
tly publishe by Wallner et al.22 The purpose of this stuy was to test the hypo
thesis that the shorter halflife of 103P versus 125I results in a shorter urat
ion of raiationrelate symptoms after prostate brachytherapy. Delivery systems
for prostate brachytherapy Ioine an pallaium sees are available loose, loa
e in Mick cartriges, in neeles, an in tissueabsorbable strans. Both 125I an
 103P sees can be elivere using prostate implant neeles (a neele an styl
et) with the help of tissueabsorbable spacers for creating appropriate spacing
between the sees. Also, one coul use Mick isposable or reusable cartriges lo
ae with raioactive sees, an implant the sees using a Mick applicator.11 Ne
eles an other accessories are available from several manufacturers for these p
roceures. Sees loae in tissueabsorbable materials with appropriate spacing
have recently become available in a presterilize format. These convenient see
containing strans are userfrienly an can be use in the operating room witho
ut any aitional workup. See
Basic an avance techniques in prostate brachytherapy
542
migration, suggeste as a potential problem with loose sees, woul also be avoi
e by using strans.23,24 Best ioine125 see (moel 2301) The Best see (moel
2301) consists of a tungsten ro with absorbe 125I encapsulate in oublewalle
 titanium. The source is laserwele at one en. Laser weling leas to sees
that are precise in iameter an length. Furthermore, Best sees are oublewalle
, an are therefore mechanically strong. The source contains a tungsten marker t
hat almost covers the entire length of the see, leaing to superior image chara
cteristics. Ioine is asorbe uniformly onto tungsten ros, incluing the ens.
Hence, the Best see is one of the most isotropic of all the 125I sources curren
tly available.19 (See Figure 40.3.) Dosimetry In accorance with American Associ
ation of Physicists in Meicine (AAPM), Task roup 43 (T43)27 requirements, th
e osimetry factors for Best ioine125 sees were measure inepenently by two
separate groups: Ali Meigooni at the University of Kentucky an Raviner Nath at
Yale University. The raial ose functions measure using micro lithium fluori
e thermoluminescence osimeters are provie in Table 40.2. For etaile escrip
tions of osimetric parameters see Nath an Yue,27 an Meigooni et al.28 Dosimet
ry measurements Doses of raioactive sees are normally measure using lithium f
luorie thermoluminescent osimeters (TLD). These TLDs are place in shallow hol
es machine in a soli water phantom material (Raiation Measurements, Miletow
n, WI). TLD chips of varying imensions are commercially available (Hershaw, Sol
on, OH). The polymer material use in the construction of the phantom material h
as been shown to have some effect on the experimental values.25 D90 is efine a
s the ose elivere to 90% of prostate volume. Stock et al have shown that a bi
ochemical cure rate correspons to the ose elivere (100160 y).9 Hence, T43
recommens the measurement of the osimetry parameters using certain establishe
proceures. The ose istribution aroun a seale source is etermine using th
e formalism accoring to T43:
Table 40.2 Raial ose function values for Best ioine 125 sees
Raial istance (cm) Nath27 (in Water) Meigooni28 (in Soli Water)
0.5 1.0 1.5 2.0 1.046 1.000 0.938 0.876 1.048 1.000 0.899 0.824
Raioactive sources for interstitial brachytherapy
543
2.5 0.773 3.0 0.693 0.683 3.5 0.596 4.0 0.528 0.522 4.5 0.458 5.0 0.406 0.358 5.
5 0.371 6.0 0.331 0.287 6.5 0.286 7.0 0.240 0.208 8.0 0.159 9.0 0.115 10.0 0.083

Dashes inicate that raial ose function values have not been mae.
(1) where SK is the airkerma strength of the source, is the dose rate constant,
G(r, ) is the geometry factor, g(r) is the radial dose function, F(r, ) is the an
isotropy function, and r0=1 cm. The dose rate constant is defined as the dose ra
te per unit air-kerma strength, and is provided in units of (Gy h1 U1) (U=unit of
the airkerma strength of the source). The other two parameters that are require
 for treatmentplanning purposes are raial ose function, g(r) an anisotropy
function, F(r, ). Radial dose function represents the tissue attenuation of photo
ns from the radioactive seeds. The formula for radial dose function is: (2) Furt
her details about this function can be found in the literature.19,28 In E (2),
the two D values are the dose rate constants measured at distances r and r0. r0,
the reference distance is normally 1 cm. G(r, ), the geometric factors, describe
s the effect of the distribution of radioactive material inside the source. Anis
otropy function Anisotropy function F(r, ) represents the variation of dose rate
around the source at each distance. According to TG-43 formalism, the anisotropy
function is: (3) Meigooni et al also recently published the Monte Carlo calcula
tions for Best 125I seeds.29 Recently, the AAPM sub-committee on ow Energy Inter
stitial Brachytherapy Dosimetry assembled the revised dosimetric data, based on
TG-43 prereuisites, useful for medical physicists while planning for brachyther
apy procedures. These data take into consideration all the available experimenta
l information, theoretical calculations, etc. The
Basic and advanced techniues in prostate brachytherapy
544
revised recommendation from the subcommittee is that the dose calculation does n
ot reuire the use of an anisotropy constant.30 The measured anisotropy factors
and estimated anisotropy constant for Best 125I seeds are provided in Table 40.3.
The values reported by Nath and Yue and Meigooni et al are within experimental
margin of error.27,28 The dose rate constants for Best seeds were determined expe
rimentally and theoretically by Nath and Yue,27 and Meigooni et al28 in independ
ent measurements. These values are provided in Table 40.4 and correspond to the
National Institute of Standards and Technology (NIST-1999) SK standard, revised
in 2000. For values to be used in clinical studies, medical physicists and oncol
ogists are urged to make informed decisions based on published data.30
Table 40.3 Anisotropy factors for Best iodine-125 seeds
Radial distance Anisotropy factor Nath and Yue28 Meigoni et al29
0.99 0.99 0.97 0.98
2 cm 0.96 4 cm 0.94 5 cm 6 cm 0.96 7 cm Anisotropy constant 0.96
Table 40.4 Dose rate constants for Best iodine125 seeds
Dose rate constant (cGy h1 U1)
Meigooni et al 1.03 Nath an Yue27 1.02 1.01 Monte Carlo stuies29 Note: values
correspon to NIST 1999 SK stanar, revise in 2000,
28
Table 40.5 Dosimetry parameters for Best pallaium103 sees
Dose rate constant (cy h1 U1)
Peterson an Thotmsen Meigooni et al32
24
Anisotropy constant*
0.71 (TLD) 0.94 0.69 (TLD) 0.89 0.67 (MC) 0.88 * Simple average metho TLD, ther
moluminescent osimeters; MC, Monte Carlo metho. Experimental values obtaine u
sing TLD measurements.

Recently, Heintz et al compare ifferent 125I sources use for permanent inters
titial implants.20 The raial ose function values for Best moel 2301 substantia
lly match Amershams 6711 sources. Furthermore, moel 2301, Best 125I sees, emit p
ure 125I spectrum. Some of the other 123I sees emit silver xrays, thereby lowe
ring the average energy.
Raioactive sources for interstitial brachytherapy
545
Best pallaium103 see (moel 2335) The imensions, ouble wall encapsulation, a
n other exterior aspects of Best 103P sees are similar to those of Best 125I se
es escribe above (see Figure 40.3). The interior of the Best 103P see moel
2335 consists of six polymer resin beas coate with 103P separate by a rectan
gular tungsten marker in the mile. The osimetric parameters for the Best 103P
source were obtaine by two inepenent groups of investigators, Peterson an T
homasen (University of Wisconsin) an Meigooni et al (University of Kentucky).2
5,31 The Monte Carlo calculations were carrie out by Meigooni et al. Some of th
e Peterson an Meigooni ata are provie in Table 40.5. Conclusions The future
of high an low ose rate brachytherapy looks promising. Accurate etermination
of isease sites using sophisticate imaging moalities, such as magnetic resona
nce imaging (MRI), positron emission tomography (PET) an PET/CT, realtime plan
ning in the operating room employing optimize osimetry, osesparing to critic
al organs using unique signal emitting evices, an combinations of brachytherap
y with new treatment moalities,32,33 are some of the noval approaches that are
currently uner investigation. References
1. Hilaris BS, Mastoras DA, Shih LL, Boner WR. History of brachytherapy. In: Na
g S, e. Principles an practice of brachytherapy. Armonk, NY: Futura Publishing
, 1997:13. 2. olberg SW, Lonon FS. Frage er Bezichungen Zaischen Bequerelstr
ahlen Hantuffectionen. Dermatologische Zeitschrift 1906; 10:457. 3. Abbe R. Rai
um in surgery. Journal of the American Meical Association 1906; 47:183. 4. Amer
ican Heart Association. 2002 heart an stroke statistical uptake. Dallas, TX: Am
erican Heart Association, 2003. 5. Henschke UK, Hilaris BS, Mahan D. Afterloai
ng in interstitial an intracavitary raiation therapy. Am J Roentgenol 1963; 90
:386395. 6. Henschke UK. The treatment of cancer with small sources of raioactiv
e iriium. In: Pack T, Ariel IM, es. Treatment of cancer an allie iseases,
Vol I, 2n en. New York: Paul B Hoeber, Harper Books, 1958:431. 7. Blasko JC, R
age H, Schumacher D. Transperineal percutaneous ioine125 implantation for pro
state carcinoma using transrectal ultrasoun an template guiance. Enocurie/Hy
pertherm Oncol 1987; 3:131139. 8. rimm PD, Blasko JC, Rage H. Ultrasounguie
transperineal implantation of ioine125 an pallaium103 for the treatment of
earlystage prostate cancer. Urol Clin North Am 1994; 2:113116. 9. Merrick S, W
allner KE, Butler WM. Permanent interstitial brachytherapy for the management of
carcinoma of the prostate glan. J Urol 2003; 169:16431652. 10. Merrick S, Butl
er WM, Lief JH, Dorsey AJ. Temporal resolution of urinary morbiity following pr
ostate brachytherapy. Int J Raiat Oncol Biol Phys 2000; 47:121. 11. Anerson LL
, Nath R, Weaver KA, et al. Interstitial brachytherapy: Physical, biological an
clinical consierations. New York: Raven, 1990:313.
Basic an avance techniques in prostate brachytherapy
546
12. For further information, visit: isotopes.lbl.gov 13. BoutrouxJaffr E In: Pie
rquin B, Marinello A, es. A practical manual of brachytherapy. Maison, WI: Me
ical Physics Publishing, 1997:3. 14. Amols HI. In Intravascular brachytherapy. W
isconsin, WI: Meical Physics Publishing, 2002:289. 15. Kuske RR Jr. Breast brac
hytherapy. Brachytherapy 1999; 13(3):543558. 16. AAPM Report No. 41. Remote after
loaing technology. New York: American Institute Physics, 1993. 17. Nag S, Beyer

D, Frielan J, et al. American Brachytherapy Society (ABS) recommenations for

transperineal permanent brachytherapy of prostate cancer. Int J Raiat Oncol Bi
ol Phys 1994; 44:789799. 18. Nath R, Melillo A. Dosimetric characteristics of a 
ouble wall 125I source for interstitial brachytherapy. Me Phys 1998; 20:14751483
. 19. Heinz BH, Wallace RE, Havezi JM. Comparison of I125 sources use for perm
anent interstitial implants. Me Phys 2001; 28:671682. 20. AAPM/Raiobiological P
hysics Center at: rpc.manerson. org an aapm.org 21. Ling CC. Permanent implan
ts using Au198, P103, an I125: Raiobiological consierations base on the
linear quaratic moel. Int J Raiat Oncol Biol Phys 1992; 23:8187. 22. Wallner K
, Merrick , Cavanaugh W, et al. Cancer J 2002; 8:6773. 23. Anerson LL, Nath R,
Weaver KA, et al. Interstitial brachytherapy: Physical, biological an clinical
consierations. New York: Raven, 1990:6571. 24. Tapen EM, Blasko JC, rimm PD, et
al. Reuction of raioactive see embolization to the lung following prostate b
rachytherapy. Int J Raiat Oncol Biol Phys 1998; 42:10631067. 25. Peterson SW, Th
omasen B. Measurements of the osimetric constants for a new 103P brachytherap
y source. Brachytherapy 2002; 1:110119. 26. Nath R, Anerson LL, Luxton , et al.
Dosimetry of interstitial brachytherapy sources: recommenations of the AAPM Ra
iation Therapy Committee Task roup 43. Me Phys 1995; 22:209234. 27. Nath R, Yu
e N. Dosimetric characterization of an encapsulate interstitial brachytherapy s
ource of 125I on a tungsten substrate. Brachytherapy 2002; 1:102109. 28. Meigooni
AS, earheart DM, Sowars K. Experimental etermination of osimetric character
istics of best 125I brachytherapy source. Me Phys 2000; 27:21682173. 29. Sowars
KT, Meigooni AS. A Monte Carlo evaluation of the osimetric characteristics of
the Best Moel 2301 125I brachytherapy source. Appl Raiat Isot 2002; 57:327333.
30. Rivar MJ, Coursey BM, DeWer LA, et al. Upate of AAPM Task roup 43 Report
: A revise AAPM protocol for brachytherapy ose calculations. Me Phys (in pres
s). 31. Meigooni AS, Bharucha Z, YoeSein M, Sowars SK. Dosimetric characterist
ics of the best oublewall 103 P brachytherapy source. Me Phys 2001; 28(12):2
5682575. 32. Ellis RJ, Soee DB, Spirnak JP, et al. Feasibility an acute toxicit
ies of raioimmunoguie prostate brachytherapy. Int J Raiat Oncol Biol Phys 2
000; 48:683687. 33. Jung M, Zhangy Y, Dimtchev A, et al. Interstitial gene elive
ry in human xenograph prostate tumors using titanium metal sees, (submitte).
41 RADIOCOIL: a coile wire brachytherapy source
Piran Sioshansi Introuction Brachytherapy by see implantation for the treatmen
t of prostate cancer has been emonstrate to be a clinical success with long te
rm followup ata showing results similar to raical prostatectomy with minimal
morbiity an lower rates of ebilitating sie effects of incontinence an impot
ence.12 Despite this success story, there are opportunities for improvement in cu
rrent generation sees that can an shoul be aresse. RADIOCOIL source is a s
econ generation linear brachytherapy source esigne with the motivation of a
ressing some of the imperfections an shortcomings in using sees for brachyther
apy with an eye on improving patient outcomes. Among the features of the RADIOCO
IL linear source are: more homogeneous ose istribution, stability in tissue, t
he ability to place the source with extracapsular extension, elimination of sour
ce migration, visibility uner ultrasoun, an a smaller profile source. The sma
ller profile in turn, allows the use of smallergauge, sharper neeles for sourc
e eployment, thus reucing eema an trauma to the patient. In aition to the
enhancement in the source performance, the RADIOCOIL source is easier to prouce
, as it is manufacture by an automate batch process, minimizing the requiremen
t for 100% inspection an assaying of the source. Manufacturing metho of a typi
cal pallaium103 see The traitional metho of making a 103P see relies on b
ombaring a rhoium foil target with an internal beam in a high current cyclotro
n. During the course of irraiation, which typically takes a week or longer, a s
mall fraction of the monoisotopic nuclei of rhoium103 (103Rh) are transmute in
to 103P by a (p,n) reaction whereby a proton is absorbe in the nucleus of 103R
h an a neutron is emitte. The one week aroun the clock bombarment yiels 103
P with an abunance of less than 0.1% in the rhoium matrix. The 103P prouce
in this way is separate from rhoium by a chemical process. The purifie 103P
is then enriche an, typically, plate on to raiopaque cyliners. The raiopa

que cyliner is place insie a titanium shell, an the shell is laserwele sh
ut to construct a seale source. The sees prouce by this metho require exten
sive quality control an are 100% teste for sealesource performance. They are
iniviually assaye for etermining the apparent activity. Figure 41.1 shows s
chematically the steps
Basic an avance techniques in prostate brachytherapy
548
involve in manufacturing an testing a see. The result of this esign is a rig
i cylinrical see measuring 0.81 mm iameter an 4.5 mm in length. Sometimes,
representative sees are inserte in a phantom for osimetry. The raial ose fu
nction an the irectional activity of the see is measure to establish the
Figure 41.1 Traitional manufacturing steps for 103P sees.
ifference between the cylinrical geometry of the see from a point source isot
ropic moel that the see is esigne to emulate. It is not surprising that the
anisotropy function of the see may inicate variations on the orer of 50%, as
very little activity is emitte from the two ens of a cylinrical see as compa
re to the miplane of the see. Manufacturing metho of the RADIOMED source Th
e manufacturing of the RADIOMED source is base on irect bombarment of an exte
ne length (typically more than a kilometer) of a fine rhoium wire wrappe on
a rum by an external beam of a oneofakin, high intensity, selfextracting c
yclotron. The beam is raster scanne on the rotating rum, creating a highly uni
form activation fiel. The rhoium wire activate by this metho, after a typica
l one week of bombarment by the proton beam, contains in orer of 100 parts per
million 103P in a rhoium matrix an, thus, is classifie inherently as a seal
e source. The activate wire prouce by this process is then coile to a imen
sion of 0.35 mm an is laser cut to integer lengths from 1 to 6 centimeters. As
escribe, the manufacturing process has very few steps an easily lens itself
to automation an remote monitoring. By virtue of increase monitoring an built
in quality assurance (QA), the nee for assaying an quality control (QC) of th
e prouct is sharply reuce. Figure 41.2 shows schemat
RADIOCOIL
549
Figure 41.2 Manufacturing steps for the RADIOMED source.
ically the few steps involve in manufacturing the RADIOCOIL source. The RADIOME
D esign The iameter of the coil in this esign is a compromise between a esir
e to keep the source imension as small as possible for flexibility an complian
ce with tissue, yet give it a imension so that the source is visible to the nak
e eye an some mechanical integrity so that the source is easily hanle. The 0
.35 mm of the coile wire esign is such that, if the source is roppe, it can
be foun an, when foun, it can be lifte with a softtippe tweezer. Source is
with this imension are inherently raiopaque an are visible uner fluoroscopy
, iagnostic Xray, compute tomography (CT), an magnetic resonance imaging (MR
I) scans. The coile esign an the extene nature of the source, serenipitous
ly, contribute to the very important feature of making it highly echogenic, as e
very turn of the coil reflects the waves an is therefore visible uner ultrasou
n. The ultrasoun visibility of the source is an enabling feature of the RADIOC
OIL source, as it allows the physician to see the source as it is being release
in the presence of neighboring sources. This characteristic of the RADIOCOIL so
urce allows the physician to correct any col spots or eficiencies in the source
resting position compare to the treatment plan while the patient is being intra
operatively monitore by transrectal ultrasoun (TRUS). Thus, in principle, the
RADIOCOIL source allows the physician to perform, via ultrasoun, a postoperativ
e treatment verification in the same session uring which the source is being im

plante. In aition, this avois the nee for an aitional visit to the hospit
al for a CT scan to complete postoperative verification. Efforts are uner way t
o complete the ultrasoun posttreatment planning of the source so that, before t
he patient is release from the operating room (OR), the physician/physicist tea
m can
Basic an avance techniques in prostate brachytherapy
550
evaluate the aequacy of the ose istribution an correct any col spots or ef
iciencies in the plan an augment any eficiency in the plan. The coile esign,
flexibility, an extene nature of the source make it conformal to the organ i
n which it is implante, stable in tissue, an nonmigrating. The source flexibi
lity is shown in Figure 41.3. Repeate evaluation of the source in a canine pros
tate moel has shown that the source is stable from the time of release, when pl
ace intracapsular with or without an extracapsular extension. Raiographic exam
ination of the source has shown that the source is stable in the prostate from t
he time of release. Furthermore, the examination of the source, after sacrificin
g the animals, has shown that tissue integrates into the coil an the source can
not be separate from the tissue in which it is embee.
Figure 41.3 Flexible coile wire RADIOMED source.
RADIOCOIL source specification The RADIOCOIL source is a flexible helical coile
wire that is inherently a seale source. It is mae from a 0.05 mm 0.200 mm cros
ssection ribbon coile to an outer imension of 0.35 mm. RADIOCOIL is initially
supplie in integer lengths from 1 cm to 6 cm. (The intention is to supply the
source, in the future, in a continuous length, allowing the physician/ osimetri
st to cut it to proper noninteger lengths in the OR.) The source activity range
is 12.1 mCi/cm with uniformity of 3% over the length of the source (as measure
by a etector place at the istance of 10 mm from the axis of the source). The
source specification is outline in Table 41.1. The source calibration is tracea
ble to National Institute of Stanars an Technology (NIST) stanars. NIST cal
ibrate sources have been sent to ADCLs (accreite osimetry calibration labora
tories) for crosscalibration of their well chambers. Hospitals can, in turn, us
e the ADCLs for making their measurement traceable to NIST stanars. Figure 41.
4a shows the appearance of the RADIOCOIL source uner 40magnification an Figure
41.4b is a schematic rawing of the source. The source esign lens itself to ei
ther a preloae neele brachytherapy proceure or intraoperative loaing techni
que. Thus, the RADIOCOIL source can accommoate the physicians preference to work
with a preplan esign base on preoperative ultrasoun etermination of
RADIOCOIL
551
Table 41.1 The RADIOMED source specification
Source iameter 0.35 mm Source lengths Integer lengths (16 cm) provie in color
coe cartriges Source activity 1.02.1 mCi/cm of coil Activity uniformity 3% over
the length of the source* * As measure by a etector place 10 mm away from th
e axis of the the source.
the prostate volume, as measure uring an earlier office visit, or an intraoper
ative approach to choose an appropriate length of the source to match the actual
size of the prostate presente in the OR. The source is supplie in a sterile p
ouch reay for use. The source kit for performing a complete brachytherapy proce
ure comes with three 1 cm representative coils inclue in each pack for onsit
e source activity measurement, if necessary. Features an clinical benefits of t
he RADIOMED source The RADIOCOIL source has a number of enabling features an en
hancement over traitional brachytherapy sees. These features an the clinical

avantages they provie are summarize in Table 41.2. Dosimetry The formal osim
etry for the RADIOMED source, base on the recommenation of the American Associ
ation of Physicists in Meicine (AAPM) Task roup (T43),3 was performe by Dr
A.S.Meigooni an his team at the University of Kentucky. The osimetry stuy, wh
ich inclues measurements in soli water as well as Monte Carlo simulation an i
n both soli an liqui water, has been submitte for publication in Meical Phy
sics.4 In aition to establishing a ose rate constant of 0.64 cGy h1 U1 for a 0.
5 cm active length (0.56 cy h1U1 for a 0.1 cm active length), the highlights of t
he osimetry stuy inclue the raial ose an anisotropy functions for 0.5, 1.0
cm coils an Monte Carlo simulation for all other lengths. Figures 41.5 an 41.
6 show the raial ose function g(r) an anisotropy function f() measured at a 2
cm distance from the source center for a 0.5 cm coil, respectively, and compare
the results with a Monte Carlo simulation and, also, a commercially available Pd
seed. It is important to point out that anisotropy function is not an appropria
te description for the RADIOCOI
Basic and advanced techniues in prostate brachytherapy
552
Figure 41.4 (a) Magnified view of the RADIOCOI source, (b) Diagram of the sourc
e. Table 41.2 Features and clinical benefits of the RADIOMED
Feature
Uniform distributed activity Coil echogenic design
Clinical benefits
Homogeneous dose distribution
Visible under ultrasound Controllable source release, easy postimplant dosimetry
Natural stranded design Can be placed extracapsular Coil flexible design Stable
in tissue and nonmigratory Inherently radiopaue Visible under fluoro, x-ray, C
T, and MRI Small diameter Can be inserted by smaller diameter needlesless trauma
CT, computed tomography; MRI, magnectic resonance imaging,
source. This term is a carryover from describing the variation of a radiation em
ission from a cylindrical seed with the isotropic emission of an ideal point sou
rce that the seed is designed to emulate. For the RADIOCOI line source, there i
s a need for the AAPM to assign a new Task Group and establish new nomenclature
appropriate for a line source. In a more practical consideration, the dose from
a uniform linear source is represented by combining 1/r dependence, where r is d
istance of the tissue from the source axis, and attenuation in the intervening s
oft tissue. In contrast, the dose from a column of (n) seeds and spacers is repr
esented by a combination:
RADIOCOI
553
Figure 41.5 Comparison of Model 200 radial dose data with measured and Monte Car
lo values obtained for 0.5 cm active length source model.
where ri is the distance of tissue from source i and the attenuation of radiatio
n in intervening distance in soft tissue from each source. The emulation algorit
hm to compare the linear source against a seed-spacer column is easy to set up a
nd is remarkably accurate. Figure 41.7a shows a comparison of a 5 cm RADIOCOI s
ource and Figure 41.7b an eually spaced 5 seed-spacer column. The isodose lines
for the two figures show that at distances larger than 5 mm from the source axi
s, the dose is identical and the tissue is incapable of distinguishing between a
n ideal (fixed spacing and isotropic point source emission) from a 5 seed-spacer
column and a 5 cm euivalent RADIOCOI source. At distances closer than 5 mm to
the source axis, the RADIOCOI source, as anticipated from a linear source, del

ivers a homogeneous dose as

Figure 41.6 Comparison of anisotropy function values for 0.5 cm source at 2 cm r
adial distance.
Basic and advanced techniues in prostate brachytherapy
554
compared to the heterogeneous dose characteristic of isolated point sources. Fur
thermore, for the euivalent volume receiving a 100%, V100, prescribed dose, the
volume exposed to 150%, V150, of the prescribed dose is larger for the 5 seed-s
pacer column. In addition, the contact dose to the tissue abutting the RADIOCOI
source is lower when compared to an isolated seed. These attributes of the RADI
OCOI source may reduce the excessive dose delivered to the urethra, nerve bundl
es, or the rectum if, unintentionally, a source is placed at close proximity to
these radiosensitive structures. Thus, the RADIOCOI source may ameliorate toxic
ity to neighboring structures and reduce the undesirable radiation damage-relate
d side effects, of brachytherapythe primary complication of the procedure. Delive
ry system The small diameter of the RADIOMED source (0.35 mm) makes it suitable
for use with a 22-gauge needle. However, the 22-gauge needle does not have the r
euired rigidity for use in prostate applications. The 17- or 18-gauge needles c
urrently used for brachytherapy (standard seeds are 0.81 mm diameter) are clearl
y oversized for the fine diameter RADIOCOI source. To reach a compromise betwee
n smaller gauge, sharper needles and large-bore, rigid needles, a special 19-gau
ge (outer diameter 1.08 mm) thick wall needle has been designed. The thick wall
of the needle allows for the design of a multifaceted needle tip with improved s
trength and sharpness ideal for perpendicular piercing of tissue, as measured by
reduced penetration force. The thick wall also gives the needle the characteris
tic rigidity reuired for reaching the intended location within the prostate for
source placement. The side bevel of the needle tip gives it the customary steer
ability while the fine tip bevel makes it ideal for piercing of the tough, yet m
obile, prostate capsule. In addition, the needle cannula near the tip is roughen
ed to make it echogenic and the shank of the needle is silicone-coated to lessen
the tissue drag forces. Figure 41.8 shows the size and design of the New Dimens
ion needle in comparison with the 18-gauge commercially available needles. Table
41.3 shows the force of penetration and factional force for the RADIOCOI sourc
e as compared to three different commercially available needles. The source is d
elivered in a self-shielded cartridge, which doubles as a shipping container. Th
e philosophy behind the design of the RADIOCOI delivery system is to give the o
perating physician the option and freedom to make the final commitment for the l
ength of the source to be deployed in a given needle position intraoperatively.
Once the needle is inserted and the length of the prostate is measured under ult
rasound guidance, the physician can decide on the source length and will choose
it from the source (loaded in a cartridge) supply provided in the OR. The cartri
dge is readily attached to the hub of the needle; a special long stylet with cen
timeter demarcation lines is used to advance the source for deployment. The cart
ridge is color-coded to indicate the length of the source. Figure 41.9 shows the
color-coded cartridge for the RADIOCOI source. The template for the RADIOCOI
source is identical to the existing brachytherapy templates with the exception t
hat the needle holes are for 19-gauge needles. The RADIOCOI cartridges are supp
lied in a sterile pouch. In most cases, the source has been assayed and certifie
d by an independent nuclear pharmacist. Additional 1 cm reference sources are su
pplied (in a non-sterile form) as part of the package for on-site
RADIOCOI
555
source-activity measurement and verification, if necessary. The source cartridge

s, needles, stylets, and calibration sources are supplied together in a kit as a

n all-inclusive package. Source deployment procedure The flexible nature of the
RADIOCOI source reuires special attention during source placement. The RADIOCO
I source should not be injected, as any attempt to inject the flexible source i
nto soft tissue will cause the coil to bend. Instead, the RADIOCOI source, when
advanced by the stylet to the tip of the needle, must be released by holding th
e stylet steady while retracting the needle and thus carefully releasing the sou
rce in the track created by the needle.
Figure 41.7 Isodose line simulation from (a) an ideal 5 seed-spacer assembly as
compared to (b) a 5 cm RADIOCOI source. At distances
Basic and advanced techniues in prostate brachytherapy
556
larger than 5 mm from the source axis, the isodose lines are identical. At close
r distances, the isolated point sources deliver a heterogeneous dose compared to
the homogeneous dose for a linear source.
Even though the RADIOCOI source can be preloaded in a needle (by using customar
y wax at the needle tip), the preferred mode is to place the needle inside the p
rostate and, under ultrasound guidance, position the tip of the needle at the di
stal surface of the prostate (or 5 mm beyond the distal surface). Once the reui
red source length based on the prostate size is determined, the brachytherapy pr
actitioner chooses the appropriate cartridge containing the anatomically correct
source length, attaches the cartridge to the needle hub, and advances the sourc
e to the tip of the needle by the long stylet supplied for this purpose. The cen
timeter demarcation on the stylet allows for accurate advancement of the source
to the tip of the needle. (For example, a 4 cm long source is advanced to the
Figure 41.8 The comparison between the thick wall 19-gauge New Dimension needle
(shown to left of figure) compared to three other commercially available 18-gaug
e needles.
needle tip by stopping at the 4 cm mark between the stylet and the needle hub.)
At this point, the source is ready to be released by carefully holding the style
t steady in one hand while withdrawing the needle with the other hand. Evaluatio
n of the RADIOCOI source in an animal host The RADIOCOI source has been evalua
ted in a canine prostate model. The primary goal of the study was to evaluate th
e stability of the coiled wire design in prostate tissue.
RADIOCOI
557
The secondary goals were to evaluate the tissue response and histology of the ti
ssue surrounding the source and also to test the delivery system for the source
and to obtain the radiographic features of the source to help design a source wi
th optimal performance. The animal study was performed at Dartmouth College unde
r the supervision of Dr Jack Hoopes at the Department of Neurosurgery and Radiat
ion. In all cases, the sources were implanted with the animal under general anes
thesia with an open laparotomy. Figure 41.10 shows the RADIOCOI source placemen
t in a dogs prostate during open laparotomy. The dogs prostate was isolated and RA
DIOCOI sources of various lengths were implanted in the different prostate lobe
s such that 5 mm of the source were extended beyond the prostate gland. The dogs
were x-rayed and CT images were taken immediately post implant, a week later, a
nd at the time of sacrifice21 days post implant. Figure 41.11 shows a series of C
T scans comparing the position of the coiled wire source on day 1 and day 21. Th
e position of the RADIOCOI source when compared relative to each other, to the
other anatomical landmarks and measured against the position of the fiducial mar
kers showed the relative and absolute stability of the sources in tissue within

the accuracy of the measurement reported to be in order of 1 mm.5 Clinical evalu

ation of RADIOMED While waiting for the infrastructure for the activation of the
RADIOCOI source to come on line, an attempt was
Table 41.3 Insertion force of the New Dimension 19-gauge needle compared to thre
e other 18-gauge needles
Needle insertion force (arbitrary units)
Point junction Heel Cannula
New Dimension 19-gauge for RADIOCOI ~0 Vendor 1:18-gauge 1.20 Vondor 2:18-gauge
2.49 Vendor 3:18-gauge 1.89. n.a., not available. 1.48 2.00 n.a. n.a. 1.15 2.16
2.84 2.11 0.06 1.64 2.16 2.24
Basic and advanced techniues in prostate brachytherapy
558
Figure 41.9 RADIOCOI source in color-coded cartridges.
Figure 41.10 The position of RADIOCOI sources (arrows) implanted inside an isol
ated canine
RADIOCOI
559
prostate in an open laparotomy procedure.
Figure 41.11 Stability of the RADIOMED source in canine studies. Within the accu
racy of the measurement, the RADIOMED source is stable and non-migratory in the
prostate gland.
made to evaluate the delivery system and the performance of the RADIOCOI design
in a clinical setting and to obtain in vivo information on the visibility of th
e source by various imaging modalities (ultrasound, x-ray, CT, etc.). Ten patien
ts were implanted with the non-radioactive version of the RADIOCOI source in th
eir prostate using the transperineal approach. The non-radioactive version of th
e RADIOCOI source, it turns out, is an excellent market that is visible under a
ll imaging modalities including ultrasound, MRI, x-ray, CT, and fluoroscopy. The
patients in this study were implanted with two coils of different lengths. One
coil was placed in the base of the prostate with the distal end marking the basa
l extent of the prostate. The other coiled wire was placed at the apex of the pr
ostate. The two coils helped to clearly mark the distal and proximal boundary of
the prostate and were used as reference points throughout the brachytherapy pro
cedures. Figure 41.12 shows the TRUS image of a typical RADIOCOI marker in a pa
tient in both longitudinal and axial orientations. The two images clearly demons
trate the echogenicity of the RADIOCOI source. Note that there is minimum evide
nce of ghost echoes present in these images.
Basic and advanced techniues in prostate brachytherapy
560
Figures 41.13 and 41.14 show the fluoroscopic appearance and the x-ray image of
the non-radioactive RADIO COI marker, respectively. These images mani-fest the
inherent radiopacity of the RADIOCOI source. Figure 41.15 shows the CT scan of
the RADIOCOI marker in four adjacent slices 5 mm apart in a prostate patient de
signated for HDR treatment. (The dark spots in each slice show the footprint of
the HDR catheters as they intersect each CT slice.) Noteworthy is the predictabi
lity of the location of the RADIOCOI marker from one slice to the next, an inhe
rent characteristic of a straightline source. This feature of RADIOCOI will sim

plify the postimplant evaluation of implant uality as compared to treatment pla

n and, in the case of detecting cold spots within the gland, facilitate the decisi
on of considering supplemental external beam radiotherapy or a second implant pr
ocedure. At the three month patient follow-up, the markers proved to be well tol
erated. Relative distance between the markers, when compared to anatomical landm
arks, shows that the markers are stable and non-migratory. Conclusions The RADIO
COI permanent implant linear source is designed to address some of the shortcom
ings of the
Figure 41.12 (a) ongitudinal and (b) axial ultrasound image of a RADIOCOI sour
ce in the prostate. The absence of a ghost (echo) appearance in the longitudinal
image is noteworthy.
RADIOCOI
561
Figure 41.13 Fluoroscopic image of the non-radioactive RADIOCOI sources in a ty
pical prostate brachytherapy patient.
traditional seeds. The source utilizes a fine coiled wire design that gives it s
ome enabling features including visibility under ultrasound, homogeneous dose di
stribution that ensures the entire gland is treated with a homogeneous tumoricid
al dose, the ability to place a portion of the source extracapsular for deliveri
ng adeuate dose to the gland margins with a higher degree of accuracy, and an
Figure 41.14 Diagnostic x-ray of a non-radioactive RADIOCOI source in a prostat
e patient in the presence of a foley catheter balloon filled with contrast media
.
Basic and advanced techniues in prostate brachytherapy
562
easier method to assess the uality of implant dosimetry intraoperatively. Furth
ermore, the source is delivered with an improved delivery system suitable for ei
ther preloaded needles based on a preplanned volumetric measurement of the prost
ate or intraoperative treatment plan options. The culmination of improved clinic
al features of the RADIOCOI source is expected to contribute to even better pat
ient outcomes as compared to permanent seed implant brachytherapy. In addition t
o the improved features, the RADIOCOI source is easier to produce and has a hig
her degree of built-in uality assurance/uality control, reliability, and repro
ducibility.
Figure 41.15 A CT scan of a high dose rate (HDR) prostate patient showing the fo
otprint (white spot) of the nonradioactive source in various slices 5 mm apart.
The dark spots are the intersection of the HDR catheters intersecting each slice
.
Acknowledgment The development of the RADIOCOI source was supported, in part, b
y a NIH National Cancer Institute SBIR grant No. 2 R44 CA78005. References
1. Kupelian P, Potters , Khuntia D, et al. Radical prostatectomy, external beam
radiotherapy <72 Gy, external beam radiotherapy 72 Gy, permanent seed implantati
on, or combined seeds, external beam radiotherapy for state T1T2 prostate cancer.
Int J Radiat Oncol Biol Phys 2004; 58:2533.
RADIOCOI
563
2. Potters , Perez CA, Beyer DC, et al, for the American College of Radiology.
Permanent source brachytherapy for prostate cancer: ACR appropriateness criteria

. Radiology 2000; 215(Suppl): 13831400. 3. Nath R, Anderson , uxton G, et al. D

osimetry of interstitial brachytherapy sources. Med Phys 1995; 22(2):209234. 4. M
eigooni AS, Zhang H, Clark JR, et al. Dosimetric characteristics of a new RADIOM
ED Pd103 wire line source for use in permanent brachytherapy implants. Med Phys
(submitted). 5. Hoopes J. Tissue effect and post implant movement of Pd-103 brac
hytherapy wires and gold fiducial in the canine prostate. Internal report at Sur
gical Research aboratories, Dartmouth Medical School, Hanover, NH, February 200
4.
42 InterSource brachytherapy seeds

John Russell and Jaclyn Collins Introduction International Brachytherapy, sa (IB

t) manufactures and markets world-wide two permanently implantable radiation sou
rces, InterSource103 and InterSource125, utilizing palladium-103 and iodine-125,
respectively* The uniue hollow seed design provides a product line including i
ndividual loose seeds, seeds loaded in Mick cartridges, InterStrand103 and InterS
trand125 seeds on a biodegradable suture at a standard 1 cm spacing, InterStrand
Special, in which seed spacing on the strand is customized to a doctors prescript
ion, and EZ-Pak, in which brachytherapy needles are preloaded to prescription, st
erilized, and shipped ready to use. A cutaway view of the InterSource brachythera
py source shows the hollow seed construction and the sealed toroidal space betwe
en the two titanium tubes comprising the capsule (Figure 42.1). The manufacture
of tiny seeds with this complex geometry was made possible because of the invent
ion and development of ink
Figure 42.1 The InterSource brachytherapy source. (Reproduced with permission fro
m International Brachytherapy.)
jet printing of intensely radioactive liuids. The x-ray marker is a ring of pla
tinum iridium. Iodine-125 sources are available from 0.2 mCi to 0.7 mCi (0.2540.8
89 U) and palladium-103 sources are available from 0.5 mCi to 1.8 mCi (0.6462.327
U). Because of the hollow tube design of the InterSource, a hydraulic pressure c
annot be supported that would move an implanted seed in the direction parallel t
o the long axis of
InterSource brachytherapy seeds
565
the seed. As a result, there are very few reported cases of migration of implant
ed InterSource seeds.1,2 Product descriptions InterSource seeds Both InterSource10
3 palladium seeds and InterSource125 iodine seeds are calibrated based on the NI
ST Wide-Angle-Free-Air-Chamber (WAFAC) standard for air-kerma strength (U) in un
its of cGy cm2/hour. This calibration (National Institute of Standards and Techn
ology; NIST-2000) is available at the Accredited Dosimetry Calibration aborator
ies so that interested institutions may transfer that calibration to their dose
measurement systems. Apparent activity can be computed, using the American Assoc
iation of Physicists in Medicine (AAPM) subcommittee interim recommendation, fro
m the formulas: For InterSource103: Air-kerma strength in cGy h1 at 1 cm= 1.293App
arent activity in mCi. For InterSource125: Airkerma strength in cy h1 at 1 cm=
1.27Apparent activity in mCi. The T43 parameters3 that escribe the raiation is
tribution aroun implante sees have been measure an calculate by two inepe
nent institutions for both InterSource103 an InterSource125.37 The two sets of
results for each isotope have been average, see Tables 42.1 an 42.2.
*After this paper was finalize, manufacture of InterSource103 pallaium103 see
s encapsulate in titanium was iscontinue in anticipation of an avance plas
tic pallaium see being introuce to the market in the first quarter of 2005.
Table 42.1 T43 parameters for InterSource103 moel 1031L brachytherapy source4,5
Anisotropy constant: a Dose rate constant: 2000=0.694 cGy h1 U1 Distance r(cm) Rai
al ose function g(r)

0.0b 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.5 1.207 1.231 1.239 1.213 1.187 1.148 1.0
97 1.047 1.000 0.782
Anisotropy factor
0.906
0.901
Basic an avance techniques in prostate brachytherapy
566
2.0 0.600 0.885 2.5 0.453 3.0 0.340 0.886 3.5 0.256 4.0 0.191 0.889 4.5 0.143 5.
0 0.105 0.889 53 0.080 6.0 0.058 6.5 0.044 7.0 0.033 7.5 0.022 8.0 0.017 8.5 0.0
13 9.0 0.010 9.5 0.007 10.0 0.005 a The ose me constant , is based on the revise
d NIST-1999 calibration standard, which is generally referred to as NIST-2000. b
The value of g(r) at 0.0 cm is indicated in italics, and was linearly extrapola
ted from the previous two calculated values.
InterStrand InterStrand consists of 10 InterSource seeds strung on a biodegradable,
0.5 mm diameter, monofilament suture
Table 42.2 TG43 parameters for InterSource125 model1 251 brachytherapy Source6,7
Anisotropy constant: a Dose rate constant: 2000=1.02 cGy h1 U1 Distance r(cm) Raia
l ose function g(r)
0.0b 0.3 0.4 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 0.812 0.938 0.980 1.005 1.0
00 0.946 0.877 0.798 0.721 0.644 0.574 0.508 0.448
Anisotropy factor
0.972 0.951 0.944 0.965 0.943 0.947
InterSource brachytherapy sees
567
5.5 0.393 6.0 0.343 0.949 6.5 0.297 7.0 0.257 0.965 7.5 8.0c 0.206 a The ose ra
te constant, , is based on the revised NIST-1999 calibration standard, which is g
enerally referred to as NIST-2000. b The value of g(r) at 0.0 cm is indicated in
italics, and was linearly extrapolated from the previous two calculated values,
c The value of g(8.0) for iodine seeds is indicated in italics, and was determi
ned with a sum of exponentials the previous data. (VariSeed Informational Notific
ation from Varian Brachytherapy, 14 January 2000 release,)
(Figure 42.2) The seeds are spaced 1 cm center-to-center and fixed in place by s
light deformations of the suture at sterile packaging in a stainless steel tube
which shields the iodine or palladium radiation. The suture maintains seed spaci
ng and minimizes seed migration. The column strength of the suture is sufficient
to easily penetrate bone wax and the monofilament suture is not adversely affec
ted by fluids. The excellent visibility of the InterSource seed is maintained.
Figure 42.2 The InterStrand comprises 10 InterSource seeds strung on a monofilamen
t suture. (Reproduced with permission from International Brachytherapy.)
InterStrand Special InterStrand Special is a customized InterStrand in which the se
ed spacing and number of seeds on a suture is customized to the treatment plan p
rescribed by the physician (Figure 42.3). This can reduce the number of needles
per patient and provide excellent coverage of the base and apex of the gland. In
terStrand Special reduces prep time.

Basic and advanced techniues in prostate brachytherapy

568
EZ-Pak Preloaded Needle Program The EZ-Pak Preloaded Needle Program includes: Inte
rStrand, InterStrand Special, or InterSource iodine or palladium seeds loaded into
needles or cartridges.
Figure 42.3 The InterStrand Special is a customized InterStrand. The seed spacing
and number of seeds is prescribed by the physician. (Reproduced with permission
from International Brachytherapy.)
Figure 42.4 The EZ-Pak Preloaded Needle Program. See text for details. (Reproduce
d with permission from International Brachytherapy.)
InterSource brachytherapy seeds
569
Radiograph to ensure accurate loading according to 10% source assay and certific
ation. the prescribed treatment plan. The Needle Program is packaged, sterilized
, and provided ready for use (Figure 42.4).
References
1. Chen Q, Blair H. Accurate and efficient detection of seed migration in prosta
te I-125 permanent implantation with a collimated gamma scintillation survey met
er [Abstract]. Med Phys 2002; 29:1356. 2. Chen Q, Blair H. Detection and reducti
on of seed migration in prostate I-125 permanent implantation [Abstract]. Med Ph
ys 2003; 30:1429. 3. Nath R, Anderson , uxton G, et al. Dosimetry of interstit
ial brachytherapy sources: Recommendations of the AAPM Radiation Therapy Committ
ee Task Group No. 43. Med Phys 1995; 22:209234. 4. Meigooni A, Sowards K, Soldano
M. Dosimetric characteristics of the InterSource103 palladium brachytherapy sou
rce. Med Phys 2000; 27:10931100. 5. Reniers B, Vynckier S, Scalliet P. Dosimetric
study of a new palladium seed. Appl Radiat Isot 2002; 57:805811. 6. Reniers B, V
ynckier S, Scalliet P. Dosimetric study of the new InterSource125 iodine seed. M
ed Phys 2001; 28:22852288. 7. Meigooni A, Yoe-Sein M, Al-Otoom A, Sowards K. Dete
rmination of the dosimetric characteristics of InterSource125 iodine brachythera
py source. Appl Radiat Isot 2002; 56:589 599.
43 The customized monofilament: a new approach to permanent prostate brachythera
py
Matthew Bouffard Introduction For the purpose of convenience, permanent prostate
seed implants were first conceived in the familiar 1 cm grid: a 4.5 mm seed fol
lowed by a 5.5 mm spacer (JC Blasko pers comm). This standard spacing became the
blueprint for all permanent implants, regardless of the specific conditions of
a given patient. This led to some undesirable results, including high doses to s
ensitive and healthy tissue. Advances in treatmentplanning software (TPS) allowe
d for special loadingneedles that were not bound by standard spacing. This aided in
reducing healthy tissue doses and preserving uality of life by permitting spar
ing of the urethra and rectal wall, as well as the penile bulb and nerve bundles
.1,2 However, the special loading needles still fit into the standard spacing fo
rmat, as the larger spacing was planned in 5 mm increments, preserving the 1 cm
grid. This attempted preservation of the 1 cm grid in the z-axis creates an inhe
rent error in all special loading needles. Since standard 5.5 mm spacers are use
d in succession to create the special loads, all spacers after the first are 0.5
mm too long, as they do not directly follow a 4.5 mm seed and there is no need
for the extra length. This results in a 10% error in the length of the spacing i
n all special loads as well as an error in the placement of the second source (T
able 43.1). In general, if d is the intended sourcetosource distance in mm, the
error in the placement of the second source is given by: (0.11)100% For example, a

special loa calling for a 3.0 cm space from the center of one source to the ce
nter of the next woul contain five consecutive spacers. While the extra 0.5 mm
on the first woul compensate for the ajacent 4.5 mm see, the final four space
rs woul each be 0.5 mm too long. There is no nee for the extra material here,
resulting in 2.0 mm of extra space an a 6.67% error in the placement
Table 43.1 Percent error between intene source to source istance (.) an act
ual source to source istance (a) associate with special loas
i (cm) a(cm) Error (%)
1.50 2.00 2.50 3.00 1.55 2.10 2.65 3.20 3.33 5.00 6.00 6.67
The customize monofilament
3.50 4.00 4.50 5.00 5.50 6.00 3.75 4.30 4.85 5.40 5.95 6.50 7.14 7.50 7.78 8.00
8.19 8.33
571
of the secon source an all successive sources, aversely affecting osimetric
outcomes.3 By the same token, an inherent error is present for special loas con
taining consecutive sees. With 4.5 mm sees place consecutively, there is no 0
.5 mm space between sees to preserve the 1 cm gri. Thus, the secon see is i
splace by 0.5 mm, an error of 10% between the intene an actual sourcetosou
rce istance. Any aitional consecutive see will be isplace by a further 0.5
mm an have the same 10% error. Again, this eviation causes misplacement an a
reuction in osimetric quality an valiity. As prostate brachytherapy progres
se, strans were introuce to allow extracapsular implantation with a reuce
risk of see migration.4,5 This new technology improve implant quality an osi
metric results, but it was not without limitations.6 Early strans suffere from
the accorion effect, causing an increase in neele jamming.7 Furthermore, these
strans were also restricte in the spacing they were capable of having, being 
esigne for stanar spacing. Lastly, these strans were generally only suitable
for use in the periphery of the glan. Description an application Recently, th
e custom monofilament, a new type of stran, was introuce: the ReaiStran. Th
is stran, ue to its inherent esign characteristics, oes not suffer from the a
ccorion effect an can be constructe for any loaing pattern, stanar or not (
Figure 43.1). It has the ability to have any number of sources place consecutiv
ely, or any length of spacing between sources, thereby eliminating the inherent
error of special loas. With these capabilities, the custom monofilament can be
use for the entire implant, not just for extracapsular extension, possibly reu
cing the risk of migration an isplacement for the entire implant. The customiz
e monofilaments ability to be constructe to any osimetric pattern raises furth
er possibilities. Infinite spacing in a reproucible prouct woul offer an ai
tional imension for planning purposes (JC Blasko pers comm). Newer versions of
some treatment planning software offer inverse optimization. This feature allows
for an optimize, customize implant to be planne accoring to what best suits
a patients nees without the restriction of the 1 cm gri, proviing the opportu
nity for an implant to be fitte to the patient, rather than fitting the patient
to a template. The evolution of the permanent see implantation process has ha
a positive effect on the overall quality of the osimetric results. In the earl
y stages of the acceptance of permanent see implants, where all neeles contain
e sources with stanar spacing, the aim was simply to kill the cancer. This ge
nerally resulte in an acceptable V100 an D90,
Basic an avance techniques in prostate brachytherapy
572
but it came at the cost of an unreasonably high V150, as well as troublesome os
es to the urethra, blaer, an rectal wall. The avancement of TPS aresse th
is problem. The V150 an the oses to the urethra, blaer, an rectal wall were

lowere via special loaing. This, however, resulte in a rop in the V100 an
D90 values. The loss was acceptable, as the focus ha shifte from killing the c
ancer to treating the cancer while maintaining quality of life. The evelopment
of strans for extracapsular extension further reuce oses that were once unre
asonably high, with little or no etriment to the V100 or D90. The seemingly uni
que characteristics of the customize monofilament coul be the next avancement
in this process. The infinite osimetric variability may allow further progress
in the reuction of sensitive an healthy tissue osage. Hot spots or col spots ca
n be create as necessitate by the patients conition. Tissueahering propertie
s purporte by the monofilament may lower the risk of migration an increase the
accuracy of see placement. These qualities may lea to a reuction in the numb
er of sees neee to ensure proper coverage. The reuce risk of migration will
allow for more extracapsular sources to be place, maintaining proper ose cove
rage while placing fewer interior sources, further reucing sensitive tissue os
age an better preserving quality of life.8,9
The customize monofilament
573
Figure 43.1 Magnifie photograph of the custom monofilament, exhibiting both sta
nar an nonstanar spacing lengths.
Basic an avance techniques in prostate brachytherapy
574
Figure 43.2 Postoperative xray images of two implants performe by the same ph
ysician. The implant to the left was performe with loose loas. The implant to
the right was one with the custom monofilament an exhibits increase linearity
an reuce bunching.
Aitional benefits reater visibility The customize monofilament seems to offe
r other application avantages. By keeping the sources linearly aligne an orth
ogonal to the ultrasoun slices, the sources shoul appear with greater visibili
ty uring postoperative analysis (Figure 43.2). Furthermore, since some later TP
S versions have the ability to perform osimetry by treating the sees as linear
sources rather than point sources, maintaining the linear alignment of the sour
ces woul increase the ability to effectively valiate the osimetric results gi
ven by the postoperative analysis. When linearity is maintaine, the ose clous
are aligne as prescribe by the treatment planning software, an the postimpla
nt osimetric calculations may more accurately reflect the actual values. This i
s especially beneficial when consiering sensitive tissue oses, as linear osim
etric evaluations have shown sensitive tissue oses to be up to 35% higher than
point source evaluation (WM Butler pers comm). Aitionally, by eliminating the
inherent error of special loas with loose sources an fixe length spacers, the
customize monofilament shoul further valiate the osimetric results. None of
these attributes shoul reuce the quality of the V100 an D90. On the contrary
, the preservation of linearity an the apparent reuce risk of migration an 
isplacement shoul actually improve the V100 an D90 values when customize mono
filaments are use (Figure 43.3). Further, linear osimetry shoul allow for mor
e
The customize monofilament
575
careful monitoring of sensitive tissue ose, helping preserve postimplant qualit
y of life. These characteristics shoul allow for a smarter implant with thoughto
ut ose planning, specifically targeting problem areas while inten tionally prot
ecting sensitive an healthy tissue, hopefully improving overall implant quality

an maximizing preservation of quality of life. Preloae strans The recent av

ailability of strans that are preloae an elivere in sterile surgical kits
coul also offer practical avantages. The customize monofilament comes preassa
ye an sterilize in preloae, raiographverifie, preplugge neeles. This e
liminates valuable onsite personnel hours spent loaing neeles, calibrating, a
n sterilizing sources, an shoul reuce the raiation exposure receive by the
staff.10,11 The customize monofilament is elivere in neeles that can assist
in improving implant quality. The monofilament neele is esigne to cause mini
mal trauma. Unlike other neeles, which bore into the perineum, the monofilaments
neele is esigne to prouce a flap when penetrating the perineum. The resulti
ng flap shoul reuce eema an allow the boy to heal more rapily; improving p
atient recovery time. The neeles are preplugge with a synthetic 1.5 mm, bioabs
orbable plug, which unlike other plug materials, oes not jam or lubricate the c
ustom monofilament upon expulsion, an allows for precise location of the first
source placement. The combination of the custom monofilament an its accompanyin
g neele eliminates the accorion effect that plagues other strane source prouc
ts. The synthetic plug offers an aitional benefit. The use of bone wax by oct
ors in the clinical setting to plug the tip of seeing neeles has been commonpl
ace. Each neele
Basic an avance techniques in prostate brachytherapy
576
Figure 43.3 Postoperative analysis of a custom monofilament case. Ultrasoun e
termine the target value to be 93.1 cm3. 276 sees were implante, resulting in
a V100 of 98.17%, a D90 of 111.41%, an D10 Urethra of 107.85%. (Images courtes
y of Dr Donal B Fuller MD.)
contains approximately 45 mm3 of bone wax at its tip resulting in the introuctio
n of 100300 mm3 of nonabsorbable bone wax into the prostate glan in a typical im
plant of 2060 neeles. While bone wax is approve for use as a hemostatic agent t
o control bleeing in orthopeic surgery, stuies have shown the use or presence
of bone wax in soft tissue can lea to the growth of sarcomal cancers.12 Conver
sely, synthetic materials have a long history of use in soft tissue. These synth
etic materials are completely absorbe into the boy via enzyme reaction. As the
growth of this practice increases, care
The customize monofilament
577
shoul be given to the selection of materials an instrumentation most suite fo
r the intene use to protect patient outcome an quality of life. Unlike other
strane source proucts, the customize monofilament oes not have to start an
en with a see. Any amount of spacing material can be ae to either en of t
he monofilament. Not only oes this allow for continue use of trailing spacers to
those accustome to using them in loose loas, but it allows for baseline loai
ng as well. In baseline loaing, a monofilament that calls for its first source
in the nonbaseline plane is constructe with the appropriate amount of spacing
material at its tip, allowing the neele to be inserte to the baseline an expe
lle at that epth, eliminating the nee for the manual retraction of such neel
es. When using this metho, there is no empty neele track before the first sour
ce, possibly reucing the chances for migration or isplacement. Furthermore, by
eliminating the process of manually retracting the neeles, the time an motion
for the proceure is reuce to approximately one minute per neele (P Sanchez,
pers comm). This technique results in reuctions in operating room time as well
as allowing for shorter anesthesia seation for the patient, improving throughp
ut for the facility, an creating the opportunity for quicker patient recovery.
Conclusions The customize monofilament may have much to offer to permanent pros
tate brachytherapy, assisting those practitioners who are entering the fiel of

this practice an minimizing the learning curve of becoming proficient in see i
mplantation. It allows for customize spacing an special loaing without being
constraine by 5.5 mm spacing increments, an eliminates the inherent error asso
ciate with special loas. It oes not suffer from the accorion effect, introuce
s no bone wax to the tissue, an can reuce the alreay low risk of migration an
 isplacement associate with strane sources. The customize monofilament may
be the next step in osimetric evolution, increasing the sparing of healthy an
sensitive tissue while potentially improving implant quality an osimetric out
comes. Furthermore, its linearity simplifies postimplant analysis, makes it iea
l for linear osimetry evaluations, an can help valiate osimetric outcomes wh
ile ensuring acceptable oses to sensitive an healthy tissue. The customize mo
nofilament is also beneficial from a practical stanpoint, coming preloae, the
reby saving time an possibly reucing exposure, an offering the capability for
baseline loaing, reucing time in motion for the proceure. As the fiel of pe
rmanent prostate brachytherapy continues to evolve, the customize monofilament
can only become more avantageous. As avancements continue in treatment plannin
g software, patientspecific, optimize, customize implants can be prouce; in
which each neele will contain a special loa, customconfigure to best suit a
given patients conitions. As prostate brachytherapy continues to vie with exter
nal beam raiotherapy (EBRT) an raical prostatectomy (RP) as the best means of
treating prostate cancer while maintaining quality of life, it is innovations l
ike the customize monofilament that will allow permanent prostate brachytherapy
to continue to be a safe an effective choice for patients.
Basic an avance techniques in prostate brachytherapy
578
References
1. Butler WM, Merrick S, Lief JH, Dorsey AT. Comparison of see loaing approac
hes in prostate brachytherapy. Me Phys 2000; 27(2):381392. 2. Merrick S, Butler
WM, Wallner KE, et al. The importance of raiation oses to the penile bulb vs.
crura in the evelopment of postbrachytherapy erectile ysfunction. Int J Raia
t Oncol Biol Phys 2002; 54(4):10551062. 3. Roberson PL, Narayana V, McShan DL, et
al. Source placement error for permanent implant of the prostate. Me Phys 1997
; 24(2):251257. 4. Merrick S, Butler WM, Dorsey AT, et al. See fixity in the pr
ostate/periprostatic region following brachytherapy. Int J Raiat Biol Phys 2000
; 46(1):215220. 5. Tapen EM, Blasko JC, rimm PD, et al. Reuction of raioactive
see embolization to the lung following prostate brachytherapy. Int J Raiat On
col Biol Phys 1998; 42(5):10631067. 6. Lee WR, euzman AF, Tomlinson SK, McCullo
ugh DL. Raioactive sources embee in suture are associate with improve post
implant osimetry in men treate with prostate brachytherapy. Raiother Oncol 20
02; 65:123127. 7. Frielan JL, Feygelman V, Haller EM, et al. Problems with rigi
 see stran loging uring prostate implantation: a propose mechanism an sol
ution. Me Dosim 1997; 22(1):1721. 8. Narayana V, Roberson PL, Winfiel RJ, et al
. Optimal placement of raioisotopes for permanent prostate implants. Raiology
1996; 199(2):457460. 9. Brown D, Colonias A, Miller R, et al. Urinary morbiity w
ith a moifie peripheral loaing technique of transperineal 125I prostate impla
ntation. Int J Raiat Oncol Biol Phys 2000; 47(2):353360. 10. Butler WM, Dorsey A
T, Nelson KR, Merrick S. Quality assurance calibration of 125I rapi stran in
a sterile environment. Int J Raiat Oncol Biol Phys 1998; 41(1):217222. 11. Bice
WS Jr, Walker ES, earty S, et al. A comparative evaluation of loaing times an
exposures for permanent prostate brachytherapy. J Appl Clin Me Phys 2002; 3(4)
:263272. 12. Morrison BA. Soft tissue sarcomas of the extremities. Baylor Univers
ity Meical Proceeings 2003; 16:285287.
Part VII Postimplant: analysis of postimplant osimetry
44 Salvage of suboptimal prostate see implantation: reimplantation of an uner
ose region of the prostate base

Lesley Hughes, Frank M Waterman, an Aam P Dicker Introuction Prostate cancer
is one of the most common male cancers in the Unite States. The scope of the i
sease is farreaching with estimates by the American Cancer Society for 2004 sho
wing prostate cancer comprising 33% of all cancers in males, surpassing lung can
cer incience.1 The number of patients iagnose with prostate cancer is increas
ing worlwie, through increase physician/patient awareness an screening effor
ts. Treatment options for the iniviual patient, epening on pretreatment char
acteristics, may inclue watchful waiting, prostate brachytherapy, external beam
raiotherapy, or raical prostatectomy. Transperineal interstitial permanent pr
ostate brachytherapy (TIPPB) for the treatment of prostate cancer has been incre
asingly utilize in the past ecae. Single institution ata have provie most
information for outcome ata an proceure technique. Prostate implant technolog
y is a complex proceure with respect to treatment planning an technical elive
ry of the raioactive sees. Prostate brachytherapy techniques are varie an co
ntinue to evolve with improvements in the ease an reliability of see elivery.
Institutions with a great eal of experience have evelope short courses for i
nstruction of clinicians in see implantation. Experts in brachytherapy generall
y accept that there is a learning curve for the prostate implant technique.2 Lee
an colleagues reporte improvement of osimetric elivery after 2530 cases one
by a single practitioner.3 Formal training programs in prostate brachytherapy, w
hether proctore or certifie, significantly shorten or eliminate the learning cu
rve for iniviual practitioners.4 Unfortunately suboptimal implants o occur but
currently there is little guiance in the literature as to how to eal with thi
s ifficulty. We will iscuss one approach to salvage of a suboptimally ose pr
ostate implant. Materials an methos The patient was a 67yearol male with a
Stage II, T1cN0MO, aenocarcinoma (American Joint Committee on Cancer; AJCC, 199
7) of the prostate with a leason score 6 (3+3) an prostatespecific antigen (P
SA) of 7.7 with an American Urological Association (AUA) urinary symptom score o
f 5, who chose prostate brachytherapy as his treatment ecision after full iscu
ssion of all treatment options available for his isease. His preimplant prostat
e volume was 36.7 cc an he i not receive hormonal therapy.
Basic an avance techniques in prostate brachytherapy
582
The ioine125 (125I) implant was preplanne to eliver a minimum ose of 150 y
to the prostate base on the recommenations of American Association of Physici
sts in Meicine (AAPM) T43 for calculating ose.5 The sees were peripherally
loae, which prouce an isoose istribution characterize by a broa ose min
imum in the central portion of the prostate encircle by a high ose region. The
implant was planne so that the 150 y isoose line was approximately 35 mm beyo
n the prostate. The implant require 21 neeles an 77 sees using a 125I see
strength of 0.54 mCi/see (National Institute of Stanars an Technology; NIST
99). The preplan V100 an D90 were 100% an 212 y, respectively. Compute tomog
raphy (CT) scans for postimplant osimetric evaluation were obtaine on the ay
of the implant an 35 ays later. These stuies were obtaine using the CT scann
er (PQ5000, Picker, Clevelan, OH) in our epartment. Axial images were obtaine
at 2.5 mm intervals using a 2 mm slice thickness. The same iniviual (FW) i
all the see localization an contouring. The urethra an rectum were contoure
beginning at the base of the prostate an extening to the last image that conta
ine sees, which in this case was 1.75 cm inferior to the apex. The postimplant
CT scan obtaine on the ay of the implant reveale that the seeing began 1.52
cm inferior to the actual base of the glan, which left the superior aspect of t
he glan significantly unerose. This also resulte in a large number of sees
being implante in the proximity of the apex an extening 1.5 cm inferior to t
he apex. The patient was informe of the suboptimal see placement immeiately a
n after a full iscussion of the options available, a reimplantation was plann
e. Planning the reimplantation presente several challenges. It is necessary t
o know the locations of the sees alreay implante an the ose istribution e

livere by these sees as a starting point for planning the reimplantation. Henc
e, the plan must be generate base on a postimplant imaging stuy in which all
of the sees can be localize. A transrectal ultrasoun (TRUS) volume stuy is n
ot suitable for this purpose because not all of the sees can be visualize. The
refore, the plan was base on the postimplant CT scan obtaine 35 ays after the
proceure. However, the use of a CT scan for planning presents another problem;
namely, the treatmentplanning system (Varisee, Varian, Palto Alto, CA) oes n
ot allow the plan to be generate from a CT scan. The treatmentplanning system
was evelope to utilize TRUS images on which the template hole pattern was alre
ay superimpose. Both the prostate contours an the template corners must be i
gitize into the computer. This problem was overcome by superimposing a gri on
the CT images an igitizing them into the treatment planning system as if they
were TRUS images. A 1 cm1 cm gri of points was first superimpose onto the CT im
ages using a tool of the ACQsim CT simulator (Philips Meical Systems, the Nethe
rlans). Har copies of the CT stuy were then generate for igitization into t
he computer. First, it was necessary to efine the corners of the template on th
e CT images. The template use to perform the implant measures 6 cm6 cm. Thus, a
6 cm6 cm template was rawn on the CT images base on the superimpose gri point
s. The exact alignment of the template relative to the prostate is unimportant a
t this stage of the planning process; therefore, the relative positions of the t
emplate an prostate were arbitrarily selecte base on the available gri point
s. The treatmentplanning software allows the alignment of the template an the
prostate contours to be moifie once the igitization process is
Salvage of suboptimal prostate see implantation
583
complete. Thus, once the igitization was complete, the template was move rel
ative to the prostate so that the prostate was centere left to right. The templ
ate was also move in the anteriorposterior irection so that the most posterio
r contour of the prostate was locate on the first row of template holes. It was
assume that the prostate coul be aligne with the template as planne at the
time of the implant or, if not, the plan coul be moifie by relabeling the row
s accoringly. Results Figure 44.1 shows the first nine images of the postimplan
t CT scan obtaine 35 ays after the initial implant. These images were acquire
at 2.5 mm intervals an show the first 2 cm of the glan, beginning at the base
. It is evient from these images that the first 1.5 cm of the glan is evoi o
f sees. Aequate seeing only appears about 2 cm from the base. Thus, one woul
expect the ose coverage to be suboptimal. The top half of Figure 44.2 is a sag
ittal plane through the central axis of the prostate base on the CT scan shown
in Figure 44.1 that graphically illustrates the unerose region of the glan.
The circles an triangles represent sees implante in this plane. As note abov
e, the seeing began 1.52 cm inferior to the base, which left the superior aspect
of the glan significantly unerose. It is also apparent that the sees an t
he ose istribution extene well beyon the apex as a result of the inferior s
hift in the see istribution. The secon implant was preplanne to boost the o
se to the superior portion of the glan. Figure 44.3 shows the osevolume histo
grams (DVH) of the first an secon implants. Note that V100 an D90 in the firs
t implant were only 46% an 49 y, respectively. The DVH inicates that the enti
re prostate, incluing the unerose region, woul receive a ose of at least 3
5 y from the original implant. Thus, the secon implant neee to eliver a max
imum ose of only 115 y to the unerose region. For this reason, the secon i
mplant was planne using a see strength of 0.42 mCi instea of 0.54 mCi, to sca
le own the total ose that woul be elivere by the secon implant. The use of
lower strength sees allowe us to maintain the same see spacing use in the i
nitial implant; however, it is not necessary to use lower strength sees. The se
con implant was performe 49 ays after the initial implant. A total of 53 ai
tional sees were implante using 16 neeles. Figure 44.4 shows the first nine i
mages of the CT scan obtaine 34 ays after the secon implant (an 84 ays afte

r the first implant). The sees ae uring the secon implant can be ientifie
 by comparison of Figures 44.1 an 44.4. The improvement in ose coverage is sh
own graphically in the lower half of Figure 44.2 an by the DVH in Figure 44.3.
The DVH, which is base on a postimplant CT scan shown in Figure 44.4, shows tha
t the reimplantation increase V100 an D90 to 98% an 201 y, respectively. Fi
gure 44.5 shows DVHs of the urethra following the first an secon implants. The
urethral ose was relatively high ue to the clustering of sees near the apex
an beyon. This is evience by the fact that 25% of the urethra receive a os
e 400 y from the first implant. In the combine implant, 25% of the urethra rece
ive a ose 500 y. The apparent increase in the urethral ose
Basic an avance techniques in prostate brachytherapy
584
Figure 44.1 Images from the compute tomographic (CT) scan obtaine 35 ays afte
r the first implant showing the suboptimal seeing at the base of the prostate.
largely reflects the fact that the superior 2 cm of the urethra (approximately o
ne thir of the contoure volume) receive very little ose from the first impla
nt. Thus, the DVH was shifte towar higher oses when the ose to this volume w
as increase by the secon implant. Figure 44.6 shows osesurface histograms (D
SH) of the surface of the rectum following the first an secon implants. The DS
H inicates that 10% of the rectal surface receive a ose equal to or greater t
han 150 y as a result of the first implant. This ose increase to about 200 y
for the combine implants. This increase reflects the fact that a large fractio
n of the rectal surface receive very little ose from the first implant. The pa
tient at one month followup for the first implant ha an AUA symptom score of 8
an was on no meication (alphablockers) for urinary problems. He was experien
cing some erectile ysfunction an was starte on silenafil citrate (Viagra). H
e continue to have erectile ysfunction with little response to silenafil citr
ate. His symptomatology remaine stable with respect to his urinary symptoms unt
il 6 months after the secon implant when his AUA score increase to 13. He was
trie on tamsulosin hyrochlorie (Flomax) with some relief of his symptomatolog
y. His PSA evaluation showe a steay ecline from 7.7 to 0.2 ng/mL with the lat
est followup 37 months
Salvage of suboptimal prostate see implantation
585
postimplant. Due to back pain, the patient was evaluate with a bone scan an pl
ain films correlate to show egenerative isease of the spine. Discussion Trans
perineal prostate brachytherapy has been increasingly utilize in the treatment
of prostate cancer. Due to the highly technical nature of the proceure there is
operator variation ue to the learning process or technical ifficulties. Fluor
oscopy uring the proceure may assist in proper neele placement an see eliv
ery. Localizing see placement in the base an apex of the glan may help to fac
ilitate see implantation.6 See fixity is also a problem with movement of sees
causing changes in the osimetry.7
Basic an avance techniques in prostate brachytherapy
586
Figure 44.2 Sagittal planes through the central axis of the prostate that show t
he isoose istribution an ose coverage following the first an secon implant
s.
Salvage of suboptimal prostate see implantation

587
Figure 44.3 Dosevolume histograms (DVH) that show the prostate osimetry follow
ing the first an secon implants.
Figure 44.4 Images from the compute tomography (CT) scan obtaine 34 ays after
the secon implant showing the reseeing of the unerose region.
Basic an avance techniques in prostate brachytherapy
588
Figure 44.5 Dosevolume histograms (DVH) that show the urethral osimetry follow
ing the first an secon implants.
Figure 44.6 Dosesurface histograms (DSH) that show the rectal osimetry followi
ng the first an secon implants.
Salvage of suboptimal prostate see implantation
589
Embee sees in suture an preloae neeles are associate with improve post
implant osimetry in a single institution stuy.8 The impact of source placement
errors epens on the see ensity. The misplacement of anterior sees in impla
nts with a lower anterior see ensity has been shown to have a greater impact o
n the postimplant osimetry than the misplacement of posterior sees. Neele iv
ergence of only small egrees (510 egrees) can cause up to 1020% reuction of min
imum target ose.10 Training of physicians, by proctoring or certification, in 
elivery of prostate brachytherapy shortens the learning curve an improves the q
uality of the osimetric plans.4 Formal training programs for new practitioners
woul be of benefit in prostate brachytherapy elivery. There is little guiance
in the literature on the salvage of suboptimal implants. Choices may inclue ex
ternal beam raiotherapy (EBRT), high ose rate implantation, intensity moulate
 raiation therapy (IMRT), raical prostatectomy, an reimplantation. The case
presente in this chapter escribes reimplantation as a salvage proceure. We
show that this is a feasible an tolerable proceure. References
1. Jemal A, Tiwari RC, Murray T, et al. Cancer Statistics, 2004. CA Cancer J Cli
n 2004; 54:829. 2. Nag S, Beyer D, Frielan J, et al. American Brachytherapy Soc
iety (ABS) recommenations for transperineal permanent brachytherapy of prostate
cancer. Int J Raiat Oncol Biol Phys 1999; 44:789799. 3. Lee WR, euzman AF, Ba
re RL, et al. Post implant analysis of transperineal interstitial permanent pros
tate brachytherapy: evience for a learning curve in the first year at a single
institution. Int J Raiat Oncol Biol Phys 2000; 46:8388. 4. Presser J, Stone NN,
Chircus JH, et al. Multicenter experience with prostate brachytherapy training [
Abstract]. Int J Raiat Oncol Biol Phys 2001; 51(suppl 1):199. 5. Nath R, Aners
on LL, Luxton , et al. Dosimetry of interstitial brachytherapy sources: Recomme
nations of the AAPM Raiation Therapy Committee Task roup No. 43. Me Phys 199
5; 22:209233. 6. Bair MC, Holt RW, Selby TL. Improvement of transperineal implan
t osimetry by intraoperative cystoscopic confirmation of prostate anatomy. J Ur
ol 2000; 164:406410. 7. Merrick S, Butler WM, Dorsey AT, et al. See fixity in t
he prostate/ periprostatic region following brachytherapy. Int J Raiat Oncol Bi
ol Phys 2000; 46:215220. 8. Lee WR, euzman AF, Tomlinson SK, McCullough DL. Ra
ioactive sources embee in suture are associate with improve post implant o
simetry in men treate with prostate brachytherapy. Raiother Oncol 2002; 65:1231
27. 9. Sihu S, Morris WJ, Spainger I, et al. Prostate brachytherapy post impla
nt osimetry: a comparison of prostate quarants. Int J Raiat Oncol Biol Phys 2
002; 52:544552. 10. Nath S, Chen Z, Yue N, et al. Dosimetric effects of neele i
vergence in prostate see implant using 125I an 103P raioactive sees. Me Ph
ys 2000; 27:10581066.

45 Can prostate brachytherapy treat potential extraprostatic isease?

Ashish Patel, Frank M Waterman, an Aam P Dicker Introuction Extraprostatic ex
tension (EPE) is an unfavorable prognostic factor for prostate cancer patients w
ith clinically organconfine isease. rowing evience suggests that EPE may exi
st in a significant fraction of favorable risk clinical stage T1T2 patients, with
numbers reporte between 10% an 50%.1,2 The extent of EPE has been the topic o
f several recent stuies, with potential implications for prostate brachytherapy
.35 The goal of brachytherapy is to eliver a uniform ose with an aequate margi
n for isease eraication. However, the steep ose graient at the periphery of
the prostate (up to 20 y/mm) brings into question the ose actually being eliv
ere to potential EPE.6 As a result, the American Brachytherapy Society (ABS) cu
rrently recommens that patients with a high risk for EPE be treate with extern
al beam raiotherapy (EBRT) with brachytherapy use as a boost.7 The raial exte
nt of EPE has been the subject of several recent stuies examining hematoxyline
osin (H&E) staine postprostatectomy specimens.35 Davis et al from the Mayo Clin
ic, reporte that EPE was present in 28% of the specimens, with a meian raial
istance of 0.5 mm.4 A subset analysis of 107 patients, who meet the criteria fo
r brachytherapy (PSA <10, leason <7, glan size <60 cc), showe EPE present in
11 cases, with a mean an maximum raial extent of 0.03 mm an 0.6 mm, respectiv
ely. Similar finings were escribe by Sohaya et al. This series foun EPE to
be within 3.3 mm in 90% of favorable risk cases (clinical stage T1T2, PSA 10 ng/mL
, leason 6).5 Both of these stuies suggest that a 35 mm treatment margin from th
e prostatic capsule woul encompass 95100% of EPE in favorable risk patients. Tre
atment Investigators from Thomas Jefferson University have recently reporte on
the ability to consistently eliver the full prescription ose 35 mm outsie the
prostatic capsule using both ioine125 (125I) an pallaium103 (103P) permane
nt prostate see implants.8,9 In the report by Butzbach et al,8 22 favorable ris
k patients, who receive 103P sees as either monotherapy (5 patients) or as a
boost following external beam raiation therapy (17 patients) were evaluate. Th
e implants were prescribe to 120 y for monotherapy an 7080 y for boost, an w
ere planne with a margin of 35 mm from the ege of the prostate. All implants we
re conucte using a peripheral loaing technique, with sees averaging 1.5 mCi/
see in strength (Figure 45.1). All patients in this stuy unerwent
Can prostate brachytherapy treat potential
591
both pre an postimplant compute tomography (CT) scans, with contouring conuc
te by a single iniviual (FW). The postimplant CT was obtaine, on average, 36
ays after the proceure. The raial istance between the prescription isoose
line an the ege of the prostate contour was measure in five locations aroun
the posterior aspect of the prostate for each transverse image (Figure 45.2). Th
ese locations were chosen base on previous reports emonstrating EPE to be most
prevalent in the posterior half of the prostate glan.1012 Results A summary of
the Butzbach et al results is shown in Table 45.1. The average istance range f
rom 4.0 mm to 6.3 mm for the five efine positions aroun the prostate. The 6 oc
lock position receive the smallest coverage, averaging 4.0 mm, which was an eff
ort to minimize rectal toxicity. When the average istance in each location arou
n the prostate was stratifie to the apex, miglan, an base, the smallest ma
rgins were locate at the base an 6 oclock position. Base on these results, pro
state brachytherapy using 103P is capable of elivering a prescription ose 35 m
m beyon the glan using a peripheral loaing technique. However, the margins i
 vary throughout the glan, ranging between 0 mm to 8 mm, which suggests
Basic an avance techniques in prostate brachytherapy
592

Figure 45.1 Treatment planning: 103P. (a) Planne prescription isoose line (12
0 y outermost line). The plan use a 35 mm margin outsie the prostatic glan (c
rosshatch). The implant was performe with either a 1.5 or 2.0 mCi see. Represe
ntative pretreatment plans with both strengths are shown using the same 40 cc pr
ostate, illustrating that the intene coverage is virtually ientical. Note tha
t fewer
Can prostate brachytherapy treat potential
593
sees are require when using a higher see strength, (b) Transverse compute to
mography (CT) image of prostate after prostate brachytherapy. Note the periphera
l istribution of sees.
that implant quality is operatorepenent with excessive source placement error
reucing or even eliminating margin over portions of the glan.13 A similar rep
ort by Patel et al stuie 60 consecutive favorable risk patients, who receive
125I prostate implants, to etermine whether implants alreay planne with a 5 m
m margin for source placement error were also capable of treating extraprostatic
extension.9 In contrast to the Butzbach et al report,8 all 60 patients receive
125I prostate implants as monotherapy. The prescribe ose in this series was 1
45 y. Similar techniques as escribe in the Butzbach et al stuy were employe
to contour the prostate an implant the sees. Previous stuies inicate that p
lanning a 35 mm margin outsie the prostatic cap
Figure 45.2 Raial istance measurements in the posterior aspect of the prostate
. Table 45.1 Mean preplan an postimplant extraprostatic margins (stanar eviat
ion) (mm) at the base, miglan, an apex of the prostate
Position lan Base Miglan Apex
4.90.4 6.80.6 5.90.5 6.30.6 4.50.4 4.90.5 5.40.5 6.90.7 6.30.4 8.40.5 Left lateral
.10.4 Left posterolateral 5.2 0.4 3.40.5 Posterior 4.00.4 2.705 Right posterolateral
5.40.4 4.00.5 Right lateral 6.30.3 4.00.5
Basic an avance techniques in prostate brachytherapy
594
sule allows for compensation for source placement errors, therefore a 5 mm margi
n was planne to allow for these errors that are inherent to prostate brachyther
apy an not to treat EPE.14,15 As in the previous stuy, the raial istance bet
ween the prescription isoose line an the ege of the prostate contour was meas
ure in five locations aroun the posterior aspect of the prostate for each tran
sverse image (Figure 45.2). A summary of the Patel et al results is shown in Tab
le 45.2. The mean postimplant margins range from 2.9 mm to 9.4 mm, with overall
mean postimplant margins at the base, miglan, an apex of 3.8, 5.0, an 7.2
mm, respectively. When pre an postimplant margins were compare, a statistical
ly significant ifference was foun at the base (p=0.00003), but not at the apex
(p= 0.6960) or miglan (p=0.31274). Therefore, the planne margins at the mi
glan an apex were not neee to compensate for source placement errors in the
se regions. However, this was not true at the base, where the plans were not exe
cute as well as in the miglan an apex. Of the measurements, 38% at the base
were <3 mm versus 15% an 12% at the miglan an apex, respec tively, which w
as approximately the same as planne. When an implant is planne with a ose mar
gin that is intene to compensate for source placement error, any margin availa
ble to treat EPE epens on the egree of source placement error. Therefore, EPE
can only be treate after compensating for source placement error. A thir stu
y investigating the extraprostatic ose istribution with permanent prostate bra
chytherapy was recently reporte by investigators from the Schiffler Cancer Cent
er.16 In this stuy, Merrick an associates examine 26 patients with low risk p
rostate carcinoma enrolle in a prospective, ranomize phase III trial comparin

g 103P an 125I sources. Similar to the stuies from Thomas Jefferson Universit
y, a 5 mm margin was planne aroun the prostate; however, a ifferent implantat
ion technique was utilize with the placement of aitional sees at the base of
the seminal vesicles an periprostatic region. These extraprostatic implants ac
counte for approximately 40% of the total sees place uring the proceure. Th
e prescribe ose was 145 y for 125I an 115 y for 103P. None of the patients
in this series receive aitional EBRT; however, 11 patients i receive 34 mon
ths of neoajuvant
Table 45.2 Mean preplan an postimplant extraprostatic margins ( stanar eviati
on) (mm) at the base, miglan, an apex of the prostate
Position
Left lateral Left posterolateral Posterior Right posterolateral Right lateral

Base Miglan Apex Preplan Postimplant Preplan Postimplant Preplan Psotimplant

5.52.7 6.33.4 4.62.3 5.73.4 5.32.4 3.73.0 4.04.0 2.94.1 3.54.7 4.63.0 5.82.9 5.92
22.4 5.72.3 5.42.5 5.52.8 .5.12.5 5.33.0 6.52.7 9.44.5 6.62.7 4.12.2 6.02.6 9.03.
.2 4.93.7 6.94.7 9.03.5
hormonal therapy
mplant osimetry
n ay 0, an the
e capsule, 100%,
.

seconary to urinary obstruction or unfavorable geometry. Posti

was base on CT scans obtaine within 2 hours of implantation o
raial istance was measure from the center of the glan to th
90%, an 75% isoose lines along 72 raial lines at 5 increments

Can prostate brachytherapy treat potential

595
A comparison of 125I an 103P osimetric characteristics yiele no clinically
significant ifferences between the two isotopes. The greatest margins were foun
 at lateral an posterolateral positions at the base an apex of the glan. Neg
ative margins were foun at the anterior of the base in both isotopes, which cor
respons to the location of the blaer neck, a structure that was intentionally
unerose. A composite polar plot emonstrating the mean sagittal prostate im
ensions an osimetric margins is presente in Figure 45.3. The overall mean 100
% isoose
Figure 45.3 Prescription isoose line measurements. Representative crosssection
of miprostate etailing location of measurements taken along the periphery of
the glan.
margin was 6.5 mm  1.8 mm. No statistically significant ifferences were observe
between the two isotopes at the 100% isoose margin; however, the margins for 1
03P were significantly smaller at the 90% an 75% isoose levels. This isotopic
ifference at lower isooses is expecte to be seconary to the sharp ose fall
off characteristic of 103 P. No statistically significant correlations were fo
un between any osimetric margin an factors, such as age, isease classificati
on parameters, number of sees, see strength, or volumetric parameters.
Basic an avance techniques in prostate brachytherapy
596
Conclusions These three stuies confirm that prostate brachytherapy is capable o
f elivering a prescription ose margin 3 mm beyon the prostatic capsule; howev
er, it is important to note that none of these stuies attempt to aress the bi
ologic necessity or appropriate ose to treat EPE. Due to variability in implant
quality an its operator epenence, it is not recommene to treat patients at
high risk for EPE with implants alone. The role of prostate brachytherapy alone
in intermeiaterisk patients will be aresse in the recently activate Raia

tion Therapy Oncology roup clinical trial, RTO 0232, which ranomizes patients
to two arms, EBRT with brachytherapy boost or brachytherapy alone. References
1. Epstein JI, Partin AW, Sauvageot J, Walsh PC. Preiction of progression follo
wing raical prostatectomy. A multivariate analysis of 721 men with longterm fo
llowup. Am J Surg Pathol 1996; 20(3):286292. 2. Partin AW, Kattan MW, Subong EN,
et al. Combination of prostatespecific antigen, clinical stage, an leason sco
re to preict pathological stage of localize prostate cancer. A multiinstitutio
nal upate. JAMA 1997; 277(18):14451451. 3. Davis BJ, Pisansky TM, Wilson TM, et
al. The raial istance of extraprostatic extension of prostate carcinoma: impli
cations for prostate brachytherapy. Cancer 1999; 85(12):26302637. 4. Davis BJ, Ha
ock M, Wilson TM, et al. Treatment of extraprostatic cancer in clinically org
anconfine prostate cancer by permanent interstitial brachytherapy: is extrapro
static see placement necessary? Tech Urol 2000; 6(2):7077. 5. Sohaya C, Kupelia
n PA, Levin HS, Klein EA. Extent of extracapsular extension in localize prostat
e cancer. Urology 2000; 55(3):382386. 6. Dawson JE, Wu T, Roy T, et al. Dose effe
cts of see placement eviations from preplanne positions in ultrasoun guie
prostate implants. Raiother Oncol 1994; 32:268270. 7. Nag S, Beyer D, Frielan
J, et al. American Brachytherapy Society (ABS) recommenations for transperineal
permanent brachytherapy of prostate cancer [Review] [132 refs]. Int J Raiat On
col Biol Phys 1999; 44(4):789799. 8. Butzbach D, Waterman FM, Dicker AP. Can extr
aprostatic extension be treate by prostate brachytherapy? An analysis base on
postimplant osimetry. Int J Raiat Oncol Biol Phys 2001; 51(5):11961199. 9. A e
taile examination of the ifference between planne an treate margins in 125I
permanent prostate brachytherapy. Brachytherapy 2003; 2:223228. 10. Blasko JC, W
allner KE, Cavanagh W. Raiotherapeutic strategies in the management of clinical
ly localize, lowrisk prostate cancer: selection, results, an the search for ans
wers. Cancer J Sci Am 1998; 4(3):157158. 11. Catalona WJ, Dresner SM. Nervespari
ng raical prostatectomy: extraprostatic tumor extension an preservation of ere
ctile function. J Urol 1985; 134(6):11491151. 12. Catalona WJ, Bigg SW. Nervespa
ring raical prostatectomy: evaluation of results after 250 patients. J Urol 199
0; 143(3):538543.
Can prostate brachytherapy treat potential
597
13. Waterman FM, Yue N, Corn BW, Dicker AP. Eema associate with I125 or P10
3 prostate brachytherapy an its impact on postimplant osimetry: an analysis ba
se on serial CT acquisition. Int J Raiat Oncol Biol Phys 1998; 41(5):10691077.
14. Yu Y, Waterman FM, Suntharalingam N, Schulsinger A. Limitations of the minim
um peripheral ose as a parameter for ose specification in permanent 125I prost
ate implants. Int J Raiat Oncol Biol Phys 1996; 34(3):717725. 15. Roberson PL, N
arayana V, McShan DL, et al. Source placement error for permanent implant of the
prostate. Me Phys 1997; 24(2):251257. 16. Merrick S, Butler WM, Wallner KE, et
al. Extracapsular raiation ose istribution after permanent prostate brachyth
erapy. Am J Clin Oncol 2003; 25(5):e178e189.
Part VIII Quality of life an posttreatment sequelae after prostate brachytherap
y
46 Healthrelate quality of life following prostate brachytherapy
W Robert Lee, Deborah WatkinsBruner Prostate brachytherapy (PB) in the treatmen
t of clinically localize prostate cancer is increasing ramatically worlwie.
This resurgence in popularity is the result of improve technology an newer tec
hniques that allow for an outpatient proceure. PB offers the potential avantag
e of convenience an ecrease morbiity compare to raical prostatectomy (RP).
There is also a wiesprea perception that PB is associate with a better quali
ty of life compare with other treatments. Healthrelate quality of life (HRQOL
) encompasses a wie range of human experience. Contemporary efinitions of HRQO
L are base on the Worl Health Organizations categorization of health as a state

of complete physical, mental, an social wellbeing an not merely the absence of
isease.1 It is important to emphasize that HRQOL, in general, involves the perc
eptions of health an ability to function as reporte by the patient involve. H
RQOL research applies the principles of psychometric test theory, a iscipline w
ith which many clinicians are unfamiliar.2 Data are collecte with HRQOL questio
nnaires (instruments) which, when possible, shoul be complete by the patients
themselves. These instruments generally consist of a number of questions (items)
that are organize into scales. Each scale is esigne to measure a ifferent a
spect (omain) of HRQOL. HRQOL instruments may contain several ozen items are
ssing a number of omains. HRQOL instruments are generally categorize as generi
c or iseasespecific. eneric HRQOL instruments measure overall wellbeing an
at the very least shoul aress the level of functioning in the physical, emoti
onal an social omains. Examples of generic HRQOL instruments inclue the RAND
Meical Outcomes Stuy 36item Health Survey (SF36),3 an the Sickness Impact P
rofile (SIP).4 Diseasespecific HRQOL instruments focus on those omains that ma
y be more clinically relevant for the population stuie. Diseasespecific HRQOL
tools have been evelope for patients with cancer, AIDS, an many other coniti
ons. A number of reliable, vali instruments to measure prostate cancer specific
HRQOL in men with early stage prostate cancer are available. Examples inclue t
he Functional Assessment of Cancer TherapyProstate (FACTP),5 the University of
California, Los AngelesProstate Cancer Inex (UCLAPCI),6 the Prostate Cancer T
reatment Outcome Questionnaire (PCTOQ),7 the Expane Prostate cancer Inex Com
posite (EPIC),8 an the prostate cancer moule of the EORTC QLCC30.9 oo HRQOL
research requires instruments that have been teste an foun to be reliable an
 vali. This review will be confine to those stuies using reliable, vali ins
truments in men treate with PB. Stuies will be classifie accoring to the typ
e of brachytherapy (low ose rate or permanent vs high ose rate or temporary).
Healthrelate quality of life
601
HRQOL following low ose rate prostate brachytherapy In one of the first analyse
s of HRQOL in men treate with PB, investigators at the University of California
, Los Angeles compare generic an iseasespecific HRQOL in men unergoing brac
hytherapy for early stage prostate cancer to those unergoing raical prostatect
omy an agematche health controls.10 The stuy incorporate the SF36 an the
UCLAPCI questionnaires in a crosssectional esign. The men with prostate cance
r complete the various questionnaires 317 (meian: 7.5) months following treatme
nt. The PB group inclue 48 men treate with PB14 receive external beam raioth
erapy (EBRT) in aition to PB an 34 i not, an the RP group inclue 74 men.
There were 134 men in the control group. Compare to men in the RP group, men t
reate with PB were oler, less healthy, an ha less formal eucation. eneric
HRQOL i not iffer greatly among the three groups. In fact, the only generic H
RQOL omain in the SF36 that iffere was physical function, with RP patients sc
oring higher than the PB or control groups. Disease specific HRQOL measures were
very ifferent between the groups. Urinary function (leakage) was worse in the
PB group than in controls but better than in the RP group. The PB group ha more
irritative urinary symptoms an worse bowel function than controls. Sexual func
tion an bother were worse in the PB group than controls, but no ifferent from
the RP group. Physical function, urinary function, an bother an the American U
rological Association (AUA) symptom inex scores improve with time after brachy
therapy. The authors also ivie the PB group accoring to whether EBRT was als
o given an foun that men who receive both EBRT an PB score worse in all is
easespecific HRQOL omains compare to those men treate with PB alone. Investi
gators from the University of Virginia have reporte a crosssectional analysis
of 242 men with clinically localize prostate cancer.11 In this report, 138 men
(57% response rate) complete an returne maile questionnaires incluing the F
ACT, International Prostate Sympton Score (IPSS), an the Brief Sexual Functio
n Inventory. Of these men, 27 ha been treate with RP, 70 receive PB combine

with 89 months of anrogen eprivation therapy (ADT), an 41 ha been treate wit
h a combination of PB, EBRT, an 89 months of ADT. When the ageajuste FACT s
cores were compare between all three groups, the scores in the RP an PB groups
were similar but the scores of men that ha receive EBRT in aition to PB wer
e consierably lower (inicating ecrease HRQOL). This paper is ifficult to in
terpret as ADT was given in all men treate with PB. Recent reports inicate tha
t even shortterm ADT combine with PB can have significant effects on sexual fu
nction (see below). Davis an colleagues from Eastern Virginia Meical School ha
ve reporte on a HRQOL in men treate with PB alone, EBRT, an RP for clinically
localize prostate cancer.12 These investigators maile the SF36, the UCLAPCI
an the IPSS questionnaire to more than 600 men following efinitive treatment.
The response rate was greater than 80% in all groups. Important emographic if
ferences between the treatment groups were ientifie. Men treate with RP tene
 to be younger, healthier, an less likely to receive neoajuvant anrogen ep
rivation (NAAD) therapy compare to men treate with PB. The meian time from tr
eatment to survey completion was
Basic an avance techniques in prostate brachytherapy
602
longer in the men treate with RP compare to the men treate with PB (37.9 mont
hs vs 22.4 months, p>0.05). The raw an ajuste SF36 scores were not significa
ntly ifferent between men treate with RP an those treate with PB. Examinatio
n of the urinary, bowel an sexual omains of the PCI inicate that men treate
with PB or EBRT ha better sexual an urinary function than men treate with RP
. Men treate with PB also reporte less sexual bother than men treate with RP.
Bacon et al have analyze a subset of men being followe in the Health Professi
onals Followup Stuy.13 The authors reporte on 842 men that ha been iagnose
with clinically localize prostate cancer between 1993 an 1998. The stuy popu
lation ha complete the SF36 an UCLAPCI questionnaires. These men receive v
arious treatments incluing: raical prostatectomy (n=421), external beam raiot
herapy (n=221), brachytherapy (n=69), hormonal treatment (n=33), watchful waitin
g (n=31), an other (n=67). The authors provie no information as to whether som
e men inclue in the PB group also receive EBRT. In aition, no information w
as provie concerning the use of ADT in combination with PB, RP, or EBRT. Agea
juste generic HRQOL scores showe very small ifferences accoring to treatmen
t group with the RP group having the highest scores. Significant ifferences wer
e observe in sexual, urinary, an bowel HRQOL accoring to treatment group. Men
treate with PB an EBRT reporte higher sexual an urinary function as well as
less sexual bother compare to men treate with RP. The men treate with PB or
EBRT, however, also reporte significantly worse bowel function, bowel bother, a
n urinary bother (in the case of PB) than men treate with RP. In a very recent
report from investigators at the University of Michigan, Wei et al have provie
 a crosssectional analysis of more than 1000 men treate with RP, PB, or EBRT b
etween 1995 an 1999.14 The PB group inclue an unreporte percent of men treat
e with a combination of EBRT an PB. Aitionally, compare to the RP group the
PB group was more likely to be treate with ADT. The PB group ha the shortest
time between treatment an completion of the HRQOL questionnaires (meian: 21 mo
nths, range: 452). These authors use a number of valiate questionnaires inclu
ing the EPIC instrument an compare the HRQOL in each group to a group of agem
atche controls. The EPIC instrument was constructe by moifying the UCLA PCI i
nstrument an aing items that aress irritative urinary symptoms, irritative
bowel symptoms, symptoms relate to anrogen eprivation, an items expaning th
e assessment of functionspecific bother in each of these new omains. Measures
of generic HRQOL (SF36 an FACT) i not iffer between controls an treatmen
t groups. Using the EPIC instrument, however, the PB group was foun to have sig
nificantly worse urinary, bowel, an sexual HRQOL compare to controls. A compar
ison of HRQOL scores in men at least one year from completion of therapy foun t
hat the PB group ha significantly worse urinary irritative, bowel, an sexual s

ymptoms than the RP or EBRT groups. By excluing the patients that receive EBRT
from the PB group sexual HRQOL was similar to the EBRT group an superior to th
e RP group, suggesting that the aition of EBRT to PB leas to ecrease sexual
function. In a recent report from the Michigan group, Hollenbeck et al have exa
mine sexual HRQOL following PB.15 The authors compare the sexual HRQOL in 84 m
en treate with PB an a similar number of agematche controls. The majority of
men treate with PB receive some form of ADT prior to PB an more than 40% of
men receive EBRT in aition to PB. Avance age an the use of ADT were inepe
nently associate with
Healthrelate quality of life
603
sexual HRQOL following PB. In men uner the age of 69, 33% reporte at least fai
r sexual function following PB compare to 19% in men treate with ADT. For thos
e men over the age of 69, at least fair sexual function was reporte by 26% foll
owing PB but only 5% of these oler men reporte fair sexual function if ADT was
use along with PB. Knowing the prevalence of erectile ysfunction after PB alo
ne or in combination with EB an/or ADT is also critical to unerstaning the be
nefit of interventions to ameliorate this ecrement in quality of life. A recent
stuy by Potters et al ocumente a ifferential response to silenafil (Viagra
) by cancer treatment combination in a subanalysis of 84 patients (from a cohort
of 1166 patients, meian age: 68 years; meian followup: 34 months) accepting
treatment for ED.16 Of the 36 patients who never receive ADT an were treate w
ith either 50 mg or 100 mg of silenafil as neee, 12/15 (80%) men treate with
PB alone an 18/21 (86%) men treate with EBRT + PB were able to achieve erecti
ons satisfactory for intercourse. In contrast, of 48 patients who ha been treat
e with neoajuvant ADT, 11/25 (44%) treate with PB alone an 11/23 (48%) trea
te with EBRT+PB were able to achieve success with silenafil. Summarizing the r
esults outline above, several statements can be offere. First, when HRQOL is m
easure with a generic instrument (SF36 for instance), there o not appear to b
e any large ifferences between men treate for prostate cancer an similarly ag
e men without cancer, or those with cancer who have yet to receive efinitive t
reatment. Secon, when HRQOL is measure with a symptomspecific instrument, the
re are often ifferences between men treate for prostate cancer an similarly a
ge men without cancer. Thir, it appears that there are treatmentspecific chan
ges in HRQOL within the urinary, bowel, an sexual omains. Specifically, it app
ears that men treate with RP have more ifficulty within the sexual an certain
urinary omains compare to men treate with PB or EBRT. On the other han, PB
an EBRT are associate with more bowel ysfunction than RP, an PB is associate
 with more urinary bother than EBRT or RP. The strength of these conclusions is
tempere by the crosssectional nature of these reports. Litwin has emonstrate
 that agematche men without prostate cancer report less than perfect sexual,
bowel, an urinary HRQOL. In aition, without a control group it is ifficult t
o unerstan the magnitue of the problem. Unlike urinary an bowel incontinence
, which are abnormal at any age, but increase in prevalence with age ue to func
tional impairments an concurrent meical isease, there is a high level of erec
tile ysfunction (ED) in the general population associate with normal aging. A po
pulation stuy of physiologic erectile ysfunction among 1290 subjects between t
he ages 40 an 70 emonstrate age to be the most significant inepenent preic
tor of ED, although other factors were foun to correlate with ED to lesser egr
ees (i.e. heart isease, hypertension, iabetes, cigarette smoking, etc.).17 Ove
rall, 17.2% of subjects reporte minimal ED, 25.2% reporte moerate ED, an 9.6
% reporte complete ED. The probability of complete ED triple from 5.1% to 15%
in men between 40 an 70 years an the probability of moerate ED ouble from 1
7% to 34%. An estimate 40% of men age 40 ha either minimal, moerate, or comp
lete ED, while that estimate rose to 67% by age 70. In sum, prospective ata col
lection, incluing baseline values, are require to examine the net effect of an
y treatment on various HRQOL omains. In one of the few prospective reports, Lee

an colleagues have escribe a prospective stuy examining HRQOL in a group of

men treate with PB.18 In a pilot stuy reporte in 2000, the authors stuie 3
1 men who ha complete the FACTP questionnaire prior to
Basic an avance techniques in prostate brachytherapy
604
PB an at several times (1, 3, 6, 12 months) following PB. All men were treate
with PB alone, no EBRT was utilize. Clinically meaningful ecreases in the FACT
P scores (inicating ecrease HRQOL) were note one month following PB with a
return to baseline by 12 months. An examination of the omains of the FACTP em
onstrate that the HRQOL ecreases occurre in the Functional WellBeing (FWB),
Physical WellBeing (PWB), an Prostate Cancer Symptom (PCS) subscales. In a late
r report from the same group at Wake Forest University, Lee compare changes in
HRQOL as measure by FACTP in men treate with PB, RP, or EBRT.19 Ninety men we
re inclue (PB, 44; RP, 23; EBRT, 23). The stuy esign i not use ranomizati
on, treatment was chosen base on the recommenation of the treating physicians
an the wishes of the patient. The authors compare the HRQOL profiles in each o
f the treatment groups. After ajusting for baseline HRQOL there were significan
t ifferences in HRQOL at one month accoring to treatment group. Men treate wi
th PB or RP experience larger eclines in HRQOL at one month than men treate w
ith EBRT. Importantly, however, by one year there were no significant ifference
s in HRQOL accoring to treatment group. In fact, the HRQOL scores for all group
s at 12 months were not significantly ifferent than baseline scores for any of
the treatment groups. HRQOL following treatment with PB alone vs treatment with
combination PB an EBRT In most of the crosssectional reports above the prostat
e brachytherapy (PB) groups containe men treate with PB alone an men treate
with a combination of PB an EBRT. In a few papers the HRQOL consequences associ
ate with the use of EBRT combine with PB were analyze. In the Braneis et al
paper, the aition of EBRT to PB preicte for poorer generic an iseasespeci
fic HRQOL along with higher IPSS scores (more urinary symptoms).10 In the Krupsk
i et al paper the group receiving EBRT an PB ha worse HRQOL compare to the gr
oup treate with PB alone.11 The Michigan paper i not specify what percent of
men ha receive EBRT in the PB group but they reporte that excluing those pat
ients that receive a combination of EBRT an PB improve sexual HRQOL in the PB
group.14 The best way to etermine whether the aition of EBRT to PB is associ
ate with a ecrease in HRQOL compare to treatment with PB alone is by way of a
ranomize trial. No trials have yet been accomplishe although the Raiation T
herapy Oncology roup (RTO) has a trial ongoing. For the time being there is at
least a suggestion that the aition of EBRT may ecrease HRQOL. HRQOL followin
g high ose rate prostate brachytherapy The number of reports aressing HRQOL f
ollowing treatment with high ose rate (HDR) prostate brachytherapy is small. A
literature search using PubMe foun only two reports using a valiate HRQOL in
strument. In the first report, Joly et al analyze 71 men with prostate cancer t
reate with a combination EBRT an HDR PB an compare them to 71 agematche co
ntrols without cancer.20 In this retrospective, crosssectional stuy all partic
ipants were maile a generic
Healthrelate quality of life
605
HRQOL instrument (Nottingham Health Profile) an a prostatecancer specific tool
(EORTC QLCC30). eneric HRQOL scores were similar between the two groups, but
a comparison of EORTC QLCC30 scores foun ifferences in the urinary an sexual
omains. Patients treate with EBRT an HDR PB were more likely to have urinary
incontinence an to be inactive sexually compare to controls. In a very recent
report Egawa an colleagues from Japan aministere the SF36 an a portion of
the EORTC prostate questionnaire to 58 men treate with HDR PB an EB.21 The aut

hors observe significant ecreases in five omains of the SF36 in the first mo
nth after HDR treatment but by 12 months, scores ha returne to baseline in all
generic HRQOL omains. A similar time course of ecline an recovery was observ
e in the iseasespecific omains measure. All iseasespecific scores ha ret
urne to baseline 12 months following treatment. Future irections The stuy of
healthrelate quality of life (HRQOL) in men with early stage prostate cancer i
s a relatively young iscipline. Several reliable, valiate instruments exist t
o measure HRQOL in men with prostate cancer. These tools have been applie to o
cument HRQOL prior to treatment, to estimate the effects of treatment on HRQOL,
an to explore relationships among changes in ifferent omains of HRQOL. Most e
vience to ate suggests that there are important ifferences in sexual, urinary
, an bowel HRQOL accoring to treatment receive. This unerscores the importan
ce of completing ranomize trials in men with clinically localize prostate can
cer. References
1. Worl Health Organization (WHO). Constitution of the Worl Health Organizatio
n, basic ocuments. eneva: WHO, 1948. 2. Tulsky DS. An introuction to test the
ory. Oncology 1990; 4:4348. 3. Stewart AL, Hays RD, Ware JE. The MOS shortform g
eneral health survey: reliability an valiity in a patient population. Me Care
1988; July:724735. 4. Bergner M, Bobbitt RA, Carter WB, ilson BS. The Sickness
Impact Profile: evelopment an final revision of a health status measure. Me C
are 1981; Aug:787805. 5. Esper P, Mo F, Choal , et al. Measuring quality of lif
e in men with prostate cancer using the functional assessment of cancer therapyp
rostate instrument. Urology 1997; 50:920928. 6. Litwin MS, Hays RD, Fink A, et al
. The UCLA Prostate Cancer Inex: evelopment, reliability, an valiity of a he
althrelate quality of life measure. Me Care 1998; 36:10021012. 7. ShraerBoge
n CL, Kjellberg JL, McPherson CP, Murray CL. Quality of life an treatment outco
mes: prostate carcinoma patients perspectives after prostatectomy or raiation th
erapy. Cancer 1997; 79:19771986. 8. Wei JT, Dunn RL, Litwin MS, et al. Developmen
t an valiation of the expane prostate cancer inex composite (EPIC) for comp
rehensive assessment of healthrelate quality of life in men with prostate canc
er. Urology 2000; 56:899905. 9. Borghee , Sullivan M. Measurement of quality of
life in localize prostatic cancer patients treate with raiotherapy: Developm
ent of a prostate cancerspecific moule supplementing the EORTC QLQC30. Qual Li
fe Res 1996; 5:212221.
Basic an avance techniques in prostate brachytherapy
606
10. Braneis JM, Litwin MS, Burnison CM, Reiter RE. Quality of life outcomes aft
er brachytherapy for early stage prostate cancer. J Urol 2000; 163:851857. 11. Kr
upski T, Petroni R, Bissonette EA, Theoorescu D. Qualityoflife comparison of
raical prostatectomy an interstitial brachytherapy in the treatment of clinic
ally localize prostate cancer. Urology 2000; 55:736742. 12. Davis JW, Kuban DA,
Lynch DF, Schellhammer PF. Quality of life after treatment for localize prostat
e cancer: ifferences base on treatment moality. J Urol 2001; 166:947952. 13. B
acon C, iovannucci E, Testa M, Kawachi I. The impact of cancer treatment on qu
ality of life outcomes for patients with localize prostate cancer. J Urol 2001;
166:18041810. 14. Wei JT, Dunn RL, Sanler HM, et al. Comprehensive comparison o
f healthrelate quality of life after contemporary therapies for localize pros
tate cancer. J Clin Oncol 2002; 20:557566. 15. Hollenbeck BK, Dunn RL, Wei JT, et
al. Neoajuvant hormonal therapy an oler age are associate with averse sexu
al healthrelate qualityoflife outcome after prostate brachytherapy. Urology
2002; 59:480484. 16. Potters L, Torre T, Fearn PA, et al. Potency after permanent
prostate brachytherapy for localize prostate cancer. Int J Raiat Oncol Biol P
hys 2001; 50:12351242. 17. Felman HA, olstein I, Hatzichristou D, et al. Impo
tence an its meical an psychosocial correlates: results of the Massachusetts
Male Aging Stuy. J Urol 1994; 151:5461. 18. Lee WR, McQuellon RP, HarrisHeners
on K, et al. A preliminary analysis of healthrelate quality of life in the fir
st year after permanent source interstitial brachytherapy (PIB) for clinically l

ocalize prostate cancer. Int J Raiat Oncol Biol Phys 2000; 46:7781. 19. Lee WR,
Hall MC, McQuellon RP, et al. A prospective qualityoflife stuy in men with cl
inically localize prostate carcinoma treate with raical prostatectomy, extern
al beam raiotherapy, or interstitial brachytherapy. Int J Raiat Oncol Biol Phy
s 2001; 51:614623. 20. Joly F, Brune D, Couette JE, et al. Healthrelate qualit
y of life an sequelae in patients treate with brachytherapy an external beam
irraiation for localize prostate cancer. Ann Oncol 1998; 9:751757. 21. Egawa S,
Shimura S, Irie A, et al. Toxicity an healthrelate quality of life uring an
 after high ose rate brachytherapy followe by external beam raiotherapy for
prostate cancer. Jpn J Clin Oncol 2001; 31:541547.
47 Rectal complications following permanent see implants
Louis Potters Introuction Ultrasounguie permanent prostate brachytherapy (P
PB) alone or in conjunction with external beam raiotherapy (EBRT) for aenocarc
inoma of the prostate is quickly growing in popularity as a treatment option for
patients with early stage, localize cancers. As experience with this technique
has grown over the last several years, reports in the literature have presente
the urinary morbiity from several centers.13 The rectal morbiity associate wi
th PPB has been less well investigate an ocumente.4,5 As patients are aske
to participate in the treatment ecisions for early stage, localize prostate ca
ncer, the morbiity of each treatment metho plays an important role in the sele
ction process for each patient. In the immeiate postimplant perio, a patient u
nergoing PPB may have some mil transient rectal iscomfort an bleeing as a r
esult of the ultrasoun probe uring the proceure. During the weeks following t
he implant, there may be changes in bowel habits in the form of iarrhea or cons
tipation, tenesmus, an rectal pressure.68 These symptoms generally respon to co
nservative symptomatic management. Late injury inclues proctitis, rectal ulcera
tion, fistula formation, an incontinence.9 The most common of these is proctiti
s, which often presents as a painless hemorrhoial type bleeing that is usually
selflimite. Bleeing from proctitis presents late, about one to two years aft
er implantation an may be exacerbate by constipation. Conservative management
is recommene with stool softeners an local steroi creams or foams. Aggressiv
e measures, such as biopsies an laser treatments, may precipitate ulceration an
 fistula formation an shoul be avoie whenever possible.7,10,11 Rectal injur
y following PPB fortunately occurs with low frequency. However, it remains the m
ost severe complication that can occur an can be associate with malpractice li
tigation. This is ue to the catastrophic nature of rectal injuries couple with
their elaye onset at a time when followup visits to the raiation oncologist
are infrequent. The importance of unerstaning rectal injury cannot be unerst
ate. Appropriate management strategies must be explaine to the patient. One qu
estion that arises frequently is whether there is a oseeffect that can explain
or be responsible for rectal injury. Clearly, the proximity of the rectal mucos
a to the posterior aspect of the prostate is associate with high raiation ose
, even when an implant ose conforms tightly to the prostate itself. Further, th
ere is little consensus as to what constitutes a high ose to the rectum. In a
ition, the efinition of the rectum is ambiguous when escribing a elivere os
e to that structure. Authors have escribe various efinitions of the rectum, e
ither as a point or volume. Conforming our efinition
Basic an avance techniques in prostate brachytherapy
608
an how it is represente on postimplant osimetric stuies is require in orer
to establish ose criteria for the risk of rectal injury. In a recent review fr
om Wallner et al, they examine several cases of severe rectal ulceration an we
re unable to istinguish osimetric characteristics that woul have ientifie a
risk for eveloping such a complication. As an attempt to review this subject,
the series from elblum an Potters will be reviewe. Materials The elblum an
Potters series reporte on 825 patients with biopsy proven aenocarcinoma of the

prostate who were treate with PPB (Table 47.1). The treatment metho for these
patients has been previously reporte.12 PPB was performe using realtime intra
operative transrectal ultrasoun (TRUS) guiance with neeles place using the p
eripheral spacing technique. Both the sagittal an axial images on TRUS were use
 to confirm the proximity of neele placement to the posterior prostate margin.
Patients who receive PPB alone were treate to a minimum peripheral ose (MPD)
of 136 y with pallaium103, an 144 y with ioine125 (post T43
Table 47.1 Patient characteristics from the elblum an Potters series
Variable
Number of patients implante Age (meian) Meian preimplant PSA <10 ng/mL 1020 ng
/mL >20 ng/mL leason score 24 56 7 89 Stage (1998 AJCC) T1c T2a T2b Prostate size
(meian) Isotope 125 I 103 P Neoajuvant antianrogen therapy Preimplant irra
iation Total activity (meian) 103 P
No.
825 67 yrs (range: 5289) 9.49 ng/mL (range: 0.656 ng/mL) 393 378 54 99 471 206 49
380 379 66 34.6 cc 240 685 173 140 116 mCi
Rectal complications following permanent see implants
125
609
I 38.2 mCi PSA, prostatespecific antigen; AJCC, American Joint Committee on Can
cer.
formalize).13,14 When external beam irraiation precee PPB, a ose of 45 y a
t 180 cy/fraction was prescribe an elivere via fourfiel technique. Anteri
or an lateral fiels measure on average 1212 cm an 1112 cm, respectively. The M
PD for 103P was 102 y an for 125I was 108 y (post T43 formalize) when com
bine moality treatment was use. At each followup appointment, patients were
interviewe with respect to their bowel function. Bowel an rectal complaints we
re ocumente using a moifie Raiation Therapy Oncology roup (RTO) rectal sy
mptom scoring scale (Table 47.2). Patients who complaine of rectal irritation w
ere treate with hyrocortisone acetate hemorrhoial suppositories three times 
aily for a minimum of 3 weeks an avise to use sitz baths. Patients with persi
stent irritation or complaints were treate with hvrocortisone retention enemas
twice aily for 2 to
Table 47.2 Moifie RTO rectal toxicity scale use in the current stuy
Moifie RTO rectal toxicity rae 1 Tenesmus, ear mucous ischarge rae 2 In
termittent rectal bleeing, erythema of rectal lining on proctoscopy rae 3 Rec
tal ulceration rae 4 Bowel obstruction, fistula formation, bloo transfusion r
equire RTO, Raiation Therapy Oncology roup.
4 weeks. Crue an actuarial analysis for the incience of rectal morbiity was
evaluate. Compute tomography (CT)base postPPB osimetry was initiate in 19
941995. The metho of rectal ose calculation has varie consierably since this
time (rectal point ose vs rectal volume) an thus meaningful ata from the ose
volume histogram were not available for that stuy. Results With a meian follo
wup of 48 months (2485 months), the crue incience of rectal toxicity in this p
atient cohort peake at 9.6% at 10 months with patients either experiencing gra
e 1 or 2 rectal toxicity. Of the patients, 58 (7%) ha grae 1 complaints while
21 (2.5%) reporte grae 2 complaints. During the entire followup perio, 77 pa
tients (9.4%) reporte grae 1 toxicity while 54 patients (6.6%) reporte grae
2 toxicity or rectal bleeing. The actuarial incience of rectal toxicity for pa
tients that unerwent PPB alone versus PPB an external beam irraiation is show
n in Table 47.3. The aition of external beam raiotherapy (EBRT) i not impac
t the incience of rectal morbiity. Aitionally there was no ifference

Basic an avance techniques in prostate brachytherapy

610
Table 47.3 Actuarial incience of rectal toxicity for patients receiving combine
 external beam raiotherapy an PPB verses PPB alone
PPB+EBRT (n=140) PPB Alone (n=685)
8.9% 6.5% 0.4%

rae 1 10.5% rae 2 7.1% rae 3 0.7% PPB, transperineal interstitial permanen
t prostate brachytherapy; EBRT, external beam raiotherapy.
Table 47.4 Univariate analysis of contributing factors for grae 1 an 2 rectal
toxicity
Characteristic rae 1 an 2 (pvalue)
Isotope 0.634 Neoajuvant hormones 0.962 External beam raiotherapy 0,086 Case
orer 0.178
in the incience of grae 1 or 2 rectal toxicity for the selection of isotope, t
he aition of hormone therapy, or case orer (Table 47.4). By 3.5 years, no pat
ients reporte rectal complaints. Rectal ulceration reporte as a grae 3 compli
cation was not reporte until 12 months following PPB (Table 47.5). Four cases o
f the 825 patients in this stuy (0.5%) reporte a grae 3 rectal complication.
Colonoscopy or proctoscopy ocumente all four patients with grae 3 toxicity. T
wo patients reporte problems with rectal ulceration at 12 months postPPB an o
ne each at 18 an 23 months after the implant. Three of the four patients were i
mplante with 125I, while one patient was implante with 103P. One of the three
patients treate with 125I receive external beam irraiation in combination wi
th PPB an only one of these patients receive neoajuvant antianrogen therapy
as part of his treatment. Two of the four patients with rectal ulcers were biops
ie at the time of routine colonoscopy for anterior rectal proctitis, with bleein
g an ulceration subsequent to the biopsy. Bot h pathology reports ientifie in
flammatory proctitis with neovascularization. The ulcerate regions were escrib
e as ranging in size from 2 cm to 4 cm in greatest imension. All four ulcers r
esolve with conservative management. No patient has ha a significant rop in h
ematocrit or require a bloo transfusion. No grae 4 toxicities were reporte i
n this stuy. Discussion A common metho for assessing rectal toxicity after ra
iation therapy is the Raiation Therapy Oncology roup (RTO) toxicity scale. In
the elblum series, a moifie scale was use an their results are similar to
others who present an absolute rectal bleeing
Rectal complications following permanent see implants
611
rate of approximately 6%. The overall incience of proctitis in the literature f
ollowing permanent brachytherapy has been reporte to range from 1% to 12%.7,15
While the 12% proctitis rate reporte by Wallner et al represents the early expe
rience with CTbase implants, subsequent refinements of the technique inicate
that proctitis rates have ecrease to about 46% overall.5,7,16,17 Merrick et al
report similar results of rectal toxicity in their small, retrospective series o
f patients treate with permanent prostate brachytherapy (PPB) alone.18 They rep
ort a rate of selflimite proctitis of 9% from a cohort of 45 patients. They ca
lculate anterior rectal wall oses base on postimplant CT scans one with a rec
tal obturator in place on postoperative ay 5 with a mean estimate ose exposur
e to the anterior portion of the rectum of 82.5% of the prescription ose from t
he implant. No correlation was foun between the average rectal ose an prostat
ic volume. They also foun no relationship of the isotope use for PPB an the i
ncience of proctitis. The aition of external beam raiotherapy (EBRT) to PPB

was also not a contributing factor for the evelopment of rectal toxicity. They
conclue from their osimetric calculations that permitting oses 85% of the pres
cribe ose to the anterior rectal wall, inepenent of isotope use is associat
e with a 9% incience of proctitis. This is ientical to the 9% quote in the c
urrent stuy.
Table 47.5 rae 3 rectal toxicities from the elbum series
Patient Isotope EBRT Hormones Implant ose
1 2 3 4
125 125
Time for ulcer formation (mths)
12 12 23 18
Time to resolution (mths)
9 8 7 Still ealing at 8 mths
Rectal biopsy
Yes Yes No No
I I 125 I 103 P
No Yes No No
No No Yes No
144 y 100 y 144 y 120 y
EBRT, external beam raiotherapy,. raiotheraphy.
A secon stuy evaluating rectal osimetry in patients treate with PPB by Walln
er et al reviewe 65 patients treate with T1/T2 prostate cancers treate with C
Tplanne prostate implants alone.19 They report a 10% incience of proctitis. Fu
rther, a correlation was note with reporte grae 1 (rectal bleeing) an grae
2 (rectal ulceration) toxicity an a rectal surface ose greater than or less t
han 100 y for patients treate with 125I. There was no significant relationship
for the onset of rectal toxicity for the total isotope activity implante, the
minimum permitte ose (MPD), or iniviual see strength. An upate of this ser
ies by Hu an Wallner reports a meian time to rectal bleeing of 8 months postP
PB.15 Of the patients from that stuy, 13 unerwent sigmoioscopy an 7 were fou
n to have rectal ulceration while the remaining 6 ha raiation proctitis. Rect
al bleeing resolve spontaneously in that stuy as well. Syner et al reporting
on grae 2 proctitis following 125I implants conclue that the incience of pr
octitis was volumeepenent (Figure 47.1).20 In fact, the risk of proctitis was
volumeepenent for each ose stuie, 80, 140, 160, an 240 y, respectively.
The 5 year actuarial risk of grae 2 proctitis was 5% if 1.3 cc or less of the
rectal volume
Basic an avance techniques in prostate brachytherapy
612
receive the prescription ose of 160 y, an 17% for >1.3 cc (p=0.001). Others
have trie to correlate the risk of proctitis relative to the elivere ose an
volume of the rectum with less success.15,21 These stuies of etaile osimetr
ic analysis ientify higher rectal oses in patients with raiation proctitis, m
aking it a potential metho of ientifying patients at higher risk of rectal inj
ury. However, to ate there has not been a single series that has ientifie an
absolute raiation ose as a threshol for eveloping proctitis. While there is
currently no specific recommenation on ose, intraoperative planning systems ma

y allow the operator to measure an limit the rectal ose uring the case.18,22
An earlier report from Blasko an colleagues reports a 2.6% incience of proctit
is following PPB with 71% resolving spontaneously.23 They suggeste an increase
risk of late proctitis following combine moality treatment (6%) verses PPB al
one (1%). The aition of EBRT to permanent prostate brachytherapy may be a risk
factor for increase rectal toxicity. While the elblum stuy was unable to em
onstrate a ifference in proctitis between patients treate with implant only ve
rsus combine external raiation an implant, a stuy from Staten Islan Univers
ity Hospital reporte an alarmingly high percentage of 2.3% for fistula formatio
n.9 While these ata raise a concern that combination therapy may be associate
with more rectal injury, to ate no other series, whether monotherapy or combine
 therapy, has reporte a fistula rate this high. Using the incience of colosto
my as a surrogate for fistulization, Benoit et al reporte a rate of 0.3% in Me
icare patients unergoing prostate brachytherapy.24
Figure 47.1 The crue Incience of rectal toxicity in all 825 patients who recei
ve permanent prostate brachytherapy (PPB).7 raes: (1) tenesmus, clear mucous
ischarge; (2) intermittent rectal bleeing, erythema,
Rectal complications following permanent see implants
613
or rectal lining on proctoscopy; (3) rectal ulceration; (4) bowel obstruction, f
istula formation, bloo transfusion require.
Nonetheless, the observation of increase rectal bother when PPB is combine wit
h external raiation has been reporte by Braneis et al in a stuy comparing qu
ality of life inicators between implant alone an when combine with external r
aiation.25 In that stuy, rectal bother was significantly higher in those patie
nts treate with combination therapy. Recent series reporting on threeimension
al conformal external beam raiotherapy (3DCRT) of the prostate present much h
igher rates of rectal bleeing an iscomfort.26,27 A report from the Fox Chase
Cancer Center escribes late rectal bleeing to be an expecte complication of h
igh ose treatment of prostate cancer.28 The cumulative incience of grae 2 an
grae 3 rectal toxicity at 12 months was approximately 40% an 25%, respectivel
y. The meian rectal ose elivere to the prostate glan in that stuy was 72 
y, with a range of 62.1180.74 y. Most rectal bleeing evelope between 6 an 12
months following the treatment for a meian uration of 3 months. The central 
ose to the prostate glan was the only treatment relate factor foun to be sign
ificant on Cox multivariate analysis. Others have reporte a combine rate of ac
ute grae 1 an 2 rectal toxicity of 14% in patients unergoing 3DCRT with the m
ajority of the patients receiving 70.2 or 75.6 y to the prostate an early ata
with short followup inicate lower rates of rectal complications when intensit
y moulate raiotherapy (IMRT) is utilize. Diagnosis of raiation proctitis is
usually mae by sigmoi or colonoscopy. It is accepte that if areas of proctit
is are ientifie in a previously irraiate fiel, surgical interventions, such
as biopsies, may precipitate further erosion of the mucosa with the subsequent
formation of an ulcer or fistula.29 This is illustrate in the elblum series wh
ere 50% of the patients that experience grae 3 rectal toxicity ha been biopsi
e prior to the evelopment of the ulcer. These patients also appear to require
a longer time to heal from their ulcers (89 months vs 34 months for patients not b
iopsie). No fistulas were reporte in this series. The use of aggressive iagno
stic measures, such as biopsies, shoul be avoie at all costs an it is paramo
unt that patients be eucate on this matter since followup with a gastroentero
logist is generally not associate with communication back to the raiation onco
logist. The mechanism of raiation proctitis appears relate to eema an fibros
is of arterioles in the luminal crypts of the colonic mucosa.29,30 As fibrosis i
ncreases, the mucosal lining becomes more friable an is clinically associate w
ith bleeing. The gastrointestinal literature has reporte an increase risk of
eveloping raiation proctitis in patients with unerlying vascular changes asso

ciate with iabetes, hypertension, an chronic inflammatory bowel iseases. Non
etheless, rann an Wallner have emonstrate that patients with inflammatory bo
wel iseases may not be at an increase risk of rectal injury when treate with
PPB.31 Several meical interventions have been reviewe in the literature inclu
ing sulcralfate enemas, systemic an perrectum sterois, an argon laser coagul
ation for large ulcers.3239 However, the optimal meical treatment for raiation
proctitis has not
Basic an avance techniques in prostate brachytherapy
614
yet been ientifie. In patients with significant bleeing, the use of 4% formal
in appears to be an effective an noninvasive measure to control raiation proc
titis. Counter et al have presente their ata treating patients with raiation
proctitis an ientifie that 100% ha initial success with cessation of bleein
g.39 Three patients out of 27 ha recurrent bleeing; none require transfusion.
One patient require repeat formalin instillation, with no further bleeing at
3 months followup. Another promising treatment for patients with persistent ble
eing is the use of the argongas laser. Fantin et al have presente their exper
ience with this technique an have shown that with a meian of two treatment ses
sions (range: 24), complete symptom relief was achieve.35 All interventions were
well tolerate without complications. During followup (meian: 24 months, rang
e: 1824 months), there was no recurrence of symptoms (bleeing, tenesmus). Most i
mportantly, with these two treatment approaches, no evience of progression was
ientifie in these stuies. PPB is a now a stanar option for most men seeking
treatment of early stage, localize prostate cancer. A review of the literature
shows a lack of information regaring the evelopment of gastrointestinal toxic
ity after PPB. In fact, the recently reporte guielines by the American Brachyt
herapy Society (ABS) fail to consier rectal morbiity at this time.40 Fortunate
ly, the incience or rectal injury is not high following PPB relative to efinit
ive external raiotherapy. Nonetheless, there is more to learn to prevent the on
e or two catastrophic injuries that can occur. Most important is that close foll
owup an patient eucation is vital at the time of initial consultation an at
each followup visit. Any symptoms shoul be treate conservatively without biop
sy or surgical intervention unless the patient is bleeing enough to require tra
nsfusion. It is also vital that the urologist unerstan the significance of rec
tal injury as they may play a more important role uring followup. Any referral
to a gastroenterologist shoul be accompanie with information that the patient
sustaine raiation via a PPB an that it is likely that erythema an neovascul
arization will be visible on colonoscopy. However, a biopsy shoul not be perfor
me. It is likely that with future stuy, the ose limitations of the rectum wil
l be better elineate an that intraoperative techniques will be available to l
imit the rectal ose. However, it is likely that rectal injury will remain part
of the management of patients unergoing PPB an that unerstaning the risks of
incorrectly treating such patients will, it is hope, limit ba outcomes. Refer
ences
1. elblum D, Potters L, Ashley R, et al. Urinary morbiity following ultrasoun
guie transperineal prostate see implantation. Int J Raiat Oncol Biol Phys 1
999; 45(1):5967. 2. Terk MD, Stock R, Stone NN. Ientification of patients at in
crease risk for prolonge urinary retention following raioactive see implanta
tion of the prostate. J Urol 1998; 160(4):1379 1382. 3. Nag S, Scaperoth DD, Baa
lament R, et al. Transperineal pallaium 103 prostate brachytherapy: analysis of
morbiity an see migration. Urology 1995; 45(1):8792.
Rectal complications following permanent see implants
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4. Vicini FA, Kini VR, Emunson , et al. A comprehensive review of prostate ca

ncer brachytherapy: efining an optimal technique. Int J Raiat Oncol Biol Phys
1999; 44(3):483 491. 5. Kleinberg L, Wallner K, Roy J, et al. Treatmentrelate s
ymptoms uring the first year following transperineal 125I prostate implantatio
n. Int J Raiat Oncol Biol Phys 1994; 28(4):985990. 6. Blasko JC, Mate T, Sylvest
er JE, et al. Brachytherapy for carcinoma of the prostate: techniques, patient s
election, an clinical outcomes. Semin Raiat Oncol 2002; 12(1):8194. 7. elblum
DY, Potters L. Rectal complications associate with transperineal interstitial b
rachytherapy for prostate cancer. Int J Raiat Oncol Biol Phys 2000; 48(1):119124
. 8. Merrick S, Butler WM, Dorsey AT, Dorsey JT 3r. The effect of constipation
on rectal osimetry following prostate brachytherapy. Me Dosim 2000; 25(4):2372
41. 9. Zeitlin SI, Sherman J, Raboy A, et al. High ose combination raiotherapy
for the treatment of localize prostate cancer [Discussion: 9596.] J Urol 1998;
160(1):9195. 10. Han BH, Wallner KE. Dosimetric an raiographic correlates to pr
ostate brachytherapyrelate rectal complications. Int J Cancer 2001; 96(6):37237
8. 11. Wallner K, Roy J, Harrison L. Dosimetry guielines to minimize urethral a
n rectal morbiity following transperineal I125 prostate brachytherapy. Int J
Raiat Oncol Biol Phys 1995; 32(2):465471. 12. Cha CM, Potters L, Ashley R, et al
. Isotope selection for patients unergoing prostate brachytherapy. Int J Raiat
Oncol Biol Phys 1999; 45(2):391395. 13. Beyer D, Nath R, Butler W, et al. Americ
an Brachytherapy Society recommenations for clinical implementation of NIST199
9 stanars for (103)pallaium brachytherapy. Int J Raiat Oncol Biol Phys 2000;
47(2):273275. 14. Nath R, Roberts K, Ng M, et al. Correlation of meical osimet
ry quality inicators to the local tumor control in patients with prostate cance
r treate with ioine125 interstitial implants. Me Phys 1998; 25(12):22932307.
15. Hu K, Wallner K. Clinical course of rectal bleeing following I125 prostate
brachytherapy. Int J Raiat Oncol Biol Phys 1998; 41(2):263265. 16. Merrick S,
Butler WM, Dorsey AT, et al. Rectal function following prostate brachytherapy. I
nt J Raiat Oncol Biol Phys 2000; 48(3):667674. 17. Battermann JJ. I125 implanta
tion for localize prostate cancer: the Utrecht University experience. Raiother
Oncol 2000; 57(3):269272. 18. Merrick S, Butler WM, Dorsey AT, et al. Rectal o
simetric analysis following prostate brachytherapy. Int J Raiat Oncol Biol Phys
1999; 43(5):10211027. 19. Wallner K, Roy J, Harrison L. Dosimetry guielines to
minimize urethral an rectal morbiity following transperineal I125 prostate br
achytherapy. Int J Raiat Oncol Biol Phys 1995; 32(2):465471. 20. Snyer KM, Stoc
k R, Hong SM, et al. Defining the risk of eveloping grae 2 proctitis followin
g 125I prostate brachytherapy using a rectal osevolume histogram analysis. In
t J Raiat Oncol Biol Phys 2001; 50(2):335341. 21. Zelefsky MJ, Yamaa Y, Cohen 
, et al. Postimplantation osimetric analysis of permanent transperineal prostat
e implantation: improve ose istributions with an intraoperative computeropti
mize conformal planning technique. Int J Raiat Oncol Biol Phys 2000; 48(2):6016
08. 22. Zelefsky MJ, Yamaa Y, Cohen , et al. Postimplantation osimetric analy
sis of permanent transperineal prostate implantation: improve ose istribution
s with an intraoperative computeroptimize conformal planning technique. Int J
Raiat Oncol Biol Phys 2000; 48(2):601608. 23. Blasko JC, Rage H, rimm PD. Tran
sperineal ultrasounguie implantation of the prostate: morbiity an complica
tions. Scan J Urol Nephrol Suppl 1991; 137:113118.
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616
24. Benoit RM, Naslun MJ, Cohen JK. A comparison of complications between ultra
sounguie prostate brachytherapy an open prostate brachytherapy. Int J Raia
t Oncol Biol Phys 2000; 47(4):909913. 25. Braneis JM, Litwin MS, Burnison CM, Re
iter RE. Quality of life outcomes after brachytherapy for early stage prostate c
ancer. J Urol 2000; 163(3):851857. 26. Shipley WU, Zietman AL, Hanks E, et al. T
reatment relate sequelae following external beam raiation for prostate cancer:
a review with an upate in patients with stages T1 an T2 tumor. J Urol 1994; 1
52(5 Pt 2):17991805. 27. Schultheiss TE, Lee WR, Hunt MA, et al. Late I an U c
omplications in the treatment of prostate cancer. Int J Raiat Oncol Biol Phys 1

997;37(1):311. 28. Teshima T, Hanks E, Hanlon AL, et al. Rectal bleeing after c
onformal 3D treatment of prostate cancer: time to occurrence, response to treatm
ent an uration of morbiity. Int J Raiat Oncol Biol Phys 1997; 39(1):7783. 29.
Rubin P, Cassarett W. Clinical raiation pathology. Philaelphia: WB Sauners,
1968. 30. Donner CS. Pathophysiology an therapy of chronic raiationinuce in
jury to the colon. Dig Dis 1998; 16(4):253261. 31. rann A, Wallner K. Prostate b
rachytherapy in patients with inflammatory bowel isease. Int J Raiat Oncol Bio
l Phys 1998; 40(1):135138. 32. Babb RR. Raiation proctitis: a review. Am J astr
oenterol 1996; 91(7):13091311. 33. Sasai T, Hiraishi H, Suzuki Y, et al. Treatmen
t of chronic postraiation proctitis with oral aministration of sucralfate. Am
J astroenterol 1998; 93(9):15931595. 34. rigsby PW, Pilepich MV, Parsons CL. Pr
eliminary results of a phase I/II stuy of soium pentosanpolysulfate in the tre
atment of chronic raiationinuce proctitis. Am J Clin Oncol 1990; 13(1):2831.
35. Fantin AC, Binek J, Suter WR, Meyenberger C. Argon beam coagulation for trea
tment of symptomatic raiationinuce proctitis. astrointest Enosc 1999; 49(4
Pt 1):515518. 36. Kochhar R, Sharma SC, upta BB, Mehta SK. Rectal sucralfate in
raiation proctitis [Letter]. Lancet 1988; 2(8607):400. 37. Charneau J, Bouacho
ur , Person B, et al. Severe hemorrhagic raiation proctitis avancing to grau
al cessation with hyperbaric oxygen. Dig Dis Sci 1991; 36(3):373375. 38. Baum CA,
Bile WL, Miner PB Jr. Failure of 5aminosalicylic aci enemas to improve chro
nic raiation proctitis. Dig Dis Sci 1989; 34(5):758760. 39. Counter SF, Froese D
P, Hart MJ. Prospective evaluation of formalin therapy for raiation proctitis.
Am J Surg 1999; 177(5):396398. 40. Nag S, Beyer D, Frielan J, et al. American B
rachytherapy Society (ABS) recommenations for transperineal permanent brachythe
rapy of prostate cancer. Int J Raiat Oncol Biol Phys 1999; 44(4):789799.
48 Sexual function following permanent prostate brachytherapy
regory S Merrick an Wayne M Butler Introuction Erectile ysfunction (ED) has
been estimate to affect up to 30 million American men, results in a eleterious
effect on quality of life (QOL) incluing physical an emotional wellbeing, mar
ital iscor, an loss of selfesteem, an is a common sequela following treatme
nt for prostate cancer.14 The National Institutes of Health (NIH) Consensus Confe
rence efine ED as the inability of attain an/or maintain penile erection suffi
cient for satisfactory sexual performance, an recommene the evelopment of rel
iable methos for assessing an evaluating treatment outcomes.1 Following potent
ially curative treatment for carcinoma of the prostate glan, potency preservati
on has generally been assume to be most likely following brachytherapy, but lon
ger followup has raise substantial oubts about the potencysparing avantage o
f brachytherapy. 57 Following either pallaium103 (103P) or ioine125 (125I) br
achytherapy with or without supplemental external beam raiotherapy (EBRT), ED h
as been reporte in 687% of cases.518 With aitional followup, continue eterio
ration in erectile function is expecte ue to the normal aging process.19 Raia
tionrelate impotence likely represents a multifactorial process.20 In the case
of brachytherapy, there is emerging evience that ED is techniquerelate an m
ay be minimize by careful attention to source placement.21 Accoringly, the oc
umentation of sexual function an ose istributions following brachytherapy may
result in refinement in treatment techniques, improve treatments for ED, an u
ltimately improve QOL outcomes. Quality of life instruments QOL instruments att
empt to uniformly report lifestyle effects an to facilitate comparisons between
ifferent moalities. Investigators have increasingly quantifie postimplant QO
L with survey instruments much more etaile than previously publishe scales, s
uch as those of the Raiation Therapy Oncology roup (RTO).22 Unfortunately, no
ne of the current QOL instruments aequately capture brachytherapyrelate sexual
ysfunction. In fact, a variety of sexual symptomatology other than ED occurs f
ollowing prostate brachytherapy.7,8 In a prospective ranomize brachytherapy tr
ial, brachytherapyspecific sexual changes were etaile by means of an inepth
survey of sexual function following
Basic an avance techniques in prostate brachytherapy

618
therapy by blening the specific erectile questions of the International Inex o
f Erectile Function (IIEF5) with a number of more etaile topics (Table 48.1).
8,23 Hematospermia, orgasmalgia (pain at the time of orgasm), an alteration in
the intensity of orgasm were reporte by 26%, 15%, an 38% of patients, respecti
vely.8 Orgasmalgia normally occurs within the first year following implantation
an is probably relate to inflammation of the terminal portion of the ejaculate
ry ucts an the urethra.24 Hematospermia can occur as both an early an late ph
enomenon an probably is a result of raiationinuce capillary fragility with
no apparent clinical significance.24 For most patients, the sie effects are of
limite uration. In aition, following brachytherapy, raiation therapy, an r
aical prostatectomy, Schover et al reporte a significant incience of ifficul
ty reaching orgasm.7 Prostate brachytherapy oes affect sexual function, an unf
ortunately, some of these changes are not aresse by stanar survey tools. Fo
r QOL instruments to accurately reflect sexual function, new valiate instrumen
ts with the inclusion of brachytherapyspecific symptomatology will be manatory
to accurately measure sexual QOL an to evaluate ifferent brachytherapy techniq
ues. The assessment of sexual potency following brachytherapy The wie ranges of
ED after brachytherapy are in part a result of ifferences in followup, efinit
ions of potency, an the moe of ata collection. Litwin an colleagues
Table 48.1 Specific erectile function questions of the International Inex of Er
ectile Function (IIEF)
1. How often are you able to get an erection 4. During sexual intercourse, how o
ften were you able to maintain your erection after you ha uring sexual activit
y? penetrate (entere) your partner! 0=No sexual activity 1=Almost never/never
0=Di not attempt intercourse 2=A few times (much less than half the 1=Almost ne
ver/never time) 3=Sometimes (about half the time) 2=A few times (much less than
half the time) 4=Most times (much more than half the 3=Sometimes (about half the
time) time) 5=Almost always/always 4=Most times (much more than half the time)
5=Almost always/always 2. When you ha erections with sexual stimulation, how of
ten were your erections 5. During sexual intercourse, how ifficult it to har e
nough for penetration? maintain your erection to completion of 0=No sexual activ
ity intercourse? 1=Almost never/never 0=Di riot attempt intercourse 2=A few tim
es (much less than half the 1=Extremely ifrtcult time) 3=Sometimes (about half
the time) 2=Very ifficult 4=Most times (much more than half the 3=Difficult tim
e) 4=Slightly ifficult . 5=Almost always/always
Sexual function following permanent prostate brachytherapy
619
5=Not ifficult 3. When you attempte sexual intercourse, how often were you abl
e to penetrate (enter) your 6. How o you rate your confience that you coul pa
rtner? get an keep an erection? 0=Di not attempt intercourse 1=Almost never/ne
ver 1=Very low 2=A few times (much less than half the 2=Low time) 3=Sometimes (a
bout half the time) 3=Moerate 4=Most times (much more than half the 4=High time
) 5=Almost always/always 5=Very high
reporte that physician ratings of patient symptoms i not correlate well with
patient selfassessment of QOL.25 To ate, only two potency stuies have exclusi
vely utilize patientaministere valiate instruments.6,7 The International I
nex of Erectile Function (IIEF5 or IIEF6) comprises 15 questions in 5 omains
(erectile function, orgasmic function, sexual esire, intercourse satisfaction,
an overall satisfaction) an has been valiate as a sensitive an specific to
ol for the evaluation of male sexual function.26,27 In contrast, Stock an colle
agues formulate a fourtiere system where erectile function was grae on a 03
scale with erectile function primarily assigne by the physician at the time of
patient interview (Table 48.2).5,10

Table 48.2 Mount Sinai fourtiere system for erectile function graing (from St
ock et al5,10)
Score Definition
0 1 2 3 No erections Ability to have erections but insufficient for vaginal pene
tration Erectile function sufficient for vaginal penetration but suboptimal Norm
al erectile function
The Mount Sinai scoring system is currently being utilize in two prospective ra
nomize prostate brachytherapy trials.23,28 In the pretreatment evaluation of e
rectile function, the Mount Sinai scoring system has been emonstrate to closel
y correlate with IIEF scores (unpublishe ata). The remainer of the brachyther
apy potency literature has either utilize a nonvaliate patient questionnaire
,13,36 or has not utilize patient questionnaires, an has efine potency by ei
ther a subjective physician interpretation of erections sufficient for vaginal p
enetration,1416,18 or without any efining criteria.9,17 Potency preservation Fol
lowing brachytherapy, wie ranges of ED have been reporte an may be the result
of ifferences in patient followup, various efinitions of ED, an the moe of
ata collection (Table 48.3). Data collection an QOL outcomes stuies shoul b
e obtaine by irect patient surveys for vali an reliable ata/information.25
In general, series with longer followup an those that have use patientamini
stere instruments have reporte
Basic an avance techniques in prostate brachytherapy
620
lower rates of potency preservation. Using the IIEF5 instrument, Merrick an co
lleagues reporte a 6 year 39% rate of potency preservation for patients unergo
ing brachytherapy with or without supplemental EBRT an/or with or
Table 48.3 Potency preservation after permanent prostate brachytherapy
Stuy Year No, patients
71 65 56 434 46 100 50 34 313 27 132 482
Potency Meian f/u Definition of preservation (mths) potency
94% at 2 yrs 94% at 2 yrs 86% at 3 yrs 85% at 4 yrs 77% at 3 yrs 62% at 5 yrs 60%
at 4 yrs 59% at 2 yrs 59% at 6 yrs 55% at 2 yrs 47% at 5 yrs 53% at 5 yrs 24 15
36 28 (m) 24 33 (m) 34 13 31 18 (m) 24 34 41 41 24 ? 03 Er
ction suffici
nt ? Er

ction suffici
nt Er
ction suffici
nt Quit
a bit V
ry much IIEF-511
Data coll
ction m
thod
? ? MD int
rvi
w ? ? ? Pt administ
r
d
Ston
9 1995 Stock10 19% Walln
r15 1996 Shark
y17 1998 Dattoli14 1996 Z
itlin16 1
998
Kit
l
y36 2002 M
rrick8 2001 Stock5 2001
Chaikin13 1996 Z
i
f
ky12 1999 Pott
rs18 M
mck6 2001 2002
181 39% at 6 yrs 181 25% at 6 yrs Schov
r7 2002 138 19% at 4 yrs f/u, follow-up;
MD, doctor; m, m
an; Pt, pati
nt.
MD phon
int
rvi
w or Pt administ
r
d 03 2/3 MD int
rvi
w, 1/3 Pt administ
r
d Er

ction MD phon
int
rvi
w suffici
nt Er
ction ? suffici
nt Er
ction MD int
rvi
w
suffici
nt IIEF-511 Pt administ
r
d IIEF-5>21 Pt administ
r
d IIEF-6>22 Pt admin
ist
r
d
without hormonal manipulation.6 In pati
nts not r
c
iving EBRT, a 52% rat
of po

t
ncy pr
s
rvation was r
port
d at 6 y
ars.6 This finding is comparabl
to prior
5 and 6 y
ar pot
ncy r
sults from Mount Sinai and M
morial Sloan-K
tt
ring for
pati
nts und
rgoing monoth
rap
utic brachyth
rapy.5,12 Using th
IIEF-6, Schov
r

t al r
port
d that 19%, 1318%, and 715% of patients undergoing brachytherapy, ra
dical prostatectomy, or external beam radiation therapy did not develop any erec
tile dysfunction.7 In contrast, Potters and colleagues, utilizing physician inte
rviews to assign potency, reported a 76% rate of potency preservation at 5 years
for patients undergoing monotherapeutic brachytherapy.18 In addition, Potters e
t al reported that 80% of brachytherapy-induced ED was apparent by 24 months,18
while two other studies reported the median time to development of brachytherapy
-induced ED was 17 months and 6 months, respectivel.6,12
Sexual function following permanent prostate brachytherapy
621
Because sexual function represents a spectrum of performance, reported rates of
potency preservation are often divergent even when the same or comparable instru
ments are used due to various thresholds of success. For example, following brac
hytherapy, Merrick and colleagues defined potency preservation as an IIEF-5 scor
e 11, which translates into the ability to successfully engage in sexual intercou
rse on almost one half of all encounters.6 In contrast, the Cleveland Clinic use
d an IIEF-6 score >22 out of 30 to select patients who did not develop any erect
ile dysfunction following potentially curative treatment. When an IIEF-5 score >
21 out of 25 was utilized to define the absence of ED following brachytherapy, t
reatment success at the Schiffler Cancer Center was comparable to that of the Cl
eveland Clinic (25% vs 19%).6,7 Following potentially curative cancer treatment,
it is uestionable whether investigators should report potency preservation as
the absence of any diminution of erectile function rather than the ability to su
ccessfully engage in sexual intercourse a certain percentage of the time. Using
patient-administered validated instruments with longer follow-up, two factors re
garding post-implant ED have become apparent. First, there is a significant inci
dence of immediate postimplant ED which is presumably related to needle trauma.
Merrick and colleagues reported that 6 of 34 patients developed severe ED (IIEF5<6 in the immediate postimplant period).8 ong-term potency in patients who exp
erienced immediate postimplant ED has not been detailed, but spontaneous improve
ment has been noted with time. The second important factor is that ED increases
with time after treatment. Series with longer follow-up have uniformly reported
lower rates of potency preservation (Table 48.3). Potency preservation rates fol
lowing all treatment approaches are significantly lower when patient-administere
d uestionnaires are used in comparison to the collection of data by physician i
nterview. The utilization of patientadministered uestionnaires following radica
l prostatectomy has resulted in dramatically differing results.7,2932 In four of
those studies, a 744% rate of potency preservation following radical prostatectom
y was reported.7,3032 In contrast, however, a study of 64 patients using a valida
ted patientadministered uestionnaire and a definition of potency as the ability
to engage in unassisted intercourse with or without sildenafil reported that 86%
of patients were potent following radical prostatectomy.29 Only 2 patients were
exclusively sildenafildependent. A possible selection bias, however, may have be
en inherent to the study because only 38% of the 59 patients who agreed to parti
cipate returned all four uestionnaires. A recent Cleveland Clinic study with a
patient participation rate of 49% and a mean follow-up of 4.3 years reported a p
otency preservation rate of 1318%.7 Factors affecting potency The mechanism of br
achytherapy-induced ED has not been definitively proven. The etiology, however,
likely represents a multifactorial process including neurogenic compromise, vasc
ular insufficiency, local trauma, and psychogenic causes.20
Basic and advanced techniues in prostate brachytherapy
622

Preimplant erectile function Stock and colleagues reported preimplant erectile f

unction to be the strongest predictor of posttreatment ED.5 Six years following
brachytherapy, 70% of patients with normal preimplant erectile function maintain
potency versus only 34% of patients with preimplant erections sufficient for in
tercourse, but considered suboptimal (Figure 48.1).
Figure 48.1 Potency preservation as a function of preimplant erectile function.
These patients were scored by Stock et al as either normal (solid line), subopti
mal, but capable of intercourse (dashed line), and no erections or erections ina
deuate for intercourse.5
Merrick and colleagues also reported that preimplant erectile function was predi
ctive of 6 year potency preservation (50% for normal erections vs 13% for preimp
lant erections considered suboptimal). Patient age Patient age may influence the
likelihood of brachytherapyinduced ED. Potters and colleagues reported that pat
ients <60, 6069, and 70 years of age had a 78%, 58%, and 50% potency preservation
rate at 5 years following brachytherapy.18 Schover et al also reported that youn
ger age was strongly associated with better sexual outcomes.7 In contrast, Merri
ck and colleagues reported that patient age was significant in univariate, but n
ot multivariate analysis (Figure 48.2),6 while Stock et al reported no relations
hip between patient age and impotence.5
Sexual function following permanent prostate brachytherapy
623
Supplemental external beam radiotherapy The addition of supplemental external be
am radiotherapy (EBRT) appears to increase the incidence of brachytherapyinduced
ED. Merrick and colleagues reported that the addition of supplemental EBRT to b
rachytherapy decreased the 6 year actuarial rate of potency preservation from 52
% to 26%.6 Potters et al reported a statistically nonsignificant reduction in po
tency when EBRT was added to brachytherapy (76% vs 56%, p=0.08).18
Figure 48.2 The effect of age at implant on potency preservation from Merrick et
al.6 The population was stratified into age cohorts of (dotted line). <60 years
(solid line), 6069 years (dashed line), and 70+ years
Neo-adjuvant hormonal manipulation Studies investigating the relationship betwee
n neoadjuvant hormonal manipulation and potency preservation have reported mixed
results. With a median follow-up of 34 months, Potters et al18 reported the use
of neo-adjuvant hormonal manipulation predicted for posttreatment ED. In patien
ts undergoing monotherapeutic brachytherapy, 76% maintained potency whereas when
neo-adjuvant hormonal manipulation was followed by brachytherapy without supple
mental EBRT, the potency preservation rate was 52%. Schover et al also reported
poor sexual outcomes with the use of neo-adjuvant hormonal therapy.7 In contrast
, two other studies failed to reveal a relationship between hormonal therapy and
ED.5,6 In univariate analysis, Stock et al reported a deleterious effect due to
neo-adjuvant hormonal therapy (potency preservation rates of 54% vs 61%, p=0.04
).5 In multivariate analysis, however, neo-adjuvant hormonal manipulation was no
t statistically significant (p=0.12). Merrick et al reported the utilization of
hormonal manipulation did not statistically affect the ultimate rate of potency
preservation (37% vs 46%, p=0.836).6
Basic and advanced techniues in prostate brachytherapy
624
Choice of isotope Of the multiple published brachytherapy potency studies, only
two have evaluated the influence of choice of isotope on potency preservation.5,
6 With 6 year results, neither study was able to discern a difference in potency
preservation based on isotope. A current ongoing prospective study comparing pa

lladium-103 (103Pd) with iodine-125 (125I) for patients with low risk prostate c
ancer (clinical stage T1b-T2a 1996 American Joint Committee on Cancer, prostatespecific antigen (AJCC, PSA) 10 ng/m, Gleason score 56) will provide invaluable i
nformation regarding the ultimate effect of choice of isotope on potency preserv
ation.23 Additional clinical and treatment parameters Of a multitude of addition
al clinical and treatment parameters evaluated, one study reported a strong corr
elation between diabetes mellitus and brachytherapy-induced ED. Patients without
and with diabetes had potency preservation rates of 49% versus 0% at 2 years an
d 41.4% versus 0% at 6 years (p=0.017).6 Mechanism of brachytherapyinduced ED Ra
diation-related impotence likely represents a multifactorial process.20 In the c
ase of brachytherapy, there is emerging evidence that ED is techniue-related an
d may be minimized by careful attention to source placement. Radiation dose to t
he prostate gland Conflicting results have been reported regarding the relations
hip between radiation dose to the prostate gland and the development of brachyth
erapy-induced ED. Stock and colleagues reported that a D90 (radiation dose deliv
ered to 90% of the prostate gland) >160 Gy for 125I or a D90 >100 Gy for 103Pd w
as predictive of brachytherapyinduced ED.5 Although statistically significant di
fferences in potency preservation were reported, the absolute differences were m
inimal (58% vs 64%, p=0.02). In contrast, Merrick et al reported no correlation
between the radiation dose delivered to the prostate gland in terms of the D90 a
nd V100/150/200 (the percent volume of the prostate receiving 100%, 150%, and 20
0% of the prescription dose) and brachytherapy-induced ED.6 Neurovascular bundle
s Following radical prostatectomy, ED has been correlated with surgical trauma t
o the neurovascular bundles (NVB).33 Excessive radiation to the NVB represents a
potential mechanism for brachytherapy-induced ED. DiBiase and colleagues report
ed that radiation doses to the neurovascular
Sexual function following permanent prostate brachytherapy
625
Figure 48.3 Schematic diagram of the location of the neurovascular bundles (NVB)
which lie approximately 2 mm outside the posterolateral lobes of the prostate.
Merrick et al. calculated additional points on a 4 mm wide ribbon to simulate th
e NVB plexus.35
bundles are in the range of 200% of the minimum prescribed dose (MPD).34 To date
, studies have failed to establish a relationship between radiation doses to the
NVB and brachytherapy-induced ED.8,35,36 Merrick and colleagues evaluated the r
adiation dose to the NVB by means of a two-dimensional geometric model based on
day 0 computed tomography (CT)-based dosimetry (Figure 48.3).8,35 Because the ex
act location of the NVB is somewhat variable, represents a plexus and not a well
-localized structure, and is not identifiable on CT, the delivered radiation dos
e to the region of the NVB was determined by both point dose measurements and do
se-surface histograms with the conclusion that the radiation dose in the evaluat
ed area is relatively homogeneic.8,35 With a median follow-up of 37 months, no r
elationship was discerned between radia tion doses to the NVB (mean dose: 217% M
PD) and the development of brachytherapy-induced ED in both retrospective and pr
ospective evaluations.8,35 Although initial reports have been negative, it is po
ssible that with longer follow-up NVB doses may be found to contribute to brachy
therapy-related ED. Penile erectile bodies There is an increasing body of data i
mplicating excessive radiation doses to the proximal penis and radiationrelated
ED.3740 The penile erectile bodies (the paired corpora cavernosa and the midline
corpus spongiosum) represent a potential candidate for sitespecific radiation do
ses related to ED (Figure 48.4).39 Followed posteriorly into the perineum, the c
orpora cavernosa separate and form the crura of the penis, which are attached to
the inferior pubic rami. Between the two crura, the corpus spongiosum enlarges
to form the bulb of the penis, which is attached superiorly to the inferior surf
ace of the urogenital diaphragm. The penile bulb is best visualized on T2-weight
ed magnetic

Basic and advanced techniues in prostate brachytherapy

626
resonance imaging (MRI) and appears as an oval-shaped hyperdense midline structu
re located 1015 mm inferior to the apex of the prostate gland (Figure 48.4).39 Wi
th day 0 CT-based dosimetric evaluation, the radiation dose delivered to 50% of
the bulb of the penis (D50) was <50 Gy and the dose delivered to 95% of the bulb
of the penis (D95) was <20 Gy for the majority of patients who maintained poten
cy while the vast majority of men who became impotent exceeded these values.37,3
8 In contrast, Kiteley et al also evaluated radiation doses to the bulb of the p
enis and were unable to correlate proximal penile doses with posttreatment ED.36
Excessive radiation doses
Figure 48.4 Transverse computed tomography (CT) and magnetic resonance imaging (
MRI) of the midbulbar region illustrating the corpora cavernosa, left and right
crura, penile bulb, pubic arch, and rectum.
to the bulb of the penis are the result of either poor planning and/or poor intr
aoperative techniue. Refinements in implant techniue, including preplanning an
d intraoperative seed placement with utilization of the sagittal ultrasound plan
e for the deposition of the apical/periapical seeds, will result in lower radiat
ion doses to the proximal penis with potential improvement in potency preservati
on. Management of brachytherapyinduced erectile dysfunction Patients with radiat
ion-related ED typically respond well to conventional erectile aids with improve
d erectile function and overall uality of life.41 Mulhall reported that a lack
of erectile activity may be deleterious to erectile function and, as such, patie
nts should be encouraged to develop regular erections with or without sexual rel
ations.42 In the absence of routine penile erections, the corporal smooth muscle
experiences chronic hypoxia with resultant loss of elasticity and distensibilit
y which may lead to a venous leak. Based on the premise that erections enhance t
issue oxygenation and suppress smooth muscle fibrosis, therapy to enhance noctur
nal erections (night-time physical therapy for the
Sexual function following permanent prostate brachytherapy
627
penis) might have a therapeutic benefit.43 Montorsi et al demonstrated that sild
enafil (Viagra), but not placebo, taken at bedtime produced a significant improv
ement in nocturnal erectile activity.44 This concept of night-time physical thera
py could also potentially reduce brachytherapy-induced ED. The majority of patien
ts with brachytherapy-induced ED respond favorably to sildenafil citrate.45 In a
brachytherapy population, the 6 year actuarial rate of potency preservation was
92% when potent patients were grouped with ED patients who used sildenafil citr
ate (Figure 48.5).6 The postimplant response to sildenafil was highly dependent
on preimplant potency.6 Patients with normal erections and erections sufficient
for vaginal penetration but considered suboptimal responded favorably to sildena
fil in 95% and 70% of cases, respectively.6 Potters et al reported that 83% of p
atients who underwent prostate brachytherapy with or without supplemental EBRT r
esponded favorably to sildenafil, while only 46% of patients who received neo-ad
juvant hormonal manipulation responded to such pharmacologic support. Radiationinduced ED also responds to vasoactive agents and penile implants.46,47 Conclusi
ons Brachytherapy-induced erectile dysfunction (ED) is more common than initiall
y reported. Although the etiology of brachytherapy-induced ED is likely multifac
torial, the
Figure 48.5 Potency preservation in a brachytherapy cohort with and without the
support of sildenafil citrate (Viagra). (Adapted from Merrick et al).6
available data strongly support the proximal penis as an important site-specific
structure. Some, but not all studies have also reported preimplant potency, pat

ient age, diabetes mellitus, the use of supplemental external beam radiotherapy,
and radiation doses to the
Basic and advanced techniues in prostate brachytherapy
628
prostate gland to be predictive for the development of posttreatment ED. The maj
ority of patients with brachytherapy-induced ED respond favorably to sildenafil.
In order to obtain the most reliable uality of life information, potency data
should be collected by validated patient-administered instruments. Continued att
empts to refine patient selection, brachytherapy planning philosophies, intraope
rative techniue, and postimplant management may result in improved rates of pot
ency preservation, improved treatments for ED and ultimately, better uality of
life outcomes. References
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, ee WR, deGuzman AF, et al. Radiation dose to the neurovascular bundles or pen
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n of radiation dose to the bulb of the penis in men with and without prostate br
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KE, Merrick GS, Benson M, et al. Penile bulb imaging. Int J Radiat Oncol Biol P
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40. Mulhall JP, Yonover P, Sethi A, et al. Radiation exposure to the corporeal b
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life and sexuality following radical prostatectomy in patients with prostate can
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Minimizing radiation-induced erectile dysfunction. J Brachyther Int 2001; 17:22
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rostate brachytherapy patients with erectile dysfunction. Urology 1999; 53:111211

16. 46. Pierce J, Whittington R, Hanno PM, et al. Pharmacologic erection with i
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49 Prostate-specific antigen bounce following prostate brachytherapy
Frank A Critz Introduction Prostate-specific antigen (PSA) bounce is an extremel
y interesting and very common phenomenon that has been recognized in men treated
for prostate cancer with brachytherapy,15 as well as external beam irradiation a
lone.6,7 PSA bounce is defined by a temporary postirradiation PSA rise of benign
etiology.1 PSA bounce causes two major issues: anxiety in men treated with irra
diation for prostate cancer and a problem for physicians who must sort PSA bounc
e from treatment failure. In our experience, anxiety caused by bounce can be min
imized by educating men about this issue before and after irradiation for prosta
te cancer. Conseuently, all physicians who have contact with men given irradiat
ion for prostate cancer should be aware of PSA bounce. This report describes PSA
bounce after brachytherapy for prostate cancer. Materials and methods From 1992
to 1998, 1658 men with clinical stage T1T2, Nx, M0 prostate cancer were treated
with simultaneous irradiation (SI), a radioactive iodine-125 (125I) seed implant
with the ultrasound-guided transperineal implant techniue followed by external
beam radiotherapy (EBRT). Men who received neo-adjuvant androgen deprivation we
re excluded from this analysis. The techniue of irradiation has been previously
described. PSA bounce is defined by a PSA increase of 0.1 ng/m or more, above
the PSA level before the bounce followed by a subseuent decrease to or below th
e prebounce nadir with PSA 0.2 ng/m as the floor. PSA fluctuations below PSA 0.
2 ng/m are not considered a bounce. For example, a PSA change from 0.1 to 0.2 t
o 0.1 ng/m is not a bounce; whereas, an increase from 0.2 to 0.3 ng/m or more
and back to 0.2 ng/m is defined as a bounce. All men in this study were treated
five or more years ago. Follow-up was performed three months postimplant, three
months later, and every six months thereafter. Men were not changed to annual f
ollow-up. Although PSAs were often obtained on a more freuent basis than every
six months when trying to sort bounce from recurrence, only PSAs obtained at the
above follow-up schedule were used in this study. PSA measurements were obtaine
d from a variety of laboratories using several different PSA assay techniues.
Basic and advanced techniues in prostate brachytherapy
632
Results Figure 49.1 describes a hypothetical case of PSA bounce which is used to
define various terms associated with bounce. Prebounce nadir means the lowest P
SA achieved prior to onset of PSA bounce. The first PSA rise of 0.1 ng/m or mor
e above the prebounce nadir defines the onset of PSA bounce and duration of PSA
bounce is defined by the time from onset to return of PSA to the prebounce nadir
level or lower. PSA bounce peak means the highest PSA achieved during bounce an
d bounce height is defined by bounce peak less prebounce nadir. A second bounce
is defined by a subseuent PSA rise after return to the initial PSA bounce nadir
. Calculated at the time of onset, the overall incidence of PSA bounce in this s
tudy is 45% (746/1658) (Figure 49.2). Figure 49.3 demonstrates the incidence of
bounce, calculated from the time of onset, according to age of men at implantati
on. Figure 49.4 evaluates the time to onset of bounce for the 746 men who experi
enced a bounce. Of bounce cases, 99% have an onset within 60 months of implantat
ion. Only 1% (7/746) had a bounce onset at 66 months or more follow-up. Figure 4
9.5 documents the bounce height of all men who experienced a bounce in this stud
y. The median height is 0.3 ng/m (0.1 11.8 ng/m) and 17% had a bounce height of
1.1 ng/m. Bounce duration is documented in Figure 49.6, including the median an
d range of bounce height according to duration. As noted, bounce duration is dir
ectly related to bounce

Figure 49.1 A hypothetical case of prostate-specific antigen (PSA) bounce used t

o define terms associated with PSA bounce called bounce characteristics.
Prostrate-specific antigen bounce
633
Figure 49.2 The time distribution of PSA bounce calculated at the time to onset
of bounce based on evaluation of all 1658 men in this study.
Figure 49.3 The incidence of bounce, calculated from the time to onset, accordin
g to age of men at implantation. Younger men have a
Basic and advanced techniues in prostate brachytherapy
634
significantly higher freuency of PSA bounce than older men.
Figure 49.4 Of the 746 men who experienced a bounce, this histrogram documents t
he time to onset of bounce. As noted, 3% of men who have a bounce experience thi
s phenomenon at the first PSA after treatment, 68% of bounces have an onset betw
een 12 and 24 months postimplant and 99% of bounces have an onset within 60 mont
hs of implantation. Only 1% of men who had a bounce had an onset at 66 months or
more following implantation.
Prostrate-specific antigen bounce
635
Figure 49.5 Bounce height for the 746 men in this study who had a PSA bounce. Th
e median bounce height is 0.3 ng/m (0.111.8 ng/m).
Figure 49.6 Duration of PSA bounce for the 746 men who experienced a bounce base
d on PSA obtained at 6 month intervals from implantation, except for the initial
PSA at 3 months postimplant. Additionally, the median and range of bounce heigh
ts is provided for bounce duration which
Basic and advanced techniues in prostate brachytherapy
636
shows a direct correlation between duration and median bounce height.
height. The prebounce nadir is evaluated in Figure 49.7. The disease-free surviv
al rate, calculated with PSA cutoff point of 0.2 ng/m, is demonstrated in Figur
e 49.8. This information is inaccurate due to the bias caused by men who experie
nce a bounce because men who have an early recurrence will not have the opportun
ity to bounce. Inherently, men who have a bounce should always have a significan
tly higher diseasefree survival rate. When adjustment is made for the bias cause
d by bounce men, disease freedom from prostate cancer is unrelated to bounce. Di
scussion To analyze prostate-specific antigen (PSA) bounce, only men treated fiv
e or more years ago should be evaluated.
Figure 49.7 Prebounce nadir for the 746 men who experienced a bounce. The median
prebounce nadir was 0.7 ng/m (0.18.9 ng/m).
Prostrate-specific antigen bounce
637

Figure 49.8 Disease freedom according to whether or not men experienced a PSA bo
unce. Although calculated correctly, the findings in this figure are inaccurate
and misleading due to bias caused by bounce men. Men who have an early recurrenc
e will not have a chance to have a PSA bounce. Conseuently, men who have a boun
ce will inherently have a better disease-free survival rate than men who do not
experience a PSA bounce. After adjustments for the bias factor created by bounce
, there is no significant difference in disease freedom between men who have a b
ounce and men who do not have a bounce.
Also, the freuency of PSAs to be evaluated should be standardized. In this repo
rt, all men were treated 5 or more years ago and only PSAs obtained at 6 month i
ntervals, based on the date of implantation, were evaluated except for the initi
al PSA three months postimplant. PSA bounce is a very common phenomenon. Overall
, 45% of men experience a PSA bounce (Figure 49.2) with a peak time to onset at
18 months postimplantation (Figure 49.4) and 68% of men have a bounce onset betw
een 12 and 24 months postimplant. Onset of PSA bounce is rare after 60 month fol
low-up and occurred in only 1% of men in
Basic and advanced techniues in prostate brachytherapy
638
this study. The freuency of PSA bounce is directly related to a mans age at impl
antation (Figure 49.3) and is 58% for men aged 60 at implant compared to a bounce
freuency of only 26% in men aged 71 or more. Sixty-five percent of men have a
bounce duration of 6 months, a single PSA rise (Figure 49.6). However, 35% of me
n have a bounce duration between 12 and 30 months and bounce duration is directl
y related to bounce height. Consistent with the time to onset of PSA bounce, the
median prebounce nadir is 0.7 ng/m (Figure 49.7). Although the median prebounc
e height in this study is only 0.3 ng/m (Figure 49.5) and does not appear impre
ssive, a PSA rise of any magnitude can cause considerable anxiety in men who are
expecting a fall in PSA after brachytherapy for prostate cancer. The peak bounc
e height in this study is 16.0 ng/m and 15% of men have multiple PSA bounces. V
arious clinical factors have been evaluated to see if there is any relationship
to PSA bounce. Neither pretreatment PSA, Gleason score, clinical stage, race, pr
ostate size, nor implant dose is related to PSA bounce.2 The only clinical facto
r related to PSA bounce is patient age at implantation. In fact, age of men has
a dramatic effect on PSA bounce. Although intuitively one might think that older
men with enlarged prostates might have a higher freuency of PSA bounce, analys
is of this database shows that PSA bounce is much more common in younger men. Of
men aged 60, 58% had a bounce compared to 40% of men aged 6170 and 26% of men age
d 71. Additionally, young men have an earlier onset of bounce, the duration is lo
nger, the bounce height is higher and have more multiple bounces than older men.
2 In other words, most men aged 60 will have a PSA bounce and bounce characterist
ics are much worse in younger men than older men. The etiology of PSA bounce is
unknown. We have noted a recurrence of urinary symptoms postimplantation that ro
ughly corresponds to the time of onset of PSA bounce which may reflect delayed i
nflammation of the prostate. Additionally, we have noted a relationship of sexua
l function to the incidence of PSA bounce which may partially explain why young
men have a higher bounce freuency than older men. However, the majority of case
s of bounce are not related to any known factor. One of the most important issue
s of PSA bounce is the relationship to disease freedom. Based on previous analys
is of our data,1,2 as well as other reports,35 PSA bounce is not related to treat
ment failure. Comparison of diseasefree survival (DFS) curves between bounce and
no bounce men will inherently show that men who experience a bounce have a bett
er DFS rate (Figure 49.8). However, this is due to the bias caused by PSA bounce
and when adjustments are made for this bias, no difference is noted in DFS rate
according to whether or not men have a bounce. In fact, all evidence indicates
that PSA bounce is unrelated to prostate cancer but is due to an effect on benig
n prostate epithelium prior to destruction by SI. No pretreatment clinical facto

rs of prostate cancer, except for patient age, are related to bounce.2 Additiona
lly, PSA bounce is rare after 5 year follow-up. Only 1% of men who had a bounce
have an onset after 5 year follow-up. This correlates with the finding that of t
he men who achieve PSA 0.2 ng/m after SI, 99% will do so by 5 year follow-up.8
Achievement of PSA nadir 0.2 ng/m suggests destruction of most, if not all, ben
ign prostate epithelium for this is the same PSA goal for a successful radical p
rostatectomy. Distinguishing PSA bounce from recurrence of prostate cancer is an
other major issue. Except for close PSA followup, there is no known method to so
rt bounce from recurrence. Malignant epithelium may resolve slowly from a histol
ogical standpoint after
Prostrate-specific antigen bounce
639
irradiation, which may confound interpretation of prostate biopsy.9 Several case
s of falsepositive prostate biopsies have been documented in men who have had a
bounce.10 The characteristics of bounce (Figure 49.1) appear to be unrelated to
monotherapy with either 125I or 103Pd implantation or EBRT before or after seed
implant for prostate cancer.3 Variation in reporting of bounce characteristics m
ay be due to different definitions of bounce, studies with less than a 5 year fo
llow-up and variation in freuency of PSA follow-up. In this study, PSA bounce i
s defined as a rise of 0.1 ng/m. Other investigators of brachytherapy have defi
ned bounce by a rise of 0.2 ng/m, 0.4 ng/m, or a percent increase in PSA.35 Def
ining bounce by a rise of only 0.1 ng/m could be criticized as being within the
background noise of the PSA assay. However, the time distribution of men with a
PSA rise of 0.1 ng/m compared to 0.2 ng/m or more is similar instead of a ran
dom event as one might expect if a PSA rise of 0.1 ng/m was due to background n
oise.2 Conclusions Prostate-specific antigen (PSA) bounce is defined as a tempor
ary PSA rise caused, it is believed, by an effect on benign epithelium and is un
related to either the cure of prostate cancer nor any clinical factors except fo
r age of men at implantation. Young men have a higher freuency and worse bounce
characteristics than older men, which is particularly disconcerting because you
ng men would be more favorable candidates for local salvage treatment should the
re be local recurrence. Except for close PSA follow-up, there is no known way to
distinguish bounce from treatment failure. References
1. Critz FA, Williams WH, Benton JB, et al. Prostate specific antigen bounce aft
er radioactive seed implantation followed by external beam radiation for prostat
e cancer. J Urol 2000; 163:1085 1089. 2. Critz FA, Williams WH, evinson AK, et a
l. Prostate specific antigen bounce after simultaneous irradiation for prostate
cancer: The relationship to patient age. J Urol 2003; 170:18641867. 3. Cavanagh W
, Blasko JC, Grimm PD, Sylvester JE. Transient elevation of serum prostate-speci
fic antigen following 125I/103Pd brachytherapy for localized prostate cancer. Se
min Urol Oncol 2000; 18:160165. 4. Merrick GS, Butler WM, Wallner KE, et al. Pros
tate-specific antigen spikes after permanent prostate brachytherapy. Int J Radia
t Oncol Biol Phys 2002; 54:450456. 5. Stock RG, Stone NN, Cesaretti JA. Prostatespecific antigen bounce after prostate seed implantation for localized prostate
cancer: Descriptions and implications. Int J Radiat Oncol Biol Phys 2003; 56:4484
53. 6. Hanlon A, Pinover WH, Horwitz EM, Hanks GE. Patterns and fate of PSA bou
ncing following 3D-CRT. Int J Radiat Oncol 2001; 50:845849. 7. Rosser CJ, Kuban D
A, evy B, et al. The prostate specific antigen bounce phenomenon after externa
l beam radiation for clinically localized prostate cancer. J Urol 2002; 168:20012
005. 8. Critz FA. Time to achieve PSA nadir 0.2 ng/ml following simultaneous irr
adiation of prostate cancer. J Urol 2002; 168:24342438.
Basic and advanced techniues in prostate brachytherapy
640
9. Crook JM, Perry GA, Robertson S, et al. Routine prostate biopsies following r

adiotherapy for prostate cancer: results for 226 patients. Urology 1995; 624:45.
10. Smathers S, Wallner K, Sprouse J, True . Temporary PSA rises and repeat pr
ostate biopsies after brachytherapy. Int J Radiat Oncol Biol Phys 2001; 50:120712
11.
50 Factors predicting for urinary incontinence following prostate brachytherapy
Tracy  McElveen, Frank M Waterman, Hayeon Kim, and Adam P Dicker Introduction P
ermanent prostate brachytherapy has evolved over the last decade as a treatment
option for early stage prostate cancer. Excellent five year biochemical control
rates have been shown, suggesting that radical prostatectomy, threedimensional c
onformal radiation therapy, and brachytherapy are relatively eually effective f
or the treatment of favorable risk disease.19 Issues about uality of life are in
creasingly important to patients faced with choosing a treatment modality and to
the physicians who counsel them. Quality of life issues often include rectal bl
eeding or diarrhea, sexual dysfunction, irritative urinary symptoms, and urinary
incontinence. Although the majority of papers have focused on the obstructive u
rinary symptoms patients have experienced, little has been published regarding u
rinary incontinence. Urinary incontinence has been associated with radical prost
atectomy and according to the National Medicare Experience over 47% of patients
have reported some degree of incontinence following this procedure.10 Rates over
a broad range of 040% following brachytherapy have been reported.4,5,1115 Many re
ports however, contain physician-acuired information, which has been shown to c
orrelate poorly with data collected with patient self-assessment uestionnaires.
10,1620 Much of the published literature on this topic lacks dosimetric informati
on and does not specifically include urinary incontinence in the grading scale.
Attempts to establish widespread use of validated self-assessment uestionnaires
to adeuately address incontinence have been unsuccessful to date. Although the
incidence of incontinence after brachytherapy is likely less than after radical
prostatectomy, it may not be as low as presently believed. We evaluated the inc
idence of urinary incontinence and attempted to determine predictive factors for
this complication. Method Our study cohort consisted of 153 consecutive patient
s with early stage prostate cancer implanted by one physician (APD) at our insti
tution from October 1996 through December 2001, giving a median follow-up of 47
months (range: 1474). Patient characteristics included a prostatespecific antigen
(PSA)10, Gleason score (GS)6, and stageT2b. Patients who received external beam ra
diotherapy (EBRT) and patients implanted with palladium103 (103Pd) were excluded
. All patients were evaluated with a history and physical examination, baseline
International Prostate Symptom Score (IPSS),
Basic and advanced techniues in prostate brachytherapy
642
and an internal pathologic review of biopsy specimens. Seven of these patients r
eceived approximately three months of androgen suppression for glandular downsiz
ing and were included in our analysis. After obtaining institutional review boar
d approval, a survey including a selfassessment uestionnaire and an IPSS form w
as sent to each of the 153 patients. The American Brachytherapy Society (ABS) re
commends that the IPSS be used for reporting urinary morbidity and that incontin
ence, dysuria, gross hematuria, and urinary retention be uantified using the Na
tional Cancer InstituteCommon Toxicity Criteria (NCICTC).2123 ABS also recommends
the use of patient-administered instruments that report actuarial and crude inc
idences of these toxicities. In an effort to comply with these recommendations w
e used the NCI-CTC version 2 to formulate a patient selfassessment uestionnaire
. For other adverse events some words were simplified, but for urinary incontine
nce, the scale appeared on the survey in the exact language used in the NCICTC v
ersion 2: grade 0 is no incontinence; grade 1 includes incontinence with coughin
g, sneezing, laughing; grade 2 includes spontaneous incontinence with some contr
ol; and grade 3 is no control. This is not a selfassessment tool, however; no se
lf-assessment instrument currently exists to provide a measure of the incidence

of incontinence in patients treated with brachytherapy. Because the exact langua

ge of the scale for incontinence was used in the survey, we believed that using
the NCICTC version 2 was the best way to report a crude incidence of incontinenc
e, while using the ABS recommended tool, and the recommended patient self-assess
ment format. Patients who reported any degree of incontinence were contacted by
phone and interviewed to determine baseline urinary continence. Preimplant compu
ted tomography (CT) scans were obtained with a foley catheter in place, which wa
s used to assess prostate volume, to evaluate for pubic arch interference, and f
or treatment planning. The prostate and intraprostatic urethral volumes were con
toured on each slice by the same individual (FMW). The implants were planned (Va
riseed, Varian, Palo Alto, CA) to deliver a minimum dose of 145 Gy to the gland
plus a symmetric 35 mm margin. 125I seeds, 0.40.6 mCi, National Institute of Stand
ards and Technology (NIST-99), were loaded peripherally keeping the seeds within
the prostate except at the apex. This techniue generated a broad dose minimum
in the central portion of the prostate encircled by a high dose region to promot
e urethral sparing. Implants were performed under general anesthesia with a fole
y catheter in place to visualize the urethra. The earliest implants, which inclu
ded approximately half of those in this study, were performed under template and
fluoroscopic guidance using a techniue similar to that described by Wallner et
al.24 The more recent implants were performed with transrectal ultrasound and f
luoroscopic guidance. Transperineal needles were placed according to the plan an
d seeds were implanted using a Mick applicator (Mick RadioNuclear Instruments, In
c, Mount Vernon, NY). In earlier years, patients were admitted overnight for obs
ervation, but more recently, were discharged the day of the procedure with the f
oley catheter in place. Catheters were self-removed the following morning. Posto
perative dosimetry was calculated in accordance with ABS guidelines from a CT sc
an performed approximately 30 days after the procedure to allow for resolution o
f prostatic edema (mean: 37.126.4 days; range: 0195 days).25 The prostate and intr
aprostatic urethral volumes were contoured on each slice by the same individual
(FMW) who also localized the implanted seeds. A urinary catheter was used for th
e
Factors predicting for urinary incontinence
643
preimplant CT scan, but not for most postimplant CT scans, which made the urethr
a more difficult to localize. At the time of the postimplant CT scan, most of th
e edema had resolved so that the pre- and postimplant volumes were similar. The
urethra was localized as a 45 mm diameter circle based on its location in the pre
implant CT scan. This localization was done on an image-by-image basis while vie
wing both studies simultaneously. A dose-volume histogram (DVH) of the volume de
fined by the contours was compiled to evaluate the urethral dose. We acknowledge
that there is a certain degree of uncertainty in the location of the urethra an
d in the urethral dose because of the absence of a urinary catheter. However, we
showed in a prior study that the urethral dose, defined as the D10, D25, or D50
is relatively insensitive to the location of the urethra when the 125I seeds ar
e peripherally loaded so as to produce a broad dose minimum in the central porti
on of the prostate.26 In that study, the dose to the urethra identified by a fol
ey catheter was compared with a surrogate urethra that was localized at the geom
etric center of the prostate. The surrogate urethral D10, D25, or D50 were highe
r by 3.55.5%, 1.06.0%, and 2.37.1%, respectively. ee et al recently showed similar
results with a surrogate urethra contoured on CT scans obtained 30 days postimp
lant.27 In this study, the urethra in the postimplant CT scan was localized at t
he approximate location of the urethra in the preimplant CT scan instead of at t
he geometric center of the prostate. Thus, we would expect the uncertainty in th
e postimplant urethral doses in this study to be even less than in the previous
studies using surrogate urethras. Eight patients received their CT scan on day 0
, the day of the procedure, instead of day 30. We showed previously that the ure
thral dose is significantly underestimated by day 0 dosimetry.28 More specifical

ly, we reported that the urethral D10, D25, D50, D75, and D90 doses increased, o
n average, by 90, 81, 67, 49, and 40 Gy, respectively, between day 0 and day 46
postimplant. Therefore, the urethral doses of patients who received their CT sca
n on day 0 were normalized to those who received a later scan by increasing the
urethral doses accordingly. Postimplant dose-volume histograms (DVHs) of the pro
static urethra were compiled for each patient and the D5, D10, D25, D50, D75, an
d D90 urethral doses were recorded. These are defined as the doses that encompas
s 5, 10, 25, 50, 75, and 90% of the volume of the urethra, respectively. Postimp
lant DVHs also were compiled for the prostate. The prostate dosimetric parameter
s recorded were V100, V200, and V300 the percentages of the prostate volume that
received a dose eual to or greater than 100%, 200%, and 300% of the prescribed
dose, respectively, and D90, the minimal dose delivered to 90% of the prostate
volume. In addition, the prostate volume, total activity of the implant, number
of needles, number of seeds, and seed activity also were recorded. Clinical char
acteristics recorded included age, stage, Gleason score, pretreatment PSA, preim
plant IPSS, length of follow-up, and the most recent IPSS collected in the surve
y. Of the 153 patients surveyed, 4 patients died of other causes and 112 (75%) r
esponded. Of these, 37 reported grade 1 or grade 2 and no patient reported grade
3 incontinence. Four patients indicated that they had some degree of incontinen
ce prior to their implant and these patients
Basic and advanced techniues in prostate brachytherapy
644
Table 50.1 Univariate analysis of clinical parameters
Parameter All patients Grade 0 Grades 1,2 p-value (n=108) (n=75) (n=33) (t-test)
Age at implant (yrs) 656 656 657 0.759 Gleason score 5.90.8 5.80.9 6.00.8 0.136 PSA 6.
21.8 6.41.7 5.82.0 0.200 Prostate volume (cc) 42.012.9 42.4 12.1 41.3+14.5 0.659 Foll
ow-up (mths) 4517 4317 50417 0.019 Preimplant IPSS 7.85.5 6.64.5 10.06.4 0.005 Postimp
lant IPSS IPSS 8.56.6 6.95.5 11.47.4 0.002 PSA, prostate-specific antigen; IPSS, In
ternational Prostate Symptom Score.
were deleted from the analysis leaving a total of 108 patients, 33 of whom state
d that their leakage began after prostate brachytherapy. Of the 33 patients, 28
(26%) reported grade 1 incontinence and 5 (5%) reported grade 2. Because the num
ber of patients who reported grade 2 was so small, the data for grades 1 and 2 w
ere combined into a single group for univariate and multivariate analysis. The r
esults of a univariate analysis of the clinical parameters of the patients that
did and did not experience incontinence are listed in Table 50.1. The two groups
were homogeneous for age, Gleason score, PSA, and prostate volume. In comparing
patients with incontinence to patients without, there was a significant differe
nce in the length of follow-up (p=0.019). However, as shown in this study, the i
ncidence of incontinence decreased markedly between 1997 and 2001 as a result of
a similar annual reduction in the urethral dose. Thus, we attribute the differe
nce noted in the length of follow-up to the fact that the majority of patients r
eporting incontinence were implanted during the early years of our study when ur
ethral doses were much higher. There also was a significant difference in the me
an preimplant IPSS of the patients who did (10.06.4) and did not (6.64.5) experien
ce incontinence (p=0.003). Mean postimplant IPSS is also listed in Table 50.1. T
he mean postimplant IPSS of the patients experiencing incontinence (11.47.4) not
only remained significantly greater (p=0.002) than patients who did not (6.95.5),
but no significant difference between the preand postimplant IPSS values was no
ted. This indicates that the patients in our study returned to their respective
baseline IPSS within the varying lengths of follow-up. Table 50.2 lists the resu
lts of the univariate analysis of the dosimetric parameters. There was no signif
icant difference in total activity implanted, number of needles used for implant
ation, and uality of the implant as defined by the prostate D90, V100, and V200
values. There was a significant difference however, in the value of V300, the p

ercentage of the prostate volume that received at least 300% of the prescribed d
ose. This is consistent with the finding described below that patients experienc
ing incontinence received a significantly higher urethral dose. Table 50.2 also
indicates that the difference in the number of seeds implanted was marginally si
gnificant (p=0.043). We attribute this to the fact that we used fewer high stren
gth seeds to implant the same prostate volume during the early years of this stu
dy when the majority of the incidences of incontinence occurred. We noted a sign
ificant difference in the urethral doses of the patients who did
Factors predicting for urinary incontinence
645
and did not experience incontinence. The urethral D5, D10, D25, D50, D75, D90 do
ses of the patients experiencing incontinence all were significantly higher, as
indicated by the pvalues in Table 50.2. In a multivariate analysis, the preimpla
nt IPSS and the urethral D10 dose were the only parameters that remained signifi
cant (p=0.003 and p=0.002, respectively). Results Urethral dose The urethral dos
e-volume histograms (DVH) of the patients who did (grades 1, 2) and did not (gra
de 0) experience incontinence were separately averaged and are plotted in Figure
50.1. This figure shows that the patients experiencing incontinence received a
significantly higher dose at all levels of coverage (Table 50.1). The most signi
ficant difference was noted at the D10 level where the mean urethral D10 dose of
patients with grade 0 was 314 78 Gy, compared with 394147 Gy for patients who exp
erienced incontinence (p=0.002). The incidence of incontinence is plotted in Fig
ure 50.2 as a function of the urethral D10 dose. These data are listed
Table 50.2 Univariate analysis of dosimetric parameters
Parameter All patients Grade 0 Grades 1, 2 p-value (n=108) (n=75) (n=33) (t-test
) Total activity (mCi) 47.410.3 47.310.1 47.610.8 0.891 No, needles 16.23.5 16.03.6 1
6.63.2 0.362 No. seeds 88.419.4 90.920.0 83.516.0 0.043 Prostate D90(Gy) 182.335.8 18
0.834.2 185.341.5 0.522 Prostate V100(%) 94.55.8 94,4 5.0 94.7 6.0 0.825 Prostate V20
0 (%) 51.616.4 49.5+16.5 55.413.9 0.075 Prostate V300 (%) 21.111.6 19.29.9 24.513.5 0
,024 Urethral D5 (Gy) 351122 32583 411170 0.003 Urethral D10 (Gy) 338110 31478 394147
0.002 Uretrtral D25(Gy) 31795 29771 363124 0.003 Urethral D50 (Gy) 28276 26758 31798 0
.004 Urethral D75 (Gy) 22957 21950 25267 0,009 Urethral D90 (Gy) 18049 171 43 19959 0.
010
Basic and advanced techniues in prostate brachytherapy
646
Figure 50.1 A plot of the averaged urethral dose-volume histograms (DVH) of pati
ents who experienced grade 0 and grades 1, 2 urinary incontinence following perm
anent prostate brachytherapy.
Figure 50.2 The incidence of urinary incontinence as a function of the urethral
D10 dose.
Factors predicting for urinary incontinence
647
Table 50.3 The incidence of incontinence as a function of the urethral D10 dose
D10 (Gy) Grade 0 Grades 1, 2 Incidence (n=75) (n=33) (%)
150249 250349 350449 450 13 41 14 7 4 12 7 10 2410 236 3310 5912
in Table 50.3. Figure 50.2 illustrates that the incidence of incontinence increa
sed with the urethral D10 dose. Although there is no clear dose threshold, the i
ncidence of incontinence increased noticeably when the D10 dose exceeded 350 Gy,

increasing to nearly 60% when the dose exceeded 450 Gy. The first three bars in
Figure 50.2 represent dose increments of 100 Gy. The last bar includes all of t
he patients who had a D10 dose 450 Gy and extends to 810 Gy, the highest D10 dose
observed. These patients were grouped together because there were only 17 patie
nts with a D10 dose 450 Gy. The error bars represent the standard deviation in th
e percentage of patients who experienced incontinence, which was calculated as =pq
/n where p and q were the percent of patient who did and did not experience inc
ontinence, re pectively, and n wa the total number of patient in that do e int
erval. Preimplant IPPS The incidence of incontinence i plotted in Figure 50.3 a
a function of the preimplant IPPS. The e data are li ted in Table 50.4. Thi f
igure indicate that the incidence of incontinence wa relatively in en itive to
the preimplant IPSS in the range of 014. When the IPPS i 15 or greater, however
, the incidence of incontinence increa ed harply to about 70%. The e re ult h
ow that patient with an IPSS15 are at a much higher ri k of incontinence. The fi
r t three bar in Figure 50.3 repre ent an IPSS increment of 5. The la t bar wa
extended to 1524 becau e there were only 14 patient with an IPSS15. Multivariate
analy i indicated that the urethral D10 do e and the preimplant IPSS are both
predictive of po timplant incontinence. Figure 50.3 include patient who may be
incontinent a a re ult of the urethral D10 do e, and doe not olely reflect t
he incontinence attributable to the IPSS. Similarly, the incidence of incontinen
ce plotted in Figure 50.2 doe not olely reflect the incontinence attributable
to the D10 do e. Although we cannot completely eparate the influence of the e t
wo variable , we can reduce the influence of the
Ba ic and advanced technique in pro tate brachytherapy
648
Figure 50.3 The incidence of urinary incontinence a a function of the preimplan
t IPSS: American Urological A ociation (AUA) core. Table 50.4 The incidence of
incontinence a a function of the preimplant IPPS
IPPS Grade 0 Grade 1, 2 Incidence (n=75) (n=32) (%)
04 59 1014 1524 30 26 15 4 9 23 7 8 247 5 25  10 10 71+12
D10 do e by deleting patient with a D10 do e 350 Gy from Figure 50.3. A hown i
n Figure 50.4, thi reduced the incidence of incontinence from about 25% to 12%
for patient with an IPSS <10, but did not alter the incidence of incontinence f
or patient with an IPSS 15. Thi finding trongly ugge t that patient with a
preimplant IPSS 15 are at a high ri k for incontinence regardle of the urethral
do e, while patient with an IPSS < 10 are at minimal ri k unle they receive
a high urethral do e. Becau e only five patient reported grade 2 incontinence,
there were in ufficient data to differentiate between the ri k factor for grade
1 and grade 2 incontinence. However, it i intere ting to note that four of the
five patient had either a very high urethral D10 do e or a high preimplant IPS
S. Two patient had urethral D10 do e of 580 Gy and 810 Gy and two patient had
preimplant IPSS of 17 and 20. The remaining patient had a D10 do e of 285 Gy an
d a preimplant IPSS of 10. The e re ult ugge t that the everity of incontinen
ce i related to the urethral do e and the preimplant IPSS.
Factor predicting for urinary incontinence
649
A total of 41 patient did not re pond to the urvey. However, the preimplant cl
inical parameter and the po timplant do imetry of the e patient did not differ
ignificantly (p0.3) from the patient that re ponded to the urvey. Mo t notabl
y, their mean urethral D10 do e wa 34691 Gy and their mean preimplant IPSS wa 9
.27.6. No incontinence wa documented on the chart of the e patient ; however, u
ntil the finding of thi tudy, we did not routinely a k about urinary leakage
on follow-up vi it . Incidence of incontinence v year of implantation Figure 50

.5a how the incidence of incontinence in our patient a a function of the yea
r that the implant wa performed. We began our implant program in October 1996,
and included thi abbreviated year in the 1997 cohort. A high incidence of incon
tinence (52%) i noted in the patient implanted in 1997. The incidence of incon
tinence remained above 30% in 1998 and 1999 and then decrea ed
Figure 50.4 The incidence of urinary incontinence a a function of the preimplan
t IPSS (AUA core) in the patient population having a urethral D10 do e 350 Gy.
Ba ic and advanced technique in pro tate brachytherapy
650
markedly to an average of 12% for the year 2000 and 2001. The overall decrea e
in the incidence of incontinence i attributed primarily to the reduction in the
average urethral D10 do e during the e year , which i plotted in Figure 50.5b.
The average D10 do e wa approximately 450 Gy in 1997, but only half that value
in 2001. Over the year a con ciou effort wa made to reduce the urethral do e
, a more information became available that a ociated urethral do e with morbid
ity. Another contributing factor i the reduction in the fraction of patient wi
th preimplant IPSS15. Figure 50.5c how the percent of patient implanted each y
ear who had an IPSS15. Approximately 25% of the patient in 1997 had a preimplant
IPSS15, but only 57% of the patient met that criterion in 2000 and 2001. A marke
d decline in both ri k factor , the urethral D10 do e and the IPSS wa noted ove
r the 5 year included in thi tudy, re ulting in a ignificant decline in the
incidence of incontinence. Our clinical experience, a well a the re ult hown
in Figure 50.250.4, indicate that the incidence of incontinence following brach
ytherapy can be minimized by patient election and by keeping the urethral do e
a low a po ible. Pro tate brachytherapy ha evolved con iderably ince it in
ception and ha demon trated excellent clinical outcome ; comparable with radica
l pro tatectomy and external beam radiotherapy (EBRT). De pite it ucce , a co
n iderable amount of controver y continue to exi t and long term data are now b
eing recorded regarding morbidity, do imetry, and quality of life. We generated
a elf-a e ment que tionnaire ba ed on the National Cancer In tituteCommon Tox
icity Criteria (NCI-CTC), ver ion 2 to evaluate urinary incontinence in our pati
ent population and found a 31% incidence of incontinence. We evaluated patient a
nd do imetric parameter to determine predictive factor for thi complication a
nd found that both the magnitude of the urethral D10 do e and the magnitude of t
he preimplant IPSS correlate directly with the incidence of urinary incontinence
.
Factor predicting for urinary incontinence
651
Figure 50.5 (a)The incidence of incontinence in patient implanted during the ye
ar 1997, 1998, 1999, 2000, and 2001. (b) The mean urethral
Ba ic and advanced technique in pro tate brachytherapy
652
do e of the patient implanted each year, (c) The percentage of the patient imp
lanted each year who had a preimplant IPSS15.
To our knowledge, only the tudy by Merrick et al evaluated patient treated wit
h brachytherapy alone and correlated urinary incontinence to do imetric and clin
ical parameter .29 They found no ignificant difference in the EPIC core (Expa
nded Pro tate cancer Index Compo ite) of implanted patient compared with newly
diagno ed control .30 They reported a mean maximal urethral do e of only 13018 Gy
on day 0. Our re ult indicate that the incidence of incontinence would be low

at thi do e level. It i probable that their lack of an ob erved do e-re pon e

i becau e of the low urethral do e delivered to their patient . Several inve t
igator have tudied the relation hip between the urethral do e and urethral mor
bidity, in which incontinence wa not a pecific endpoint.16,3133 Wallner et al f
ound that urinary morbidity wa related to the maximum central urethral do e and
to the length of the urethra that received a do e greater than 450 Gy.31 With i
ntraoperative planning, Zelef ky et al were able to limit the median urethral do
e to 201 Gy compared to 378 Gy with conventional planning and noted that acute
urinary ymptom re olved more quickly with the lower do e .33 De ai et al repor
ted increa ed urinary toxicity core in patient receiving higher urethral do e
.16 Merrick et al found that the mean membranou urethral do e wa a predictive
factor for urethral tricture .32 The e report ugge t a do e-re pon e for the
urethra but to date no reported erie ha demon trated an a ociation between
urethral do e and urinary incontinence. Our data clearly demon trate a do e-re p
on e for the urethra and indicate that the urethral D10 do e i a predictive fac
tor for urinary incontinence. We found on multivariate analy i that the preimpl
ant IPSS al o wa predictive of urinary incontinence. Although the IPSS wa deve
loped initially by the American Urological A ociation in 1992 for evaluating pa
tient with benign pro tatic hypertrophy, it ha become a u eful tool for a e
ing patient for ba eline urinary function and for tracking po tradiation urinar
y ymptom .34,35 U ing thi core to tratify patient at higher ri k for po tim
plant complication , however, i omewhat controver ial.36,37 Several inve tigat
or have correlated preimplant IPSS with acute urinary toxicity and urethral tr
icture in implanted patient .12,34,38,39 Merrick et al did not find a relation
hip between the preimplant IPSS and incontinence with a mean antecedent IPSS of
only 5.69.29 In our erie , the mean preimplant IPSS al o wa low (7.85.5) but we
noted a ignificant difference (p=0.003) between the core of patient who did
not report incontinence (6.6 4.5) and tho e who did report incontinence (106.4).
In our tudy population, 13% had an IPSS 15 and the incidence of incontinence inc
rea ed dramatically in the e patient . A higher ba eline urinary function core
appear to be predictive for tho e patient more likely to experience urinary in
continence. In addition to determining predictive factor for incontinence we o
ught to evaluate the incidence of thi complication in our patient population. T
he ri k of urinary incontinence following pro tate brachytherapy ha not been cl
early defined with reported incontinence rate ranging from 0% to 40%.4,5,1115,40
Available report are limited and difficult to interpret becau e of differing i
mplant technique , the variety of tool u ed to
Factor predicting for urinary incontinence
653
a e thi complication, the variou definition of incontinence, and the diffe
rence in the mode of data collection, uch a phy ician grading or patient elf
-a e ment. The technique of pro tate brachytherapy have evolved con iderably
over the la t decade and a greater empha i ha been placed on urethral paring.
Talcott et al reported a 40% incidence of incontinence in patient with implant
performed u ing a uniform eed di tribution.11 Po timplant do imetry wa not o
btained for the e patient ; however, thi type of eed di tribution produce hig
h central do e within the pro tate and likely re ulted in high urethral do e
imilar to tho e delivered in our earlier implant when our incidence of incontin
ence ranged from 30% to 50%. At the other extreme, Merrick et al reported no dif
ference in EPIC core of patient following brachytherapy compared with newly d
iagno ed control with implant carried out u ing a technique that limited the m
ean maximal urethral do e to 130 Gy.29 Our re ult reflect thi evolution of tec
hnique in that our incidence of incontinence decrea ed ignificantly between 199
7 and 2001 a we reduced the urethral do e delivered. Difference in implant te
chnique that have an impact on the urethral do e likely contributed to the wide
range of incontinence reported in the literature. The variety of tool u ed to a
e incontinence al o contribute to the confu ing re ult reported in the lit

erature and to the wide range of reported rate . Several inve tigator u ed the
Radiation Therapy Oncology Group (RTOG) toxicity cale or a modification to grad
e morbidity and reported 05% incidence of incontinence.3,1215 Thi cale, however,
doe not pecifically include incontinence a an endpoint or detail it everit
y. It i not urpri ing then that the incidence of incontinence i generally low
in tudie that u e thi cale. The IPSS ha been hown to be u eful for monito
ring urinary morbidity, but it al o doe not include incontinence. The EPIC i a
frequently u ed elfa e ment quality of life tool developed by expanding the
Univer ity of California, Lo Angele -Pro tate Cancer Index (UCLA-PCI) and a k
four que tion regarding urinary incontinence.20,30 Both the EPIC and the UCLA-P
CI are caled to tandardized value from 0 to 100, with 100 being the mo t favo
rable outcome. Thi tool provide an evaluation of the impact of incontinence on
the patient quality of life, but it doe not de cribe the crude rate of incont
inence in the patient population, making compari on with tudie u ing thi ca
le difficult. Another rea on for the controver y about the incidence of incontin
ence following pro tate brachytherapy i the definition of incontinence. Incontine
nce i con idered a any involuntary leakage of urine, but thi general term enc
ompa e occa ional leakage of a few drop , to complete lo of the capacity to
contain urine. Ragde et al, for example, reported the incidence of incontinence
to be 0% in patient without a hi tory of tran urethral re ection of the pro tat
e (TURP).3 However, patient were only cla ified a incontinent if they require
d the u e of a protective pad. Many patient who reported incontinence in our t
udy would have been regarded a continent u ing the e criteria, which would have
re ulted in a ignificantly lower incidence of incontinence. How inve tigator
define incontinence ha a ignificant effect on the incidence reported. Differen
ce in the mode of data collection, be it phy ician grading or patient elfa e
ment, have al o been hown to ignificantly affect re ult . Some reported tudi
e are ba ed on phy ician a e ment of patient ymptom while other are ba ed
on elfa e ment. In the CaPSURE databa e of more than 3900 patient , phy ician
rated patient in everal area , including urinary incontinence. The e data we
re compared with
Ba ic and advanced technique in pro tate brachytherapy
654
patient-completed elfa e ment que tionnaire and ignificant difference were
noted in all quality of life and clinical domain .18 The incidence of incontine
nce, then, varie with different mode of data collection and i likely to be an
under-reported complication in tudie ba ed olely on phy ician a e ment. Wh
en evaluating incontinence in brachytherapy patient , it i important to know th
e incidence of thi problem in the general population. In tudie of men without
pro tate cancer or patient who opted for ob ervation of their cancer, the inci
dence of incontinence ha not been a low a could be expected. In a population
of normal older men, Litwin found that 33% had ome degree of urinary leakage.41
Similarly, in a review of the literature including over 21 tudie and 12 000 m
en, Thorn found the prevalence of incontinence among community-dwelling older me
n to be 1134% with a median of 17%.42 Report in the literature indicate that inc
ontinence i not uncommon in the general population and highlight the fact that
a portion of brachytherapy patient would be expected to experience ome level
of urinary leakage independent of their diagno i or it treatment. Serie that
report extremely low incontinence rate may be undere timating thi ymptom for
any of the rea on noted in thi di cu ion. It i under tandable, con idering a
ll of the above, that a wide variation exi t in the incidence of incontinence f
ollowing pro tate brachytherapy a reported in the literature. More tandardizat
ion i needed in the definition of incontinence, in the tool that are u ed to a
e incontinence, and in the manner in which the urethral do e i calculated a
nd reported. Inve tigator al o need to report the urethral do e and preimplant
IPSS when reporting the incidence of incontinence. Conclu ion Similar to exual
dy function, urinary incontinence i a feared complication of pro tate cancer t

reatment and i , therefore, an important endpoint to evaluate when inve tigating

treatment outcome and quality of life. There are everal e tabli hed type of
incontinence including urge incontinence (detru or overactivity), neurogenic inc
ontinence (detru or underactivity), overflow incontinence (urethral ob truction)
, and tre incontinence (urethral incompetence). The mechani m of incontinence
in patient that have received pro tate brachytherapy i not well defined. Our
tudy doe not attempt to peculate on the mechani m of incontinence in our pop
ulation nor doe it indicate how incontinence may change with time, age, medicat
ion, or urgical intervention. An attempt to collect thi information wa made v
ia telephone interview ; however, the e detail were difficult for patient to r
ecall, precluding reliable conclu ion . The data pre ented identify two ri k fac
tor for urinary incontinence, urethral D10 and the preimplant IPSS. The e facto
r hould be u ed to guide phy ician recommendation but do not repre ent contr
aindication to implantation. In electing patient for thi procedure, a global
picture including tage of di ea e, Glea on core, pro tate pecific antigen (PS
A), medical comorbiditie , prior urological procedure , medication , urinary ym
ptom , and preimplant do imetry hould be evaluated. Limiting the urethral D10 d
o e and electing patient with preimplant IPSS le than 15 will minimize the i
ncidence of urinary incontinence in patient receiving pro tate brachytherapy.
I
n the timeline of our memorie , major tragedie tand out a pike . Mo t people
remember clearly what they were doing when they learned that hijacked airliner
had cra hed into the World Trade Center. Tho e of u who are old enough can pin
point preci ely where we were when we heard that Pre ident Kennedy had been hot
. Being diagno ed with pro tate cancer i comparable. I wa itting at my de k a
t the Stanley Medical Re earch In titute at 10 a.m. on May 4, 2004. The immediat
e problem wa how to per uade re earch taff member who do not like one another
to work together. I wa not expecting the call, becau e my urologi t had aid t
he pathology report after my biop y would take even to ten day to compile, and
thi wa only day ix. The arrival of the call did not unduly alarm meat r tbecau
e I wa rea onably certain that the lump in my pro tate wa benign and the biop
y would be negative. My pro tate peci c antigen (PSA) wa only 3.3, well within t
he normal range, and the urologi t had not een any u piciou -looking area on
ultra ound at the time of the biop y. Be ide , I wa a healthy 66 year old and
planning to live at lea t twenty more year . I had only recently acquired a few
harbinger of old age: forgetting name , a dimini hed urinary tream, and plaque
on the kin. And I hadnt yet pa ed the de nitive landmark
2
ON BEING DIAGNOSED
of the aged: ending obituarie of old friend to other old friend . Cancer wa
not omething I wa uppo ed to get; cancer wa for other people. A one man put
it: Pro tate cancer wa a remote from the po ibilitie I imagined for my elf a
a voyage to the moon.1 The urologi t did not preface it a bad new , but announced in
a traightforward way that the biop y had been po itive, that I had cancer in t
hree of the nine core ( ample ) taken during the biop y, and that the Glea on
core wa 7. In an effort to wrap my mind around thi meteor that had ju t broken
through the roof and landed on my de k, I a ked what the Glea on core meant. T
he only Glea on I knew wa old-time comedian Jackie Glea on, but I doubted he ha
d anything to do with pro tate cancer. I al o wondered to my elf why I had not d
one any reading on pro tate cancer, o that I could interpret Glea on core and
number of po itive core , but then remembered that I had been virtually certai
n I didnt have it, o why wa te my time? A Glea on 7, he aid, wa an intermediate t
age of cancer. It meant that the cell were not a benign a a Glea on 4, 5, or
6 but not a malignant a an 8, 9, or 10. I did not like the ound of

ON BEING DIAGNOSED
3

An Unwelcome Surpri e When my doctor aid, General, you have pro tate cancer, I wa
thru t into an immediate and fearful tate of confu ion. I can till recall my i
nability to move a mu cle for what eemed like an eternity after hearing my diag
no i . Gen. H. Norman Schwarzkopf U.S. Army, retired I have cancer? Ye . In the left l
be. So now, at la t, I know, I thought, and my ground-floor tudy, urrounded on three
ide by a yard and tree , in which I had taken hi call, eemed to darken. Every
thing ha changed. From now on cancer i nt out there. It the enemy in ide me, eat
ing away at omething vital. I experienced the clich inking feeling, a if omethi
ng had uddenly happened to my blood pre ure. Charle Neider, Adam Burden
intermediate; it reminded me of the C I had gotten in intermediate algebra. I al o r
emembered all too clearly from my hi tology cour e in medical chool what intermed
iate cancer cell looked like under a micro cope. They were ugly and mi hapen, no
t a bad a the gro ly deformed cell that would qualify a Glea on 9 or 10, bu
t certainly not to be confu ed with normal, orderly-looking cell . And the urolo
gi t wa telling me that the ugly cell were in ide me, now, a I at at my de k
. Unbelievable. I wa deeply re entful and felt that I had been urreptitiou ly
violated. The remainder of the workday had an illu ory quality. I called my wife
, but aid nothing to anyone el e. I needed r t to under tand the new my elf. My
tronge t memorie of the day are of li tening to other people complain about t
hing and being orely tempted to cut
4
ON BEING DIAGNOSED
them off, aying, Why are you complaining, Ive got cancer! In the late afternoon, I l
i tened a a re earch colleague wallowed in hi per onal and profe ional woe f
or almo t an hour. I wondered brie y whether, if I trangled him, a jury would nd m
e not guilty becau e of mitigating circum tance . Then, driving home, I got caug
ht in the wor t traf c jam I had encountered in month . Sitting there, I thought o
f Job. Man i born to trouble, a the park y upward, it wa written. Thi had to be
the archetypal bad day. My immediate ta k wa to learn a much a I could about
pro tate cancer and my option for treatment. I realized that I, a trained phy
ician, knew almo t nothing about the pro tate. It appeared to be, in fact, the m
o t ob cure and lea t intere ting organ in the body. In anatomy cla , my group
of four tudent had had a female cadaver to di ect, while the group at the nex
t table had had a male. Our in tructor had told u to change table periodically
o that we would under tand the anatomy of both exe . It hadnt happened. Our al
mo t all-male cla had a per onal a well a a profe ional intere t in female
anatomy and, more often than not, eight of u were working at our table. Im not
ure I even aw a pro tate. How long did I have to make a deci ion? One book on p
ro tate cancer advi ed you to commit to a therapeutic trategy within about three
to ix month after youre diagno ed, but ooner if your cancer i large and high grad
e. One tudy reported that delay in treatment of more than three month increa ed
chance of recurrence ten year later. Another tudy found that pro tate cancer
for which treatment wa begun an average of two month after diagno i did not
have an increa e in recurrence three year later. A large 2005 tudy wa the mo
t rea uring, nding that urgical delay of up to a year after biop y did not in
crea e chance of recurrence ve year later; therefore, men who wait everal month
after biop y before undergoing RP [ urgery] are not jeopardizing their probabil
ity of cure.2 Thi lack of profe ional con en u on important pro tate cancer i
ue would, I would oon di cover, turn out to be common. In general, I am neithe
r laid back nor indeci ive, and I found that with cancer growing in ide of me, a
ll en e of lei ure di appeared. De pite reported a urance that pro tate cance
r i low growing, I heard only the word growing, not the word low. Each day th

at pa ed meant that my cancer had grown, and perhap pread. One day it
ON BEING DIAGNOSED
5
A New Land I en ed that I had cro ed ome invi ible line into a new pha e of m
y life, the way a traveler might feel when cro ing a frontier and entering a di
fferent country, with a trange language, new cu tom , different rule , ign th
at made no en e, policemen with unfamiliar uniform pacing in pair up and down
the railway platform . Michael Korda, Man to Man
would be contained within the pro tate gland and perhap curable, but there woul
d come a day when it would not be. Which day would that be? It eemed obviou th
at the thing I needed mo t wa accurate information. I found my elf identifying
with retired general Norman Schwarzkopf, who, on being diagno ed with pro tate c
ancer, aid: For me it wa like war. Fir t thing you do i learn about the enemy.3 A
a re earcher, I had dozen of que tion I wanted an wered, taking into con ide
ration all available tudie . A a per on with cancer, I wanted them all an were
d ye terday. My earch of book and profe ional paper told me that, according
to the American Cancer Society, I wa one of 230,110 men in 2004 who were being
diagno ed with pro tate cancer. Di couragingly, that number had not changed much
in recent year . It meant that 630 men were being diagno ed every day, 26 every
hour, 1 every 2.3 minute , Sunday and holiday included. Two hundred thirty th
ou and, one hundred ten men i equivalent to the entire male and female populati
on of Danbury, Connecticut, or Olympia, Wa hington. I had been in Danbury and Ol
ympia, and that eemed like a lot of people. I al o learned that 1.6 million Ame
rican men currently have, or have had, pro tate cancerwhich i more than the metr
opolitan population of Columbu , Memphi , Milwaukee, Sacramento, or San Antonio.
Mi ery love company, but having all that company didnt make me feel any better.
6
ON BEING DIAGNOSED
No, the man of the hou e i not at home.
My reading al o informed me that I had joined the rank of ome notable men who
have had pro tate cancer, including athlete uch a Stan Mu ial, Arnold Palmer,
and Len Daw on, and political leader uch a Bob Dole and Rudy Giuliani. Even
Andrew von E chenbach, director of the National Cancer In titute from 2002 to 20
05, ha had pro tate cancer, and that didnt eem promi ing. All the e men were me
mber of the Pro tate Cancer Club. Joining uch a club provided little con olati
ongood grief, I had not even applied for member hip. I wa even more di couraged
by the li t of former club member , now decea ed. It included educated, importan
t, and wealthy men with acce to the be t available medical re ource . The mo t
di heartening name on the li t wa that of Willet Whitmore, who had been chief
of urology at Memorial Sloan-Kettering Cancer Center and regarded a one of the
nation expert on pro tate cancer. Ultimately, my earch for information about p
ro tate cancer wa di appointing on three count . Fir t, I wa appalled to learn
how little wa known with certainty about the cau e , natural cour e, and the r
elative merit of variou treatment option . Hadnt Pre ident
ON BEING DIAGNOSED
7
Select Member of the Pro tate Cancer Club (identified by occupation or po ition
for which they are be t known) Current Member Ma on Adam Ed A ner Du ty Baker
Marion Barry Harry Belafonte Jim Berry Saxby Chambli Sean Connery Len Daw on

Robert De Niro Bob Dole Loui Farrakhan Dan Fogelberg George Foreman Rudy Giulia
ni Robert Goulet Merv Griffin Andy Grove Je e Helm Charlton He ton Hamilton Jo
rdan George Karl Herb Kelleher John Kerry Michael Korda Jame Leach
Actor Actor Ba eball manager, Chicago Cub Mayor, Wa hington, DC Entertainer Car
tooni t U.S. enator, Georgia Actor NFL quarterback, Kan a City Chief Actor U.
S. enator, Kan a Leader, Nation of I lam Singer Heavyweight boxer Mayor, New Y
ork City Actor TV producer Bu ine man, cofounder and chairman of Intel U.S. en
ator, North Carolina Actor White Hou e chief of taff Coach, Denver Nugget CEO,
Southwe t Airline U.S. enator, Ma achu ett Editor in chief, Simon and Schu
ter Member of Congre , Iowa
8
ON BEING DIAGNOSED
Marv Levy Jerry Lewi Nel on Mandela Michael Milken Roger Moore Robert Mueller R
upert Murdoch Stan Mu ial
NFL coach, Buffalo Bill Actor Pre ident of South Africa Wall Street financier A
ctor Director, FBI Media owner Ba eball player, Saint Loui Cardinal Robert Nov
ak Journali t Arnold Palmer Profe ional golfer Richard Petty na car driver Sidn
ey Poitier Actor Colin Powell U.S. ecretary of tate Pat Robert on Televangeli
t Norman Schwarzkopf General, U.S. Army Richard Shelby U.S. enator, Alabama Pau
l Steven U.S. Supreme Court ju tice Ted Steven U.S. enator, Ala ka Joe Torre
Ba eball manager, New York Yankee De mond Tutu South African cleric Andrew von
E chenbach Director, National Cancer In titute Andrew Young Mayor, Atlanta Pa t
Member Who Died from Pro tate Cancer, with Age at Death Don Ameche (85) Wayne C
alloway (62) Stokely Carmichael (Kwame Ture) (57) William Ca ey (68) Actor Chair
man, Pep ico Black activi t Director, CIA
ON BEING DIAGNOSED
9
Silvio Conte (69)
Member of Congre , Ma achu ett Gregory Cor o (70) Poet Hume Cronyn (91) Actor
Glenn Davi (80) Hei man Trophy winner Dean Gallo (59) Member of Congre , New
Jer ey John Gardner (89) Founder, Common Cau e Ayatollah Khomeini (89) Iranian c
leric Herbie Mann (73) Jazz fluti t Spark Mat unaga (73) U.S. enator, Hawaii Fr
anoi Mitterand (79) Pre ident of France Marion Motley (79) NFL player, Cleveland
Brown Jerry Orbach (69) Actor Jo eph Papp (70) Director, New York State Shake
peare Fe tival Johnny Ramone (55) Guitari t, founder of punk band Bobby Rigg (7
7) Tenni player Steve Ro (65) Chairman, Time Warner Corneliu Ryan (54) Write
r Telly Savala (70) Actor William Shockley (79) Phy ici t and Nobel laureate Le
o Szilard (66) Scienti t and writer Pierre Trudeau (80) Prime mini ter of Canada
Robert Penn Warren (84) Writer Willet Whitmore (78) Expert on pro tate cancer
Nixon declared war on cancer more than thirty year ago? What had my colleague
been doing all thi time? Second, the mo t u eful information that wa available
wa cattered in variou profe ional paper , book , and web ite . Much of it w
a out of date and contradictory, and ome wa factually wrong. Moreover, promin
ent urologi t trongly di agreed with one another, publicly and not alway poli
tely, thu giving the appearance (ironic, given their profe ion) of an adole ce
nt urinating conte t. If
10
ON BEING DIAGNOSED

I, a a trained medical profe ional, wa having trouble orting it out and deci
ding what to do, what mu t nonprofe ional experience? Finally, I wa urpri ed
to realize that I wa the per on who wa expected to a e the treatment optio
n and decide which one to elect. I had grown up in an era when doctor u ually
made trong recommendation . Now, a noted by the New York Time , patient are
awa h in information, but many nd the job of being a modern patient, with it log
through medical uncertainty, to be lonely, frightening, and overwhelming.4 De pite
having an extremely upportive wife, an excellent interni t and urologi t, and
many highly knowledgeable medical friend , I felt very much alone with my deci i
on. Thi book, then, i the book I wi h had been available to me when I wa diag
no ed with pro tate cancer. I hope it will lighten the load for other men who re
ceive the ame diagno i .
C H A P T E R
2
How Seriou I Your Cancer?
everal que tion immediately came to mind on the day I wa diagno ed with pro ta
te cancer. How eriou i it? I it likely to kill me? If o, when? Thi chapter
provide information you need to begin an wering uch que tion . Tho e who wi h
to kip the more technical di cu ion will nd a ummary at the end of the chapt
er. Pro tate cancer i cancer. It affect the walnut- ized gland that it benea
th the bladder and contribute ome of the uid making up the emen; a detailed de
cription of the pro tate can be found in Appendix A. Pro tate cancer hould not
be confu ed with benign pro tatic hypertrophy (BPH), the other commonindeed, alm
o t univer alpro tate af iction of older men. For benign pro tatic hypertrophy, the
operant word i benign. It i an enlargement of the pro tate that can cau e ym
ptom uch a frequency (having to urinate often), urgency (dif culty in holding y
our urine), a weak ow, and tarting and topping of the ow. Benign pro tatic hyper
trophy can be treated with medication or urgery. Sometime , in the cour e of
urgery for BPH, it i di covered incidentally that the enlarged pro tate ha can
cer a well, but the two condition are unrelated. The following di cu ion a u
me that the type of pro tate cancer with which you have been diagno ed i an ad
enocarcinoma. More than 95 percent of all pro tate cancer are of thi type. The
S
12
HOW SERIOUS IS YOUR CANCER?
other 5 percent include mall cell, quamou cell, arcoma , and other rare type
; all tend to be more aggre ive and to have wor e progno e than adenocarcinom
a . The three mo t widely u ed indicator of everity in men with newly diagno e
d pro tate cancer are total pro tate peci c antigen (PSA) level; whether the canc
er can be felt on digital rectal exam (often abbreviated a DRE) and an e timate
of the cancer volume; and the appearance of the cancer cell obtained by biop y
(Glea on core). Other indicator of everity are not a widely u ed but may al
o be very u eful; the e are de cribed below.
WHAT IS YOUR PSA? Pro tate peci c antigen i a protein produced by cell in the p
ro tate and, in very mall amount , by cell el ewhere in the body. PSA wa di c
overed in 1979 and became widely u ed a a mea ure of pro tate di ea e in the la
te 1980 . It i increa ed in mo t ca e of pro tate cancer, when the pro tate i
enlarged by benign pro tatic hypertrophy and by in ammation of the pro tate (pro
tatiti ). Thu , PSA i a general mea ure of pro tate pathology and i not peci c
for cancer. Orga m and ejaculation may increa e PSA, and for thi rea on men are
advi ed not to ejaculate for two day prior to having a PSA te t. The level may

al o be elevated by ma aging the pro tate, a occur during treatment for pro
tatiti , but hould not be elevated by a normal digital rectal exam. There have
been claim that vigorou bicycle and motorcycle riding may increa e PSA by putt
ing eat pre ure on the pro tate, but other tudie do not agree. PSA i al o p
artially determined by one gene , o ome men have higher PSA level on a geneti
c ba i alone. Conver ely, PSA level may be decrea ed in men who are obe e and
in men who are taking na teride (Pro car) for BPH or na teride (Propecia) to preve
nt baldne . Pro car and Propecia are the ame drug, but Pro car contain a high
er do e of na teride (5 mg) than Propecia (1 mg). All the e factor need to be ta
ken into account when evaluating PSA level. There i little agreement on the nor
mal PSA level. However, there i agreement on one fact: PSA i a continuum, and
the lower it i , the better off you are. Since mo t men pro tate increa e in i
ze a they age, what i con idered to be a normal PSA level increa e with age.
A
HOW SERIOUS IS YOUR CANCER?
13
What I the Upper Limit of Normal PSA? Many urologi t now u e age-corrected lim
it for normal PSA level , recognizing that mo t men pro tate increa e in ize
a they age, thereby increa ing their PSA. The following have been recommended:
Age 4049: Age 5059: Age 6069: Age 70 and above: Upper limit of 2.5 Upper limit
of 3.5 Upper limit of 4.5 Upper limit of 6.5
Not everyone agree with the e figure ; ome urologi t u e lower level .
level of 4 nanogram per milliliter of erum (hereafter written a PSA 4) ha tr
aditionally been regarded a the upper limit of normal, but increa ingly urologi
t are u ing age-corrected upper limit ( ee box). The mo t important a pect of
PSA, however, i the fact that it i a continuum, not po itive or negative like
a pregnancy te t. Thu , there i virtually no difference between a PSA of 3.9 a
nd 4.1, but a va t difference between 3.9 and 0.9. Approximately 85 percent of m
en with pro tate cancer have PSA level higher than 4. However, that mean that
15 percent of men with pro tate cancer have PSA level lower than 4. For example
, General Schwarzkopf had a PSA of only 1.2 when he wa diagno ed with pro tate
cancer. My own PSA level when I wa diagno ed wa 3.3, well within normal limit
for a 66-year-old man. Well-de cribed ca e exi t in which men with PSA level
of le than 1.0 have pro tate cancer that have already pread to their lymph n
ode and bone ; fortunately, uch ca e are unu ual.1 If your PSA i elevated, w
hat are the chance that you have pro tate cancer? If your PSA i between 4 and
10, the likelihood i about 25 percent. The majority of uch elevation are cau
ed by benign pro tatic hypertrophy or by infection of the pro tate. If your PSA
i
14
HOW SERIOUS IS YOUR CANCER?
more than 10, the chance of your having cancer are over 50 percent. If your PSA
i greater than 50, not only do you probably have cancer but it i likely to ha
ve already pread beyond the pro tate. Charle William , in hi book That Black
Men Might Live, reveal that hi PSA at the time of diagno i wa 172. In ca e
of meta tatic di ea e in which the cancer ha pread widely throughout the body,
PSA level can go higher than 1,000. Becau e of wide pread di ati faction with
the u e of total PSA a a marker for pro tate cancer, attempt are being made t
o re ne the te t. One method i to eparate the total PSA into it component , pe
ci cally the PSA fraction that i bound to other protein in the blood and the fra
ction that i not bound (called free PSA). A a general rule, cancer cell make
le free PSA than healthy pro tate cell do. Therefore, the higher the fraction

of free PSA in your blood, the greater the chance that you are free of cancer.
One tudy reported that if the free PSA i more than 25 percent of the total PS
A, the likelihood of having cancer i only 8 percent. At the other extreme, if t
he free PSA i le than 10 percent of the total PSA, the probability of having
cancer i 56 percent. Michael Dor o, a phy ician who wrote about hi own pro tat
e cancer, knew he wa in trouble when hi free PSA report came back a 4 percent
; he correctly noted that thi wa not a good number!2 Free PSA mea urement are mor
e dif cult to take than total PSA and thu are not available in ome laboratorie .
Thi te t i therefore underutilized. Another promi ing u e of PSA te ting i t
o ob erve how rapidly the PSA i ri ing. Thi i called the PSA velocity. Any ri
e in PSA i a cau e for concern, but a low ri efor example, le than 0.75 in a
ny given yeari le worri ome.3 A PSA ri e of more than 0.75 per year i more wo
rri ome, and a ri e of more than 2.0 in a year (for example, 1.3 to 3.4, or from
0.9 to 3.1) i very worri ome. Studie report that men with a PSA increa e of m
ore than 2.0 in the year prior to diagno i of their cancer had a much higher de
ath rate from their cancer, de pite having treatment by urgery or radiation.4 T
he problem with PSA velocity, however, i that it i nece ary to have had PSA m
ea urement on an annual ba i in order to u e it. Mo t men do not undergo PSA t
e ting that often, and many men do not have their PSA te ted at all. The potenti
al importance of PSA velocity in predicting pro tate cancer i a major rea on wh
y every man hould have
HOW SERIOUS IS YOUR CANCER?
15
PSA Velocity Save a Senator An excellent example of the importance of PSA veloc
ity i Senator John Kerry, who ran for Pre ident in 2004. In November 2002, duri
ng an annual checkup, Kerry had a PSA of 3.2, within the normal range for hi ag
e. However, Kerry wife, Tere a Heinz Kerry, noted that hi PSA had increa ed fro
m 2.0 in December 1999 and 2.7 in February 2001 and urged him to have additional
te t . In December 2002, a digital rectal exam wa normal and hi PSA wa 3.4.
Since Kerry father had died of pro tate cancer and becau e of the ri e in hi PS
A, a biop y wa done, which found pro tate cancer in five of the ample . Kerry
ub equently underwent a radical pro tatectomy and ha had no recurrence of hi
cancer to date. Thi ca e illu trate the importance of PSA velocity a a marker
for pro tate cancer. It al o illu trate the benefit of having a wife who aggre
ively monitor her hu band health and, in thi ca e, may have aved hi life. a
dapted from L. K. Altman New York Time , October 3, 2004
a ba eline PSA te t in hi 40 and a regular PSA te t from age 50 on ( ee Chapte
r 15). Since PSA te ting varie omewhat between laboratorie , it i advi able t
o have the te ting performed by the ame laboratory whenever po ible.
IS YOUR CANCER PALPABLE? The econd indicator of everity that i widely u ed to
evaluate men with newly diagno ed pro tate cancer i whether the cancer can be
felt on a digital rectal exam. The fact that 15 percent of pro tate cancer have
PSA of le than 4 i uf cient rea on for middle-aged and older men to have uc
h an exam a part of their annual phy ical. The procedure, de pite being undigni e
d and mildly uncomfortable,
16
HOW SERIOUS IS YOUR CANCER?
Getting a Second Opinion Robert Hitchcock, in hi book Love, Sex, and PSA, tell
of a man who a ked hi urologi t which finger he had u ed to examine the pro ta
te during hi digital rectal exam. The doctor replied that he had u ed hi middl
e finger. Would you repeat the exam and u e your index finger thi time? a ked the p
atient. Why would you want me to do that? replied the doctor. Becau e I would like to

get a econd opinion, aid the man.

can di cern many early- tage cancer that would otherwi e go undetected until th
ey became larger. The majority of pro tate cancer begin in the po terior portio
n of the pro tate gland, the area clo e t to the wall of the rectum. Thu , many
mall cancer can be perceived by an examining nger. The procedure ha aved many
live , perhap including my own. When phy ician doing rectal examination feel
omething in the pro tate that may be cancer, they try to e timate it ize (vo
lume) and al o whether it occupie one or both ide of the gland. Such e timate
are impreci e but nonethele provide information regarding everity. Pro tate
cancer volume ha been hown to be very important in predicting future cour e.
In one tudy, men who e cancer involved 10 percent or more of the pro tate had a
10 percent chance of recurrence, wherea men who e cancer involved 5 percent or
le had a 5 percent rate of recurrence. In general, the larger the volume of t
he cancer, the greater the chance it ha pread out ide the pro tate. Thoma Sta
mey, a pro tate cancer re earcher at Stanford Univer ity, ha argued that pro ta
te cancer volume and Glea on cell type ( ee below) are the two mo t important pr
edictor of future cour e.5 Cancer volume al o correlate rea onably well with P
SA level, with larger tumor producing higher PSA .
HOW SERIOUS IS YOUR CANCER?
17
Te ting for Pro tate Cancer There nothing like a battery of ho pital te t to ma
ke you feel vulnerable, naked; a if your very life depend on them. A , indeed,
it may. I had been living a quiet life, minding my bu ine , not breaking the l
aw. My time had felt free; which wa what I had been u ed to; which I had alway
wanted; and till wanted. And now, uddenly, I wa caught up in the fullfledged
machinery of cancer; the machinery of what felt like a WAR. Te t . Que tion . D
ebate . Crucial deci ion . What wa the enemy local trength, po ition, logi tic
, tactic ? What wa hi trategy? Above all, who would win? Charle Neider, Adam
Burden
E timating the ize of the pro tate cancer i part of what i called taging the
cancer. The Tumor, Node, Meta ta i y tem, u ually ju t abbreviated the TNM y
tem, i mo t widely u ed. In thi y tem, nonpalpable pro tate tumor are label
ed tage T1. Palpable tumor are labeled tage T2 and further divided a follow
: T2a. Palpable, but appear to involve le than half of one lobe T2b. Palpable
, and appear to involve more than half of one lobe but not both lobe T2c. Palp
able, and appear to involve both lobe Thi numerical de ignation i included i
n the Partin table , explained below, which are u ed to e timate the po ible p
read of the cancer. The main problem with thi taging y tem i that it i impr
eci e. A omewhat better e timate of tumor volume can be obtained from the ultra
ound examination done at the time of biop y. It i common for phy ician to hav
e patient with pro tate cancer undergo bone can after the initial diagno i h
a been made. Thi procedure involve injecting a mall amount of radioactive tr
acer into the blood tream. The tracer i electively ab orbed by the bone , and
the patient i examined by nuclear canner; any pread of
18
HOW SERIOUS IS YOUR CANCER?
the cancer to the bone i vi ible a a hot pot. Bone can are negative for more t
han 95 percent of pro tate cancer patient when r t diagno ed, becau e in mo t ca
e the cancer ha not pread or, if it ha pread, it i too mall to be detect
ed on the can. If it ha pread, a different cour e of treatment i followed. I
per onally found the bone can to be a benign procedure; lying on the canning
table in a emidarkened room eemed a ne opportunity for a hort nap.

WHAT IS YOUR GLEASON SCORE? The third indicator of everity u ed to evaluate men
with newly diagno ed pro tate cancer i the appearance of the cell obtained by
biop y. Of the three indicator of everity, thi i the mo t important. In 196
6 Donald Glea on, a pathologi t at John Hopkin Univer ity, propo ed a cla i cat
ion of pro tate cancer by appearance of the cell . He ugge ted grade 1 to 5, w
ith 1 being the mo t benign and 5 the mo t malignant. Grade 1. Cancer cell are
well differentiated, with relatively normal architecture and clearly defined bor
der . They are arranged in a compact ma .
HOW SERIOUS IS YOUR CANCER?
19
Grade 2. Cancer cell are till well differentiated, their arrangement i more i
rregular, and occa ional cell clu ter are een to invade urrounding ti ue . G
rade 3. Cancer cell are only moderately differentiated, their arrangement i qu
ite irregular, and many cell group are een to be invading urrounding ti ue.
Thi grade i the one mo t commonly een in biop y pecimen . Grade 4. Cancer ce
ll are poorly differentiated, have di torted hape , and progre ive inva ion o
f urrounding ti ue by cell clu ter i evident. Grade 5. Cancer cell are gro
ly di torted, appear to melt together, bear no re emblance to normal cell , an
d are no longer arranged in any formal clu ter . Cell that are intermediate bet
ween normal and grade 1 are commonly referred to a pro tatic intraepithelial ne
opla ia, or PIN, meaning that the pro tate hould be watched for po ible proble
m . Thought to be a precur or of future pro tatic cancer, PIN i found in about
5 percent of all pro tate biop ie . When it i ob erved in an otherwi e normal p
ro tate, mo t urologi t recommend a rebiop y to make ure that an area of cance
r ha not been mi ed. It i becoming increa ingly clear that, from the point of
view of progno i , the Glea on grading of cancer cell i not a continuum. The
majority of men with pro tate cancer will be found to have grade 3 cancer cell
on biop y at the time of their initial diagno i . The gap between grade 1 and
2 and grade 2 and 3 are not large, and individual with all three cell type ha
ve very favorable progno e . By contra t, the gap between grade 3 and 4 i larg
e; men with grade 4 cell have a igni cantly wor e progno i than men with grade
3 cell , and the more grade 4 cell that are pre ent, the wor e the outlook. In
one tudy, men with grade 4 compared to grade 3 cell had an almo t threefold gr
eater chance of having their cancer pread, and pread more quickly. Men with gr
ade 5 cell have an even wor e progno i , but uch men are fortunately few in nu
mber. One tudy reported that the percentage of grade 4 and 5 cell in a tumor w
a the ingle be t predictor of recurrence of the pro tate cancer.6 Cla ifying
the cell in pro tate cancer become more complicated, however, becau e almo t a
ll men with pro tate cancer have foci of cancer cell at everal different ite
in the pro tate gland. In
20
HOW SERIOUS IS YOUR CANCER?

Seeing Your Own Cancer Come take a look at thi , the pathologi t aid. He and the ur
ologi t were peering through the eparate eyepiece of a teaching micro cope. I
looked at the pecimen, tained a delicate pink and blue, and followed a he ho
wed me the ne t of cancer cell infiltrating the normal architecture of the ur
rounding pro tate gland. A I looked at the pread of the e malignant interloper
, I felt dizzy. You guy go on without me, I aid. Ill meet you back in the room. T
a a gloomy examining room in the nearby urology clinic, adorned with picture o
f di ea ed pro tate . So I went and at, till a bit un teady, on the examining
table. The pro tate ti ue wa mine, the brightly colored product of a biop y do
ne 2 week earlier. My colleague at Univer ity Ho pital a umed that I would wa
nt to join them in reviewing the biop y. The biop y wa critical in electing th

e be t treatment option; the virulence, extent, and di tribution of the cancer w

ould determine whether I had a chance of being cured. But their fa cinating inte
llectual exerci e wa my potential death entence. Roger Ro enblatt, M.D., Getting
the New , Annal of Family Medicine
other word , pro tate cancer, unlike mo t other cancer , i multifocal; therefor
e, almo t every man affected can be aid technically to have pro tate cancer , n
ot pro tate cancer. A he wa devi ing hi cla i cation of cancer cell , Glea on
wa aware of thi multifocal nature of pro tate cancer. Accordingly, he propo ed
that the tumor hould be cla i ed twice: the r t number grade would be for the ce
ll type that wa mo t frequent, and the econd number grade would be for the cel
l type that wa econd mo t frequent. Thu wa born the Glea on core, ranging f
rom 2
HOW SERIOUS IS YOUR CANCER?
21
(1+1) to 10 (5+5). Glea on core of 2, 3, and 4 are rarely een, becau e uch c
ancer do not u ually increa e the PSA enough to call attention to them elve an
d are therefore not biop ied. A Glea on core of 6 (3+3) i the one mo t commonl
y reported in pro tate biop ie ; it mean that only grade 3 cell were een unde
r the micro cope. A Glea on core of 7 i al o common and pre ent problem for
the Glea on coring y tem. If the Glea on 7 core i a 3+4, it mean that grade
4 cell con titute le than 50 percent; they could be a low a 5 percent and
a high a 45 percent (grading are done in increment of 5 percent). If, on the
other hand, the Glea on 7 core i 4+3, then grade 4 cell are in the majority
and could be a high a 95 percent. Grade 4 cell are igni cantly more malignant
than grade 3 cell , o it i important to pecify whether a Glea on 7 core i 3
+4 or 4+3. A Glea on 7 (3+4) tumor, with few grade 4 cell , could have a relativ
ely favorable progno i clo e to that for a Glea on 6 cancer. Conver ely, a Glea
on 7 (4+3) tumor, with a high percentage of grade 4 cell , could have a relativ
ely poor progno i clo e to that for a Glea on 8 cancer. In one tudy of more th
an two thou and pro tate cancer , tho e graded Glea on 7 (4+3) had a recurrence
rate at even and ten year double that for tho e graded Glea on 7 (3+4).7 Remem
ber that the cla i cation of cancer cell type in men who have been recently diagn
o ed with pro tate cancer i ba ed on the biop y pecimen . There i no guarante
e that the biop y material wa obtained from repre entative ection of the mult
ifocal tumor. In fact, when Glea on core taken from biop y pecimen have been
compared with Glea on core taken from the complete cancer after it ha been r
emoved at urgery, it i found that the actual Glea on core i higher (for exam
ple, Glea on 6 on biop y but Glea on 7 on urgical pecimen) twice a often a i
t i found to be lower. Thi phenomenon i referred to a undergrading. Another
problem in a e ing your Glea on core i lack of agreement among pathologi t ;
a 2005 tudy concluded that inter-rater agreement wa only fair.8 In ummary, t
hree indicator of everity are commonly u ed to evaluate men with newly diagno
ed pro tate cancer. Although the total PSA i moderately helpful, it may be affe
cted by other condition . The percentage of free PSA and the rate of PSA ri e (v
elocity) are probably more u eful but are le widely available. The econd indi
cator, whether the cancer i palpable on digital rectal exam and
22
HOW SERIOUS IS YOUR CANCER?
Table 1. Percentage of Men with Cancer That Have Already Spread Out ide the Pro
tate to the Seminal Ve icle and/or Lymph Node at the Time of Diagno i (Abbre
viated Ver ion of Partin Table ) Cancer appear to involve More than half of one
Le than half lobe Both lobe of one lobe (T2b) (T2c) (T2a) PSA 02.5 56 7 (3+4) 7
(4+3) 810 0% 3 3 5 1% 7 7 10 3% 10 11 15 2% 11 14 18

Glea on core
Cancer not felt on rectal exam (T1a)
PSA 2.64.0 56 7 (3+4) 7 (4+3) 810 1 5 5 7 2 9 8 13 3 12 11 17 PSA 4.16.0 56 7 (3+4) 7
(4+3) 810 1 5 6 8 2 9 11 14 4 14 15 19 PSA 6.110.0 56 7 (3+4) 7 (4+3) 810 2 10 10 1
6 5 16 16 22 7 22 21 27 PSA ] 10.0 16 39 40 46 8 23 24 29 5 18 20 23 3 13 13 18
56 7 (3+4) 7 (4+3) 810
NOTE:
6 18 21 28
10 30 31 36
19 46 48 53
All number are approximation , within 95 percent confidence limit , and are ba
ed on pecimen from 5,079 men who underwent urgery at John Hopkin Ho pital b
etween 1994 and 2000.
HOW SERIOUS IS YOUR CANCER?
23
an e timate of the pro tate cancer volume, depend in part on the kill of the e
xaminer. The third indicator, the Glea on core, i probably the ingle be t pre
dictor of everity but ha ome limitation , e pecially for Glea on 7 cancer . T
he e three indicator of everity are commonly combined into table to predict w
hether a given cancer ha pread beyond the pro tate. The table were developed
by Alan Partin at John Hopkin Univer ity, ba ed on 5,079 urgical pecimen ex
amined between 1994 and 2000. They are u ually referred to a Partin table and
are found in mo t book on pro tate cancer. The abbreviated ver ion hown in Tab
le 1 allow a man to elect the table covering hi PSA core, then to elect the
tage of hi cancer (whether it i palpable and, if o, it ize), and nally to
elect the correct Glea on core. The re ult i a number that i the percentage
chance that the cancer ha pread beyond the pro tate to involve the eminal ve
icle and/or lymph node . Given the limitation of the data that go into the Par
tin table , it i evident that the predictive value of the e table may or may n
ot be accurate for any peci c man. The e three indicator of everity are al o u
ed to divide pro tate cancer into level that predict the likelihood that the c
ancer will recur after treatment, a will be di cu ed in Chapter 11. The mo t w
idely u ed categorie are tho e promoted by the National Comprehen ive Cancer Ne
twork and the American Cancer Society: Low chance of recurrence Not palpable on
rectal exam (T1) or, if palpable, occupie le than half of one lobe (T2a); and
Glea on core 6 or lower; and PSA below 10. Intermediate chance of recurrence O
ccupie both halve of one lobe (T2b) or both lobe (T2c); or Glea on core 7; o
r PSA 1020. High chance of recurrence Tumor ha pread beyond pro tate cap ule; o
r Glea on core 810; or PSA above 20.
24
HOW SERIOUS IS YOUR CANCER?
Very high chance of recurrence Tumor ha pread to eminal ve icle , lymph node
, or more di tantly to bone or other organ .
ADDITIONAL PREDICTORS OF SEVERITY It would be helpful if we had more accurate in
dicator of everity for men with newly diagno ed pro tate cancer . Our treatmen

t deci ion , and even whether to pur ue treatment at all, are currently ba ed on
inadequate data. If we were better able to predict which cancer are likely to
remain quie cent and which are likely to progre , many men would not have to un
dergo treatment and could thereby avoid complication uch a incontinence and i
mpotence. The fact that we do not have valid indicator of everity re ult from
the failure of pro tate cancer re earch ( ee Chapter 14). Additional predictor
of everity are available from pro tate biop ie but are underutilized. The e i
nclude information on the number of pecimen (core ) po itive for cancer, the p
ercentage of each po itive core occupied by cancer, and the percentage of Glea o
n grade 4 or 5 cell in each core. For example, my own biop y included 9 probe ,
5 on the right ide (where the cancer had been felt) and 4 on the left ide. On
the right, 3 of the 5 had cancer, and on the left 0 of 4 did. Thu , overall, 3
of 9 were po itive. Several tudie have reported that the percentage of biop y
probe that are po itive for cancer i a better predictor of recurrence than the
PSA level. In a large tudy of 1,149 pro tate cancer biop ie , only 1 core wa
po itive for cancer in 47 percent of ca e ; 2 in 24 percent; 3 in 18 percent; an
d 4 or more in 11 percent. One tudy focu ed peci cally on Glea on 7 cancer and
reported that when one third or fewer core were po itive for cancer, chance of
cancer recurrence after urgery were igni cantly lower than if one half or more
core were po itive. Another tudy reported that the percentage of biop y core
po itive for cancer wa the be t predictor of cancer recurrence for men undergoi
ng beam radiation treatment.9 Biop y report al o u ually include information on
what percentage of each po itive core i occupied by cancer cell . In two of my
po itive core , cancer involved 20 percent of the pecimen; in the third core,
it involved 5 percent. Furthermore, Glea on grade 4 cell
HOW SERIOUS IS YOUR CANCER?
25
occupied 40 percent of the cancer in one core, 20 percent in another core, and t
here were no grade 4 cell in the third core. Studie ugge t that the percentag
e of cancer in the po itive core and the percentage of the cancer that i Glea
on grade 4 and 5 cell are both predictive of outcome. One tudy reported that i
f cancer in any po itive core occupie more than 50 percent of the core, the cha
nce of cancer recurrence i 15 percent higher at ve year and 30 percent higher
at ten year than if the cancer occupie le than 50 percent of the core.10 And
for Glea on 7 cancer , the greater the percentage of grade 4 cell in the po it
ive core , the more eriou the cancer. Attempt to identify additional and more
accurate indicator of everity in newly diagno ed pro tate cancer continue. S
uch indicator include peci c protein in the blood and urine, a well a mea ure
ment of gene that have become activated; the e will be di cu ed in Chapter 15.
Advance in thi re earch area could profoundly affect how we treat pro tate ca
ncer. In ummary, the be t predictor of having pro tate cancer and of the ever
ity of uch cancer are the following: PSA level: below 4 i favorable; 410, you
have a 25 percent chance of having cancer; 1020, you have a 50 percent chance of
having cancer; the higher the level over 20, the greater the chance of your havi
ng cancer and the more eriou the cancer i likely to be. Free PSA: the lower t
he free PSA a a percentage of total PSA, the more likely it i that you have ca
ncer; over 25 percent i a good ign; under 10 percent i not a favorable ign.
PSA velocity: the fa ter the PSA ri e , the more likely it i that you have canc
er; a very rapid increa e in PSA ugge t a rapidly growing cancer. In order for
thi mea ure to be u eful, you mu t have had multiple PSA mea urement over tim
e. If the cancer i large enough to be felt on digital rectal exam, that i not
a favorable ign. The larger the ize (volume) of the cancer, the more eriou i
t i likely to be. If cancer i felt in only one-half of one lobe of the pro tat
e, that i good. If it i felt in both halve of one lobe, the outlook i not qu
ite o favorable. If it i felt in both halve (both lobe ) of the pro tate, tha
t i le favorable.

26
HOW SERIOUS IS YOUR CANCER?
A Glea on core (the combination of two number ) of 5 or le i very good. A c
ore of 6 i till good. A core of 7 (3+4) or 7 (4+3) i not a good, but the e
riou ne depend on the percentage of grade 4 cell pre ent. A core of 8 or mo
re i not favorable. Each biop y con i t of a number of core . The fewer core
po itive for cancer, the better your chance of having a curable cancer. Each bi
op y core that ha cancer i a e ed for the percentage of the total core that
i cancerou . If the core that ha the mo t cancer ha le than 50 percent, tha
t i good. Any ign of pread of cancer out ide the pro tate i definitely not f
avorable.
C H A P T E R
3
Surgical Treatment
T
he urgical removal of pro tate cancer ha for many year been the mo t common t
reatment of the di ea e. It ha been controver ial, with proponent arguing that
urgery i the only real hope for permanently curing the cancer, and opponent
claiming that the main effect of urgery i to produce incontinence and impotenc
e without any clear evidence, compared to other form of treatment, that it actu
ally lengthen men live . The urgical removal of enlarged pro tate , both tho e
cau ed by benign pro tatic hypertrophy and tho e re ulting from cancer, ha a l
ong hi tory. Until the middle of the twentieth century, the operation wa carrie
d out through an inci ion in the perineum, the area between the back of the cro
tum and the anu . During the pa t halfcentury it ha become more popular to urg
ically remove cancerou pro tate through an abdominal inci ion, an operation re
ferred to a a radical retropubic pro tatectomy. The term radical i u ed becau
e lymph node and other ti ue urrounding the pro tate are routinely removed b
y the urgeon a well a the pro tate it elf. Becau e of it location, urgical
removal of the pro tate i technically a dif cult operation, although in the kill
ed hand of an experienced urologi t it ha become fairly routine.
28
SURGICAL TREATMENT
WHO ARE GOOD CANDIDATES? The be t candidate for urgery are younger men who e c
ancer i in a relatively early tage. An example would be a man in hi ftie with
a cancer detected by PSA, not yet palpable on rectal exam, and a Glea on core
of 5 or 6. One tudy reported that men 50 year old or younger had igni cantly lowe
r recurrence rate than did older men and that recurrence rate increa ed with the
age of the patient at the time of urgery.1 Younger men with pro tate cancer ar
e much more likely to be offered, and to elect, urgical treatment. In a Canadi
an tudy, 59 percent of men younger than 60 year of age cho e urgery, compared
to only 6 percent who cho e radiation. By contra t, for men aged 60 to 69, the
percentage who cho e urgery and radiation wa the ame.2 The fact that younger
men with pro tate cancer are more likely to choo e urgery i problematic in ofa
r a the e are the men for whom impotence, a common ide effect of urgery, i a
pt to be the mo t keenly felt; but it i al o true that younger men will more pr
obably recover exual function following urgery. Some urologi t have argued th
at urgery i mo t likely to be effective for men who e cancer i at an intermed
iate tage of developmentlikely to pread but having not yet done o. Example of
uch ca e are palpable tumor with a Glea on core of 6 and a PSA level of 10
to 20; Glea on 7 (3+4) tumor ; and po ibly Glea on 7 (4+3) tumor in which grad

e 4 cell occupy the majority but not mo t of the tumor. Surgical removal in uc
h ca e , it i argued, can truly be life aving. Surgical removal of the pro tate
i a major operation that u ually take two to three hour but may take longer;
therefore, men with other eriou di ea e are not con idered to be appropriate
candidate . One tudy reported that men with heart di ea e, di ea e of their ar
terie , chronic lung di ea e, or evere kidney di ea e all had an elevated death
rate following pro tate cancer urgery; but uch men are poor candidate for an
y type of major urgery. Further, a will be di cu ed in Chapter 8, the princip
al urvival advantage of urgical treatment for pro tate cancer, compared to ra
diation, may not become evident until fteen year or more after urgery. Thu , ca
ndidate for urgery hould have a remaining life expectancy of at
SURGICAL TREATMENT
29
lea t fteen more year . In the Canadian tudy cited above, only 3 percent of men
aged 70 to 79 cho e urgical treatment. When John Hopkin Univer ity urologi t
Patrick Wal h wa a ked whether he would ever con ider urgical treatment for a
man with pro tate cancer who wa 80 year old, he replied: Only if he were brought
in by hi parent .3 Mo t urgeon re trict candidate for urgery to tho e in who
m the cancer i thought to be con ned to the pro tate. If the cancer ha already
pread, it i rea oned, urgical removal will accompli h nothing. Not all urgeon
agree, however, and ome remove the pro tate in ca e in which the cancer i k
nown to have pread, under the theory that removal will low additional pread a
nd make hormone and/or radiation treatment more effective.
THE PROCEDURE A pro tatectomy nece itate an average ho pital tay of two day
and a minimum of three week recuperation. It can be carried out under general an
e the ia or with a local block ( pinal or epidural) in which the patient i awak
e but feel nothing. When a local block i u ed, the patient i al o given edat
ion and u ually leep through the operation. Some urgeon and ane the iologi t
have trong preference regarding which form of ane the ia to u e, wherea oth
er let the patient decide.
30
SURGICAL TREATMENT
I Laparo copic or Robotic Surgery Right for You? Laparo copic and robotic pro t
atectomie are new; thu few follow-up data are available. Advocate and critic
make the following point : All agree that there i u ually le blood lo , le
pain, horter ho pitalization, quicker recovery, and maller car . Some claim
the camera provide better vi ion. Other claim the urgeon cannot ee the who
le field and, becau e their finger are not u ed in the operative field, they ca
nnot feel cancerou ti ue that i palpable but not vi ible in lymph node or u
rrounding ti ue . Some claim the computerized y tem permit a more preci e di
ection of the nerve next to the pro tate and thu improve the chance of reta
ining erection . Other doubt thi . There i a yet no long-term follow-up to a
certain the fact . Some claim that preliminary tudie with the e method have r
eported a higher rate of po itive margin , meaning that ome cancer may have bee
n left behind. Other ay thi occur only with inexperienced urgeon . All agre
e that the co t of urgery u ing the robotic y tem i extremely high and may no
t be fully covered by medical in urance. All agree that laparo copic and robotic
urgery i technically very demanding and difficult to learn. Men electing thi
option hould choo e a urgeon who ha performed at lea t thirty uch urgerie
. (The que tion i , on whom hould tho e thirty urgerie be done?)
SURGICAL TREATMENT

31
In mo t ca e a vertical, abdominal inci ion i made, tretching from the umbili
cu to ju t above the peni ; thi i the retropubic pro tatectomy. A perineal ap
proach, in which a mall horizontal inci ion i made between the back of the cr
otum and the anu , i al o po ible; it can be u eful for men who are obe e, but
may pre ent problem if the tumor i very large. A perineal pro tatectomy i mu
ch le commonly performed now than it wa in the pa t. In the la t decade, lapa
ro copic pro tatectomie have been introduced in which the procedure i carried
out with long in trument through multiple mall abdominal inci ion . Thi urge
ry i technically very demanding and i pre ently performed in only a few center
, but it i rapidly becoming more widely available. Proponent claim that it pr
oduce le blood lo , le po toperative pain, and quicker recovery. All the
ame, laparo copic pro tatectomie have been de cribed by one urgeon a being li
ke backing a tractor trailer around a curve by looking through the rearview mirror
.4 A variant of laparo copic urgery i urgery performed by a robot, which the u
rgeon guide by u ing a computer. The urgeon may be eated everal feet from th
e patient, or theoretically could even operate while itting in the next room. T
hree computer-controlled robotic arm do the actual urgery: one hold a video c
amera, and the other two hold tiny tool for cutting, uturing, and o on. The r
obotic
32
SURGICAL TREATMENT

y tem mo t widely u ed i the da Vinci y tem developed by the U.S. military; t

he robot co t approximately $1.2 million. Surgical removal of the pro tate can
be carried out in many ca e via a nerve- paring procedure. Thi approach, devel
oped in 1982 by Patrick Wal h at John Hopkin Univer ity and Pieter Donker in t
he Netherland , involve careful di ection of the nerve to the peni that run
immediately next to the pro tate gland. When the nerve are pre erved, the chanc
e of retaining erection after urgery are increa ed. In 10 to 20 percent of pr
o tate cancer ca e , it i not po ible to pre erve one or both nerve , becau e
the cancer i immediately adjacent to them, and pre erving the nerve would ri k
leaving cancer cell behind. Mo t urgeon like to wait at lea t four week aft
er the biop y before doing urgery; thi delay give the rectal wall a chance to
heal and decrea e the likelihood of injuring the wall a the pro tate i remov
ed. In addition, it provide the man who ha decided on urgery an opportunity t
o take care of nece ary ta k . All blood-thinning medication including warfari
n (Coumadin), a pirin, and a pirin-containing over-the-counter medication houl
d be topped at lea t ten day prior to urgery. Many urgeon a k patient to d
onate two unit of blood for u e during urgery, ince the average blood lo i
between one and one and a half unit . Some urgeon a k men to take iron pill
to build up their blood before the urgery. Men hould al o di cu ane the ia w
ith the urgeon and/or ane the iologi t. One of the thing I per onally found he
lpful before urgery wa planning how I would pend my po t-op recuperation period.
I went to a video tore and made a li t of old movie I wanted to ee. My wife r
ecommended comedie , but I elected mo tly the tragedie of Ingmar Bergman; they
eemed more in keeping with the occa ion. I al o did all the yard work I could,
knowing that it would be depre ing to it on the porch during my recovery and
look at the job not done. Another pre urgical ta k I found helpful wa to vi ua
lize my elf entering the ho pital, taking off my clothe , putting on a urgical
gown (it hardly warrant uch a de ignation), and being wheeled into the operati
ng room. It wa al o helpful to have vi ited the ho pital for my pre urgical pro
cedure . Although I am a phy ician, my
SURGICAL TREATMENT
33

Thing to Do Prior to Surgery Check your in urance coverage. Draw up and ign a
living will if you do not already have one. If you are employed, check your ick
-leave policy and arrange coverage of your ta k for at lea t three week follow
ing the urgery. Stop warfarin (Coumadin), a pirin, and all medication containi
ng a pirin (Anacin, Bufferin, and the like) at lea t ten day prior to urgery.
Donate two unit of blood for u e during your urgery, and take iron pill if yo
ur urgeon recommend it. Di cu ane the ia option with your urgeon and/or an
ane the iologi t. Plan for your initial week of recuperation. If you live alon
e, make arrangement to have omeone tay with you for the fir t week after urg
ery. Vi ualize your elf going to the ho pital and into urgery. The image will d
ecrea e your anxiety when you actually do o.
relation hip with urgeon and ho pital wa a di tant one on the be t of day .
My only per onal urgical experience were having had my ton il removed a a ch
ild under ether ane the ia (which I clearly recall produced a feeling of uffoca
tion), and the urgical repair of a fracture under local nerve block ane the ia
that did not really block the nerve. Thu , I could identify with General Schwarz
kopf who, in di cu ing hi own lack of enthu ia m for urgery, aid, I go into a
kung-fu attack po ition when I go through the door of a ho pital.5 Men who have pr
eviou ly undergone other major urgery are likely to have an ea ier time with pr
o tate cancer urgery than tho e who have not. On the day prior to urgery, you
are re tricted to a clear liquid diet, and the night before, you are in tructed
to give your elf a Fleet
34
SURGICAL TREATMENT
enema. Entering the ho pital for the urgery proved to be le onerou than I ha
d anticipated, thank to having practiced vi ualizing it and to the upport of m
y wife. Neverthele , the temptation remained to uddenly declare that watchful
waiting wa the better treatmentand bolt. I wa cheduled a the r t ca e of the d
ay, an arrangement I recommend o that you do not have to lie around waiting. My
urgeon greeted me; I brie y contemplated a king him to replace my degenerating h
ip while he wa working on my pro tate, ince it wa anatomically clo e by. Howe
ver, I decided that although I wa certain about hi urgical kill , I wa unce
rtain about hi en e of humor. In the operating room, I urveyed the operating
room taff, rea ured my elf that they appeared alert, then uddenly wa a leep.
I vaguely recall awakening in the recovery room and being a ked que tion . I do
not recall feeling much pain, but I have a high pain thre hold, o I may not be
an accurate gauge. I apparently an wered que tion correctly, becau e I wa whe
eled to my room and my waiting pou e. I con idered rolling my eye up in my hea
d and letting my tongue hang out the ide of my mouth a a novel greeting but ca
lculatedcorrectly, I wa later toldthat even a trong marriage might not with tand
uch a hock. De pite being groggy from the ane the ia, I felt it nece ary to
reconnoiter my anatomy. I had an intravenou tube in one arm, and my leg were e
nveloped in pneumatic tocking that effectively tied my leg to the bottom of t
he bed. Every minute or o, the mechanized tocking lowly ma aged my leg , a
proce de cribed by one man a coiling upward , queezing you from ankle to thigh
like two pet boa in e tru .6 The tocking decrea e the probability of blood clo
t in your leg . My lower abdomen wa covered with a large dre ing, and an adja
cent maller dre ing covered the opening for the urgical drain. A urinary cath
eter exited my peni and ran to a bag attached to the bed. My peni and the urr
ounding ti ue were partially black-and-blue. I wa beginning to under tand the
meaning of the major in major urgery. And then, with a hock, I noticed: my peni
looked a little horter! I recalled a pa age in Dr. Patrick Wal h Guide to Surv
iving Pro tate Cancer:
SURGICAL TREATMENT

35
Note that the gap between the bladder and urethrawhere the pro tate u ed to bei n
ow filled by the bladder. Some men worry that the peni will be hortenedthat the
urgeon will pull it up to meet the bladder. Thi doe nt happen; in tead the bla
dder i mobile, and can ea ily be pulled down to meet the urethra.7 The book rea
urance notwith tanding, the vi ual evidence wa compelling. Mo t men pay clo e
attention to uch detail . My initial reaction wa a mix of incredulity and amu
ement, and I wondered how many other thing that I had read about pro tate urg
ery were mi taken. A few week later, I urveyed the medical literature regardin
g thi phenomenon. Two tudie reported that, when mea ured before pro tate urg
ery and three month later, two third of men howed a decrea e in penile length
. In the majority of ca e , the decrea e wa minimal, but in ome men it wa 15
percent or more. One man wrote that hi genital had hrunk and receded into [hi ]
body to uch an extent that they urely would have been afe from Lorena Bobbit
t.8 Over time, the decrea e in penile length become le noticeable and doe not
eem to affect penile function. Some urologi t have attributed the hortening t
o di u e or bro i of the peni , but thi i obviou ly not the ca e, ince the h
ortening i vi ible immediately po t-op. A more likely explanation i that it i
a reaction to the inevitable mall-nerve damage during urgery. The bottom line
i that nobody eem to know the cau e, but denying that the decrea e may occur
i not helpful when the fact are otherwi e. On the day of urgery, I received
intravenou ketoralac (Toradol) for pain and did not feel much di comfort. Torad
ol i a trong, non teroidal anti-in ammatory drug that hould not be taken for mo
re than ve day becau e of it propen ity for cau ing peptic ulcer or ga trointe
tinal bleeding. It can al o generate nau ea or diarrhea. It made me drow y, o
I lept much of the day and woke up at two in the morning. Fortunately, I had br
ought headphone and CD with me and pent mo t of the night li tening to cla i
cal mu ic, plea ed that I had urvived the ordeal. My private room wa well wort
h the extra expen e and allowed my wife to pend the night there a well. The fo
llowing morning, twenty-four hour after urgery, one of
36
SURGICAL TREATMENT
the urologi t checked me and a ked if I wanted to go home. Thi eemed extraord
inary, but i typical of ho pital tay in the era of managed care. I allowed th
at I thought I would tay for another day; if I had been living alone, I would h
ave tayed longer. Neverthele , becau e of the ri k of ho pital-acquired infect
ion , ho pital are dangerou place to pend time unnece arily and no one hou
ld tay longer than i ab olutely e ential. I pent the day walking the hall wi
th my catheter bag, accompanied by other pro tatectomy patient . In My Pro tate
and Me, William Martin de cribe a imilar cene a the few, the humiliated, the U
rine Corp and add : One look at my new peer group moved me to ugge t that it might
be be t if would-be vi itor ju t ent a note or called. Men look better in pow
er uit .9 By day 2 after urgery, I wa ready to go home but had two urgent que t
ion for my urologi t: What did the pathology report ay about whether my cancer
wa con ned to the pro tate, and had he aved one or both nerve ? The econd que
tion wa quickly an wered: one (a I had u pected would be the ca e from the i
ze and po ition of the cancer). To an wer the r t que tion, my urologi t went to
the pathology department him elf and reviewed the lide with the pathologi t. T
he cancer had penetrated the cap ule but had not gone through it and had a negativ
e margin, meaning that the cancer appeared to have been con ned to the ti ue remove
d at urgery. There wa al o no pread to the eminal ve icle or lymph node . T
hi wa all po itive new . The r t two week of recuperation were urpri ingly pl
ea ant. My wife and I read, took hort walk , and watched movie each afternoon
before having a drink. I wa urpri ed at how ea ily I became fatigued and I too
k a nap each day. I al o li tened to a CD erie on the hi tory of cience and

at in the un. I wa fortunate to have only occa ional pain, uch a when I nee
zed and thereby increa ed the abdominal pre ure; I u ed no pain medication afte
r leaving the ho pital. Some nur e recommend a rm laugh pillow to hold again t your
abdomen when you laugh, neeze, or cough. It i al o important to not become con
tipated, which can be cau ed by pain medication uch a codeine, by iron pill
, and by inactivity; con tipation can be avoided by taking laxative . (I elected
not to take iron pill
SURGICAL TREATMENT
37
Of Dog and Catheter Geoff Barnard of Flag taff, Arizona, wrote the following a
ccount of hi adventure with a catheter after having a radical pro tatectomy fo
r pro tate cancer: Several night ago here at home I woke up to our moke alarm
going off. That cared the dog, Copper, who dove under the bed and in o doing g
ot my catheter tube wrapped around her neck. So imagine my fun, grabbing the tub
e with a forty-five-pound terrified dog pulling in the other direction, creamin
g to Diane to hold the dog and get it unwrapped, with the moke alarm going off
all the while. It will be hard to top that with an encore!
after urgery, a had been recommended to build up my blood count, in order to a
void po ible con tipation.) My main po t-op problem wa the catheter, which rub
bed my peni and made it ore. Anchoring the catheter tube with afety pin to m
y pant leg eemed to help. Other have recommended putting K-Y jelly or antibiot
ic ointment around the opening of the peni to minimize irritation. You are give
n a mall catheter bag that trap to your leg to u e during the day; it allow
you to take walk and move about freely. At night, you hook a large catheter bag
to the ide of the bed. Bert Gottlieb, writing of hi experience in The Men Clu
b, aid that he chri tened hi mall catheter bag Rover, becau e it followed him eve
rywhere.10 My recuperative turning point came with the removal of the catheter a
nd uture clip at ten day ; contrary to what I had been told, the r t did not hu
rt, but the econd did, brie y. Some urgeon leave the catheter in for up to thre
e week . It removal meant that I wa on my own to either recover urinary contin
ence or not. My urologi t had advi ed me to bring to the ho pital what i e ent
ially an adult diaper ( ee Chapter 10) to wear home, but even a I did o,
38
SURGICAL TREATMENT
I felt a if I wa getting well. I re umed driving, which al o made me feel bett
er. Recovering urinary function after pro tate urgery i an intere ting experie
nce. It i a if you have been living in an old hou e for many year and are fam
iliar with the ound of it plumbing. You know when omeone down tair u he the
toilet and when omebody up tair i taking a hower. When your hou e i comple
tely renovated with new plumbing, all the old familiar cue are gone and you hav
e to learn new one . I wa fortunate that my external phincter wa up to the ta
k. Continence returned quickly. I di carded the adult diaper the r t day and the
reafter u ed pad of varying ize for three week . After that, I needed nothing
, and in fact had le dribbling than I had had prior to urgery. Some men are n
ot o fortunate; Michael Korda account in Man to Man de cribe the problem that
occur when continence doe not return quickly. After three week I returned to
work but re tricted my hour . Some men recover more lowly and take more time of
f. My trength gradually returned. At ix week po t-op, at which time my urolog
i t aid I could re ume normal activitie , my wife and I went on a planned vacat
ion to Newfoundland to do ome gentle ea kayaking. It wa not until approximate
ly three month after urgery, however, that I felt that my full trength had re
turned.

SURGICAL TREATMENT
39

COMPLICATIONS Surgical removal of the pro tate carrie the ame ri k of complica
tion a doe all major urgery. The e include infection, po t-op bleeding, and
the ultimate complication, death. The death rate in the r t thirty day following
pro tatectomy, according to a 2005 Canadian tudy, i le than 2 in 1,000 for
men under age 60, and 6 in 1,000 for men 60 to 79.11 The mo t important cau e of
death i thrombo i (clot ) of the vein in the leg , which often cau e tender
ne in the calf or leg welling; when clot break free, they may travel to the
lung and heart a emboli and cau e hortne of breath, che t pain, and ometim
e udden death. The be t prevention i to get patient up and walking oon afte
r urgery and to maintain regular walking and leg exerci e for everal week . W
hile the catheter i in place, a mall number of men have bladder pa m , which
are painful contraction of the bladder a it trie to expel the catheter. Korda
de cribe the e in Man to Man; in mo t ca e , the pa m can be alleviated with
medication. Some men develop a narrowing of the urethra where it i urgically
attached to the bladder, and thu a narrowing of the urinary tream. Severe ca e
of thi bladder neck ob truction require urgical dilation, which can be done
a an outpatient procedure. The three mo t common complication of all treatment
for pro tate cancer are urinary incontinence, impotence, and bowel dy function
. Incontinence rate after urgical removal of the pro tate vary widely and are
a ource of pirited debate among urologi t . Some of the difference are due to
variable level of kill among urgeon . Some re ult from different de nition of
incontinence; for example, ome re earcher u e frequent leakage a a de nition, wher
ea other u e wear pad . One man de nition of frequent and leakage may diffe
from another man de nition , and one man may wear a pad for occa ional, minimal l
eakage while another may not wear one de pite having copiou leakage. Incontinen
ce rate al o vary depending on whether the phy ician i making the a e ment o
r whether the information come from que tionnaire elf-report from the patient
. A one publication noted: Treating phy ician in the e tudie report complicat
ion
40
SURGICAL TREATMENT
rate that are generally low, but may be inaccurate becau e patient may minimiz
e complication of treatment to their doctor , who in turn may ubcon ciou ly di
count patient report of ymptom .12 Finally, incontinence rate vary depending on
the patient group being followed, with young men with low-grade tumor having t
he fewe t problem . The lowe t rate of urinary incontinence following pro tate
urgery have been claimed by the John Hopkin Univer ity group who, for a mall
group of fty-nine patient , reported that only 7 percent were wearing pad eight
een month after urgery. Thi wa a highly elect group of patient , however, w
ith an average age of 57 and early tage tumor (88 percent Glea on 6 or le ; 8
5 percent PSA le than 10). A much more repre entative group of patient i the
1,291 men from ix different area in the United State urveyed by the Pro tat
e Cancer Outcome Study. At two year following pro tate urgery, 22 percent wer
e wearing pad . Thi group wa ub tantially older (72 percent age 60 and older)
and had had more advanced cancer (only 56 percent Glea on 6 or le ) than the
John Hopkin cohort. Other tudie that have a e ed urinary continence in men
following pro tate urgery have reported re ult clo er to tho e of the Pro tat
e Cancer Outcome Study.13 It thu appear that ome degree of urinary incontine
nce i a common complication following pro tatectomy. Approximately 20 percent o
f men u e pad for at lea t occa ional leakage, although tudie ugge t that th
e incontinence i evere in fewer than 10 percent of men. There i al o con en u
that the incontinence i mo t likely to occur in the early month after urger
y and u ually improve over time. Impotence i a common complication of pro tate

 urgery and, like incontinence, it a e ment i made more dif cult by varying d
e nition and the election of patient . Should impotence be de ned a inability to
have an erection at all? An erection rm enough to have intercour e? With or witho
ut pharmacological help? When we review the varied and contradictory tudie on
impotence that have been publi hed, three nding tand out. Fir t, the younger a
man i at the time of urgery, the better are hi chance of regaining potency.
Second, a man who had ati factory exual function prior to urgery i much more
likely to have a favorable outcome
SURGICAL TREATMENT
41
after urgery. Third, erectile function can return very lowly following urgery
; according to one review, it continue to improve after radical pro tatectomy up
to at lea t 2 year after treatment.14 Thi low rate i con i tent with what i k
nown about nerve regeneration after nerve have been traumatized, even when they
have not been evered. That being aid, let me empha ize that, in the word of
one re earch group, few men undergoing radical pro tatectomy eventually achieve th
e preoperative level of erectile function.15 Impotence i a major complicationfor m
o t men, the major complication of pro tate urgery. The reported rate of impote
nce vary widely, however, and are hotly di puted. At one end of the pectrum i
the John Hopkin Univer ity tudy de cribed above, which included a mall, ele
ct ample of young men with early- tage pro tate cancer. Wal h and hi colleague
claimed that the impotence rate among the e men wa 62 percent at three month
po t-op; 46 percent at ix month ; 27 percent at twelve month ; and 14 percent
at eighteen month . El ewhere, Wal h ha claimed that, if both nerve are pre er
ved during urgery, the rate of impotence hould be no higher than 20 percent fo
r men in their fortie and ftie , and no higher than 40 percent for men in their
ixtie .16 Other re earcher have claimed that the John Hopkin number repre e
nt elect patient and are unreali tically optimi tic. The large and more repre
entative Pro tate Cancer Outcome Study reported an overall rate of impotency of
60 percent eighteen month after urgery; the rate for men who had had both ner
ve pre erved wa 56 percent, only lightly better than the overall rate. Other
tudie have reported even higher rate of impotence, including rate of 75 perc
ent and 80 percent at twelve month po t-op.17 Mo t of the e tudie de ned men a
potent if they were able to achieve an erection uf cient for intercour e, with o
r without the a i tance of ildena l (Viagra) or other oral medication. It i cle
ar that nerve- paring urgery i effective in decrea ing the rate of po t urgica
l impotence. Although little advantage wa reported by the Pro tate Cancer Outco
me Study, other tudie have di agreed. A large tudy of 1,014 men aged 60 to 7
0 reported that 92 percent of the men were impotent when neither nerve wa
42
SURGICAL TREATMENT
pre erved during urgical removal of the pro tate, but that only 66 percent were
impotent when one or both nerve were pre erved. The author concluded: Mo t of t
he tudie u ing patient-report, validated, que tionnaire methodology have corro
borated that nerve paring technique i a ociated with better exual . . . recov
ery after radical pro tatectomy than the non-nerve paring technique.18 Becau e of
the ize of the cancer, it i ometime not po ible to pare both nerve . In
ummary, impotence i a eriou problem for men following pro tate urgery. For m
en in their 60 in whom one or both nerve have been pre erved, the impotence ra
te range between 40 and 80 percent. Younger men fare omewhat better, e peciall
y tho e who had ati factory pre urgical exual function. Men in whom both nerve
have been cut during urgery will almo t all be impotent. The third major comp
lication of pro tate cancer treatment i di turbance in bowel function, uch a
crampy pain, diarrhea, and bowel urgency. For individual who elect urgery, thi

 i u ually not a igni cant problem. A large tudy of 1,296 men who had urgical
treatment for pro tate cancer reported minor bowel ymptom in ome men and fou
nd that thi i hort-lived, and during the r t 3 month after urgery bowel functi
on improve igni cantly and e entially return to normal.19
OUTCOME Many men believe that if their pro tate cancer i removed urgically and
if there are no ign that the cancer ha pread, then they are cured. Thi bel
ief ha been fo tered by enthu ia t for pro tate cancer urgery and by media pr
e entation . The author of a 1996 Time magazine article about pro tate cancer cl
aimed that urgical treatment i the only one that can virtually guarantee a cureif
the cancer ha not meta ta ized. . . . If the cancer ha not pread beyond the
pro tate wall and the gland i removed, the cancer i gone. Period.20 Unfortunatel
y, that i not alway true. Recurrence of pro tate cancer following urgery i n
ot a rare event. Two factor trongly in uence the chance of recurrence: the age o
f the man and the tage of the cancer. The younger the man i at the time of ur
gery, the better hi chance that the cancer will be curable. In one tudy of
SURGICAL TREATMENT
43
more than three thou and patient , the chance of cancer recurrence ten year af
ter urgery were 24 percent in men 41 to 50; 29 percent in men aged 51 to 60; 34
percent in men 61 to 70; and 37 percent in men over 70. In another tudy in whi
ch all the men had PSA between 4 and 6, only 13 percent of men aged 40 to 50 we
re con idered to be not cured (de ned by the pathological characteri tic of the c
ancer), compared to 19 percent of men 51 to 60 and 26 percent of men 61 to 73. I
n thi tudy, the age of the man wa a better predictor of outcome than wa the
pre urgical PSA level.21 The tage of pro tate cancer i determined by the Glea
on core and other pathological feature , a de cribed in Chapter 2. In one tud
y that compared men who all had nonpalpable tumor , tho e who had a Glea on cor
e of 6 or le and a PSA of 10 or le had only a 4 percent chance of cancer rec
urrence at ten year ; by contra t, tho e who had a Glea on core of 7 or more an
d a PSA over 10 had a 27 percent chance of cancer recurrence. In another tudy o
f men followed for more than twenty year after urgery, tho e who e cancer wa
con ned to the pro tate at the time of urgery had a 27 percent rate of recurrence
; tho e who e cancer wa not o con ned had an 83 percent rate of recurrence.22 An
important que tion in re earch on the outcome of pro tate cancer treatment i h
ow the outcome i mea ured. Following urgery, the PSA level hould drop to virt
ually zero, ince the pro tate ha been removed and the amount of PSA made by ot
her ti ue i negligible. For thi rea on, it i common practice among urologi
t to a ume that any PSA level of 0.2 or higher mean that ome cancer cell p
read beyond the pro tate at the time of urgery and that the cancer ha recurred
. Some urologi t u e a PSA of 0.4 or higher rather than 0.2 a an indication of
recurrence. A will be di cu ed in Chapter 11, recurrence mea ured by PSA alon
e doe not nece arily mean that the cancer will progre . Another mea ure of ca
ncer recurrence i that it ha pread (meta ta ized) to the lymph node , bone ,
or other organ . Such cancer u ually continue to pread and eventually lead to
death if the man doe not die from another cau e. The ultimate mea ure of treatm
ent for pro tate cancer i how often the cancer kill . Another way to look at ou
tcome i to a e whether treatment lengthen a patient life beyond what he wou
ld have lived naturally or would have lived with
44
SURGICAL TREATMENT
Table 2. Follow-up Studie of Recurrence Rate and Pro tate Cancer Death Follow
ing Surgery for Pro tate Cancer
Hou ton: Baylor Number of men: Average age: Year of urgery: Average follow-up:

% recurrence mea ured by PSA 10 year : 15 year : % meta ta e 10 year : 15 year

: % dead becau e of pro tate cancer 10 year : 15 year : 1,000 63 19831998 4.4 ye
ar St. Loui : Wa hington Univer ity 3,478 61 19832003 5.4 year Roche ter, Minn.
: Mayo Clinic 3,170 66 19661991 5.0 year Baltimore: John Hopkin 2,404 58 198219
99 6.3 year
25
32
48 60 18 24
26 34 10 18
16
NA
2
3
10 18
6 10
SOURCE: G. W. Hull, F. Rabbani, F. Abba , et al., Cancer control with radical pr
o tatectomy alone in 1,000 con ecutive patient , Journal of Urology 67 (2002): 5
28534; K. A. Roehl, M. Han, C. G. Ramo , et al., Cancer progre ion and urvival
rate , Journal of Urology 172 (2004): 910914; H. Zincke, J. E. Oe terling, M. L.
Blute, et al., Long-term (15 year ) re ult after radical pro tatectomy for clin
ically localized ( tage T2c or lower) pro tate cancer, Journal of Urology 152 (1
994): 185057; M. Han, A. W. Partin, C. R. Pound, et al., Long-term biochemical di
ea e-free and cancer- pecific urvival following anatomic radical retropubic pr
o tatectomy, Urologic Clinic of North America 28 (2001): 555565.
pro tate cancer if he had not been treated at all; both of the e factor are di
cu ed in Chapter 8. Four large tudie a e ed the recurrence rate and cancer
-related death rate following urgery for pro tate cancer. Becau e the tudie
included men of different age and with different tage of cancer and al o u ed
different outcome mea ure , it i dif cult to compare them. The tudie were carr
ied out at Baylor College of Medicine in Hou ton, Wa hington Univer ity School o
f Medicine in St. Loui , the Mayo Clinic in Minne ota, and John Hopkin Univer
ity in Baltimore. On the ba i of follow-up period averaging between 4.4 and 6.
3 year , the percentage of men at ten or fteen year following urgery who would
have evidence of cancer recurrence a mea ured
SURGICAL TREATMENT
45
by PSA and by the pread of the cancer (meta ta e ) wa calculated. Death due t
o the pro tate cancer were al o e timated. The re ult of the tudie are ummar
ized in Table 2 and are con i tent with the re ult of mo t maller tudie . The
John Hopkin tudy included more men who were younger (average age 58) and who
had earlier, and thu more curable, tage of cancer. The number of men with no
npalpable cancer in the John Hopkin tudy wa 44 percent, compared to only 7 p
ercent in the Mayo Clinic tudy. In contra t to the other tudie , the John Hop
kin tudy excluded eventy- ve men from follow-up becau e of evidence of advanced
cancer and the need for additional treatment; uch exclu ion improve recurrenc

e rate . Given the data, what can be aid about outcome following pro tate urg
ery? It eem evident that recurrence of pro tate cancer i not a rare occurrenc
e. At ten year after urgery, at lea t one quarter of men will have a recurrenc
e of cancer a mea ured by PSA, and at lea t 10 percent will have meta ta e . At
ten year , the death rate from pro tate cancer i le than 10 percent. Fifteen
year after urgery, there i more evidence of cancer, and the death rate from
the cancer varie from 10 percent in younger men with le evere di ea e to 18
percent in older men with more evere di ea e. Finally, the urgical treatment o
f pro tate cancer ha one triking advantage over all other treatment , which i
that it provide men with the mo t information. After urgery, men know their e
xact Glea on core ba ed on the entire cancer, not ju t the biop y, and they kno
w whether cancer ha pread out ide the pro tate to eminal ve icle or urround
ing lymph node . And becau e the entire pro tate i removed at urgery, monitori
ng the po t urgical level of PSA provide an accurate and unambiguou mea ure of
cancer recurrence. Thu , urgical treatment remove many uncertaintie from the
follow-up; the new may not nece arily be favorable, but at lea t the patient
know what the new i . Perhap for the e rea on a comparative tudy of the men
tal health of men treated by urgery, beam radiation, or watchful waiting report
ed that the men treated by urgery worried igni cantly le after treatment than
men in the other two group .23
C H A P T E R
4
Radiation Treatment
A
fter urgery, radiation i the mo t frequent treatment of pro tate cancer in the
United State . It popularity increa ed after Andy Grove, chairman of Intel, pu
bli hed a 1996 Fortune magazine cover tory account of why he had elected radia
tion treatment for hi pro tate cancer. An analy i of Medicare data found that
radioactive eed therapy (brachytherapy) i replacing radical pro tatectomy a the
treatment of choice for early- tage pro tate cancer.1 Radiation i , of cour e, u
ed to treat many form of cancer. It work by di rupting the deoxyribonucleic ac
id (DNA) of cancer cell , which grow more rapidly than do normal cell ; both typ
e of cell are damaged, but cancer cell are damaged more everely. Radiation h
a been u ed to treat pro tate cancer ince the early year of the la t century.
One of the originator of the idea wa Alexander Graham Bell. In 1903, while pr
e ident of the National Geographic Society, Bell wrote to a phy ician who wa tr
eating cancer: There i no rea on why a tiny fragment of radium ealed in a ne gla
tube hould not be in erted into the very heart of the cancer, thu acting dir
ectly upon the di ea ed material. Bell ugge tion wa implemented, and by 1917 rep
ort began to be publi hed on the ef cacy of radium, in erted directly into the tu
mor, for treating pro tate cancer.2 The u e of external radiation to treat pro t
ate cancer, called beam therapy, did not become wide pread until the 1960 .
RADIATION TREATMENT
47
Both form of radiation treatmentplacing the radioactive ub tance into the cance
r, and beaming it into the cancer from out ide the bodyare widely u ed. The forme
r i now widely referred to a eed therapy. Of cially, it i called inter titial
radiotherapy or brachytherapy, brachy being the Greek word for hort and implying th
at the radiation i placed a hort di tance from the cancer. The eed may be im
planted permanently or ju t for everal hour ; the latter i known a high do e
rate (HDR) brachytherapy. Radioactive form of iodine, palladium, and iridium ar
e pre ently u ed to generate the radiation. Radiation can be beamed into the bod
y from an external ource in a variety of way . The newer method utilize comput

er and hightech equipment to focu the radioactivity harply on the cancerou t

i ue, thereby cau ing le damage to the urrounding ti ue and fewer complica
tion . Beam therapy i of cially referred to a external beam radiation therapy (E
BRT), and the newer variation are called three-dimen ional conformal radiation
therapy (3DCRT) and inten ity-modulated radiotherapy (IMRT). Proton and neutron
beam radiation are in experimental tage . The technology of both eed and beam
radiation treatment i continuou ly evolving; it i likely to improve further, e
pecially in the ability to focu the radiation peci cally on the cancerou ti u
e.
48
RADIATION TREATMENT
Way Radiation I U ed to Treat Pro tate Cancer A. By placing the radioactive u
b tance directly into the cancer. Thi i called eed therapy, brachytherapy, or
inter titial radiotherapy. 1. Permanent: Seed are in erted and left in permane
ntly. 2. Temporary: Seed are implanted for everal hour , then removed. Thi i
called HDR (high do e rate) brachytherapy and i ometime referred to a the A
ndy Grove method, ince it wa popularized by the Intel chairman. B. By beaming
the radiation into the cancer from out ide. Thi i called external beam radiati
on therapy, or EBRT. 1. Three-dimen ional conformal radiation therapy (3DCRT):
pecial computer produce a preci e focu of the beam. 2. Inten ity-modulated rad
iotherapy (IMRT): a high-tech ver ion of 3DCRT in which radiation come from mul
tiple direction . 3. Conformal proton beam radiation therapy: a above, but u in
g proton rather than X-ray .
WHO ARE GOOD CANDIDATES? Radiation treatment, like urgical treatment, work be
t on men with le evere form of pro tate cancer. Ideal candidate are thu me
n who e tumor i not palpable ( tage T1c), or palpable but occupying le than h
alf of one lobe ( tage T2a); who e PSA i le than 10; and who e Glea on core
i 6 or le . For Glea on core of 7 or more, eed therapy i not recommended u
nle it i u ed together with beam therapy. One tudy howed that the be t cand
idate for beam therapy are tho e with the lowe t percentage of cancer-po itive
core (one third or le ) on biop y; thi nding i imilar to the recommendation
for good candidate for urgical treatment.3
RADIATION TREATMENT
49
A Man Who Cho e Beam Therapy If I have urgery, Ill be inactive for a long time.
Im in good hape for eventy-eight and want to tay that way a while longer. I do
nt know what ide effect Ill have with radiation but Im optimi tic, I feel Ill be a
ble to continue my power walk , travel and work. . . . I need to be in Boulder
for an Antarctic work hop May 7 to 9. Charle Neider, Adam Burden
Men who do not wi h to have urgery or who cannot have urgery for medical rea o
n are al o candidate for radiation therapy. Some men imply do not wi h to ub
ject them elve to a major urgical procedure with it attendant ri k and exten
ded recovery period. Other have heart, lung, kidney, or other medical condition
that increa e the ri k of major urgery. Seed therapy require minor urgery a
nd thu can be carried out on many of the e men; beam therapy require no urger
y at all and i often the r t choice of uch men. It can al o be argued that radi
ation therapy i a wi e choice for older men, e pecially tho e with a life expec
tancy of le than fteen year . A will be di cu ed below, the long-term urviva
l of men treated with radiation appear to be a high a that of tho e treated
urgically for at lea t the r t ten year following diagno i of their cancer; if
urgical treatment ha an advantage in regard to life expectancy, it probably be
come manife t only after that period. Therefore, if a man i not likely to live

more than ten year , it i logical to elect a treatment without the burden and
complication of pro tate urgery. In practice, older men are more likely to ch
oo e radiation over urgical treatment; in one tudy of men in their 70 , radiat
ion wa cho en even time more frequently than urgery.4 Finally, beam radiatio
n therapy i increa ingly being u ed for men who have undergone urgical removal
of their pro tate cancer but in whom, following urgery, it i di covered that
not all the
50
RADIATION TREATMENT
A Man Who Cho e a Combination of Seed Therapy and Beam Therapy Whatever therapy
I cho e, I would be willing to accept an increa ed ri k of dying, if I could pre
erve my exuality. I al o vowed to do whatever I could to avoid becoming a urol
ogical cripple. That determination would become my compa , a I worked to et a
cour e inwhat wa for methe uncharted wilderne of cancer. Michael Dor o, Seed o
f Hope
cancer wa removed (there i a po itive margin, or the cancer ha pread through the
pro tate cap ule). A 2005 European tudy demon trated de nitively that beam thera
py following uch urgery igni cantly improved the outcome.5 Finally, men may el
ect radiation treatment becau e they believe that it ha fewer ide effect , e p
ecially incontinence and impotence, than urgical treatment. Some men are not vi
able candidate for radiation treatment, e pecially tho e with large pro tate (
over 50 to 60 gram ). A normal pro tate in a young man weigh approximately 20 g
ram , then increa e gradually a he age . Large pro tate require more eed , a
fact that increa e the treatment ide effect . Large pro tate are al o dif cult
to irradiate adequately by beam therapy without injuring the urrounding ti ue
and without leaving untreated area of the pro tate (commonly referred to a col
d pot ). In ome ca e , the ize of large pro tate can be reduced by r t giving h
ormone treatment. Many urologi t believe that men with di ea e of the urinary
tract or di ea e of the colon, uch a ulcerative coliti , are not appropriate
candidate for radiation treatment becau e radiation may exacerbate tho e di ea
e . Finally, eed therapy i generally not u ed for men who have previou ly had
part of their pro tate removed urgically to relieve ymptom of BPH; car ti u
e complicate the placRADIATION TREATMENT
51
ing of the eed . For recurrent cancer , eed therapy by it elf i not u ed, alt
hough beam therapy i u ed, often in conjunction with hormone therapy ( ee Chapt
er 11). In the pa t, eed therapy and beam therapy have each been u ed by them e
lve . Today, increa ingly, they are being u ed together or in conjunction with o
ther treatment . Thi i true not only for advanced cancer but al o for earlyand intermediate- tage cancer , ome of which are currently being treated with b
oth eed and beam therapy or with beam and hormone therapy. Such u e of combined
treatment remain controver ial; ome radiation advocate argue that eed or b
eam radiation therapy by it elf i uf cient, while other argue that combined the
rapy produce better outcome . The data to re olve thi controver y do not yet e
xi t.
THE PROCEDURE Seed Therapy Compared to other treatment for pro tate cancer, ee
d therapy i convenienta major rea on for it increa ing popularity. U ually, onl
y two outpatient vi it are required. On the r t vi it, the radiation oncologi t
place an ultra ound probe, imilar to that u ed in pro tate biop ie , in the re
ctum and then carefully map the pro tate. Thi allow for a calculation of exac
tly where eed hould be placed and how manyu ually fty to one hundredwill be need

ed. On the econd vi it, the man i given a regional block ( pinal or epidural)
or general ane the ia for a procedure that la t approximately an hour. While he
i on hi back with hi leg elevated and pread, long needle are in erted int
o the pro tate through the perineum, the area between the back of the crotum an
d the anu . The radioactive eed , which are maller than grain of rice, are in
erted into the pro tate through the needle . It i vital that the eed be plac
ed evenly, o that no area of cancer are left untreated; therefore, ultra ound,
CT can , and/or uoro copy are u ed to guide the placement. An antibiotic i giv
en to minimize the chance of infection. Following the procedure, the patient i
taken to the recovery room for a few hour , then allowed to go home. A urinary
catheter i
52
RADIATION TREATMENT
Lawyer and Seed Therapy John Bla ko, M.D., i a pioneer radiation oncologi t in
Seattle and a leader in promoting eed therapy. He advi e men to take a week o
ff from work following the implantation procedure. He i al o quoted a aying: Th
e only people I end back to work right away are lawyer . The e people are u ed
to being a pain in the a . Michael Dor o, Seed of Hope
u ed during the procedure but i u ually removed before di charge. Po toperative
pain can be controlled by ice pack and pain pill . A graphic r t-per on account
of thi procedure i found in Michael Dor o Seed of Hope. Following implantati
on of the eed , the patient can re ume normal activitie within day . One man c
laimed to have run a halfmarathon two week after the procedure!1 Mo t men have
ome continuing di comfort, including frequency and urgency of urination, abdomi
nal tenderne , and pain in the rectum. The e ymptom generally re olve over e
veral week , although they may recur later. After implantation of the eed , men
are mildly radioactive for up to two month . During that time, they are advi ed
not to tand within ix feet of a pregnant woman, not to allow mall children t
o it on their lap , and not to try to conceive a child. Some phy ician al o a
k them to wear a condom during intercour e in ca e a radioactive eed i expelle
d with the ejaculate. Modern creening technique at airport may al o detect ra
diation from the eed , o men may wi h to carry a note from their phy ician. By
the end of the two month , the eed are no longer radioactive and remain in th
e man pro tate for the re t of hi life. The follow-up for men with eed therapy
generally con i t of an appointment two to three week after implantation to a
e the do e of radiation. Another appointment a month later a e e
RADIATION TREATMENT
53
Recovery from Seed Therapy A I wa dre ing, I realized that my crotum wa twi
ce it normal ize, and deep purple! That tartled me. I had an ache in my groin
, but my crotum looked like I hould be feeling eriou pain! That evening wa nt
too bad. My job wa to lie in the hotel bed with an ice bag in my crotch. Pain
pill made it tolerable a I watched TV. I had a con tant urge to urinate, and w
a up frequently trying to pee, but with limited ucce . Sometime in the middle
of the night I pa ed an ob tructing blood clot through my peni followed by a u
rge of pent up urine. Now that got my attention! That wa probably the mo t unco
mfortable event of the entire therapy. Michael Dor o, Seed of Hope
54
RADIATION TREATMENT
Beam Therapy Here in thi urreali tic chamber wa the be t that modern medicine

had to offer. It would be here that I mu t fight my battle with the killer in m
y groin. . . . There I wa , alone in thi temple of technology. Everyone el e ha
d fled the radiation that would oon be flooding the room. A mental image of a
acrificial lamb on the altar fla hed through my mind. . . . Even though I knew I
would feel nothing when the radiation hit me, I had to re i t the urge to cring
e. I felt a need to lighten up, a I lay there taring up at the x-ray machine.
I cho e to addre the one-eyed Cyclop . Take me to your leader, alien! It mu t have
heard me! Suddenly it wa alive and buzzing. Michael Dor o, Seed of Hope
po ible complication . A PSA and rectal exam are u ually done every four to ix
month for the next ve year , then annually.
Beam Radiation The procedure for beam therapy i ub tantially more onerou than
for eed therapy, de pite the fact that beam therapy involve no ane the ia, u
rgery, or pain and i performed completely on an outpatient ba i . Before beam t
herapy can be tarted, everal vi it to the ho pital radiology department are r
equired. During the e vi it , a cradle-like ca t i made of the man body; during
each radiation treatment, he will lie in that ca t to en ure that he i ab olut
ely immobile and unable to move during the treatment . Small black tattoo are p
laced on the lower abdomen o that the radiation beam can be correctly aligned e
ach time. The lower abdomen i carefully mapped by putting a contra t material i
nto the bladder and rectum and taking X-ray
RADIATION TREATMENT
55
picture of it; thi help en ure that the radiation beam will be directed preci
ely at the pro tate and not at another organ. The actual treatment are given ve
day a week for ve to nine week . Thu , men mu t be prepared to follow a rigid t
reatment chedule for everal week and are not allowed to mi a treatment. Thi
regimen may po e no problem for men who work in metropolitan area where they
can top by the ho pital each day on the way to work. But for men who live in mo
re rural area , it may involve taying in a hotel in the city for the entire tre
atment period. The treatment them elve are quick and painle . The man lie in
hi cu tom-made ca t on the X-ray table in a emidarkened room for about fteen m
inute . Depending on the peci c type of beam therapy being u ed, the huge X-ray m
achine may be tationary overhead or it may move during the treatment. La er bea
m , often from everal direction , give the room a Star War ambience; one half
expect to ee Han Solo and Luke Skywalker come through the door.
COMPLICATIONS Two minor ide effect of beam therapy are pubic hair lo and fat
igue. The hair lo may be temporary or permanent, but the radiation doe not af
fect hair on the head. Fatigue, which may be marked, u ually begin three to fou
r week after the treatment cour e begin . Exerci e can help; one tudy reported
that men who walked for thirty minute each day during the treatment period exp
erienced le radiation fatigue.6 Symptom of urinary dy function are common in
men who undergo radiation treatment , both immediately following the treatment a
nd month or even year later. Frequency, urgency, pain on urination, trouble t
arting the urinary tream, narrowing of the tream, and inability to empty the b
ladder completely are all common. Relatively le frequent are ymptom of incon
tinence. Men undergoing beam therapy are a e ed for urinary ymptom a number
of time . At ve to ten month after treatment, 12 percent of men complained of fr
equency, and 18 percent of urgency. Two to four year later, the e ymptom of u
rinary irritation continued to be reported by 15 to 44 percent of men in differe
nt tudie , with evere ymptom in 2 or 3 percent. One tudy that a e ed
56
RADIATION TREATMENT

thirty-nine men an average of thirteen year after beam therapy reported that ha
lf of them continued to report urinary ymptom , e pecially epi ode of blood in
their urine (hematuria).7 It can be argued, however, that the e men were treate
d with technologically earlier ver ion of beam therapy that were le focu ed o
n the pro tate and did more damage to the bladder. For eed therapy, report of
irritative urinary ymptom are imilar to, if not more evere than, for beam th
erapy. One tudy that directly compared the two form of radiation approximately
two year after treatment reported that tho e who had undergone eed therapy ha
d igni cantly more urinary ymptom . Several tudie have reported that men who a
re treated with a combination of eed and beam therapie have more urinary ympt
om than men treated with either therapy alone; thi nding i not urpri ing inc
e the combination yield a higher do e of radiation than either therapy alone.8
It i important to note that although irritative urinary ymptom are common, y
mptom of urinary incontinence are relatively unu ual in men treated with radiat
ion. One large tudy reported that only 4 percent of men were till wearing pad
ve year after beam therapy.9 The irritative urinary ymptom can often be ameli
orated with medication. Symptom of impotence and erectile dy function are commo
n in men who undergo either beam or eed therapy. Rate of impotence immediately
following beam therapy are low but then increa e progre ively during the follo
wing ve year to 50 to 60 percent, owing to radiation effect on the nerve and
mall arterie going to the peni . The ame pattern i een after eed therapy, w
ith a low increa e in erectile dy function in the r t ve year after treatment. P
ain during orga m (26 to 40 percent) and blood in the ejaculate (15 to 17 percen
t) have al o been de cribed following eed therapy. Although eed therapy repute
dly cau e le impotence than beam therapy, two of the three tudie that direc
tly compared the two treatment reported more impotence for men who had undergon
e eed therapy, while the third tudy reported impotence to be lightly higher f
ollowing beam therapy. It ha been ugge ted that drug like ildena l (Viagra) ar
e more
RADIATION TREATMENT
57
Urinary Complication Ive al o had to deal with an irritable bladder. Mine wa nt h
appy being caught in the radiation bathing my pelvi . It now eem to regi ter f
ull at three ounce , and with un eemly in i tence. That make it difficult to i
t through a movie or more than a hundred mile in a car. A hopping trip ha to
be planned with a pit top in mind. Im certainly becoming aware of where all the
bathroom are in town. Michael Dor o, Seed of Hope
likely to be effective in improving erection in men who have had eed therapy t
han in tho e who underwent beam therapy. There i al o evidence, a would be exp
ected, that men who undergo a combination of beam and eed therapy have more imp
otence than men undergoing either treatment alone. Two tudie that compared old
er and newer form of beam and eed therapie both reported lower rate of impot
ence with the newer form of radiation; other complication may decrea e a well
.10 Bowel dy function i a third major complication of radiation treatment for p
ro tate cancer. Thi dy function include frequency (diarrhea), urgency that may
lead to fecal oiling, cramping, pain in the rectum or painful hemorrhoid , and
bleeding. Such ymptom can often be treated with medication and decrea e over
time in mo t patient . In one tudy at ve to ten month following beam therapy, o
ne third of men had bowel frequency and urgency, 18 percent had bleeding, and 16
percent had cramping. A tudy ve year after treatment reported that 16 percent
of men were continuing to have diarrhea, and 13 percent to have rectal bleeding.
A tudy of men thirteen year after beam therapy found that 13 percent were ti
ll experiencing ome rectal bleeding.11 Although rare, another bowel complicatio
n of radiation to the pro tate i rectal cancer. A 2005 tudy found that beam ra
diation,

58
RADIATION TREATMENT
Bowel Complication Morning are the wor t time, becau e of bowel movement . Eve
n on a low-bulk diet, I have two or three each morning, and the anu burn fierc
ely both during the movement and for a couple of hour afterward . The pain i f
atiguing. . . . The oncoming of a bowel movement i it elf unplea ant, and very
different from a normal oncoming one. I feel a perva ive, increa ing malai e, th
en increa ing anxiety. Only later do I feel ignal in the bowel, which come ud
denly and urgently while my phincter warn me I lack normal control over it. Cha
rle Neider, Adam Burden
compared to urgery, increa ed the chance of developing rectal cancer by 70 per
cent.12 Thi gure wa widely reported in the medical new media. It i crucial to
put uch report in proper per pective. In the tudy, the chance of developing
rectal cancer among men treated with beam therapy wa 1 in 246 ca e , compared
with 1 in 386 ca e among men not treated with beam therapy; thu , the ri k of r
ectal cancer increa ed mode tly but not enough to be an overriding factor in el
ecting treatment. Studie of rectal complication following eed therapy, althou
gh le numerou , report imilar nding . In one tudy ve year following treatment
, 9 percent of the men were experiencing rectal bleeding. Two tudie have compa
red bowel dy function in men who had beam or eed therapy; one of them reported
more dy function with beam therapy, the other reported the oppo ite.13 There i
broad con en u that higher do e of radiation, whether by eed or by beam, lea
d to more complication . In one tudy, higher do e of radiation produced urinar
y ymptom in 13 percent and rectal ymptom in 14 percent of men ve year after
treatment, wherea the comparable gure for lower do e of radiation were 4 perce
nt and 5 percent.14 All compari on between eed and beam
RADIATION TREATMENT
59
therapie are tentative, ince both form of radiation treatment are till being
developed.
Outcome A e ing the outcome of radiation treatment for pro tate cancer i both
dif cult and confu ing. The ultimate outcome mea ure for the treatment of all for
m of cancer i the number of people who die from it, but pro tate death may no
t occur until ten, fteen, or more year after treatment. Becau e the average age
of men with pro tate cancer who are treated with radiation ha traditionally bee
n older than that of men treated with urgery, many will die from other cau e b
efore living long enough to a certain whether or not their pro tate cancer would
have killed them. In lieu of u ing death a an outcome mea ure, mo t re earcher
on radiation treatment u e a ri ing PSA level, a do tho e who mea ure the out
come of urgical treatment. However, there i a major difference: Following urg
ical removal of the pro tate, the PSA level i expected to drop to zero; followi
ng radiation treatment, thi i not alway the ca e. Radiation i expected to ki
ll all the cancer cell , but not nece arily all the normal pro tate cell . The
ame i true in radiation treatment for cancer of the brea t or pituitary glan
d; radiation i expected to kill all cancer cell but not all normal cell , o t
he brea t and pituitary continue to function after radiation treatment have bee
n completed. Following radiation treatment for pro tate cancer, the PSA i expec
ted to fall, but the level to which it i expected to fall i widely debated. So
me re earcher ay it hould become le than 1.0, other 0.5, and other 0.3. I
n an attempt to e tabli h a tandard, the American Society for Therapeutic Radio
logy and Oncology (ASTRO) decreed in 1997 that, following the fall of the PSA to
it lowe t level (nadir), a recurrence of pro tate cancer hould be aid to hav

e occurred when the PSA then ri e on three con ecutive mea urement . In practic
e, the de cent of the PSA to it nadir after radiation treatment may take two or
more year . Since the PSA i u ually mea ured only two or three time per year,
it i often not until three, four, or more year after radiation treatment that
the failure of the treatment become evident.
60
RADIATION TREATMENT
A e ing the recurrence of cancer following radiation treatment i till more c
omplicated, however, becau e of what i called the PSA bounce. In approximately
one third of men treated with radiation, PSA level increa e one to three year
after treatment, then return to a lower level. Thi ri e doe not ignify the re
currence of cancer but i in tead thought to be cau ed by a delayed relea e of P
SA from irradiated cancer cell . The PSA increa e a ociated with the bounce may
la t for a long a a year. During thi time, there i no way to tell whether t
he PSA increa e i merely a PSA bounce that ha no clinical igni cance, or whethe
r it indicate a failure of radiation treatment and a recurrence of the cancer.
If it i a PSA bounce, it will go back down; if not, it will continue to ri e. D
e pite the problem in a e ing the effectivene of radiation treatment u ing
the PSA, there i trong evidence that the lower the PSA goe after radiation, t
he le are the chance of recurrence. In one tudy, men who e PSA wa 1.0 or lo
wer following beam therapy had only a 4 percent chance of meta ta e eight year
later, wherea men who e PSA did not go below 2.0 had a 39 percent chance of ha
ving meta ta e eight year later. In another tudy, men who e PSA wa 0.2 or le
had only a 1 percent chance of having cancer recurrence eight year later, co
mpared to a 16 percent chance for tho e with higher PSA .15 Thu , the ab olute l
evel of the PSA nadir following radiation treatment i important in predicting r
ecurrence. A noted in Chapter 2, another igni cant predictor of outcome followin
g beam radiation therapy i the pretreatment PSA velocity. Men who had had PSA i
ncrea e of 2.0 or more in the year prior to the diagno i of their pro tate ca
ncer had a much higher rate of recurrence and death, compared to men who had ha
d PSA increa e of le than 2.0.16 Comparing the outcome of different tudie
of radiation treatment al o generate problem . Some tudie u e the ASTRO guide
line cited above, while other tudie modify tho e guideline or u e an ab olut
e PSA nadir, uch a 1.0 or 0.5. Stati tical problem are abundant: ome tudie
u e the actual number for the follow-up period and other e timate future numb
er ba ed on the follow-up period (actuarial number ). A tudy may follow men fo
r ve year after treatment and then e timate the ten-year recurrence rate ba ed o
n the nding from the
Table 3. Outcome of Seed Therapy: Percentage of Patient with Evidence of Cancer
Recurrence a Mea ured by Ri ing PSA Average follow-up period Recurrence rate 2
0% 5 year Clinical information 2.5 year Period at which recurrence i e timate
d
Study 392
Number of men
Grado et al., 1998 Scott dale, Ariz. 147 12.2 year 34% 10 year
Ragde et al., 2000 Seattle 695 4.3 year 29% 5 year
Beyer and Brachman, 2000 Scott dale, Ariz. 230 3.5 year 18%
Bla ko et al., 2000 Seattle 125 4.3 year 13%
9 year

Grimm et al., 2001 Seattle

10 year
median age 70.4 20% Glea on ?7 median PSA 7.3 average age 70.5 0% Glea on ?7 ave
rage PSA 8.8 median age 74 16% Glea on ?7 31% PSA ]10 median age 69 40% Glea on
?7 median PSA 7.3 median age 70 0% Glea on ?7 22% PSA ]10
SOURCE: G L. Grado, T. R. Lar on, C. S. Balch, et al., Actuarial di ea e-free u
rvival after pro tate cancer brachytherapy u ing interactive technique with bip
lane ultra ound and fluoro copic guidance, International Journal of Radiation On
cology, Biology, Phy ic 42 (1998): 289298; H. Ragde, L. J. Korb, A.-A. Elgamal,
et al., Modern pro tate brachytherapy: Pro tate pecific antigen re ult in 219
patient with up to 12 year of ob erved follow-up, Cancer 89 (2000): 135141; D.
C. Beyer and D. G. Brachman, Failure free urvival following brachytherapy alone
for pro tate cancer: Compari on with external beam radiotherapy, Radiotherapy a
nd Oncology 57 (2000): 263 267; J. C. Bla ko, P. D. Grimm, J. E. Sylve ter, et al
., Palladium-103 brachytherapy for pro tate carcinoma, International Journal of
Radiation Oncology, Biology, Phy ic 46 (2000): 839850; P. D. Grimm, J. C. Bla ko
, J. E. Sylve ter, et al., 10-year biochemical (pro tate pecific antigen) contro
l of pro tate cancer with 125I brachytherapy, International Journal of Radiation
Oncology, Biology, Phy ic 51 (2001): 3140.
Table 4. Outcome of Beam Therapy: Percentage of Patient with Evidence of Cancer
Recurrence a Mea ured by Ri ing PSA Average follow-up period Recurrence rate 4
3% 3.5 year Period at which recurrence i e timated 5 year Clinical informatio
n
Study 253
Number of men
Kupelian et al., 1997 Cleveland 1,527 3.4 year 31%
Beyer and Brachman, 2000 Scott dale, Ariz. 1,100 5.0 year
5 year
Zelef ky et al., 2001 New York
15% low ri k 45% int. ri k 62% high ri k 30% by PSA 7% meta ta e
5 year
Hanlon et al., 2002 Philadelphia
615
5.3 year
5.3 year
median age 71 31% Glea on ?7 58% PSA ]10 median age 74 26% Glea on ?7 54% PSA ]1
0 average age 69 ri k defined by Glea on core, PSA, and tage average age 69 22
% Glea on ?7 average PSA 15
578 205 8.6 year 51% by PSA 18% meta ta e 22% 5 year 10 year
Klein and Kupelian, 2003 Cleveland Zeitman et al., 2004 Bo ton 104 4.5 year

4.3 year
30%
8 year
DAmico et al., 2004 Bo ton
38% Glea on ?7 42% PSA ]10 average age 72 31% Glea on ?7 48% PSA ]10 median age
73 74% Glea on ?7 median PSA 11
SOURCE: P. Kupelian, J. Katcher, H. Levin, et al., External beam radiotherapy ve
r u radical pro tatectomy for clinical tage T12 pro tate cancer: Therapeutic im
plication of tratification by pretreatment PSA level and biop y Glea on core
, Cancer Journal from Scientific American 3 (1997): 7887; D. C. Beyer and D. G.
Brachman, Failure free urvival following brachytherapy alone for pro tate cance
r: Compari on with external beam radiotherapy, Radiotherapy and Oncology 57 (200
0): 263267; M. J. Zelef ky, Z. Fuk , M. Hunt, et al., High do e radiation deliver
ed by inten ity modulated conformal radiotherapy improve the outcome of localiz
ed pro tate cancer, Journal of Urology 166 (2001): 876881; A. L. Hanlon, H. Dirat
zouian, G. E. Hank , et al., Po ttreatment pro tate- pecific antigen nadir highl
y predictive of di tant failure and death from pro tate cancer, International Jo
urnal of Radiation Oncology, Biology, Phy ic 53 (2002): 297303; E. A. Klein and
P. A. Kupelian, Localized pro tate cancer: Radiation or urgery? Urologic Clinic
of North America 30 (2003): 315330; A. L. Zeitman, C. S. Chung, J. J. Coen, et
al., 10-year outcome for men with localized pro tate cancer treated with externa
l radiation therapy: Re ult of a cohort tudy, Journal of Urology 171 (2004): 2
10214; A. V. DAmico, J. Manola, M. Loffredo, et al., 6-month androgen uppre ion
plu radiation therapy v radiation therapy alone for patient with clinically l
ocalized pro tate cancer, Journal of the American Medical A ociation 292 (2004)
: 821827.
64
RADIATION TREATMENT
r t ve year . It i known that the horter the period of real followup, the le a
ccurate the actuarial e timate are apt to be. It ha al o been demon trated tha
t actual and actuarial number may differ con iderably. In addition to the e pro
blem , different tudie of radiation treatment outcome include men of differen
t average age , different everity of cancer , different type of eed and beam
therapie , and different amount of radiation. If we keep the e eriou limitati
on in mind, what are the re ult of radiation outcome tudie for pro tate canc
er? The major tudie are ummarized in Table 35. Following eed therapy (Table
3) the recurrence of cancer, a mea ured by a ri ing PSA, i approximately 15 to
30 percent at ve year po ttreatment and appear to be approximately the ame at
ten year , depending in part on the number of men in the tudy with high PSA o
r high Glea on core . However, mo t men in the e tudie had le evere form
of pro tate cancer; in the ve tudie cited, an average of only 15 percent of the
men had pretreatment Glea on core of 7 or higher. Thu , mo t men in the e tu
die would be expected to have a low rate of recurrence. For beam therapy (Table
4), the recurrence of cancer, a mea ured by a ri ing PSA, i approximately 25
to 40 percent at ve year po ttreatment, and approximately 35 to 50 percent at te
n year . The men in the e tudie had igni cantly more advanced cancer at the ti
me of treatment than the men in the eed therapy tudie ; for example, approxima
tely half of them had Glea on core of 7 or higher. Men treated with a combinat
ion of eed and beam therapy (Table 5) appeared to have a comparatively favorabl
e outcome, imilar to that for eed therapy. A noted previou ly, ome re earche
r believe that adding beam therapy to eed therapy for men with low- and interm

ediate-grade cancer improve the outcome; other re earcher di agree. In ummar

y, three fact tand out. Fir t, multiple tudie have demon trated that the hig
her the do e of radiation given, the lower the recurrence rate. Second, it i ev
ident that the higher the do e of radiation given, the more eriou the complica
tion affecting the urinary tract, the ability to have erection , and the rectal
function. The e two fact put men and their oncologi t quarely between a rock
and a hard place. The third fact i that the election of patient for any outc
ome
Table 5. Outcome of Seed and Beam Therapie When Given Together: Percentage of P
atient with Evidence of Cancer Recurrence a Mea ured by Ri ing PSA Average fol
low-up period Recurrence rate 28% 5 year 2.0 year Period at which recurrence i
e timated Clinical information
Study 62
Number of men
Grado et al., 1998 Scott dale, Ariz. 75 12.2 year 21% 10 year
Ragde et al., 2000 Seattle 689 4.0 year 12%
Critz et al., 2000 Decatur, Ga. 119 5.4 year 2%
5 year
Merrick et al., 2004 Wheeling, W.Va.
7 year
median age 70.2 37% Glea on ?7 median PSA 8.7 median age 70.4 18% Glea on ?7 ave
rage PSA 14.7 median age 66 23% Glea on ?7 27% PSA ]10 median age 58.1 82% low r
i k by Glea on, PSA, and tage
SOURCE: G. L. Grado, T. R. Lar on, C. S. Balch, et al., Actuarial di ea e-free
urvival after pro tate cancer brachytherapy u ing interactive technique with bi
plane ultra ound and fluoro copic guidance, International Journal of Radiation O
ncology, Biology, Phy ic 42 (1998): 289298; H. Ragde, L. J. Korb, A.-A. Elgamal,
et al., Modern pro tate brachytherapy: Pro tate pecific antigen re ult in 219
patient with up to 12 year of ob erved follow-up, Cancer 89 (2000): 135141; F.
A. Critz, W. H. William , A. K. Levin on, et al., Simultaneou irradiation for
pro tate cancer: Intermediate re ult with modern technique , Journal of Urology
164 (2000): 738743; G. S. Merrick, W. M. Butler, K. E. Wallner, et al., Permanen
t inter titial brachytherapy in younger patient with clinically organ-confined
pro tate cancer, Urology 64 (2004):754759.
66
RADIATION TREATMENT

tudy profoundly affect the re ult . At one end of the patient election pectru
m i a tudy in which 67 percent of the men had a Glea on core of at lea t 7 or
a PSA of at lea t 10, with 28 percent having PSA core of over 20; a ve-year re
currence rate of 43 percent i not urpri ing among men with uch eriou form
of cancer. At the other end of the pectrum i a tudy that included only younge
r men (average age 58.1) with le eriou form of cancer. Thi tudy reported
a recurrence rate of 2 percent at even year po ttreatment. In evaluating claim
of better re ult for individual treatment center or peci c treatment , we mu
t alway examine the election of men who were included in the treatment tudy.
One nal mea ure of outcome i how ati ed men are retro pectively with the treatme

nt they cho e. Relatively few tudie have been made on thi que tion, and mo t
men ay they are ati ed with their deci ion. One tudy reported that, approximat
ely three year after treatment, fteen of ninety- ix men wi hed they had cho en a
different treatment. Tho e who were di ati ed included three of fty- ix (5 perce
nt) who had had urgery; one of eleven (10 percent) who wa pur uing watchful wa
iting; three of ixteen (19 percent) who had cho en beam therapy; and eight of t
hirteen (62 percent) who had undergone eed therapy.17 The e number are mall a
nd the difference not tati tically igni cant; more uch tudie are needed in o
rder to obtain a u eful mea ure of outcome.
C H A P T E R
5
Hormone Treatment
T
e to terone, the male ex hormone produced by the te ticle , timulate the grow
th of pro tate cancer . It wa di covered more than a century ago that urgical
removal of the te ticle ca trationimprove ymptom for men with advanced pro tat
e cancer. Charle Huggin , a urologi t at the Univer ity of Chicago, undertook e
xperiment in 1941 to bring about the ame effect by giving men with pro tate ca
ncer female ex hormone to block te to terone. Thi work, which in e ence prod
uce a chemical ca tration, wa the r t ucce ful treatment of any form of cance
r u ing medication. For hi re earch, Huggin wa awarded a Nobel Prize in Medic
ine in 1966. For the pa t half-century, hormone treatment ha been commonly u ed
to treat pro tate cancer, mo tly for advanced ca e in which the cancer ha pr
ead to the bone or other organ . In the 1990 , the u e of hormone to treat pro
tate cancer broadened to include earlier tage of cancer, e pecially in conjun
ction with radiation beam therapy. In 1989, only 10 percent of men being treated
by beam therapy were al o given hormone treatment; by 2001, thi gure had increa
ed to 75 percent.1 The reduction of te to terone by hormone treatment both low
the growth of cancer cell and kill them. The olde t and mo t
68
HORMONE TREATMENT
direct method of reducing te to terone i urgical ca tration. Another method i
giving a drug that block te to terone from getting into the cancer cell . Such
drug are called antiandrogen ; example are bicalutamide (Ca odex) and utamide
(Eulexin). A third method of reducing te to terone i cutting off the hormone (c
alled luteinizing hormone, or LH) that timulate the te ticle to produce te to
terone. Thi LH i made by the pituitary gland in re pon e to in truction from
the hypothalamu in the brain. A group of drug called luteinizing hormonerelea
ing hormone (or LHRH) agoni t and antagoni t trick the hypothalamu into not
ending in truction to the pituitary. The pituitary in turn fail to end LH to
the te ticle , o no te to terone i produced. Leuprolide (Lupron) and go erelin
(Zoladex) are example of LHRH agoni t , and abarelix (Plenaxi ) i an example
of an LHRH antagoni t. Figure 1 illu trate the e chemical method for reducing
te to terone.
WHO ARE GOOD CANDIDATES? The be t candidate for hormone therapy are men who e p
ro tate i large or who e cancer ha pread locally, beyond the pro tate. Thi t
reatment i al o u ed for men who e cancer ha pread di tantly to bone ( ee Ch
apter 11). Hormone are routinely u ed for men who e cancer ha pread locally t
o lymph node near the pro tate, and they are being u ed increa ingly often, tog
ether with radiation, for men
HORMONE TREATMENT

69
Michael Milken Pro tate Cancer In 1993, Michael Milken, a forty- ix-year-old for
mer Wall Street financier, wa diagno ed with pro tate cancer. Hi PSA wa 24, a
nd cancer had already pread to hi lymph node but not to hi bone . He wa imm
ediately tarted on combined hormone therapy, con i ting of both an antiandrogen
and an LHRH agoni t. Within ix month , hi PSA had fallen to 3, and he wa giv
en radiation treatment. U ing continuing hormone treatment and trict dietary co
ntrol, Milken cancer ha continued to remain in remi ion. adapted from Leon Jaro
ff, The man cancer, Time, April 1, 1996
who have high-grade cancer ( uch a Glea on type 8, 9, and 10) that are likely
to pread. In men who have very large pro tate , hormone treatment hrink the
pro tate o that eed or beam radiation therapy will be more effective. Finally,
hormone may be u ed a the only treatment for elderly men with pro tate cancer
who are unable or unwilling to undergo urgical or radiation treatment . Men wi
th low- and intermediate-grade pro tate cancer are not thought to be viable can
didate for hormone treatment. However, hormone are being u ed more and more fr
equently in uch ca e , de pite the fact that no tudie demon trate that they a
re of any value. Some phy ician tart men on hormone treatment immediately afte
r their diagno i while the patient are deciding what de nitive treatment they wi
h to have; the ide effect of uch treatment mu t be weighed again t the dubio
u bene t of it u e.
THE PROCEDURE The earlie t form of hormone treatment, removal of the te ticle ,
can be done urgically on an outpatient ba i . It i of cially called an
70
HORMONE TREATMENT
Drug U ed for Hormone Treatment of Pro tate Cancer Antiandrogen (taken a pill
) bicalutamide (Ca odex) flutamide (Eulexin) nilutamide (Nilandron, Anandron) L
HRH Agoni t and Antagoni t (given by injection) leuprolide (Lupron, Eligard, V
iadur implant) go erelin (Zoladex) triptorelin (Trel tar) abarelix (Plenaxi )
orchiectomy, from the Greek word orchi , for te ticle. The procedure en ure that no
te to terone will be available to timulate pro tate cancer cell . It advantag
e include not having to take monthly injection , and it low co t compared to o
ther form of hormone therapy; it main di advantage i that it i permanent and
thu cannot be u ed like intermittent hormone therapy to decrea e ide effect .
Hi torically, ca tration ha been carried out for other rea on a well. Ca tra
ted men were called eunuch in ancient Per ia, India, and China and were put in
charge of harem ; often, they ro e to governmental po ition of high authority.
Ca trati have al o been valued for their inging voice . Many men today elect me
thod of te to terone uppre ion other than orchiectomy for pro tate cancer. Su
rgical ca tration i a deeply emotional i ue for men, and there are very few ha
rem po ition available to provide con olation! Antiandrogen drug are taken ora
lly. Flutamide (Eulexin) mu t be taken three time a day, but the other can be
taken once daily. The LHRH agoni t , by contra t, mu t be taken a injection , u
ually in the buttock. They can be given once a month or every three month ; leu
prolide (Lupron) ha an additional formulation that can be injected every four m
onth . A different formulation of leuprolide, Viadur, can be implanted under the
kin and la t for one year. Abarelix (Plenaxi ) i an LHRH antagoni t; it act
ion i different from
HORMONE TREATMENT
71

Drug Co t The following are approximate monthly co t of the hormone treatment

commonly u ed for pro tate cancer, a reported in 2004 by the Medical Letter (4
6:2223). Many men take both an LHRH blocker and an antiandrogen. In 2004, Medicar
e decrea ed the reimbur ement rate to doctor for admini tering the LHRH blocker
; the expen e for patient ha increa ed accordingly. Antiandrogen bicalutamid
e (Ca odex) flutamide (Eulexin) nilutamide (Nilandron, Anandron) LHRH blocker l
euprolide Lupron Depot Eligard Viadur go erelin (Zoladex) triptorelin (Trel tar
Depot) abarelix (Plenaxi )
$417 $444 $344
$685 $612 $474 $470 $420 $944
that of the agoni t , but the re ult are the ame. Becau e of eriou ide effe
ct , it i given only to men who cannot take the other drug . The co t of the an
tiandrogen and the LHRH blocker , everal hundred dollar per month, i covered
by mo t medical in urance plan . Some companie making the e drug have program
that upply them to men who are not covered by in urance. In the pa t, diethyl
tilbe trol (DES) wa u ed to uppre te to terone. It i no longer u ed becau
e of it ide effect . Ketoconazole (Nizoral) and mege trol (Megace) are occa io
nally u ed, and other drug are being developed. Many controver ial i ue exi t
with regard to how hormone treatment hould be given. One debate concern whic
h i more
72
HORMONE TREATMENT
effective, an antiandrogen or an LHRH blocker. Another addre e whether the two
type of hormone treatment hould be given together (a i commonly done) a a co
mbined androgen blockade. Other i ue include the optimal duration of treatment w
hen u ing hormone at the ame time a radiation treatment, and whether the horm
one treatment i mo t effective if given before, during, or after the radiation
treatment. In addition, concerning hormone treatment for men who e urgical or r
adiation treatment ha failed, it i debated whether hormone treatment hould be
tarted a oon a the PSA increa e or whether it i better to wait until clin
ical or radiologic ign indicate that the cancer i actually preading. There i
al o lively argument about whether hormone treatment hould be intermittently
topped, to give the body a chance to recover, or whether it i mo t effective i
f given continuou ly. Intermittent hormone therapy con i t of tarting hormone
treatment when the PSA exceed , ay, 4.0; continuing it until it fall below 1.0
; then topping until the PSA again exceed 4.0. Preliminary tudie ugge t tha
t intermittent therapy produce outcome imilar to continuou hormone treatment
but with fewer ide effect ; therefore it i being increa ingly u ed.2
COMPLICATIONS Hormone treatment ha eriou ide effect . A man hould be tarte
d on hormone therapy only when it i likely that the bene t of treatment will out
weigh the ide effect . The mo t important ide effect are the following: Hot a
he : Similar to tho e experienced by women undergoing menopau e, the e occur in
approximately half of men on hormone therapy. They vary from mild u hing to drenc
hing per piration and may la t from a minute to an hour. Medication , including
mege trol (Megace), low-do e e trogen compound , venlafaxine (Effexor), gabapent
in (Neurontin), and paroxetine (Paxil), may provide relief. Brea t tenderne an
d enlargement: Thi ymptom occur in approximately one third of men and i more
common in tho e taking antiandrogen , e pecially after being on the medication
for everal month .
HORMONE TREATMENT

73
Hot Fla he Menopau e i ju t another word in the dictionary for mo t men. While om
e make a token effort to re earch and empathize with their mate , in mo t ca e ,
men feel that it enough for them to how tolerance a women go through the change
. It difficult for men to relate. Few even try. Sometime nature ha a way of putt
ing the proverbial hoe on the other foot. Ive learned the hard way. Even in the
biting cold of winter, I find my elf having to leep with both a ceiling fan and
a floor fan on trying to cool the hot fla he that I experience throughout the
night. Charle William , That Black Men Might Live
74
HORMONE TREATMENT
Brea t Effect It i now even month ince I began my hormone uppre ion thera
py. I had no idea that I would mi my te to terone o much. Ive nicknamed thi t
herapy Rever e Steroid . Thi i not ju t ome therapeutic idea that i to be ta
ken lightly. My body i definitely changing. My brea t are enlarging. If thi g
et any wor e, Ill need to try on ome training bra ! Michael Dor o, Seed of Hope
Impotence: Dif culty with erection occur in almo t all men on hormone therapy, e
pecially men who have undergone orchiectomy or are taking LHRH blocker . Impote
nce occur omewhat le frequently in men taking antiandrogen , particularly in
the initial month of treatment, ince the e men continue to produce te to tero
ne. Decrea ed libido: Following urgical and radiation treatment for pro tate c
ancer, erection are frequently impaired becau e of damage to nerve , arterie ,
and vein , but the libido remain intact. Following hormone therapy, however, bo
th erection and libido are affected. In the r t in tance, men may be impotent an
d care; in the econd, they may be impotent and not care. O teoporo i : Thi i
one of the mo t eriou ide effect of hormone treatment, and it incidence inc
rea e with time. Studie have hown that men on hormone therapy lo e 8 to 10 pe
rcent of their bone ma in the r t two year of treatment, then approximately 2
percent per year thereafter. Con equently, 20 percent of men on long-term hormon
e treatment experience a bone fracture within ve year .3 Thi ri k can be minimiz
ed by regular exerci e, calcium, and vitamin D upplementation. Medication call
ed bipho phonate can off et the effect of hormone therapy and rebuild bone ; t
he be t- tudied drug are pamidronate (Aredia), alendronate (Fo amax), and zoled
ronic acid (Zometa). In 2006, however, report linked the e drug to eriou pro
blem in the jawbone. Men who will be taking hormone therapy for everal year
hould have a ba eline bone mineral den ity can.
HORMONE TREATMENT
75
Lo t Libido Some change have not been o ubtle. It been at lea t ix month i
nce Sherry and I were able to have exual intercour e. It a if the wire from m
y brain to my peni have been di connected. Come to think of it, that exactly wh
at ha happened! I embrace my wife, and nothing tir in my loin . Fortunately,
Ive had a concomitant lo of libido, o I dont eem to mi the ex too much. Mich
ael Dor o, Seed of Hope
Fatigue: Approximately half of men taking hormone treatment experience fatigue
and weakne . The cau e may be a lo of mu cle ma or anemia, both of which ma
y be ide effect of the treatment. Regular exerci e can help, and anemia hould
be treated if it occur . Mental change : Some men on hormone treatment experien
ce increa ed moodine . Depre ion ha been frequently reported, but it i uncle
ar whether it i attributable to the hormone , the ide effect of the hormone ,
or the cancer it elf. Earlier tudie ugge ted that men on hormone treatment h

ave problem with their memory, but a 2003 tudy found that, contrary to what wa
expected, the memorie of the men on hormone igni cantly improved.4 Wor ening
of ymptom : When LHRH analog drug are r t tarted, they produce a brief outpour
ing of te to terone that may temporarily wor en the pro tate cancer ymptom . Th
i equence can be e pecially problematic if the cancer ha already pread to th
e bone . The temporary wor ening of ymptom i called a air; it can be blocked b
y giving an antiandrogen for at lea t one week prior to the r t injection of the
LHRH analog. Other ide effect of hormone treatment may include weight gain, e
levation of chole terol, diarrhea (with utamide), and impairment of night vi ion
(with nilutamide). In addition, hormone treatment affect the PSA level, cau ing
it to become le u eful a a mea ure of recurrence of the cancer.
76
HORMONE TREATMENT
Benefit of Hormone Treatment The [hormone] treatment had one unexpected benefit
: it improved my driving. No kidding! It made me le aggre ive, which i one o
f the accidental ble ing of the whole thing. I dont know if it wa the hormonal
therapy, or ju t facing my mortality, but I found my elf le judgmental than I
u ed to be. Don, in David Bo twick et al., Pro tate Cancer
OUTCOME De pite the igni cant ide effect of hormone treatment, it can be effect
ive in treating pro tate cancer. For men with meta tatic di ea e where the cance
r ha pread to other ite , hormone therapy may improve both the quality and qu
antity of their live ( ee Chapter 11). For men with intermediate and advanced g
rade where the cancer ha pread locally or i at high ri k of doing o, hormon
e treatment in conjunction with radiation i proving to be highly effective. The
bene t of hormone therapy how up in a tudy carried out by the European Organiz
ation for Re earch and Treatment of Cancer (EORTC). A total of 415 men with pro
tate cancer were randomly a igned to be treated either by beam therapy alone or
by beam therapy plu three year of hormone treatment with an LHRH agoni t. Onl
y 11 percent of the men had early tage of cancer; the remainder had intermedia
te or advanced tage , many with local exten ion of the cancer out ide the pro t
ate but without di tant pread. At a follow-up period 5.5 year later, men treat
ed with the combination of beam plu hormone had le evidence of clinical di ea
e (26 percent ver u 60 percent), le evidence of di tant meta ta e (10 perce
nt ver u 29 percent), and fewer death due to pro tate cancer (12 ver u 42) th
an men treated by beam therapy alone.5 A imilar tudy wa carried out by the Ra
diation Therapy Oncology Group (RTOG), a con ortium of U.S. cancer treatment cen
ter . A total of 945 men, all of whom had pro tate cancer that had pread
HORMONE TREATMENT
77
locally, were randomly a igned to receive either beam therapy alone or beam the
rapy plu an LHRH agoni t that wa tarted during the la t week of radiation and
continued inde nitely. At the end of ix year , men treated with the combined the
rapy had fewer di tant meta ta e (27 percent ver u 37 percent) and fewer death
from their cancer (65 ver u 80). Other RTOG tudie have demon trated that a
combination of radiation plu hormone therapy i more effective than radiation a
lone for men with very large pro tate cancer and for tho e with Glea on core
of 8 to 10. Still another U.S. tudy reported that radiation plu hormone therap
y, compared to radiation alone, bene ted men with a Glea on core of 7 or more and
a PSA of at lea t 10.6 It eem e tabli hed, therefore, that adjuvant hormone t
herapy, when given with radiation beam therapy, improve the outcome in ome men
with intermediate and advanced tage of pro tate cancer. The optimal type and
duration of hormone treatment and the do e of radiation are till being debated;
everal ongoing trial are attempting to an wer the e que tion . It i le cle

arly e tabli hed, however, that adjuvant hormone therapy i u eful for men with
earlier tage of pro tate cancer. Nine ongoing tudie that combine hormone tre
atment with radical pro tatectomy, radiation, or watchful waiting are trying to
re olve thi i ue. The large t of the e, the Bicalutamide Early Pro tate Cancer
Study, i following 8,113 men in North America, Europe, I rael, Au tralia, and
Mexico; it preliminary nding ugge t that hormone therapy may be u eful in thi
group.7 If any bene t i demon trated, it will have to be carefully weighed again
t the ide effect of taking hormone . When hormone therapy ha been given to m
en with pro tate cancer prior to urgery, it ha not hown any bene t.8 Many urge
on , in fact, note that hormone treatment prior to urgery can di tort normal an
atomical landmark and make the urgery more dif cult.
C H A P T E R
6
Cryotherapy
T
he idea of killing cancer cell by freezing them ha a long hi tory. The techniq
ue i widely u ed for ome form of kin cancer. Cryotherapy, al o called cryoab
lation, wa r t tried for pro tate cancer in the 1960 , but the re ult were di a
ppointing. Thi approach wa revived in the early 1990 and, with improved techn
ology, ha gradually gained adherent .
WHO ARE GOOD CANDIDATES? The be t candidate are men who are relatively young an
d who do not have evere form of pro tate cancer. Cryotherapy i al o commonly
u ed for individual who have been treated with radiation but who e cancer ha r
ecurred. Surgery cannot be performed on mo t men who have already had radiation
treatment, becau e the radiation de troy anatomical landmark and cau e car t
i ue to form; cryotherapy i therefore one of the few option available. In ord
er for it to be effective, the cancer mu t be con ned to the pro tate; men with ad
vanced cancer are not candidate . Men who e pro tate weigh more than approxima
tely 40 gm are al o not appropriate candidate unle the pro tate can r t be do
wn ized with hormone therapy.
CRYOTHERAPY
79
THE PROCEDURE The procedure i carried out in a ho pital, and the patient u uall
y remain overnight. It i performed under local ( pinal or epidural) or general
ane the ia, with technique imilar to tho e u ed in implanting eed in radiat
ion eed therapy. Long needle are in erted into the pro tate through the perine
um. A freezing ub tance (liquid nitrogen wa utilized in the pa t, but argon ga
i now u ed) i in erted through the needle and the pro tate i frozen into w
hat i memorably referred to a an ice ball. An ultra ound probe i in erted int
o the rectum o that the urologi t can carefully place the needle and en ure th
at ti ue out ide the pro tate i not frozen. Becau e the urethra run directly
through the pro tate, a catheter carrying warm, circulating water i in erted th
rough it o that the urethra will not al o be frozen. Following the procedure, a
urinary catheter i left in place for approximately two week , during which tim
e mo t men do not go back to work.
COMPLICATIONS The complication of cryotherapy are fewer now than in the pa t. T
he mo t dangerou po ibility i freezing the wall of the rectum, which i immed
iately behind the pro tate, thereby creating a tula (hole) between the bladder a
nd the rectum. Thi complication i very
80

CRYOTHERAPY

eriou , even life-threatening, and the wall mu t be urgically repaired. In the

pa t, it occurred in 2 percent of ca e but today i much le common. Urinary
retention and long-term incontinence al o u ed to be common, occurring in up to
two third of ca e , but more recent tudie have reported an incontinence rate
of le than 10 percent. The mo t frequent complication i impotence, ince it i
virtually impo ible to freeze the pro tate without freezing the adjacent nerv
e , arterie , and vein running to the peni . One tudy reported an impotence ra
te of 87 percent among men who had been potent prior to cryotherapy. A one revi
ew ummarize the data, Cryotherapy i a ociated with higher rate of impotence t
han are mo t other localized treatment alternative .1
OUTCOME The long-term outcome of cryotherapy i not yet known, for the technolog
y i till evolving. In one analy i , the cancer recurrence rate two year after
treatment, a mea ured by ri ing PSA, wa 40 percent for low-ri k, 55 percent f
or intermediate-ri k, and 64 percent for high-ri k cancer . In another tudy, th
e recurrence rate at even year were reported to be 39 percent, 32 percent, an
d 39 percent for men with low-, intermediate-, and high-ri k cancer .2 A pecial
concern in cryotherapy i that cancer cell that lie immediately adjacent to th
e urethra, which i being warmed during the freezing proce , may not become fro
zen and will thu urvive.
C H A P T E R
7
Alternative and Experimental Therapie
A
lternative therapie for medical di order have become increa ingly popular in t
he United State , with 42 percent of adult reporting having u ed one or more.1
The increa ing popularity of alternative therapie ha been fueled in part by ra
pid di tribution of information on the Internet and in part by di ati faction w
ith an increa ingly dy functional traditional medical care y tem. Men with pro
tate cancer are e pecially u ceptible to claim made for alternative therapie ,
ince exi ting treatment produce a plethora of unfortunate complication . Alte
rnative medicine cover a broad pectrum of therapie , ranging from pecial diet
, vitamin and mineral upplement , and herbal therapie to hyperthermia, ultra
ound, light therapy, gene therapy, magnet , acupuncture, moxibu tion, yoga, ma
age therapy, relaxation exerci e , and prayer. There i ome evidence that peci c
dietary factor may be u eful in preventing or lowing the growth of pro tate c
ancer; the e factor , along with vitamin and mineral , are di cu ed in Chapter
13. The pre ent chapter will focu on herbal therapie a well a other experim
ental therapie being tudied for the po ible treatment of newly diagno ed pro
tate cancer. Drug and other experimental treatment for recurrent pro tate canc
er are examined in Chapter 11.
82
ALTERNATIVE AND EXPERIMENTAL THERAPIES
HERBAL THERAPIES Herb are eed plant who e tem wither after each growing ea
on. Herbal therapie are e pecially attractive to men with pro tate cancer, in
that they are widely u ed in China, where the incidence of pro tate cancer i ve
ry low. Many men a ume, therefore, that ome cau e-and-effect relation hip mu t
exi t. Herb are generally con idered to be natural, pure, and afe, although t
hi i not alway the ca e. Advocate point out that a quarter of all exi ting m
edication , including digitali and morphine, are derived from plant . Herbal th

erapie are al o popular becau e the ingredient are available in health food t
ore and on the Internet without a pre cription. Many con umer do not realize t
hat a long a the product i not adverti ed to treat a peci c di ea e, e ential
ly no regulation or te ting of the compound take place. The number of men with
pro tate cancer who u e herbal therapie i impre ive, ranging from 10 to 22 p
ercent in variou urvey . A tudy of 238 men treated by urgery or radiation fo
r pro tate cancer in Charlotte ville, Virginia, reported that 12 percent were al
o u ing herb , and a que tionnaire returned by 1,099 men being treated at ix m
ajor urology treatment center in the United State revealed that 16 percent wer
e u ing herbal therapy. Studie of men being creened for pro tate cancer how a
high utilization of herbal therapie a well, ranging from 21 percent in Denver
to 29 percent in Toronto.2 The herb that i mo t popular among men with pro tat
e cancer i aw palmetto, an extract of the dwarf palm that grow in the outhea
tern United State . It i aid that Native American u ed it berrie for urina
ry problem . Extract of the berrie are prepared in a variety of way and conta
in a mixture of fatty acid , terol , avonoid , and other compound . The compo it
ion of aw palmetto preparation may vary, ince it manufacture ha not been t
andardized. Saw palmetto i thought to have ome ability to block te to terone.
Te ted widely in men with benign pro tatic hypertrophy (BPH), it ha been found
to increa e urine ow, decrea e urinary ob truction, and decrea e the number of ti
me the individual ha to get up at night.3 In one tudy, aw palmetto wa a ef
fective a na teride
ALTERNATIVE AND EXPERIMENTAL THERAPIES
83
(Pro car), which i widely u ed to treat BPH; in another tudy, however, aw pal
metto wa only one third a effective. Saw palmetto ha not been demon trated to
have any effect on cancer cell but may provide mall ymptomatic relief by inc
rea ing urine ow. Neverthele , it i widely u ed for pro tate cancer; two tudie
in Canada reported that 12 and 20 percent of patient were taking aw palmetto
, and a tudy of ix American treatment center reported 16 percent. Saw palmett
o i al o popular among men who fear getting pro tate cancer; in one tudy 15 pe
rcent of brother of men with pro tate cancer were u ing it.4 Several tudie of
alternative therapie have noted that men who are relatively young and tho e wh
o have more education, more money, and more advanced pro tate cancer than other
are more likely to u e the e therapie . A tudy in San Franci co comparing the
u e of alternative therapie by ethnic group found that herbal remedie were u
ed almo t equally by white (14 percent), Hi panic (17 percent), African American
(19 percent), and A ian (19 percent) men with pro tate cancer.5 The majority of
men u ing herb or other alternative therapie do not inform their treating phy
icianbut they hould do o, for herb may interact with other medication . A maj
or problem in evaluating the effectivene of herbal therapie for treating pro
tate cancer i that mo t men do not take ju t one herb. Many who take aw palmet
to, for example, al o take pygeum, which come from the bark of a tree native to
Africa, where it ha traditionally been u ed to treat urinary problem . Evaluat
ion become even more dif cult when men are u ing combination of herb , uch a r
a agenthi lehyam, a traditional Indian treatment for cancer that contain 38 diffe
rent botanical . . . and 8 inorganic compound , all prepared into a pa te in a
palm ugar and hen egg ba e.6 Men who u e herbal therapy are likely to be imultan
eou ly u ing other form of alternative medicine. Shark cartilage i an example,
intermittently u ed for many year in cancer patient with the mi taken belief
that hark do not get cancer. In a tudy in the United State , 8 percent of men
with pro tate cancer were taking hark cartilage, while in a Canadian tudy, 24
percent were u ing it. One tudy of hark cartilage in men with pro tate cancer
reported that it had no effect; other tudie are currently under way, funded b
y the National
84

ALTERNATIVE AND EXPERIMENTAL THERAPIES

Well, ye , ome of them do have ide effect .
A Novel Treatment One of my favorite example of a patient trategy come from a
man I know who al o ha pro tate cancer: In tead of imagining hi good cell at
tacking hi bad cell , he goe to Europe from time to time and impo e Continent
al image on hi bad cell . He remind me that in an earlier, more holi tic age,
doctor u ed to advi e ick people to go abroad for their health. Anatole Broyar
d, Intoxicated by My Illne
Center for Complementary Medicine under the National In titute of Health.7 Alte
rnative therapie may, of cour e, have ide effect . For aw palmetto, the e inc
lude headache, ga trointe tinal di tre , and increa ed blood pre ure. Other he
rbal product have been reported to
ALTERNATIVE AND EXPERIMENTAL THERAPIES
85
have eriou complication in ome individual , including liver failure from kav
a kava, hepatiti from jin bu huan, and eizure from yohimbe. The ri k of uch
complication i increa ed by the fact that there i virtually no regulation of
the manufacturing or content of the e preparation .
PC-SPES: A CAUTIONARY TALE The mo t popular herbal treatment for pro tate cancer
ha been PCSPES, the name being an abbreviation of pro tate cancer and the Lati
n word for hope. It wa commercially available from 1996 until 2002, when it manufa
cturer, BotanicLab in Brea, California, abruptly hut down. At the time, it wa
e timated that approximately ten thou and men with pro tate cancer were taking P
C-SPES and a bottle of ixty cap ule wa elling for $108. The recommended do e
ranged from ix to twelve cap ule per day, depending on the everity of the ma
n cancer.8 If all the men had been taking ix cap ule a day, ale would have t
otaled $3.2 million each month. PC-SPES wa formulated by Sophie Chen, a Taiwane
e immigrant who trained in chemi try and initially worked at everal large drug
companie . She al o held an adjunct faculty po ition at New York Medical Colleg
e. Her principal collaborator wa Xuhui Allan Wang, an herbali t who claimed that hi
great-grandfather had been court phy ician to a Chine e emperor.9 Chen and Wan
g formulated PC-SPES a a combination of ix Chine e herb , a Chine e mu hroom,
and the American herb aw palmetto. The e herb are believed to have mild e trog
enic activity, thu uppre ing te to terone. Together with Chen brother, John,
they began to manufacture and ell their new formulation. They adverti ed it a
merely being u eful for pro tate health, not peci cally for cancer or any di ea e con
dition, thu avoiding all regulation that require te ting for afety and ef cacy.
The formulation wa an immediate ucce . Men with pro tate cancer noted a rapi
d decrea e in their PSA, and word pread quickly through the Internet and pro ta
te cancer urvivor group . In 1999 The Herbal Remedy for Pro tate Cancer wa pub
li hed by Jame Lewi , a Ph.D. in education admini tration who him elf had pro t
ate cancer. It extolled PC-SPES and acknowledged that Sophie Chen had read
86
ALTERNATIVE AND EXPERIMENTAL THERAPIES
the
ent
PES
ael
an

entire manu cript and offered her ugge tion which provide accurate a e m
of it content .10 By 1999, pro tate cancer re earcher had begun te ting PC-S
and reporting that it did indeed lower the PSA and hrink the pro tate. Mich
Milken Pro tate Cancer Foundation gave Chen a grant of $150,000 and provided
additional $500,000 for re earcher to te t it. The National Center for Comp

lementary and Alternative Medicine, part of the National In titute of Health, a

l o provided fund . Some of the re earcher who were involved in te ting PC-SPES
were, like Sophie Chen, on the faculty of New York Medical College and from her
received mall amount of company tock.11 From the earlie t u e of PC-SPES, certai
n ide effect were noted. Mo t prominent wa brea t enlargement, which wa nearly
univer al; in ome ca e brea t grew o large that men had to have them urgically
removed. Al o noted were blood clot in the leg thromboemboli mwhich occurred in appr
oximately 5 percent of patient . Several people, including ome of the re earcher
who were te ting PC-SPES, noted the imilarity of it ide effect to tho e of
diethyl tilbe trol (DES), an e trogen compound that effectively uppre e te to
terone but that ha eriou ide effect , including thromboemboli m. In 2001 an
additional ide effect of PC-SPES wa noted when everal men who were taking it
began to bleed my teriou ly. In one ca e, reported in a medical journal, a man
had turned up in an emergency room in Idaho bleeding from every ori ce, and the ho p
ital had barely aved him.12 The denouement of the PC-SPES tale wa provided not b
y the National In titute of Health or the Food and Drug Admini tration, but rat
her by a Connecticut hou ewife.13 Her hu band had been taking PC-SPES with effec
tive lowering of hi PSA until 2001, when the formulation uddenly topped worki
ng. She decided to have variou lot of PC-SPES te ted for adulteration, and the
te t came back po itive: the lot of PC-SPES that had been effective had been
adulterated with DES, and the lot that were no longer effective did not contain
DES. She po ted the information on the Internet, and it wa quickly con rmed by o
ther laboratorie . In January 2002, the tate of California opened an inve tigat
ion, and it wa hown that PC-SPES had been adulterated with DES ince it earli
e t manufacture in 1996. It wa al o found to be adulterated
ALTERNATIVE AND EXPERIMENTAL THERAPIES
87

with indomethacin, an anti-in ammatory drug, and warfarin, an anticlotting drug pr

e umably added to try to counteract the DEScau ed thromboemboli m. In a further
inve tigation by California of cial , the tate found adulteration with ome pharmac
eutical agent in every BotanicLab product that it could te t.14 BotanicLab a a co
mpany pleaded no conte t to a felony charge and hut down. Sophie Chen and her b
rother pleaded no conte t to mi demeanor charge , were ned nearly $500,000, and w
ere barred from the dietary upplement bu ine in California. M . Chen denied a
ny wrongdoing: I am ju t a cienti t, he aid. I am only trying to nd a cure for canc
r.15
EXPERIMENTAL TREATMENTS A variety of alternative therapie for the primary treat
ment of pro tate cancer are in the experimental tage . One uch therapy i hype
rthermia, in which the pro tate gland i warmed; thi treatment i thought to im
prove the effectivene of radiation therapy. The warming may be done by in erti
ng mall tube (inter titial radiofrequency) or mall eed (thermo eed ) direct
ly into the pro tate. An alternative form of hyperthermia i high-inten ity focu
ed ultra ound (HIFU), which i being u ed in Europe and Canada. A probe i in e
rted into the rectum and, over a period of everal hour , end out ultra ound h
eat wave that de troy the cancer cell . The
88
ALTERNATIVE AND EXPERIMENTAL THERAPIES
main drawback of all form of hyperthermia i the danger of heat damage to the r
ectum, urethra, and other adjacent ti ue. Whether uch treatment will re ult in
fewer complication or better outcome remain to be a certained. A form of lig
ht therapy i al o being tudied. A chemical derived from plant chlorophyll i i
njected intravenou ly. A la er light ource i then beamed into the pro tate, ca
u ing the chemical to de troy the cancer cell . Gene therapy i being developed

to treat both primary and recurrent pro tate cancer . A viru injected into the
body goe to the cancer cell , where it deliver a peci c gene. Thi gene make t
he cancer cell more u ceptible to drug , which are then admini tered to the pa
tient. In addition, variou drug are being examined for po ible u e again t pr
o tate cancer. Included are drug that directly attack cancer cell , drug that
make the cancer cell more u ceptible to other form of treatment, and drug th
at cut off the blood upply to the cancer. Mo t experimental drug are being dev
eloped for recurrent pro tate cancer, but a few are directed at primary pro tate
cancer .
C H A P T E R
8
Treatment Deci ion
T
homa Stamey, a pro tate cancer re earcher at Stanford Univer ity, wrote that wh
en people are faced with a eriou illne beyond their comprehen ion, [each of u
] become childlike, afraid, and looking for omeone to tell u what to do.1 In th
e ca e of pro tate cancer, men are frequently advi ed by different phy ician to
do entirely different thing and then told: But in the end, it i up to you to de
cide. There i probably no other major di ea e in which the burden of treatment de
ci ion i placed o fully on the houlder of the patient . In addition to havi
ng to make the treatment deci ion , men are warned that the e deci ion mu t be
made in a timely manner. Some tudie have hown that delay in initiating treat
ment, e pecially for men with advanced tage of cancer, increa e the chance of
cancer recurrence. Judd Moul, a pro tate cancer peciali t at Duke Univer ity,
coun el : I have a referral academic practice in which a lot of patient are getti
ng econd and third opinion . Some of the e patient oat from one doctor to anoth
er eeking the magic an wer. . . . There are a lot of men who become well inform
ed, but thi can ometime lead to paraly i .2 Thi chapter will ummarize ten fac
tor that hould be con idered when choo ing a treatment for pro tate cancer, an
d will pell
90
TREATMENT DECISIONS
out the advantage and di advantage of the major treatment option . Before we c
ommence thi di cu ion, however, we need to addre the que tion that cro e e
very man mind after being diagno ed with pro tate cancer: What will happen if I
do nothing?
WATCHFUL WAITING Pro tate cancer are, in general, among the lowe t-growing hum
an cancer . Since they u ually occur in men who are middle-aged or older, the qu
e tion ari e whether the cancer will kill you before you die from omething el
e. Several tudie have examined the natural cour e of pro tate cancer in men wh
o were not treated. Mo t were carried out in Scandinavian countrie , where medic
al follow-up i excellent and where it i cu tomary to treat pro tate cancer muc
h more con ervatively than in the United State . In mo t European countrie , in
fact, watchful waiting ha been a common approach to pro tate cancer, although t
hi tradition i gradually changing, e pecially in Germany, where radiation trea
tment ha become increa ingly popular. In countrie with waiting li t for elect
ive urgery, uch a England and Canada, a period of watchful waiting i often a
dmini tratively nece ary. The Scandinavian tudy that ha followed men longe t
wa carried out in rebro County, Sweden. There, between 1977 and 1984, 223 men we
re diagno ed with pro tate cancer. Their average age wa 72, their cancer were
all thought to be localized to the pro tate, and the cell type wa poorly differ
entiated (that i , more malignant) in only nine ca e ; thi cla i cation wa prio

r to wide pread utilization of the Glea on grading y tem or availability of the

PSA. The men therefore fell into the category of having pro tate cancer for wh
ich watchful waiting i con idered a rea onable option. They were clo ely follow
ed for an average of twenty-one year , by which time 91 percent of them had died
.3 The percentage of living men who e pro tate cancer pread beyond the cap ul
e during ve-year follow-up period were a follow : ve year , 32 percent; ten year
, 47 percent; fteen year , 55 percent; and twenty year , 64 percent. A total of
16 percent of the men died from pro tate cancer, but in men who were aged 70 or
younger at the time of diagno i , the pro tate cancer death rate wa 22 percent.
TREATMENT DECISIONS
91
The mo t urpri ing nding wa that the pread of the cancer and the cancer-relate
d death were relatively con tant for the r t fteen year after diagno i but then
increa ed harply. A ummarized by the re earcher : An approximately 3-fold high
er rate wa found both for progre ion and death during follow-up beyond 15 year
. . . . If uch patient are in their 60 or younger, di ea e progre ion that
occur after 15 or more year may be a real concern, arguing for early local tre
atment with a curative intent.4 In another Swedi h tudy, 695 men diagno ed with p
ro tate cancer between 1989 and 1999 were randomly a igned to either watchful w
aiting (348 men) or pro tate urgery (347 men). The average age of men in both g
roup wa 65. In the watchful waiting group, 74 percent of the cancer were palp
able but had no known pread, and 30 percent had Glea on core of 7 or higher.
At an average follow-up of 6.1 year after diagno i , among the men being follow
ed by watchful waiting the cancer had pread out ide the pro tate in 31 percent
of ca e , had pread to di tant organ in 16 percent, and had killed 9 percent o
f the men. The comparable rate for men treated by urgery were 12, 10, and 5 pe
rcent, re pectively.5 The mortality rate in the e Swedi h tudie for men who w
ere not treated for their cancer are comparable to rate reported in earlier re
earch. A ummary of ix tudie calculated that 13 percent of men with Glea on
core of 7 or lower who were not treated had died from pro tate cancer by ten y
ear after diagno i . For men with Glea on core of 8 or higher, the death rate
wa 66 percent.6 The mo t u eful American tudy of watchful waiting ha been th
e Connecticut tudy of 767 men who were aged 55 to 74 at the time their pro tate
cancer wa diagno ed. All elected watchful waiting and were followed for twenty
year , by which time 96 percent had died. The men Glea on core were highly pr
edictive of their likelihood of dying from pro tate cancer: men with a Glea on
core of 5 or le had a 10 percent chance of dying from their pro tate cancer; G
lea on core 6, a 27 percent chance; Glea on core 7, a 51 percent chance; and G
lea on core 8 to 10, a 66 percent chance. The likelihood of dying from pro tate
cancer, of cour e, varied widely by the age of the men at the time of diagno i
; that i , men who were older were more likely to die from other cau e before t
heir cancer could kill them. For example, among men with a Glea on
92
TREATMENT DECISIONS

core of 7, 73 percent who were 64 or younger at the time of initial diagno i d

ied from pro tate cancer, but among men 65 or older, only 37 percent died from p
ro tate cancer. Thi tudy, authored by Peter Albert en and hi colleague at th
e Univer ity of Connecticut, wa publi hed in 2005 and hould be reviewed by all
men who are con idering watchful waiting a a treatment option.7 The tudie ci
ted above a e ed the quantity of life for men who e pro tate cancer were init
ially not treated. Other re earcher have attempted to a e the quality of lif
e for uch men. In the r t Swedi h tudy, for example, after four year of watchf
ul waiting, 21 percent of the men had frequent urinary leakage (with half of the
m wearing pad ), and 45 percent complained of erectile dy function. Similarly, a

Dani h tudy followed fty-two men, average age 69, of whom one third had relativ
ely malignant cell type . By approximately three year after beginning watchful
waiting, 31 percent had experienced urethral tricture, 31 percent had required
a tran urethral re ection of the pro tate for urinary ymptom , 21 percent were
u ing pad becau e of ymptom of incontinence, 44 percent had been treated with
hormone , 8 percent had required radiation treatment for meta ta e , 77 percent
had impaired erection , and 12 percent were impotent. On the other hand, an Ame
rican tudy of 310 men, average age 75, reported ome cancer-related decrea e in
exual function but no increa e in urinary or bowel complaint ve year after th
e on et of watchful waiting.8 Thu , for men with intermediate or evere form of
pro tate cancer, it appear that the an wer to the que tion, What will happen if
I do nothing? i rea onably clear. A ummarized by one re earch group: It i like
ly that with watchful waiting, roughly 30% to 40% of men with Glea on um 6 canc
er and well over 50% of men with Glea on um 7 cancer will be dead of pro tate
cancer or uffering from progre ing, hormonally refractory meta tatic di ea e
within 10 to 15 year if not treated definitively, with ome additional time ([5
year ) added if a patient ha tage T1C [not palpable on rectal exam] di ea e a
t diagno i . The e gure are ba ed on the a umption that men who elect watchful
waiting do nothing to decrea e the chance of their pro tate canTREATMENT DECISIONS
93
cer progre ing. A 2005 tudy of forty-four men on watchful waiting who adopted
major life tyle change , including a vegan diet, exerci e, and tre management
, ugge ted that uch change can igni cantly low the progre ion of the cancer.
9 Thi tudy i di cu ed at greater length in Chapter 13. Depending on one per
pective, the outcome of watchful waiting can be viewed a a gla either half em
pty or half full, and men with pro tate cancer till regularly choo e watchful w
aiting a a treatment alternative. A noted in Time magazine: Faced with thi bewi
ldering array of draconian treatment and their humiliating ide effect many older
men and ome younger one opt for watchful waiting. The number of men choo ing thi
option i , however, decrea ing; in 19931995, 20 percent of men cho e watchful w
aiting, but in 19992001, only 8 percent did o.10 The be t candidate for watchfu
l waiting are men who are over age 70 and who e cancer i in the early tage ; f
or example, nonpalpable on rectal exam, Glea on core of 5 or le , and PSA of 5
or le . Such cancer are tati tically likely to grow lowly. Other rea onable
candidate for watchful waiting are elderly men with igni cant health problem ,
a life expectancy of le than ten more year , and an early- tage cancer. A tud
y of 1,158 men who cho e watchful waiting found that three quarter of them were
over age 65 and had a Glea on core of 6 or le .11 Mo t men who choo e watchfu
l waiting get repeat PSA and digital rectal exam at lea t twice a year and rep
eat biop ie yearly to watch for progre ion of the cancer. A di advantage of wa
tchful waiting i living with uncertainty. You are e entially placing a bet, an
d if you bet wrong, you may lo e the window of opportunity to cure the cancer. A
Patrick Wal h ob erved: When cancer e cape from the pro tate, it doe nt end out
a pre relea e announcing the event; it ju t goe , a ilently a it appeared
in your body in the r t place. One man who bet wrong wa Willet Whitmore, chief of
urology at Memorial Sloan-Kettering Cancer Center in New York and regarded a on
e of the nation pro tate cancer expert . When he him elf got pro tate cancer, he
cho e watchful waiting, but hi cancer progre ed. Before he died in 1995, it wa
reported that he aid he regretted the fact that he had waited too long before
actively treating hi di ea e. Ironically, Whitmore i mo t quoted today for hi
uccinct expre ion of the dilemma of
94
TREATMENT DECISIONS

Watchful Waiting Morty i a eventy-four-year-old retired magazine editor who e

pro tate cancer wa diagno ed two year ago. Hi PSA at diagno i wa 11 ng/mL a
nd it ha tayed there, plu or minu a point, for the pa t year. Hi DRE [digit
al rectal exam] i normal, he ha no ymptom , and hi can are clean. After my i
nitial diagno i , I con ulted a lew of urologi t and radiation peciali t , he
ay . They all told me I hould get carved or get zapped. But I decided in tead ju
t to keep an eye on thing . Since then, Ive been in good health. I leep through
the night without having to get up to pee. I till enjoy the good life, exually
, emotionally, and in every other way. In fact, my life i even better, becau e
Ive become vividly aware of how preciou each day i . David Bo twick et al., Pro tat
e Cancer
pro tate cancer treatment: If cure i nece ary, i it po ible, and if cure i po
ible, i it nece ary?12 In actual practice, watchful waiting i evolving today
into nothing more than delayed treatment for many men. The rea on, according to
Judd Moul, i that the men ju t cant tand to ee their PSA value going up. Either
the patient , their doctor , or a combination of both together get cold feet. Thu
, one tudy of men who cho e watchful waiting reported that 53 percent had aban
doned the trategy and ought treatment within two year ; other tudie too have
reported igni cant dropout rate from watchful waiting.13 Additional in ight on
the effectivene of watchful waiting a a treatment trategy hould come from t
he large Pro tate Cancer Intervention Ver u Ob ervation Trial (PIVOT) being run
by the federal Department of Veteran Affair . Thi trial regi tered 731 men be
tween 1994 and 2002, and re ult hould be available by 2008.
TREATMENT DECISIONS
95
TEN FACTORS TO CONSIDER Choo ing a treatment for your pro tate cancer will be ea
ier if you y tematically con ider each of the following ten factor : 1. The e
verity of your cancer: A de cribed in Chapter 2, pro tate cancer can be divide
d into four group on the ba i of ri k of recurrence and progre ion: Low ri k:
Not palpable on rectal exam (T1) or, if palpable, occupie le than half of on
e lobe (T2a); Glea on core 6 or below; and PSA le than 10. Intermediate ri k:
Palpable and occupie more than half of one lobe (T2b) or i in both lobe (T2c
); or Glea on core of 7; or PSA of 10 to 20. High ri k: Cancer ha pread beyon
d the cap ule of the pro tate but not to the eminal ve icle (T3a); or Glea on
core of 8 to 10; or PSA greater than 20. Very high ri k: Cancer ha pread to
eminal ve icle (T3b) or lymph node (N+), or ha meta ta ized to bone or other
di tant organ (M+). The everity of the cancer can be further re ned by con ider
ation of the number of biop y core po itive for cancer, the percentage of each
core containing cancer, and the velocity with which the PSA ha ri en. The ever
ity of your cancer hould be the ingle large t determinant of your treatment ch
oice. 2. Your life expectancy: will treatment extend it?: How long you are likel
y to live i the econd-mo t important factor in making a treatment deci ion. A
noted in the accompanying box, treatment deci ion for a 50-year-old man who i
expected to live an additional twenty- ve or more year may be quite different fr
om deci ion for a 70-year-old man who e life expectancy i le than fteen more
year . Be aware that the e life expectancy projection are average for the enti
re American male population, and that many medical and life tyle factor modify
the e number . For example, tudie have
96
TREATMENT DECISIONS

hown that a 40-year-old man will lo e 3.1 year of life if he i overweight a

mea ured by body ma index (BMI 2529) and 5.8 year of life if he i obe e (BMI
30 or more), compared to men of normal weight. If he i both overweight and a m

oker, he will lo e 6.7 year , and if obe e and a moker, he will lo e 13.7 year
.14 In a review of the tudie on cancer recurrence, rate of meta ta i , and de
ath attributable to pro tate cancer, it eem very likely that active treatment
extend the life of mo t, but not all, men who choo e it over watchful waiting.
At thi time, there i no evidence that either urgery or beam radiation ha an
advantage over the other in thi regard; the lower death rate reported with u
rgery appear to occur becau e urgery i more likely to be offered to younger me
n with le eriou form of cancer. In uf cient information i available to deter
mine whether eed radiation extend life more readily than the other treatment ,
but data o far do not ugge t that it doe . 3. Your willingne to live with u
ncertainty: Some men are more willing than other to live with uncertainty. Reti
red General Norman Schwarzkopf i one who i not willing, a he emphatically exp
lained: Im not a type-B per onality who know I have a cancer growing in ide of me
and can live with the knowledge. Not urpri ingly, Schwarzkopf cho e urgery a hi
treatment. He fall into the category of men who ay, I want the cancer out, pre
ferably by ye terday.15 Surgery provide the mo t information about pro tate cance
r: it actual ize; the true Glea on core ba ed on the entire cancer; whether t
he cancer extend into or beyond the cap ule; and whether it ha already pread
beyond the margin of re ection or to the eminal ve icle or lymph node . Equal
ly important i the fact that the PSA following urgery can be u ed a a predict
or of cancer recurrence. By contra t, radiation treatment provide no informatio
n about the exact pathologic tage of the cancer, and the po tradiation PSA i a
le accurate predictor of recurrence, e pecially in light of the po ibility o
f a PSA bounce. Hormone treatment may al o interfere with the accuracy of the PS
A a a predictor of recurrence. For ome men, having thi information i le im
portant, and they are willing to accept ome uncertainty in return for what they
perceive to be the advantage of other form of treatment. The problem of uncer
tainty following pro tate cancer treatment
TREATMENT DECISIONS
97
Life Expectancy Table The following are U.S. life expectancy data ba ed on 2002
mortality tati tic and publi hed by the Center for Di ea e Control and Prevent
ion in 2004. Life expectancie in Canada and We tern Europe are approximately on
e year longer. Your life expectancy i age: White male Black male 77.4 77.9 78
.5 79.3 80.3 81.6 83.3 85.3 87.7 90.7 94.1 72.8 73.5 74.6 76.0 77.6 79.6 81.8 84
.5 87.5 90.8 94.5
If you are age: 40 45 50 55 60 65 70 75 80 85 90
Note: Life expectancie are average for the entire white and black male populat
ion. If you have heart problem , hyperten ion, high chole terol, diabete , other
eriou illne , or are overweight, a moker, u e alcohol exce ively, do not e
xerci e, and/or your parent and grandparent died relatively young, deduct a fe
w year from the life expectancy table. If you have none of the e factor , add a
few year . SOURCE: E. Aria , United State Life Table , 2002, National Vital St
ati tic Report 53 (2004): 16.
98
TREATMENT DECISIONS
wa a e ed in a tudy of fear of recurrence in men undergoing urgical, extern
al beam radiation, or eed implant radiation treatment. Prior to treatment, the
men in all three group had approximately the ame level of fear. Two year afte
r treatment, the men who had undergone urgery had the lea t fear of recurrence,
and tho e who had been treated with either beam or eed radiation cored approx
imately 10 percent higher on the fear a e ment cale. The e re ult are imila

r to the nding of better po t-op mental health in men who were treated by urger
y, compared to tho e treated by beam radiation, a de cribed in Chapter 3.16 4.
Quantity ver u quality of life: Treatment deci ion for pro tate cancer often i
nvolve deciding between the po ibility of living longer but not a well and liv
ing well but not a long. For example, watchful waiting for a man with a low-ri
k cancer promi e a relatively ati factory quality of life at lea t initially,
but a de nite po ibility that he will not live a long a if he were treated. Con
ver ely, choo ing urgery may provide a longer life but one with a high probabil
ity of at lea t partial impotence and other ide effect . 5. Sexual function: Th
e relative importance of exual function mu t be con idered when making treatmen
t deci ion . For ome men, it may be the ingle mo t igni cant factor. It i know
n that, following treatment for pro tate cancer, men are more likely to retain a
cceptable exual function if they are younger and if they functioned well prior
to treatment. However, for the majority of men, the progno i i poor; a ummar
ized by one re earch group, in reality, mo t [pro tate cancer] urvivor experienc
e evere and la ting exual dy function and di ati faction.17 Being diagno ed wit
h pro tate cancer i , in fact, le traumatic for men who already have ome impo
tence, a oppo ed to men who till have active ex live ; the former have le t
o lo e. For a man who e r t priority i to pre erve exual function, none of the
choice are attractive. Even watchful waiting carrie a long-term ri k of increa
ing exual dy function, either from the expanding tumor or from the hormone or
radiation therapy that u ually become nece ary a the cancer progre e . Howev
er, men who opt for watchful waiting do pre erve their exi ting exual function
for the immediate future, in contra t to all other treatment option .
TREATMENT DECISIONS
99
Figure 2. Percentage of Men Reporting Impotence Following Beam Radiation and Sur
gical Treatment for Pro tate Cancer. SOURCE: Data from A. L. Poto ky, W. W. Davi
, R. M. Hoffman, et al., Five-year outcome after pro tatectomy or radiotherapy
for pro tate cancer. Journal of the National Cancer In titute 96 (2004): 135867.
When treatment option other than watchful waiting are compared, radiation treat
ment pre erve exual function better than urgery for at lea t the r t two year
. Fourteen or more tudie have compared exual function after beam radiation an
d urgery, with the former being found uperior every time. Although far fewer
tudie have compared eed radiation to urgery, they al o ugge t that eed radi
ation ha an advantage in pre erving exual function for at lea t the r t year fo
llowing treatment.18 For the longer term, the advantage of radiation treatment o
ver urgery in pre erving exual function appear to decrea e. The multi ite Pro
tate Cancer Outcome Study followed 981 urgery and 286 beam radiation patient
for ve year after treatment. The tudy de ned impotence a an erection in uf cient fo
r intercour e. At two year , 82 percent of men who had had urgery were impotent c
ompared to 50 percent of tho e who had had beam radiation. At ve year , 79 percen
t of men who had had urgery were impotent compared to 64 percent of tho e who h
ad had beam radiation.19 The e re ult , hown in Figure 2, re ect the growing awar
ene that urgery produce immediate impotence but then gradual improvement tha
t
100
TREATMENT DECISIONS
may continue for two year or longer, wherea radiation treatment produce a lo
w decline in exual function that may continue for ve year or more. Although ee
d therapy, ba ed on early report , wa thought to produce le exual dy functio
n than either beam therapy or urgery, thi reputation ha not held up over time
. A tudy comparing exual function in 154 men treated with eed therapy and 60

men treated with urgery reported that exual function wa better with BT [brachyt
herapy] initially but the e difference did not per i t at a longer follow-up. In
a tudy that compared all three therapie , men who had undergone eed therapy ra
ted them elve lightly lower on exual quality of life than men who had undergo
ne either beam therapy or urgery.20 One rea on may be patient ati faction, whi
ch i included in quality-of-life cale . If men choo e eed therapy becau e the
y expect to retain exual function but then are di appointed, thi di ati facti
on will be re ected in low core on elf-rating que tionnaire . Conver ely, men w
ho choo e urgery expecting to have evere exual problem ometime nd that the
problem are not a bad a they feared, and uch men may be comparatively ati ed
. A eriou problem in comparing eed therapy with other therapie on any outcom
e mea ure i that eed therapy increa ingly i accompanied by beam therapy. In 1
999 half of all men treated with eed therapy al o had beam therapy. Hormone the
rapy i al o being u ed increa ingly often with both eed and beam therapy. In
uch ca e exual function i invariably lower, u ually much lower, than without
the econd therapy. In a ummary of tudie of men under age 60, beam therapy al
one produced an impotence rate of 50 percent, but beam therapy plu hormone pro
duced a rate of 80 percent. Similarly, eed therapy alone produced an impotence
rate of 57 percent, but for eed therapy plu hormone , the rate wa 86 percent.
21 What about Patrick Wal h claim that at John Hopkin . . . 86 percent of men w
ho undergo urgery are potent? Thi frequently cited number i the re ult of a ma
ll tudy of relatively young men, median age 57, who had early- tage cancer . In
almo t all ca e (89 percent), it wa po ible to remove the cancer and pre erv
e both nerve . A total of fty-nine men provided que tionnaire data over an eighte
en-month period, but only twenty-four men returned all que TREATMENT DECISIONS
101
tionnaire ; the tudy ha been criticized on other methodological ground a wel
l.22 What thi tudy doe how i that young men with early- tage pro tate cance
r retain exual function when both nerve are pre erved; unfortunately, mo t men
with pro tate cancer are not thi young, and it i ometime not po ible to pr
e erve both nerve becau e of the ize of the tumor. In ummary, if pre erving
exual function for the near term i your r t priority, your be t bet i watchful
waiting combined with a lot of luck. Your next-be t option i beam or eed radia
tion alone, which will provide a mode t advantage over the urgical option for a
t lea t a year or two, although not nece arily for long period . With urgery,
pre erving both nerve , if po ible, provide a rea onable chance for exual fun
ction, e pecially for younger men, wherea pre erving ju t one nerve i not au p
iciou . The wor t option for exual function are urgery with neither nerve pre
erved, beam and eed radiation together, and any hormone therapy; the incidence
of impotence with all of the e i very high. 6. Urinary function: Problem with
urinary function hould al o be con idered when making treatment deci ion . For
many men, the po ibility of being partially or completely incontinent cau e g
reater fear than impotence. Even dripping urine and wearing a pad may have, acco
rding to a urvey of Medicare patient , a more ignificant effect on patient than
lo of exual function.23 Multiple compari on of urinary function following ur
gery and beam therapy have hown that urgery produce igni cantly more urinary l
eakage. In one tudy, 29 percent of men who had had urgery wore pad to tay dr
y compared to only 4 percent of men who had had beam therapy. Another tudy repo
rted that urinary function wa wor e in men who had had urgery immediately foll
owing treatment, but then gradually improved during the r t year to almo t equal
the urinary control of po tbeam radiation patient .24 Although leaking and other
ymptom of urinary incontinence are unu ual following radiation treatment, irri
tative urinary ymptom are not. Such ymptom include frequency, urgency, havin
g to get up everal time at night, and trouble tarting the urinary tream. Suc
h ymptom appear to be e pecially common following eed therapy. In one compara
tive tudy two year following treatment,

102
TREATMENT DECISIONS
men who had had eed therapy complained of irritative urinary ymptom much more
often than men who had undergone beam therapy or urgery.25 In ummary, leaking
urine and other ymptom of incontinence are common following pro tate urgery,
but u ually improve over time. Irritative urinary ymptom are more common with
radiation treatment , e pecially eed therapy, and the e may per i t. 7. Bowel
function: Pro tate cancer treatment may injure the bowel wall, which i located
immediately adjacent to the pro tate. Comparative tudie have con i tently ho
wn that thi damage i more likely to happen during radiation, e pecially eed t
herapy, than during urgery. Bowel ymptom may include diarrhea, urgency, fecal
oiling, cramping, and bleeding. In one comparative tudy of men two year foll
owing treatment, 17 percent of tho e who had been treated with eed therapy a e
ed bowel dif cultie a being a moderate or eriou problem, compared to 5 perce
nt of men who had undergone beam therapy and 3 percent of men who had had urger
y.26 Bowel ymptom are wor e immediately after radiation, then improve over tim
e but may take a long a two year to tabilize. 8. Acce to a competent docto
r: The acce ibility of competent urologi t , urgeon , radiologi t , and oncolo
gi t i another factor to con ider when making a treatment deci ion. If you hav
e acce to only one uch peciali t, or if your health in urer dictate your ch
oice, your treatment deci ion may be made for you. Relatively few men have the r
e ource to call the phy ician of their choice and ay, I would like to be chedul
ed for treatment by you next week and, incidentally, I would like to donate $100
,000 to your department re earch fund. The men who are able to do o alway get an
appointment. You hould look for a treating phy ician who i competent and cari
ng, who i intere ted in you and your problem. He or he hould be board certi ed
and not have been ubject to any medical di ciplinary action . Thi information
i available from tate medical board and appear on ome Internet web ite . Th
e be t way to nd a competent phy ician i to a k for recommendation from everyon
e you know who ha any connection to the medical profe ion. Widely adverti e th
e fact that you have pro tate cancer, o that your friend ,
TREATMENT DECISIONS
103
and your friend friend , will help you identify the mo t competent phy ician . O
ne book on pro tate cancer cleverly ugge t calling the ecretarie and nur e
in the local department of urology and a king, Who at your in titution would you c
hoo e to treat your father?27 Another ource of helpful information i the local p
ro tate cancer upport group. Further, you want a treating phy ician who perform
thi procedure on a regular ba i . Urologi t hould be doing at lea t twenty- v
e radical pro tatectomie per year, and radiation oncologi t hould be doing at
lea t fteen eed or beam therapie per year. Multiple tudie have hown that hi
gh-volume urologi t generate igni cantly fewer immediate and long-term po topera
tive complication . At the far end of the high-volume pectrum are urologi t u
ch a William Catalona at Wa hington Univer ity, Patrick Wal h at John Hopkin
Univer ity, and Peter Scardino at Memorial Sloan-Kettering Cancer Center, who ha
ve done, re pectively, at lea t 3,478, 2,494, and 1,000 radical pro tatectomie .
28 It i both legitimate and important to a k a potential treating phy ician app
roximately how many procedure he or he did la t year, and for how many year h
e or he ha been doing them. It i unfortunately not po ible to a k your poten
tial treating phy ician ome of the mo t crucial que tion . The e include: How m
uch do you u ually drink the night before you treat patient ? Are you having an
affair with a nur e or technician in the operating room or radiology department
that will di tract you? Have you done thi procedure o often that you are bored
and pend mo t of the time thinking about your golf game or inve tment portfoli

o? For men having urgery, one other que tion to a k your phy ician i , Who will
a i t you in urgery? A radical pro tatectomy i a technically dif cult procedur
e that may la t four hour or more. The ideal a i tant i another board-certi ed
urologi t. In univer ity teaching ho pital , the a i tant i often a urology or
general urgery re ident in training who may or may not be competent. If you ar
e having your urgery at a univer ity medical center, you may have to accept tha
t fact. For men choo ing urgery, it i u eful to a k your phy ician about hi o
r her opinion of nerve paring. Thi procedure ha been practiced for more than
twenty year and i known by all board-certi ed
104
TREATMENT DECISIONS
Select a Urologi t, Not a Philo opher In Intoxicated by My Illne , Anatole Broy
ard aid that he would like to di cu hi pro tate with hi urologi t not a a di
ea ed organ but a a philo opher tone. . . . I there an Urde ire, an archeolo
gy of pa ion that antedate or predate the pro tate? You are about a likely to
find uch a per on a you are to find a lawyer who play Bach Goldberg Variation
while helping you with your will, or a plumber who di cu e T. S. Eliot poetr
y while fixing your ink. They exi t, but they are very rare creature . Competen
t urologi t , rather, are highly killed craft menand that i exactly what you wa
nt. They may or may not be good at talking to you, although you hould expect th
em to an wer your que tion . Medical tudent who are good at talking often beco
me p ychiatri t , and, a a p ychiatri t, I can tell you that you certainly do n
ot want one of u operating on your pro tate.
urologi t . Whether or not one or both nerve can be pared depend on the ize
and po ition of the cancer, which will not become known until the pro tate i be
ing removed. Beware of blanket promi e . Beware al o of tati tic . If your trea
ting phy ician overwhelm you with data on how killed he or he i , or how illu
triou the department of urology or radiology i , get another opinion. It i po
ible to produce advantageou treatment tati tic by accepting only ea y ca e
, and ome urologi t have followed thi cour e. If you are married, it i u efu
l to have your wife join you for interview with po ible treating phy ician ; w
omen often have better in ight than men do. All treating phy ician are bia ed
toward their own treatment, but you want to avoid tho e who are zealot . 9. Acce
to a good ho pital: It i vital to have your pro tate cancer treated in a ho
pital that perform many uch procedure each year.
TREATMENT DECISIONS
105
The e high-volume ho pital have been hown in tudie of radical pro tatectomy
to have fewer urgical death and fewer po toperative complication ; the ame i
pre umably true for radiation treatment a well. The quality of nur ing and ane
the ia hould al o be a matter of eriou intere t. For men undergoing radiatio
n treatment , a k how modern the equipment i and compare with what you are told
at other ho pital ; radiation equipment i con tantly being improved and upgrad
ed. The fty be t ho pital department of urology, a publi hed in 2005 by USNew ,
are li ted in the box nearby. Be aware that the ranking depend heavily on repu
tation, which among medical profe ional re t largely on re earch tatu . Thu
, it i po ible to have a highly rated ho pital that ha excellent re earch but
not nece arily excellent clinical care. Another u eful li t of ho pital i th
at of cancer center de ignated by the National Cancer In titute. The e are ho p
ital where major cancer re earch i taking place; it will u ually, but not alwa
y , include pro tate cancer re earch. A of late 2005, there were ixty-two de i
gnated cancer center , including the well-known Dana-Farber Cancer In titute in
Bo ton, Memorial Sloan-Kettering Cancer Center in New York, M. D. Ander on Cance

r Center in Hou ton, and Fred Hutchin on Cancer Re earch Center in Seattle. A li
t of the e center by tate can be acce ed on the Internet by going to the hom
e page of the National Cancer In titute (cancer.gov) and clicking on Treatment, then
on Treatment Facilitie . Still another li t of cancer treatment facilitie i the N
ational Comprehen ive Cancer Network, nineteen af liated facilitie that are de cr
ibed on the web ite of the coordinating organization (nccn.org). All of the e l
i t , however, uffer from the ame limitation: exemplary cancer re earch i not
nece arily accompanied by exemplary clinical care. 10. Convenience and co t: T
he convenience and co t of pro tate cancer treatment option are practical but c
rucial con ideration . Compared to radiation, urgery entail a more prolonged r
ecovery, during which time work and regular activitie mu t be curtailed. The ac
ce ibility of the treatment unit hould be weighed; having to tay in a hotel i
n a di tant city for everal week to have beam therapy will be
106
TREATMENT DECISIONS
The Fifty Be t Ho pital for Urology in 2005 Thi ranking i ba ed on a variety
of factor , including reputation among medical profe ional , ratio of nur e to
patient , mortality ratio, and equipment. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12
. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. John Hopkin Ho pital
, Baltimore Cleveland Clinic Mayo Clinic, Roche ter, Minn. UCLA Medical Center,
Lo Angele New YorkPre byterian Univ. Ho p. of Columbia and Cornell Barne Jewi h
Ho pital/Wa hington Univer ity, St. Loui Ma achu ett General Ho pital, Bo ton
Memorial Sloan-Kettering Cancer Center, New York Duke Univer ity Medical Center
, Durham, N.C. Stanford Ho pital and Clinic , Stanford, Calif. Univer ity of Tex
a , M. D. Ander on Cancer Center, Hou ton Univer ity of California, San Franci c
o Medical Center Methodi t Ho pital, Hou ton Univer ity of Michigan Medical Cent
er, Ann Arbor Northwe tern Memorial Ho pital, Chicago Clarian Health Partner (I
U and Methodi t Ho pital ), Indianapoli Vanderbilt Univer ity Medical Center, N
a hville Ho pital of the Univer ity of Penn ylvania, Philadelphia Lahey Clinic,
Burlington, Ma . Univer ity of Iowa Ho pital and Clinic , Iowa City NYU Medica
l Center, New York Univer ity of Virginia Medical Center, Charlotte ville Willia
m Beaumont Ho pital, Royal Oak, Mich. Parkland Memorial Ho pital, Dalla YaleNew
Haven Ho pital, New Haven, Conn. Shand at the Univer ity of Florida, Gaine vill
e
TREATMENT DECISIONS
107
27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46.
47. 48. 49. 50.
Univer ity of Miami, Jack on Memorial Ho pital Chri t Ho pital and Medical Cente
r, Oak Lawn, Ill. St. Luke Medical Center, Milwaukee Univer ity of Wi con in Ho
pital and Clinic , Madi on Univer ity of Pitt burgh Medical Center Ohio State Un
iver ity Ho pital, Columbu Sentara Norfolk General Ho pital, Norfolk, Va. Abbot
t Northwe tern Ho pital, Minneapoli Univer ity of North Carolina Ho pital , Cha
pel Hill Ru h Univer ity Medical Center, Chicago Henry Ford Ho pital, Detroit Sa
ra ota Memorial Ho pital, Fla. Univer ity Ho pital of Cleveland Texa Heart In
titute at St. Luke Epi copal Ho pital, Hou ton Univer ity of Colorado Ho pital,
Denver Brigham and Women Ho pital, Bo ton Univer ity Ho pital, Cincinnati Advoca
te Lutheran General Ho pital, Park Ridge, Ill. Lanca ter General Ho pital, Pa. U
niver ity Medical Center, Tuc on, Ariz. F. G. McGaw Ho pital at Loyola Univer it
y, Maywood, Ill. Univer ity of Minne ota Medical Center, Minneapoli Memorial He
rmann Ho pital, Hou ton St. Elizabeth Ho pital Medical Center, Young town, Ohio
SOURCE: Be t Ho pital 2005, Urology, USNew , u new (click on Ranking and Guide

, then on Be t Ho pital , then on Urology).

108
TREATMENT DECISIONS
le plea ant and more expen ive than having eed therapy or urgery at a local
ho pital. Except for watchful waiting, all treatment for pro tate cancer are ex
pen ive. An analy i of total co t for the initial workup, treatment, and ix m
onth of follow-up in the mid-1990 reported them to be a follow :29 Treatment
Seed radiation Beam radiation Surgery Seed plu beam radiation Surgery plu beam
radiation Total co t $15,301 $15,937 $19,019 $24,407 $31,329
Since the e co t were ba ed on data from 1993 to 1996, the co t today would be
till higher. A certaining who will pay the co t of treating your pro tate can
cer varie from complex to Byzantine. If you are 65 or older, you are covered by
Medicare, part A, for the co t of ho pitalization; you may al o be covered by
Medicare, part B, for the co t of the treating phy ician . However, you have to
pay a deductible and approximately 20 percent of the co t (the copayment), and
you have to u e phy ician who accept Medicare a ignment (mo t do). If you are
an armed force veteran, you may be eligible to u e program at military or Vete
ran Admini tration ho pital (the treatment program at Walter Reed Army Ho pita
l in Wa hington, D.C., for example, i excellent). Or you may be able to u e Tri
care coverage for mo t treatment co t by other provider who participate in the
Tricare program. If you have private in urance, uch a Blue Cro /Blue Shield
or Aetna, coverage for co t varie widely by your peci c plan and your location.
The in urance plan of large companie u ually offer more generou coverage tha
n the plan of mall companie . In ome plan , you are re tricted to u ing a el
ect li t of ho pital and phy ician , wherea other plan allow you to choo e yo
ur own. Virtually all plan pay only a rea onable and cu tomary fee, which i determ
ined by zip code and varie by region. You will almo t certainly
TREATMENT DECISIONS
109
pay a portion of the bill, and the more expen ive the phy ician you elect, the
higher your portion of the bill i likely to be. Managed-care companie al o re
trict your option . One tudy found that Health Maintenance Organization (HMO )
were more likely to have men treated by radiation than by urgery, ince radiat
ion entail lower ho pitalization co t .30 For men under the age of 65 who do no
t have health in urance, Medicaid i the be t mean of coverage. Quali cation dif
fer widely by tate. Medicaid coverage i roughly imilar to that of Medicare, a
nd you mu t u e phy ician who accept Medicaid, which many do not. A u eful anal
y i of payment y tem for covering pro tate cancer treatment can be found in P
ro tate Cancer: A Survivor Guide, by Don Kaltenbach and Tim Richard (Seneca Hou
e Pre , 2003). Once the e ten factor have been con idered, many men look for
a de nitive treatment trial to tell them what to do. Unfortunately, the de nitive tr
eatment trial i a myth. Becau e the treatment of pro tate cancer i con tantly
changing and becau e pro tate cancer progre e o lowly, the information provi
ded by mo t treatment trial i out of date by the time it become available. In
my ca e, I wi hed that twenty year ago re earcher had tarted a compari on of
urgical ver u beam radiation treatment o that I would know the long-term out
come regarding death rate, ide effect , and the like. Of cour e, uch a compar
i on i never going to happen, becau e the
110
TREATMENT DECISIONS

treatment technology for pro tate cancer i a moving target. Many comparative tr
eatment trial have been tarted in recent year , but the re ult will not be kn
own for fteen or twenty year . By that time urgical and radiological technique
will have evolved, combination of treatment will be routinely u ed, and additio
nal treatment option will be available. And at that time, we will wi h that com
parative treatment trial had been tarted now, u ing the technology of the futu
re.
ADVANTAGES AND DISADVANTAGES OF TREATMENT OPTIONS In Seed of Hope, Michael Dor
o decried the contradictory and confu ing advice he found in the medical literat
ure on pro tate cancer treatment . Urologi t advocated urgery, radiologi t ad
vocated radiation treatment, and cancer peciali t who were neither urgeon nor
radiation therapi t were plit between the two treatment modalitie . Moreover, a
panel of expert in the American Urological A ociation tried to objectively et
tle the treatment i ue but found the data inadequate for valid compari on of tr
eatment. . . . Ba ically they gave up! Dor o, him elf a phy ician with pro tate ca
ncer, concluded in a note of exa peration: What a mother to do?31 That i a very rea
onable que tion. If a phy ician with pro tate cancer ha dif culty orting out th
e treatment option , how can a layman be expected to do o? And yet that i the
me age given by mo t pro tate cancer peciali t : In the nal analy i , Mr. Smith,
the deci ion i up to you. In an effort to help men a e the option , the follow
ing ection ummarize ome advantage and di advantage of each of the major tr
eatment option . The e ob ervation hould be regarded a general con ideration
to which there are alway exception .
Watchful Waiting Candidate : Men with early- tage cancer (nonpalpable; PSA and G
lea on core 5 or le ) who are over age 70 or who expect to live le than ten
more year . Some men choo e thi option if hortterm pre ervation of exual func
tion i paramount.
TREATMENT DECISIONS
111
Chance of cure: Remote. Convenience: Excellent; a rectal exam, a PSA te t, and p
o ibly a biop y every few month are u ually all that i nece ary. Sexual ide
effect : None unle the cancer progre e . Urinary ide effect : None unle t
he cancer progre e . Bowel ide effect : None unle the cancer progre e . Oth
er ide effect : None. Follow-up treatment if needed: PSA i only moderately u e
ful a a marker, ince the cancer can pread even with a low PSA. The PSA veloci
ty may be more u eful. All treatment option remain open unle the cancer prea
d out ide the pro tate. Uncertaintie : Many; you know you have an untreated can
cer growing in ide you. Unknown : Rate of growth; whether the cancer ha pread;
preci e Glea on core.
Surgery Candidate : Any man up to approximately age 75 if he i in good health,
ha a life expectancy of at lea t ten more year , and there i no evidence of ca
ncer out ide the pro tate. Many cardiac and other eriou medical condition mak
e men ineligible for urgical treatment. Chance of cure: Good, if the cancer ha
not pread beyond the pro tate. Convenience: Highly inconvenient in that it inv
olve major urgery with ane the ia and everal week of recuperation. Sexual i
de effect : Rate of impotence 40 to 90 percent depending on the man age and whet
her nerve paring i po ible. Maximum impotence immediately after urgery, then
may lowly improve for two year or longer. Urinary ide effect : High rate of
incontinence immediately after urgery, then u ually improve . U e of pad after
two year i 5 to 10 percent. Occa ional urinary tricture. Bowel ide effect :
Minimal. Other ide effect : Weakne and ri k of blood clotting (thrombo i ) f
ollowing urgery.
112

TREATMENT DECISIONS
Follow-up treatment if needed: Beam radiation and hormone therapy commonly u ed.
Seed radiation not po ible. Uncertaintie : Le worry than any other treatment
. Surgery provide accurate information on the cell type, ize, and margin of t
he tumor and whether the cancer ha pread to the lymph node , eminal ve icle ,
and other organ . Po t-op PSA i an accurate indicator of recurrence. Unknown :
The po ibility that the cancer ha pread beyond the pro tate, not detected at
urgery.
Beam Radiation Therapy Candidate : Any man of any age, even if he i not in good
health, ha a life expectancy of le than ten year , or ha cancer that ha p
read beyond the pro tate. Not appropriate for very large pro tate unle initia
lly hrunk by hormone therapy, nor for men with chronic bowel di ea e. Chance of
cure: Good, if cancer ha not pread beyond the pro tate. Convenience: Depend
on the proximity of the ho pital or treatment unit, ince it require daily outp
atient treatment ve day a week for ve to nine week . No ane the ia, no urgery, n
o ho pitalization, and no pain. No re triction on activitie . Sexual ide effect
: Initially minimal, but impotence lowly increa e over everal year to 40 to
60 percent. Rate higher if combined with eed or hormone therapy. Urinary ide
effect : Incontinence rate lower than for urgery but till a mall ri k. Irrita
tive urinary ymptom and bleeding may be evere and per i tent. Bowel ide effe
ct : Frequency, urgency, cramping, and bleeding occur in 10 to 20 percent of ca
e and may be per i tent. Other ide effect : Occa ional lo of pubic hair. Fat
igue during treatment common. Rectal tula now rare. Follow-up treatment if need
ed: Surgical treatment dif cult becau e radiation di tort anatomical landmark an
d cau e carring of ti ue ; other option open. Uncertaintie : Provide little
information on extent of cancer and po ible pread. Po ttreatment PSA ambiguou
a a predictor of recurrence, e pecially with PSA bounce.
TREATMENT DECISIONS
113
Unknown: Chance of re idual pro tate cancer in remaining pro tate cell ; likeli
hood of another form of cancer econdary to radiation effect ; chance that the
cancer ha already pread beyond the pro tate.
Seed Radiation Candidate : Men of any age but with a Glea on core 6 or le and
a PSA le than 10. Minor urgery required, o may not be appropriate for ome
men with evere medical condition . Not ugge ted for very large pro tate unle
initially hrunk by hormone therapy, nor for men with chronic bowel di ea e. N
ot appropriate for men who have had urgery for BPH. Chance of cure: Appear fav
orable o far, but long-term data are not yet available. Convenience: Very conve
nient, u ually requiring only outpatient urgery with rapid recovery. No re tric
tion of activitie except for the r t few day . Sexual ide effect : Initially mi
nimal, but impotence lowly increa e over everal year to 40 to 60 percent. Ha
the un upported reputation of cau ing le exual dy function than beam therap
y. Painful orga m and blood in ejaculate have been reported. Rate of impotence m
uch higher if beam therapy or hormone therapy i u ed with eed therapy. Urinary
ide effect : Incontinence rate lower than urgery but till 5 to 10 percent. I
rritative urinary ymptom may be evere and per i tent, more o than for beam t
herapy. Bowel ide effect : Frequency, urgency, cramping, and bleeding occur in
10 to 20 percent of patient and may be per i tent. Other ide effect : Men are
mildly radioactive for up to two month . Follow-up treatment if needed: Surgical
treatment dif cult becau e radiation di tort anatomical landmark and cau e ca
rring of ti ue; other option open. Uncertaintie : Provide little information
on extent of cancer and po ible pread. Po ttreatment PSA i unclear a a predi
ctor of recurrence, e pecially with PSA bounce. Unknown : Chance of re idual pr

o tate cancer in remaining pro tate cell ; likelihood of another form of cancer
econdary to radi114
TREATMENT DECISIONS
ation effect ; chance that the cancer ha already pread beyond the pro tate.
Hormone Therapy Candidate : Any man, even tho e not in good health or with a lif
e expectancy of le than ten more year . Can be u ed to hrink large pro tate
prior to radiation treatment; u ed with radiation treatment for men with cancer
likely to pread (Glea on core 8 to 10) or that have pread locally, and for
cancer that have pread di tantly to bone or other organ . Chance of cure: Re
mote; hormone low cancer growth but u ually do not kill all the cancer cell .
Convenience: Antiandrogen are taken daily a pill ; LHRH agoni t are given by
intramu cular injection every one, three, or four month . Sexual ide effect :
Impotence and lo of libido in almo t all ca e if given long term; effect occ
ur more lowly with antiandrogen alone. Urinary ide effect : None. Bowel ide
effect : None. Other ide effect : Hot a he ; brea t tenderne and enlargement;
o teoporo i , with po ible fracture ; fatigue; anemia; weight gain; increa ed c
hole terol; hort-term wor ening of cancer ymptom ( air) when tarting on LHRH a
goni t . Follow-up treatment if needed: All option open. Uncertaintie : Hormone
interfere with PSA level , o not u eful a a marker for cancer progre ion. U
nknown : Rate of growth; whether the cancer i continuing to pread.
Cryotherapy Candidate : U ed mainly a a econdary treatment for men who have ha
d radiation treatment, who e cancer ha recurred but not pread beyond the pro t
ate. Not u eful for men with large pro tate .
TREATMENT DECISIONS
115
Chance of cure: In uf cient data. Convenience: Done under ane the ia, u ually wit
h overnight ho pital tay. Man u ually off work for two week . Sexual ide effec
t : Impotence very common, up to 90 percent. Urinary ide effect : Incontinence
approximately 10 percent. Bowel ide effect : Low incidence of injury to rectal
wall, including tula, which can be dif cult to repair. Other ide effect : None. F
ollow-up treatment if needed: Surgical treatment extremely dif cult, other option
open. Cryotherapy can be repeated multiple time . Uncertaintie : Whether all ca
ncer cell have been killed, e pecially tho e clo e to the urethra. Unknown : Lo
ng-term effectivene ; chance that the cancer ha already pread beyond the pro
tate.
HOW I MADE MY DECISIONS My own bigge t problem initially wa fully comprehending
that I had cancer, with it po ible implication for my life. I felt mortal in
a way that I never had before. I noticed it in little thing , like a brief he i
tation over whether I hould renew a medical journal ub cription for one year o
r three year .
116
TREATMENT DECISIONS
By talking to my wife and medical colleague , I developed a plan of attack. I re
ad two of the mo t widely u ed book on pro tate cancer and checked a few web it
e , but wa di appointed with what I found. Much of the information eemed bia e
d toward one treatment or another, and ome of the web ite were openly commerci
al. In di cu ing the cancer with my family and friend , I realized that I had a

 trong upport y tem already in place. I next a e ed the everity of my canc

er. Having a Glea on core of 7 wa not advantageou , but the fact that it wa 3
+4 and not 4+3 wa helpful. Three of my nine biop y core contained cancer cell
, which occupied 20 percent of the core in two ca e and 5 percent in the third.
Glea on grade 4 cell con tituted 40 percent of the cancer cell in one core an
d 20 percent in the econd, and there were no grade 4 cell in the third. The fa
ct that all three po itive core were in the right half of my pro tate eemed to
con rm the impre ion of my urologi t that the cancer wa con ned to the right lobe
but occupied more than half the lobe; thu , the cancer wa probably a tage T2b
. The fact that my PSA wa only 3.3 eemed promi ing. My previou PSA had been 2
.0 but had been mea ured almo t four year previou ly; I wa deeply embarra ed
to realize that I had gone that long without a PSA te t and thu could not a e
my PSA velocity over the pa t year. Overall, my a e ment of the everity of
the cancer produced a mixed outcome: intermediate wa the of cial term, which tran lat
ed into it could be wor e, but it certainly could be better. In a e ing treatment
option , I quickly ruled out watchful waiting and eed radiation therapy a not
appropriate for my Glea on 7 cancer. Hormone therapy by it elf al o eemed inapp
ropriate, ince it would not cure the cancer and brought with it eriou ide ef
fect . That left beam radiation therapy and urgery a the logical option . I ca
lculated my life expectancy to be more than fteen year . A major factor in my ult
imate deci ion wa a di tinct unwillingne to live with uncertainty. Surgery fo
r me offered the advantage of nding out the preci e everity of the cancer and a
l o being able to u e the PSA po toperatively to a certain po ible recurrence.
I thought carefully about the po ible ide effect of urgery. The odd of urin
ary incontinence beyond the r t few week eemed relatively low, and I thought th
at I could live with pad if nece ary.
TREATMENT DECISIONS
117

I did not like the threat of impotence at all, but I had had a long and ati fyi
ng ex life and wa willing to barter it if nece ary for a po ible cancer cure
and additional year of productive life. Given the apparent ize and po ition o
f my cancer, I wa not optimi tic about aving both nerve but wa hopeful that
one could be aved, thereby providing a rea onable chance of continuing to have
a ex life. I di cu ed the po ible outcome exten ively with my wife, who wa e
xtremely upportive and agreed with my deci ion. Having been happily married for
thirty- even year eemed a great advantage in thi ituation. Once I had decid
ed on urgery, the next que tion were who and where. My medical colleague were extr
ly helpful in thi regard, and I quickly a certained that there were at lea t th
ree Wa hington-area urologi t who were highly regarded. One of them, Nichola C
on tantinople, wa the urologi t who had performed my biop y and whom I liked. I
al o explored the po ibility of going to John Hopkin Ho pital in Baltimore,
where I had clo e re earch tie and vi ited regularly. I had a cordial telephone
conver ation with Patrick Wal h, who wa then chief of urology, and inve tigate
d clinical a pect of the ho pital. I al o a certained that my medical in urance
plan would cover the co t of treatment and that I had abundant ick leave avail
able. After weighing all the option , I decided to have my urgery done by Dr. C
on tantinople at Sibley Memorial Ho pital, a highly regarded community ho pital
a mile from my home. A medical colleague on taff there had veri ed it reputation
for excellent ane the iologi t and nur e and a ne radiology department with th
e late t equipment in ca e I needed follow-up beam radiation after urgery. I li
ked the idea that my urologi t would be a i ted in urgery by one of hi boardcerti ed urologi t partner , not by a re ident in training. In my medical training
year , I had performed my r t biop y, appendectomy, and Cae arian ection under
the watchful eye of an attending urgeon and under tood that uch upervi ory
urgery i nece ary. When it came to uturing my evered urethra to my bladder i
n a manner that might determine my lifetime urinary continence, however, I opted
for proven experience. In retro pect, I would make the ame deci ion again, ex

cept that I would probably buy more good red wine to help me with the deci ion m
aking.
C H A P T E R
9
Your Support Sy tem
I
t i aid that being diagno ed with cancer change a per on forever. My interni
t, who i a per onal friend and who di covered my cancer, aid exactly that to m
e. I did not doubt him then, and I certainly do not doubt him now. A cancer diag
no i i one of life de ning moment , a new tint to one gla e that put the worl
d in a different light. Cancer doe nt ju t change the per on, however. It al o ch
ange the per on relation hip with family member , friend , coworker , and prof
e ional colleague . It i not only that you ee the world in a different light
but al o that other ee you in a different light. You are till the per on they
knew before, but you have omething added, the big C, a it i often called. It i
not a tigmatizing a the embroidered A that He ter Prynne had to wear in Nathaniel
Hawthorne tory, a he tood in the public pillory holding her illegitimate ch
ild, but it occa ionally feel that way.
YOURSELF The mo t important part of your upport y tem, by far, i your elf. Th
i i true even if you have been well married for thirty- even year , have a dau
ghter and on-in-law who are both phy ician , and have
YOUR SUPPORT SYSTEM
119
Sometime I have a feeling people know we have pro tate cancer.

clo e friend , a I wa fortunate to have at the time of my diagno i . It i equ

ally true if you are living alone, e tranged from your family, and have no clo e
friend , a i too often the ca e. Regardle of your relation hip with other
, cancer affect your inner elf in a way that other cannot ee. Every cancer c
an be aid to pread to your oul, even if it never pread to any other organ.
To utilize your own inner trength in dealing with pro tate cancer, men mu t wre
tle with four i : the myth of immortality, accepting immode ty, the fear of incontin
ence, and the fear of impotence. Tho e who ucce fully confront the e demon wi
ll nd the going much ea ier. Tho e who fail to do o will be plagued by a fth i indeci
ion. They will al o be prone to denial and depre ion. The latter can become a m
ajor problem; a tudy in Florida reported that uicide among men with pro tate c
ancer wa four time more common than among all men in their ame age group.1 Th
e myth of one own immortality confront everyone with cancer, not ju t men with
pro tate cancer. It i the realization, u ually for the r t time, that mortal ha two
meaning : a human being, and one who i ubject to death. Before being diagno e
d with cancer or another potentially fatal di ea e, we ecretly believe that we
are amaranth ; the diagno i of cancer rapidly wilt our petal , and death becom
e real. A Richard Handy noted after being diagno ed
120
YOUR SUPPORT SYSTEM
My Clo e t Po e ion The diagno i change everything. . . . Now cancer will be
my clo e t po e ion, going with me from office to hou e, to conference and d
inner partie , a I go my elf. I have got to get u ed to having it alway here.
I have got to think about what influence it may a ume in time, not only over me

but on my family, friend , and work. Corneliu Ryan, A Private Battle

with pro tate cancer, Cancer permanently evered me from the long youthful tie of
emotionally believing my elf to be immortal de pite all of aging warning that I
wa nt.2 I wa urpri ed by ome of my own thought in reaction to the reality of dy
ing. Under tandably, I wondered whether I would ee my two grandchildren graduat
e from college, and whether my profe ional re earch would come to full fruition
. But I al o wondered about more mundane thing , like whether I would live to e
e the Red Sox beat the Yankee and win a World Serie . Miraculou ly, ve month la
ter it happened! Then I aid to my elf, but what about the Chicago Cub ? Confron
ted with the reality of death, many men react initially with anger and elf-pity
. Corneliu Ryan, a ucce ful writer, wa diagno ed with advanced pro tate canc
er at age 50 and re ected uch thought : I feel uch a terrible en e of inju tice
. What did I do to de erve thi ? Yet, that ju t the kind of que tion Ive got to e
liminate from my mind. I do not exactly tru t my ability to maintain objectivity
publicly unle I can relea e the body quake and hock in private. And how ca
n you make people who havent got cancer under tand what happened to you without h
aving to endure their pity a well? Im damned if I want pity. Self-pity i bad en
ough. Anger i e pecially prominent in individual who feel unful lled profe iona
lly and tho e who have deferred activitie they enjoy until
YOUR SUPPORT SYSTEM
121
Irony Now at la t I under tand the conditional nature of the human condition. Ye
t, unlike Kierkegaard and Sartre, Im not intere ted in the irony of my po ition.
Cancer cure you of irony. Perhap my irony wa all in my pro tate. Anatole Broya
rd, Intoxicated by My Illne
their hoped-for golden year of retirement. Although anger i a natural reaction
to being diagno ed with cancer, it erve no u eful purpo e. A one man noted,
uch anger i like a catheter in erted in your oul, draining your pirit.3 The myth
of one immortality can be re olved in a variety of way . The mo t common i thr
ough religiou belief, a conviction that your cancer i part of a divine plan an
d that dying i merely a way tation to a life hereafter. Thi approach i well
illu trated by Chuck and Martha Wheeler in their book Af rming the Darkne . Chuck
wa diagno ed with advanced pro tate cancer at age 65. He lived eight painful,
complication- lled year but ma tered hi fear of death through hi religiou beli
ef. Another approach i through intellectual acceptance. In the word of Franci
Bacon, It i a natural to die a to be born. In thi regard, it i helpful to have
had an acquaintance with death. For individual who e ole experience with deat
h ha been that of a beloved pet or an elderly grandparent, the initial confront
ation with their own potential demi e i likely to be deeply troubling. Some of
u , by contra t, have met death early and often. I lo t my father when I wa a c
hild, my own child and two very clo e friend in my early twentie , and a few ye
ar later my mother and a i ter to cancer. A a young phy ician, I cared for pe
ople who were dying, pronounced people dead, and had the ta k of informing their
relative . Death never become one friend, but experience can at lea t make it
an acquaintance. A uch, it eem le demonic. The econd challenge in trengt
hening the elf i to accept immode ty a a reality. Mo t men are reluctant even
to talk about their
122
YOUR SUPPORT SYSTEM
genital and their ex life, to ay nothing of di playing their genital to the
world. Having pro tate cancer, however, make thi nece ary. Phy ician probe y
our rectum with their nger . The nger are replaced by in trument and what ound

like a taple gun during your biop y. And then, during treatment, you get to h
ow your genital to a variety of technician , nur e , and phy ician . A Richard
Handy de cribed it: My peni and anu , the part of my body I had mo t carefully
clo eted, were now open to in pection by anyone dre ed in white who wanted to l
ook at them. They were no longer protected by mode ty or guarded by will.4 For ind
ividual who have been taught that their genital are dirty and di gu ting, thi
openne can be a problem. Corneliu Ryan experienced uch feeling : I wa emba
rra ed by what wa happening in a way that difficult to de cribe. Perhap being
fa tidiou about that part of the body, one cant help having a feeling that what
wa happening wa repul ive. I felt that I wa dirty. I didnt put my underwear i
n with the hou e laundry and I couldnt let Katie wa h it. It wa omething I had
to do my elf. I didnt want anyone, not even Katie, to witne the evidence of my
di gu t and humiliation.5 The reality, of cour e, i that medical per onnel who
are dealing with urological patient ee genital all day, every day. You may be
lieve that your are pecial becau e they belong to you, but other will probabl
y not give your genital more than a pa ing glance. If you can accept immode ty
a your new way of life, the diagno i and treatment of your pro tate cancer wi
ll go in nitely more moothly. Per onally, I found it comforting to imagine my elf
on a nude beach in California where, after the r t few minute , nobody paid much
attention to anybody el e. The third and fourth i ue that mu t be confronted
to trengthen one elf in dealing with pro tate cancer are the two principal comp
lication of treatment: urinary incontinence and impotence. Both may vary from b
eing minor annoyance to being major problem , depending on one age, choice of t
reatment, and luck of the draw. And for both, treatment are available, a will
be di cu ed in Chapter 10. Urinary incontinence i e entially a plumbing probl
em and hould be regarded a uch, imilar to having a chronically leaky
YOUR SUPPORT SYSTEM
123
bathroom faucet. Urine i , in mo t individual , imilar to terile water. It i
not harmful in any way, and in di a ter , uch a being tranded on a mountain l
edge or an i olated i land, many have aved their live by drinking their own ur
ine. Although men may be deeply embarra ed to leak urine, tho e clo e t to them
may be le concerned. De iree Howe, in writing about her hu band po t-pro tate
ctomy incontinence, put the problem in proper per pective: Continence wa way do
wn the li t of characteri tic I ought in a man. Integrity, compa ion, intelli
gence, humor, courage, etc., were Dick qualitie , all of which were high on my c
heck-li t. It didnt hurt that he wa very good looking, too. Solution for incont
inence were available, but cure for poor character are more difficult, if not i
mpo ible, to find.6 Singer-actor Robert Goulet al o di played a remarkable atti
tude toward hi urinary incontinence. Three week after having urgery for pro t
ate cancer, he rejoined the ca t of Camelot, wearing a diaper under hi pant . H
e later recounted on national televi ion what happened on tage during the r t h
ow: Well, right in the middle of the how, there a cene where Im all alone on ce
nter tage, tanding on top of a mall hill, potlit, with my leg pread, ingi
ng my heart out. I hit one particular high note, and, ure enough, my bladder gu
hed away. I could feel my eye widening. I didnt think the audience could actual
ly tell what had happened, but after the how, I a ked Patricia Keye , my co tar
, if he noticed anything, well, trange, during that ong. And he aid, There wa
nothing obviou , Robert, but when I aw that twinkle in your eye, I knew it me
ant a tinkle down your thigh.7 Compared to incontinence, the problem of impotence
i both more common and more dif cult to deal with. Erection de ne being a man from
adole cence on, and for many men the inability to have erection ugge t that
they are no longer men. The problem i e pecially trouble ome for younger men, f
or men who believe they have not had their hare of exual adventure and plan to
do o in the future, and for older men who have ju t married a 30 omething wife
.

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YOUR SUPPORT SYSTEM
Talking About It There wa al o another fear: that omehow my incontinence would
be obviou to people. Peculiar to fear thi , Margaret aid, con idering my tend
ency to talk too much about it, to bring people attention to it when there wa n
o need to. It i an occupational hazard of cancer, whatever form it take , thi
need to talk about it. Michael Korda, Man to Man
It i factor uch a the e that incline ome men toward watchful waiting, defer
ring de nitive treatment of their pro tate cancer while they continue to have an a
ctive ex life. Again t thi mu t be weighed the ri k of having the cancer prea
d. Rudy Giuliani, then mayor of New York City, ummarized thi dilemma in a nati
onal televi ion interview: The r t thing youve got to come to term with i if you d
ont live, you cant have exual function.8 Similarly, Leon Prochnik titled hi book a
bout pro tate cancer You Cant Make Love if Youre Dead. Per onally, I regarded the
po ibility of impotence following pro tate cancer treatment a appalling. Howev
er, I regarded the po ibility of death from pro tate cancer it elf a more appa
lling. I al o took great ati faction in knowing that I had had a full and ati
fying ex life and realized that I had tored up uf cient memorie to la t for th
e re t of my life. If nece ary, I could relive and replay them endle ly, lowl
y turning them over in my mind, like Marcel Prou t petit madeleine in hi Reme
mbrance of Thing Pa t.
FAMILY AND FRIENDS For a man who i married or living with a partner in a long-t
erm relation hip, the other per on will be affected by the pro tate cancer almo
t a much a he will. A one wife phra ed it: He got the diagno i , but we both ha
ve pro tate cancer.9 It i therefore imperative
YOUR SUPPORT SYSTEM
125
to include the other per on from the beginning in di cu ion of your cancer; me
n who initially keep the new from their partner invariably nd they have made a m
i take. Wive and partner can be extremely upportive and helpful in uch itua
tion . They can collect and organize information on treatment option , e peciall
y given the plethora of book and web ite available. Many men have found it u e
ful to have their wive accompany them to meeting with their urologi t , ince
it i dif cult to remember everything they are being told. Women ometime a k que
tion men are reluctant to rai e becau e of embarra ment or timidity. Another
ta k partner can help with i organizing and paying the medical bill . The admi
ni trative complexity of the American medical care y tem i beyond belief. The
New York Time noted that when you become a patient in the United State , you ente
r a world of paperwork o urreal that it belong in one of Kafka tale of the t
riumph of facele bureaucracy.10 Even though I am a phy ician and hould theoreti
cally under tand the medical payment y tem, nobody really can do o. I wanted t
o focu on my treatment and recovery, o my wife took full re pon ibility for o
rting out what had been, or needed to be, paid by Medicare, Tricare, Blue Cro ,
or u . De pite the fact that he i an economi t, the ta k proved to be extreme
ly complex and confu ing; the bill took more than a year to ort out. I conclud
ed that having the urgery wa , in fact, ea ier than orting out the payment . T
he ultimate mea ure of wive value in helping hu band wa demon trated by a 1996
tudy howing that married men with pro tate cancer, on average, live 40 percen
t longer than ingle men; the tati tic on divorced men fell between the two.11
The importance of a wife a a upport y tem i al o illu trated by per onal ac
count , uch a Chuck and Martha Wheeler Af rming the Darkne and Michael Korda M
an to Man ( ee Appendix B). In a table marriage, confronting pro tate cancer to
gether can trengthen the hu band-wife bond. My own wife wa , and continue to b

e, a wonderful help in getting through my pro tate cancer experience. In thirty even year of marriage we had hared many challenge , including rai ing childre
n and living for a year in a remote Arctic village. Prior to marrying, we had ea
ch worked for two year in
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YOUR SUPPORT SYSTEM
A Wife Anger I wa angry that Charle had not gone in for treatment earlier; ang
ry becau e he refu ed to get a econd opinion, and angry that through all of hi
uffering, he didnt eem eriou enough about getting to the root of hi problem
. I wa angry that the fir t phy ician didnt give him a more thorough examination
, and that he didnt demand more te t after Charle didnt improve from the medicat
ion he pre cribed. Valerie William in Charle William and Vernon William , That
Black Men Might Live
the Peace Corp in Africa. We viewed my pro tate cancer a an unwelcome but nove
l challenge, one that we would confront and manage together. If I had had to do
o, I could have managed it a a ingle man, but having a upportive partner mad
e the experience in nitely ea ier. Of cour e, not all marriage are table, and in
uch ituation pro tate cancer, like any other eriou illne , can exacerbate
marital problem . The wife may blame her hu band for not having gotten a regula
r medical checkup or PSA te t. She may re ent the lo of income made nece ary
by her hu band illne , or having to take care of him. She may feel cheated out
of her quiet retirement year ; a one wife put it, Both of u are retired and we h
ad hope . . . of doing all the thing we planned. Even in olid marriage pro tat
e cancer tend to divide couple . A another wife put it: The doctor nding had alr
eady begun to make a difference. Cancer had eparated u . In pite of phy ical c
lo ene a barrier, invi ible but everla ting, had come between u .12 Impotence in
evitably affect wive , which i another rea on for involving them in treatment
deci ion . The a umption i generally made that a wife view her hu band impote
nce a a great lo . U ually thi i true, e pecially for younger wive , but in
ce pro tate cancer occur mo t commonly after the age of 60, ome wive in that
age
YOUR SUPPORT SYSTEM
127
group have a dimini hed intere t in ex and therefore may not view their hu band
impotence a a major lo . In a tudy comparing hu band and wive on their deg
ree of concern about impotence and urinary incontinence following pro tate urge
ry, wive were ub tantially le concerned than hu band .13 All of the foregoin
g, and mo t book on pro tate cancer, a ume that the man partner i a woman. Ga
y men, of cour e, have men a partner , who are equally a affected by the pro t
ate cancer a wive or girlfriend are. In one of the few piece written about p
ro tate cancer in gay men, Thoma Blank at the Univer ity of Connecticut di cu
ed difference in the two type of ituation. For example, po ible rectal ide
effect may be more eriou than urinary ide effect for ome gay men, inclinin
g them away from radiation a a treatment.14 A Gay Man Guide to Pro tate Cancer,
edited by Gerald Perlman and Jack Dre cher, include much u eful information. O
ther family member and friend are al o integral part of a man upport network
. I wa fortunate in having friend who are medical profe ional , everal of wh
om were helpful in obtaining information about urologi t , ane the iologi t , th
e advantage and di advantage of variou ho pital , and other factor that went
into my deci ion making. Their per onal upport wa extremely important and ta
nd out in my mind a one of the high point of the period preceding and followi
ng my urgery. In deciding how to handle the new of pro tate cancer with other
family member and clo e friend , be aware that their reaction i likely to depe

nd on your de nition of the ituation. If you eem to have the problem under contr
ol and can di cu it in a calm, factual way, they will u ually accept it. If, o
n the other hand, you are unable to di cu it at all, or break down each time y
ou do, their reaction may not be helpful. One man wrote that what a man with pro
tate cancer want mo t from friend i not merely love but al o an appreciative g
ra p of hi ituation, what i known now in the literature of illne a empathet
ic witne ing. 15 Of cour e, ome family member and friend have multiple problem
of their own and may not be able to react appropriately. Corneliu Ryan de crib
ed uch a reaction when he told a couple, previou ly clo e friend , that he had
pro tate cancer:
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YOUR SUPPORT SYSTEM
I know I didnt imagine the udden change in atmo phere. I en ed that Katie notic
ed it a well. It wa a though I had, in an in tant, become a totally different
per on in our friend view. I felt a though I had committed ome unpardonable g
affe, ome eriou breach of ocial etiquette. They averted their eye . There wa
ilence and then they embarra edly expre ed regret . Michael Korda al o reco
rded the reaction of friend to hi new . The more people were ob e ed with thei
r own health and piritual well-being, I di covered, the more likely they were t
o be unable to deal with the ubject of cancer. Richard Handy older brother never ca
lled or even ent a card, and a colleague with whom he had worked for eighteen yea
r never called or wrote and to thi day ha never acknowledged my illne . And Vict
or Newton ob erved that a few long-time friend topped calling. . . . Some, I thi
nk, were imply afraid of cancer.16 After I had been diagno ed with pro tate cance
r, I looked over my holiday card regi ter and my Rolodex and made li t . Family
member and everal clo e friend I called per onally. Other I informed by a co
llective email, providing information about the biop y re ult and my tentative
plan for treatment. Mo t of the re pon e were enormou ly warm and upportive b
ut, like other , I experienced a few people who appeared unable to deal with the
ubject in any form. You try to not take uch reaction per onally by rea oning
that it i their problem.
COWORKERS AND BUSINESS COLLEAGUES For many people, e pecially tho e living alone
, their primary upport network may be coworker with whom they pend ve day a w
eek. They and bu ine colleague mu t be informed of your pro tate cancer, and
they can be very helpful. In your earch for an appropriate doctor or ho pital,
for example, a coworker or bu ine colleague may have a i ter-in-law who work
in a ho pital or an uncle who had pro tate cancer. Such individual can markedl
y enlarge your information-gathering capacity. But they cant help with your cance
r if they dont know you have it. Handling new of your pro tate cancer in the wor
kplace i omeYOUR SUPPORT SYSTEM
129
what different from handling it among your friend . If you are a upervi or, the
reaction of ubordinate may vary from feeling threatened by the lo of their
protector to being ecretly plea ed that they will be rid of you, at lea t for a
while. A an employee, your medical leave i likely to put more work and re pon
ibility onto other . For a man till in the prime of hi career, a diagno i of
pro tate cancer may make him a poor ri k for further advancement. In general, t
hi i not a major problem, for mo t people are aware that pro tate cancer grow
very lowly. One tudy howed that one year following treatment for pro tate ca
ncer, the employment rate for affected men did not differ from tho e not affecte
d.17 The American with Di abilitie Act, pa ed in 1990, prohibit di criminati
on again t employee for medical rea on , but the act i dif cult to enforce. Afte

r I had decided on a cour e of treatment and had a date for urgery, I per onall
y told tho e with whom I wa working mo t clo ely and then ent an email to the
re t. It read a follow : To: All Concerned Thi i to let you know that I will
be on medical leave for three week or o tarting June 1. I will be having urg
ery for cancer of the pro tate, which i not my fir t choice of how to pend tim
e but better than other po ible problem that come with age, uch a Alzheimer .
It appear to be in a relatively early tage, o the long-term outlook i good.
I concluded with ome humor, aying that I expected to receive getwell card fr
om two organization that everyone wa aware were the la t organization that wo
uld ever end card to me. A general announcement of thi ort eemed an effecti
ve way to avoid the whi pering and rumor that routinely circulate in an of ce whe
n ome people know and other do not. It put everything on the table at once, an
d I found that within two or three day my working relation hip with my coworker
had returned to normal. Some coworker and profe ional colleague , of cour e,
will be uncomfortable with any di cu ion of cancer. The difference may be cul
tural, ince in ome culture and ubculture talking about cancer i till not
polite. I di covered thi my elf when, three month after having urgery, I wa
at a profe ional meeting in Europe. Two of my European colleague , whom I have
known for many year , ca ually
130
YOUR SUPPORT SYSTEM
a ked how I wa , and I told them that I had recently been treated for pro tate c
ancer. Their expre ion and embarra ed reaction were imilar to what I might
have expected if Id aid Id recently turned into a werewolf. When confronted with
profe ional colleague and ca ual acquaintance I had not een for a while, the
i ue of di clo ure continued to be a dilemma. In re pon e to the que tion How ar
e you doing? I had to calculate how well I knew the per on, whether they were like
ly to have heard about my cancer from other , and whether they even wanted to kn
ow. I found that there wa no ea y olution but that it became le of a problem
over time.
SUPPORT GROUPS For many men, pro tate cancer upport group become their main u
pport y tem. Properly run, uch group can be extremely helpful in providing in
formation, friend hip, and a place to di cu problem and ide effect of treat
ment with other who have been there. Many men with pro tate cancer derive ati
faction from helping other who are o affected. The e group meet monthly or mo
re often in a meeting room made available to them by a local ho pital, community
center, church, or ynagogue. Some are led by profe ional , u ually a nur e or
ocial worker, while other are led by the member them elve . Some group are
re tricted to men only, wherea other include wive and partner . Group may p
ecialize in one area, uch a men who have been recently diagno ed, men with rec
urrent cancer, or men with problem of incontinence or impotence. There are two
principal network of pro tate cancer upport group in the United State . U To
o, a network of over three hundred group , i coordinated by U Too Internationa
l in Downer Grove, Illinoi . Local upport group can be identi ed on the U Too w
eb ite, u too.org, or by calling the Illinoi of ce at 1-800-8087866. The other ne
twork of upport group i Man to Man, coordinated by the American Cancer Societ
y. It wa tarted in 1989 by a man who had grown tired of going to general cance
r upport group in which mo t of the member were women. I didnt want to hear
YOUR SUPPORT SYSTEM
131
Support Group I think it very valuable to belong to a upport group. The more y
ou talk about a problem you have, the ea ier it i to live with it. And you lear
n from other people. And if you talk about a ubject, youre going to be more at e

a e, youre going to find out what be t to do and what not. Charle Neider, Adam Bu
rden
about their cervix and they didnt want to hear about my pro tate wa hi impetu to
begin the all-male group . Local af liate can be identi ed on the American Cancer
Society web ite, cancer.org, or by calling the ociety chapter in your area. Man
y communitie offer the two upport group , and men eeking help may wi h to try
both to ee which better meet their need . Support group in Canada can be ide
nti ed on the web ite of the Canadian Pro tate Cancer Network, cpcn.org. In ummar
y, a upport y tem i e ential to a i t you through pro tate cancer. However,
you are the key to developing that upport, and it i primarily your re pon ibi
lity to put it together. Family, friend , coworker , and upport group member c
an all be extremely helpful if you let them. The variou member of your upport
y tem, in fact, are the initial element in your healing proce . Richard Hand
y de cribed it nicely when he likened healing to the weaving of a tape try: When
a weaver fir t thread hi loom, tho e individual bit of color dotted at diff
erent location eem to make no en e. But in time, with the addition of more t
rand and different loom etting , inchoate figure begin to emerge and grow int
o definite hape . Only when the tape try of my healing had been completed and I
had tepped back everal year later to ee it completely, did the intricate ar
chitecture of it pattern , their interwoven theme and feeling , become clearer
and better under tood.18
C H A P T E R
10
Major Complication and Their Treatment
I
ncontinence and impotence are the two mo t-feared complication of pro tate canc
er treatment. They are the Scylla and Charybdi of pro tate voyager , and rare i
the man who ucce fully ail by both without being affected by one or the ot
her. Even men who elect watchful waiting a their option may experience them a
their cancer increa e in ize. Although incontinence and impotence have been br
ie y di cu ed in preceding chapter , their importance for men with pro tate cance
r merit a chapter of their own. It i vital, however, to place incontinence and
impotence in proper per pective. For men who e cancer ha grown beyond the pro
tate or pread to other organ , incontinence and impotence do not loom o large.
A Anatole Broyard noted: In my own ca e, after a bru h with death, I feel that j
u t to be alive i a permanent orga m.1 Argument abound about which i wor e, inc
ontinence or impotence. Wal h and Worthington, in their book Dr. Patrick Wal h G
uide to Surviving Pro tate Cancer, argue that recovery of urine control i far mor
e important and . . . ca t a far greater hadow on your life. If omething wron
g with your ability to urinate, youll be reminded of it everal time a dayor wor
e, everal time an hour not ju t a few time a week or month. On the other hand, m
any men hare the opinion of Charle William , who wrote that not
MAJOR COMPLICATIONS
133
even the threat of death chilled me to the bone a much a the pro pect of never
being able to make love again. Many urologi t agree that once the du t ettle on
i ue of cancer control and incontinence, erectile dy function i the lingering
quality of life compromi e for many men.2 For any peci c man, the choice of which
i wor e i u ually ea y to make: it i whichever affect him more.
URINARY INCONTINENCE: THE PROBLEM Incontinence i a problem becau e the male ure
thra, which carrie urine from the bladder to out ide the body, run directly th

rough the pro tate. Thu , when the pro tate i being de troyed by urgery, radia
tion, or cryotherapy, the urethra i inevitably affected. A detailed in Appendi
x A, urine ow in male i controlled by two phincter an internal one immediately
above the pro tate, where the urethra exit the bladder, and an external one ju
t below the pro tate. During urgery for pro tate cancer, the internal phincter
i de troyed, becau e it i anatomically contiguou to the pro tate; to pre erv
e the internal phincter ri k leaving behind ome cancer cell . That effectivel
y leave one working phincter to do a job previou ly done by two. The average d
aily urine ow i approximately one-half gallon, o the ta k i demanding. Further
, radiation and cryotherapy treatment may damage either or both phincter . The
magnitude of urinary incontinence a a problem in any particular man depend on
everal factor . Mo t important i the function and trength of the external ph
incter, the development of
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MAJOR COMPLICATIONS

An Incontinent Hou e One man noted that, while he wa having problem with urina
ry incontinence, the plumbing in hi hou e al o developed problem . The di hwa her
leaked and tained the ceiling of the room below, the kitchen drain tarted dri
pping, a toilet eal had to be replaced, and the ink in the ma ter bath joined
the dripping choru . He concluded, My hou e i incontinent! Aubrey Pilgrim, A Revoluti
nary Approach to Pro tate Cancer
which varie in different men. Like all mu cle , it weaken with age. Urologi t
with le er kill may inadvertently damage the external phincter; the inconti
nence in uch ca e i cau ed by incompetence. The two principal type of urinar
y incontinence are tre incontinence and urge incontinence. Stre incontinenc
e occur when pre ure in ide the abdomen increa e , a during coughing, neezin
g, blowing the no e, laughing, inging, pa ing ga , or exerci ing. The increa e
d abdominal pre ure cau e the bladder to contract and thereby exert more pre
ure on the external phincter, which, if it i weak, let urine pa through. A
full lower bowel will al o cau e thi type of incontinence, ince the bowel pre
e on the bladder. Urge incontinence occur when the man cannot hold hi urine
long enough to get to the bathroom. Thi may re ult from irritation or pa m of
the bladder. It occur frequently in men with benign pro tatic hypertrophy and
following radiation treatment for pro tate cancer. Although mo t book di cu
tre incontinence and urge incontinence a if they were di tinctly eparate ent
itie , in real life many men have combination of both type . A e ing the eve
rity of urinary incontinence i dif cult becau e men vary widely in their reaction
to it. Intermittent dribbling, nece itating the wearing of a thin pad, may be
of no con equence for one man but a con tant calamity for another. For thi rea
on, Mark Litwin
MAJOR COMPLICATIONS
135
Thi look too complicated for meI ugge t you call a urologi t.
and hi colleague at UCLA Medical Center developed a brief que tionnaire that c
over both the magnitude of the problem and the extent to which it bother the m
an ( ee Table 6). The que tionnaire i now widely u ed becau e the correlation b
etween the magnitude of the problem and how much it bother men turn out to be
quite low; that i , men with a mall problem may be very bothered by it, and men
with a big problem may not be.
URINARY INCONTINENCE: THE SOLUTIONS The good new about urinary incontinence cau
ed by treatment for pro tate cancer i that in mo t ca e the problem improve

over time. Thi i e pecially true of incontinence due to urgery, which initial
ly may be a eriou dif culty. Mo t tudie ugge t that incontinence continue to
be a long-term major problem for approximately 5 to 10 percent of men treated f
or pro tate cancer but can be improved with help.
136
MAJOR COMPLICATIONS
Table 6. UCLA Pro tate Cancer Index for Urinary Function 1. Over the LAST 4 WEEK
S, how often have you leaked urine? Every day . . . . . . . . . . . . . . . . .
. . . . . . . . . 1 (Circle one number.) About once a week . . . . . . . . . . .
. . . . . . 2 Le than once a week . . . . . . . . . . . . . . . 3 Not at all
. . . . . . . . . . . . . . . . . . . . . . . . . . 4 2. Which of the following
be t de cribe your urinary control during the LAST 4 WEEKS? No control what oev
er . . . . . . . . . . . . . . 1 (Circle one number.) Frequent dribbling . . . .
. . . . . . . . . . . . . . 2 Occa ional dribbling . . . . . . . . . . . . . .
. . 3 Total control . . . . . . . . . . . . . . . . . . . . . . . 4 3. How many
pad or adult diaper per day did you u ually u e to control leakage during the
LAST 4 WEEKS? 3 or more pad per day . . . . . . . . . . . . . . 1 (Circle one n
umber.) 12 pad per day . . . . . . . . . . . . . . . . . . . 2 No pad . . . . .
. . . . . . . . . . . . . . . . . . . . . . 3 4. How big a problem, if any, ha
each of the following been for you? Very mall Small Moderate Big (Circle one n
umber No problem problem on each line.) problem problem problem 0 1 2 3 4 a. Dri
pping urine or wetting your pant ? . . . . . . . . . 0 1 2 3 4 b. Urine leakage
interfering with your exual activity? . . . . . . . 5. Overall, how big a probl
em ha your urinary function been for you during the LAST 4 WEEKS? No problem .
. . . . . . . . . . . . . . . . . . . . . . 1 (Circle one number.) Very mall pr
oblem . . . . . . . . . . . . . . . . . 2 Small problem . . . . . . . . . . . .
. . . . . . . . . 3 Moderate problem . . . . . . . . . . . . . . . . . 4 Big pro
blem . . . . . . . . . . . . . . . . . . . . . . . 5
SOURCE: Web ite of the UCLA Department of Urology (uclaurology); u ed with permi
ion.
MAJOR COMPLICATIONS
137
That brand ha excellent ab orbency!
The r t tep i to control the leakage with ab orbent pad of ome kind. A wide v
ariety are available at pharmacie and medical upply tore . They range from wh
at are e entially adult diaper to thin pad that can be tucked into one underp
ant . The adult diaper are variou ly labeled a ab orbent underwear, undergarment ,
d brief and are old under brand name uch a Attend , Depend , Poi e, and Seren
ity. The next level down i underpant with an ab orbent pouch in front, for in
tance, Sir Dignity brief . A men develop increa ing continence, they graduate t
o ab orbent pad imilar to tho e u ed by women during their men trual period .
The pad come in a variety of ize , hape , and level of ab orbency, from extr
a plu , extra, and regular to light and ultra thin. Many have attachment and ca
n be tted ea ily into jockey- tyle underpant . The next tep in improving contine
nce i to a k your urologi t to rule out a urinary tract infection that could be
making the problem wor e. Thi can be done readily by checking the urine. In ca
e of evere and per i tent incontinence, the urologi t may want to carry out a
dditional te t , uch a putting a dye in the bladder and then viewing the bladd
er by X-ray (a cy togram) or by placing a thin tube up the peni to view the bla
dder (a cy to copy). Minimizing caffeine intake i a valuable tep in controllin
g
138

MAJOR COMPLICATIONS
incontinence. Caffeine increa e the frequency and urgency of having to urinate;
thu , eliminating coffee, tea, and caffeine-containing oft drink may improve
matter . Medication being taken for other medical condition may al o wor en in
continence. Tho e known to do o include alpha-adrenergic blocker u ed to treat
hyperten ion, including doxazo in (Cardura), proza in (Minipre ), and terazo i
n (Hytrin). Individual taking the e medication hould a k their phy ician to
witch them to another type of antihyperten ive medication. Mo t urologi t enco
urage patient undergoing pro tate cancer treatment to do Kegel exerci e to inc
rea e continence. The e exerci e were developed by Arnold Kegel in the 1940 fo
r u e by women who wanted to trengthen the mu cle in the pelvi after childbir
th. The dif culty i locating the correct mu cle to be exerci ed. One et i u ed
to top urine ow; halting the ow in mid tream and holding it for everal econd
i the recommended way to identify the e mu cle . The other et i u ed to tight
en the buttock . One author ugge t imagining that youre trying to hold a quarter
between your cheek , while another, perhap to greater effect, ugge t the follow
ing: Imagine that you are tanding on top of a hill, naked, with a $1,000 bill t
ucked between the cheek of your buttock . You are not able to u e your hand , b
ut you need to hold onto the bill during high gu ty wind . That queezing of you
r buttock , pulling up internally and tightening down with your pelvic mu cle ,
i a Kegel exerci e.3 The true ef cacy of Kegel exerci e for men apparently ha n
ever been formally te ted, o recommendation vary widely. Some urologi t advi
e doing them at lea t every hour for ve minute , while other ugge t much le often
. Some urologi t ay they hould only be done tanding up, while other urge do
ing them in any po ition, while watching TV, driving a car, itting in church, or
anywhere at any time. Intriguingly, ome claim that Kegel exerci e are al o great f
or improving virility and achieving greater ejaculation and arou al control. In 20
04 the National In titute of Health funded a re earch project that i tudying
the be t way to teach men to do Kegel exerci e .4 Sometime urinary continence c
an be mode tly improved with medication. Deconge tant , uch a tho e u ed for c
old , have been
MAJOR COMPLICATIONS
139
recommended; an example i p eudoephedrine (Sudafed). Imipramine (Tofranil), a c
ommonly u ed antidepre ant, may help the mu cle tone of the phincter. Oral ant
icholinergic drug are widely u ed to improve continence in elderly per on and
are worth trying; the mo t frequently pre cribed are tolterodine (Detrol) and ox
ybutynin (Ditropan, Oxytrol). Dry mouth, blurred vi ion, con tipation, and leep
ine are common ide effect of anticholinergic . Men hould alway check with
their urologi t before tarting on any of the e drug and a certain that the new
medication doe not interact with tho e being taken for other condition . De pi
te all of the above ugge tion , urinary incontinence will continue to be a prob
lem for 5 to 10 percent of men following pro tate cancer treatment. Injection o
f collagen through the urethra may trengthen the phincter for approximately ha
lf of the men who try it, but it i expen ive and the effect are u ually not la
ting. Out of de peration, ome men u e a foam rubber penile clamp, which can be
relea ed when the man wi he to urinate, or a condom catheter (widely known a
a Texa catheter), which i worn on the peni and collect the urine, but both
olution bring their own complication and are not recommended by mo t urologi t
. The de nitive olution for evere, prolonged urinary incontinence i an arti cial
phincter. It can be urgically implanted around the urethra and operated by a
mall bulb placed in the crotum. When the man wi he to urinate, he queeze th
e bulb, which open the urethra; after a minute or two the phincter automatical
ly clo e again. De pite it high co t and po ible complication , uch a infec
tion and the need for replacement, the arti cial phincter ha given a normal life

back to many men previou ly plagued by incontinence. My own experience with inc
ontinence wa fortunately brief. I found it trange and embarra ing to buy what
i e entially an adult diaper but wa impre ed by how much pharmacy helf pa
ce wa allotted to the e item ; I wa rea ured that I had plenty of company! Lu
ckily, my external phincter took charge almo t immediately after removal of the
catheter, and I wa able to di card the adult diaper and heavy pad within a da
y. By the end of three week I did not require any pad, and thereafter have had
only occa ional minor dribbling when I increa e my abdominal pre ure. The dribb
ling i le than what I experienced in the year immediately prior to urgery.
Al140
MAJOR COMPLICATIONS
The Artificial Sphincter: A Paean to No Longer Being Peed On It i nt until after
a hower the next morning that I begin to feel like a whole per on again. Save f
or the quirky little maneuver I have to go through to urinate, it i a heady fee
ling to feel like the ame man I wa before. No more a pri oner locked in olita
ry, I can walk in the unlight, free of the Velcro hackle . Im neither a nudi t,
nor have I fla her tendencie , but it i a thrill to be able to walk around wit
hout anything on. Not to be able to perform the imple, mo t natural act of walk
ing naked from a hower to a bedroom a few feet away can only be de cribed a li
ving in a cell without wall ; it i day before I can allow my elf to appreciate
their di appearance. . . . I leep deeply, dream-free and, not ince the night
before the fir t operation, in the raw. The nightmare i over. I think I can hea
r the fat lady inging. Bert Gottlieb, de cribing what it like to have an artific
ial phincter, in The Men Club
though radical pro tatectomy i much more likely to cau e urinary incontinence t
han to improve it, tudie have hown that for ome men the urgery doe , in fac
t, improve urinary ymptom .5
IMPOTENCE: THE PROBLEM Impotence, or erectile dy function (ED), a it ha become
known, i the gorilla in the pro tate cancer clo et. It lurk ju t out of ight
but never out of mind. Choo ing between variou treatment for pro tate cancer
ba ed on the likelihood of impotence i a little like choo ing how you wi h to d
iethe outcome of all the choice i remarkably imilar. Normal male exual functi
oning i complex. A man mu t feel
MAJOR COMPLICATIONS
141

ome de irelibidoand thi urge i in tigated by a mix of te to terone and p ycholo

gical factor . The peni mu t become erect, which i determined by the arterie ,
vein , and nerve that upply it. Orga m i initiated in the brain and involve
the ejaculation of perm and eminal uid through the contraction of mu cle . And
all of thi mu t be orche trated moothly if it i to be ucce ful. The proble
m in ca e of pro tate cancer i that the arterie , vein , and nerve that contr
ol erection run along ide the pro tate. To treat pro tate cancer by any method
mean ri king damage to one or more of the e. The arterie are needed to carry b
lood to the peni . The nerve cau e the mooth mu cle in the peni to relax o
that it can ll with blood. Thi lling, in turn, hut the vein , thereby trapping
blood in the peni . An erection, therefore, i imply a peni engorged with bloo
d, and erectile dy function occur when blood doe not ll the peni a it i upp
o ed to. Even when a nerve- paring procedure i u ed in the urgical removal of
the pro tate, arterie and vein may till be damaged, with re ultant erectile d
y function. More mi information probably exi t about exual function than about
any other a pect of pro tate cancer. One man wa told that treatment would have n

o phy iological effect on my libido or en ation of orga m. . . . I would till

be able to achieve orga m, and it would feel much the ame a before. A widely rea
d book rea ure men that after a radical pro tatectomy, they will have normal en
ation, normal ex drive, and can achieve a normal orga m. And, even if they do ha
ve erectile dy function, it can alway be treated.6 The truth i quite different. Ce
rtain men have been hown to experience a decrea e in libido following treatment
for pro tate cancer, although it i u ually not marked. Some of the decrea e ma
y be due to a le ening of te to terone, although the mechani m for thi decrea
e i not clear. Dimini hed libido may al o be due to depre ion econdary to ere
ctile dy function or other factor . Ejaculation i aboli hed by pro tate urgery
, ince the eminal ve icle , which upply mo t of the eminal uid, and the va a
deferentia, which carry the perm, are both evered. A one man noted, ejaculati
on wa vi ible proof of my manhood. . . . And now that integral part of my exual
expre ion wa going to be taken away from me forever. . . . No emen, no ejacul
ation! It wa a cut and dried a a beheading. A George Burn de cribed it when,
in hi early 90 ,
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MAJOR COMPLICATIONS

he wa a ked about hi exual performance: I come du t. One man who had pro tate ur
gery looked on the humorou ide of not ejaculating: Now that I would no longer pr
oduce my ejaculate, I might even be able to fake an orga m.7 Ejaculation i al o d
ecrea ed by radiation treatment , although thi effect take place more lowly.
For mo t men the quality of orga m i le inten e after treatment for pro tate
cancerhardly urpri ing, given the number of pelvic tructure affected by the
urgery. The ame i true after radiation treatment. Michael Dor o, a phy ician w
ho elected radiation treatment for hi pro tate cancer, wrote: I really mi my pr
o tate gland! . . . What I didnt realize i that it produce about 80% of the ple
a ure of an ejaculation. That feel-good pul ing in my groin during orga m i gon
e!8 The principal lo in exual function after pro tate cancer treatment i the a
bility to achieve an erection uf cient for intercour e. Charle Neider call it h
aving joined the Limp Peni Club.9 A noted in previou chapter , it now eem clear
that the majority of men who undergo pro tate cancer treatment of any kind will
uffer ome degree of erectile dy function. Are there any predictor regarding
which men are more likely to develop uch dy function? The r t key predictor i a
ge. A men grow older, they progre ively lo e ome of the nerve that upply th
e peni ; in addition, the arterie , like arterie el ewhere in the body, may fun
ction le well. According to one e timate, by age 60, a man only ha about ixty
percent of the nerve he wa born with.10 The econd major predictor of exual fun
ction following treatment for pro tate cancer i the man exual functioning prio
r to treatment. Studie have reported that approximately 20 percent of adult men
are unable to achieve erection uf cient for vaginal intercour e, and an additio
nal 30 percent have dif culty maintaining erection .11 The reality, therefore, i
that at lea t one third of men uffer ome degree of erectile dy function at the
time they are diagno ed with pro tate cancer; uch men are certainly not going
to function better after treatment. The cenario remind me of the man who a ked
hi urologi t whether he would be able to play the piano after having pro tate
urgery. I dont ee why not, aid the urologi t. Why, that wonderful, aid the man
could play before. The third predictor of po ttreatment erectile dy function i
MAJOR COMPLICATIONS
143
I It Po ible to Accurately A e Erectile Dy function after Pro tate Cancer T
reatment? It i difficult to get credible figure on the incidence of erectile d
y function following pro tate cancer treatment, in part becau e many men do not
an wer uch que tion hone tly. A e ment al o vary depending on the definitio

n u ed. Doe a man qualify a having an erection if hi peni , which normally h

ang at 7 oclock, reache 8:30 or 9 oclock? Doe it depend on how tiff it i ? Or
how long the erection la t ? Or whether he can only get an erection while tandi
ng up? Or only with the a i tance of medication? Some have defined an erection
a one ufficient to achieve vaginal penetration, but that depend in part on th
e partner anatomy and cooperation. And what about frequencyif one achieve an ere
ction once every ix month , i that ufficient to ay that erectile dy function
doe not exi t? Urologi t who advocate for one or another form of treatment a
e erectile dy function differently in attempt to make their number look goo
d. Their data are often not reliable.
whether the man ha other cau e of dy function. Diabete often contribute , and
many medication cau e a degree of erectile dy function, including ome of tho
e taken for hyperten ion and depre ion. It i often po ible for a man to ub t
itute another medication that ha fewer uch ide effect . The nal predictor of e
rectile dy function following pro tate cancer treatment i the type of treatment
the man received. Surgical pro tatectomy produce immediate erectile dy functio
n in almo t all ca e but then a low recovery of function, peaking at eighteen
to twenty-four month . Recovery i mo t likely when both nerve were pared but,
at lea t in one tudy, almo t a ati factory when only one nerve i pared.12
Radiation produce little erectile dy function
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MAJOR COMPLICATIONS
initially, but uch dy function increa e over the following month and even yea
r . Cryotherapy and hormone therapy produce the mo t erectile dy function. One o
ther a pect of erectile dy function following pro tate cancer treatment i rarel
y di cu ed: the po ible lo of the man exual fanta y life. A tudy among for
ty-eight men who had been treated by urgery, beam, and eed radiation therapy r
eported the following: The men became no talgic when they de cribed how they onc
e enjoyed thinking about ex, now a lo t pa time. They di clo ed that they no lo
nger enjoyed exual feeling in re pon e to eeing an attractive woman. They al
o expre ed a profound en e of lo a ociated with a lo of a fanta y life in
which they were able to imagine them elve a potential exual partner . They w
ere adly con ciou of their dimini hed libido .13 The e men had previou ly enjo
yed eeing attractive women in their everyday live and fanta izing about them.
The experience wa omething that they had identi ed a part of their live a men. Im
potence, however, rendered the e fanta ie unreali tic. Thi tudy make clear t
hat a man lo of potency following pro tate cancer treatment lead to more than
erectile dy function; it may lead to fanta y dy function a well. For ome men, thi
can be a eriou lo .
IMPOTENCE: THE SOLUTIONS The importance of having an active ex life varie wide
ly among men, e pecially among men of different age . For a man who i diagno ed
with pro tate cancer in hi 40 and who ha a younger wife, the threat of impot
ence may appear cata trophic. For a man in hi 60 , with a wife of imilar age,
the threat of impotence following treatment for pro tate cancer may be omething
he can live with, e pecially if he believe that the treatment ha cured hi ca
ncer. Thi willingne to accept partial or full impotence in exchange for a uc
ce ful cancer treatment may explain why, in one urvey, nearly half the men who e
xperience erectile dy function after pro tate cancer therapy choo e not to eek
treatment for their impotence.14 It
MAJOR COMPLICATIONS
145
Adju ting to Impotence I could not predict ju t how the lo of . . . exual pot

ency might affect me. I wa certain it would not enhance my elfimage, but perha
p I would handle it rea onably well. Important a it wa in my life, exual act
ivity did not involve large block of my time on a daily ba i . Mo t of what giv
e meaningfamily, friend , teaching, reading, writing, movie , bicycling, qua h,
kiing, good foodwould till be there and there wa little rea on to think that
my capacity for any of them, with the hort-term exception of bicycling, would b
e dimini hed in any way. William Martin, My Pro tate and Me Patient can have exc
ellent quality of life even though erection and urinary control may not be perf
ect. Im ure that there are many patient who have artificial hip and knee who
cannot run and dance like they once did but are plea ed with the outcome. Patrick
Wal h, letter, Journal of Urology, 2004
may al o explain the wide di parity reported by re earcher between men who are
deeply di tre ed by po ttreatment exual dy function and men who are little bot
hered by the dy function. A critical tep in nding olution to the impotence pro
blem i open and frank di cu ion between the man and hi partner. Michael Dor o
advi e that the ilent male model doe nt work here and add : Men eem to have more
trouble than women talking about their intimate feeling and erotic need . You w
ill have to overcome your chauvini tic leaning . It eem to be one of life para
doxe , that ometime a frank exual di cu ion can be mo t difficult with the
146
MAJOR COMPLICATIONS
one haring our bed! Bizarre, i nt it? You may have looked into her eye a you
hared thou and of orga m , and yet find it difficult to look into her eye and
di cu your new exual concern .15 There are many way to enhance whatever erec
tile function remain after pro tate cancer treatment. Direct phy ical timulati
on of the peni by the man or hi partner i more effective than vi ual or fanta
y timulation. It can be a i ted by lubricant uch a K-Y jelly, A troglide,
or, lea t expen ive of all, aliva. A tanding po ition often help erection , m
aking it more dif cult for blood to e cape from the peni and return uphill to the
heart. Another mean of lowing the e cape of blood i to place a rubber band o
r erection ring, available in ex hop , around the ba e of the peni prior to f
oreplay. Blood can then enter the peni through the central arterie but can le
readily e cape through the weaker-walled vein , which are con tricted by the b
and . Even men who have complete erectile dy function can have a ex life after
pro tate cancer treatment. Since orga m begin in the brain, not the peni , they
are till po ible, although they may be more dif cult to achieve. A in all a pe
ct of exual function, practice make perfect. People phy ical need and re pon
e differ. A one author note : Ju t a all arti t have to practice with differe
nt bru he and mixing color and how to apply them to get the de ired re ult, if
we want to improve our lovemaking, we have to do the ame.16 It ha been aid tha
t a we deal with the problem of a exible peni , it i be t not to be too rigid;
in fact, exibility often produce greater rigidity. Article , book , and web ite
advice abound regarding how to improve exual functioning following pro tate can
cer treatment. Among the be t ource of information i The Lovin Aint Over: The C
ouple Guide to Better Sex after Pro tate Di ea e, by Ralph and Barbara Alterowi
tz ( ee Appendix B). The premi e of the book i that you can have a loving and at
i fying exual relation hip without having an erection. They ugge t: Dont focu on
the erection. In tead, focu on loving and deriving plea ure from it. When erec
tion were ea y, we tended to focu olely on them. In reality, mo t of u mi e
d out on other mean of deriving plea ure, which could have made loving much mor
e plea urable even then.
MAJOR COMPLICATIONS
147

Ma turbation: Sweet Irony! Authoritie agree that exerci ing the peni early and
often following treatment for pro tate cancer increa e the chance for return
of function. The peni need an abundant blood upply to nouri h the ti ue , an
d the arterie to the peni are often damaged during treatment. Book advocate a
ctivitie uch a frequent direct exual timulation and penile ma age. Many of u o
r men remember hearing in our youth that ma turbation wa elf-abu e and would make
u become blind. I recall the tory of the boy who wa caught ma turbating by h
i mother and told to top. But mother, cant I continue ju t until I need gla e ? he
replied. But now, weet irony! In our youth, we were told that ma turbation wou
ld cau e a deterioration of our body. In our old age, we are now told that not m
a turbating will cau e a deterioration of our body.
Similarly, Michael Dor o note that making love doe not have to equal ex; furthe
rmore having ex doe not have to equal intercour e. Women know thi better than m
en do and, judging from comment po ted on pro tate cancer web ite , it eem to
be a dif cult le on for men to learn. Dor o wife put it mo t uccinctly: Michael,
I married you for who you are, not for your peni .17 For men who want to enhance t
heir erection with arti cial mean , the ve main option are oral medication , peni
le injection , vacuum pump , nerve graft , and urgically implantable penile pro
the e . Of the e, oral medication are by far the mo t popular and mo t widely
u ed. It i , in fact, dif cult to watch a porting event on televi ion without ee
ing adverti ement for ildena l (Viagra), vardena l (Levitra), or tadala l (Ciali ).
The e medication are helpful for many men, but they do not live up to the promi
e of in tant pharmaceutical nirvana that the ad ugge t. In one tudy of their
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MAJOR COMPLICATIONS
Oral Medication to Enhance Erection ildenafil (Viagra) 25 mg., 50 mg., and 10
0 mg. rectangular blue tablet peak action 30 minute to 2 hour ; may la t up to
6 hour food affect action, o hould not be taken le than one hour before o
r within two hour after eating vardenafil (Levitra) 2.5 mg, 5 mg, 10 mg, and 20
mg round white tablet peak action 30 minute to 2 hour ; may la t up to 12 hou
r okay to take with food except for high-fat meal , which delay on et of action
tadalafil (Ciali ) 5 mg, 10 mg, and 20 mg oval and-colored tablet peak action
30 minute to 6 hour ; may la t up to 36 hour okay to take with food Side effe
ct : All three medication can have imilar ide effect : flu hing, headache, di
zzine , indige tion, and tuffy no e. Sildenafil al o occa ionally give a blue
tinge to vi ion. Rarely, the medication may produce a prolonged erection (pria
pi m), which hould be treated with ice pack and, if needed, a vi it to the eme
rgency room, or they may cau e ome lo of vi ion, which may be permanent. DO N
OT TAKE the e medication if you are al o taking nitrate medication for angina
or che t pain. The combination can cau e a evere fall in blood pre ure and eve
n death. (The nitrate drug include i o orbide, Dilatrate, I ordil, Sorbitrate,
I mo, Imdur, nitroglycerin, Deponit, Minitran, Nitro-Bid, Nitrodi c, Nitro-Dur,
Nitro tat, and Tran derm-Nitro.) You hould tart at a low do e and take the new
drug cautiou ly if you are al o taking any of the following: medication to lo
wer blood pre ure, e pecially doxazo in (Cardura)
MAJOR COMPLICATIONS
149
medication for benign pro tatic hypertrophy, uch a tam ulo in (Flomax) medica
tion for AIDS erythromycin, ketoconazole (Nizoral), itraconazole (Sporanox) ant
idepre ant in the elective erotonin reuptake inhibitor category (SSRI )
ef cacy in men who had had a radical pro tatectomy, they were aid to be helpful t
wo third of the time in men younger than age 55 but only one third of the time

in men 60 or older.18 The oral medication , r t introduced in 1997, work by block

ing an enzyme (pho phodie tera e type 5) in the peni , thereby allowing the moo
th mu cle to relax and the peni to ll with blood. They do not produce an erecti
on by them elve , but only when accompanied by erotic timulation. The effective
do e varie among men; ome men, but not all, get a better re pon e at a higher
do e. The e medication hould never be taken by men who are al o taking nitrat
e for angina or che t pain; the drug interaction can be fatal. Another rare but
important ide effect i their propen ity for cau ing blurred vi ion and/or par
tial blindne , u ually in one eye, a condition of cially called nonarteric i chem
ic optic neuropathy (NAION). At lea t forty ca e have been reported, and the ey
e change are u ually permanent. Men who have hyperten ion, diabete , hyperlipid
emia, and/or a mall optic cup (which can be a e ed via an exam by an ophthalm
ologi t or optician) are at higher ri k for thi ide effect.19 No controlled t
udie have yet been done comparing the three medication . If a man doe not re p
ond to one of them, it i not clear what hi chance are of re ponding to anothe
r one. All three medication are priced imilarly, at approximately $10 per pill
. Some re earch ha ugge ted that oral medication to enhance erection not onl
y have an immediate bene ta tronger erection but may al o have longer-term bene t by
preventing bro i and
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MAJOR COMPLICATIONS

atrophy of penile ti ue. Biop ie of penile ti ue were performed at ix-month

interval in two group of men: one group took ildena l every other night for ix
month and the other did not.20 If additional, longer-term bene t are de nitely pr
oven, the e medication will be routinely pre cribed to pre erve penile function
in men undergoing treatment for cancer of the pro tate. Another form of the am
e type of medication can be placed directly into the urethra a a uppo itory. C
alled MUSE (Medicated Urethral Sy tem for Erection), it ha been u ed le ince
the oral medication became widely available. Men for whom oral medication are
not effective may choo e to try injection directly into the peni . The medicat
ion dilate the blood ve el , allowing the peni to ll with blood, and may produ
ce a erviceable erection la ting for about an hour. Self-injection i not for a
ll men, however, and, a noted by one author, obviou ly i not ideal for men who c
ant ee well [and] men with poor hand-eye coordination.21 Penile elf-injection ar
e well de cribed by Robert Hitchcock in Love, Sex, and PSA and by Michael Korda
in Man to Man ( ee Appendix B). The other three option for erection enhancement
are vacuum pump , nerve graft , and penile pro thetic implant . The pump i pla
ced over the peni prior to intercour e and, by creating a vacuum, draw blood i
nto it. Some men nd it quite ati factory, while other do not; for example, Char
le Neider complained that the peni doe nt eem to be en itive and it erect, but n
tiff.22 Nerve graft are till experimental and are u ually done at the time of
urgery on men in whom neither nerve can be aved. Penile pro thetic implant mu
t be in erted by a urgeon and come in a variety of tyle . Chuck Wheeler in Af rm
ing the Darkne de cribe having uch an implant. Vacuum pump and implant are
both explained in detail in Sheldon Mark Pro tate and Cancer and Paul Lange an
d Chri tine Adamec Pro tate Cancer for Dummie ( ee Appendix B). My own experien
ce with erectile dy function ha been rea onably ati factory. Like mo t men, I
had almo t complete erectile dy function immediately following urgery. After a
few week , I wa able to achieve orga m, but, a noted by other , it wa le in
ten e than before. The initial orga m were accompanied by minor pelvic pain and
ome lo of urine; both have been de cribed by other men, and both re olved p
ontaneou ly.
MAJOR COMPLICATIONS
151

I found vardena l (Levitra) to be the mo t helpful of the three available oral med
ication . Other aid were a tanding po ition, which force the blood in the pen
i to run uphill to the heart; a rubber band at the ba e of the peni to partial
ly con trict the vein and thereby low the out ow of blood; and exual activity i
n the morning hour , when it i known that te to terone level , and thu libido,
are highe t. I noted a low improvement in erectile function between three and
ix month po t urgery, at which time I ucce fully had vaginal intercour e. I
had mentally prepared my elf to po ibly never have thi experience again, o it
wa a memorable event. Between ix and twelve month , erectile progre wa mor
e rapid. According to one tudy, the maximal erection recovery following pro tat
e cancer urgery doe not occur until after an average of eighteen month , but i
t can continue for two year or longer.23 De pite all of the available aid for
recovering erectile function, the ingle mo t important factor, in my experience
, i having a loving and under tanding wife or partner. In thi , I feel truly fo
rtunate and would wi h the ame for every man who mu t confront recovery from pr
o tate cancer.
C H A P T E R
11
What Happen if the Cancer Spread or Come Back?
iving with pro tate cancer, aid one man, i like being trapped in ide a cage with
a baby lion. In the beginning, the lion i mall and nonthreatening, but you know
that the lion will grow and may eventually devour you. Thi man cancer did pre
ad and in the end did devour him.1 Prior to the PSA era, by the time of diagno i th
e pro tate cancer would have already pread beyond the pro tate in the majority
of ca e . By the 1990 , thi number had been reduced to one third, and by now it
i pre umably igni cantly lower than that. Pro tate cancer that have pread at
the time of diagno i pre ent many of the ame treatment problem a cancer tha
t recur after the initial treatment. In ome ca e , the recurrence i expected b
ecau e of a Glea on core of 8 to 10, a PSA over 20, or other indicator of a la
rge and eriou cancer. In other ca e , the recurrence of the cancer i unexpect
ed, a when a man ha a low Glea on core, a low PSA, and a mall tumor. Some of
the e men are told by their urologi t that they are cured following their initial
urgical or radiation treatment , and recurrence of the cancer, when it occur ,
can be a cruel hock. It i helpful to keep in mind the magnitude of the recurre
nce problem. There are approximately 1.6 million men in the United State who ha
ve been diagno ed with and, in mo t ca e , treated
L
IF THE CANCER SPREADS OR COMES BACK
153
for pro tate cancer. They con titute 17 percent of American who are living with
cancer. The majority of the e men have regular PSA check at lea t annually for
ten to fteen year . Each time they are te ted, they wait nervou ly for the re ul
t will thi be the time their PSA i elevated, indicating the recurrence of cance
r? Since the recurrence of pro tate cancer i often without ymptom , there i a
Kafkae que quality to the repeat PSA te t , a my teriou internal proce over
which a man ha no control.
WILL IT KILL ME, AND IF SO, WHEN? The recurrence and pread of pro tate cancer u
ually follow certain pathway . If the cancer wa removed urgically, micro copi
c bit of cancer may have been left behind where the pro tate lay (called the be
d of the pro tate). If the cancer wa treated with radiation, it may recur in po
rtion of the pro tate that did not receive enough radiation to kill all the can
cer cell . At any point, the cancer may pread beyond the pro tate to the emina

l ve icle , adjacent lymph node , or bladder; thi i called local pread. It ma

y al o pread more di tantly, called meta ta i . Bone , e pecially the pine, ar
e favorite ite for pro tate cancer meta ta e , but late in it cour e the canc
er may al o meta ta ize to the kidney or virtually anywhere in the body. The pr
edictor of pro tate cancer pread include the ame factor
154
IF THE CANCER SPREADS OR COMES BACK
A Clerk in a Great Court I try not to think about the number but I cant help doin
g it. PSA i like a clerk in a great court itting in judgment on me, on my heal
th or illne , my life or death. What terrible or wonderful me age will thi cl
erk deliver? Will I be condemned? Or re cued? And will the tumor be maller? If
o, how mall? Will my ide effect have been experienced in vain? Did I make a
eriou mi take in not electing a pro tatectomy? Or watchful waiting? Wait, wai
t, wait and ee. Charle Neider, Adam Burden
that predict a eriou cancer. Men who have a PSA greater than 20, a Glea on co
re of 8 to 10, or a large tumor that occupie both lobe of the pro tate are at
high ri k for recurrence. Thu , a man with a PSA between 4 and 10 ha an 11 perc
ent chance of cancer recurrence at ve year , wherea a man with a PSA over 20 ha
a 40 percent chance; in addition, the man with the higher PSA will have hi can
cer recur ooner. Similarly, a man with a Glea on core of 6 or le ha a 3 per
cent chance of recurrence at ve year , wherea a man with a Glea on core of 8 to
10 ha a 38 percent chance. A man who e cancer occupie more than 20 percent of
the pro tate ha three time the ri k of recurrence a a man who e cancer occup
ie le than 10 percent of the gland.2 Several effort have been made to put va
riou factor together into a ingle chema to predict pro tate cancer pread fo
llowing urgery or radiation treatment. The e chema are referred to a nomogram
, which are merely graphic repre entation of ri k ba ed on the pa t experience
of imilar men. The imple t nomogram for men following urgery con i t of the
following:3 Take your numerical Glea on core. Add 1 if your PSA wa 4 to 10. A
dd 2 if your PSA wa 10.1 to 20.
IF THE CANCER SPREADS OR COMES BACK
155

Add 3 if your PSA wa over 20. Add 2 if your eminal ve icle were po itive for
cancer at urgery. Add 2 if the cancer had po itive margin at urgery. Subtract
4 if you had add-on (adjuvant) hormone treatment. Subtract 2 if you had add-on
(adjuvant) radiation treatment.
Add the number . Your chance that your cancer will recur, a mea ured by ri ing
PSA ve year following urgery, are hown in Table 7. Additional nomogram , deve
loped by Michael Kattan and hi colleague at Memorial Sloan-Kettering Cancer Ce
nter in New York, are widely u ed by phy ician and are commonly referred to a
Kattan table .4 The nomogram utilize information uch a ize of the cancer and
evidence of inva ion at urgery, or radiation do e and length of radiation ther
apy. A major hortcoming of all y tem i that they are ba ed on the experience
of men treated in pa t year , wherea treatment are continuing to improve and
pa t experience may not nece arily be applicable. There are three main tage i
n the recurrence and pread of pro tate cancer: 1. Recurrence a mea ured by PSA
: A noted in Chapter 3 and 4, a ri e in the PSA after urgical or radiation tr
eatment indicate recurrence of cancer. In the medical literature, thi increa e
i commonly referred to a a biochemical failure. If the PSA i going to ri e i
n a po t urgical patient, it will do o within the r t ve year in 72 percent of c

a e ; between ve and ten year in 23 percent of ca e ; and between ten and fteen y
ear in 5 percent of ca e . It i rare to have a recurrence of cancer beyond ftee
n year . Or we might ay that the average time from urgery to a ri ing PSA i t
hree and a half year .5 Once the cancer ha recurred, a indicated by a ri e in
the PSA, it progre ion i highly variable. If no treatment i given, the PSA w
ill continue to ri e relatively rapidly in approximately half of all men, lowly
in a third of men, and very lowly in the remainder. In 7 percent of ca e , the
PSA will not reach a level of 10 in le than twenty year .6 Thu , ome men wil
l have a continuou ly ri ing PSA and progre ion of their cancer, while other w
ill have almo t no progre ion for many year de pite receiving no treatment.
156
IF THE CANCER SPREADS OR COMES BACK
Table 7. Nomogram for Predicting Recurrence of Cancer Five Year Following Surge
ry Total point 5 or le 6 7 8 9 10 11 12 13 or more Percent recurrence 6% 10 1
8 23 33 40 48 60 68
2. Progre ion from PSA ri e to meta ta i : The ingle be t predictor of the con
tinuing progre ion of recurrent pro tate cancer i the PSA doubling time. A th
e name implie , thi i the time it take for the PSA level to go, for example,
from 1.1 to 2.2 or 4.2 to 8.4. The horter the doubling time, the wor e the prog
no i for men treated by either urgery or radiation. If the PSA double in le
than ix month , it i likely that the cancer ha already meta ta ized to bone
or other organ ; if it double in more than twelve month , the cancer i probab
ly till localized near the pro tate bed. Charle Pound and hi colleague at Jo
hn Hopkin Univer ity developed prediction of how long it take recurrent pro
tate cancer to meta ta ize once the PSA ha ri en, ba ed on the PSA doubling tim
e, Glea on core, and whether the initial PSA ri e occurred ooner than two year
after urgery ( ee Table 8).7 Having a horter PSA doubling time double the c
hance that a man will have meta ta e by even year after the PSA ri e, other
thing being equal. Note that the e prediction are a be t-ca e cenario, ince
they repre ent the outcome for 304 men who were an average age of 58 when operat
ed on for pro tate cancer. There i evidence that younger men with pro tate canc
er have a better progno i , o older men are likely to have a higher rate of met
a ta i than the e number predict. Among the men followed at John Hopkin , the
median time from PSA ri e to meta ta i wa eight year , but varied from two to
twelve year . 3. Progre ion from meta ta i to death: The interval between the
IF THE CANCER SPREADS OR COMES BACK
157
Table 8. Percentage of Pro tate Cancer That Will Have Spread to Bone or Other
Organ , a Predicted by PSA Doubling Time, Glea on Score, and How Quickly the PS
A Ro e Following Surgery PSA doubling time: Glea on core 57; PSA ri e more than
two year after urgery at 3 year more than 10 month le than 10 month PSA
doubling time: 5% 18% at 5 year 14% 31% at 7 year 18% 40%
Glea on core 57; PSA ri e le than two year after urgery at 3 year at 5 yea
r 24% 65% at 7 year 41% 85%
more than 10 month le than 10 month
21% 19%
Glea on core 810; PSA ri e more than two year after urgery at 3 year 23% at
5 year 40% at 7 year 53%

Glea on core 810; PSA ri e le than two year after urgery at 3 year 47%
NOTE:
at 5 year 69%
at 7 year 79%
The number are ba ed on 304 men who had a ri e in their PSA following radical p
ro tatectomy at John Hopkin Ho pital between 1982 and 1997. The average age of
the men at urgery wa 58. Younger men generally have a more benign cour e than
older men; thu , the e prediction may be overly optimi tic for older men. SOUR
CE: C. R. Pound, A. W. Partin, M. A. Ei enberger, et al., Natural hi tory of pro
gre ion after PSA elevation following radical pro tatectomy, Journal of the Ame
rican Medical A ociation 281 (1999): 159197.
development of pro tate cancer meta ta e and death average three to ve year in
variou tudie . In the John Hopkin tudy cited above, the median time wa ve
year , but a noted, it wa a tudy of younger men. The Pound group found that t
he horter the interval between urgery and the development of meta ta e , the
horter the time to death wa likely to be. For example, among men who developed
meta ta e within three year of urgery, 87 percent had died by ve year after t
heir urgery.8
158
IF THE CANCER SPREADS OR COMES BACK
Stage of Di ea e The di tinction between tage of di ea e i an arbitrary one.
There i no ingle moment when Victor cancer cea ed to be omething he lived wi
th and became, in tead, omething that wa killing him. It wa killing him from
the beginning and he lived with it until the end. Still, there i ome rea on to
divide the experience into period . There wa a time when Victor lived a more o
r le normal life de pite the di ea e, and it wa followed by a period that wa
not like normal life at all, when we under tood that hi life it elf wa nearin
g it end. There wa no defining moment that eparated one period from the other
, but they were nonethele different. Audrey Newton, Living with Pro tate Cancer
In ummary, the ooner the PSA ri e following treatment for pro tate cancer, th
e wor e the progno i . Thereafter, the fa ter the PSA doubling time, the ooner
the man i likely to develop meta ta e . And the ooner he develop meta ta e ,
the ooner he i likely to die. The duration from initial treatment to ri e in P
SA average three to four year but may be a long a fteen year . The duration f
rom PSA ri e to meta ta i average ix to eight year , and from meta ta i to d
eath three to ve year . Thu , for men who e cancer appear not to have pread at
the time of initial diagno i , the average time between initial treatment and de
ath will be approximately fteen year for tho e who are de tined to die from thei
r cancer. Thi prediction i not valid for men who e cancer ha already pread a
t the time of initial diagno i . Alway keep in mind, however, that prediction
are merely prediction . A in all recurrent cancer , one ubgroup progre e ver
y rapidly, one ubgroup progre e hardly at all, and the va t majority of men f
all between the e two extreme . There are alway tho e who defy prediction . Con
ider the 45-year-old man who in 1976 wa diagno ed with pro tate cancer that ha
d already meta ta ized to hi
IF THE CANCER SPREADS OR COMES BACK
159
bone and di tant lymph node . He wa treated with hormone therapy and twenty- e
ven year later wa till doing ne.9 All of u who have pro tate cancer hope that

we too will do better than the average prediction .

TREATMENT OPTIONS FOR RECURRENT CANCER There are two goal in treating pro tate
cancer that ha pread: (1) improve the quantity of the man life, and (2) improv
e the quality of the man life. The dilemma in treatment ari e when attempt to
improve quantity make the quality wor e, not better. Hone t a e ment of treat
ment option are crucial to re olving the e dilemma . The main tay for treating
recurrent pro tate cancer i hormone therapy, a de cribed in Chapter 5. Pro tat
e cancer u e te to terone to grow, o blocking te to terone low the growth. P
ro tate cancer contain cell that are en itive to te to terone and other cell
that are not en itive. Over time the te to terone-in en itive cell become pre
dominant and hormone therapy become ineffective. The cancer i then called andr
ogen independent. The average duration of effectivene for hormone therapy for
a pro tate cancer that ha already meta ta ized i approximately two year ,10 bu
t during that time men often have comfortable remi ion . One of the two major c
ontrover ie in providing hormone therapy to men with recurrent pro tate cancer
concern when to tart the treatment: a oon a the PSA ri e , or not until the
cancer meta ta ize or otherwi e cau e ymptom of growth. Proponent of immed
iate treatment point to two tudie reporting that immediate hormone therapy pro
duced better outcome . In one tudy, men treated with immediate hormone therapy,
compared to tho e for whom hormone therapy wa delayed, developed meta ta e mo
re lowly, had half the number of eriou complication (pathological fracture ,
pinal cord compre ion, ob truction of the ureter), and urvived longer. In t
he other tudy, at even year after treatment only 6 percent (3 of 47) of the m
en who had received immediate hormone therapy had died from pro tate cancer comp
ared to 31 percent (16 of 51) of the men who had received delayed therapy.11 Pro
ponent of delayed hormone therapy point to an older tudy that had re ult exac
tly oppo ite to the two tudie cited above. They
160
IF THE CANCER SPREADS OR COMES BACK
al o argue that giving hormone to men before there i evidence of cancer pread
mean ubjecting them unnece arily to the multiple ide effect of hormone . P
atrick Wal h, a taunch opponent of immediate hormone therapy, ay that it will a
ctually take life out of the year a man ha to live, without adding any year t
o that life. Wal h al o note that the pharmaceutical indu try ha trongly in uence
d urologi t to tart hormone therapy early becau e the indu try make at lea t a
billion dollar a year on hormonal agent for the treatment of pro tate cancer.12
Several tudie are under way that perhap will re olve the immediate ver u del
ayed treatment debate. The other active hormone therapy controver y i whether t
o give hormone continuou ly or intermittently (in other word , treat the man fo
r a few month until hi PSA fall and then top treatment for everal month un
til the PSA ri e again). The two argument for intermittent therapy are that it
give the man a better quality of life by giving him a vacation from the hormone th
erapy ide effect , and that intermittent therapy delay the proce of the canc
er becoming androgen independent. Some but not all tudie upport the e argumen
t . A European tudy reported that only 7 percent of men on intermittent hormone
therapy had had progre ion of their cancer at the end of three year compared
to 39 percent of the men on continuou hormone therapy. A U.S. tudy found no di
fference in progre ion rate between the two group . There doe appear to be co
n en u that the quality of life i better for men on intermittent therapy, inc
e they have period free from the ide effect of the therapy;13 therefore, inte
rmittent therapy i being increa ingly u ed. Two large tudie comparing continu
ou and intermittent hormone therapy are in progre . Beam radiation i the eco
nd mo t commonly u ed treatment for men who have recurrent pro tate cancer. It c
an be utilized in one of three way . Fir t, a what i commonly referred to a a
djuvant therapy, beam radiation can be u ed immediately following urgery to rad
iate the pro tate bed in men at high ri k for cancer recurrence ay, with a Glea

on core of 8 to 10. At lea t ten tudie have ugge ted that thi therapy may r
educe, or at lea t delay, the chance of recurrence of cancer.14 Radiating the p
ro tate bed after urgery, however, carrie with it an inevitable increa e in in
continence,
IF THE CANCER SPREADS OR COMES BACK
161
Intermittent Hormone Therapy I wa fir t diagno ed with pro tate cancer at the t
ender age of 36. Sub equently, . . . I went through the conventional treatment
of radical pro tatectomy and . . . radiation therapy to the pro tate bed. . . .
Five year later in 1989 I wa faced with a new problem: It became apparent that
my PSA . . . wa ri ing ignificantly. . . . The conventional wi dom at the tim
e wa for me to undergo immediate ca trationa more than terrifying pro pect e pec
ially for a p ychiatri t like my elf with ome background in tudying Freud. . .
. [A urologi t ugge ted intermittent hormone therapy.] In tead of my facing a
permanent blockade of te to terone, thi protocol la t for only nine month at
a time, followed by two year of normal te to terone level . Hi recommendation
ha proved to be objectively correct. Friend and colleague who underwent perma
nent ca tration when facing a imilar bind at the ame time have all ince died.
Paul Steinberg, Safety in Number , Wa hington Po t, Augu t 24, 2004
impotence, rectal bleeding, and other ide effect . And ince it i not known wh
ich cancer are going to recur, many men are unnece arily irradiated. The econ
d u e of beam radiation for cancer recurrence i called alvage radiation therap
y. It i utilized once the PSA ri e , indicating that recurrence ha taken place
. The radiation i directed at the pro tate bed, where it i a umed ome pro ta
te ti ue i growing. Thi approach ha become increa ingly popular a the r t ch
oice for treatment when cancer recur after urgery, with many men aving hormon
e therapy for later u e if needed. Although no tudie have yet demon trated tha
t alvage beam radiation prevent the development of meta ta e or lengthen lif
e, everal tudie have reported promi ing re ult : one large tudy found that 4
5 percent of the men
162
IF THE CANCER SPREADS OR COMES BACK
I God Be otted with Irony? I do get profoundly ad thinking of what ha been an
d i no longer: people, place , thing . And in my art Ive tried again and again t
o find way to hold on to the preciou in the pre ent, to memorialize what i e
ential to me a an ob erver of my time, to make hi tory come alive o that oul
long dead will till have meaning. Indeed, i it not wonderfully ironic that I
worked for twenty- ix year writing a book which inve tigate a uicide, while
during the pa t eight and a half year Ive been fighting to tay alive becau e my
pro tate cell refu e to commit uicide (which i what healthy cell do)? I Go
d be otted with irony? Gordon Sheppard, a Canadian writer with meta tatic pro tat
e cancer, The Wondrou World of Pro tate Cancer, unpubli hed e ay
had no progre ion of their cancer during the four-year follow-up period. Not u
rpri ingly, the men mo t likely to re pond to alvage beam radiation are tho e w
ho have a low Glea on core, who have a prolonged PSA doubling time, and who beg
an treatment immediately after their PSA ro e.15 The third type of beam radiatio
n for pro tate cancer recurrence i palliative therapy. It i u ed for cancer t
hat have pread locally or meta ta ized to bone or other organ . The intent i
to hrink the tumor and thereby provide relief of pain. Such radiation i common
ly u ed in the late tage of the cancer. Hormone therapy and beam radiation are
the mo t commonly u ed treatment for recurrent pro tate cancer. Surgery i lit
tle u ed except in a few center , becau e removal of the pro tate after beam or

 eed radiation therapy i technically very dif cult and carrie a high incidence o
f incontinence and other ide effect . Seed radiation therapy ha been tried fol
lowing beam radiation or cryotherapy failure, but the re ult have not been prom
i ing. Cryotherapy i ometime
IF THE CANCER SPREADS OR COMES BACK
163
utilized for recurrent cancer following eed radiation, but it u e following be
am radiation or urgery ha not been encouraging. In a e ing all the e econda
ry treatment , we need to carefully weigh po ible bene t again t the virtually c
ertain eriou ide effect . Chemotherapy i often tried in the late tage of r
ecurrent pro tate cancer but with limited bene t. It i relatively ineffective for
pro tate cancer, in contra t to many other human cancer . Chemotherapy work be
t on cancer cell that are dividing very rapidly, and pro tate cancer cell div
ide lowly compared to other cancer . The mo t promi ing chemotherapeutic regime
n for pro tate cancer to date ha been docetaxel (Taxotere), a drug that ha bee
n u ed to treat brea t cancer, in combination with e tramu tine (Emcyt) or predn
i one. In late 2004 the e combination were hown in clinical trial to lengthen
the life of men with advanced pro tate cancer by approximately two month .16 Th
i wa the r t time that any chemotherapeutic agent had been demon trated to hav
e an effect on pro tate cancer urvival. Many other drug are being te ted, incl
uding etopo ide (VePe id), mitoxantrone (Novantrone), paclitaxol (Taxol), carbop
latin (Paraplatin), and vinbla tine (Velban). Generally the e drug are u ed in
combination with each other or with hormone therapy. Chemotherapeutic agent do
have igni cant ide effect : hair lo ; fatigue; nau ea and vomiting; bone marrow
uppre ion, cau ing anemia and increa ed u ceptibility to infection; and alle
rgic reaction . E tramu tine, one of the mo t widely u ed drug , can al o cau e
blood clot , which can be fatal. Mo t of the e drug mu t be given intravenou ly
every three to ix week . Some men with advanced pro tate cancer decide to enro
ll in clinical trial of experimental drug other than chemotherapeutic agent .
Such trial are u ually carried out at univer ity medical center in three pha e
: pha e I a e e whether the drug i afe to be given to human , pha e II a
e e it ef cacy do e in a few men, and pha e III te t it ef cacy in a large numb
er of men. Among the mo t promi ing ongoing clinical trial are tho e te ting va
ccine that tell the body immune y tem to attack the cancer. In 2005 the re ult
of a trial of one vaccine, Provenge, appeared to be moderately hopeful. Other
drug being tudied are tho e that inhibit growth factor , cut off the cancer bl
ood upply (angiogene i inhib164
IF THE CANCER SPREADS OR COMES BACK
itor ), promote the death of cancer cell (apopto i ), and inhibit peci c enzyme
needed by the cancer cell (for example, cyclooxygena e inhibitor uch a COX2).17 The be t way for men to tay current on the e tudie i by peru ing the w
eb ite recommended in Appendix C, e pecially tho e marked with a teri k . It ha
proven to be relatively dif cult to per uade men with advanced pro tate cancer t
o enroll in clinical trial . It wa e timated in 2003 that four time more women
(34,757) were enrolled in brea t cancer trial than men (8,309) in pro tate can
cer trial .18 A major rea on i that mo t men with advanced pro tate cancer are
older than mo t women with advanced brea t cancer, and they are not a willing t
o try to extend their live for a few additional month . Such trial , however, a
re e ential to nding new and better treatment . Men who wi h to identify ongoing
clinical trial for which they may be eligible can do o by going to the web it
e of the National Cancer In titute (cancer.gov) and clicking on Clinical Trial . The
y can al o acce thi information directly by going to a web ite developed by t
he National Library of Medicine, clinicaltrial .gov (type in Pro tate Cancer and you

r city). Another ource of information on clinical trial i a commercial web it

e, centerwatch , which include a li t of drug trial being pon ored by pharmac
eutical companie (click on Oncology, then on Pro tate Cancer).
WEIGHING QUANTITY VERSUS QUALITY OF LIFE Pro tate cancer ha the reputation of b
eing a low-growing and indolent form of cancer. An oft-repeated aying i that
many more men die with pro tate cancer than die from pro tate cancer. For every
hundred men diagno ed with pro tate cancer thi year, only thirteen will die. We
are lulled into thinking of pro tate cancer a a rather benign male rite of pa
age into old age. Fortunately, for the majority of men, it i . But for a minori
ty of men who get pro tate cancer, it i anything but a benign rite. In 2005 it
killed more than thirty thou and men in the United State , accounting for 10 per
cent of all male cancer death ( econd only to lung cancer).
IF THE CANCER SPREADS OR COMES BACK
165
Di cu ion of Death Nothing i more inevitable than my death; nothing i more fi
nal, irrever ible, or irrevocable. For tho e fortunate enough to have the time t
o contemplate their own death , a I have, nothing i more preoccupying. Very fe
w people eem willing to di cu or even think about the e thing . Morbid, they ay,
a they change the ubject. It ha nt been my good fortune to find other willing
to hare their thought and feeling about their own death . Chuck Wheeler, Affi
rming the Darkne
Pro tate cancer death are u ually not plea ant death . Meta ta e of the cancer
to bone can cau e bone pain that may become evere and i often wor e at night
. Fracture of bone econdary to the meta ta e are not uncommon; if the fractu
red bone i a vertebra, it may cau e compre ion of the pinal cord, a true medi
cal emergency. The enlarging cancer may al o block the urethra or the ureter , p
roducing kidney failure. Weight lo , anemia, and extreme fatigue are common. St
udie of men with pro tate cancer in the la t year of life have reported a low
but teady decline in the quality of life throughout the entire twelve month .19
Men who have derived a much bene t a they are likely to get from the multiple f
orm of pro tate cancer treatment hould addre four ta k . The r t i to put th
eir bu ine affair in order, including writing or updating a will, drawing up
a living will in ca e they become mentally incapacitated, and perhap de ignatin
g omeone to have power of attorney. The econd ta k i to thoroughly di cu th
e i ue of pain control with their phy ician . Undermedicating patient with adv
anced cancer ha been a evere problem in the United State . Opiate , nerve bloc
k , and pot beam radiation to treat bone meta ta e hould be readily available
for the relief of pain. Injection of trontium-89
166
IF THE CANCER SPREADS OR COMES BACK
Developing a Style When youre ill you in tinctively fear a dimini hment and di fi
gurement of your elf. It that, more than dying, that frighten you. Youre going t
o become a mon ter. I think you have to develop a tyle when youre ill to keep fr
om falling out of love with your elf. It important to tay in love with your elf
. That known a the will to live. And your tyle i the in trument of your vanit
y. If they can afford it, I think it would be good therapy, good body narci i m
, for cancer patient to buy a whole new wardrobe, mo tly elegant, ca ual clothe
. Anatole Broyard, Intoxicated by My Illne
(Meta tron), a radioactive i otope that i electively taken up by cancer cell
in bone , can be highly effective in alleviating pain; it take four to ix week
to take effect, but may provide relief for up to ix month . It i crucial to

become knowledgeable about pain control before you need it and to convey your wi
he to your phy ician clearly and conci ely. If you think they are not hearing
you, put your wi he in writing. The third ta k i to a e your upport netwo
rk. Studie have hown that married men decline more lowly than unmarried men.2
0 Carefully think through the ource of your own phy ical and moral upport. Vi
iting nur e and ho pice can be extremely helpful in thi regard and are tron
gly recommended. Part of your moral upport i being able to di cu death with
other , but tho e clo e t to you often avoid the ubject. Your pro tate cancer
upport group or member of the clergy can often be helpful in thi regard, a ca
n book on death and dying. Death and Dying by Eli abeth Kubler-Ro , How We Die
by Sherwin Nuland, and Final Gift by Maggie Callahan and Patricia Kelley are w
idely u ed. Book on cancer in general, uch a Stephen Her h Beyond Miracle : L
iving with Cancer, contain much u eful information on the late tage of
IF THE CANCER SPREADS OR COMES BACK
167
Dying with Style In July 2005, Jame Smith died at age fifty-five of pro tate ca
ncer. Since he wa a devout Pitt burgh Steeler football fan, the viewing at the
funeral home had Smith body in a recliner in front of a TV that played continuo
u Steeler highlight film . He wa dre ed in pajama and bathrobe in Steeler c
olor , had a Steeler blanket on hi lap, a beer and pack of cigarette at hi i
de, and had the TV remote in hi hand. Smith i ter noted that it wa ju t like he
wa at home. Wa hington Po t, July 7, 2005
cancer and dying. The two be t book on dying from pro tate cancer are Chuck and
Martha Wheeler Af rming the Darkne and Anatole Broyard cla ic Intoxicated by M
y Illne . The nal ta k i to carefully weigh the quantity of your remaining mont
h or week again t their quality. Broyard urged men to develop a tyle when facing
death. How people die i their urvivor nal memory of them; it ha been called love
at la t ight.21 Corneliu Ryan, facing death from pro tate cancer, elected to ght
it: I may have a rendezvou with death at ome di puted barricade. The econd to la
t word i important: the word di puted. Come tomorrow, every in tinct, every nerve,
every fiber in my body ha now got the me age, I hope that even in ane the ia
my mind will di pute that barricade. I will even di pute the Man Up tair about
itif I have to.22 Charle Neider, by contra t, cho e to accept it: But I wa read
y to go, if need be. Only a young man could have written Dylan Thoma Rage, rage a
gain t the dying of the light. Raging in thi context trike me, at eventy-eight
, a gracele . I admire Socrate , who drank hi cup of hemlock with tyle, and
168
IF THE CANCER SPREADS OR COMES BACK
Robert Falcon Scott, who died quietly and bravely in the tent on the va t Ro I
ce Shelf in Antarctica.23 It i not important what tyle you choo e, merely that
you choo e. For men in whom pro tate cancer appear to have won, it i a la t c
hance for both a per onal victory and a victory over the cancer.
C H A P T E R
12
What I Known About the Cau e ?
nce a man ha been diagno ed with pro tate cancer, he inevitably a k him elf: W
hat cau ed it? Per onally, I very much wanted to know. Saying that I had a di ea
e that trike randomly, like a bolt of lightning, wa not very ati fying. It
would be better if I could under tand it antecedent , even if my own behavior h
ad omehow contributed to it cau e. Yet when I began earching the medical lite

rature for an wer , I wa di appointed. A 2003 review of the ubject tated that
the etiology [cau e] of pro tate cancer remain virtually unknown.1 Given that a fe
deral war on cancer had been declared in 1971, thi a e ment wa di quieting. Here
we are, thirty- ve year later, knowing little more than we knew then about the m
o t common cancer to affect American men.
O
CLUES In earching for cau e of pro tate cancer, cienti t have major clue to
work with. Seven of the mo t important are the following: 1. It increa e with
age. Pro tate cancer i the mo t age-dependent of all human cancer . It i very
uncommon in young men; almo t three quarter of ca e
170
WHAT IS KNOWN ABOUT THE CAUSES?
A Brief Hi tory of Pro tate Cancer The fir t ca e of pro tate cancer in the medi
cal literature wa publi hed in London in 1817. Pro tate cancer wa aid to be a v
ery rare di ea e in 1853, and a total of only fifty ca e had been de cribed world
wide a late a 1893. During the fir t half of the twentieth century, the incide
nce of pro tate cancer increa ed markedly until it became the mo t common male c
ancer in Europe and North America. Part of the rea on for the dramatic ri e i t
hat men are living longer, and pro tate cancer increa e with age. Thi account
for only ome of the increa e, however; the other rea on are unknown. In recen
t decade , the incidence appear to have leveled off.
are diagno ed after age 65. However, when pro tate ti ue i randomly examined f
rom younger men who died from other cau e , early ubclinical, cancer-like chang
e , often micro copic in ize, are commonly found. In a tudy of autop ie in De
troit, 30 percent of men in their 30 and 50 percent of men in their 50 howed
ome early, cancer-like change in their pro tate .2 So apparently whatever init
iate the cancer-cau ing proce doe o many year before the cancer manife t
it elf clinically. Thi ugge tion wa trengthened by a 2005 tudy reporting th
at a man PSA level in hi 30 predict hi chance of later developing pro tate
cancer.3 2. It true incidence in the United State ha apparently not changed o
ver the pa t three decade . In the early 1990 , there wa great intere t in what
appeared to be a harp increa e in the incidence of pro tate cancer. It i now
evident that thi apparent pike wa due to the increa ingly wide pread u e of t
he pro tate peci c antigen (PSA) blood te t, introduced in 1986, which wa detect
ing more early ca e . Allowing for the increa e expected with an aging populatio
n, the actual age-corrected incidence of pro tate cancer in the United State do
e not appear to
WHAT IS KNOWN ABOUT THE CAUSES?
171
Table 9. Ethnic Difference in Pro tate Cancer in the United State , 2004 (age a
dju ted per 100,000 population) Incidence rate African American White Hi panic A
ian American Native American
SOURCE:
Death rate 73 30 24 14 22
272 164 137 100 54
Cancer Fact and Figure , 2004 (Atlanta: American Cancer Society, 2004).
have changed in recent decade . Thi fact ugge t that whatever i cau ing the

cancer ha not changed either. 3. There are marked difference in incidence amon
g ethnic group in the United State . Ethnic difference in the incidence of pro
tate cancer are among the greate t for any form of cancer. The incidence and de
ath rate (mortality) for 19962000 are hown in Table 9. Thu , African American
have an incidence of pro tate cancer twice a high a Hi panic and ve time a h
igh a Native American , although all are minority group . Minority group tatu
per e doe not eem to explain the difference in incidence. The incidence rat
e for African American i 66 percent higher than for white . The comparable rat
e for 1973 1977 al o how a 66 percent difference, indicating that the di parity
did not change between the 1970 and the 1990 .4 Po ible rea on for the high
incidence of pro tate cancer among African American men have been widely debated
; ugge tion have included gene , diet, and te to terone level.5 Their high dea
th rate i more under tandable, ince African American men are le likely to ge
t PSA te ting or rectal exam and have le acce to pecialized medical profe
ional .6 However, even when African American and white men have equal acce t
o care, a in the Veteran Admini tration medical y tem or with prepaid medical
plan , tudie report that African American till go to their phy ician with
more advanced tage of pro tate cancer.7 Whether thi di crepancy i due to a r
eal difference or to a greater reluctance among African American to eek approp
riate care remain to be a certained. The remarkably low rate of pro tate cancer
among Native
172
WHAT IS KNOWN ABOUT THE CAUSES?
Table 10. Pro tate Cancer Death Rate per 100,000 Men (age adju ted, for the yea
r 2000) Trinidad and Tobago Sweden Norway Denmark Cuba Ireland New Zealand Chile
France United Kingdom Germany Au tralia United State Canada Mexico Croatia Spa
in Greece Ru ia Japan Kazakh tan Turkmeni tan China
SOURCE:
32.3 27.3 26.8 23.1 22.1 21.6 21.2 19.9 19.2 18.5 18.4 18.0 17.9 17.1 16.6 15.3
15.0 10.7 6.8 5.5 5.2 1.8 1.0
Cancer Fact and Figure , 2003 (Atlanta: American Cancer Society, 2004).
American one third the rate of white and one fth the rate of African American ha
received relatively little attention from re earcher . The low pro tate cancer r
ate among Ala kan native ha been con rmed in everal tudie over the pa t halfcentury and i upported by report of very low Arctic-area rate among Inuit me
n in Greenland and Sami (Lapp) men in northern Norway.8 Given the unu ual diet
of the e group , they would eem to be worth inve tigation by pro tate cancer re
earcher . 4. There are marked difference in the incidence of pro tate cancer i
n different countrie . Although acce to health care differ among countrie , t
he incidence of pro tate cancer varie markedly even among countrie with imila
r acce . The highe t rate are for Scandinavian countrie
WHAT IS KNOWN ABOUT THE CAUSES?
173
We mu t be doing omething right!
(Sweden, Norway, Denmark) and Caribbean countrie (Trinidad and Tobago, Cuba). C
on rmation of the high rate in the latter came from a tudy in Jamaica that report
ed an incidence of pro tate cancer among the highe t in the world.9 The high rat
e of pro tate cancer among African American and African Caribbean rai e the q
ue tion of the rate in African nation . A limited number of tudie have been do
ne, but tho e ugge t intermediate rate compared to other countrie .10 At the l

ower end of the incidence pectrum are countrie uch a Japan and China, who e
rate have been veri ed in everal tudie . There i ome evidence, however, that
the incidence of pro tate cancer i increa ing in A ia; in both Japan and Singap
ore, the rate doubled between 1978 and 1997.11 In ummary, national difference
in pro tate cancer death rate vary more than thirtyfold. Such ma ive differenc
e are found in few other di ea e except tho e that are known to be cau ed by i
nfectiou agent . 5. When men move from a low-incidence country to a high-incide
nce country, their ri k of getting pro tate cancer increa e . Studie of migrant
continue to intrigue re earcher . Japane e immigrant to the United State hav
e an incidence of pro tate cancer four time higher than Japane e men in Japan.
Whether the Japane e
174
WHAT IS KNOWN ABOUT THE CAUSES?
men emigrate to the United State early in life or later in life doe not eem t
o make a difference. Japane e American and Chine e American men who were born in
the United State have twice a high an incidence of pro tate cancer a Japane
e and Chine e men who are immigrant . Similarly, Chine e immigrant to Au tralia
have pro tate twice a large, a mea ured by ultra ound, a tho e of Chine e m
en in China.12 6. Dog are the only mammal be ide human that frequently devel
op pontaneou pro tate cancer. Pro tate cancer in dog i imilar to that in hu
man in many way . It i age dependent and become clinically manife t a cancer
at a time corre ponding to human cancer.13 Dog pro tate gland how imilar can
cer-like change in younger dog . Finally, pro tate cancer in dog ha a tendenc
y to pread to lymph node and bone , ju t a it doe in human . One of my colle
ague , on reading a draft of thi book, a ked whether anything i known about th
e relative incidence of canine pro tate cancer in different countrie . Ala , the
an wer i apparently no. 7. The incidence of pro tate cancer among men with ch
izophrenia i low. Five tudie of cancer incidence among individual with chiz
ophrenia in Denmark, Finland, and I rael have reported an unu ually
WHAT IS KNOWN ABOUT THE CAUSES?
175
low rate of pro tate cancer; in the Scandinavian tudie , the rate wa approxima
tely half the rate expected. A follow-up tudy ugge ted that antip ychotic medi
cation may exert a protective effect.14 Gene , diet, and decrea ed expo ure to o
ther po ible cau ative agent hould al o be con idered. Given the known fact ,
what might be cau ing pro tate cancer? In recent year , ve major theorie have r
eceived the mo t re earch attention. Let u now con ider each of them.
GENES It i clear that gene play a role in cau ing pro tate cancer, ju t a the
y are thought to play a role in cau ing mo t other form of cancer. Family tudi
e , e pecially of twin , how that if your brother or father ( r t-degree relative
, who hare half your gene ) or uncle or grandfather ( econd-degree relative ,
who hare a quarter of your gene ) had pro tate cancer, your chance of getting
it are approximately twice a high a if they did not. An affected brother incre
a e your ri k a little more than an affected father, and an affected brother or
father increa e your ri k a little more than an affected uncle or grandfather.
15 If two of your r t-degree relative had pro tate cancer, your chance of getti
ng it are ve time greater than if none of them had it. If three r t-degree relati
ve had pro tate cancer, your chance of getting it are ten time greater. In on
e family, ve out of ix brother developed pro tate cancer.16 Such family clu ter
are rare, but when they occur, the cancer tend to appear at a young age. If y
our family member had cancer in organ other than the pro tate, doe that incre
a e your ri k? Some tudie have ugge ted that having brea t or brain cancer in
the family increa e your ri k of pro tate cancer, but not all tudie agree. T

he pro tate cancer ri k appear to be greater if your female relative had the ra
re, genetic-type brea t cancer cau ed by BRCA gene , which are known to predi po
e to brea t cancer. Studying cancer in twin i another way to look for genetic
clue . Since identical twin hare the ame gene but fraternal twin hare onl
y half their gene , like all other brother , one would expect pro tate cancer to
appear in both identical twin more often than in both
176
WHAT IS KNOWN ABOUT THE CAUSES?
fraternal twin if gene play a major cau ative role. And that i what we nd. Stu
die performed in Sweden, Denmark, and Finland, where national twin regi trie e
nable uch re earch, how that if the r t twin ha pro tate cancer, the econd tw
in in a fraternal pair will get it 3 percent of the time, but the econd twin in
an identical pair will get it 18 percent of the time.17 The e tudie how that
gene play ome role in cau ing pro tate cancer. Re earcher have tried hard to
identify gene that might be involved and have reported u piciou candidate gene
on almo t every chromo ome. Example include the RNASEL gene on chromo ome 1, th
e MSR1 gene on chromo ome 8, and the ELAC2 gene on chromo ome 17. Evidence linki
ng the e peci c gene to pro tate cancer, however, i till rather weak. Another
type of genetic abnormality, the fu ion of two gene , wa reported in late 2005
to occur in 80 percent of pro tate cancer but not in normal pro tate ti ue; th
e que tion remain whether thi i a cau e of the cancer or an effect. The late
t trend in genetic re earch i to look at polymorphi m of candidate gene . If
a gene were a car, a polymorphi m would be a car with a dented fender. It i a p
articular variant of a gene and help determine how active the gene i . Polymorp
hi m may be inherited and have therefore been invoked to try to explain why om
e ethnic group are more predi po ed than other to pro tate cancer. Gene being
clo ely tudied for polymorphi m include tho e regulating te to terone and oth
er androgen that, a will be explained hortly, may play a igni cant role in pro
tate cancer. An example of a recently reported gene polymorphi m thought to be
a ociated with pro tate cancer i the KLF6 gene, which function to uppre ce
ll growth. A recently ummarized by one re earcher, the evidence point toward a
much more complex genetic ba i of pro tate cancer than initially anticipated.18 G
ene certainly play ome role, but the role may be more mode t than many re earc
her originally expected. Some inve tigator have concluded that only 5 to 10 pe
rcent of pro tate cancer have a hereditary ba i .19 The fact i that among iden
tical twin pair , who hare the ame gene , only one twin get pro tate cancer i
n the va t majority of ca e . And men who migrate from low-ri k countrie to hig
h-ri k countrie rapidly increa e their ri k without changing their gene . Nonge
netic factor are obviou ly very important.
WHAT IS KNOWN ABOUT THE CAUSES?
177
VIRUSES AND OTHER INFECTIOUS AGENTS The infectiou theory of pro tate cancer wa
fa hionable twenty year ago, but mo t contemporary book do not even mention i
t. The neglect of thi line of re earch i urpri ing, ince approximately 15 pe
rcent of all cancer worldwide are cau ed by infectiou agent . For example, Hel
icobacter pylori bacteria i a ociated with tomach cancer, hepatiti B viru w
ith liver cancer, human papillomaviru with cervical cancer, Ep tein-Barr viru
with na opharyngeal cancer, and human T-lymphocyte viru with ome leukemia and
lymphoma . Pro tate cancer i al o a type of cancer that increa e in incidence
in individual who e immune y tem i uppre ed; thi correlation i con i ten
t with an infectiou proce . Furthermore, when biop ie of pro tatic ti ue are
examined under the micro cope, in ammation i frequently pre ent, con i tent with
infection. For all the e rea on , infectiou agent hould be eriou ly con ide
red a po ible cau e of pro tate cancer. The 1970 aw much intere t in herpe

 implex viru in pro tate cancer. In 1973, for example, a re earch group at the
Univer ity of Florida reported nding particle of thi viru in pro tate cancer
cell .20 Sub equent report have been both po itive and negative, with the latte
r predominating. Since 2002 there ha been a re urgence of intere t in viru e a
po ible cau e of pro tate cancer. Cytomegaloviru , Ep tein-Barr viru , human
herpe viru 8, human endogenou retroviru E, and human polyomaviru e have all
been reported in pro tate ti ue. It ha been aid that the pro tate i a complex
habitat where mixed infection with oncogenic [cancer cau ing] DNA viru e freq
uently occur and [thi ] open the di cu ion to the potential role of the e viru
e in the cancer of the pro tate.21 Drawing the mo t attention a a candidate for
cau ing pro tate cancer i the human papillomaviru (HPV). Thi viru i known
to cau e many ca e of cervical cancer in women a well a ome cancer of the p
eni and rectum in men. To date, more than twenty tudie have a ked whether HPV
cau e pro tate cancer; the re ult have been contradictory, with the majority
being negative.22 One dif culty in doing re earch on thi viru , however, i that
there are at lea t one hundred different human ubtype of HPV.
178
WHAT IS KNOWN ABOUT THE CAUSES?
The big problem with re earch on viru e and other infectiou agent i a certai
ning cau e and effect. Infectiou agent can be found in many bodily ti ue , bu
t the fact that they are found there doe not nece arily mean that they are cau
ing cancer.
SEXUAL ACTIVITY The idea that pro tate cancer may be cau ed by peci c form of e
xual activity ha a puritanical appeal, e pecially for people who believe that
ex wa be towed on humankind exclu ively for procreation, not recreation. Conver
ely, the idea that pro tate cancer i cau ed by exual activity may al o appeal
to men inclined toward hedoni m, cancer thu becoming for them a badge of a lif
e well lived, a paean to Priapu . The po ibility that certain form of exual a
ctivity may lead to pro tate cancer ha been downplayed by many. One expert clai
med that there i no good rea on to believe that having an active ex life could
timulate the pro tate to grow, or cau e pro tate cancer.23 Another wrote that tudie
attempting to demon trate a link between pro tate cancer and variou exual i
ue have univer ally truck out.24 The e conclu ion are urpri ing, given the nu
mber of tudie that have reported a ociation between peci c exual activitie
and the development of pro tate cancer. One example i exually tran mitted di e
a e . Between 1971 and 2000, thirty-eight tudie on STD and pro tate cancer we
re publi hed. One ummary of thi re earch concluded that the data ugge t an elev
ated relative ri k of pro tate cancer among men with a hi tory of exually tran
mitted infection . Another rea oned that the available epidemiological evidence doe
upport a po ible link between STD and pro tate cancer. Some of the tudie rep
orted having had ex with pro titute a a ri k factor. Other focu ed on the li
fetime number of female exual partner . For example, a tudy of 753 men with pr
o tate cancer in Seattle reported that ri k e timate increa ed directly with a li
fetime number of female exual partner ; men with fteen or more partner lifetime h
ad approximately twice the ri k of developing pro tate cancer a men with only o
ne partner.25 Not all tudie have, however, con rmed the e re ult , and ome cont
radict each other. Some report an early age of r t intercour e to
WHAT IS KNOWN ABOUT THE CAUSES?
179
I Wouldnt Change a Thing It not unnatural for the patient to think that it ex th
at i killing him and to go back over hi amatory hi tory for clue . And of cour
e thi i plendid material for peculation, both lyrical and ironical. Im tempt
ed to ingle out particular women and particular practice that trike me now a

more likely to be carcinogenic than other . Coitu interruptu , which wa widel

y practiced before the Pill, eem a likely u pect, and oral ex come to mind
a putting greater train on the pro tate. But after aying thi , I want to make
clear that I certainly dont hold my cancer again t the e women whatever I did, it
wa worth it. I have no complaint in that direction. I wouldnt change a thing,
even if I had known what wa coming. Anatole Broyard, Intoxicated by My Illne
be a ri k factor, imilar to the nding for cervical cancer in women, but other
tudie do not corroborate thi . Nor do re earcher agree on what hould be mea u
red: frequency of intercour e? frequency of ejaculation? frequency of ex in pe
ci c age period ? number of partner ? number of extramarital affair ? epi ode of
STD in the man? STD in the partner ? expo ure to pro titute ? u e of condom ?
A po ible problem i bia in data collection: Do men with pro tate cancer exagg
erate their exual activity when a ked about it retro pectively, after they have
been diagno ed with cancer? A 2005 tudy of thi que tion reported that men do
not exaggerate the age of their r t exual activity or the lifetime number of the
ir exual partner .26 Another problem ha been reliance on a ingle tudy of Cat
holic prie t that reported their pro tate cancer rate to be lightly higher tha
n that of nonprie t control .27 Under the a umption that prie t are celibate,
thi tudy ha been widely cited a having proven that exual activity i not a
cau e of pro tate cancer. There are way around the e methodology problem . For
180
WHAT IS KNOWN ABOUT THE CAUSES?
example, in one tudy howing that having had yphili or gonorrhea increa ed ch
ance of getting pro tate cancer, re earcher al o collected blood pecimen fro
m the ubject and con rmed their hi tory of yphili by mea uring antibodie in t
he blood.
HORMONES In 1941 Charle Huggin demon trated that blocking the production of te
to terone low the growth of pro tate cancer (a di cu ed in Chapter 5). Sixt
y- ve year after Huggin nding , we are not much further ahead; we are till blocki
ng te to terone to low the growth of pro tate cancer, e pecially in ca e when
the cancer ha pread. Additional evidence that te to terone i involved in cau
ing pro tate cancer come from a variety of ource . Rat given high do e of te
to terone develop pro tate cancer. Eunuch and other male who are ca trated pr
ior to puberty do not produce te to terone and do not develop pro tate cancer. M
en with cirrho i of the liver, a condition that increa e their female ex horm
one, which uppre e te to terone, have a lower incidence of pro tate cancer. T
he cumulative evidence trongly ugge t a role for te to terone. Although te to
terone, made largely by the te te , i the mo t important of the male ex hormo
ne (called androgen ), it i metabolized in the pro tate to dihydrote to terone
(DHT) by an enzyme. The further metaboli m of te to terone and DHT within the p
ro tate i complex and involve everal other androgen , enzyme , and receptor .
The eventual product i the activation of gene , protein ynthe i , and prolife
ration of cell in the pro tate. Thi proce can go wrong at many place , po i
bly re ulting in pro tate cancer. At lea t twelve tudie have mea ured te to te
rone and DHT in individual with pro tate cancer, but only one wa able to how de n
itely that men with higher erum level of te to terone have a higher ri k of pr
o tate cancer.28 Among the many problem with the e tudie i that we do not yet
know which androgen, enzyme, or receptor i the crucial one to mea ure. Another
dif culty i that mea uring the e hormone in a per on blood may not accurately re e
ct what i going on in the pro tate. The timing of the blood draw i al o import
ant; erum level of te to terone vary during the day from a peak in the morning
, when libido i u ually highe t, to a low point in
WHAT IS KNOWN ABOUT THE CAUSES?

181
the evening. In addition, erum te to terone level may be affected by urgery,
tre , and diet; for example, a high-fat diet increa e te to terone. Mo t impo
rtant, we know that pro tate cancer begin many year before it i detected, o
the critical hormonal change may take place many year before they are mea ured
, perhap during puberty. It ha even been peculated that pro tate cancer begin
during the prenatal development of the pro tate. If, indeed, the critical horm
onal event cau ing pro tate cancer occur during adole cence or earlier, mea uri
ng hormone forty or more year later i unlikely to provide de nitive an wer . It
ha been que tioned whether difference in male hormone may explain the ethnic
difference in the incidence of pro tate cancer. In one tudy of 525 black and
3,654 white , the te to terone level among black were 3 percent higher. A tu
dy of Japane e men, who have a low incidence of pro tate cancer, found a compara
tively low level of DHT and of the enzyme that convert te to terone to DHT. Oth
er tudie have reported virtually no difference in te to terone or a ociated
androgen among ethnic group .29 It thu remain to be een whether ethnic diffe
rence in hormone are real and, if o, whether they are related to ethnic diffe
rence in pro tate cancer.
DIETARY FACTORS Studie on fat and other dietary factor a po ible cau e of
pro tate cancer have been ongoing for over thirty year . Currently uch theorie
are fa hionable, with one author declaring atly that pro tate cancer i an apparen
t ca ualty of the edentary We tern life tyle and it notoriou ly unhealthy dietr
ich in animal fat, proce ed fare, fa t and other junk food, and poor in fre h v
egetable and fruit .30 A major impetu behind dietary theorie of pro tate cancer
i the fact that migrant from low-ri k to high-ri k countrie , a from Japan t
o the United State , rapidly increa e their incidence of pro tate cancer; it ha
been peculated that thi i mo t likely a re ult of their changed diet. The pr
imary evidence u ed to link fat intake to pro tate cancer i tudie howing tha
t countrie in which people eat large amount of
182
WHAT IS KNOWN ABOUT THE CAUSES?
fat are al o countrie with high rate of pro tate cancer. In one tudy of thir
ty-two countrie , for example, the author concluded that mortality from cancer of
the pro tate i highly correlated with total fat con umption.31 To date, at lea t
forty retro pective tudie have been done on fat intake and pro tate cancer. S
ome have examined ubtype of fat, uch a aturated, monoun aturated, and polyu
n aturated. Other have taken into account the ource of the fat, uch a meat,
dairy product , and egg . The geographical a ociation tudie , uch a tho e de
cribed above, have been remarkably con i tent in howing an a ociation between
total fat intake and pro tate cancerwith ome notable exception . E kimo , for e
xample, have a very high fat intake but a low incidence of pro tate cancer. On t
he other hand, pro pective tudie , in which dietary information i collected an
d the ubject are then followed to a certain the incidence of pro tate cancer,
have almo t all been negative. In a Norwegian tudy of 25,708 men followed for m
ore than ten year after dietary information had been collected, no a ociation wa
found between energy-adju ted intake of total fat, aturated fat, mono-un atur
ated fat, or poly-un aturated fat and incidence of pro tate cancer. For uch rea o
n , according to expert in thi eld, the fat-cancer a ociation [ha ] now become m
ore tenuou .32 In addition to fat , exce dietary calcium and zinc have been clai
med to promote pro tate cancer. In both ca e , extremely high
WHAT IS KNOWN ABOUT THE CAUSES?
183

quantitie were needed to produce the a ociation, and other tudie have not co
n rmed the re ult . Zinc i of pecial intere t; the pro tate, for unknown rea on
, contain a higher concentration of zinc than any other organ. All dietary tud
ie have problem : People do not eat fat, calcium, or zinc alone, but a part of
a varied diet containing many food . Fat may promote cancer not becau e of the
ir content but rather becau e of the way the meat i cooked. Fat are al o known
to increa e male ex hormone , o the actual mechani m of cancer enhancement ma
y be hormonal. And perhap mo t important, ince pro tate cancer i thought to b
egin many year before it become manife t, the relevant dietary information may
be what the per on ate many year earlier. In addition to dietary factor that
may promote pro tate cancer, recent attention ha focu ed on food that may prot
ect again t it. The mo t impre ive data have been for tomatoe , on which over a
hundred tudie have been publi hed. Other dietary factor under tudy include
green tea, red wine, oybean , yellow and green vegetable , ber, vitamin A, vitam
in D, vitamin E, and elenium ( ee Chapter 13).
OTHER POSSIBILITIES The ve factor di cu ed above are the leading candidate a
contributor to the cau ation of pro tate cancer. A multitude of other factor h
ave been inve tigated, but none appear to be a promi ing. The mo t eriou atte
ntion ha been given to moking, alcohol, va ectomy, and expo ure to dioxin or c
admium. Smoking. Since moking i a known ri k factor for lung and bladder cance
r , it ha been exten ively tudied for other cancer a well. At lea t ixty- ve
re earch group have examined moking and pro tate cancer, with the majority rep
orting no relation hip.33 Men who moke are more likely to con ume a high-fat di
et, which may explain re earch that ha found a relation hip. A few tudie ugg
e t that moking ha ten the pread of pro tate cancer once it develop , but oth
er tudie have not con rmed thi . Alcohol. Heavy alcohol u e i a ociated with c
ancer of the liver, larynx, and e ophagu . At lea t thirty- ve tudie have exami
ned
184
WHAT IS KNOWN ABOUT THE CAUSES?
alcohol u e and pro tate cancer. The conclu ion of one review wa that moderate al
cohol con umption up to about three drink per day doe not appear to in uence pro
tate cancer ri k.34 Equivocal upport wa found for the po ibility that heavy al
cohol u e (eight or more drink a day) may increa e the ri k. On the other hand,
two tudie of autop y ti ue taken from men who died from cirrho i of the liv
er reported fewer in tance of pro tate cancer than expected. Cirrho i increa e
the production of e trogen , which block te to terone, and thu low the grow
th of pro tate cancer. Severe alcoholi m may therefore decrea e the chance of d
eveloping pro tate cancer. Va ectomy. Surgical cutting of the va a deferentia, t
he duct that carry perm from the te ticle , i a common form of male contracep
tion. Studie carried out in the 1980 and early 1990 ugge ted that men who ha
d had a va ectomy, e pecially if it had been performed when they were relatively
young, were at increa ed ri k for pro tate cancer. Sub equent tudie have refu
ted thi nding; a 1998 review of fourteen tudie , for example, concluded that no c
au al a ociation wa found between va ectomy and pro tate cancer.35 It i now bel
ieved that earlier tudie did not correct for election bia ; men who had had v
a ectomie were more likely to go to urologi t for follow-up, and urologi t in
turn would have been more likely to look for pro tate cancer in the e men. Diox
in. A component of ome herbicide u ed by farmer and al o of Agent Orange, a d
efoliant u ed during the Vietnam War, dioxin ha been linked to a variety of can
cer . Many, but not all, tudie of occupational ri k expo ure have reported tha
t pro tate cancer occur at a di proportionately high rate among farmer . A prel
iminary tudy of Vietnam veteran claimed that men with pro tate cancer were app
roximately twice a likely a men without pro tate cancer to report having been
expo ed to Agent Orange.36 A larger con rmatory tudy i needed. Cadmium. The fact
that a be to cau e me othelioma, a form of cancer, ha led to a u picion tha

t indu trial carcinogen may cau e other cancer . In addition, feeding variou c
hemical to rat can produce pro tate cancer in them. In 1965 it wa reported th
at an exce number of ca e of pro tate cancer had occurred in a factory where
worker were expo ed to cadmium. Thi nding wa e pecially interWHAT IS KNOWN ABOUT THE CAUSES?
185
e ting ince cadmium inhibit zinc, which i in high concentration in the pro ta
te. Exten ive tudie have ub equently been carried out on the incidence of pro
tate cancer among worker expo ed to cadmium with the conclu ion that there i no
indication for an increa ed ri k of pro tate cancer among the worker .37
SUMMARY OF CAUSES De pite having pent almo t $200 billion ince 1971 for a war on
cancer, we know little more about the cau e of pro tate cancer now than when war wa
declared. At lea t even clue hould help direct our re earch.
1 Pro tate cancer i found predominantly in older men. 2 It incidence in the Un
ited State ha apparently not changed in 3
4 5 6 7
recent decade . There are marked ethnic difference in incidence and death rate
in the United State , with the rate for African American being high, for whit
e and Hi panic intermediate, and for A ian American and e pecially Native Ame
rican low. The incidence i very high in Scandinavian and Caribbean nation and
very low in A ian nation ; the United State i intermediate. Men who migrate f
rom low-incidence to high-incidence countrie rapidly acquire the higher prevale
nce of their new country. Other than human , dog are the only mammal that freq
uently get pro tate cancer. The incidence of pro tate cancer among men with chi
zophrenia i low.
Gene play ome role in cau ing pro tate cancer, a indeed they do in cau ing mo
t cancer and mo t chronic di ea e . However, it doe not appear that a man inh
erit gene that directly cau e the cancer; rather, it eem likely that he inhe
rit everal gene that make it more likely that he will get cancer if he i exp
o ed to a factor or factor who e identity i till unknown. Such gene are call
ed predi po ing gene . Infectiou agent hould be carefully tudied a po ible
cau e of pro tate cancer. To date, everal u pect have been brought in for
186
WHAT IS KNOWN ABOUT THE CAUSES?
que tioning but were relea ed owing to lack of rm evidence. They continue to be u
nder u picion. Similarly, ome a pect of exual activity appear to play a role
in cau ing pro tate cancer, perhap through having more exual partner or incr
ea ed expo ure to exually tran mitted di ea e . Male ex hormone undoubtedly p
lay a part in cau ing pro tate cancer, but their peci c role i uncertain. The ho
rmonal change could be a primary cau e of the cancer or a econdary effect of d
iet, infectiou agent , or other primary cau e . Exce dietary fat ha been ext
en ively inve tigated a a cau e of pro tate cancer; recent tudie ugge t that
it i a le likely trigger than previou ly believed. Exce calcium and zinc h
ave al o been tudied a ri k factor , with inconclu ive re ult . It i likely t
hat ome of the e factor interact with other . For example, both dietary factor
and infectiou agent may affect male hormone , which in turn may increa e ex
ual activity and re ult in exually tran mitted di ea e that affect the pro tat
e. In a genetically predi po ed individual, uch infection may re ult in cancer
. The bigge t impediment to di covering the cau e of pro tate cancer i that th
e important event may occur early in life, even decade before the cancer becom

e manife t.
C H A P T E R
13
Factor That May Prevent Emergence or Recurrence
G
iven that one in every ix American men i expected to be diagno ed with pro tat
e cancer during hi lifetime, we might anticipate that major re earch would have
been undertaken to prevent it emergence or recurrence. The National Cancer In
titute and other re earch group neglected prevention re earch for o many year
that today we know remarkably little. Mo t pro tate cancer prevention trial we
re initiated only within the pa t ve year and will therefore not yield u eful da
ta for many year to come. For example, trial of elenium and vitamin E (the SE
LECT trial); betacarotene and vitamin C and E (Phy ician Health Study II); and
the anti-in ammatory drug rofecoxib will not be completed until 2012 or later. Th
eorie about the prevention of pro tate cancer and it recurrence fall into four
categorie : they are ba ed on dietary factor , vitamin and mineral , medicatio
n , and life tyle change . Except for the r t of the e, remarkably few hard data
are available to help men make deci ion about what to do. Indeed, in relatively
few major di ea e do anecdotal data and per onal opinion o outweigh factual
data. The prevention of pro tate cancer i a ubject that generate far more hea
t than light.
188
PREVENTING EMERGENCE OR RECURRENCE
DIETARY FACTORS Dietary factor have been linked to pro tate cancer, both a po
ible cau e of the di ea e and a way to prevent it emergence or recurrence.
Red meat, fat, and exce ive zinc are example of dietary item that are po ibl
e cau e , a di cu ed in Chapter 12; reducing your intake of uch item may red
uce your chance of getting pro tate cancer. Dietary factor linked to po ible
prevention are in a different category. The e food item are not thought to cau
e the cancer but rather to po ibly prevent it emergence or continued growth. M
any author , in di cu ing dietary factor , confu e cau e and prevention, writin
g a if the failure to con ume uf cient preventive dietary item , uch a oy and
green tea, cau e pro tate cancer. Thi i almo t certainly not true; mo t Inui
t E kimo , for example, do not eat oy or drink green tea and yet have a very lo
w incidence of pro tate cancer. The dietary factor that have been mentioned mo
t prominently a po ibly having a preventive action again t pro tate cancer inc
lude the following:
Tomatoe The be t evidence for a food that may low the development of pro tate
cancer i available for tomatoe . Among ixteen tudie carried out to a certain
the relation hip between tomato intake and reduced pro tate cancer, ix found a
igni cant relation hip, three found a trend that wa not tati tically igni cant,
and even reported no relation hip. In a tudy of 47,000 health profe ional ,
men who con umed more than 10 half-cup erving of tomato product per week had a
35% lower ri k of developing pro tate cancer compared to men who never ate tomat
o product . Similarly, among 14,000 Seventh Day Adventi t hou ehold , men con uming
more than 5 erving of tomatoe per week had a 43% lower ri k of pro tate cance
r compared to men who ate le than 1 erving of tomato product per week. Al o im
pre ive are two tudie in which men ate large amount of tomato product in th
e week between being diagno ed with pro tate cancer and having their pro tate
urgically removed; in both tudie their PSA level declined.1
PREVENTING EMERGENCE OR RECURRENCE

189
John Kerry Diet Following Senator John Kerry diagno i of pro tate cancer and u
rgery in 2002, hi wife, Tere a, per uaded him to change hi diet. According to
one account: She ha worked hard, he aid, to educate her hu band to eat martand to
break him of hi habit of eating pa ta, ice cream, and bag of chocolate chip co
okie from the Faneuil Hall market in Bo ton that are full of butter. He alway did
eat alad, but he wouldnt eat cooked green , he aid. Now he alway ha broccoli
and love bru el prout . He al o eat more alad and tomatoe , green pea , le
ntil , bean and other vegetable , he aid. L. K. Altman, New York Time , October
3, 2004
What i not known i the ingredient in tomatoe that i re pon ible for thi app
arent effect. It ha been widely a umed to be lycopene, a plant carotenoid abun
dantly pre ent in tomatoe , but in one animal tudy, tomato powder wa more effe
ctive than puri ed lycopene in hrinking the tumor .2 Studie al o ugge t that to
mato auce, pa te, or cooked tomatoe are more effective a anticancer agent th
an are raw tomatoe . One hould not inve t heavily in tomato-farm tock yet, ho
wever. Dietary tudie are notoriou ly dif cult to carry out becau e people do not
accurately recall what they ate in the pa t. More eriou i that dietary infor
mation in mo t tudie i gathered for men in middle and old age, when in fact t
he critical dietary intake for pro tate cancer development may occur much earlie
r in life. In addition, American are aid to con ume an average of ninety-one p
ound of tomatoe per year, mo tly a pa ta auce, ketchup, pizza, chili, and a
l a. If tomatoe are truly effective in preventing pro tate cancer, why i the d
i ea e o prevalent? Becau e of uch problem , the Food and Drug Admini tration
in late 2005 rejected a reque t by
190
PREVENTING EMERGENCE OR RECURRENCE
tomato product manufacturer to adverti e their product a having cancer-relate
d bene t .
Green Tea Indigenou to Southea t A ia, tea wa introduced into Europe and Ameri
ca in the eventeenth century. Other than water, it became the mo t widely con u
med beverage in the world; it played a igni cant role in the American Revolution,
and it importation made a few men, including John Jacob A tor, wealthy. Worldw
ide, the drinking of black tea (80 percent) far out trip that of green tea (20
percent), but in China the percentage are rever ed. Green tea i made in uch a
way that the chemical compo ition e entially remain imilar to that of fre h
leave , wherea black tea ha a omewhat different chemical compo ition. Except
for tomatoe , the evidence to upport green tea a po ibly preventing pro tate
cancer i tronger than for any other dietary factor. In animal tudie , green t
ea ha been hown both to prevent arti cially induced pro tate cancer and to reduc
e the ize of exi ting
PREVENTING EMERGENCE OR RECURRENCE
191
cancer . The extremely low incidence of pro tate cancer in China, where green te
a i drunk in large quantitie , ha led many to u pect a direct relation hip be
tween the e two fact . In Zhejiang Province, where green tea i widely grown, it
i typically the only nonalcoholic beverage con umed by men, e pecially older men
, throughout their lifetime. Patient with pro tate cancer were compared to patien
t with other di ea e on the frequency, duration, and quantity of green tea con
umption. More control than pro tate cancer patient were green tea drinker (8

0 percent ver u 55 percent); the control al o drank more tea and had been drin
king it for more year . Drinking fre h tea wa e pecially important, o that incre
a ing the number of new batche brewed per day to 2 or more wa a ociated with
a 76% reduced ri k of cancer.3 De pite uch tudie , in 2005 the Food and Drug Adm
ini tration denied reque t to label green tea a an effective cancer prevention
agent. It i uncertain what ingredient of green tea i re pon ible for it po
ible effect on pro tate cancer. Many re earcher u pect it i the polyphenol ,
which have been hown to have antioxidant propertie and al o to decrea e the le
vel of androgen . Studie ugge t that black tea, the kind widely con umed in A
merica and Europe, offer ome degree of pro tate cancer prevention, but not a
much a green tea.
Soy Soybean are indigenou to Southea t A ia and con umed exten ively in Japan
and Korea, mo tly a tofu (bean curd), natto (fermented oybean ), and oymilk.
Soybean contain e trogen-like compound called avonoid , one of which, i o avone,
i thought to have anticancer propertie . Soy product may al o be preventive ag
ent , becau e until recently pro tate cancer wa rare in Japan. It i intere tin
g that the incidence of pro tate cancer increa e harply among Japane e men who
migrate to the United State , where they pre umably eat a modi ed diet that, amon
g other factor , include le oy. I o avone have been tudied in animal model
of pro tate cancer with mixed re ult . In ome but not all tudie , i o avone app
eared to decrea e the on et of cancer or reduce the ize of exi ting
192
PREVENTING EMERGENCE OR RECURRENCE
cancer . Mo t human tudie in which oy intake wa compared for patient with p
ro tate cancer and for control have hown little or no difference; one tudy pu
bli hed in 2004 did how a tati tical difference for the con umption of tofu an
d natto.4 If oy product do have an effect on pro tate cancer, it may be becau
e of their e trogen-like propertie , which would block te to terone.
Red Wine and Red Grape There are ugge tion that red wine and red grape may p
rovide ome protection again t pro tate cancer. A tudy conducted in Seattle com
pared 753 men with pro tate cancer to 703 matched control . In an exten ive diet
ary urvey, the control were found to have con umed more red wine but not white
wine, beer, or liquor. For each gla of red wine con umed per week, there wa
a 6 percent reduction in ri k for pro tate cancer. According to the re earcher ,
con umption of 8 gla e or more of red wine per week igni cantly reduced the rela
tive ri k of more aggre ive pro tate cancer by 61%.5 Like oy, red wine contain a
vonoid that may exert an anticancer effect through their e trogen-like properti
e . Red grape contain re veratrol, a compound clo ely related to avonoid that a
l o ha antioxidant and anti-in ammatory propertie . The e tudie hould be con rme
d before men make major change in their alcohol intake.
PREVENTING EMERGENCE OR RECURRENCE
193
Michael Milken Diet Michael Milken wa a prominent Wall Street financier who wa
impri oned for ecuritie fraud in the 1980 . After hi relea e, he wa diagno
ed in 1993 with pro tate cancer that had pread to hi lymph node . He had hormo
ne therapy and radiation, and began a trict dietextremely low fat and containing
large quantitie of oy, tofu, green tea, antioxidant , and vitamin E. Accordin
g to one account: A typical lunch, prepared by hi private dietitian, con i t of
mu hroom barley oup, a tofu egg- alad andwich (the egg i actually tofu with mu
tard and pice ) with carrot and lettuce, and a black-bean-and-corn alad with
a oy-ba ed drink. One of Milken favorite , an Egg McNothing, con i t of a fatfree crumpet with oy chee e, vegetarian Canadian bacon and crambled egg white

. Milken and hi chef have even publi hed The Ta te of Living Cookbook, pecifical
ly for men with pro tate cancer. In addition to hi diet, Milken u e meditation
, yoga, e ame-oil ma age , and aromatherapy to timulate hi immune y tem. Tw
elve year after being diagno ed, Milken continue to work and do well. Few men,
however, would be willing to follow uch a diet and regimen, and almo t none ha
ve their own per onal dietitian and chef. Leon Jaroff, The man cancer, Time, April 1,
1996
Other A ingle tudy of 1,294 men with pro tate cancer and 1,451 men without can
cer reported that the control had con umed igni cantly more vegetable, but not f
ruit or cereal, ber.6 Dietary ber ha been identi ed a a po ible protective factor
for other form of cancer, peci cally colon, brea t, and ovarian.
194
PREVENTING EMERGENCE OR RECURRENCE
Vegetable uch a cabbage, cauli ower, turnip , broccoli, kale, and bru el pro
ut were linked to the prevention of pro tate cancer in one tudy.7 The effectiv
ene of dietary approache in preventing the emergence or recurrence of pro tat
e cancer i till to be determined. Each man ha to decide what trade-off he i
willing to make, weighing the importance of giving up food he really like and
eating more of ome food he doe not like. What for one man i a en ible, hea
lthy diet may for another man be dietary ma ochi m.
VITAMINS AND MINERALS Vitamin and mineral have attracted much attention a po
ible preventive factor for pro tate cancer, with many web ite featuring them
prominently. But anecdote heavily outweigh fact . The vitamin and mineral tha
t have been mo t tudied in relation to the prevention of pro tate cancer are e
lenium, vitamin E, vitamin A, beta-carotene, and vitamin D. Selenium. Selenium i
a trace metal and a nece ary component of everal enzyme , e pecially one (gl
utathione peroxida e) thought to prevent free-radical damage to cell tructure .
Selenium i thought to work clo ely with vitamin E and i theorized to have ant
ioxidant propertie , enhance immune function, and decrea e te to terone. A longi
tudinal tudy of aging men in Baltimore reported that men with lower level of b
lood elenium were more likely than other to develop pro tate cancer. In contra
diction, however, the area of the world where men are mo t likely to be elenium
de cient i China, where the incidence of pro tate cancer i very low. A large t
udy (SELECT: the Selenium and Vitamin E Cancer Prevention Trial) i under way to
a e elenium and vitamin E a preventive factor for pro tate cancer, but th
e re ult will not be known until 2013. Vitamin E. The main component of vitamin
E i alphatocopherol, which i believed to be an antioxidant that work in conj
unction with elenium. Vitamin E wa previou ly thought to help prevent heart at
tack , but thi property i now in que tion; one tudy even concluded that high
do e of vitamin E may increa e heart attack . A tudy in Finland in which men w
ho were moker
PREVENTING EMERGENCE OR RECURRENCE
195
were given alpha-tocopherol to ee if it would prevent lung cancer found no effe
ct on lung cancer but a 32 percent reduction in pro tate cancer. Other tudie o
f vitamin E and pro tate cancer have yielded highly con icting re ult .8 Fifteen t
hou and phy ician are being tudied in the United State to a e the effect
of vitamin E and C and beta-carotene on the incidence of pro tate cancer, but t
he re ult will not be available until 2012. Vitamin A and beta-carotene. Vitami
n A i e ential for cell in the eye having to do with vi ion. A precur or of v
itamin A, betacarotene i metabolized to a compound that function a vitamin A.
Beta-carotene i clo ely related to lycopene, the ingredient in tomatoe though

t to be re pon ible for their pro tate-cancer preventive effect. Studie linking
vitamin A and beta-carotene with pro tate cancer have been contradictory, with
ome reporting a preventive effect and other claiming an increa e in the incide
nce of pro tate cancer. A trial of beta-carotene to prevent lung cancer re ulted
in an increa e in lung cancer; in recent year intere t in the e compound a c
ancer preventive agent ha markedly decrea ed.9 Vitamin D. Formed in the kin b
y expo ure to unlight, vitamin D i e ential for bone formation. Intere t in v
itamin D and pro tate cancer ari e from epidemiological ob ervation that pro t
ate cancer i , with ome exception , more prevalent in northernlatitude nation
that get le unlight and among per on with dark kin that ab orb le unlig
ht. One tudy reported that high-do e calcitriol, a form of vitamin D, decrea ed
PSA level in patient with advanced pro tate cancer.10 Additional trial are i
n progre .
MEDICATIONS The idea that peci c medication may prevent the emergence or recurre
nce of pro tate cancer aro e primarily from ob ervation on na teride (Pro car).
Thi ub tance block the enzyme that convert te to terone to dihydrote to tero
ne (DHT), thereby reducing the ize of the pro tate. It ha proven e pecially ef
fective a a treatment for benign pro tatic hypertrophy (BPH). A trial of na teri
de in the prevention of pro tate cancer began in 1993 with over eighteen thou an
d men (Pro tate Cancer Prevention Trial). The re ult , publi hed in 2004, howed
that na teride did
196
PREVENTING EMERGENCE OR RECURRENCE
Caution: The Molire Principle Molire, the eventeenth-century French playwright, o
nce wrote: Nearly all men die of their remedie , and not of their illne e . Thi pr
inciple i important to remember when attempting to prevent pro tate cancer. Exc
e ive con umption of elenium may produce abdominal pain, arthriti , emotional
in tability, hair lo , and liver dy function. Exce ive con umption of vitamin
E may increa e a bleeding tendency and the incidence of troke . Exce ive con u
mption of vitamin D may produce kidney damage, and exce ive expo ure to unligh
t a a way of increa ing vitamin D ab orption may lead to kin cancer . Exce iv
e con umption of vitamin A may produce hair lo , elevated blood lipid level , n
eurological ymptom , and liver damage.
indeed igni cantly reduce the prevalence of pro tate cancer but did o at a di tu
rbing price: the po ibility that if cancer i detected, it may be of a higher p
athological grade.11 The reduction in pro tate cancer wa 25 percent, but thi goo
d new wa off et by the bad new that the cancer that did occur were more mali
gnant than expected. Thu , it i not recommended that na teride be u ed to preven
t pro tate cancer. Te t of other medication to reduce pro tate cancer are in p
rogre , including a trial of duta teride, a drug imilar to na teride, in eight
thou and men (the REDUCE tudy). Statin , u ed to lower chole terol, have hown
ome promi e; one tudy reported that tatin u e decrea ed PSA, and another foun
d that it decrea ed the incidence of pro tate cancer. However, in 2006 a large
tudy of tatin concluded that they had no effect on any cancer , including canc
er of the pro tate.12 Al o being inve tigated are drug that decrea e in ammation;
tudie of long-term a pirin u er ugge t that a pirin may lower the ri k of d
eveloping pro tate cancer.13 A tudy of rofecoxib (Vioxx), a cyclooxygena e (COX
-2) inhibitor, wa under way
PREVENTING EMERGENCE OR RECURRENCE
197
until the drug wa withdrawn in 2004 becau e of it cardiac ide effect . Re ear
ch on another COX-2 inhibitor, celecoxib (Celebrex), i planned.

LIFESTYLE CHANGES Life tyle change are recommended for the control and preventi
on of all cancer , including pro tate cancer. Like motherhood, the e recommendat
ion are hard to di agree with. The relation hip between phy ical activity and p
ro tate cancer ha been examined in at lea t twenty-eight tudie , with incon i
tent re ult . Two reported that phy ical activity and exerci e did not reduce th
e incidence of pro tate cancer but reduced the everity of the cancer that devel
oped.14 Weight control i de irable for many rea on , but it effect on pro tate
cancer i ambiguou . One tudy reported that obe e men were more likely to ee
progre ion of their cancer ; another tudy found that cancer wa le likely to
be detected in obe e men becau e their pro tate gland are larger; till anothe
r tudy claimed that middle-aged obe e men have a decrea ed ri k of pro tate can
cer.15 Not moking and drinking alcohol only in moderation are univer ally recom
mended for cancer prevention. Regarding diet, the American Cancer Society advoca
te the following tep to reduce the ri k of cancer. The e guideline are con i
tent with recommendation for preventing heart di ea e, diabete , and other con
dition : Eat five or more erving of vegetable and fruit each day. Choo e who
le grain in tead of proce ed (refined) grain and ugar . Limit con umption of
red meat , e pecially high-fat and proce ed meat . Choo e food that help main
tain a healthful weight. When diet and life tyle change are combined and adhere
d to faithfully, there i evidence that they may low the progre ion of pro tat
e cancer. A controlled tudy, publi hed in 2005, randomized into two group nine
ty-three men who had early- tage pro tate cancer and who had elected watchful wa
iting. The men in one group
198
PREVENTING EMERGENCE OR RECURRENCE
continued their u ual diet and life tyle. Tho e in the other group went on a veg
an diet with oy, h oil, elenium, vitamin C and E, exerci e, tre management
technique , and a weekly group meeting. At the end of one year, the men on the
vegan diet had a 4 percent decrea e in their PSA (from an average of 6.23 to 5.9
8), wherea the diet-a -u ual group had a 6 percent increa e (from an average of
6.36 to 6.74).16
SO WHAT SHOULD YOU DO? Taking into con ideration everything that i known about
the emergence and recurrence of pro tate cancer, what hould you do? Mo t import
ant, realize that remarkably little i known with certainty, and what i unknown
far outweigh what i known. Weigh quantity of life again t quality of life. Ma
king ome dietary or life tyle change after being diagno ed with pro tate cance
r may not be dif cult, wherea you may nd making other change to be quite hard. I
have modi ed my own diet mode tly but not radically. I eat tomatoe in one form or
another with each dinner, and I drink tomato juice with lunch. Red grape are n
ow a taple nack item in our home, alway available. I have not given up red me
at altogether but have continued to decrea e my con umption of it, a trend I had
begun everal year before being diagno ed with pro tate cancer. I increa ingly
drink green tea and may, if I live long enough, come to like it. I am exploring
red wine and have been urpri ed to di cover that many of them, e pecially the
more expen ive one , are very good; my pa t experience wa apparently limited b
y having bought only inexpen ive one . However, for me they will never completel
y replace ne Belgian ale or California porter . I continue to take low-do e a pi
rin and multivitamin each day, a I had been doing for many year , although I
witched from a multivitamin that had upplemental zinc to one without. I topped
taking upplemental vitamin E, ba ed on the mo t recent cardiac tudy, and have
not tarted taking upplemental eleniumalthough I will watch the emerging tudi
e and could be per uaded to do o. Regarding oy and tofu, I draw the line and
will wait until I di cover a ta ty chocolate chip cookie made from oy.
C H A P T E R

14
Science and Politic
ro tate cancer i a major threat to men health. Adult men in the United State h
ave a 1 in 6 chance of being diagno ed with it in their lifetime. Thi compare
with a 1 in 13 chance of being diagno ed with lung cancer, 1 in 17 with colon ca
ncer, 1 in 68 with leukemia, and 1 in 81 with tomach cancer. Once diagno ed wit
h pro tate cancer, 1 of every 5 men will die from it. In 2005 thi tran lated to
an e timated 29,528 death , one every eighteen minute . Since the American popu
lation i aging, the problem will almo t certainly wor en. In 2005, approximatel
y 234,000 men were initially diagno ed with pro tate cancer; in 2025, the number
i projected to be 384,000 men, and in 2045, to be 452,000 men. The projected f
uture co t of pro tate cancer are a tronomical. Given the e number , one might
expect that the United State government would long ago have organized a eriou
effort to uncover the cau e of, and better treatment for, pro tate cancer. In
1971, in hi State of the Union me age, Pre ident Nixon propo ed an inten ive ca
mpaign to nd a cure for cancer. He af rmed that the time ha come when the ame kind of
concentrated effort that plit the atom and took man to the moon hould be turn
ed toward conquering thi dread di ea e. In what ub equently came to be known a
the war on cancer, the federal government poured billion
P
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of dollar into the e effort , with private indu try and donation through organ
ization uch a the American Cancer Society adding more billion . According to
a 2004 e timate of cancer re earch expenditure , American have pent, through tax
e , donation , and private R and D [re earch and development], clo e to 200 bill
ion, in in ation-adju ted dollar , ince 1971.1 Pro tate cancer ha received le th
an it hare of re earch fund and attention. In the mid-1990 , brea t cancer re
earch received approximately even time more in federal re earch fund than pr
o tate cancer, de pite the fact that approximately the ame number of women and
men were o diagno ed each year. By 2005 thi di crepancy had been reduced, o t
hat brea t cancer received approximately twice a much federal funding per diagn
o ed ca e. Pro tate cancer re earch in the United State i upported almo t exc
lu ively by three organization : the National Cancer In titute; the Pro tate Can
cer Re earch Program, under the U.S. Department of Defen e; and the private Pro
tate Cancer Foundation, founded by nancier Michael Milken.
THE NATIONAL CANCER INSTITUTE The National Cancer In titute (NCI) i part of the
National In titute of Health (NIH), located in Bethe da, Maryland. Federally f
unded, it budget for 2006, at more than $5 billion, ha doubled ince 1997. De
pite it ma ive budget, many have criticized the NCI for the ame rea on other
NIH in titute have been criticized: the application proce for re earch grant
i unnece arily cumber ome and lengthy; it focu e too much on ba ic re earch
and not enough on re earch that i likely to help tho e who currently have canc
er; and it i extremely con ervative, o that re earcher who are innovative and
think out ide the box do not get funded. A noted in one analy i of the NCI: S
omehow, along the way, omething important ha gotten lo t. The earch for knowl
edge ha become an end unto it elf rather than the mean to an end. And the re e
arch ha become increa ingly narrow, o much o that phy ician- cienti t who wa
nt to think y temically about cancer or the organi m a a wholeor who might have
completely new approache often cant get funding.2
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201
Another hortcoming of the NCI ha been it lack of leader hip or coordination o
f cancer re earch effort at the national level. Each univer ity and cancer re e
arch center get a piece of the federal budgetary pie and goe off to it own pr
ivate corner to eat it. Nobody ha been putting the piece together, which ha
lowed down progre on pro tate cancer a much a on other form of cancer. Pro t
ate cancer ufferer were hopeful that more progre would be made between 2002
and 2005, when Andrew von E chenbach wa the director of the NCI. He had been di
agno ed with pro tate cancer and hi father had died from it. Changing the cour
e of thi federal behemoth, however, proved to be a dif cult ta k.
THE PROSTATE CANCER RESEARCH PROGRAM The Pro tate Cancer Re earch Program i run
by the U.S. Army under the Department of Defen e. It origin are a cla ic Wa
hington tale. In 1992, women advocacy group for brea t cancer re earch were put
ting pre ure on the federal government to increa e funding. Repre entative Patr
icia Schroeder, at that time, chaired the Hou e Armed Service Committee, over e
eing pending by the Department of Defen e. Since expenditure for the National
Cancer In titute were re tricted by federal regulation , Schroeder arranged to g
ive $25 million to the Army to coordinate additional brea t cancer re earch. Ove
r the next three year , Congre added $390 million more and a ked the In titute
of Medicine to evaluate the program. The evaluating committee publi hed it rep
ort in 1997, calling the Department
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of Defen e brea t cancer re earch a unique and valuable entity, e pecially for it pot
ential to focu on innovation, in way that go beyond what traditional in tituti
on like the National In titute of Health are able to do.3 Ob erving the ucce
of the brea t cancer lobby, advocate for pro tate cancer re earch decided to fo
llow uit. A dinner wa arranged to which, according to one participant, everal m
ember of Congre were invited who ju t happened to have pro tate cancer. In 1997
Congre allocated $45 million to the Department of Defen e for what became kno
wn a the Pro tate Cancer Re earch Program (PCRP). Since 1997 Congre ha conti
nued to fund thi re earch; in 2006, the allocation wa $80 million. Through 200
5, a total of over $600 million had been pent on more than fourteen hundred re
earch project . A prominent part of the program i the Center for Pro tate Di ea
e Re earch, an excellent clinical program for military veteran with pro tate c
ancer directed by Col. David E. McLeod, M.D., at the Walter Reed National Medica
l Center. Another important component i a ti ue bank in which blood and pro ta
te ti ue are collected from men with pro tate cancer and then made available to
re earcher . A pro tate cancer databa e regi try that include more than eleven
thou and men with pro tate cancer i al o available to inve tigator . Whether t
he Pro tate Cancer Re earch Program will ultimately be any more ucce ful than
the National Cancer In titute in funding innovative re earch or making a major b
reakthrough on pro tate cancer remain to be a certained, but to date it i well
regarded by re earcher in thi eld.
MICHAEL MILKENS PROSTATE CANCER FOUNDATION Michael Milken, a umma cum laude grad
uate of the Univer ity of California at Berkeley and a graduate of the Wharton S
chool of Bu ine , ro e to billionaire prominence in the 1980 a the Wall Stree
t king of junk bond , high-yield debt ecuritie . Rudy Giuliani, then a New York pro
ecutor, went after him for ecuritie fraud, and Milken eventually pleaded guil
ty to ix count of violation related to market manipulation.4 He paid $600 mil
lion in ne and
SCIENCE AND POLITICS

203

pent twenty-two month in pri on. Ironically, both Giuliani and Milken were ub
equently diagno ed with pro tate cancer and became friend and national advocat
e for pro tate cancer ufferer . Milken wa diagno ed with pro tate cancer in 1
993, hortly after hi relea e from pri on. Ju t 46 year old, he had a Glea on
core of 9, a PSA of 24, and the cancer had already pread to hi lymph node . H
e wa treated with beam radiation and hormone and began a very trict diet; thi
rteen year later he remain in remi ion. Milken approached pro tate cancer in
the ame manner he had approached Wall Street ecuritie . I decided that I had to
change the cour e of hi tory, he recall , and propo ed a Manhattan Project for pro tat
e cancer to di cover the cau e and better treatment .5 He pledged $25 million o
f hi own fund and in 1993 began CaPCURE (cancer of the pro tate cure), a found
ation that in 2003 wa renamed the Pro tate Cancer Foundation (PCF). Milken ha
accompli hed a remarkable amount in pur uit of hi goal . Between 1993 and 2003,
hi foundation rai ed $230 million and funded over twelve hundred pro tate canc
er re earch project . In contra t to the National Cancer In titute, where gettin
g a re earch grant funded often take eighteen month or more, Milken award are
given in three month . Al o in contra t to the NCI, the re earch project funde
d by the foundation are more focu ed on nding better treatment and le oriented
toward ba ic cellular re earch. Milken foundation ha coordinated a pro tate ti
ue bank to di tribute ti ue to re earcher and ha et up a genetic project
to collect blood from familie with three or more member who have pro tate canc
er. It ha al o put together a con ortium of eight leading pro tate cancer re ea
rch center and hold an annual meeting to bring leading re earcher together to
exchange idea . Perhap mo t remarkably, hi foundation require it grantee t
o cooperate with one another and to openly di cu their re earch nding , a true
accompli hment in a eld that ha at lea t it hare of prima donna . In addition
to hi re earch effort , Milken ha attempted to rai e the pro le of pro tate canc
er. He ha lobbied Congre and worked with other advocacy group , uch a the N
ational Pro tate Cancer Coalition, to organize public event . Since 1996 the Pro
tate Cancer Foundation ha worked with major league ba eball team to
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SCIENCE AND POLITICS
A Great Teacher Cancer i a har her teacher than in pirational be t eller or ad
for vitamin-mineral upplement . It remind u that we are not immortal, that
our time i limited, that di ea e and death are till out there waiting for u .
. . . Cancer i the worm in the apple of jaunty optimi m about life; the banana
peel on which even the healthie t and fitte t of u lip ; a great teacher, if y
oure lucky enough to urvive the le on. Michael Korda, Man to Man

pon or a PCF Home Run Challenge that rai e public awarene and generate re e
arch fund . A laudatory article in Fortune in late 2004 concluded that Milken ha ,
in fact, turned the cancer e tabli hment up ide down.6
THE NATIONAL PROSTATE CANCER COALITION The National Pro tate Cancer Coalition (N
PCC) i the foremo t advocacy organization on pro tate cancer. It began in 1996,
when a group of men who had pro tate cancer met in Texa and decided to form an
organization modeled after the women advocacy group for brea t cancer. Mary Lo
u Wright of the Mathew Foundation for Pro tate Cancer Re earch, a pioneer in th
e eld, wa in trumental in getting it tarted; Michael Milken, the American Cance
r Society, and Zeneca Pharmaceutical (which later became part of A traZeneca) w
ere al o helpful. Located in Wa hington, D.C., the NPCC ha a taff of twelve an
d operate with a combination of corporate pon or hip and donation . It twofol
d mi ion i promoting re earch on, and awarene of, pro tate cancer. To accomp
li h the r t, it i active on Capitol Hill and ha clo e relation hip with many
member of Congre who have pro tate cancer ome of whom have publicly acknowledg

ed
SCIENCE AND POLITICS
205
their illne and other who have not. Senator Ted Steven of Ala ka and former
Senator Bob Dole have for many year been the leading congre ional voice on pr
o tate cancer. Both the Senate and the Hou e of Repre entative have informal ca
ucu e compo ed of legi lator who have family member with ome form of cancer.
Member of Congre who have been con i tent upporter of pro tate cancer re e
arch include Senator Michael Crapo (Idaho), Byron Dorgan (N. Dak.), Diane Fein
tein (Calif.), Tom Harkin (Iowa), Kay Hutchin on (Tex.), Harry Reid (Nev.), and
Jeff Se ion (Ala.), and Repre entative Sherrod Brown (Ohio), Loi Capp (Cali
f.), Je e Jack on (Ill.), Peter King (N.Y.), Kendrick Meek (Fla.), and Deborah
Pryce (Ohio). In a variety of way NPCC attempt to increa e awarene of pro ta
te cancer, and e pecially of how important it i that men be te ted. It ha work
ed with major league ba eball on an education campaign, Take a Swing Again t Pro t
ate Cancer; with the National Hockey League on a Hockey Fight Cancer program; and wit
h NASCAR driver uch a Dale Earnhardt, Jr., and Jimmie John on on a NASCAR Young
Gun Con umer Challenge, in which fan can donate at NASCAR race to ght pro tate
cancer. The NPCC ha promoted September a Pro tate Cancer Awarene Month, an e
xten ion of work by the Pro tate Cancer Education Council, a maller advocacy gr
oup in Denver. In many of the e activitie , NPCC work with other organization ,
including U Too and Milken Pro tate Cancer Foundation. Much of NPCC in uence com
e from Aware, it free biweekly on-line new letter, which ha over forty- ve thou and
ub criber . The new letter include ummarie of breaking new on pro tate can
cer re earch and link to the torie . De pite the fact that it carrie pharmace
utical company ad and thu cannot be truly objective on drug development , Aware i
the mo t u eful and widely read online new ource for men with pro tate cancer
.
OTHER PLAYERS The olde t exi ting pro tate cancer group i the Pro tate Cancer E
ducation Council (pcaw), founded in 1988. It originated
206
SCIENCE AND POLITICS
Like, I totally agree! Thi political correctne ha gone waaay too far!
Pro tate Cancer Awarene Week and ha worked hard to make men aware of the nece
ity of having regular PSA te t and rectal exam . It too ha corporate pon or
hip . Other cancer organization erve individual with all type of cancer, no
t ju t pro tate cancer. The large t and olde t i the American Cancer Society (c
ancer.org), which began in 1913. It ha both tate and local of ce and rai e bot
h public awarene and fund for re earch and prevention. The National Coalition
for Cancer Survivor hip (canceradvocacy.org), founded in 1986, i the olde t ca
ncer organization led by cancer urvivor them elve . It work clo ely with the
media to promote education about cancer. Both of the e organization accept dona
tion . Finally, there are the pharmaceutical and radiation-related indu trie , w
hich tand to gain nancially from the wide pread u e of hormone and radiation the
rapie . The e companie pon or pro tate cancer awarene event and cancer cre
ening program . In late 2004 the Theragenic Corporation, manufacturer of the e
ed mo t comSCIENCE AND POLITICS
207

monly u ed for radiation eed therapy, per uaded Congre to pa a re olution enc
ouraging doctor to inform pro tate cancer patient of all the proven treatment
option available, including brachytherapy.7 Congre thereby put it elf into the
medical information bu ine . The pharmaceutical indu try, however, doe not hav
e an e pecially di tingui hed record in the pro tate cancer eld. For example, TAP
Pharmaceutic ha paid over $1 billion ince 2001 to ettle a erie of civil c
laim and criminal charge . Jointly owned by Abbott Laboratorie and Takeda Chem
ical Indu trie , TAP i the manufacturer of leuprolide (Lupron), one of the two
major LHRH blocker widely u ed in hormone therapy. The company wa accu ed of b
ribing urologi t to witch their patient from Zoladex (go erelin) to Lupron. O
ne cheme wa to give doctor free Lupron ample and then encourage them to bil
l Medicare or other third-party payer . In another cheme, the company gave the fe
deral government an in ated whole ale price for Lupron and then old the drug to h
undred of doctor at a far lower price. On billing Medicare, the doctor could
expect to get $100 or more in illicit pro t on every hot.8 It i alleged that hund
red of urologi t cooperated with the company, ome netting $30,000 or more. Ha
advocacy been effective in rai ing awarene of pro tate cancer and increa ing
fund for re earch? It unque tionably ha been on both count . Re earch fund h
ave increa ed more than tenfold ince 1993. Fund for pro tate cancer re earch,
however, till are only approximately half tho e devoted to brea t cancer on a p
er patient affected or per death ba i . Pro tate cancer i di advantaged in it
advocacy effort in that mo t of tho e affected are older men, and it i dif cult
to organize them to march on Congre . Over the la t decade pro tate cancer, oft
en abbreviated PC, ha become omewhat more politically correct, but it till ha
a long way to go.
C H A P T E R
15
Advice for Men Who Do Not Have Pro tate Cancer
C
hico, one of the comedian Marx Brother , wa once een by hi wife ki ing a cho
ru girl. When pre ed for an explanation, he quickly replied: I wa nt ki ing her,
I wa whi pering in her mouth. We men have a remarkable ability to deny the obvio
u and create excu e indeed, it may be one of our greate t trength . Failing to
get regular checkup to detect pro tate cancer i imply another illu tration of
denial vain, and coy excu e, a Milton long ago called it.
SHOULD I BE TESTED? Many tudie of men who have undergone regular exam for pro
tate cancer, including digital rectal exam and PSA te ting, ugge t that uch
precaution ave live . Compari on of 173 men in Minne ota who died of pro tate
cancer with 346 matched control reported that only 61 percent of the men who di
ed had had a digital rectal exam in the previou nine year , compared to 81 perc
ent of the men in the control group. The author of the tudy concluded that a cr
eening [exam] may have prevented 50 percent to 70 percent of death . . . due to
pro tate cancer. A tudy in Wa hington State of 171 men who died of pro tate canc
er and 342 matched control imilarly found that men who have been creened with D
RE [digital
ADVICE FOR MEN WITHOUT PROSTATE CANCER
209
They ay I have a pecial kill in diagno ing pro tate cancer.
rectal exam] and/or PSA are at lower ri k of mortality from pro tate cancer than
men who have not been creened.1 The mo t convincing data, however, come from a
tudy in Au tria in which PSA te ting wa made available without charge to men in

one Au trian tate but not in other . Within ve year , more than two third of m
en aged 45 to 75 in the tate with free PSA te ting had been te ted one or more
time . Two year after the tudy began, the death rate from pro tate cancer bega
n falling in that tate igni cantly more than in the other tate ; the re earcher
claimed that thi trend wa the r t evidence . . . that the policy of making PSA
te ting univer ally available and at no co t may have led to a reduction in deat
h from pro tate cancer in that population.2 In contra t to the e tudie , a highly
publicized 2006 tudy of 501 men who died from pro tate cancer in New England r
eported that creening by PSA or digital rectal exam did not ave live . The aut
hor ugge ted that phy ician hould not u e the reduction of mortality a a ju
ti cation for endor ing routine te ting of a ymptomatic men. An accompanying editoria
l, however, noted that 78 percent of male primary-care phy ician and 95 percent
of male urologi t aged 50 and over do have their PSA te ted. Two large tudie
of PSA te ting are currently in progre . One, the PLCO Cancer Screening Trial,
ha regi tered 74,000 men in the United State ; the other, the European Randomi
zed Study of Screening for Pro tate Cancer,
210
ADVICE FOR MEN WITHOUT PROSTATE CANCER

PSA Te t a a God end All the debate notwith tanding, PSA te t are a god end.
. . . You hould know your PSA number ju t a you know your chole terol count.
. . . When I it in meeting at work and look at group of men who are my contem
porarie , I want to hout at them, Do you guy know what your PSA i ? Andy Grove, Taki
g on Pro tate Cancer, Fortune, May 13, 1996
ha regi tered 172,000 men at ve European ite .3 Re ult of the two tudie are
expected in 2006 and 2008, re pectively. In the meantime, men mu t decide whethe
r to heed the exi ting evidence or await de nitive re ult . Becau e of uch di cre
pant re ult , wide pread PSA te ting continue to be controver ial. Some authori
tie argue that ince three quarter of PSA between 4 and 10 turn out not to be
cancer, uch te ting lead to unnece ary biop ie , expen e, and anxiety for th
e men involved. They point out that PSA te ting i not en itive enough, ince i
t mi e 15 percent of men who have igni cant cancer even though they have PSA o
f le than 4. They argue that it ha not yet been conclu ively proven that PSA
te ting actually ave live . Finally, they note that autop y tudie of men dyi
ng from other cau e have found mall foci of pro tate cancer in almo t all olde
r men, o technically almo t all men have ome cancer. Opponent of PSA te ting
received further upport in 2004 when Thoma Stamey and hi colleague at Stanfo
rd Univer ity publicly claimed that the PSA era i probably over for pro tate canc
er in the United State .4 Predictably, other re earcher immediately re ponded tha
t the PSA era i not over for pro tate cancer.5 They pointed to data ugge ting that
PSA creening ha been the main rea on for the decrea e in advanced tumor . For
example, between 1983 and 1985, only 75 percent of men diagno ed with pro tate
cancer were alive ve year
ADVICE FOR MEN WITHOUT PROSTATE CANCER
211
Kojak Mi take Telly Savala , the bald actor who played the televi ion detective Ko
jak, had come in with a PSA of 10, he recalled. Dr. Skinner cheduled the biop y, bu
t Savala had a Chri tma pecial and cancelled it. Then he needed a vacation. I
called to tell him how important it wa , but he never came back. Four year lat
er he wa gone. David Horowitz, The End of Time
later. Between 1995 and 2000, 99 percent of men o diagno ed were alive ve year
later. Becau e of the continuing di agreement about the value of wide pread PSA
te ting, an independent panel of expert wa a embled under the Agency for Heal

thcare Re earch and Quality. They could not agree either and concluded that the ju
ry i till out on the value of routine creening and that patient hould talk with
their clinician to make individualized deci ion .6 The cycle i complete: men ha
ve for year been told that they mu t make the deci ion regarding what treatment
to pur ue if they get pro tate cancer. Now, men are al o being told that they m
u t make the deci ion whether even to be te ted to determine whether they have p
ro tate cancer. At r t glance, it all ound very democratic, but the truth i th
at few men have uf cient information to make informed deci ion . I it any wonder
that many ignore the whole bu ine and avoid te ting altogether, baf ed by the p
lethora of fact and the indeci ion of expert ? To me it eem elf-evident that
PSA te ting, combined with a digital rectal exam, i not only worthwhile but on
e of the few thing a man can do to diagno e cancer early and po ibly ave hi
life. Much of the confu ion come from thinking of PSA te ting a either negativ
e (3.9 or lower) or po itive (4.0 or higher). We aw in Chapter 2 that PSA i a
continuum and become valuable a a diagno tic tool
212
ADVICE FOR MEN WITHOUT PROSTATE CANCER
I think the dog ate my la t PSA appointment card.
when mea ured on a regular ba i o that the rate of change (PSA velocity) can b
e calculated. The argument that almo t all older men have micro copic foci of ca
ncer cell in their pro tate gland doe not negate the value of checking PSA; t
he te ting i an attempt to identify men who have a micro copic focu that, for
whatever rea on, i growing into a full-blown cancer.
BIOPSIES If the word embarra wa intended to de cribe any ingle act, it urel
y applie to a pro tate biop y. You lie on your ide, your back ide naked to the
world, while your urologi t in ert a probe into your rectum, then remove tiny
liver of ti ue from your pro tate with what ound like a taple gun. The ma
gnitude of the pain i remarkably minimal compared to the magnitude of the indig
nity. When hould a man have a biop y? If your phy ician feel anything u picio
u on digital rectal exam, that i a certain indication.
ADVICE FOR MEN WITHOUT PROSTATE CANCER
213
Surviving a Biop y Tying up the gown i the harde t part of the di robing exerci
e becau e it ha to be done backward , and a everybody know , the behind-your-ba
ck- kill gene i not carried on the male chromo ome the way color-blindne i . S
imply put, a man cannot do thi ta k. . . . Thankfully, Im facing away from the a
ction, a if Im on one ide of the room, my behind on the other. But from that po
ition I cant watch the monitor with it jerky black and white picture of my pro
tate, and o cant make futile attempt at humor, which alway buoy my pirit an
d ma k my agitation. I it a boy or a girl, doctor? Bert Gottlieb, The Men Club
Any igni cant ri e in your PSA level, a de ned below, i al o an indication. In pr
eparation for a pro tate biop y mo t, but not all, urologi t a k you to top ta
king a pirin compound a week to ten day prior to the procedure. The night befo
re, you are a ked to drink a olution that clean out your bowel by cau ing diar
rhea. Alternatively, you are al o a ked to give your elf a Fleet enema prior to
the procedure valuable practice for the humiliation of the biop y it elf. The uro
logi t r t in ert into your rectum the ultra ound probe, equivalent in ize to a
bout two nger . The probe end out ound wave that outline the pro tate on a m
all creen, allowing the urologi t to ee exactly where to biop y. Sometime the
ultra ound how dark area that may indicate cancer, but often it fail to ho
w cancer even when it i pre ent. At one time it wa hoped that pro tate ultra o

und would become a u eful a the mammography u ed to detect female brea t cance
r, but it ha proven di appointing for that purpo e; it main u e i to guide th
e biop y and determine pro tate volume.
214
ADVICE FOR MEN WITHOUT PROSTATE CANCER
The biop y it elf involve the u e of very thin needle that go through the wall
of the rectum and into the pro tate, where they remove a thin liver of ti ue.
Each uch piece i called a core. In the pa t, it wa u ual to take ix core ,
but it i now common to take eight to twelve. Lying on the table, you will know
exactly how many have been taken by counting the di tinctive click of the biop
y gun. The pain i a momentary ting, likened by one urologi t to a rubber band be
ing napped again t the kin,7 although men vary widely in their pain thre hold. T
he entire procedure take about a half hour. Following the biop y, many urologi
t have men take an antibiotic for three day to minimize the likelihood of infe
ction. It i normal to have mall amount of blood in the emen, urine, or tool
for a few day , and there are no re triction on activitie other than riding a
bicycle. Since the biop y i merely ampling ection of the pro tate and not t
he entire gland, it i po ible that cancer i pre ent and i mi ed by the biop
y. If the urologi t i u piciou , if the biop y how precancerou cell (pro
tatic intraepithelial neopla ia, or PIN), or if the PSA continue to ri e, the b
iop y may have to be repeated at a later date. In the end, o to peak, the info
rmation provided by a pro tate biop y i extremely u eful. If the biop y i nega
tive, the chance are high (approximately 7080 percent) that cancer i not pre en
t. If it i po itive, you will know your cell type (Glea on core) and you will
al o have information on the ize of the cancer from the ultra ound picture, the
percentage of po itive core , and the percentage of cancer in each po itive cor
e. From the e you can a e the eriou ne of your cancer and begin making rat
ional treatment deci ion .
TEN RECOMMENDATIONS The American Cancer Society currently recommend that men ha
ve a digital rectal exam and PSA te t annually, beginning at age 50, if they hav
e a life expectancy of at lea t ten more year . For men who have a family hi tor
y of pro tate cancer and for African American men, with their higher rate of pro
tate cancer, the recommendation i to begin annual te ting at age 45. Ba ed on
a review of the exi ting literature, my recommendation are a follow . They are
ba ed on tudie howing that men who
ADVICE FOR MEN WITHOUT PROSTATE CANCER
215
Living with Cancer Life doe nt end when you have cancer. In tead, it become va t
ly more preciou . I find my elf reluctant to wa te any cheri hed moment. I will
no longer vegetate in front of the TV, a I have in the pa t. Im le willing to
pend any preciou time in anger, argument, long committee meeting , or unnece
ary trip . Michael Dor o, Seed of Hope
have very low PSA value are unlikely to develop pro tate cancer in the immediat
e future.8 Knowing what I know now, here i what I per onally would do.
1 I would get a ba eline PSA by at lea t age 40 to 45. A 2005 tudy
2 3
4
5

reporting that a man PSA level in hi 30 predict the ri k of later pro tate ca
ncer ugge t that getting a ba eline PSA even earlier may be u eful.9 If the PS
A wa le than 1.0, I would not get an additional PSA for five year . If I had
a family hi tory of pro tate cancer or wa African American, I would reduce the
interval to two year . A long a my PSA remained le than 1.0, I would continu
e thi practice for ucce ive five-year period until age 60 unle I developed
ymptom of an enlarging pro tate. I would get a digital rectal exam each time
I had a PSA te t. From age 60 on, or if my PSA had ri en to between 2.0 and 3.0
before age 60, I would increa e the frequency of PSA te ting to every year. I wo
uld keep a per onal record of my PSA te t re ult o that I could a e the PSA
velocity. It i one of the mo t u eful predictor of pro tate cancer. Knowing y
our PSA i a important a knowing your chole terol level. If my PSA ro e more t
han 0.7 in any given year, I would fir t repeat the te t to verify it accuracy.
If accurate, I would get a free PSA determination ( ee Chapter 2), which i a w
ay to identify noncancer
216
ADVICE FOR MEN WITHOUT PROSTATE CANCER
6
7
8
9 10
cau e of increa ing PSA (for in tance, pro tatiti or benign pro tatic hypertro
phy). If at any time my PSA indicator were worri ome (PSA velocity greater than
0.7 in a year, free PSA le than 20 percent), I would have a biop y. If the bi
op y turned up any que tionable finding , including pro tate intraepithelial neo
pla ia, I would have a repeat biop y in a few month . If I got to age 75 to 80 w
ithout evidence that my PSA wa increa ing more than the minimal ri e expected w
ith age, I would top having PSA te ting. If, at any point, my life expectancy w
a le than ten more year , I would top having PSA te ting. Even though there
i not yet definitive evidence, I would try to modify my diet along the line re
commended in Chapter 13, pecifically reducing my fat intake and increa ing toma
to product , green tea, red wine, and perhap oy. For mo t of u , thi i much
more difficult than undergoing PSA te ting and biop ie .
FUTURE RESEARCH De pite di agreement about the value of PSA creening for pro t
ate cancer, all urologi t agree on one fact: We need better te t . The PSA and
digital rectal exam are u ed becau e they are the be t we have; both will be re
garded in the future a relatively primitive. Thank to the effort of the advoc
acy group and elect member of Congre , a de cribed in the previou chapter,
re earch i now under way to de ign more accurate te t for diagno ing pro tate
cancer and for identifying which pro tate cancer are likely to remain quie cen
t and which are likely to pread. Thi re earch involve protein found in the b
lood, protein found in the pro tate ti ue, gene , and imaging technique . Amon
g the candidate in the blood for predicting pro tate cancer are protein with n
ame uch a caveolin1, in ulinlike growth factor binding protein (IGFBP 2 and 3
), interleukin 6 and it receptor (IL6 and IL6R), kallikrein 2, telemera e, and a
tran forming growth factor (TGF beta 1). An e pecially promi ing approach repor
ted in late 2005 u e everal protein that are part of the immune y tem re pon
e to cancer; according to the report, the panel of
ADVICE FOR MEN WITHOUT PROSTATE CANCER
217

[protein ] performed better than did pro tate- peci c antigen (PSA) in di tingui h
ing between the group with pro tate cancer and the control group.10 Several protei
n found in the pro tate ti ue it elf, available by biop y or urgery, appear p
romi ing for predicting the everity of the cancer. The e include alpha methylac
yl-coenzyme A racema e (AMACR), annexin , Bcl2 and other factor that control the
death of cell (apopto i ), E-cadherin, enhancer of ze te homolog 2 (EZH2), hep
in, and thymo in-beta 15.11 Men earching pro tate cancer web ite for new of
current re earch will ee frequent reference to the e protein . Probably the mo
t promi ing future technique for predicting pro tate cancer i the u e of gene
and protein expre ion. With microarray technique , thou and of gene and prot
ein are put onto a microchip. Ti ue from a pro tate cancer i then al o put on
to the chip, howing which peci c gene or protein are activated. The hope i th
at peci c combination of gene and protein , commonly referred to a gene expre
ion pro le , will be identi ed and will be u eful in predicting the probable future
cour e of any given cancer. In late 2004 thi technology became available for p
redicting the recurrence of brea t cancer, and it i likely to become available
for pro tate cancer in the near future. Another approach to improving prediction
ha been to re ne imaging technique o that they can be u ed to a e the ize
of pro tate cancer and predict their future cour e. To date, the u e of traditi
onal computerized tomography (CT) and magnetic re onance imaging (MRI) ha been
di appointing. Re earcher have begun u ing nanoparticle called lymphotrophic
uperparamagnetic nanoparticle (LSN ), which enter into lymph cell and can be
een on MRI; whether thi procedure can predict the pread of pro tate cancer to
lymph node i being inve tigated. The variety of re earch offer much hope for
the future. Men in their 50 or younger are almo t certainly going to bene t and w
ill have far more preci e mean to both diagno e and predict the cour e of their
cancer. Tho e of u who are older may or may not bene t, but we have the ati fac
tion of knowing that our on and grand on will not experience the uncertaintie
that we have faced. Thi i no mall progre .
A P P E N D I X
A
The Anatomy and Function of the Pro tate Gland
ichelangelo David notwith tanding, the anatomy of the human male leave a lot to
be de ired. Our organ of reproduction and recreation are hopele ly intertwine
d with our organ of liquid wa te di po al. A one ob erver facetiou ly noted, Onl
y a Civil Engineer could have de igned the body [ ince] who el e would de ign a
wa te di po al line through a recreational area?1 Sitting in the center of thi an
atomical a emblage i the pro tate, a reddi h-brown organ approximately an inch
and a half in diameter. It lie at the bottom of the pelvi , with the peni bel
ow, the bladder above, the pelvic bone in front, and the rectum behind (Figure 3
). Thi la t juxtapo ition i e pecially important, becau e feeling the pro tate
through the rectal wallthe much-maligned digital rectal exami the only way a phy
ician can phy ically a e thi organ. Fortunately, the majority of cancer de
velop in the po terior portion of the pro tate, thereby making them detectable t
o a killed examining nger. The pro tate ha been variou ly de cribed a looking
like a walnut, a che tnut, or a mall plum. In a young man, it weigh approximat
ely 20 gm, then increa e in ize a the man age . It contain mu cle and gland
, the latter ecreting uid that a i t and protect male perm. One component o
f the pro tate uid i pro tate peci c antigen (PSA), de cribed in Chapter 2.
M
220
APPENDIX A

The pro tate contribute approximately one third of the uid that make up the em
en. The other two third come from the paired eminal ve icle , mall gland th
at it at the ba e of the bladder and drain through the pro tate into the urethr
a. Joining the eminal ve icle duct in the pro tate are the paired va a deferen
tia, which carry perm from the te ticle . The va a deferentia, eminal ve icle
duct , and duct from the internal pro tate gland all join together at the uret
hra in the middle of the pro tate (Figure 4). Thu , the pro tate center i the G
rand Central Station of the male reproductive y tem. There, at the time of orga
m, perm and the accompanying uid begin their journey down the urethra, through
the peni , and with luck up the vagina and uteru , perhap to nd a waiting egg in
the fallopian tube . According to Dr. Peter Scardino Pro tate Book, the perm t
ravel at the tartling rate of 28 mile per hour, which, by amu ing coincidence, i
al o the top peed a worldcla human runner can achieve during a print.2 Thi
complicated y tem would work ne, except for the fact that the urethra ha a eco
nd function, a the exit for urine. Filtered by the kidney and then depo ited i
nto the bladder, which it on top
ANATOMY AND FUNCTION OF THE PROSTATE
221
What a Curiou Organ I would like to it down with my doctor and talk to him abo
ut the pro tate. What a curiou organ. What can God have been thinking when He d
e igned it thi way? Anatole Broyard, Intoxicated by My Illne
of the pro tate, urine pa e via the urethra directly through the middle of the
pro tate. To avoid a con tant dribble of urine, the mu cle at the ba e of the
bladder function a an internal phincter (although technically they are not a t
rue phincter). The urethra al o ha an external phincter, a true phincter, ju
t below the pro tate. Both phincter mu t relax for urination to take place. W
hen one or both phincter are damaged, a during urgical
222
APPENDIX A
removal of the pro tate, or are not working properly, which may happen a a ide
effect of medication, the re ult may be ome degree of incontinence. During org
a m and ejaculation, the external phincter open to allow the emen to leave, w
hile the internal phincter clo e to prevent emen from owing upward into the bl
adder. When the phincter do not work properly, emen may ow into the bladder; t
hi i called retrograde ejaculation. It i harmle , but obviou ly doe not re
ult in the fertilization of female egg . Alternatively, if the internal phincte
r doe not clo e during orga m, urine may de cend through the urethra, a common
but u ually temporary complication of urgical removal of the pro tate ( ee Chap
ter 3). But thi anatomical anarchy actually i even wor e. Running clo e to the
ide of the pro tate are tiny arterie , vein , and nerve that go to the peni
and control erection. If they are damaged during urgical removal or irradiatio
n of the pro tate, the man may achieve only a partial erection or no erection at
all. Thi impotence i a common ide effect of pro tate cancer treatment, a di
cu ed in Chapter 10. Given thi anatomy, it become apparent why enlargement o
f the pro tate, due to either benign pro tatic hypertrophy (BPH) or cancer, may
cau e eriou problem . An enlargement of the pro tate may queeze the urethra,
thereby making the urinary tream progre ively maller and urination more dif cul
t. Thi proce occur commonly in aging men, ince the pro tate begin to enlar
ge during middle age. The rea on for thi enlargement i unknown; it i cau ed b
y te to terone timulation but appear to erve no u eful evoluANATOMY AND FUNCTION OF THE PROSTATE

223
tionary purpo e other than to make urology a nece ary medical pecialty. Given
thi anatomy, it al o become apparent why treating pro tate cancer can o often
lead to incontinence and impotence. It i virtually impo ible to urgically ex
ci e, irradiate, or otherwi e remove a cancerou growth in the pro tate without
damaging urrounding tructure . A one ob erver ummarized the ituation: God pe
cialty wa humanity, not urology.3 Mo t people a ume that pro tate cancer i a di
ea e exclu ively of men. Thi i not quite true. In the r t few week of develop
ment in utero, male and female are exactly alike. The anatomy of both develop
from the ame tructure . Therefore, there are remnant of female tructure in
male , and remnant of male tructure in female . In women, the remnant of th
e pro tate are tiny, paired paraurethral gland that lie be ide the urethra. Rar
ely, the e gland can become cancerou , the re ult being a female equivalent of
pro tate cancer. Approximately ixty uch ca e have been de cribed in the medic
al literature. Thu , we have equality between the exe : men can (rarely) get br
ea t cancer, and women can (rarely) get the equivalent of pro tate cancer.
A P P E N D I X
B
Evaluation of Book About Pro tate Cancer
he following, li ted alphabetically by author, are a e ment of forty- even bo
ok about pro tate cancer publi hed in the pa t even year (a teri k indicate
tho e I have found mo t valuable). Al o included are a few volume publi hed ear
lier that are of pecial intere t. Book on pro tate health in general are not i
ncluded. Alterowitz, Ralph, and Alterowitz, Barbara. The Lovin Aint Over: The Coup
le Guide to Better Sex After Pro tate Di ea e. We tbury, N.Y.: Health Education
Literary Publi her, 1999. Written by a man who ha had pro tate cancer and hi
wife, thi book focu e exclu ively on impotence. It explain the complexitie o
f erection and orga m and outline option for couple faced with varying degr
ee of impotence. Baggi h, Jeff. Making the Pro tate Therapy Deci ion (rev. ed.)
. Lo Angele : Lowell Hou e, 1998. Originally publi hed in 1995, thi book wa
aid to have been revi ed in 1998. The change , however, appear to have been mini
mal and the book i now outdated. Barrett, David M. (ed.) Mayo Clinic on Pro tat
e Health. Roche ter, Minn.: Mayo Clinic, 2000. Thi lim (166-page) book i inad
equate in mo t re pect . Only half of it i about pro tate cancer: a little bit
about everything, not enough about anything. It prioritie are dif cult to under
tand. For example, it allocate only four page to radiation beam and eed thera
py combined, but eight page to complementary treatment . No reference or note
are given.
T
BOOKS ABOUT PROSTATE CANCER
225
Bodai, Ernie. I Flunked my PSA! Severna Park, Md.: B2Z Publi hing, 2002. Thi th
in volume wa written by a urgeon who had pro tate cancer. Although adverti ed
a a pro tate cancer primer, it provide too little information on mo t ubject . Re
ading the book i rather like being erved a mall appetizer when you are hungry
for the main cour e. Bo twick, David G.; Crawford, E. David; Higano, Cele tia S
.; and Roach, Mark (ed .). American Cancer Society Complete Guide to Pro tate Ca
ncer. Atlanta: American Cancer Society, 2005. Thi revi ed and updated edition o
f the American Cancer Society 1996 book i olid a far a it goe . However, man
y men will want more detailed data. For in tance, no ve-, ten-, or fteen-year urv

ival rate gure appear; little mention i made of treatment tudie that compare
one type of treatment with another; and no numbered reference allow one to foll
ow up on any given fact or gure. Thu , the book lack peci c information to help a
man choo e which treatment i be t for him. The e hortcoming may be an inevit
able con equence of having ixty-two author and trying not to offend anyone. *B
royard, Anatole. Intoxicated by My Illne . New York: Ballantine Book , 1992. Th
i book i a little cla ic. The author wa a book critic for the New York Time
when, at age 69, he wa diagno ed with advanced pro tate cancer. He died fourte
en month later, after having written the r t four e ay in thi book. He yearne
d for an untamed, beautiful death and ugge ted that we hould have a competition in d
ying, ort of like Halloween co tume . . . . Let give a prize for the mo t beaut
iful death. We can call it heaven. Bubley, Glenn J., with Conkling, Winifred. What
Your Doctor May Not Tell You About Pro tate Cancer: The Breakthrough Informatio
n and Treatment That Can Help Save Your Life. New York: Warner Book , 2005. Thi
book wa written by a Bo ton oncologi t who e practice include many patient
with recurrent pro tate cancer. For uch men it i a u eful book, ince it conta
in excellent chapter on experimental treatment and clinical trial . The chapt
er on natural remedie (herb , vitamin , and mineral ), dietary factor , and u
pport group are al o recommended. The author ummarize traditional treatment
in an unbia ed but omewhat ketchy manner. The weake t element i the hi trioni
c titlethe book i , in fact, much better than it cover would ugge t. Centeno, A
rthur, and Onik, Gary. Pro tate Cancer: A Patient
226
APPENDIX B
Guide to Treatment. Omaha: Addicu Book , 2004. Written by a urologi t and a rad
iologi t, both of whom pecialize in pro tate cancer, thi book too offer a lit
tle of everything but not enough of anything. It tend to minimize the eriou ne
of ide effect and complication of the variou treatment . It i ympatheti
c to cryo urgery; Onik wa one of the developer of thi treatment. Connell, Wil
l. Pro tate Cancer Treatment Option : A Guide to the Ba ic . Gra Valley, Calif
.: Edconco Pre , 1997. Written by an engineer who had pro tate cancer, thi boo
k trength i that it help men rationally think through their treatment option
and decide what i be t for them. It weakne i that it i omewhat out of da
te. Dattoli, Michael; Ca h, Jennifer; and Kaltenbach, Don. Surviving Pro tate Ca
ncer Without Surgery. Sara ota, Fla.: Seneca Pre , 2005. Thi book i a compani
on to Pro tate Cancer: A Survivor Guide, by Kaltenbach and Richard . Both are pu
blication of Dattoli cancer treatment center, which pecialize in radiation e
ed therapy combined with beam radiation. A uch, the author extol the merit o
f brachytherapy (Side effect with [ eed] implant are u ually mild and rever ible)
and denigrate urgical treatment. *Dor o, Michael A. Seed of Hope: A Phy ician
Per onal Triumph over Pro tate Cancer. Battle Creek, Mich.: Acorn Publi hing, 20
00. Thi book wa written by an emergency room phy ician who, at age 54, had a G
lea on 6 pro tate cancer and elected to u e a combination of hormone , beam radi
ation, and radiation eed therapy. He write hone tly and well about hi deci io
n-making tep , the advantage and di advantage of hi approach, and the effect
of the cancer on hi wife and him elf. It i an e pecially u eful book for men
con idering radiation eed therapy. Ell worth, Pamela; Heaney, John; and Gill,
Cliff. 100 Que tion and An wer About Pro tate Cancer. Sudbury, Ma .: Jone an
d Bartlett, 2003. Written by two urologi t and a man who cho e urgery for hi
pro tate cancer, thi book include u eful information but i oddly organized. T
he que tion-and-an wer format make it dif cult to nd the information you want and
lead to a great deal of repetition. Much of the information i pre ented in cha
rt that I per onally found terile and un ati fying. Fi her, William L. How To
Fight Pro tate Cancer and Win. Baltimore: Agora Book , 2001. Thi book i mi tit
led and include relaBOOKS ABOUT PROSTATE CANCER

227
tively little about pro tate cancer. It cover benign pro tatic hypertrophy and
pro tate health in general, empha izing herbal and dietary approache . *Gottlieb
, Bert, and Mawn, Thoma J. The Men Club: How To Lo e Your Pro tate Without Lo i
ng Your Sen e of Humor. Oxnard, Calif.: Path nder Publi hing, 1999. Thi i an ent
ertaining account of a radical pro tatectomy followed by ongoing incontinence an
d implantation of an arti cial phincter. Injecting humor into uch a equence i
a major challenge, but the author ucceed. Gottlieb wa a 61-year-old adverti i
ng executive when diagno ed with a Glea on 5 cancer, and alternate ection are
written by Mawn, hi urologi t. Patient and phy ician developed a clo e and mutu
ally affectionate relation hip. Gottlieb wife play a igni cant role in getting h
er hu band through the ordeal with both love and levity. She even get Mawn to p
romi e that he will have no more than one gla of wine the night before the ur
gery. Gray, Ro . Pro tate Tale : Men Experience with Pro tate Cancer. Harriman
, Tenn.: Men Studie Pre , 2003. Thi collection of hort torie about men wit
h pro tate cancer i imilar to tho e to be found on many pro tate cancer web it
e . The e, however, have been edited by the author, a re earcher in Toronto, and
are much more readable. The author ay that he wanted to make the truggle and
triumph of men with pro tate cancer more vi ible to men them elve and more und
er tandable to other ; he ha ucceeded in doing o. Grimm, Peter D.; Bla ko, John
C.; and Sylve ter, John E. (ed ). The Pro tate Cancer Treatment Book. New York:
McGraw Hill, 2004. A an up-to-date guide to treatment option , thi book i u
eful. It focu e e pecially on radiation eed therapy and work at the Seattle Pr
o tate In titute, the home of the editor . A uch, it ha a cheerleader tone, w
ith an implication that everything important i done at their headquarter . Each
chapter i multiauthored by a different group, o the writing tyle vary con i
derably. The book include no information about cau e , focu ing exclu ively on
treatment . Handy, Richard Y. Pro tate Cancer: Treatment and Recovery. Amher t,
N.Y.: Prometheu Book , 1996 (originally publi hed in 1988). Diagno ed with pro
tate cancer at age 52, the author had a pro tatectomy followed by radiation that
left him permanently impotent. Hi account focu e on the effect of the impote
nce on hi wife and
228
APPENDIX B
him elf, and he recount with ruthle hone ty the indignitie , depre ion , and
vulnerabilitie that followed. Hi preoccupation with hi peni i a Freudian d
elight, and men who uffer impotence a a con equence of their cancer will nd the
book u eful. Hennefent, Bradley. Surviving Pro tate Cancer Without Surgery. Ro
eville, Ill.: Ro eville Book , 2005. Thi i an odd book in that it main me ag
e i a diatribe again t urgical treatment. For example: Con ider the magni ed humil
iation of radical pro tatectomy patient . Not only ha their money been taken, b
ut their peni e have been crippled and their ex live have been largely de tro
yed. The author, an emergency room phy ician, wa in pired to write the book by hi
uncle, who died from hi pro tate cancer treatment. *Her h, Stephen P. Beyond Mira
cle : Living with Cancer. Santa Ana, Calif.: Seven Lock Pre , 2000. Of the man
y book aimed at urvivor of cancer in general, not ju t pro tate cancer, thi
i among the be t. Written by a p ychiatri t and peciali t in cancer patient a
nd pain management, it encourage patient to take an active role both in managi
ng their cancer treatment plan and in managing tre . Hitchcock, Robert. Love,
Sex, and PSA. San Diego: TMC Pre , 1997. Thi i a brief, light book written by
a 61-year-old playwright who got pro tate cancer (Glea on 5, PSA 7.7). It inclu
de a traighttalking and ometime humorou ection on impotence and it po ib
le treatment (the author u ed elf-injection ucce fully). Horowitz, David. T
he End of Time. San Franci co: Encounter Book , 2005. Horowitz wa 62 year old
when he wa diagno ed with pro tate cancer (Glea on 7, PSA 6.0) and had a radica

l pro tatectomy and po t-op radiation. Thi book cover very little about hi ca
ncer but rather i a erie of meditation on life, death, and love. It i nicel
y written but of limited value to men looking for information. Howe, De iree Lyo
n, Hi Pro tate and Me. Hou ton: Winedale Publi hing, 2002. Written in a chatty
tyle by the wife of a man with pro tate cancer, the book, a the title ugge t
, i aimed at wive . It tart lowly, but the econd half include an exten ive
and u eful account of how Howe coped with her hu band po t urgical incontinence
and impotence. For women confronted with the e ituation , the book will be hel
pful. Kaltenbach, Don, with Richard , Tom. Pro tate Cancer: A Survivor Guide. Sa
ra ota, Fla.: Seneca Hou e Pre , 2003 (originally publi hed in 1996). The autho
r, who wrote thi book with the help of a
BOOKS ABOUT PROSTATE CANCER
229
profe ional writer, wa a lawyer who wa diagno ed with pro tate cancer in hi
early 40 . He cho e radiation eed therapy, and the book i trongly bia ed towa
rd thi treatment. Kaltenbach, in fact, now work for a urgical group pecializ
ing in thi treatment, o the text ha ome of the avor of an adverti ement. The
mo t u eful part i a ection on co t and in urance coverage of pro tate cancer
treatment. Klein, Eric A.; Jamnicky, Leah; and Nam, Robert. So Youre Having Pro
tate Surgery. Hoboken, N.J.: John Wiley, 2003. Written by two urologi t and a u
rology nur e, thi lim volume i targeted at men who have elected to have urge
ry. Thu , it i urpri ing that a third of the book cover other i ue . It ha
a folk y tyle (We knowyoure tough and can handle a lot). The page are printed in gre
en on white, which may appeal if you are Iri h. Overall, moderately helpful but
not e ential reading. *Korda, Michael. Man to Man: Surviving Pro tate Cancer. N
ew York: Vintage Book , 1996. Thi i a widely read account of the author radica
l pro tatectomy. He wa a 61-year-old publi hing executive with pro tate cancer
(Glea on 6, PSA 22), for which he elected urgery. The author i brutally hone t
in detailing hi problem and reaction , and when the book wa originally publi
hed, it wa one of the r t uch book available. Korda had major po t-op problem
with incontinence and thu make pro tatectomy ound wor e than many other men
have reported. Still, the text contain much that i helpful. *Lange, Paul H.,
and Adamec, Chri tine. Pro tate Cancer for Dummie . New York: John Wiley, 2003.
Written by a well-known urologi t who him elf had pro tate cancer and a medical
writer who e hu band had pro tate cancer, thi i one of the mo t widely read bo
ok . It i u er friendly, with a detailed (ten-page) table of content , lot of
boxe , and icon indicating technical material that can be kipped. Lewi , Jame
. The Herbal Remedy for Pro tate Cancer. We tbury, N.Y.: Health Education Litera
ry Publi her, 1999. Thi book i e entially a 200-page adverti ement for PC-SPE
S, an herbal remedy promoted for u e in pro tate cancer. Since PC-SPES wa ub e
quently taken off the market by the Food and Drug Admini tration becau e it had
been adulterated with dangerou medication , the information in thi book i now
moot. Lintzenich, Jo eph W. Oh No, Not Me. San Jo e, Calif.: Writer
230
APPENDIX B
Club Pre , 2001. The author wa a 55-year-old pilot in 1992 when he wa diagno
ed with pro tate cancer and ub equently had urgery. Thi i hi accountplea ant
and chatty, but omewhat longwinded. Reader are not really intere ted in how t
he author name hi dog or thi level of detail: John blew the horn at 2:35 and P
at handed me my coat and I wa out the door. In it favor, the author provide the
mo t detailed account available of hi day immediately after urgery. He deal
frankly with hi incontinence and impotence, both of which were re olved; a u
ch, the book i a counterpoint to Korda Man to Man. Loo, Marcu H., and Betancou
rt, Marian. The Pro tate Cancer Source Book. New York: John Wiley, 1998. Thi i

a practical and wellwritten ummary of treatment option for pro tate cancer, e
mpha izing urgery. It i e pecially worthwhile on little detail that mo t book
overlook, uch a recommending that you bring a CD player to the ho pital for
your tay and, if po ible, chedule your urgery a the r t ca e in the morning
o that you wont have to wait. Unfortunately, the work i now out of date on many
treatment i ue . *Mark , Sheldon. Pro tate and Cancer: A Family Guide to Diagn
o i , Treatment, and Survival (3rd ed.). New York: Per eu , 2003. Originally pub
li hed in 1995, thi i one of the be t book available on pro tate cancer. It i
u er friendly, with forty chapter in que tion-and-an wer format and a helpful
index. Like mo t book written by urologi t , it primarily cover treatment i
ue and include little on cau e (except nutrition), re earch, and other i ue
. Martin, William. My Pro tate and Me. New York: Cadell and Davie , 1994. Thi a
ccount of pro tate cancer wa written by a ociology profe or in hi 40 . With
a Glea on core of 7 and a PSA of 8, he elected to have a radical pro tatectomy.
Hi account i u eful but rather long-winded and would have bene ted from a rm edi
tor. McClure, Mark W. Smart Medicine for a Healthy Pro tate. New York: Avery Pen
guin Putnam, 2001. Written by a urologi t who upport complementary and holi ti
c medicine, only one third of thi book i about pro tate cancer. It cover in d
etail diet, vitamin , herb , and life tyle change that ome men have found help
ful. Neider, Charle . Adam Burden: An Explorer Per onal Ody ey Through Pro tate
Cancer. Lanham, Md.: Madi on Book , 2001. The author, an adventurer and writer,
wa diagno ed with pro tate canBOOKS ABOUT PROSTATE CANCER
231
cer (Glea on 6, PSA 16) at age 78 and died from it at age 86. The diary account
of hi beam radiation treatment may be u eful for men con idering thi treatment
. The book could have u ed a thorough editing, a mo t reader may not be intere
ted in what the weather wa each day, what the author wa reading, and the like
. Newton, Audrey Currie. Living with Pro tate Cancer. Toronto: McClelland and St
ewart, 1996. Written by hi wife, thi book tell of a Canadian man who at age 5
9 wa diagno ed with pro tate cancer that had pread to hi lymph node . With ra
diation treatment and hormone uppre ion, he lived ten more year . Two year a
fter being diagno ed with pro tate cancer, he wa diagno ed with bowel cancer. H
i per onal tory i inter per ed with chapter of factual material, now outdate
d, about pro tate cancer. Nixon, Daniel W., and Gomez, Max. The Pro tate Health
Program: A Guide to Preventing and Controlling Pro tate Cancer. New York: Free P
re , 2004. Thi book wa written by a phy ician who i pre ident of the In titu
te of Cancer Prevention and by a cience reporter. A one might urmi e, the maj
ority of the book focu e on healthy diet and life tyle . It ha a worthwhile c
hapter on alternative treatment for pro tate cancer but i comparatively weak o
n tandard treatment and their complication . O terling, Jo eph E., and Moyad,
Mark A. The ABC of Pro tate Cancer: The Book That Could Save Your Life. Lanham,
Md.: Madi on Book , 1997. Written by a urologi t who e father died of pro tate
cancer and a public health educator, thi widely read book i now out-of-date. I
t ha many trength , including te timonial by famou and not- o-famou people
who have had pro tate cancer. However, it i cumber ome and omewhat repetitiou a
nd a pharmaceutical company logo on the back rai e que tion about objectivity.
Perlman, Gerald, and Dre cher, Jack (ed .). A Gay Man Guide to Pro tate Cancer.
Binghamton, N.Y.: Haworth Pre , 2005. Thi book i e ential reading for gay m
en who are diagno ed with pro tate cancer. The erie of torie written by gay
men of varying age and profe ion include a ocial worker, p ychologi t, p yc
hiatri t, economi t, and urologi t. All of them frankly di cu gay concern tha
t bear on their diagno i , including concurrent AIDS and their exuality. Many o
f the author have al o been involved in Malecare, Inc., a nonpro t upport group
( ee malecare).
232

APPENDIX B
Pienta, Kenneth J., and Moyad, Mark A. Pro tate Cancer from A to Z. Ann Arbor, M
ich.: Media Group, 2004. Thi uneven book provide rea onably full information o
n ome ubject (PSA interpretation, hormone treatment) but bare bone on other
ubject ( urgery, radiation treatment). It over impli e urvival tati tic and
include no reference for men who want to delve in more detail. The book al o p
rovide no per pective on the relative importance of many ubject . For example,
hark cartilage, with no evidence that it i u eful in pro tate cancer, i li t
ed along ide lycopene, for which evidence of it utility doe exi t. Pilgrim, Au
brey. A Revolutionary Approach to Pro tate Cancer. Pitt burgh: SterlingHou e, 19
97. Written by a chiropractor who had pro tate cancer, thi book ha a chatty t
yle but i omewhat di organized. The type i very mall and there are few vi ua
l . Like mo t pro tate book , it focu e almo t exclu ively on diagno i and tre
atment. It pecial trength are an intere ting chapter on quackery and a lengthy l
i t of re ource . Prochnik, Leon. You Cant Make Love if Youre Dead. Lo Angele : A
ri Pre , 2000. Written by a Hollywood creenplay writer with pro tate cancer (G
lea on 6, PSA 11), thi brief book recount hi earch for the right treatment.
Predictably, each doctor he con ulted ugge ted a different cour e. Hi ultimate
advice came from hi aunt, who recommended urgery, her rea oning later being u
ed a the title of the book. Ryan, Corneliu , and Ryan, Kathryn Morgan. A Priva
te Battle. New York: Simon and Schu ter, 1979. Corneliu Ryan wa a ucce ful w
riter when, at age 50, he wa diagno ed with advanced pro tate cancer. The re ul
t i thi book written by Ryan and hi wife, al o a writer, about the ub equent
four year until he died. The event took place from 1970 to 1974, and what i
triking i how different, and yet how much the ame, everything i . We forget t
he far greater tigma urrounding cancer thirty- ve year ago; the Ryan initially
kept the diagno i a ecret from even their clo e t friend . On the other hand,
each urologi t Ryan con ulted urged a different cour e of treatment, imilar to
the ituation many men encounter today. During hi battle with cancer, Ryan wro
te A Bridge Too Far, which, at the time of hi death, wa econd on the nationwi
de be t- eller li t. Ryan editor for that book wa Michael Korda, who, two decad
e later, would write a book about hi own pro tate cancer.
BOOKS ABOUT PROSTATE CANCER
233
*Scardino, Peter T., and Kelman, Judith. Dr. Peter Scardino Pro tate Book: The C
omplete Guide to Overcoming Pro tate Cancer, Pro tatiti , and BPH. New York: Ave
ry, 2005. With the retirement of Patrick Wal h a the de facto dean of American
pro tate cancer, Peter Scardino appear to be applying to a ume that role. The
imilar book title , the cowriting of both book with profe ional writer , and
many allu ion to the treatment uperiority of Scardino Memorial Sloan-Kettering
Cancer Center in New York over Wal h John Hopkin Medical Center in Baltimore
and other center all ugge t a u urper to the pro tate cancer throne. What the
two book hare i a trong bia toward urgery a the be t treatment for mo t m
en. That being aid, there i much valuable information in the Scardino book plu
a u eful glo ary and index, and the information i up-to-date a of late 2004
. The hortcoming include weak ection on cau e , hormone treatment, and re ou
rce , and few per onal torie . Although well written, there i much duplication
of information, which contribute to it almo t ve-hundred-page bulkyet another f
eature it hare with the Wal h and Worthington book. Strum, Stephen B., and Pog
liano, Donna. A Primer on Pro tate Cancer: The Empowered Patient Guide. Hollywoo
d, Fla.: Life Exten ion Foundation, 2002. Thi vi ually appealing book ha pictu
re , diagram , and even ome multicolor text. Strum i an oncologi t and a pione
er in hormone therapy. The book i almo t exclu ively devoted to diagno i and t
reatment and in part i quite technical. Publication wa partially funded by th
e Pro tate Cancer Re earch In titute, cofounded by the enior author, and the bo

ok appear to be bia ed toward radiation and hormone therapy. Wainrib, Barbara,

and Haber, Sandra. Men, Women, and Pro tate Cancer. Oakland, Calif.: New Harbing
er, 2000. Thi i a mode tly updated edition of a 1996 book, Pro tate Cancer: A
Guide for Women and the Men They Love. It i written by two female p ychologi t
who previou ly publi hed a book on brea t cancer. Aimed at the wive of men aff
ected with pro tate cancer, it i e pecially heavy on emotional i ue and tayi
ng p ychologically healthy. The be t chapter ha ugge tion for re ponding to a
man impotence. *Wal h, Patrick C., and Worthington, Janet F. Dr. Patrick Wal h
Guide to Surviving Pro tate Cancer. New York: Time Warner, 2001. Thi i one of
the mo t widely read book on pro tate cancer. Although it ha many trong point
, it i wordy, redundant, and weighty (443
234
APPENDIX B
page ). Worthington i a cience writer at John Hopkin Medical Center, and Wal
h i the former chief of urology there. The book trongly re ect the urgical in
tere t of the enior author and in ome ection ha the tone of a cheerleader.
It include much valuable information but ha ome omi ion ( uch a the po i
ble role of exually tran mitted di ea e ) and trong bia e (it i overly enthu
ia tic about dietary cau e ). It i al o unduly anguine about outcome and po
toperative complication following pro tatectomy. *Wheeler, Chuck, and Wheeler,
Martha. Af rming the Darkne : An Extended Conver ation About Living with Pro tate
Cancer. Beverly, Ma .: Memoir Unlimited, 1996. Thi i a book in which the hu
band and wife, married for fty year , kept eparate diarie . Chuck Wheeler wa d
iagno ed with a Glea on 9 pro tate cancer at age 65 and died eight year later.
Thi account of tho e year include hi urgery, penile pro the i , orchiectomy
, meta ta i , and pain control. At one point Chuck write : You know, honey, Ive nev
er died before. Thi i a brand-new experience for me. It a con tant preoccupati
on. Thi excellent book about dying from pro tate cancer hould be read by anyone
who doubt that the di ea e can be na ty and deadly. William , Charle R., and W
illiam , Vernon A. That Black Men Might Live. Ro coe, Ill.: Hilton Publi hing, 2
003. Rev. Charle William ha written a cautionary tale aimed at black men but
applicable to all men. He initially ignored multiple ign and ymptom that om
ething wa eriou ly wrong with hi urinary tract. When he nally had a PSA taken,
it wa 172. What did he do next? He ignored it for ve additional month . When at
la t he had a biop y, the pro tate cancer had already pread to hi bone . Tryi
ng to alvage omething from thi per onal di a ter, Rev. William dedicated the
remainder of hi life to educating other black men about the importance of regu
lar PSA te ting. The writing i uneven, but the me age come through loud and c
lear.
A P P E N D I X
C
U eful Web ite on Pro tate Cancer
ike many men of my era, I am Internet challenged and relatively web ite illitera
te. I do not under tand, for example, why you click Start on the computer when w
hat you want to do i Stop. The following evaluation of web ite on pro tate can
cer i therefore ba ed on my amateur tatu ; accompli hed Internet expert may d
i agree with my conclu ion . Literally dozen of web ite provide information on
pro tate cancer. Like all web ite , they come and go, o do not be urpri ed if
you are unable to nd ome of tho e mentioned here. There i much u eful informat
ion on the Internet; there i al o much mi information. One of the bigge t chall
enge in evaluating web ite i to a certain who pon or them. Many of the pro
tate cancer web ite are pon ored by peci c treatment center , type of treatmen
t, or pharmaceutical companie . The e ite therefore have commercial intere t ,

although ometime not obviou ly. Valuable information may be pre ented but, by
de nition, you are not being given a balanced point of view becau e the web ite h
a omething to ell. The following a e ment thu include only tho e web ite
that do not appear to be unduly in uenced by commercial intere t . The only excep
tion are ite that include unique information not available el ewhere, uch a
centerwatch (information on drug trial ) and ghtpro tatecancer.org (advocacy).
L
236
APPENDIX C

Speci cally excluded are the following: Web ite that advocate a pecific treatmen
t, for example, cryocarepca.org, which promote cryo urgery, or ecpcp.org, which
promote alternative therapie . Web ite that tout pecific treatment center ;
example are oncolink.upenn.edu, which promote the cancer treatment center at t
he Univer ity of Penn ylvania, and prci.org (al o acce ed by pro tate-cancer.or
g), which promote the Pro tate Cancer Re earch In titute in Lo Angele . Web it
e that promote pecific drug , device , and/or publication , and where commerci
al intere t would be expected to influence the information they provide, for in
tance, pro tateforum, cancerfact , and eekwellne . Web ite that accept paid
adverti ing. An example i p ari ing, which claim that paid adverti ing deci ion
are kept eparate from editorial content and new placement; it i a worthy ideal
, but in the real world he who pay the piper get to call the tune. Many pro ta
te cancer web ite include electronic new letter , mailing li t , me age board
, blog , li t erv , chat room , and online di cu ion group . The e can be very
helpful, e pecially for men who are geographically i olated from upport group
and comprehen ive librarie . But again, beware of commercial intere t ; if a web
ite i trying to ell you omething, you have clicked your way into a bu ine
and not a cla room. Many of the mo t valuable web ite accept donation . Helpin
g them out i an effective way to keep them independent. The following, then, ar
e the mo t u eful web ite on pro tate cancer among the more than forty ite I
examined. Tho e marked with an a teri k (at the beginning of the li t) are tho e
that appeared to me to be the be t. *cancer.gov (al o acce ed through nih.nci.
gov) Thi i the ite of the National Cancer In titute, a component of the Natio
nal In titute of Health. Well de igned, it allow you to immediately elect the
type of cancer on which you want information. The ection on treatment under Can
cer Topic are excellent and provide a imple Patient Ver ion, a more detailed Health
ofe ional
USEFUL WEBSITES
237

Ver ion, and an En E paol ver ion. The ection on Clinical Trial allow you to p
r type of cancer and the tage. When you input your zip code, the ite give you
all the trial within a given number of mile (which you pecify) of that zip c
ode. The ection Treatment Facilitie provide the name and addre e of the of cial
cancer center , de ignated by the National Cancer In titute, a explained in Cha
pter 8. Another ection on Cancer Stati tic give you more than you ever wanted to
know. Overall, thi web ite i the be t for reliable information on pro tate can
cer. *cancer.pro tate-help.org (formerly pro tate-help.org) Thi private ite wa
created by Don Cooley of San Jo e, California, after he wa diagno ed with pro
tate cancer in 1997. He adverti e it a the large t noncommercial, individually
owned and operated ite, and it i indeed huge. It i well organized and ea y to n
avigate with gateway ( uch a Patient Helping Patient ) that lead to peci c topic
(Surgical Removal of the Pro tate) that in turn lead to ubtopic (Who Should/Should N
ot Have Surgery). In early 2006 the gateway were under revi ion to make it even e
a ier to nd what you want. The ite include group and chat room . Cooley pride

him elf on including only proven cienti c data on hi ite and doe not hy away
from controver ie . Overall, thi web ite i a labor of love on which Cooley cl
aim to have pent over 6 year of 10- to 16hour day . He accept donation , but the
e are not tax deductible becau e he doe not have tax-exempt tatu . *phoenix5.
org Thi private web ite wa created in 2000 by Robert Young after he wa diagno
ed with pro tate cancer (with a PSA of over 1,000). It i exceptionally practic
al, with, for example, a li ting of pro tate cancer upport group by tate and
evaluation of other web ite . It principal a et are per onal torie written
by pro tate cancer urvivor and their wive /partner , but it al o include lin
k to new torie on pro tate cancer. The ite i e pecially trong on the effe
ct of pro tate cancer on ma culinity and exual function. *u too.org Thi i th
e be t ite for locating a pro tate cancer upport group. It i maintained by U
Too International Inc. in Downer Grove, Illinoi ,
238
APPENDIX C
which adverti e it elf a the olde t and large t cancer education and upport net
work. It claim 330 upport group , which can be identi ed by clicking on individual
tate . The ite i le u eful than ome other for obtaining objective treatm
ent information, becau e it i linked with web ite that have commercial intere
t . cancer.org Thi web ite of the American Cancer Society i not well de igned
but, with patience, yield much u eful information. It tronge t feature i Medic
al Update , which include ummarie of important pro tate cancer new torie of
the pa t few year . When I acce ed it in 2005, however, thi feature appeared t
o be relatively de cient in recent torie . The web ite include me age board an
d, if you look hard enough, the location of the American Cancer Society pon ored
Man to Man upport group . Finding a group in my area required a great deal of
trial-and-error hunting; I nally located one under the heading In My Community. cpcn.
org Thi i the web ite of the Canadian Pro tate Cancer Network (CPCN), which i
the national a ociation of pro tate cancer upport group . For Canadian who a
re looking for a upport group, here i the place to begin. hypertext.org Thi
ite wa organized in 1997 by Bill Dycke , who wa diagno ed with pro tate cancer
and elected to have radioactive eed implant . In acknowledgment for the ite,
Dyke thank hi urologi t who, by eeming to have o little intere t in my illne
, forced me to learn about it for my elf. Information on the ite i in Spani h
and Portugue e a well a Engli h. Ba ic information i available, with link to
many other ite for more detailed information. A nice feature i a pull-down m
enu that allow the u er to elect peci c topic . However, when I acce ed the i
te in 2005, ome topic had not been updated in more than two year . nccn.org Th
i i the ite of the National Comprehen ive Cancer Network, an organization of
nineteen leading cancer treatment center in the

2
The traditional term o teopathic le ion ha been largely uper eded by the term
omatic dy function. Although thi i not trictly ynonymou , it i in accord w
ith the World Health Organization International cla ification of di ea e . When
the original definition of an o teopathic le ion wa coined by Andrew Taylor St
ill the concept of the po ibility of a dy function in the mu culo keletal y te
m without the pre ence of di ea e wa revolutionary. It ha taken many year for
the medical profe ion to accept that it i po ible to have altered function i
n the pine or peripheral joint . It ha taken even longer to accept that the e
dy function can often be treated ucce fully by manual method . Many different
profe ion are u ing manual technique in treatment of the e condition , and t
heir model of the cau e, type of approach nece ary and underlying principle v
ary lightly. O teopathic concept originally looked on joint le ion in relatio

n to po ition of di placement. Technique were de igned to be correction or ad

ju tment to thi perceived malpo itioning. A more under tanding of the phy iol
ogy, patho-phy iology and pathology ha developed, o the thinking in relation t
o the le ion ha changed. In 1959 Alan Stoddard, in hi Manual of O teopathic Te
chnique, defined the condition. An o teopathic pinal le ion i a condition of
impaired mobility in an intervertebral joint in which there may or may not be al
tered po itional relation of adjacent vertebrae. When altered po ition i pre e
nt, it i alway within the normal range of movement of that joint. He went on
to tate that although the definition wa limited, it related pecifically to th
e condition that be t
re pond to manipulation. Thi definition, and the follow-on tatement relating
to it remain relevant today, and form the ba i of the approach u ed by mo t o
teopathic manipulative practitioner . Some chool of thought till relate to dy
function by their po ition of fixation. Some are more intere ted in their inab
ility to move in certain direction . Some are more concerned with the relative h
ypermobility of adjacent egment required to compen ate for the re tricted area
. The ite of maximum tenderne and ymptom i often found to be the relative
ly hypermobile area and i thu notoriou ly unreliable in diagno i . Local dy fu
nction, if of recent origin, may be tender and en itive, but quickly ub ide t
o a low level of irritability, leaving the compen ating area to become increa i
ngly en itive. There i , therefore, a time-frame to the tenderne and thi mu
t be con idered in diagno i and a e ment. It i important to di tingui h betw
een a primary and a econdary dy function. Old o teopathic terminology de cribed
primary and econdary le ion in relation to their character and po tural relat
ion hip. A econdary le ion wa one that had undergone certain change in the ti
ue , making it nece ary to perform two tage correction . Thi wa not only a
very complex way of looking at the problem , but wa taught differently by diff
erent teacher according to their own per onal model of under tanding. In attemp
ting to implify matter without totally lo ing the ba ic thinking, the BSO ha
adopted a notation ba ed on what i thought to be a logical phy iological and an
atomical ba i . A le ion may be primary or econdary. A
Somatic dy function primary dy function can be due to trauma, either a a ingle
incident or a a re ult of a erie of micro-traumata. If the train are due t
o the pre ence of ome dy function el ewhere in the body, then we have a econda
ry le ion or dy function. There i , therefore, a primary le ion at one or more
ite with a palpable dy function el ewhere that ha been dependent on the primar
y for it production. Sometime the primary ha re olved naturally or due to the
intervention of treatment. If the homeo tatic mechani m are capable of re tora
tion of normality in the econdary dy function, then it will pontaneou ly corre
ct. If they are not, due to weakne , mu cular imbalance or habit, what wa a e
condary le ion become a elf-maintaining entity of dy function in it own right
. The econdary dy function i really then an adaptation to ome intrin ic fault
in the tructure. An adaptation that cannot fully balance will go on to become
a compen ation. Adaptation i a normal phy iological re pon e to tre . It i o
nly un ucce ful adaptation proce e that lead to the need for compen ation. Th
ere i much debate a to what convert an adaptation to a compen ation. Some thi
nk that it i due to pattern learnt by mu cle . Thi can lead to perverted fu
nction that eventually lead to phy iological modification that become patho-ph
y iological and eventually pathological or at lea t partly irrever ible. Thi en
tail the alteration of the material propertie of the collagenou and ela tic t
i ue that make up the tendon , ligament , fa cia and other oft ti ue tructu
re urrounding an articular unit. Some of the e change are not capable of pon
taneou re olution once they have pa ed a certain tage. It i before thi poin
t that manual technique have the mo t u e in re toration of normal function. If
mu cle or fa cia i too hort due to compen atory mechani m , there will be a t
endency for reduced movement in adjacent joint coming under the control of the
e oft ti ue . The bone and joint are only able to function a well a permit
ted by the oft ti ue attached

5
to and overlying them. Joint work be t in the middle of their range, and the re
duced movement will be accompanied by time when the joint will inevitably meet
their end of range too ea ily and train will develop. Not all omatic dy fun
ction tate are at end of range, but qualitative change occurring in cap ule
and ligament tend to make a joint with re tricted mobility much more u ceptibl
e to mechanical problem . If the demand of body mechanic are for flexibility,
there will be a tendency for compen atory hypermobility in ome area with it a
ttendant ymptomatology. Hypermobile joint tend to have a characteri tic chroni
c aching on being a ked to maintain a tatic po ition, and are likely to have re
current acute locking epi ode a well. The chronic aching i often due to u ta
ined mu cle hypertonia in an attempt to tabilize the area. Mu cle i not de ign
ed for thi u tained contraction and will change omewhat according to the e t
re e . Adaptive change in the mu cle required to maintain thi tate include
partial fibro i and change in the fa cial and myofa cial ti ue to become  t
ringy or ropy. The o-called fibro iti and per i tent mu cular irritability
need little de cription. There i another type of econdary le ioning that i du
e to altered neurology from the pre ence of perverted vi cero- omatic reflexe e
manating from a dy functional or di ea ed vi cu . The nature of the palpable fin
ding in the e ca e will be omewhat different from the impler mechanical dy f
unction . The ti ue have a characteri tic doughy nature, and the palpable jo
int dy function that follow the oft ti ue irritability will have a more  pri
ngy nature. If the condition i maintained for long enough, however, there may
be adaptive change in the omatic ti ue that will not re tore to normal pont
aneou ly when the primary irritation from the nervou y tem ha gone. The other
characteri tic that i different about thi type of econdary le ioning i that
it tend to have an indurated
6
Somatic dy function y tem to produce ymptom el ewhere. It i often difficult
to define what i relevant in the objective finding . Quality of any di turbance
i een to have far greater ignificance than quantity. Imbalance of action aro
und a moving part ha to be examined with the part in motion. Static examination
i an e ential part of te ting, but moving analy i u ing active, pa ive and
po ibly re i ted movement i nece ary. The aim of treatment can be directed
to ymptom , ign , a balance between the two, or imply the re toration of func
tion. It could be directed to helping the patient tructure become more able t
o adapt it elf to the environment. It i a umed in thi re pect, that a tructu
re that i working harmoniou ly i more efficient and i le likely to be a ou
rce of ymptom . A imple tatement to encap ulate thi i that function govern
comfort.
feel. Normal mechanical dy function tend to bulge out with mu cle contraction p
roducing a palpable welling of the belly of the mu cle. The mu cle affected by
the econdary dy function related to a vi ceral condition are often flatter mu
cle that tend to pull in rather than bulge out when they contract. It i very e
a y to mi the pre ence of thi phenomenon. Any patient who ha the mi fortune
to have a eriou vi ceral condition, if examined before, during and after, will
how evidence of thi . It i a finding that may only become evident in retro pe
ct, but thi hind ight will enable the experience to be u ed to recognize the i
tuation the next time it happen . A ymmetry i the rule when dealing with the hu
man tructure. Neverthele , a ymmetry that i dy functional i ignificant a i
t i often a ource of ymptom or a tre or to the
DIAGNOSIS
3

The criteria on which a manipulative pre cription i ba ed and the choice of tec
hnique to be u ed are governed by the diagno i or pre-treatment a e ment. In
conventional medicine, diagno i relate to the di covery of di ea e and thi le
ad automatically to a choice of treatment method . The approach to treatment wi
ll vary lightly according to the knowledge, philo ophy and y tem of the practi
tioner, but will u ually fit in with e tabli hed guideline . Diagno i in o teop
athy i imilar, in that it relate to the di covery of di ea e in much the ame
way a orthodox medicine, but then hould lead the practitioner along a choice
of everal different path . The fir t path i the choice whether to treat or not
, and a to whether the ca e i uitable for o teopathic intervention. For a ca
e to be uitable there mu t be ome evidence of mechanical dy function pre ent t
hat can be changed or improved by phy ical treatment. If it i not uitable, po
ibly due to the pre ence of eriou di ea e that would be better attended by an
other di cipline, the practitioner mu t have enough knowledge about the conditio
n to be able to refer the patient to a uitable authority. Thi would avoid wa t
ing the patient time and money on fruitle phy ical treatment. He mu t under
tand the contra-indication to treatment, and have enough knowledge of pathology
to be able to make a rea oned judgement a to po ible danger . Hi knowledge a
nd competence to decline to treat mu t be ufficient to avoid being negligent, y
et mu t be tempered by a en e of reali m. He mu t decide whether there are any
po ible benefit from hi therapy. If a ca e i deemed uitable for o teopathic
treatment, a reali tic a e ment of the likely outcome or progno i i e entia
l a a afety mea ure. A progno i of a good outcome that i not backed up by th
e re ult of treatment i clearly a wrong progno i , and although the cho en pat
h may prove to be the correct one, the time- cale may be wrong. The next path i
into a deci ion a to the be t choice of treatment technique , and the order in
which they are to be carried out. The choice of technique to be u ed i govern
ed by the examination finding at the time, and the pretreatment a e ment at e
ach occa ion. There are no ab olute ; a with all y tem different practitioner
will have varied approache according to a multitude of criteria. Choice of ap
proach will depend on the under tanding of the requirement of the ca e, and on
the ability of the practitioner to carry out the cho en procedure. It would be f
ooli h to believe that every practitioner i capable of equal kill in each tech
nique. The wi e practitioner will maximize hi trength , but will con tantly be
endeavouring to overcome hi weakne e and add to hi trength . There will be
a hort-term and long-term aim to treatment, and it i e ential to involve the
patient in the diagno i deci ion and the rea oning for the approach cho en. So
me patient only want a quick fix and then get back to work without a thought
for the longterm ituation. Some really want to achieve a longer-la ting re ult,
and are prepared to perform any nece ary exerci e routine and maintenance trea
tment chedule. They are the cu tomer, and have the right to choo e. If a ther
api t feel that a cho en way i not to hi liking, he then ha to decide whethe
r to obey the patient wi he or to decline to treat
8
Diagno i the treatment and the technique u ed in that treatment, there mu t be
a choice of method , frequency, duration and inten ity o that the treatment te
chnique fit the dy function. The fir t requirement of the diagno tic proce in
the mechanical en e i to find the ti ue cau ing the ymptom . The next requi
rement i to a e the fault in that ti ue or ti ue . The rea on for that bre
akdown i relevant a are the predi po ing and maintaining factor . In other wor
d why did thi particular per on get thi particular problem at thi particula
r time? Diagno i in thi en e require a knowledge of the normal behaviour of
ti ue and the type of ymptom that can be produced when they are tre ed. It
i very important to di cover aggravating and relieving factor o that the pre
ci e ti ue or tructure can be di covered. Patho-phy iology i the tudy of ti
ue that are dy functional, but not yet in a tate of true di ea e. Ultimately

we are looking for what ha been called the manipulable le ion. The three proc
e e of hi tory, examination and palpation hould ideally lead to the ame conc
lu ion . Separate operator performing the three part of the overall a e ment
hould be able to make an independent deci ion ba ed on their finding with eac
h method. In practice, naturally, one operator i u ually re pon ible for the wh
ole operation, but attempting to make an a e ment on one a pect only i a very
good exerci e in thorough analy i . A an example of thi , con ider a patient w
ho pre ent with lower back ache with ome radiation into hi right thigh po ter
iorly a far a the knee. He ha no en ory phenomena of pin and needle or num
bne and ha not noticed any weakne . He tate that it ha come on for no par
ticular rea on but on clo e que tioning he had been rather vigorou in cleaning
hi car the day before. He i righthanded. He admit to having had imilar di co
mfort after ten pin bowling. He i relieved by lying down, although he feel ti
ff afterward . Walking eem to ea e the pain, but if he walk for more than 20
minute , the pain
and pa the patient on to omeone el e who i prepared to follow the patient
choice. It i the practitioner duty to point out the pitfall of any choice, b
ut he mu t ultimately bow to the wi he of the patient. In practice mo t patient
will realize the de ire of the practitioner to help them in the be t way, and
will choo e the method directed. However, there i the con tant problem of the f
inancial con traint placed upon any patient required to undergo private treatme
nt, and the practitioner mu t be reali tic in hi pre cription if it may entail
long-term treatment. The diagno tic proce , in a medical en e, require tanda
rd clinical method kill and under tanding of ymptom , ign and the normal c
our e of di ea e proce e . In an o teopathic en e there i the broader orthopa
edic type of a e ment and there i the o teopathic thinking, ba ed almo t enti
rely on applied anatomy. Orthopaedic and neurological te ting are e ential to d
i cover warning and precaution and mu t precede o teopathic examination. Once
a particular ca e ha had pathological entitie excluded, and ha been deemed u
itable for o teopathic care, then the true o teopathic diagno i will begin. O t
eopathic treatment and the technique u ed can be either general a a form of t
uning the mechanical tructure , or pecific to a particular complaint. General
treatment i a cla ical o teopathic approach taught at many chool of o teopa
thy, and hould not be an excu e for lack of diagno i . It hould be performed f
or a pecific rea on and although it doe not depend on pecific individual tru
ctural diagno i , it mu t, neverthele , depend on palpatory finding . Specific
or regional treatment i ba ed on a hi tory, examination and palpation of the pe
rceived dy function and technique are cho en that have the be t chance of re to
ration of function in a mechanical en e. Some operator have a model of improve
ment of circulation, ome of relea ing tiffne and tightne . Some are mainly
intere ted in relief of pain. Whatever the chief rea on for
Diagno i tart to increa e, particularly if he i carrying anything. He find
the di comfort increa e when he it for a prolonged period and ha noticed tha
t it i difficult to tie hi right hoelace. He ha light increa e of pain when
coughing or neezing and get a tab of pain if he trie to bend too far. He no
tice that when he fir t tand after itting he i lightly idebent to the lef
t, but that thi di appear after a minute or o. Hi job involve walking a lot
, but he find that if he ha to tand till, he get a lot of lower back aching
. He ha had ome lower back di comfort before, but it ha alway been on the le
ft ide and ha never travelled into the lower extremity. The e epi ode are inc
rea ing in frequency and often di able him for a few day at a time. Hi medical
hi tory i uneventful, but he ha had minor meni cal dy function with the right
knee and hi left foot eem to be enlarged lightly. He ha noticed that he of
ten catche hi left heel in hi right trou er turn-up. After taking the hi tory
the patient i a ked to undre to hi underpant and the examination begin . H
e i a ked to tand and we ob erve hi po ture and carriage of weight from behin
d, in front and from the ide. He appear to be rea onably ymmetrical but tand
with a light bend in hi right leg at the knee. When he i a ked to tand wit

h the leg traight he appear to have hi left iliac cre t lower than the right
. The wai t fold i lightly deeper on the right than the left. Hi right hould
er i lightly lower than the left. He ha very light varico e vein evident on
the left calf. Active movement into flexion how ome guarding in the lumbar a
rea and he upport hi weight on hi thigh a he return from flexion. Sideben
ding to the left i full, but attempt to idebend to the right cau e increa e o
f di comfort. Exten ion cau e no problem but he i rather tiff. Rotation i
lightly fuller to the right than the left. If he i a ked to let one knee ag o
that he bear mo t of hi weight on one leg, the right ide of the pelvi drop
far ea ier than the left. In a itting po ition he tend to lump and
9
to upport hi weight on hi hand placed behind him. In upine lying, the pa i
ve hip rotation on the left i much more limited than the right although the rig
ht i the ymptomatic ide. Hyperexten ion in the right knee i much more limite
d than in the left. Pa ive movement te ting of the acro-iliac joint in thi p
o ition how that the left ide i much more rigid than the right. In a idelyi
ng po ition pa ive movement te ting how that the lumbo- acral egment i very
re tricted and tend to idebend to the left with ea e but not to the right. Ne
urological te ting i normal a are the pul e in the lower extremitie . At fir
t ight thi may eem a confu ing jumble of unconnected fact and irrelevant fin
ding . However, from all thi it i po ible to make ome a umption and come t
o a rea oned working hypothe i a to the cau e of the pre enting yndrome. Thi
working hypothe i al o lead to an under tanding of the ca e and a trategy fo
r treatment, management and choice of technique . If we look at the relevance of
all the e factor , everal thing become evident. Every one of the e ymptom g
ive another clue to the overall diagno i . Hi ti ue are telling a tory; we
mu t decipher and unravel the relevance of each of the e factor . The conclu ion
from the hi tory hould accord with the finding on examination and palpation.
Some a pect of the pattern of the problem eem to relate to a mild degree of di
c degeneration and ub equent right- ided facet yndrome. If we treat the ca e
from thi imple viewpoint we are going to have a mea ure of ucce with the h
ort-term pattern, but the recurrence will have to be dealt with a a repeated c
ri i management. Some patient are happy to do thi , but if the underlying cau
e of the problem can be orted out, they are often much happier. The analy i of
each finding follow : 1. The nature of the pain i much more typical of a refer
red pain yndrome rather than a true nerve root pre ure.
10
Diagno i leg ide ha become an over tretch of the oft ti ue on the other i
de. The repetitive nature of the previou epi ode on the other ide and the rap
id re olution, point to imple facet dy function and joint train . The tran fer
of the pain to the other ide and the tendency for it to move more di tally ind
icate po ible di cal irritability. The meni cal dy function in the right knee m
ight be unconnected, but could indicate a tendency to leave that knee in light
flexion when tanding, thu predi po ing to knee train . The enlargement of the
left foot may be incidental, but if he tend to tand with greater weight-beari
ng on the left leg than the right, the foot will pread and enlarge. The catchin
g of the heel of one foot in the turn-up of the other leg i ometime indicativ
e of a hort leg.
2. A right-handed per on cleaning a car doe a lot of idebending to the right,
thu compre ing the right ide of the pine and po ibly cau ing facet appo iti
on on the right. 3. Ten pin bowling involve lifting and quite evere rotation t
o the left; a back that cannot rotate o well to the left may be trained. 4. Pa
in relieved by re t but that feel tiff afterward i typical of di cal irritab
ility with ub equent mu cle guarding. 5. Pain ea ed by walking that increa e w
ith prolonged walking indicate that the pine doe not like idebending and rot

ation. 6. The increa e of pain with carrying weight when walking implicate the
di c again. 7. Prolonged itting increa e irritability in the po terior tructu
re due to the increa ed tretch on the po terior a pect of the di c, ligament
and mu cle . 8. Difficulty in tying the right hoelace point to a re triction o
f right idebending combined with flexion. 9. The cough reflex i indicative of
ome di cal involvement due to the increa e of intra-abdominal pre ure. 10. Pai
n on bending too far i indicative of over tretch of the po terior ti ue . 11.
A ymptom of being idebent on fir t tanding up after itting i indicative of
di cal irritability or facet joint welling producing a protective po ture. That
it ub ide after a few moment indicate that the inflammatory element i not
too evere. 12. Pain on prolonged tanding and po tural ny tagmu  can be indic
ative of poor proprioceptive control of mu cle and i common in ligamentou over
tretch and early di c degeneration. 13. The movement of the pain from one ide
to the other could be due to many cau e ; in thi ca e it appear that a imple
facet yndrome ha been converted to a di c pattern a the compre ion on the h
ort
14.
15.
16.
17.
The finding on examination hould help in the a e ment: 1. He tend to tand
with more weight on the left leg. Thi could indicate a hort leg on the left an
d a compen ation of weight tran fer to the hort leg. 2. Hi increa e of wai t f
old on the right on ymmetrical tanding indicate a hort leg on the left. 3. T
he lower right houlder indicate a mild left convex lateral colio i which i
in accord with the hort leg theory and compen ation to thi . 4. The varico e ve
in on the left calf only are indicative of increa ed weight-bearing on that id
e and poorer venou return. 5. The guarding in flexion i typical of mild di c o
r facet yndrome. 6. The upporting of the weight on the thigh i typical of a
di cal irritability and can indicate nuclear movement and antalgic protection of
the area. 7. The limited and painful idebending to the right might indicate a
facet yndrome,
Diagno i or could be due to lateral di placement of nuclear material in a di c.
The tiffne on exten ion i often due to reluctance to compre the po terior
element of the di c and po terior ligament . The ea ier rotation to the right
accord with the mild colio i produced by the left leg tanding habit and tend
ency of the pine to rotate away from the idebending under prolonged tre . Th
e knee drop te t, where he i a ked to let the weight drop onto one leg at a tim
e, i much ea ier to the left, indicating a tendency to tand with greater weigh
tbearing on the left, confirming the hort leg and di torted po ture theory once
again. The need to lump when itting i indicative of poor control of po tural
mu cle. The need to upport the weight on the hand i indicative of di cal irr
itability. The re tricted hip rotation on the left i indicative of increa ed we
ight-bearing on that ide, further confirming the one ided tanding habit. The l
ack of hyperexten ion of the right knee i indicative that thi knee i often al
lowed to flex lightly. Thi follow the ame theory of one- ided tanding. The
rigidity in the left acro-iliac i further confirmation of the one- ided po tur
e producing a riding up and tiffening of the acro-iliac. The re triction of th
e lumbo- acral joint on pa ive movement te ting accord with the mixed facet/di
c yndrome. The ea ier idebending to the left help confirm the po tural habit
and ub equent di c damage.
11

8.
9.
10.
11.
12.
13.

lightly flexed for o long that it ha been predi po ed to meni cal dy function
and lack of hyperexten ion. The lumbo- acral di c ha tood the on laught of al
l thi for ome time, but ha now become mildly degenerate, and there are ign
of a mild di cal irritability or even herniation. The final traw of the car wa
hing and evere right idebending have jammed the facet of the lumbo- acral egm
ent on the right and the di c at that level ha reacted to the compromi ed movem
ent po ibility and ha become ymptomatic. How can we relate the e finding and
conclu ion to po ible treatment and choice of technique ? Each different di c
ipline of practitioner will naturally have varied idea on how to deal with any
particular ca e. There will be different opinion a to the relevance of the fin
ding and even a to their exi tence at all. A phy ician might be inclined to de
al with thi ca e with re t, mild antiinflammatory medication and advice a to p
o ture and activity. A urgeon might advi e the patient to ee a phy iotherapi t
for traction for the di c and exerci e for the mu cle . A p ychologi t would b
e intere ted in what the tre e are on the patient that have cau ed hi back t
o become dy functional at thi particular time. How would an o teopath deal with
it? The hort-term management would be imilar to the other di cipline a the
need of the ca e are for re toration of function, reduction of pain and rea ur
ance. A uming that there were no other contra-indication , the patient might be
treated a follow . 1. Short-term cri i management of acute pain. 2. Mid-term
improvement of function for re olution of the pre enting yndrome. 3. Long-term
treatment and education for prevention of recurrence . The hort-term cri i man
agement might be advice to u e hot or cold pack or alternation of the e. Gentle
oft ti ue, cro -fibre kneading of the area to relax ome of the pa m
14.
15.
16.
In ummary we have a patient who ha a difference in leg length, hi left leg be
ing horter than the right. He tend to tand with more weight on the left leg t
han the right and ha had repeated epi ode of left- ided facet yndrome. Hi ri
ght knee ha been kept
12
Diagno i pre ion factor in the facet joint and vertical adju tment i particu
larly u eful for thi . Firmer articulation of the left hip and acroiliac would
be u ed a time progre e to increa e mobility there. The lack of exten ion of
the right knee would need to be addre ed, u ing acce ory movement articulation
into medial and lateral gapping and traction. Further advice about po ture and
exerci e to tretch the tight left ide and po ibly trengthen the lumbar erec
tor pinae might be nece ary. The long-term approach would be to extend the tre
atment interval a the ymptom improve and give natural healing a chance to et
tle the di c. The po ture would be reempha ized to en ure that the patient wa
tanding more ymmetrically. An a e ment would need to be made a to whether a

heel lift wa indicated. So, we can ee that a whole lot of eemingly irrelevant
and confu ing finding can be integrated into an a e ment, a diagno i , a pro
gno i and a choice of technique to be u ed in treatment.
could be performed with the patient idelying. Thi po ition would be better tha
n prone a it would avoid exce ive exten ion that might occur in the prone po i
tion. Supine articulation to both acro-iliac might follow. If the pain i not
too evere, leg tug high velocity thru t (HVT) to the left acroiliac to mobiliz
e it and help de- tre  the right could be u eful. Sidelying flexion articulat
ion to the lower lumbar area to increa e drainage and free movement could be u e
d. It would be helpful to the patient to explain why the injury had occurred and
to di cu the po ible longer-term management. The mid-term approach would be
to try to re tore full function to the lumbo- acral egment with pecific HVT di
rected to the facet joint . Minimal lever approache would be u ed to avoid tor
ioning the di c. The HVT would be performed mo tly into compre ion. Providing t
he patient could extend without exce ive pain, a vertical adju tment would be h
elpful to reduce pre ure on the di c and to gap the facet in traction. Onelegg
ed tanding habit often lead to a comCLASSIFICATION OF OSTEOPATHIC TECHNIQUES
4
The original cla ification of o teopathic technique wa evolved at the turn of
the twentieth century and wa divided into the general term of oft ti ue, ar
ticulation and thru t. While the e were u eful, they were limited and cau ed om
e problem . They were re tricted in term of dialogue between practitioner , or
or in ability, to communicate the exact purpo e of the technique being performed
. Variou attempt were made to recla ify the different approache , and in the
early 1970 a team teacher at the BSO formed a new cla ification li t. Althoug
h the e have been modified lightly ince that time, they remain broadly the am
e today. Other y tem of cla ification exi t, but I have cho en to keep to the
BSO y tem in thi work. When omething i cla ified, it i unfortunately limi
ted by that very cla ification. De pite thi , it i clearly nece ary to have
ome form of definition of the actual procedure u ed o that teaching can be y
tematized. It m u t be realized however, that any cla ification cau e ome p
roblem , and will not nece arily be in accord with the particular approach of a
n individual practitioner. For the e rea on , the cla ification that have been
defined have been de igned in an attempt to tate what i actually being applie
d rather than what i trying to be achieved. In conventional medical practice it
i relatively ea y to define a particular medication, and the do age. Thi i f
ar le imple in manual medical y tem . If one practitioner ay he ha perfor
med a particular procedure, who i to define the efficiency and effectivene of
thi tatement? Thi i not to ay that we hould not be trying to rationalize w
hat we are doing. It i only too ea y to ay that it i an art, and therefore ca
nnot be cla ified. It wa felt, therefore, that we hould make our cla ificati
on for perceived re ult in tead of application. The cla ification being u ed to
day i broadly divided into three main grouping . There i inevitably ome overl
ap, and ome particular method are u ed far le often than other . Neverthele
, they are defined o that they can be de cribed in a way to make communication
ea ier. Education of tudent i al o much more efficient with a tructured cla
ification, although they quickly realize that no cla ification cover every e
ventuality. The three grouping are: (i) rhythmic technique , (ii) thru t techni
que , and (iii) low velocity tre technique .
RHYTHMIC TECHNIQUES The e are technique where the u e of rhythm i fundamental
to the procedure. They are repetitive in nature but are not imply the ame move
ment repeatedly; they are varied by all the u ual modifying factor . All o teopa
thic technique i varied according to the re pon e from the ti ue and tructur

e being worked. According to the perception of the mechani m of a particular te

chnique, o it will vary in rate, rhythm, direction, force, duration and number
of repetition . There will al o be variation in all the other factor po ible
to produce the optimum re ult in the particular circum tance . In many way the
14 Cla ification of o teopathic technique control and variable in o teopathic
technique are dependent on palpatory awarene a much a perceptual diagno tic
factor . Palpatory kill will naturally vary between different individual , but
the main objective of all form of manual therapy i to improve function. Impro
vement of function i de igned to increa e comfort, reduce pain and re tore norm
ality to part of the neuro-mu culo keletal y tem that are found to be working
at le than their optimum efficiency. Thi will vary according to the demand o
f the individual and their environment. O teopathy ha been variou ly defined ov
er the year , but one definition of Littlejohn, a tudent of A.T. Still, and the
founder of the B S O , wa , O teopathy i the cience of adju tment. There ar
e many philo ophical argument relating to thi tatement, but the objective of
technique i to allow the optimum ability for the body homeo tatic mechani m to
perform thi adju tment. Rhythmic technique , by their nature are de igned to
lowly re-e tabli h movement, circulatory flow, and drainage, and to remove blo
ckage in function in the omatic tructure . Rhythmic technique can be u ed a
treatment by them elve or a preparatory procedure for other categorie of te
chnique. The choice of when to apply a given technique in a treatment e ion na
turally depend on the purpo e of the technique and the de ired effect. Traditio
nally rhythmic technique are u ed to loo en ti ue o that high velocity techn
ique can be applied more ea ily. Thi relegate them to a po ition of econdary
procedure , which i unfortunate. They are powerful in their own right and hou
ld have a much more important role than imply padding out a treatment while pre
paring for more pectacular technique . Suitably applied rhythmic technique can
be a whole treatment in it elf, and form the main tay of mo t o teopathic treat
ment e ion . For the ake of rea onable brevity the de cription of each techn
ique will not repeatedly tate the variou modification po ible. The reader i
referred to the ection relating to modifying factor and a ked to con ider eac
h of the e a applied to each technique cla ification. A rhythmic technique i
divided into eight categorie . 1. 2. 3. 4. 5. 6. 7. 8. Kneading Stretching Artic
ulation Effleurage Inhibition Springing Traction Vibration
KNEADING Kneading can be defined a a rhythmic queezing and ma aging of mu cle
and other oft ti ue to relax them by reducing fluid conge tion and tonic ir
ritability. The mechani m by which thi produce a re ult i open to que tion. S
ome ay that the effect i one of reflex balancing. Some think that the re ult c
ome from fluid interchange and lymphatic drainage. There i al o a powerful p y
chological and oporific effect. The technique i applied in a low and careful
way to produce the be t re ult. The be t rhythm and tempo for any given ca e wil
l only be found by adju ting the common pattern lightly to uit the particular
patient. The amount of pre ure applied, and the duration of the technique will
al o vary according to the re ult obtained. Many different applicator can be u
ed. The mo t common way i to u e all or part of the heel of the hand or ometim
e the pad of the finger . The technique can be a pu hing or a pulling, and the
hand often work in oppo ite direction o that the amplitude of each i reduce
d according to the quantity applied by the other. The direction i u ually acro
the fibre of the mu cle, but can be diagonal or even longitudinal in ome ca
e . The mo t critical element i the finite u e of time when the pre ure ha be
en applied. If a mu cle i imply kneaded repeatedly without
Rhythmic technique attention to the duration of pre ure, ome change will be p
roduced. If the kneading force i applied, and then held for a few econd , the
mu cle will be felt to relax and melt. Thi en ation can be likened to a hot
knife through cold butter. The initial pre ure produce little change, and then
the knife ink in and i felt to work it way through the butter a it melt a
head of the blade. Kneading hould not be painful, but a en e of pre ure i in

evitable. Good handling to produce the be t patient cooperation and relaxation i

very important. The operator hould be aware of any di comfort being produced
and modify the pre ure accordingly to be at a balanced point ju t hort of pain
. The dividing line between di comfort, pain and effective pre ure i often dif
ficult to judge, and con tructive comment from a fellow tudent or practitioner
are u eful. The depth of the pre ure hould be governed a much by body weight
a by the force applied with the hand. The en e of relaxation i gained by the
operator proprioceptive mechani m becoming aware of the collap ing ten ion r
ather than any tactile en ory input. The operator hould lightly ten e hi han
d and arm to effectively enhance thi proprioceptive awarene . Very light i om
etric ten ion u ed in thi way give a much better feedback of ti ue re pon e.
15
imum cooperation and relaxation, and the technique i applied ju t hort of pain
, but at the border of di comfort to produce the be t re ult. The duration of t
retch applied will vary in each ca e according to the re pon e from the ti ue ,
but hould be long enough to allow change to take place. If tretching i appli
ed and relea ed too quickly, a tretch re pon e will be induced, and an oppo ite
effect from that de ired will take place. If a tretch i maintained, there ho
uld be a en e of collap ing ten ion, and a en e of give from the tructure
a they relax and lengthen. The direction of tretch i often felt to change a
thi take place and the operator may find the path changing quite a lot at the
end of the technique. If thi happen , the technique may become a myofa cial typ
e of relea e, but thi i quite acceptable and how an awarene of ti ue adap
tation during treatment. Stretching technique hould be relea ed lowly to avoi
d reactive ten ion in the ti ue from the tretch. Rhythm and tempo are al o va
riable that need to be con tantly adju ted to uit the patient. ARTICULATION Th
i old o teopathic term ha been retained and relate to the u e of repetitive p
a ive movement, u ually employing a lever and fulcrum. The u e of the lever all
ow the effect to be enforced without the application of high level of force. T
he main difference between articulation and imple pa ive movement i that the
operator hould be con tantly en ing the re pon e from the ti ue under hi ha
nd. He i then able to mea ure the requirement and inten ity of pre ure accord
ing to what he i feeling. Articulation can be performed over a wide arc or over
a very mall amplitude of movement according to the requirement of the ca e. A
n example of a wide arc would be taking a houlder from part abduction to full a
bduction. An example of a mall amplitude
STRETCHING Stretching can be defined a a eparation of both end of a mu cle or
oft ti ue tructure from each other to lengthen it. The purpo e i to attempt
to improve function and allow better mobility in the tructure being worked a
well a the articular tructure that they may limit by their exce ive ten ion
. Stretching can be applied with a very hort lever to influence pecific intraarticular or egmental tructure . It can be applied u ing a long lever to influ
ence the extra-articular tructure over a larger area. Good handling i critica
l to en ure max16 Cla ification of o teopathic technique would be articulation of a lumbar e
gment from almo t full flexion to it complete range. Many practitioner will ad
d a mall empha i of movement at the end of range. Thi very mall bounce can
be a u eful way of producing more rapid change in ti ue a well a allowing a
more accurate a e ment of their reactivity. Articulation can be in ingle dir
ection of movement or u e multiple vector of force. If multiple vector are u
ed, it i ea ier to approach a motion barrier which i not at the end of true ra
nge and i , therefore, more under operator control. Working at thi manufactured
barrier would eem to be inefficient, but will be found to produce the fa te t
change in mobility of the actual barrier when ub equently te ted. The movemen
t doe not produce the ame di comfort a cap ular tretch, but will, neverthele
, influence joint receptor mechani m in a very efficient way. The chief gover

ning factor in thi type of articulation i good operator control. Thi can only
come from a firm yet comfortable grip that take command of the part in uch a
way a to promote confidence. Although the vector of force can be de cribed ind
ividually, they are be t applied in a combined way to ab orb the free play and
lack in the ti ue . Thi prevent the natural tendency of the ti ue to e cape
from the induced ten ion tate. may be applied to be counter-irritant. Effleura
ge can be u ed a a light introduction to gentle kneading technique that increa
e in inten ity a the ti ue relax and conge tion decrea e . Mo t o teopath d
o not u e lubricant when performing technique a their u e can make ati factor
y grip difficult. A common exception to thi i when effleurage i applied, a
ome form of kin oil or ma age cream can help prevent orene and may make th
e technique ea ier to perform. Several proprietary type are available with a va
riety of claimed therapeutic propertie . If it i de ired to have a light lubric
ant effect, a fine talcum powder may uffice.
INHIBITION Inhibition i the one exception to the rule of tating what i being
applied in tead of tating the perceived effect of a technique. The term i reta
ined a it i an hi torical o teopathic cla ification. In practice it con i t
of pre ure applied for fairly long period . The pre ure i lowly applied and
maintained for up to a minute or o and then lowly relea ed. It i u ually appl
ied over a mall area or point. The rhythm i alway low and need a con tant f
eedback from ti ue re pon e and reaction. It i applied with increa ing pre ur
e a the patient exhale , and i maintained until uitable change i perceived.
An example would be pre ure with the pad of the thumb over the lower attachment
of the levator capulae in a ca e of acute torticolli . Con idering recent re e
arch, inhibition may have it effect on ti ue fluid interchange rather than by
balancing the afferent and efferent outflow a wa previou ly thought. Thi doe
not negate the benefit of the technique, but imply explain in a different way
what we can feel with our hand . Exce ive pre ure will have the oppo ite effe
ct in that irritation can be produced. Clearly thi i unde irable and good palp
atory awarene hould avoid thi problem.
EFFLEURAGE Effleurage ha been borrowed from the armamentarium of the ma age pr
actitioner. There are everal ituation when a rhythmically applied movement of
light force and low rate i nece ary. It i u ually u ed on the uperficial t
i ue to produce a drainage effect on lymphatic channel . In traditional ma
age, the central area i cleared fir t to make room for the fluid, and then the
effleurage i applied from peripheral to central. It i often u ed over area wh
ere conge tion i greate t to produce a circulatory re pon e to aid deconge tion
. It
Thru t technique If  timulation a di tinct from irritation i de ired, a fa
ter o cillation will be required, but active contraction technique are probably
more valuable than manual procedure . SPRINGING Springing refer to repetitive
graduated pre ure over a bony point. It i ometime combined with very hort l
everage . Springing i u ually lowly applied and relea ed u ing the propriocept
ive re pon e from the applicator and the operator arm to en e the optimum pre
ure. It can be u ed a a diagno tic procedure over pinou proce e to a e
their reactivity, tenderne and re i tance. The direction of force in pringin
g i often into acce ory range of movement. A given joint may be maintained in
a certain po ition by operator control, and then pringing i applied in anothe
r direction to improve quality of movement. It i u ually applied again t the re
i tance of the table o that operator body weight can be employed more ea ily.
It i u ually performed from a point approaching end-of-range up to end-of-range
rather than through a long amplitude. Springing i , in effect, very hort lever
articulation.
17
being tractioned again. The effect will be one of drainage and circulatory inter

change a well a opening of intervertebral foraminae to attempt to relea e nerv

e root pre ure. Manual rhythmic traction i unlikely to be a trong a mechani
cal traction, but will be much more ea y to control a the operator ha a con ta
nt feedback from the tructure . Some operator u e trap to a i t in the e te
chnique , and ome find mechanical traction apparatu beneficial if more u tain
ed traction i nece ary. Many modern mechanical traction table have an intermi
ttent etting de igned to mimic the manual rhythmic traction. The main advantage
of the mechanical traction table are that it i po ible to u e more power tha
n can be applied by hand and that the table doe not tire a doe a human operat
or.
VIBRATION Some practitioner u e manual vibration technique over hollow organ
or inu e in an attempt to increa e drainage and enhance circulatory flow. Vibr
ation i difficult to u tain for long period , but when u ed, i u ually applie
d at a fairly fa t rate for everal hort interval in a treatment e ion. Con
iderable mu cle control of the operator arm i required, and the o cillation
i performed by rapid alteration of mu cle ten ion or by fa t light tapping.
TRACTION Rhythmic traction i manually applied traction performed to a point or
area with ome relea e and repeated everal time . When applied to a point, the
operator attempt to block adjacent joint from movement, and then u ing a uita
ble grip, trie to eparate the target joint urface with the traction. When ap
plied to an area, a comfortable grip i gained, and then the traction i applied
until the area i felt to be affected. The amplitude i an important element a
the traction mu t reach the target ti ue or area. Rhythmic traction of thi o
rt i u ually applied from a point of ome tretch to a further point rather tha
n from no tretch to full tretch. The ti ue are only partly relea ed from the
tretch before
THRUST TECHNIQUES Thru t technique can be defined a technique u ing a ingle
application of force u ing high velocity and low amplitude. The objective of the
technique i to direct force to a pecific point, area or tructure. It i not
u ually nece ary to perform thru t technique at the end of a range of movemen
t. By combining many component , a barrier i formed which i at a cumulative en
d-of-range rather than at an anatomical end of range. If ufficient peed can be
attained, the inertia of
18 Cla ification of o teopathic technique the ti ue can form enough re i tan
ce to permit efficient thru t procedure . They will reach a target ti ue or tr
ucture without reaching the end of range of the joint. Thi i much le potenti
ally traumatic and uncomfortable, and allow a chance to u e thi category of te
chnique in a wider range of patient . It i accepted that not all practitioner
can achieve thi ideal of ultra rapid acceleration and very controlled braking f
orce. Inevitably ome thru t technique will be of a lower or intermediate veloc
ity, but the aim of minimal amplitude remain con i tent. A force hort of adequ
ate joint eparation i not going to be traumatic; an exce ive force or amplitu
de i potentially dangerou . Thru t technique are u ually performed parallel or
at right angle to the plane of the joint and in a direction de igned to break
joint fixation in the mo t efficient way. The barrier to motion in a well-po iti
oned thru t technique ha a characteri tic feel of potential or dynamic ten ion.
It i nece ary to balance all the available component of a particular thru t
technique to achieve the be t barrier, which i not nece arily the maximum ba
rrier. Original o teopathic thinking wa traditionally de igned to rever e the
path of a le ion. Diagno i wa concerned with di covering the pathway of the l
e ion, and thu the correct path of correction. Although thi i an attractive
concept it ha become accepted that many ca e do not require thi type of anal
y i . A imple breaking of fixation in the direction of optimum barrier i uffi
cient to improve range and quality, and po itional correction i often unnece a
ry and exce ively complex. Attempt to cla ify fixation by direction of le i
on can be extremely limiting, particularly for tudent who e palpatory awarene

 i not able to perceive the fine difference . There are certainly ome ca e w
here pecific direction play a part, but the e are relatively rare, and re ult
are ju t a good if thi element i largely ignored. Thi approach anger ome
traditionali t . It i intere ting, however, that their detailed analy i and de
ci ion of le ion correction direction will be found to give the ame path of opt
imum barrier en e achieved with far le effort by the method de cribed here.
The palpatory cue of barrier en e are le likely to give the operator a wrong
me age than a conceptional model of le ion correction analy i . Thi mean tha
t traditional de cription of a rotation le ion or a idebending le ion are beco
ming le common in chool adopting thi principle. There may be a de cription
of a egment that i reluctant to rotate or idebend, but thi information can b
e immediately introduced into the choice of vector of force mo t likely to brea
k fixation. If an accurate palpatory a e ment i not po ible in a tatic or d
ynamic examination, the patient can be placed in a thru t po ition, and the vect
or and component adju ted to find the optimum barrier. The re i tance to parti
cular movement pathway will then become more evident a much of the joint play
will have been ab orbed, and the be t path can be cho en. Thi place greater em
pha i on good operator control and balance of available component , and i not
an excu e for poorly applied technique kill. If anything, it require greater
kill, a the deci ion about the be t pathway to produce the relea e will change
lightly a the patient i moved into the technique po ition. Neither i thi an
excu e for merely popping joint . The purpo e of any technique i improvement
or re toration of normal function, not imply the completion of the technique.
Re-a e ment of function, range and quality i the critical element. Traditiona
l manipulation i performed at the end-of-range, and then by the application o
f overpre ure beyond the point of control of the patient. Well-controlled o teo
pathic thru t technique i not u ually performed in thi way. The act of inducin
g multiple component produce a point of u eful ten ion that i hort of the en
d of anatomical range. The thru t i performed in a cho en direction
Thru t technique while the econdary component are maintained by operator cont
rol to make the barrier available. The ame amplitude of the primary lever appli
ed without the econdary component would not be effective. It i , therefore, th
e under tanding and control of the e econdary component which make thi o teop
athic approach different from manipulation a it i u ually defined. Current cla
ification divide thru t technique into five broad heading . 1. 2. 3. 4. 5. C
ombined lever and thru t Combined lever and thru t u ing momentum Minimal lever
and thru t Non lever and thru t Non lever and thru t u ing momentum
19
performed coincident with a pull on the wri t . In many technique it may be dif
ficult to eparate the exact principle , and thi i not a problem if everything
i going according to plan. If, however, the technique i not producing the de
ired re ult, analy i according to ba ic principle may reveal the rea on and, t
herefore, po ibly the olution. COMBINED LEVER AND THRUST USING MOMENTUM Thi c
ategory of thru t technique i , in reality, a ubdivi ion of the previou one de
cribed above. The momentum component i introduced for everal rea on . Some pa
tient may find the po ition for a combined lever and thru t technique can be th
reatening and uncomfortable and the u e of momentum help to avoid thi . Some op
erator find that it i difficult to accelerate ufficiently rapidly from a tat
ic po ition to achieve enough controlled power to cau e facet gapping. Momentum
will aid the e ca e a it i much ea ier to overcome the inertia of the ti ue
if a gentle o cillatory movement i u ed in the primary lever direction. Care m
u t be taken to avoid the natural tendency to u e momentum in more than one para
meter of lever direction. If thi i allowed, it i far too ea y to take the par
t into a compound lever po ition which i beyond that de ired. The momentum mu t
be u ed in the accumulation of optimum lever po ition and not u ed to empha ize
everal lever at once. Momentum can be u ed while the part i held in a near o
ptimum po ition by rolling the body back and forth. Thi allow the fine tuning

to take place on the move, and the final en e of be t barrier can be palpated m
ore ea ily. An example of thi might be a typical lumbar roll type of thru t man
ipulation. The patient i po itioned for the thru t, and then gently rocked back
and forth while maintaining the lever component almo t con i tent. Thi i not
an increa e and decrea e of
COMBINED LEVER AND THRUST Thi variation u e a thru t applied at or near the dy
function or le ion, with or without application of exaggeration of the leverage
. A an alternative a thru t could be applied at or near the extremity of a leve
r remote from the le ion. A tatic fulcrum may be created by pre ure or fixatio
n at or near the le ion. A combination approach i al o po ible. A an example
of the e different type con ider: 1. Thru t at the le ion point. A typical cerv
ical thru t technique u ing idebending to one ide and rotation to the other. T
hi could be performed in a variety of patient po ition : upine, idelying or
itting. 2. Thru t at the extremity of a lever arm. An example of thi might be a
upine midthoracic thru t where the operator hand i placed under the patient
 body and the thru t i applied to the arm cro ed in a variety of way over
the che t. 3. Combination thru t at a le ion point and at the extremity of a lev
er arm. An example of thi variation would be a itting mid-thoracic, knee in th
e back technique. It would be performed with the operator hand cla ped around
the patient wri t , and a imultaneou pu h with the knee i
20 Cla ification of o teopathic technique lever , more a rolling of the body w
hile the lever are held. At the optimum point in the rolling, the lower compone
nt can be rotated toward the operator in the u ual way more ea ily a the body i
already moving in thi direction. The u e of momentum al o enable increa ed f
orce to be applied when ab olutely nece ary. The barrier can be accumulated in
the u ual way, and the momentum introduced a an increa e of acceleration in the
cho en direction. Thi often prove to be of greater efficiency for mall opera
tor , particularly when working on large patient . Naturally, due con ideration
mu t be paid to precaution again t applying an exce ive leverage in uch a ca
e. Although thru t technique i normally performed in traight line , there are
no traight line in the body, and momentum inevitably induce ome curving a pe
ct to the force. Thi may help to find the optimum direction for any given thru
t. MINIMAL LEVER AND THRUST Minimal lever and thru t can be further divided into
two broad ubdivi ion . There i the true minimal lever and thru t where the pa
rt i po itioned in a little lever po ition a i po ible, and an extremely ra
pid thru t i performed. Thi i de igned to break fixation before other ti ue
deform under the pre ure and require very rapid application and braking by th
e operator. Thi type of thru t method i probably the mo t difficult to perform
, and many operator find it difficult to acquire the particular kill nece ary
to make thi method effective. There i al o the more common method of minimal
lever and thru t which employ multiple component in an attempt to limit the am
plitude of each. The more component u ed, the le range will be nece ary and
the final thru t po ition can become clo er to the midline. Thi i de irable be
cau e it i far more comfortable, and hould become potentially le traumatic.
It i nece ary to be aware of the variou component available and to apply the
m y tematically while te ting the primary lever direction to en e the accumula
tion of compound barrier. If the primary lever i not felt to accumulate to a u
itable barrier, more component can be introduced, or lightly greater amplitude
introduced to tho e already in u e. The primary lever hould then be felt to be
come firmer and the re i tance more cri p and available to the thru ting hand. A
lthough thi can be thought of in term of minimal lever, it i rather maximal n
umber of lever , but minimal quantity of each. It i important to leave ome fre
e play available o that the variou component can be balanced again t each oth
er in the be t combination for the patient, the operator and the technique. Acqu
i ition of kill in thi category of thru t technique i well worth the con ider
able effort required. Re ult are often fa ter, with le di comfort during and
after treatment, and ti ue reaction and po ible trauma are con iderably reduc
ed. Thru t technique that gap facet joint without training urrounding ti ue

 i much more comfortable, and in the pre ence of local ti ue hortening may b
e the only way that they can be reached. To be effective, treatment con i t of
a combination of approache , not only thru t technique , and uitable mobilizing
u ing other categorie of technique will be nece ary to get a good and la ting
re ult. U ing thi type of thru t technique it i al o po ible to manipulate m
any ca e where conventional full lever technique might be impo ible. An examp
le might be a ca e where there i a di c prolap e. If full rotatory thru t i ap
plied there i con iderable danger of further impinging a nerve root, or of allo
wing further extru ion of di c material. Minimal lever technique might allow the
facet joint to be gapped a the di c i not tor ioned anywhere near a much a
in full lever technique . Relea ing the facet might be a u eful way of helping
to promote fa ter healing. Caution
Low velocity tre technique i clearly nece ary, a although the technique m
ay then be po ible, i it a de irable procedure? NON LEVER AND THRUST Non lever
and thru t technique i a type of thru t where the operator applie a force to
a bony point aiming to break fixation in a facet or joint tructure. Some prelim
inary compre ion may ub titute for the u e of lever , and high peed i often
nece ary to overcome the re i tance of the facet before the force i di ipated
through other ti ue . Thi type of approach i more commonly u ed by chiroprac
tor , but many o teopath find it of u e in ome ca e . An example might be a pr
one thoracic thru t technique where the operator u e the hand applied to oppo
ite ide of the ame or adjacent vertebrae. The thru t i made directly toward
the table or into a idebending or rotation direction according to the need of
the ca e.
21
cranio- acral technique may be more acceptable. The BSO cla ification embrace
all the e method under the broad heading of low velocity tre technique. The
four ubdivi ion of the e technique are: 1. 2. 3. 4. U ing U ing U ing U ing
u tained u tained u tained u tained leverage traction pre ure articulation
The common factor i the maintenance of a u tained po ition, pre ure or moveme
nt and the waiting for a re pon e from the ti ue . They are all lowly applied
u ing en e of optimum relaxation and change for the better in ti ue function.
They are reactive technique rather than direct action technique and the reader
i referred to the ection relating to indirect technique for fuller detail . U
SING SUSTAINED LEVERAGE An example of a low velocity tre technique u ing u t
ained leverage might be a gently held combined lever and thru t po ition where t
he operator wait for a en e of ti ue relea e and collap ing of barrier. Somet
ime the barrier will be felt to ea e in one direction, and further barrier can
be addre ed in lightly different direction . Thi can be likened to peeling a
way the layer of the dy function. Example from the categorie of indirect tech
nique might be functional technique or ome type of mu cle energy technique. U
SING SUSTAINED TRACTION An example of low velocity tre technique u ing u tai
ned traction might be a di engagement of a cap uliti in an acute houlder. A
firm traction might well irritate the ti ue , but u ing a careful hold and main
taining a very light traction until ti ue relea e i felt might be more appropr
iate. Example from the categorie of indirect technique might be ome of the ho
ld u ed in myofa cial technique.
NON LEVER AND THRUST USING MOMENTUM A thru t u ing non lever force and u ing mo
mentum recognize that ome operator find momentum aid the di covery of the op
timum en e of barrier in their patient. The technique i the ame a any other
non lever thru t, except that o cillatory momentum i induced in the direction o
f the final thru t. At the optimum moment, the thru t can be applied a an empha
i of one of the e o cillation . LOW VELOCITY STRESS TECHNIQUES The term low ve
locity tre technique may be unfamiliar to many practitioner . The term mu cl
e energy technique, functional technique, train and counter- train technique, m

yofa cial technique, harmonic technique, pecific adju ting technique, gentle th
erapeutic manipulation, neuro-mu cular technique and
22 Cla ification of o teopathic technique USING SUSTAINED PRESSURE An example
of low velocity tre technique u ing u tained pre ure might be pre ure appl
ied over a mu cle and fa cial area until a en e of relea e i perceived. Althou
gh thi overlap with inhibition from the ection relating to rhythmic technique
, there are ubtle difference . Pre ure i u ually lighter than that u ed in in
hibition, and the force can be directed at bony tructure in tead of purely oft
ti ue tructure . Example from the categorie of indirect technique might be
ome of the hold u ed in train and counter- train technique. USING SUSTAINED A
RTICULATION Low velocity tre technique u ing u tained articulation i really
a conglomerate of the previou ubcategorie and might include many of the cran
io- acral technique . Some functional technique al o fall into thi heading, a
doe harmonic technique.
CONTRA-INDICATIONS AND PRECAUTIONS
5
All y tem of medical treatment have contraindication to their u e, or the tim
ing and do age of their u e. There i more than one way of looking at contra-ind
ication to o teopathic technique and treatment. Although there are ome deficie
ncie in the y tem, thi book will continue u ing the notation of the de cripti
on of ab olute and relative contraindication . It provide ome u eful guideline
that are not too difficult to follow. Some authoritie feel that the manipulat
ive pre cription mu t be guided only by which type of manipulative technique i
uitable and po ible for the ca e. The idea i that no ca e i beyond the aid o
f o teopathic treatment. The choice of technique will be made with due con idera
tion for all the factor pre enting. Procedure will be u ed which cannot be exp
ected to produce any damage, whatever the condition. In thi way, treatment of e
ven a terminally ill patient can be undertaken. If there i any mall benefit ga
ined, even for a very hort time, thi can be ju tified. I have ome ympathy fo
r thi type of thinking, but feel that it i inappropriate for a tudent to be e
xpected to work in thi way. For thi rea on the con ideration of ab olute and
relative contra-indication remain in u e. A in all kill it i po ible to de
velop a ixth en e for warning and precaution . If an experienced operator put
hi hand on a patient, there will often be an immediate awarene of a di tur
bed ti ue tate when there i any rea on for caution. Thi ixth en e i a com
bination of learning and experience. Many hidden cue are being u ed that cannot
even be identified on a con ciou level. It can become a very reliable method of
deciding to top and re-con ider in a given ca e. Thi mu t be in addition to r
ea oning and normal caution, but mu t not become a ub titute. It i far better
to be too cautiou and to be wrong, than to be not cautiou enough, and be wrong
! Hind ight give 20/20 vi ion, but thi i no comfort to a patient hurt or, wor
e, permanently or even fatally damaged. Until thi ixth en e ha been develop
ed over a con iderable pa age of time, ome rule are advi able. Ab olute contr
a-indication relate to ituation where certain technique hould not be u ed.
It would be rare, however, for no treatment at all to be po ible. Relative cont
raindication relate to certain ca e that require pecial caution for an identi
fied rea on a to the choice of technique. A experience grow , ome technique
previou ly con idered unwi e may become po ible with uitable precaution for t
he ca e. Thru t technique are virtually out ide the active control of the patie
nt and joint may be taken to their limit of anatomical movement where ligament
and cap ule are put at ri k of damage. Thi mean that contra-indication to t
hru t technique will generally be more pecific than for other type of techniqu
e. However, trong articulation, pringing, or very firm oft ti ue kneading ar
e powerful in their own right and al o require caution. Adhe ion around joint
tructure are often tronger than the normal ti ue and force applied may damag

e normal ti ue before adhe ion are broken. In the pre ence of adhe ion , forc
e i
24
Contra-indication and precaution reduced. In my view it i dangerou to place
ab olute reliance on thi . In ome ca e , particularly when u ing violent techni
que , thi may be true. Traction, in it many gui e , i a valid and u eful meth
od of treatment, but to u e it to render afe an otherwi e un afe procedure eem
an inadvi able approach. Exce ive reliance on any ab olute rule uch a thi
i re pon ible for a rigid approach with no po ibility of variation for differe
nt circum tance . The o teopathic approach i to make con tant ubtle change ac
cording to the need of the ti ue at the time they are being worked. ABSOLUTE
CONTRA-INDICATIONS Situation where a particular technique i ab olutely contraindicated include tho e in which there i the po ibility of ti ue damage if th
e technique i performed. It i better to think of ti ue rather than condition
, a there may be condition that are unknown and that are, therefore, not diag
no ed. If ti ue are the main con ideration, applied anatomy and phy iology are
the guiding factor . A an example of thi , con ider a patient who complain of
increa ing back pain the longer he i upright. He get ome relief when lying d
own, but i much wor e when carrying any weight. He ha increa ed pain when de c
ending in an elevator a it draw to a top and find that even lifting an arm o
ut in front of him cau e back pain. Thi pattern of ymptom i typical of a we
ight-bearing tructure giving pain on increa ed loading. The two main weight-bea
ring tructure in the lower lumbar pine are the di c and the vertebral bodie
. Di c di ea e i not in it elf a contra-indication to treatment, but will alter
the choice of technique. Vertebral body damage i clearly a condition where tre
atment mu t be withheld until a clear diagno i ha been e tabli hed. If there i
bony weakne due to a econdary cancerou depo it, any technique putting forc
e through that egment would be unwi e. Many other cenario
often increa ed in an attempt to break them down and the ri k factor increa e p
roportionately. Generally, the horte t amplitude po ible con i tent with the p
roduction of the de ired re ult i to be encouraged. Even the highe t velocity,
if combined with ultra hort amplitude can be made relatively afe. Naturally, th
i i a kill-dependent factor that require time to develop and not every pract
itioner i going to be able to acquire thi kill. The increa ing u e of the cat
egory of minimal leverage technique help to reduce ri k and many ca e previou
ly incapable of being thru t can be treated in thi way. Caution i till nece
ary, however, a force generated can till damage vulnerable ti ue . A deci io
n mu t be made a to whether a given ca e i treatable at all and if it i , whic
h technique are mo t likely to help deal with the pre enting problem. If a ca e
i deemed to be untreatable u ing any category of technique, uitable con idera
tion mu t be given to method of referral and the patient mu t be informed why t
hi deci ion i being made. If thi i not done, there i a danger of them going
to another manipulator who may not recognize the contra-indication, treat the p
atient and cau e them damage. A ca e which i not going to be helped by treatmen
t i contra-indicated, not becau e harm will be done, but becau e no good can be
done. In the pre ence of a eemingly hopele  ca e where many other therapie
have been u ed, there may be a ca e for ome trial treatment. It i a good id
ea to make a contract with the patient to try a few treatment and if there i
no good re ult, to review and recon ider. There i then le of a problem about
treating apparently hopele ca e . There i a chool of thought that feel tha
t the u e of high level of traction can render all manipulative procedure  af
e. They feel that a joint urface are eparated and foraminae opened, the cha
nce of nerve or blood ve el damage and impingement i
Ab olute contra-indication could be de cribed to illu trate ti ue analy i but
applied anatomy and knowledge of ti ue behaviour in health and di ea e i the
key. The mo t dangerou condition are tho e that could weaken tructure and o

could po ibly lead to a fatality or at be t a evere injury. The greate t liab

ility to thi po ibility i in the upper cervical region where inherent weakne
in the vertebral ligamentou upporting tructure may lead to nerve or ve el
damage if un uitable force are applied. The vertebro-ba ilar y tem i particu
larly vulnerable to damage if exce ive tor ion i u ed. The re ult of thi cou
ld be cata trophic and ome te t exi t to a e the po ibility of potential t
rauma. No te t are an ab olute afety creen, but may help to eliminate the mo
t obviou ca e from having ill-advi ed technique and treatment. There i no ex
cu e for in ufficient diagno tic care and lack of precaution that could amount
effectively to negligence. Every precaution that i nece ary mu t be taken befo
re undertaking any treatment to vulnerable area . The te t mo t often quoted a
u eful creening in the upper cervical pine follow: 1. EXTENSION TEST Have the
patient eated and lowly bring hi head and neck into exten ion. Then idebend
and rotate it to one ide and then the other. Any evidence of ny tagmu , dizzin
e or dy arthria hould cau e the operator to abandon all attempt at thru t te
chnique and hould lead to an inve tigation of the rea on for the ymptom . Th
e e are cla ical ymptom of po tural hypoten ion which i indicative of compro
mi ed vertebro-ba ilar circulation. 2. HAUTANTS TEST Have the patient eated an
d a k him to tretch both arm out in front at houlder height. Have him clo e h
i eye and then extend the neck and idebend and rotate it to either ide. If o
ne arm ink and pronate ,
25
thi i a ign of impaired circulation in the vertebro-ba ilar y tem.
3. DE KLEJNS TEST Have the patient lie upine with the neck in maximum exten io
n. Slowly increa e the exten ion and add idebending and rotation to ee if ther
e i an increa e of ymptom a de cribed in the exten ion te t.
4. FLEXION TEST If extreme cervical flexion produce ymptom of parae the ia, p
articularly in the lower limb , the po ibility of a cervical bar mu t be con id
ered. A pondylotic bar can occur in patient of a generally younger age group t
han may be expected. Thi imple te t i a u eful diagno tic creen for thi con
dition. Clearly any vigorou cervical manipulation would be unwi e in the pre en
ce of thi ymptom. Ab olute contra-indication would include any ca e where the
re i evidence of evere nerve or cord compre ion. Where there are cauda equina
ign or evidence of neuropraxia, further inve tigation i e ential. Lack of d
iagno i , or even of a rea onable working hypothe i , i al o an ab olute contra
-indication. It i very ea y to fall into the trap of treating by recipe withou
t even thinking about the po ible implication of the technique cho en and tre
atment in general. A the human body i o ready to accept the benefit of manual
treatment, many practitioner are tempted to take the ea y path of treating fir
t and diagno ing afterward , if at all. Mo t time they will get away with it,
but eventually hidden pathology or anomaly will catch them out. Some ort of wor
king y tem i een to be e ential to avoid thi tendency. Extreme pain or re i
tance hould be another warning that mu t be heeded. There
26
Contra-indication and precaution ADVERSE REACTIONS Adver e reaction to treatm
ent could be due to a hyper en itive patient, an undi covered pathological tate
or exce ive force or do age in the previou treatment. An exce ively empha iz
ed hi tory and hi trionic reaction may be purely due to fear and di tre or lo
w pain thre hold. Any of the e mean that choice of technique mu t be modified ac
cordingly. If uitable care i taken to grade the treatment from the lighte t po
ible force and only increa e force according to the need of the ca e, then t
he chance of adver e reaction and over-treatment i minimal. De pite the mo t me
ticulou care, a evere reaction to treatment in general, or particular techniqu
e , can occa ionally occur. There are many way of reducing the effect of adver

 e reaction . Some practitioner advi e re t and heat. Some advi e analge ic or

antiinflammatory medication . Some will apply gentle effleurage technique and
yet other will carefully repeat the treatment plan previou ly applied. My per o
nal approach i to examine the patient thoroughly u ing examination procedure w
hich are ba ically treatment manoeuvre and try to find the rea on for the react
ion. If there i an area of inflammation and conge tion, then apply gentle techn
ique from the indirect categorie to ettle the ituation down. I generally the
n a k the patient to report in a couple of day by telephone or in per on. The m
ere fact of making contact and di cu ing the ituation eem to defu e the itu
ation and re tore the patient confidence. DOSAGE AND TIMING Do age and timing
i important in con idering relative contra-indication . A evere reaction may
ettle in a day or o to a ituation of a con iderable improvement in ign and
ymptom . If the ame patient were to be een during that pha e of orene from
treatment, the practitioner might have wrongly a umed that he had approached th
e earlier
mu t be a re-evaluation of the ign and ymptom if thi i met. It hould neve
r be nece ary to force again t pain or re i tance. Find another way, or another
day. The few occa ion when manipulative therapy ha been re pon ible for injur
y are mo tly tho e where the practitioner ha continued in the pre ence of ub t
antial re i tance from the patient. The patient ti ue know be t and if they
are giving a me age, take notice of it. The le the experience of the operator
, the greater the need to con ider the ab olute contra-indication in the light
of pain or re i tance. With increa ing experience it may be po ible to allow o
me of the ab olute category to move toward the relative contraindication categor
y.
RELATIVE CONTRA-INDICATIONS Under thi heading could be included a complete li t
of pathologie that may affect bone, di c, ligament, etc. Any patient who ha a
pathological condition may al o, incidentally, have mechanical dy function in t
he mu culo keletal y tem. A long a the object of the technique i to deal wit
h the mechanical di order and uitable care i taken to protect the patient we
lfare, contra-indication may be con idered relative in that ca e. The main rea
on for grading o teopathic technique into their variou different varietie and
different modification i that it hould be po ible to make a pre cription of
a particular approach to technique that would uit a given patient. A combined
leverage and thru t technique u ing a long leverage in an 80-year-old with a ti
ff neck would clearly be unwi e. Thi doe not mean that a very light and delica
te minimal leverage thru t technique or mu cle energy technique could not be car
ried out in uch a ca e. Relating the technique to the phy ique, age, general he
alth and tate of the ti ue at the time i part of the diagno tic proce . It
hould form part of the pre-technique a e ment in each ca e.
Relative contra-indication treatment wrongly. For thi rea on, daily treatment
i generally to be di couraged. If the operator i prepared to perform only mini
mal treatment on each occa ion and i ready to accept the po ibility of an accu
mulating adver e reaction, then thi may be acceptable. There may be rea on of
urgency due to impending travel, porting event or important life event that mak
e daily treatment e ential. From experience, I would ay that ab olute pecific
ity i nece ary in the e ca e . Short, purpo eful treatment, aimed at one peci
fic target ti ue on each vi it, eem to produce the be t re ult without exce
ive reaction. DISCS The vexed que tion of whether to manipulate in the pre ence
of evidence of di c herniation or prolap e will be the ubject of di cu ion a
long a manual therapy i in u e. The u e of magnetic re onance imaging (MRI) ha
hown that many ca e with di c di ea e have been manipulated with great ucce
. Had the MRI been performed earlier and a deci ion taken not to manipulate, t
he patient may not have had uch a good re ult! Once again each ca e i individu
al and all factor mu t be con idered in coming to a conclu ion about the be t a
pproach. In the author experience, providing there are no ign of extreme ner
ve pre ure, either before treatment, or when applying the technique po ition

lowly, then di c prolap e i a ign for caution, not abandonment. Powerful rotat
ory technique in the pre ence of radiological evidence of large o teophyte are
be t avoided for fear of nerve impingement. Forced flexion technique are unwi
e where there i evidence of di c prolap e in the lumbar pine a increa e of pr
olap e can occur.
27
menced. Over-zealou attempt to force a joint to become fully functional in thi
ituation will often lead to extreme reaction . Inflammation due to ankylo ing
pondyliti or Reiter di ea e i a ituation where exce ive active treatment
i be t avoided. Gentle ettling technique can be of u e to reduce the irritab
ility enough o more powerful procedure can be u ed later. The timing and elec
tion of when to treat can only be gained by thought, con ideration and experienc
e.
ILLNESS Although the original purpo e of o teopathic treatment wa for the treat
ment of y temic di ea e through the medium of the mu culo keletal y tem, o teo
pathic medicine of thi type i not a widely u ed a o teopathic treatment for
mechanical dy function yndrome . Thi may not be the choice of work for many pr
actitioner , but i governed by elf- election of patient . Ca e where there i
evidence of y temic di ea e can be treated either a a complement to conventio
nal medical care, or a an alternative. If a patient i unwell y temically and
i eeking treatment for the y temic di order there will be a difference in app
roach from the ame patient a king for treatment for a mechanical di order. A pa
tient who ha influenza, for example, may not get much help from treatment for t
heir low back condition a their mu cle and connective ti ue may not be able
to take the benefit of the treatment. The general illne will be making the y
tem weak and normal mechanical treatment will often be wa ted. It will tire the
patient and the ti ue will often be reactive and ultra en itive. Thi would th
en be a rea onable contra-indication to treatment at that time. Some diabetic m
ay find their in ulin balance di turbed by o teopathic treatment. Providing they
are warned to keep a clo e check on their gluco e level , thi hould not be a
problem.
INFLAMMATION A joint that i acutely inflamed i be t left to re t for a while u
ntil treatment i com28
Contra-indication and precaution treatment. If the ymptom are directly refer
able to the pondyloli the i and there are cord ign , then o teopathic techniq
ue directed to the egment are be t avoided a there i the po ibility of cau
ing further damage. However, many ca e have the condition a an artefact that h
a no bearing on the ti ue cau ing the ymptom . It exi tence then doe not p
reclude the u e of carefully applied technique. Technique that are pecific and
are applied to adjacent egment may well reduce tre on the un table egment
and can be very u eful in the overall management of the ca e. Mobilization of a
n un table pondyloli the i i unwi e and if it i due to a recent trauma, ther
e i ome evidence that complete immobilization can produce healing of thi tre
fracture condition. A congenital defect will not re pond to temporary immobil
ization.
PREGNANCY During pregnancy, any technique that might jeopardize the health of th
e mother or developing infant mu t be avoided. The chief danger, of producing a
pontaneou abortion, occur in the fir t trime ter. The po ibility of producin
g a mi carriage with properly applied o teopathic care i o light a to be alm
o t di counted; neverthele , in a patient with a hi tory of pontaneou abortio
n care i e ential. Stati tically, the highe t incidence of mi carriage i in t
he 12th and 16th week of pregnancy. The e are time when treatment i be t with

held in uch a ca e. Thi i not becau e it could cau e a mi carriage, but more
becau e there i the po ibility of being accu ed of producing it. Treatment dur
ing the later tage of pregnancy will nece itate technique modified omewhat
from normal method . Hormonal relaxation in ti ue make mo t o teopathic proce
dure ea ier to complete and light technique can be mo t effective. With uffic
ient care a patient can be treated right through her pregnancy, and in certain c
a e o teopathic care ha even been given during parturition it elf with benefit
. The BSO ha an expectant mother clinic that undertake exten ive work on preg
nant women with excellent re ult . ARTERIAL CALCIFICATION Calcification of the a
bdominal aorta i not in it elf a total contra-indication to treatment, but clea
rly mu t dictate choice of technique. Caution i e ential and extreme rotatory
technique be t avoided. In general, patient with thi condition will be of an
age where thi type of technique i unwi e anyway. Calcification of the aorta in
a younger patient may be due to extreme hyperten ion and thi mu t be taken int
o con ideration in the choice of technique. SPONDYLOLISIS AND SPONDYLOLISTHESIS
The exi tence of p o n d y l o i or pondyloli the i i not in it elf a r
ea on to withhold
LIGAMENTOUS LAXITY There are everal ituation when ligament may be exce ivel
y lax. Inflammatory di order uch a rheumatoid arthriti not only affect perip
heral joint . The ligamentou apparatu upporting the den of the axi again t
the atla can become di rupted. Any exce ive force in thi area could be di a t
rou in the e ca e . Long-term u e of y temic teroid can produce o teoporo i
, but can al o have a di organizing effect on ligamentou tructure , particular
ly in the upper cervical pine. Therefore, once again the po ibility of ligamen
tou laxity in the upper cervical column i pre ent. It i impo ible to tate w
hat i a  afe do e, but any patient taking more than 10 mg daily over a period
of more than 6 month hould be treated with caution. Anticoagulant have been
incriminated in producing ligamentou weakening due to breaking down the fibrin
matrix of the connective ti ue. Thi eem to target the upper cervical ligamen
t again, o any vigorou technique in thi area mu t be tempered by extreme ca
ution. Any patient who i taking
Relative contra-indication anticoagulant i going to be omewhat more u cepti
ble to micro-trauma, bleeding and lower healing in ti ue , o the category of
technique cho en mu t be of the lowe t inva ivene . Patient do not take the e
drug unle they have quite eriou underlying pathological condition and it i
nece ary to evaluate the overall approach to treatment with great care. MEDIC
ATION Several perfectly normal, valid medication that patient may be taking ca
n change the effect of manual treatment. I have already mentioned the effect o
f y temic teroid and anticoagulant in the ection on ligamentou laxity, but
other ub tance mu t be con idered. Analge ic clearly modify the re pon e to
pain and although they cannot obliterate it, they can reduce it to a point where
patient feedback i inaccurate. Due con ideration mu t be made for thi effect.
Antidepre ant will modify pain re pon e and ome patient will have apparentl
y trange reaction to phy ical treatment when they are taking them. Any patient
who i taking powerful medication of any kind hould be que tioned a to the re
a on for the pre cription. If they are un ure of the rea on, the practitioner h
ould inve tigate the action of, ide-effect of and reaction to the ub tance
o that he can be aware of the medical a pect of the ca e. There may be good r
ea on a to why the medical attendant ha not given much information. Probably
the mo t important medication that a patient may be taking i the one with which
the therapi t i not familiar. It hould be tandard procedure to inve tigate e
very medication that patient have pre cribed o that important precaution can
be identified. In a very hort pace of time the common drug will be under tood
and memorized. It i al o wi e to know the effect of the common recreational d
rug , alcohol and tobacco. Patient taking large amount of alcohol will have di
fferent
29

re pon e to pain and may become deficient in ome of the B vitamin , for exampl
e. Heavy moking ha been hown to reduce pinal circulation to the extent that
ome practitioner have in i ted that patient do not moke for 2 hour before o
r after treatment. They feel that much of the benefit of treatment will be lo t
if the patient doe moke in thi time. They al o feel that bringing the ubject
to the patient notice in uch a graphic way can help trigger a thought proce
in tho e who want to top to help them along the path. Every practitioner will
have to con ider whether their con cience allow them to work in thi rather mo
rali tic way; neverthele , the ill-effect of moking on pinal health are beco
ming more evident a further re earch take place. SCOLIOSIS Mild degree of co
lio i are common but extreme colio i require modified technique . If an orga
nic colio i i rea onably table, it would be unwi e to attempt to correct i
t. Vertebral bodie will have changed hape and there will often be calcificatio
n of ligament and fa cial attachment . There will often be o teophytic outgrowt
h and evere tor ional technique may cau e problem of nerve impingement. Vigo
rou technique are generally not indicated. SPONDYLOSIS AND SPONDYLARTHROSIS Th
e pre ence of the e common ageing proce e are not rea on to withhold treatmen
t, but the thoughtful operator will naturally modify approache according to the
degree of degeneration. Some categorie of technique will be perfectly acceptab
le and other not. Expect the wor t reaction, and then if you are wrong it will
not be too di a trou . MALIGNANCY In any patient who ha had a pa t hi tory of m
alignancy, the mo t thorough and earching
30
Contra-indication and precaution obviou thing and a thi ha not produced th
e re ult, the patient ha moved on to omeone el e. Every practitioner want to
give the patient a good re ult; if drawn in to a ituation where the patient i
very importunate for treatment, it can be very difficult to re i t. Several trea
tment later, if there i no re ult, it can be very difficult to extricate one e
lf from the ituation unle a contract ha been e tabli hed in the fir t plac
e. Thi i the type of ca e where it i wi e to e tabli h an agreement to a re-e
xamination after a predetermined number of treatment . If the de ired change ha
ve not taken place, it i be t to avoid further treatment until further inve tig
ation or a econd opinion have been ought. FEEL If omething ju t doe not fee
l right, then thi i a good rea on to perform more thorough inve tigation or ex
amination. The ixth en e of thi combination of learned fact , experience and
temporarily forgotten a pect can be very reliable. If omething ju t feel wron
g, top and recon ider. If ign and ymptom do not match, thi i the time to
think again. There may be hidden pathology or anomaly. There may be p ychologica
l factor , or the patient may be overempha izing their problem for ome other re
a on. It i clearly nece ary to find out more accurately what i going on. The
whole ubject of contra-indication i dependent on accurate a e ment of the c
au e and nature of the pre enting condition. If an accurate diagno i ha been m
ade, the contra-indication hould be obviou . Slow, deliberate technique while
getting feedback from the ti ue i probably the guide that mo t practitioner
rely on. It i not a finite cience, but rather an application of cience, art a
nd rea oning.
examination hould be carried out. It i e ential to have an accurate diagno i
of the ti ue cau ing ymptom . If the ti ue ymptom i of a mechanical nature
, then there i no rea on why treatment cannot be carried out. Symptom that mig
ht originate from econdary malignancie may well appear to re pond in the hort
term to phy ical treatment, but the benefit will only be temporary and i proba
bly due to circulatory and p ychological factor . VERTIGO Some type of vertigo
can be helped ub tantially by phy ical treatment, particularly to the neck and
upper thoracic area. Some type can be aggravated. Meniere di ea e and po tura
l hypoten ion can be irritated by overvigorou and rapid technique . Both may we
ll be helped, however, by appropriate technique for the ituation. Both ca e m

ay prefer to be treated itting up rather than fully recumbent. PSYCHOLOGY P ych

ological dependence on phy ical treatment i not uncommon and providing the oper
ator ha a ured him elf that the patient doe have a true phy ical cau e for hi
ymptom , then treatment can be valid. It mu t be aid, neverthele , that thi
type of patient become an expert in empha izing minor phy ical di turbance .
The le experienced practitioner may well find them elve treating uch a patie
nt who ha been the round of other . Any patient who ha een everal other pra
ctitioner hould be looked on with extreme caution. It i ea y to a ume that t
he other practitioner ha mi ed omething obviou . They may have done, but it i
far more likely that they have done the
INDIRECT TECHNIQUE
6
Although the teaching at mo t o teopathic training e tabli hment world-wide ha
been predominantly tructural in approach, there i a con iderable intere t, an
d validity, in many of the more newly developed method . Indirect method are ge
nerally le inva ive in application, although they may be no le effective in
action. They require ju t a long to perfect a direct method , but give the tu
dent or practitioner who i not experienced in direct technique an ability to tr
eat the patient immediately in a generally afe and comfortable way. Indirect me
thod hould not be an excu e for inefficiency in technique. They hould be a u
pplement and complement to primary method , and add a dimen ion to treatment tha
t cannot be gained a ea ily with tructural approache . There are ome ca e th
at will only re pond to ome of the indirect technique , but the e are rare, and
conventional tructural technique remain the main tay of o teopathic practice
for mo t practitioner . Increa ingly, a knowledge of the e method pread , the
y are being integrated with the more common tructural method a adjunct , a
upplement , or a a ub titute in ome ituation . Mo t experienced practitioner
uncon ciou ly u e a combination of approache . No ingle method ha all the an
wer to all mechanical dy function problem in the neuro-mu culo- keletal y te
m. The rea on for the development of indirect technique are intere ting and var
ied according to location. In ome countrie it ha been due to a backla h again
t the powerful manipulative method in common u age, and i een to be more hol
i tic and cooperative rather than being confrontational. In ome
quarter it ha been due to the ever increa ing fear of litigation when manipu
lation i performed and an articular ound i heard. Thi can lead to accu atio
n of po ible harm and wrong re ult . That a well-con tructed treatment ba ed o
n good technique u ing tructural method hould not be painful or traumatic i
clearly not con idered by individual who u e powerful and uncomfortable method
. There may be many rea on why practitioner continue to u e uch approache . T
hey may not have been trained ufficiently in gentle and effective technique, or
have not been able to acquire the kill adequately. They may feel that firm fo
rce i an e ential requirement, or that it i what the patient expect . In my v
iew, exce ively high force i generally a ign of weakne in approach. Force c
onceal weakne . Thi i not to ay that force i not nece ary, but imply to
tate that the minimum force capable of producing the required re ult hould be
the aim of the thinking, caring practitioner. The variou categorie of indirect
technique therefore have many u e and po ible advantage . In thi ection the
de cription of the e method i not de igned to be a full expo ition, but impl
y an introduction. Experienced practitioner of the e variou method may be om
ewhat irritated by my attempt to implify and abbreviate the y tem , but thi
implification i the whole purpo e of thi ection. The reader i referred to t
he bibliography for more detail of literature relating to each of the variou t
ype of technique and treatment. There are many other method in u e, and thi l
i t i not a comprehen ive a ome might like; however, I have tried to include
the main method in wide u e by

32
Indirect technique With the combination of the e principle , and a range of hold
and technique , the cranio acral y tem and dy function in movement pattern
can be addre ed. The head i held in a variety of predetermined hold , and the
practitioner tune in to the cranial rhythmic impul e (CRI). Thi rhythmic imp
ul e i de cribed a being an ebb and flow of approximately 10-12 cycle per min
ute. He may find di turbed movement or flow. If thi i the ca e he can apply ge
ntle force to timulate the potency of the y tem and the fluid drive to perfor
m any correction deemed nece ary. The acrum can imilarly be treated with hold
that allow it to float on the fluid drive and free it elf, guided by the pract
itioner. The dural attachment to the acrum can be freed to allow better functi
on at the upper end of the y tem in the cranium. A time ha pa ed, the concep
t ha been developed to make it applicable to mo t tructure of the body via th
e CRI. The impul e i be t located in the cranium, but can be felt to a greater
or le er extent all over the body. Treatment u ing thi principle can be applie
d to many tructure remote from the head. For thi rea on, many practitioner n
ow like to refer to treatment of the involuntary mechani m, rather than cranial
o teopathy. Although cranio- acral technique were at one time the exclu ive pro
vince of o teopath , they are now (like mo t technique ) being u ed increa ingly
by other manual therapi t . However, the fuller implication of cranial work wi
th whole body health eem only to have been con idered in detail by o teopath .
O teopath u ing cranio- acral technique often treat many condition other than
local head ymptom and many good re ult are produced. There i ub tantial in
tere t, and increa ing evidence of the particular benefit in neonate and mall
children, although the technique can be u ed with efficacy in all age group .
Of all the y tem of indirect technique, cranio- acral method are probably the
one approach u ed by ome practitioner a an exclu ive treatment techo teopathic practitioner . Although no method belong  to any y tem, ome appr
oache are more commonly practi ed by other manual therapi t uch a chiropract
or and phy iotherapi t . I have tried to remain e entially o teopathic and to
try to reflect the main method in u e in Great Britain rather than that in worl
d-wide. From my travel and ob ervation el ewhere, I u pect that the Briti h i
tuation clo ely re emble mo t other countrie . There may be regional and nation
al difference that are due largely to the lack of availability of technique tea
cher . I have tried not to be judgemental in my comment , but only to peak a a
tructural o teopath who ha eclectically incorporated many other method into
my armamentarium a I have found them u eful. There are ome method that I rare
ly u e, but I may incorporate ome of their principle , if not the technique th
em elve . The variou method are li ted alphabetically. CRANIOSACRAL TECHNIQUE
Cranio- acral technique wa fir t de cribed by William Garner Sutherland D O . H
e wa a tudent of Andrew Taylor Still, o clearly got ome of hi knowledge fro
m the fountainhead of o teopathy. He po tulated the po ibility of movement betw
een the cranial uture and bone and that they could become dy functional. He p
ut forward the idea that the e dy function were amenable to manual manipulative
method . The technique rapidly grew to include ome ba ic concept . The e were:
1. The exi tence of an inherent motility of the central nervou y tem. 2. An i
nherent motility and pul atile nature of the cerebro- pinal fluid. 3. The exi te
nce of reciprocal ten ion membrane , namely the meninge , particularly the falx
cerebelli and the tentorium. 4. The mobility of the cranial bone around articul
ar axe . 5. The mobility of the acrum between the ilia.
Gentle therapeutic manipulation nique. Thi how the effective nature of the ap
proach. If it did not produce good re ult , they would not be able to u tain a
private practice with it ole u age. FUNCTIONAL TECHNIQUE Functional technique
wa evolved by Bowle and Hoover, DO in the USA in the fir t half of the twenti
eth century. It ha been exten ively developed by Profe or William John on DO,
in Michigan. It i a y tem of o teopathic technique ba ed on the premi e that
egmental dy function i palpable a an increa ing re i tance to motion demand in

certain direction in a le ioned egment. Thi ha been called the bind en ati
on. Any dy function will have certain pathway of ea e, where re i tance to mo
vement i felt to progre ively collap e. The bind tate can be treated by fi
nding a pathway of lea t re i tance in the egment. If all direction and pathwa
y of ea e can be found thi hould allow quietening of proprioceptive feedb
ack and re- etting of Gamma gain in tendon and joint receptor . The dy functio
n can be detected by a variety of method including palpatory awarene of limit
ation of movement quality rather than range of motion. He then a e e the re p
on e to find which one bind, and which one ea e. Wherea conventional manipul
ation challenge barrier to break them down, functional technique ea e the
egment into a multiple movement pathway of accumulating ea e. Thi aim to aid r
elaxation of the adnexial ti ue and produce more harmoniou mobility. It i ba
ed on the concept that afferent feedback to the pinal cord i di turbed in a d
y functional egment. The proprioceptive mechani m are maintaining thi di turb
ance, and by pa ing the egment into a pathway of ea e, the afferent will quie
ten. Thi hould lead to a reduction of efferent firing. The um effect of thi
i to cau e a new etting of the afferent to efferent balance, and a more harmon
iou movement pattern. Although the phy iological explanation i open to que 33
tion con idering more up-to-date thinking, the validity and effectivene of the
method i not in doubt. Hold have been developed for treatment of mo t articul
ar tructure . Although the principle of moving away from a barrier are oppo it
e to conventional tructural technique, they can be integrated with other treatm
ent technique very ea ily to form a combined y tem. The y tem i guided purel
y by palpatory feedback from the ti ue and i , therefore, dependent on good pa
lpatory kill. Conventional manipulative kill give the practitioner the abilit
y to perceive accumulating barrier . Functional technique require the kill to
palpate di integrating barrier . Thi may take ome conver ion of thinking and a
warene , but mo t can acquire it fairly quickly with uitable in truction.
GENTLE THERAPEUTIC MANIPULATION Gentle therapeutic manipulation (GTM) i a y te
m of active patient movement guided by the practitioner in pecific direction a
nd in pecific order. The active movement of the patient i monitored by gentle
pre ure in particular direction over the dy functional area by the operator 
fingertip . It wa fir t demon trated in Great Britain by John Spence D O , in 1
994. The y tem originated in New Zealand, and like many of the e indirect appro
ache , evolved from a combination of other method . Immediate change in perceive
d mu cle ten ion and fa cial irritability i apparent after ucce ful treatment
u ing thi method. Relatively little i known of thi method in Europe o far,
but intere t i growing a re ult are een. It i commonly combined with pecif
ic advice a to ice pack and pecific exerci e a a follow-up to treatment. Alt
hough it employ hardly any applied force, there may be ti ue reaction due to t
he change produced. The rationale i thought to be of joint and ti ue relea e
by way of guided pathway of active movement pro34
Indirect technique natural recoil to return the thigh to neutral, would be a typ
ical harmonic technique. The range of movement might change only lightly, but t
he quality of movement hould improve markedly if the correct harmonic ha been
found. There will al o be circulatory change timulated by better relaxation an
d fluid interchange. The technique i not merely an o cillation back and forth.
There are certain direction that are e ential for the y tem to produce the be
t re ult , and amplitude i al o important. There are everal po ible explanat
ion for the effect of harmonic technique. There are evidently neurological, hyd
raulic, mechanical and p ychological rea on why it work .
ducing a return to more normal harmoniou pattern . HARMONIC TECHNIQUE Harmonic

technique i a y tem of approach that u e a harmonic pre ure again t differen

t area of the body, and allow a natural recoil to take place. It i a re-di co
very of ome of o teopathy olde t method . The early pioneer of o teopathic t
echnique u ed harmonic o cillating movement a part of their treatment. It becam
e le u ed a time went by and more linear method became popular. Although mo
t chool of o teopathy taught ome form of rhythmic technique, it wa not gener
ally taught a a y tem in it elf. E. Lederman DO re-di covered, cla ified and
re-introduced harmonic method , and ha exten ively re earched the phy iological
effect of thi , and other method of manipulative approach. The patient allow
the part being worked to be pu hed, pulled or moved in a pecific direction, an
d the natural recoil and rebound of the ti ue return it to neutral. Thi actio
n i repeated in a harmonic fa hion until an o cillation i taking place where t
he operator i acting a a cataly t. The be t balanced harmonic rhythm i when t
he effort on the behalf of the practitioner become lea t. The operator tart t
he o cillation and change peed until thi dynamic balance i found. The rate o
f o cillation will vary according to which part of the body i being worked and
length of the lever. The technique i continued until a en e of relaxation i p
erceived in the area being worked. Hold have been developed for mo t body area
, and the technique can be u ed a a treatment in it elf, or a a preliminary to
other categorie of technique if de ired. It i een a mo t u eful where the r
hythmic pattern of movement of a part ha been lo t. An example might be a in a
evere arthro i of the hip. Conventional tretching will clearly make ome cha
nge, but a gentle rolling of the thigh, allowing the
MUSCLE ENERGY TECHNIQUE Mu cle energy technique (MET) wa fir t de cribed by Fre
d Mitchell Sr. DO, in Michigan USA in 1958, and wa developed exten ively by hi
on Fred Mitchell Jr. Much further development ha taken place under Ed Stile
DO, al o of Michigan. It i a y tem of therapeutic approach that relie on diag
no i of a mechanical dy function, and treatment with active patient re i tance
in certain direction again t the re i tance applied by the operator. The barrie
r to joint motion or mu cle function i found and the operator hold the part ag
ain t thi barrier. He then get the patient to pu h either away or toward the b
arrier depending on the required re ult and effect. After a few econd , the pat
ient i in tructed to relax, and the barrier hould have been found to have move
d. The new motion barrier i found, and the activity repeated everal time . The
mechani m i perceived to be a re-balancing of the afferent to efferent feedbac
k from the pinal cord. There are four main method of approach: i otonic; i ome
tric; i okinetic; i olytic. I otonic mu cle energy technique i where the operat
or re i t the patienf active mu cle
Myofa cial technique contraction and allow light movement in the direction of
mu cle contraction to maintain the mu cle in the ame ten ion. The retraining
of mu cle i perceived to change the tone and body awarene of the mu cle o th
at it can learn a new pattern of u eful contraction. I ometric mu cle energy tec
hnique i where the operator re i t the patienf active mu cle contraction and
doe not allow movement. Thi cau e the mu cle to remain at the ame length th
roughout. Thi i u ed to either trengthen the mu cle, or to allow the practiti
oner to u e the reflex relaxation after contraction to apply a tretch. It will
al o produce a reflex relaxation in the antagoni t mu cle which can then be tre
tched more ea ily. I okinetic mu cle energy technique i where the operator re i
t contraction of a mu cle or group of mu cle in the patient and allow a grad
ual lengthening of the mu cle during the re i tance. Thi i perceived to act a
a re-education to the mu cle to normal movement and contraction pattern . I oly
tic mu cle energy technique i where the operator re i t the contraction of a m
u cle or group of mu cle in the patient and overcome the re i tance to breakd
own the mu cle. The patient mu t re i t with enough effort to cau e the mu cle
to contract, but at the ame time mu t allow the operator to overcome the re i t
ance to the effort. The effect of thi i for the mu cle or mu cle group being a
ffected to lengthen under the tretch while it i contracting. Thi i de igned
to have the effect of breaking down re i tance to movement cau ed by mu cle prot

ection and allowing greater freedom of function. Each one i de igned to have a
different action on mu cle and can be performed again t or with a barrier. Altho
ugh thi concept may now be found wanting phy iologically, re ult of recent re
earch how that the method i in wide- pread and effective u e. MYOFASCIAL TECHN
IQUE
35
Myofa cial technique ha developed relatively recently a a conglomerate of eve
ral different approache . It ha ome imilarity in approach to ome of the cran
io- acral approache . However, it can be applied to all area of the body a it
work on the mu cle to fa cia and fa cia to bone and vi cera interface. It may u
e ome of the principle of mu cle energy technique in that re i tance i utili
zed. It u e perception of fa cial plane ten ion in diagno i and treatment proc
edure . There are variou common method of application. The technique i often
applied by the operator finding an area of perceived dy function and gently guid
ing it toward more harmoniou working with adjacent area . The pre ure will var
y, and the time taken will depend on the peed of relea e perceived by the opera
tor palpating hand. Thi i generally a very low and gentle method that i pe
rceived a relating to the neurological control of the mu cle and fa cial attach
ment . Thi concept relie on accurate feedback from the fa cia and mu cle tate
a to the optimum relaxation pha e of the area. Control of the pha e of breathi
ng i al o empha ized. Some myofa cial technique approache u e quite trong tr
etch along the fa cial plane with either ingle or multiple, lowly applied tr
etche . Some approache u e diagonal tran fa cial tretche . One of the chief cr
itical element of myo-fa cial technique ucce i the u e of ufficient time i
n the hold po ition to allow adequate fluid interchange and oft ti ue creep
to take place. Mu cle are e entially fluid in life, and a fluid i noncompre
ible, ome of the action i probably produced by u tained pre ure allowing fl
uid to change it po ition within the fa cial tube . The two model of approach
may eem at variance, but like many other approache will depend on many factor
a to their applicability in any given ca e. A in mo t y tem of approach, pr
actitioner u ually polarize to a
36
Indirect technique who wa a chiropractor and o teopath. He combined the current
thinking of the two profe ion at the time to introduce a y tem of o teopathi
c manipulative method that u ed chiropractic thinking on malpo itioning and rep
lacement. The technique i mo tly a thru t approach u ing extremely rapid yet ge
ntle force that i often performed ju t hort of a facet gapping. It i de igned
to replace or retrace the pathway of le ioning po ition, and relie on pecific
po itional diagno i and pecific direction of adju tment. The y tem wa litt
le known or taught for ome year until being re-introduced to o teopathic educa
tion by Tom Dummer DO at the European School of O teopathy in Maid tone. It i n
ow being carried on by ome of hi pupil . STRAIN AND COUNTER-STRAIN Strain and
counter- train technique wa fir t de cribed by Lawrence Jone DO in 1981. He pu
t forward the notion that many omatic dy function yndrome are accompanied by
pecific trigger point . The e point are often very remote from the le ion and
may not accord with conventional anatomical thinking, but are con i tent and hav
e a characteri tic en itivity and tenderne The technique con i t of locating
the trigger point relevant to the particular dy function and then po itioning t
he patient in uch a way a to cau e the trigger point to become painle . The p
o ition i then held for ome 90 econd and the patient i then lowly returned
to a normal po ture. If the technique ha been ucce ful, the trigger point wi
ll have di appeared, or at lea t ub tantially reduced. The holding of the po it
ion will often be accompanied by a en e of relea e, heat and freedom. Te ting t
he articular le ion hould al o how a return of function in term of mobility.
The technique can be applied to any area where the trigger point ha been identi
fied, and even if it i not u ed a a ole method, form a good preparation to f

urther tructural technique . It may eem omewhat nonparticular method. Some may find one method ea ier; ome may prefer the rational
e of one method; ome may feel one i more effective. NEURO-MUSCULAR TECHNIQUE N
euro-mu cular technique wa developed by Stanley Lieff DO and Bori Chaitow DO,
Engli h o teopath . It employ a y tem of progre ively earching liding pre
ure de igned to find dy functional area and then treat them with varied direct
ion of deeper pre ure . It attempt to normalize the e area by allowing imp
roved circulation and re- etting of the neural control of tendon and mu cle .
It main action i on connective ti ue , fa cia and mu cle attachment . In the
pinal area the practitioner u e thumb pre ure on the para-vertebral gutter ,
directly over the facet joint and u ing the finger a a fulcrum, o cillate o
ver each level until the le ion i detected. He then ometime u e a lubricant
uch a a ma age oil to apply mo tly longitudinal pre ure repeatedly over the
egment in que tion and one or two adjacent egment . Neuro-mu cular technique c
an al o be applied along the whole length of the pine, concentrating on area o
f dy function with firmer pre ure. It i al o ometime u ed on the abdomen and
limb . There i a complete y tem of approach that ha been documented to apply
it to a wide range of condition . Although thi technique i not a widely u ed
now a it wa in the fir t half of the twentieth century, it ha a certain foll
owing. It i now not taught in many o teopathic chool . There are ome imilari
tie with Rolfing and ome other deep ma age technique . Practitioner u ing th
i method feel that it can produce longer-la ting re ult than ome of the artic
ular-directed technique .
SPECIFIC ADJUSTING TECHNIQUE Specific adju ting technique wa evolved mo tly by
the late Parnell Bradbury DO DC
Vi ceral technique dynamic a the po ition held for 90 econd i not in any way
rhythmic, but neverthele it i often ucce ful in relea ing pecific fixatio
n . There have been ome attempt to horten the time, and although the e are no
t in accord with the origin of the technique, they are more acceptable to ome
practitioner . To allow the hortening of the time it i nece ary to perform th
e hold with omewhat more pre ure or empha i . The thinking behind the rea on
for the exi tence of the trigger point and it preci e location may vary with di
fferent practitioner . The perception i that the le ion i due to an aberrant p
athway of movement and i maintained by mu cle pa m. The part mu t be taken bac
k through the le ion pathway until an antalgic po ition i found. The part i ma
intained in thi po ition for 90 econd , which i long enough for the proprioce
ptive feedback from the Gamma gain mechani m to quieten down. The proprioceptor
then re- et, and the pa m reduce , allowing normal articular pattern of move
ment to be re tored. The part ha thu been taken back to the po ition in which
the le ion occurred and allowed to recover lowly, guided by the operator pre u
re and control. VISCERAL TECHNIQUE The origination of vi ceral technique i uncl
ear, although Barral in France i often credited with fir t de cribing it. It i
a form of functional technique a applied directly to the vi cera and their fa
cial attachment . It ha many imilaritie to cranio- acral technique, in that t
he barrier en e i u ed in ea ing perceived dy function with extremely gentle
force guided by the operator li tening hand. The hand i at one and the ame
time a diagno tic in trument and a therapeutic tool. The re i tance to motion or
function i ought, and the appropriate technique u ed to normalize the lack of
function. All vi cera are perceived to have one or everal axe of
37
movement. The practitioner applie a hold to the vi cera with or again t the axi
or axe . He very gently te t the range and quality of movement in all availab
le plane . If nece ary, he then applie a corrective force again t a perceive
d barrier, or applie an ea e direction of combined pathway to allow greater
freedom of movement and relea e of exce ive tethering. Barral ha tated that t

he force applied need rarely exceed 300 gram . Vi ceral technique ha become ver
y widely u ed in the continent of Europe, and i now being taught in the United
Kingdom at undergraduate and po t-graduate level. The work ha been developed an
d documented well by everal authoritie , but Jean Pierre Barral from France and
Stephen Sandler from the UK need pecial mention here for their logical de crip
tive text and de cription of the technique . Many chool now integrate the te
aching of vi ceral technique with all the other approache to the body that wil
l have an influence on the vi ceral y tem . Thi i a logical development from
looking at vi ceral technique in i olation. An intere ting development in the fi
eld of vi ceral technique ha been the tudy of the relation hip between the or
gan and their upporting ti ue a well a the adjacent omatic tructure . Gra
y Anatomy and other tandard textbook refer to the fold in the peritoneum tha
t connect organ a peritoneal ligament . The e o-called ligament , uch a the
ga tro-colic ligament, provide potential for organ re triction if they are thic
kened or fibro ed, thu potentiating the eventual dy function of the organ. Vi c
eral technique taught at the Briti h School of O teopathy involve diagno ing an
d treating the e vi ceral re triction . Their manipulation involve holding and
relea e technique a well a guiding and functional technique which make u e o
f the fact that a tructure they contain mu cular element a well a collagen
ou fibre . It i hoped that, by re toring a normal functional relation hip betw
een the organ and their neighbouring tructure (both
38
Indirect technique technique are nece arily brief, they hould help to give a
ta te of the variou method . Thi may give the reader ufficient intere t to pe
rmit further tudy to be undertaken. To thi end the recommended reading li t h
ould be of ome u e.
vi ceral and omatic), one can move away from dy function toward better health.
Thi concept of working on vi cera that are dy functional a oppo ed to patholo
gical ha been found to be both afe and efficaciou . Although the e de cription
of indirect
MODIFYING FACTORS IN TECHNIQUE
7
Any p ychomotor kill can be varied by everal modifying factor . From the port
ing world let u u e a tenni hot a an example. The direction of the feet, the
bend of the knee , the ize of the racquet and the twi t of the wri t are modif
ying factor . They will be determining the ucce or failure of the troke. If
the player direct hi feet acro the direction of the ball trajectory rather t
han in the ame direction, the troke will have more power. If the knee are li
ghtly bent, the recoil of the trike again t the ball will direct the force into
the ball, and not into the player, thu increa ing the potential power of the
troke. It can be een, therefore, that any of the modifying factor will interco
nnect with the other to make up the compo ite whole that we might call a tenni
troke. The principle of under tanding the modifying factor and u ing them to
the advantage of the activity i without que tion, although ome are clearly mor
e important than other . If we relate thi to o teopathic technique, everal imp
ortant factor come to mind. Po ture and handling have been eparated and warran
t complete ection to them elve a they are o critical, but the other are on
ly lightly lower in the cale of relevance. A li t of the factor that mo t peo
ple vary when modifying a technique follow . There are everal other that will
come to mind with a little thought. The exerci e of doing thi will be of ome b
enefit a the effort of identifying them often reveal vital factor not previou
ly con idered. 1. Speed or velocity. 2. Duration or length of time. 3. Amplitud
e or di tance travelled.

4. Plane or direction of force. 5. Force or quantity of effort. 6. On et or the

point at which the u eful part of the technique begin . 7. Arre t or the method
and point of end of a technique. 8. Compre ion or the u e of clo e-packing in a
technique. 9. Primary and econdary lever or the identification of the princip
al direction of force, and the vector that help tabilize the part to make the
primary lever more efficient. 10. Re piration or the pha e of breathing when the
force of the technique i to be applied. 11. Re i tance or awarene of accumul
ating barrier en e. 12. Contact point pre ure or local clo epacking of ti ue
and pu hing a ide of unwanted tructure . 13. Hunch or the ability to develop a
ixth en e that omething need or doe not need a particular approach. SPEED
OR VELOCITY The peed at which a force i to be applied for a given technique wi
ll be dictated by the type of technique. A rhythmic technique will have a lower
peed than a thru t, but will be repeated everal time wherea a thru t i u u
ally only performed once at a given ite. There mu t be ome determination of wh
ether the peed i the ame throughout the technique, or if there i an accelera
tion and ub equent deceleration, and the rate and amplitude of the e change . I
t can be een immediately, therefore, that other modifying
40
Modifying factor in technique Effective technique need le time to perform bu
t the duration of a particular treatment e ion it elf i not really a function
of technique but i another matter completely. Every o teopath eem to have hi
idea of the duration of treatment nece ary to produce the appropriate amount
of change in the ti ue and the condition. I have known variation between 5 an
d 60 minute for a treatment e ion. My feeling i that there are different nee
d to every ca e, but due to the con traint of mo t practitioner  appointment
y tem , thi i not a very practical tatement. Experience hould allow mo t pr
actitioner gradually to reduce the time needed to work on each ca e in an effec
tive way. There will naturally be an optimum for each practitioner and hi et-u
p of con ulting room and changing area , etc. Patient eem to want a long trea
tment giving good value for money and the maximum involvement, but they do not l
ike a long waiting li t! The two are not u ually mutually compatible. AMPLITUDE
The amplitude, or di tance travelled by the applicator, i governed by ome of t
he other modifying factor . In a rhythmic technique the amplitude will tend to b
e fairly long unle it i a pringing technique when the o cillation i very h
ort. In a thru t technique the amplitude mu t be very clo ely controlled to give
the technique a hort an amplitude a po ible con i tent with the ucce ful
completion of the procedure. High velocity, hort amplitude remain the main tay
of manipulative thru t technique . A compo ite lever accumulate at a point th
e en e of re i tance hould cau e the amplitude to reduce until the force are
focu ed accurately. Thi en e of re i tance or barrier i critical to manipulat
ive technique. PLANE The plane of force in a technique will be governed by the p
urpo e of the technique
factor of on et, arre t and amplitude are immediately varied by the con iderati
on of peed. A u eful rule about peed i that rhythmic technique hould be perf
ormed lowly enough for ti ue change to be palpated during the procedure. Suita
ble change can then be made to find the optimum path for ti ue re pon e. Thru
t technique i u ually performed at a high a velocity a po ible. Once the ten
ion ha been accumulated in the target ti ue minimum effort i then di ipate
d into other ti ue , and the maximum effect of the technique i applied to the
de ired ite. The critical thing about u e of peed i that it hould alway be
under the tight control of the operator. Do not u e the patient ti ue a the
brake to the force. The control mu t be directed by the operator u ing hi mu c
le a a brake. Thi require clo e-packing of the ti ue and a thorough under
tanding of the vector nece ary for the ucce ful completion of the technique.
DURATION The duration of application of force in a given technique will vary aga
in according to the type of the technique and the re pon e from the ti ue . A r

hythmic technique will be repeated everal time until a en e of change ha tak

en place. It i ea ier to over-treat than to under-treat. Many tudent , and om
e practitioner , feel that they want to give the patient the maximum benefit and
will work for too long in a given way on a part of the body. Too much i po ib
ly wor e than too little. A technique performed for too hort a time may not pro
duce much benefit, but it i unlikely to make the ituation wor e. However, a te
chnique performed for too long may well make a pain yndrome more uncomfortable
by fatiguing the ti ue . A u tained relea e technique will be held until a en
e of relea e occur . A thru t technique may be primed everal time , but the ac
tual technique will only take a long a i nece ary to perform the thru t it e
lf.
Force and the direction of the ti ue being worked on. In a cro -fibre kneadin
g technique, the plane will be acro the mu cle. In a tretching technique, the
plane will be along the mu cle ligament or fa cia concerned. In a thru t techni
que the plane will either be acro the joint to gap it, or along the joint to p
roduce a liding force. It can be een, therefore, that an accurate knowledge of
the anatomy of the part i e ential, a i a good en e of accumulating re i t
ance to applied force . Unfortunately, anatomical variation are the rule not th
e exception, and although certain principal direction are going to be accurate
within certain limit , there may be con iderable variation from the expected pla
ne in many ca e . There are no traight line anywhere in the body, and all for
ce hould have a lightly curving vector o that they are more likely to reach
the correct plane for the technique in que tion. An analogy might be to attempt
to drop a mall pebble into a funnel. The ideal would be to drop it traight dow
n the centre. However, it would be more normal to drop it in and ee it rattle a
round until it find it own way down the centre by running around in ever-decre
a ing circle until it drop . Technique i often performed by applying force in
a particular direction and then making many mall adju tment until the optimum
direction can be found. If the force i applied without the e minor adju tment
, the quantity will often need to be very high. The correct plane ha not been a
cce ed and force i being u ed to overcome the re i tance rather than accuracy.
Thi i unwi e, uncomfortable and potentially dangerou . The plane of a given t
echnique may change a the force are applied and ti ue deform in front of the
applicator. Thi i not uncommon, and it i nece ary to allow for thi po ibi
lity and not be too rigid in approach o that the variation can take place guide
d by the ti ue . FORCE
41
Force i a variable that again integrate with all the other to make a techniqu
e pecific, ucce ful and accurate. High force hould not be u ed a a ub titu
te for accuracy. The aim hould be to u e a little force a po ible that will
achieve the de ired effect. Generally it i better to generate force by good con
trol of lever and component than to apply high level of force to make up for
inefficient technique. The higher the force, the greater the chance of injury an
d the greater the control nece ary. When higher force technique are nece ary,
it i e ential to keep the target ti ue in mind o that the force i directed
accurately and pecifically rather than allowing force to di ipate in the ur
rounding ti ue . A uitable analogy i the difference between a hotgun and a r
ifle. The power of the hotgun will have a major effect at clo e range, but at a
ny di tance it will di ipate and the effect of the force will be minimal at any
given point. The rifle, however, will be ineffective in hitting anything except
the object in the direct line of fire, but will be extremely powerful in it ef
fect on that object. The part of the hand applying the force, the length of the
lever, the amplitude and the harpne of the arre t will all modify the actual
force applied. Re-duplication of force will have an intere ting effect according
to the maintained pre ure after the initial force. If a force i applied and r
emoved the natural recoil of the ti ue will allow deformation to take place at
the target ite and in the urrounding ti ue or tructure . If there i a dy

function, with fixation and hypertonu , the fixed point may not relea e a the o
ther ti ue are relatively more compliant and can ab orb the applied force of a
technique. However, if a force i applied, relea ed and then re-applied immedia
tely afterward and held, the effect will be quite different. The force will go
into the ti ue , and recoil out but will meet the fixed part of the operator
42
Modifying factor in technique and a mall applicator would be u ed. Therefore,
within the handhold that u e the whole of the palm and finger to produce conta
ct, there might be an area of the hand, uch a the pi iform, that perform the
cardinal part of the technique. If a larger area of contact i required, uch a
in a kneading technique applied to a mu cle group, the whole hand would be appl
ied, and le empha i placed on focu ing to a mall part of the hand. Even in t
hi ca e, there might be the need to focu with the heel of the hand at the culm
ination of the technique o that the part of the mu cle needing the pre ure cou
ld be worked more efficiently.
hand or arm. Having met thi re i tance the force will not be able to di ipate,
and will recoil back into the ti ue . If the operator hold thi po ition for
a few econd there will be an induced o cillation of force within the tructure
that may allow relea e to take place. It i difficult to find an analogy for t
hi phenomenon, but imagine a ball bouncing and then your hand placed directly i
n the trajectory of the ball. If the ball hit your hand before the whole of it
energy ha been lo t, it will peed up it bounce. It will al o u ually e cape
to one ide a the energy ha to find an outlet. U ing thi in technique i a li
ttle different. The patient i po itioned in uch a way that the only direction
the ti ue can go i again t the hand and the force i , therefore, thrown direc
tly back to the target ite. Thi type of technique i often u ed by chiropracto
r in cla ical toggle-recoil thru t . The principle can be equally well u ed
by o teopathic practitioner although chiropractor often u e pecifically de i
gned table with drop ection to enhance the effect. Force i al o modified p
artly by the ize of the applicator. A ume that the palm of the hand i u ed to
tran mit a force, and i , let u ay, 4 inche acro and 4 inche long. A ume
al o that the force applied i of 16 pound pre ure. Simple arithmetic will re
veal that there will be an applied force of 1 pound per quare inch. However, if
the area of the applicator i reduced to 2 quare inche , by the operator gripp
ing in uch a way that only a mall part of the hand i performing the effective
part of the technique, the force will have increa ed to 8 pound per quare inc
h. De pite u ing the ame quantity of overall force, the effect will be ome 8 t
ime a powerful, and directed much more pecifically to a point or area. Natura
lly, there will be time when a mall area of contact i preferable, and other
when a much larger area of contact would be more appropriate. A thru t technique
u ing very high pecificity might require great pecificity
ONSET The on et, or point at which the effective part of the technique i to be
applied, i naturally a ubdivi ion of many of the other modifying factor . If t
he on et of the effective part of the technique i made too early, the force wil
l be di ipated before the technique ha had a chance to reach the target ti ue
. Thi effect can be u ed purpo ely to minimize the tre on the ti ue , but i
f it u ed without realizing that i happening, naturally the benefit of the tech
nique will be reduced. If the on et of the technique i delayed until toward th
e end of a movement troke, the power will inevitably increa e a there will be
much le room for the ti ue to deform ahead of the hand. Mo t categorie of t
echnique can have their effect enhanced or reduced by mall variation of the on
et of the effective part of the procedure. The difficult thing i en ing at wh
at point to tart the critical part of the technique. Con ider for a moment a cl
a ical lumbar roll thru t procedure. A thru t early on in an o cillating moveme
nt of the patient will have a far weaker effect than one performed at the end of
a rotatory o cillation. A large patient might need the thru t applied much late

r in
Compre ion the technique than a mall and frail one. However, exce ive attempt
to control thi variable can lead to lack of confidence, and there can be a te
ndency to become fixed and unable to move at all. The variation of on et i happ
ening whether on a con ciou level or not; it i merely nece ary to become awar
e of it o that it can be varied lightly when nece ary. ARREST The point at wh
ich a technique fini he can be referred to a the arre t. Thi can be graduated
, a in a oft ti ue technique, or harp, a in a thru t technique. There can b
e a udden relea e of ten ion, a low relea e of the accumulated ten ion, or a r
e-duplication of force a previou ly de cribed. Each one will have a varied effe
ct and mu t be con idered with all other variable . The method of arre t and rel
ea e from ten ion al o need to be con idered. If the arre t i low and then th
e applicator i rapidly removed from the contact point, there will be a differen
t effect from that produced if the applicator i removed lowly. Even if thi ef
fect i p ychological, it i till relevant to con ider it. If a technique i ar
re ted ju t hort of the relea e of ten ion, and then re-applied, there will be
another difference in the rate and quantity of the di tortion of the ti ue tha
t will inevitably affect fluid interchange. Although the e may eem minor factor
, they can make the difference between a ucce ful technique, and one that i
only partly ucce ful. The rate of change in a target ti ue i of importance i
n do age of treatment and will influence the overall re ult. Many operator hard
ly pau e to con ider how they fini h a given technique. However, even a little t
hought given to thi variable may allow a moother integration of one technique
into another. The choreography of treatment will be that much moother and a it
uation where the patient can relax will be much ea ier to produce. Naturally, th
e re ult i the objective,
43
but the vehicle to reach thi re ult i only lightly le important. COMPRESSIO
N The term compre ion need ome explanation. Compre ion i not the oppo ite o
f traction. Indeed, it can be combined with traction within the ame technique.
Compre ion can be applied in everal different direction , either eparately or
in combination. The direction mo t often u ed i a compre ion into a clo e-pa
cked po ition. The act of applying compre ion ha everal intere ting effect .
If compre ion i con idered imply a another vector of leverage and force it
can be brought into per pective. In the ame way that adding component to a com
po ite lever will reduce the quantity of each component, compre ion can act to
reduce lever amplitude. If you apply any technique in the u ual way and then add
a compre ion between your hand , the patient ti ue , and your , will meet a
point of re i tance. Thi re i tance, or barrier, will be that much earlier tha
n if the compre ion wa not u ed. Let u con ider ome example . Apply a cro fibre kneading technique to the lumbar erector pinae with a patient prone a yo
u normally would. Try adding a compre ion force into the table, and a compre i
on of your che t and arm mu cle by a mall i ometric contraction before the tr
oke lateral from the pine. You hould find that the troke i of a maller ampl
itude a you have ab orbed ome of the lack with thi pre-loading of the ti
ue . Thi will have everal effect . Fir tly, the amplitude will be le ; econd
ly, the force can be reduced according to how much compre ion i applied; third
ly, the en e of relea e in the ti ue hould be ea ier to feel. The cue houl
d become much cri per. A another example, con ider a lumbar roll type of thru t
manipulation. Po ition the patient in the normal way and then, in tead of incre
a ing the rotation at the moment of thru t, intro44
Modifying factor in technique afety. I would go o far a to ay that compre
ion i far more u eful a it allow a ub tantial reduction of force and amplitu
de. I feel that the traction rule ha many potential pitfall . By eparating joi

nt urface and putting urrounding ligament on ten ion, there i definitely a

afety component introduced, but amplitude generally need to be greater, and th
e di comfort of thi negate the benefit . Any attempt to protect di c and ot
her tructure u ing thi method i laudable, but prone to a po ible fal e en
e of ecurity. To ome extent thi rule developed out of the belief that all joi
nt dy function yndrome were due to mall di c fragment , and that traction hel
ped to replace the e. MRI and more ophi ticated diagno tic method have found t
hi belief wanting, and the u e of traction in manipulative procedure for thi
rea on ha been lowly uper eded. There i no doubt that traction ha it u e ,
but it i not a panacea.
duce a compre ion force toward the table and toward your elf. The patient i , t
herefore, andwiched firmly between your pelvi and the table. If you now apply
the rotary force, you hould find that the range or amplitude of the movement ha
been ub tantially reduced. Take thi further, and tart the technique with ev
en le rotation and it hould till be po ible to attain a ati factory lever
po ition to focu to the target joint without the common di comfort induced arou
nd the patient rib and houlder. It can be een that compre ion work , but w
hat i the mechani m? There i no definitive an wer to thi que tion, but there
are everal po ible explanation . The effect of compre ion may be due to quee
zing the fluid within fa cial tube : the fluid i naturally non-compre ible and
will, therefore, become relatively olid. There will, therefore, be a reduction
of range of movement a the relative ma of the part increa e . Thi po ibili
ty relate to the hydraulic effect of fluid pre ure and ten ion . The effect
may be due to the compre ion acting a a tabilizing of the part. Thi may be d
ue to an increa e of mu cle ten ion that i produced in a imilar way to the eff
ect of trapping an un table joint. It ha been hown that trapping a joint act
by increa ing the re ting tone of the area, and by  witching on the proprioc
eptor that have cea ed to function at their optimum. There may be effect due t
o the elimination of fa cial lide that, therefore, focu e the force to the ar
ticular tructure in a more efficient way. There may be effect due to the que
ezing having a p ychological action by the patient becoming aware of operator co
ntrol and, therefore, having a greater ability to relax and cooperate. There are
everal other po ible explanation that make en e. Not having a logical one,
I am, neverthele , happy to recommend the continuation of the method, a it i
o effective, comfortable and u eful. Some authoritie in i t that all manipulat
ive technique be applied in traction to increa e
PRIMARY AND SECONDARY LEVERS The primary lever can be identified a the principa
l or executive vector of force within a particular technique. The econdary leve
r , or component of the compo ite lever, are tabilizing and enhancing vector
that allow the greate t efficiency in the primary lever direction. The primary l
ever in a thru t technique i u ually in a liding or a gapping direction in rel
ation to the joint urface in que tion. Sliding force generally need le forc
e than gapping, and are preferable when po ible. Although the inclination of f
acet plane conform broadly to certain pre-defined direction , there will often
be ome variation. The direction of a primary lever i determined partly by a kn
owledge of the anatomy, but ultimately by palpatory awarene . The greater the n
umber of econdary lever , the le i the required contribution of each one, an
d the nearer to the midline the part can be while the technique i performed. Cl
early the nearer to the midline, the le the di tortion
Primary and econdary lever and tre on the ti ue , and the greater the comf
ort. With greater comfort, the patient i better able to relax, and there i le
likelihood of trauma in treatment and adver e reaction afterward . To illu tra
te primary and econdary lever I will again u e ome example . Flex your wri t
until it can go no further without di comfort. Become aware of the quality of re
i tance from the ti ue to thi minor tre . U e the other hand to pu h the h
and further into flexion, and become aware of the immediate increa e of normal r
e i tance and train. Return the wri t to the re ting po ition, and put it into

a degree of ulnar deviation. While maintaining thi ulnar deviation, add flexion
until re i tance i felt. It will be noticed that the point of re i tance in fl
exion i at a le er point than before, and that the quality of the re i tance i
different in feel from that induced before. Now try gripping around the wri t
and hand with the other hand and repeating the exerci e. Thi add a compre ion
vector to the overall leverage. The feel of the compo ite lever will be quite d
ifferent on a ubjective level in the wri t being levered, a well a in the han
d doing the levering. Thi imple example illu trate the u e of a compo ite lev
er with a primary force of flexion, and econdary lever of ulnar deviation and
compre ion. The quality of the re i tance felt will not only be different, but
will be le uncomfortable than a imple flexion. Additionally, if you were to u
e it a an articulation movement repeated everal time , even though the joint
are not being taken to their normal articular limit, there would be an increa e
of range produced. Thi effect can be u ed freely in treatment and make techni
que very much le uncomfortable. With practice, pecificity improve al o, a t
he variable allow the de ired tructure or ti ue to be targeted accurately. An
other example might be a cla ical thru t technique in the cervical pine. The p
rimary lever i u ually rotation in an attempt to lide one facet on the affecte
d ide forward on the
45
one below. The normal method of locking i to induce ome idebending to the aff
ected ide and to rotate to the other ide. Thi ocalled phy iological locking
obey Fryette law and add pecificity to the technique. U ing the principle
ju t outlined you could add everal other econdary lever to minimize the ampl
itude of the rotation. The one mo t commonly u ed in thi ca e would be ide hi
fting, compre ion and anterior hifting. The total of all the vector would rea
ch a 100% locking po ition, allowing the facet to be freed in the de ired direct
ion. However, the quantity of each could be reduced a the number i increa ed.
All thru t technique require a little free play o that the optimum plane can b
e found. The accumulating barrier will be felt at an earlier point than without
the econdary lever , and the potential for a ucce ful technique will be enhan
ced. To an out ide ob erver, the manipulative po ition would not eem effective
a the neck i not tor ioned a in the common method of only two or three vector
. The operator would al o need to be in much more control than normal a the ti
ue would tend to e cape from the induced ten ion. Body ti ue are like a pr
ing, and if allowed freedom to take their own path, will u ually take the ea ie
t direction. Thi ea y path i one that allow the minimum di tortion and the ma
ximum ability to di ipate force over a large area. If the technique i applied
without the optimum ten ion it i unlikely to ucceed, but it i al o extremely
unlikely to cau e pain or di tre . In every technique there will be an executiv
e or primary lever, and there will be econdary or tabilizing lever or compone
nt of the compo ite lever. Some complex technique may even have what might be
termed tertiary lever that come into play only rarely if uitable barrier en e
cannot be achieved. Even though I have avoided terminology relating to le ion p
o itioning, the deci ion about a primary lever can be taken on thi ba i if de
ired. The y tem of
46
Modifying factor in technique flexible ubject who e ti ue may be able to di
ipate the force before it reache the target ite. However, even in a flexible
ubject, at the end of exhalation the ti ue are at the point of minimum abili
ty to relea e further, o technique performed at that point can be extremely pe
cific. If technique i performed on largely inflexible ubject who happen to be
at the end of an exhalation pha e, there will be much more po ibility of traum
a and di comfort. Thi will particularly be the ca e when working on the thoraci
c pine and rib cage. RESISTANCE The re i tance to a manipulative procedure hou
ld come from the target ti ue or tructure. There hould not be more than the m

inimum re i tance from other tructure urrounding it. Indeed, the zone of effe
ctive re i tance to the force hould be a mall a po ible to en ure that the
force reach the de ired tructure in the mo t efficient way. If progre ively a
ccumulating force are applied in a general, coar e way, there i the di tinct p
o ibility of training urrounding tructure . The other po ibility i of the
force being di ipated in the other tructure and not having the de ired effect
on the part in que tion. Adhe ion around a dy functional egment will often be
tronger than the urrounding normal ti ue , and force applied will di tort t
he normal ti ue and not affect the dy function unle the force are accuratel
y localized. Imagine two piece of wood joined by a trong glue joint. Attempt
to eparate them with force might ju t a likely break the wood a the glue join
t. If, however, the glue joint it elf were to be truck with a hammer, there i
a good chance that it would eparate without a much damage to the re t of the w
ood, the maximum re i tance to the force having been formed exactly at the joint
it elf. It i difficult to acquire the kill nece ary to be able to en e the
optimum tate of
identification of lever vector i u able in both approache . Even an operator w
ho ha no knowledge of thi po itional thinking will inevitably be u ing it, a
the optimum po ition for any technique i made up of everal different force . O
nce the con ideration of primary and econdary lever ha been identified, it h
ould be po ible to return to a technique that i not working and identify what
i mi ing. Like all the other modifying factor , lever identification mu t be i
ntegrated with all other a pect to make it fully u eful. RESPIRATION The active
u e of re piration in technique i not new, but the identification of the diffe
rent pha e of breathing that can be integrated in technique to per onalize each
procedure to the patient may be. Naturally, it i not nece ary to perform ever
y technique at a pecific pha e of re piration. It would be far too ea y to conf
u e and up et the patient with con tant in truction about when to breathe! Neve
rthele , there are time when the active u e of breathing can be u eful. Many p
ractitioner a k their patient to take a deep breath and then to breathe out ju
t before a thru t technique i to be performed. Thi i de igned to produce the
optimum tate of relaxation in the ti ue and the patient. Thi i ometime u
eful, but if thi i the only u e of breathing, many other po ibilitie are bei
ng ignored. Exhalation certainly can produce relaxation, but in very flexible u
bject thi in it elf can be a problem. Patient who are generally hypermobile a
nd have a fixed area needing technique applied, might be better addre ed when
they are inhaling, to produce a en e of focu ing in the ti ue by firming them
up. During inhalation the normal phy iological curve of the pine are lightly
flattened, and thi fact can be u ed in technique. During exhalation there will
be more recoil and give in the ti ue and thi may prove to be a di advantage.
Thi i particularly true in
Hunch re i tance in a joint or other tructure while maintaining the other truc
ture in a tate of minimum ten ion. It i not difficult to en e an increa ing
barrier by being aware of exce ti ue di tortion and train. Thi i very diff
erent from an accurately applied force u ing controlled force to a target egme
nt within a zone of minimum induced ten ion. Generally the clo er the applicator
can be applied to the tructure being worked on, the better. The optimum re i t
ance depend on the be t accumulation of force controlled by u ing everal of t
he modifying factor in combination. The be t en e of re i tance i very depend
ent on the next modifying factor de cribed, contact point pre ure.
47
The total force would be imilar, but the initial pre ure of tanding on it wou
ld bring the tructure nearer to a breaking point without o much impact. Natura
lly, the u e of contact point pre ure can be exce ive a well a in ufficient,
but like mo t of the other modifying factor , can be decrea ed a well a incre
a ed a nece ary. HUNCH Some will ay that the u e of hunch or ixth en e hou

ld have no place in a cientific activity like o teopathic technique or treatmen

t. All activitie that require kill or knowledge, and rely on variable factor ,
are liable to o many variation that ixth en e i inevitably u ed. Rather th
an denying thi , it eem to make more en e to u e it con tructively. Thi doe
not mean that one hould rely purely on feeling and not u e rea oning and logi
c. It i normal for thi ixth en e to develop, and one can u e it con tructive
ly providing it i backed up by ome rea oning. When an experienced practitioner
i a ked why he i doing omething in a particular way, the an wer i often, b
ecau e it feel like it need it. If we analy e thi tatement it i po ible t
o ee that there i a combination of learning, forgotten experience , con ciou
compari on and previou mi take and ucce e . Experience can be defined a th
e ability to recognize common pattern . The recognition i one thing; being able
to act in an appropriate way i another. Hunch i really all of the e. It i ne
ce ary to guard again t the trap of the alway , never yndrome, where the two
word are u ed a an excu e for not thinking omething through thoroughly. If a
practitioner find him elf aying, I alway . . ., he may not be flexible eno
ugh to allow the e ential variation that allow the technique and the treatment
to vary ubtly to uit the particular ca e. The ame i true of I never . . .
Providing thi i u ed for contraindication , I have no argument with it; if it
i u ed in other ituation too often, it how
CONTACT POINT PRESSURE The amount of pre ure with the applicator, and the metho
d of application of that pre ure, are other critical variable . It i almo t im
po ible to produce the be t re i tance in a joint or tructure unle enough pr
e ure ha been u ed to remove the lack from the overlying tructure . Thi pre
ure mu t be very carefully applied if it i not to become painful, and the par
t of the hand or other tructure u ed mu t be con idered carefully. The method o
f application a well a the total quantity i al o important, and depending on
the type of the technique, the pre ure rarely remain tatic, but increa e and
decrea e a the technique progre e . All technique have a beginning and an e
nd, and the point of on et and arre t ha already been di cu ed, but variation
in pre ure will influence the other factor to a large extent a well. A an a
nalogy, vi ualize attempting to break a piece of wood laid acro two brick . If
the force were to be applied from a di tance, con iderable impact might be nece
ary. If, however, you were to tand on the wood and imply bounce up and down,
there would be a good chance that it would break under the train.
48
Modifying factor in technique aware of them or not. By bringing them to a level
of analy i and con ciou thought, it hould be po ible to u e them con tructi
vely to tailor a given technique to the particular patient.
a rigidity of mind that cannot fit with good manipulative kill that i tailored
to the individual patient. It can be een, therefore, that the modifying factor
are being u ed whether an operator i
HANDLING
8
The term handling refer to more than ju t the placement of the operator hand
on the urface of the patient body. The term hould encompa all a pect of
ti ue ympathy from the initial contact right through to the very la t touch of
any treatment or technique. The initial touch in any given technique hould be
mooth, gentle yet firm and impart confidence to the patient. Con ideration mu t
be given to the choreography of the treatment e ion o that one move can be
moothly integrated with the next, in an apparently eamle way. An a pect oft
en not ufficiently con idered i the initial application of the hand. If the wh
ole hand i applied in one action thi may be uncomfortable and threatening to t

he patient. It i often better to apply fir t the fingertip , then the palm and
la tly the heel of the hand. Depending on the part being worked, it may be more
appropriate to u e the ulnar part of the hand ub equently preading to the palm
, heel and then finger la t of all. The time for thi gradual introduction of t
ouch will vary with different operator but the thoughtful operator will u e com
ment from a fellow tudent or practitioner to judge the optimum in a variety of
ituation . An average for mo t operator i about 3 econd from initial touch
to full contact. Thi may eem rather a long time but feedback will reveal that
it i about right. The amount of pre ure in any ituation will al o vary. With
in rea on, the firmer the contact, the more confidence the patient will have and
, therefore, the better they will be able to relax. However, the chief guiding f
actor in thi contact point pre ure i how much of the hand i in u e. Again, a
rule i to apply a much of the hand a po ible within the particular technique. Sub equently the operator make one pecific part of the hand
the executive applicator of force to be applied for that technique within the o
verall hand contact. Thi type of con ideration eem to be needle ly detailed
and complex. However, clo e attention to thi a pect can improve patient complia
nce, relaxation and ultimately ucce in any given technique or treatment. Hand
ling not only refer to the u e of the hand but mu t include all other part of
the operator body, particularly tho e coming into contact with the patient. F
or example, where the patient head i cradled in the hand of the operator, it
i important to en ure that the patient ear and eye are not being ob tructe
d by part of the operator clothing or arm . When patient are itting, mo t of
the movement of their body i often performed by pre ure from the operator b
ody rather than the hand . In thi way the communication of the required po itio
n or movement i tran mitted efficiently without exce ive pre ure on a po ibl
e painful ite. A imple rule relating to efficiency in handling i for the oper
ator to examine carefully the po ition of hi elbow . In mo t technique , at the
culmination of the procedure the elbow hould be a clo e to hi body a i pr
acticable. To check on thi element it i a good idea to a ume a variety of tec
hnique po ition and then ee if it i po ible to modify the hand and elbow po
ition for greater comfort and effectivene . Comment from a cooperative fellow
tudent will prove valuable in thi re pect. Clearly it i important to avoid d
i comfort produced by the pulling of hair, exce ive kin drag and digging in of
wri twatche ,
50
Handling by one practitioner over another. Although each practitioner will devel
op their tyle of approach, ome adju tment can be made; there i no better way
of di covering u eful modification than giving treatment to a colleague who i
empowered to make con tructive critici m of the handling u ed. It could be very
intimidating to have one work crutinized in thi way, but if the purpo e i
for elf-improvement it i very worthwhile. Even the fine t port per onalitie
in the world have their coache who e job it i to make uch minor adju tment
in method to improve performance. We are naturally intere ted in the re ult, but
the comfort, efficiency and effectivene of the method are open to ub tantia
l variation and without con tructive comment we are unlikely to know how to adju
t them. Care hould al o be taken in the u e of equipment. If a hydraulic table
i u ed, find the be t way of elevating and lowering it to avoid exce ive jarr
ing. Avoid banging into the table when walking from one ide to the other and mo
ve pillow with due con ideration for patient comfort to avoid the head being je
rked to one ide. The term handling al o relate to all a pect of patient care
including the operator-patient relation hip. It i not ugge ted that exce ive
formality i nece ary, but that a truly profe ional relation hip will help the
patient to develop tru t. Patient education about the nature and purpo e of dif
ferent a pect of treatment i a u eful part of practitioner hip. It i not prac
tical to give exact de cription of each manoeuvre. However, involving the patie
nt in the treatment proce by way of information and di cu ion i clearly a wa

y of helping them to under tand what i happening, and i likely to avoid mi und
er tanding .
ring or long fingernail . Operator long hair hould be uitably tied back, an
d frontfa tening clinic coat avoided. It hould not be nece ary to mention tha
t breath melling of alcohol, garlic or cigarette , and finger tained and mel
ling of nicotine are unprofe ional and, therefore, not acceptable. In many tech
nique it may be nece ary for the operator body to be in clo e proximity to t
he patient. If thi i likely to cau e potential embarra ment due to the part o
f the body being u ed, then a uitably interpo ed pillow or rolled towel may com
e in u eful. Female operator will, no doubt, be e pecially aware of the need to
avoid embarra ing brea t contact with the patient and may choo e other techniq
ue when nece ary to overcome thi problem. Patient may feel extremely vulnera
ble if they are aware of contact with the operator genital area. Thi mu t be
avoided. Belt with large buckle can dig into the patient a can other item of
jewellery uch a necklace and name badge and it i a well to remember thi
in clinical practice. The power of touch hould not be undere timated, and in em
otionally un table patient can produce trong reaction and re pon e . In uch
ca e it i e ential to be ympathetic and yet firm and rea uring in uch a wa
y a to how care without exce ive involvement. It i very ea y for the caring
practitioner to fall into the trap of palliative troking that may give thi typ
e of patient a fal e impre ion. However, neither i it acceptable to be heavyha
nded in an attempt to avoid eeming exce ively ympathetic. From the point of v
iew of patient ati faction, ome practitioner will be able to build and mainta
in a practice far fa ter than other . Some of thi may be due to ubliminal awar
ene by the patient of uperior handling
OPERATOR POSTURE AND STANCE
9
Factor relevant to operator po ture are: weight in relation to gravity; the con
tact with the floor or table; the po ition of hi head; and all other a pect of
weight di tribution con i tent with the ability to tran mit force in the be t
way for each technique. The term tance relate more to the po ition of the oper
ator in relation to the table, and the proximity to the patient. Although the tw
o are to ome extent interdependent, there are difference . For example, an oper
ator can be in a good po ture from the point of view of balance and ability to c
ontrol the amount of weight he i to carry, but be in a totally inappropriate pl
acing for the technique to work well. He might have hi feet pointing in uch a
direction that when he applie the culmination of the technique, the force di i
pate away from the intended path. He i then not in ufficient contact to contr
ol the target area a hi foot po ition prevent him directing the force in the
de ired direction. If one operator trie to copy the po ture of another exactly,
purely by imitation, he i unlikely to make every procedure a ucce . The morp
hology of each practitioner i inevitably different. It i clearly e ential to
learn to control the operator body with the force he i to apply. Thi will l
ead to good control of the a pect of the force needed in treatment of the pati
ent for maximum benefit and minimum effort in technique. The patient-operator in
terface, that con titute the u e of the operator weight, balance, and grip or
hold, mu t be efficient if it i to be effective. There are many element of th
i particular modifying factor in technique that can be adju ted by ome imple
but ubtle change . Once the idea and the
rea on for them have been con idered, it take a little practice to make each o
ne a natural flowing part of the technique. Thi effort will be amply rewarded i
n le long-term effort, train, and po ible phy ical problem for the operator
, a well a increa ing the effectivene of each technique for the patient. All
technique employ force applied to the body of the patient. The e force can b
e extremely delicate and light, or can progre to quite high force when nece

ary for a pecific objective. Clearly the lighter the force applied, the le th
e potential trauma to the ti ue being worked, but if heavier force are nece
ary, they mu t be very carefully controlled to avoid exce ive di comfort. After
all, the main purpo e of manipulative treatment i an attempt to relieve pain,
not cau e more! If we analy e operator po ture from the feet up there are many
imple thing that can alter the way technique are applied. If the feet are plac
ed parallel, any force applied to the patient will require the operator to pu h
with an equal and oppo ite force to avoid the tendency of hi body to be pu hed
away from the plinth. If the feet are in an inefficient po ition it may be nece
ary to tand further from the plinth, and then be in a poorer balance po ition
. Thi will require an increa e of mu cle ten ion in hi body that immediately t
ake away ome of the control of the technique. The imple expedient of having o
ne foot lightly ahead of the other mean that it i ea ier to generate force t
hat have more control and balance than if the feet are parallel. To make one foo
t the front foot and the
52
Operator po ture and tance bent. If one knee i kept traight, movement of the
operator mu t pivot around that traight knee, and thi will artificially limit
the ability to perform the technique. Slightly bent knee al o permit movement i
n the antero-po terior plane and up and down movement a well, thu allowing a f
ar greater flexibility of approach. Sometime , however, it will be found that on
e knee traighten at the end of a given technique. Thi give the ability to fi
rm up the technique at the la t moment, and can often be a u eful inclu ion. In
a imilar way mo t technique are performed better if at lea t one of the operat
or hip i omewhat flexed. Any technique that require the hip to be extende
d will inevitably be extending the operator back. Although there may be time
when thi i nece ary, exce ive exten ion repeated too often can be ju t a mu
ch of a problem a exce ively repeated flexion. The u e of a front and a rear l
eg allow one hip to be flexed while the other may be extended. Thi inevitably
put the operator pine in a twi ted po ture which, when exce ive, can be pro
blematical. Neverthele , ome twi t mean that the technique can be performed w
ith the operator in a firmer po ition to allow better tran mi ion of force . Th
e operator own lower back i be t held in a near neutral po ition a po ible
, and if it i nece ary to bend over the patient, then the flexion u ually come
from the hip while keeping the lower back traight. To avoid train the mall
amount of twi t already mentioned mu t not be allowed to become an exce ive to
r ional movement to end-of-range. Single technique uch a thru t technique h
ould be le of a train to perform than rhythmic technique that may need to go
on for ome time, but the po ibility for over train of the operator i alway
pre ent. A ingle application technique performed many time in a week can becom
e a repetitive train if inefficient method become a habit. If the lumbar pine
i held in a neutral lordo i directly over the centre of gravity between
other the rear doe not nece arily require the operator to tep back with one o
r forward with the other. All that i nece ary i to wivel lightly on the bal
l of the feet o that the feet are pointing either to one of the top or bottom
corner of the table depending on the direction nece ary for the technique. In
very few technique will the feet be pointing traight acro the table. If the
reader will look carefully at ome of the picture later in thi book he will fi
nd that in mo t ca e there i a front foot and a rear foot. Further tudy will
reveal that in mo t ca e , if a rhythmic technique i being u ed, the weight i
being u ed to tran fer the force to the patient through the hand on the ame id
e a the front foot. However, if the technique i a thru t technique, then the w
eight i being tran ferred to the patient through the hand on the ame ide a t
he rear foot. Thi i not an ab olute rule, but it applie with very few excepti
on . Further tudy will reveal that the operator i ometime not in contact wit
h the table at all. At other time he ha hortened the lever by leaning with o
me part or part of hi lower extremitie again t the table edge. Shortening the

lever by leaning on the edge of the table i far more common in rhythmic techni
que than in thru t technique . In mo t thru t technique it i ea ier to accele
rate rapidly and then form the harp braking action nece ary to produce the ho
rt amplitude movement if the heel of the rear foot i elevated lightly off the
floor ju t before the thru t i applied. The ub equent contact of the heel with
the floor form a trigger for the completion of the thru t when the operator dr
op onto it. In mo t technique the feet hould be tending to point in approxima
tely the ame direction. If they are ub tantially different in direction there
i the tendency to lock the operator po ture a the technique progre e and o
nly to be able to move in a downward direction. Mo t technique are ea ier to p
erform with ome flow if the operator knee are lightly
Operator po ture and tance the feet, then the po ition i likely to be the mo t
efficient in the majority of ca e . The operator middle back i be t held in
a neutral po ition without exce ive train into either flexion or exten ion. So
me degree of tor ion i po ible a the mu cle are capable of controlling artic
ular tre , and thi ha the effect of firming up the technique. It hould be n
oted that with the mid back the operator own rib play an important part in ma
ny technique . If the operator i in firm contact with the patient body, a li
ght twi t in hi back will bring the ide of the operator rib cage in contact
more than the pringy part of the ternum. Thi al o allow the operator arm
greater freedom to move a the one foot forward po ture can tend to eparate the
m lightly from hi body. The operator upper thoracic area i placed under con
iderable ri k of mechanical tre a the arm are being u ed all the time. The
mechanical leverage of arm i tran mitted to the re t of the body through the
houlder and upper thoracic area. For thi rea on, it i clearly important to a
lway keep the houlder and upper thoracic area a relaxed a po ible. A good
rule i that if the operator can ee hi houlder from the po ition he i in to
perform the technique, they are probably too high for maximum efficiency. A ma
ll amount of i ometric ten ion in the operator lati imu dor i mu cle will e
n ure that the houlder are pulled down far enough o that he ha a better chan
ce of increa ing the compactne of hi body. Thi will allow tran mi ion of th
e force to the patient in the mo t efficient way. I ometric ten ion and breath-h
olding mu t be u ed with caution to avoid locking the operator rib cage exce
ively. It i perfectly under tandable to want to ee what one i doing in techni
que, but the head i a heavy object. If the operator allow hi head to incline
forward exce ively, increa ed train i being cau ed to the upper thoracic area
. The weight of the head i being placed in front of the centre of gravity line
and greater mu cular effort i required to
53
maintain the po ition. Thi doe not mean that the operator mu t look up at the
ceiling, but that he mu t be aware of the need to keep hi head a high a i re
a onably practicable. Simply keeping the neck long i often enough. Thi imple
a pect i enough to control almo t the whole of the pinal po ture of the operat
or. The operator elbow are an important part of many technique . Mo t procedu
re are performed better, and with a greater degree of accuracy, if the operator
pull hi elbow a clo e to hi ide a i fea ible. Thi need not be taken t
o extreme , but if any technique i not working a intended, a imple adju tment
of po ture of elbow i often all that i needed to fine tune it to a better
efficiency. Elbow po ition i a imple thing to con ider in thi regard. Even in
technique where it i impo ible to bring the elbow clo e to the ide , a ma
ll i ometric pull downward and inward of the operator arm will have a benef
icial effect of firming up the hold, and increa ing the proprioceptive awarene
of the operator. The lack of the operator own joint play i al o reduced if
thi mall  etting of the arm i undertaken. The hand of the operator will b
e applied to the patient but over train can be avoided by minimizing flexion or
exten ion of the wri t. When technique require the u e of the thumb for pre ur
e, thi i be t applied in a little abduction a po ible. Some i ometric etti

ng of the hand will allow the  witching on of the joint proprioceptor in the
hand and arm which will enhance the palpatory awarene of the operator con id
erably. Thi al o ha the effect of lightly protecting the hand from over trai
n by giving a en e of graduated re i tance to all force being applied. It i
ometime ea y to fall into a habit of ulnar deviation and allowing the ligament
on the radial ide to be repeatedly tre ed. Although thi may not produce a t
rue hypermobility, there i ome indication that it may produce di comfort if fr
equently repeated. Female operator may have problem , in
54
Operator po ture and tance exhalation a the operator rib articulation are i
n a vulnerable po ition to train. Breathing al o change po ture; on inhalation
the pine lengthen and traighten lightly, and the rever e happen on exhala
tion. It i ea ier to tran mit a en e of relaxation to the patient if the opera
tor i relaxing him elf. A controlled outward breath will often help to produce
an optimum en e of ti ue relaxation in the patient. If the operator a k the p
atient to breathe out at the ame time, thi how how another modifying factor
of patient re piration can be combined with operator po ture and breathing to go
od effect. The ideal te t of whether a po ture i balanced for any given techniq
ue i to attempt to perform the manoeuvre without a patient. Thi may eem very
artificial, but it demon trate balance, control of the operator body, and the
ability to perform the technique without exce ively leaning on the patient. Al
o, the under tanding of how much ten ion the operator need to put into hi han
d i often poorly con idered. If the hand i merely applied to the urface of t
he body and pre ure i increa ed until mild di comfort i felt by the patient,
there will be a completely different en ation than if the operator put hi own
hand and arm into a light degree of i ometric ten ion and then repeat the am
e exerci e. Thi type of dynamic ten ion in the operator mu t be practi ed to ac
quire the correct level for each technique. Some technique require quite firm i
ometric ten ion in the operator, other very light. An example relating to othe
r activitie that may make thi more ea ily under tood, i to imagine threading
a needle. Exce ive ten ion in the hand would make thi a very difficult exerci
e, but in ufficient ten ion would make it equally difficult, but in a different
way. Another example would be pu hing a drawing pin into a re i tant urface. T
hi would clearly require a different type of ten ion and control to the threadi
ng of a needle with cotton. The ability to take the grip in a firm but comfortab
le way, promoting confidence in the patient, and yet not cau ing unnece ary

ome technique , with the proximity of their brea t to the patient. If the oper
ator i required to place their che t again t the patient, which may be a ource
of embarra ment or difficulty, then a pillow or uitable pad can be interpo ed
. Similarly operator of both exe will want to avoid exce ive proximity of th
eir groin area to the patient. The tru t built up between practitioner and patie
nt can ea ily be de troyed if the e imple common en e a pect are not con ider
ed. It i ea y to forget that mo t patient are going to be omewhat apprehen iv
e or nervou about treatment. They may already feel vulnerable by being partly c
lothed, and a thoughtle operator who doe not realize the implication of hi p
roximity to the patient i at ri k of problem in thi regard. Ten ion en e, or
barrier en e, i acquired not only through the hand of the operator. In many
ca e the friction with the patient through the operator own che t tran mit a
much feel for the be t time to apply a thru t a doe the feeling through the
hand . One i looking for interpretation of the ubtle difference between ti ue
tre , and ti ue potential for activation of force when a technique i applie
d. The ubtle cue that come from a joint placed in a po ition of potential ar
e felt through the accumulation of force en ed by the whole body of the operat
or. The pha e within the breathing cycle of the operator when technique are to
be performed i ometime of importance. Some operator tend to hold their breat
h and train again t the fixed abdomen and thorax by thi temporary Val ava mano
euvre. Thi ha the effect of firming up the technique, but al o ha the unde ir

able effect of rai ing the operator blood pre ure. Thi i clearly be t avoid
ed. There are time when thi explo ive force of breath-holding i u eful, but i
t hould not be u ed too frequently. Some operator tend to breathe out when per
forming thru t technique to help peed up the move. Although thi can be u eful
, and avoid the tendency to hold the breath, it i better not to perform the te
chnique at the extreme of
Operator po ture and tance di comfort, i not ea y to acquire, but with a helpf
ul fellow tudent can be uitably developed. If any po ture i cau ing exce ive
fatigue for any rea on the cau e mu t be found. Performing a technique once in
i olation i not how treatment i carried out in practice. Treating many patient
in a day require the operator to ma ter the bu ine of minimizing train on
hi tructure if he i to be able to carry on the work without injury and exce
ive fatigue.
55
In any activity requiring p ychomotor kill, the expert make thing look very e
a y. Thi i true in porting or other activity uch a manipulative technique.
The rea on that it look o ea y i that the practitioner ha learned to avoid t
he thing that make it look difficult! Mo t of the e a pect are related to the
way force are tran mitted or developed. Good control of po ture and, therefore,
the practitioner him elf i one of the key way that thi can be done in o teop
athic technique.
APPLIED TECHNIQUE
10
There i a blurred dividing line between what i technique and what i treatment
, and the term applied technique relate to fitting the cho en technique to the
patient and the ituation that ha pre ented. There are everal factor that nee
d to be con idered in addition to the modifying factor already identified. Some
of the previou ly mentioned modifying factor al o need amplifying: 1. 2. 3. 4.
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. Handling Po itioning Po ture Weight-taking O
perator relaxation Hold Apparatu Approach Planning Difficult ituation Re pon
e to reaction Category of technique Interval between treatment Advice and aft
ercare
POSITIONING
HANDLING
In addition to the factor identified in the ection devoted to handling, i the
need to con ider the tran ition of hold from one technique to another. It i no
t ufficient to have good handling in a technique if the tran ition pha e involv
e a rough and irritating approach. Patient in acute pain, in particular, need
ympathetic handling, preci ion and care to avoid further unnece ary pain. Pati
ent who are nervou or embarra ed need extra care, and judiciou u e of gown
or other uitable covering hould be con idered.
Po itioning or moving the patient on the table mu t be performed lowly and purp
o efully, particularly if they are in acute pain. It may be nece ary to perform
a whole treatment with the patient in one po ition, and thi can try the ingenu
ity of even the mo t inventive practitioner. If it i nece ary to move the pati
ent, it i often better to guide the movement, after de cribing what i required
, rather than try to do all the work your elf. Acute pain i be t controlled by
the patient initiating the motion rather than the therapi t. Once they have tar
ted to move, then operator a i tance i u eful. In thi way they do not feel o
threatened by being moved into a painful po ition and can be much more in contr

ol of their pain. Mo t technique can be performed in a variety of po ition . It

make en e to find the mo t comfortable for patient and operator providing thi
doe not require the acute patient to move exce ively. It i po ible to redu
ce operator fatigue by alternating, ay, idelying with prone lying in ub equen
t patient . In thi way you can u e your pu hing mu cle in one patient, and you
r pulling mu cle in the next. Alternating in thi way can be a u eful method of
being inventive and adding variety to the day. It will often allow the di cover
y of different and u eful way of performing a particular treatment in different
type of ca e.
POSTURE
I have already commented exten ively on po ture but will reiterate here the crit
ical
Apparatu factor of afety for patient and operator, and effectivene in techn
ique by the u e of efficient po ture. Many operator have uffered injury from
train in treating patient . Much of thi could have been avoided by more care o
ver po ture. Good po ture allow the operator to reduce train on hi own truct
ure, and to reduce the po ibility of exce ive force being applied unwittingly
to the patient. Small and light operator of both exe become extremely efficie
nt at utilizing leverage rather than weight when performing technique. When lear
ning technique it i a good idea to have ome in truction from a mall operator
to help to acquire thi kill. Virtually every operator will meet a patient one
day who i much larger than normal. It i only then that the problem that the
mall operator have daily, become evident. Strength i really no ub titute for
kill.
WEIGHT-TAKING
57
movement that can become almo t balletic and apparently choreographed if practi
ed efficiently.
HOLDS
It i clearly wi e to reduce to the ab olute minimum any technique that involve
taking the patient weight. On ome occa ion there i no other way, but if th
e table can be made to do ome of the work, phy ical tre can be minimized. Av
oid breath-holding if it i nece ary to lift a patient. Learn from the weight-l
ifter who exhale when lifting. Obey the advice you give the patient about keep
ing a traight back when lifting!
The hand placement or hold for a given technique can be varied in everal differ
ent way . The thoughtful operator hould not grip exce ively with the fingertip
. It may be difficult to avoid exce ive ten ion in the grip when performing a
new hold for the fir t few time . The balance between a firm and po itive hold,
and one that i painful, uncomfortable or irritating, i very delicate. It i a
good idea to con tantly re-evaluate the approach to any particular technique; th
e method that ha become mo t comfortable by frequent u age may not be the mo t
effective for the purpo e intended. Mo t operator re trict them elve to a rela
tively limited range of technique , and can have great difficulty when called up
on to perform them in a different way. Thi i avoided if variety i added a a
regular feature.
APPARATUS
OPERATOR RELAXATION
It i important to be aware of the nece ity to relax when performing o teopathi
c technique and treatment. A tate of ten ion not only tran mit it elf to the p

atient and their ti ue , but reduce the appreciation of palpation, leverage, p

re ure, tretch and other en ory perception. Fatigue i al o increa ed if the
operator i ten e during treatment. Thi i not an excu e for loppy technique,
but rather a rea on for mooth and controlled
It i u eful to experiment with different type of apparatu uch a tool , pil
low , pad and plinth type , etc. A height-adju table plinth i extremely u eful
to allow the patient to be at the optimum height o that different area can be
worked without exce ive bending or tretching. In a multi-practitioner etting
an adju table plinth or table i e ential. A ri ing head-piece i u eful, but
failing thi pillow of variou ize can be a ub titute. Any plinth that i he
ight-adju table hould have the minimum free play in the adju tment mechani m to
allow force to be tran mitted to the patient and not the hydraulic of the tab
le. If the table i too pringy the force u ed in technique will need to be hard
er and harper to overcome the bounce of the mechani m. If the only table availa
ble i a fixed height one, it i better to be too low than
58
Applied technique rather than being targeted to the de ired ti ue. However firm
or oft the table padding, mo t patient prefer having a pillow under the abdom
en when lying prone. Thi avoid exce ive pre ure on rib , brea t and genital
. It al o allow the lumbar pine to be taken out of exce lordo i . Exce ive
lordo i can produce pain if pre ure i applied to the lumbar area. The paddin
g of the plinth hould extend over the ide o that the patient doe not have d
i comfort on the in ide of the arm when lying prone with the arm over the edge
. Many technique require the operator to lean again t the ide of the table. I
f it i padded thi will be more comfortable. For hygiene, the plinth i now gen
erally covered with a pla tic material, wherea cloth wa often u ed in the pa t
. Cloth will harbour kin debri and du t and i not really uitable, although i
t doe have the advantage that towel or paper cover do not lide on it a they
do on pla tic and vinyl. If the patient i a ked to lie directly on the pla tic
it will be ticky and unplea ant, o ome covering i u ually placed over it. T
owelling i ideal, but will tend to lide. Fitted ela ticated cover avoid thi ,
a will tho e with tring tie under the table, although they are rather fiddly
to tighten and tie when the cover i changed. Paper cover heet are hygienic a
nd look good when fre h, but are expen ive and create a di po al problem. They a
l o look awful when rucked up and crea ed and torn after the patient ha turned
over a few time . Pillow hould be covered with a uitable paper towel or ervi
ette. Some female patient wear large bra with multiple fixing . It may be pref
erable to a k them to remove the e, and, for the ake of propriety, to provide a
rear-fa tening urgical type of gown. Mo t practitioner wear a clean clinic co
at of their preferred tyle. Although ome operator choo e to work in le form
al clothe , there i ome evidence that many patient prefer their practitioner
to be dre ed like a practitioner. They find it ea ier to relate to them and
too high. It i far ea ier to lo e height by preading the leg than to tand on
tiptoe and be off-balance. However, a table that i too low will be very tiring
to work on. Pillow of different ize and den ity are nece ary, partly to pro
p the patient up when lying in different po ition , and partly to act a fulcra
for ome technique . There are advantage and di advantage in different width
of table. Patient generally feel more comfortable on a wider table, but a they
automatically tend to lie in the middle of the table, they may be o far away t
hat many technique can place a train on the operator. Table width can be 18-28
inche (46-71 centimetre ). The narrow table will tend to be un table, and will
produce unea e in elderly patient who may be fearful of falling. The wide t wi
ll be preferred by patient but, in addition to the problem tated above, it wil
l be impo ible to place the patient a tride for itting technique . If the pati
ent i itting acro the table with the back of the knee re ting on the edge,
they will till be a long way from the operator. Sitting technique will requir

e the operator to reach a long way to apply any hold, and that can place hi own
back at ri k. All table are a compromi e. One way to get around thi problem i
to have a table that taper from about a third of the way from the head end to
a narrow foot end. Thi mean that a tride technique are po ible at the foot
end, and that itting acro technique can be performed toward the foot end. Wh
en the patient i lying down they will have the impre ion that the table i wid
e, a the part they can ee i wider than the re t. The padding hould be den e
enough to prevent the combined weight of the patient and the pre ure of the tec
hnique from bottoming out the foam. Thi i of particular importance in prone
technique where the co to-chondral junction are at ri k of damage if the table
padding i too firm. If the padding i too oft, it will be difficult to ab orb
enough of the pring when treating, and ome of the effort of technique will be
lo t in the padding
Difficult ituation develop a relation hip ba ed on tru t and under tanding. Th
ere are a few practitioner who choo e to wear a uit and tie, and although thi
may be acceptable for a ho pital con ultant, it i not really practical for a t
herapi t who i required to get into ome contorted po ition on occa ion ! It i
clearly better to avoid the u e of exce ive jewellery that can catch on the p
atient or their hair.
APPROACH
59
The operator hould cultivate an unhurried, calm, rea uring approach to the pat
ient. Thi i particularly important with new patient , the very nervou , the el
derly and mall children. Exce ive formality hould be avoided, but an over-fri
endly approach could be mi interpreted a flippancy or ugge tivene and thi m
u t be guarded again t. The operator mu t appear to be calm and in control even
when highly pre urized by the telephone, late patient and hi own problem . La
ck of confidence i en ed by the patient very quickly and i taken a a ign of
weakne . However, if the patient i taken into the confidence of the operator
and involved in the deci ion proce , they often appreciate thi immen ely.
for the patient who may be in great di tre when performing rotational movement
. It i often better to work toward a part that i in acute pain rather than go
traight to it. Neverthele , a thorough palpatory inve tigation of the ite of
di comfort i rea uring for the patient and can give clue about the type of
technique mo t likely to influence the affected ti ue . In ufficient force to
achieve the required re ult can be remedied by a re-duplication of the technique
u ing a graduated approach until the re ult i obtained. Much experience i req
uired to judge ju t the right degree of force in any particular ca e, and it i
better to be ineffectual than dangerou . An ineffective technique can be repeate
d and altered until it i effective; a dangerou one may be di a trou . It i un
wi e to per evere with any technique, particularly a thru t technique, in the pr
e ence of extreme pain or re i tance. Similarly, if a patient i unable to relax
, there may be hidden pathology or anomaly.
DIFFICULT SITUATIONS
PLANNING
It i not a good idea to give ca ual, off the cuff, unplanned treatment. It i v
ery common to be pre urized into fitting omeone in to an already full chedule
for ju t a quick look. Some practitioner eem able to do thi with no proble
m , but many will be prone to make mi take and error of judgement. Empirical t
reatment will tend to have very mixed re ult . It i better to have a pecific o
bjective in mind ba ed on a diagno i and backed up by ome idea of progno i . T
he thoughtful operator hould be con idering the immediate and longer-term objec
tive of the technique that are being u ed. A planned treatment avoid unnece

ary re-po itioning

There are numerou difficult ituation that can occur in practice, and it i no
t my intention here to con ider any except tho e related to technique. Patient
can be very importunate of time and energy and encourage a practitioner to perfo
rm procedure he may con ider unwi e. It i better to politely decline than to p
erform a technique one i unea y about and then have problem due to reaction o
r injury. It i naturally important to avoid medico-legal complication , and in
thi re pect the be t rule i that of informed con ent. It i not po ible or de
irable for the patient to be informed of every ingle procedure that i to be a
pplied and it percentage ri k factor. However, the overall likelihood of ucce
, and a broad outline of what i going to be done, and the tacit permi ion to
perform the treatment technique i a nece ary preliminary to treatment. In ca
e where a practitioner ha been accu ed of wrongful
60
Applied technique reaction. Providing a thorough examination ha been performed,
and no ini ter cau e for the reaction can be found, a gentler repeat of the a
me procedure can often rever e the effect of the reaction and provide relief fr
om ymptom . Mo t patient will accept an adver e reaction if they are forewarne
d, but may not believe it i normal if they are told after the event.
treatment, a very common factor i lack of communication and information. The Ge
neral Council & Regi ter of O teopath ha gone o far a having a con ent form
available o that patient can put in writing their agreement to treatment in wh
at might be con idered en itive area of the body. The e a pect of treatment m
u t be con idered more and more in the light of litigation experienced by practi
tioner in the USA. Ca e that may eem frivolou matter to European are omet
ime fodder for legal action .
CATEGORY OF TECHNIQUE
RESPONSE TO REACTIONS
Some di comfort after treatment i common. If thi i exce ive it might be term
ed a reaction to treatment or technique. The degree of inva ivene in the cho e
n technique will clearly have ome influence on the frequency of reaction , but
ome patient will have evere reaction however careful the approach. A feedbac
k from the patient a to reaction will give con iderable guidance a to the adv
i ability of continuing treatment or the need to make a re-a e ment. Reaction
out of proportion to tho e expected hould alway give cau e for re-evaluation.
Like any other medicine, manipulative treatment ha to be given in the correct
do age. An overdo e can cau e adver e reaction , wherea undertreatment may be u
nable to produce the de ired ymptomatic and objective change . It i , neverthel
e , a far more common mi take to over-treat than to under-treat. If in doubt, h
old back. Due to the mu cle memory phenomenon it i often nece ary to repeat
a given procedure on ub equent occa ion . It i unwi e to attempt miracle treat
ment in one e ion. Making allowance for variation in fitne , re ilience,
en itivity, and tate of general health, different patient will react different
ly on variou occa ion . An accurate record of treatment and technique performe
d i not only a legal requirement; it i wi e a it give po ible clue to the
cau e of any adver e
Choice of the be t category of technique to u e and the order of technique u age
i ba ed upon the requirement of the ca e and the duration of the problem. The
lea t inva ive approach i naturally the be t, but if ati factory re ult are
not being obtained ubjectively and objectively, that i the time that mo t prac
titioner will increa e the inten ity of method. However, imply u ing firmer me
thod in the ab ence of a good re ult i not nece arily wi e. A good rule would

be to u e the gentle t procedure likely to give the de ired re ult con idering
all factor in the ca e. A ha been tated el ewhere, the effect of a technique
may be out of proportion to the application of force. Some amazing re ult have
been obtained with the gentle t of approache when a patient ha already been t
reated un ucce fully by other practitioner u ing violent method .
INTERVALS BETWEEN TREATMENT
The interval between treatment i often critical. The deci ion a to the pacin
g of treatment i only arrived at by a con ideration of all the relevant factor
relating to the chronicity, acutene , urgency and practical factor of di tanc
e, etc. If a patient report benefit out of all proportion to the technique u e
d, it i generally better to u pend treatment for a while a it i po ible to
rever e the ituation. Even though it may mean ending the patient away with no
treatment and po ibly no fee, thi i preferable to cau ing a return of the
Advice and aftercare ymptom by treating them ju t becau e they have an appoint
ment. Many condition are uncomfortable after treatment, then feel much better,
and then lowly wor en a normal life pattern take over. If treatment i undert
aken in the good pha e it i far too ea y to tir up problem . The be t timing
i probably ju t a the ymptom are tarting to recur. Naturally thi doe not
alway fit in with appointment chedule , but a rea onable compromi e can be fo
und. Maintenance treatment at extended interval of everal month ha been foun
d to reduce the frequency, inten ity and duration of acute mechanical dy functio
n attack . Many practitioner undertake thi ort of work, although ome feel th
at it i not getting fully to the bottom of any particular yndrome. Being reali
tic, occupational, po tural and degenerative tate mean that it i not alway
po ible to fully correct any dy function, and ome maintenance make en e. The
patient mu t be involved in the deci ion to undertake thi type of approach. If
the patient i ymptom free on a maintenance vi it, it would be unwi e to perfo
rm forceful technique a ymptom could be cau ed. However, ome ay that thi
i the be t time to be doing firmer work a the patient  ti ue are unlikely t
o be o reactive. The deci ion mu t be individual to each ca e and each operator
.
ADVICE AND AFTERCARE
61
Mo t patient a k for exerci e . Mo t patient , however, do not do the exerci e
pre cribed! Providing they are really motivated to do them, carefully de igned
exerci e of a tretching or trengthening nature make en e. Many patient find
that exerci e rather than exerci e i preferable. They may be per uaded to wal
k, wim, do yoga or dance rather than perform pecific exerci e . Some will a i
duou ly follow in truction . Some will overwork them elve in an attempt to get
a quicker re ult, and ome will do nothing and feel guilty about it. Each ca e m
u t be judged on it merit . Patient are generally very keen to have advice a
to prevention of recurrence and quicker re ult in treatment, o long a it doe
not involve them in any effort! Many practitioner are not aware of the re pec
t the patient ha for their advice, and it mu t be carefully given o a not to
cau e unnece ary di tre by their being unable to carry out that advice. Unwi
e advice to change job, for example, might cau e lo of re pect for the operato
r who give it a it may be completely impractical. Advice given with the be t i
ntention and con ideration for the patient welfare i fine, o long a it i
tempered with reali m.
PRINCIPLES OF LOCKING
11
All technique that work by focu ing force to a pecific joint need ome method

of producing that pecificity. The u ual method i to combine lever in uch a

way a to produce a locking or focu ing. Thi mean that other adjacent tructur
e are excluded from being the target for the force and are, therefore, to ome
extent, protected. Exce ive attempt to protect them, however, are ju t a l
ikely to harm, from over-locking which can produce di comfort and potential tr
auma. How i locking produced? The u ual method in the pine i to induce ome t
or ion with a combination of idebending to one ide and rotation to the other.
Thi o-called phy iological locking i in accord with Fryette law , and i
the main tay of manipulative work utilizing thru t technique . There are everal
other way that a combination of lever can be produced to focu force . Compre
ion, in combination with much maller quantitie of the other lever , can be e
ffective, a can the u e of accelerating rhythm and o cillation. Method of prod
ucing thi focu ing have traditionally been taught u ing the term phy iologica
l locking and ligamentou locking. The difference between the e two method i
that phy iological locking implie facet appo ition and ligamentou locking im
plie progre ive ti ue ten ion. In practice both of the e method are u ed in
combination. A thoughtful operator will u e the combination of everal complex l
everage to po ition the part in an optimum placing o that only a light empha
i of leverage will achieve a focu at the de ired point. Locking need not con i
t of a teady wind up to a point and then the application of
overpre ure. Thi will often be very uncomfortable for the patient. If held for
more than a few econd , the di comfort will cau e the barrier to increa e and
there can be a en e of over-locking. An ab olute point where the force come to
gether need to be found. If thi can be done without actually applying the full
nece ary leverage until the la t po ible moment, the patient will feel more r
elaxed and better able to cooperate with the procedure. Thi can only be done if
the operator i aware of the feel of the ti ue a the barrier accumulate , bu
t ju t hort of full ten ion. Thi allow him to tea e the ten ion point everal
time from lightly different angle until the optimum can be found. If the am
e exerci e were to be performed at the ab olute point of full ten ion and lockin
g, the di comfort would probably be unacceptable. If the accumulation of ten ion
wa to 8 0 9 0 % , the comfort level would be acceptable. The la t 1 0 - 2 0 %
or o can be added for a hort a duration a po ible to fini h the technique.
The kill nece ary to work in thi way require the ability to palpate a pathwa
y of re i tance rather than an ab olute po ition. Thi ability i well worth dev
eloping. Some authoritie in i t on the need to lock down to or up to a poin
t or egment. The inter egmental change taking place in thi ort of applicatio
n of force are omewhat complex. The ituation i al o governed by normal varia
tion in anatomy and phy iological function of the part. Al o relevant are the d
i tortion of anomaly and pathological proce e . The e will produce di torted p
attern and altered quality of movement , particularly at the end -of-range. The
phy iological movement of the pine
Principle of locking are uch that mo t manipulative technique require compo i
te lever to achieve the tabilization nece ary to localize force. Mo t techniq
ue can be performed with a variety of lever , and quantitie of tho e lever . A
an example, con ider a technique where 30 of flexion i combined with 60 of rota
tion and 20 of idebending. If the flexion i applied fir t, there will be a tend
ency to u e more than the de ired 30. The technique will till work, but it will
be found that the other lever have been decrea ed by the ame amount a the fle
xion ha been increa ed. Conver ely, if the rotation i put in fir t, it tend t
o become an executive part of the technique a the mind i focu ed on maintainin
g the rotation a the other lever are applied. The e intere ting phenomena can
be u ed to advantage by playing with the order a well a the quantity of the di
fferent component until the optimum application i found. Experienced operator
u ually apply any given leverage a a compo ite movement and are con tantly var
ying the quantitie of all the direction a the ti ue yield or otherwi e in e
ach direction. Locking doe not then become full leverage and overpre ure. It a
chieve the de irable ituation of being a near a neutral point a po ible unt

il the critical time when the technique i finalized a an arc of movement. The
term locking i actually a mi nomer. Nothing i actually locked; rather the forc
e are concentrated more at one point than any other. Extreme movement in any di
rection followed by overpre ure can tretch, deform and po ibly damage ti ue
, particularly if combined with high application of force. U e of locking help
to focu the localization without any lever reaching it maximum range, althoug
h the primary, or executive, lever i taken further than all the other . The com
mone t method in the pine i to u e idebending to one ide, and rotation to th
e other. The addition of mall quantitie of flexion or exten ion, compre ion o
r traction, etc., will further aid the localization. The way
63
any particular operator build up the lever to a given technique will vary lig
htly from patient to patient. There will be a number of other variable factor
uch a table height, patient po itioning on the table and different ability with
either hand. Neverthele , the principle obey certain ground rule . Any partic
ular area of the pine can be manipulated u ing thru t technique in a whole var
iety of po ition and way . There are certain direction that will be mo t effec
tive, lea t traumatic, and mo t comfortable for operator and patient. A u eful g
uide i that locking can be produced mo t effectively when con ciou ly u ing mov
ement lea t available in the particular egment or area. Conver ely, the greate
r the available range, the more the variety of locking procedure that will have
broadly imilar efficiency. A an example, con ider the mid cervical pine. Thi
area ha a wide range of flexibility in mo t direction in a normal ubject, a
nd o it will u ually be po ible to po ition in mo t combination of leverage .
However, the lower cervical pine ha le range of movement due to the variati
on in facet angle and other factor . The range of exten ion particularly i lim
ited, and therefore manipulation u ing exten ion will produce a locking with muc
h le amplitude of leverage than flexion. In the upper cervical pine, the move
ment lea t available i idebending, and therefore idebending will produce the
quicke t and mo t efficient locking without u ing large range of movement. Natu
rally, if too much idebending i u ed, over-locking and pain would re ult ju t
a ea ily a if any other lever were applied exce ively. Having identified thi
principle it i po ible to evaluate the particular direction that thi theory
will upport in each area. Naturally each ca e mu t be con idered on it merit
, with regard to other factor uch a age, condition, ize, etc: 1. Upper cervi
cal - empha ize idebending, although the neck a a whole can be in
64
Principle of locking flexion or exten ion. Exce ive exten ion in the upper cer
vical area i unde irable a there i the potential for tre on the vertebro-b
a ilar tructure . Mid cervical - all po ition are po ible, therefore u e a ba
lance between them all, but generally empha ize rotation a it i lea t uncomfor
table. Lower cervical - u e exten ion in addition to main leverage of rotation.
Flexion i al o effective, but need a greater range than exten ion. Upper thora
cic - mo t range are limited o u e whichever combination i mo t effective in
the particular ca e; exten ion combined with a primary lever of rotation i u ua
lly the choice. Mid thoracic - depending on the degree of kypho i , u ually exte
n ion combined with idebending. Thoraco-lumbar - exten ion produce quicke t lo
cking, although extreme flexion i effective, if more uncomfortable. Mid lumbar
- a thi i a generally freemoving area, a neutral po ition empha izing rotatio
n produce be t effect . Lower lumbar - a the facet angle are o prone to vari
ation, and the lumbo- acral angle varie o much, it i difficult to make any u
eful rule . However, idebending applied fir t, followed by the rotation element
u ing flexion in the lordotic pine, and exten ion in the flat pine will u ual
ly be effective. you are till performing left rotation at a pinal level. Thi
may eem elf-evident, but if con idered carefully can alter the approach to a p
articular technique and po ibly make it effective where it may not be otherwi e

. The focu ing of force to a pecific point in a comfortable way for the patien
t and the operator i the e ence of kill in manipulative technique. If a parti
cular combination of leverage i applied in a teadily progre ive way to a poi
nt of full ten ion, not only will the patient be uncomfortable, but the operator
i unlikely to be in an optimum po ition to complete the technique efficiently.
However, if the build-up of the accumulating ten ion i en ed, and the ab olut
e point of maximum ten ion i tea ed, it hould be po ible to accelerate thro
ugh to the thru t po ition more efficiently. Thi mean that the point can be ap
proached from everal different angle , and allowed to increa e and decrea e to
find the be t balance of all the component . Thi playing with the joint i no
t a vague earching, but i a purpo eful, con idered variation of all the compon
ent and direction of force. There hould be a con tant earch for the optimum
in a po ition of lea t train on the ti ue . In the peripheral joint imilar m
ethod can be u ed, but thi time the focu ing i be t performed by u ing compre
ion a a focu ing force. Compre ion act a a limiter of amplitude and can be
u eful to bring effective barrier en e nearer to the midline, thu avoiding pa
inful tor ional leverage . The other con ideration that i particularly effectiv
e in the peripheral keleton i the u e of induced lever by taking both hand i
n oppo ite direction . Con ider the force nece ary to wring the water out of a
odden cloth if one hand wa to remain till while the other did all the work. C
on ider how the force could be reduced by both hand working in oppo ite directi
on . Some chool of thought in the manipulative field in i t that one hand be f
ixed, while the other doe all the work. Although thi eem to make en e, the
effort,
2.
3.
4.
5.
6.
7.
8.
Any general principle are open to comment and critici m, but the analy i behin
d the e idea hould allow the development of per onal approache and individual
development. When applying locking force there are ome other con ideration t
hat need clarifying. Stand with feet parallel and turn the upper body to the lef
t. You are performing left rotation in the pine. Now tand with the upper body
fixed and perform a wivelling of the feet and pelvi to the right. De pite the
fact that the body i being turned to the right,
Principle of locking di comfort and train i much more than if both hand har
e the work. It doe take quite a lot of time and practice to make thi effective
in finding thi pathway of optimum potential without the guidance of full ten i
on. The time taken to acquire thi kill i well worthwhile in that it can produ
ce quicker re ult , le adver e reaction to treatment, and better patient ati
faction. If thi method eem intangible at fir t, carry on u ing full leverage
a with traditional train65
ing and gradually refine the method being u ed by introducing the e principle
a little at a time. It i very ea y to fall back on method , which although effe
ctive, are uncomfortable, procedural and require higher force, effort and train

for patient and operator. To quote a cliche, If you keep looking back, you wil
l end up going there. There are no ab olute in o teopathic approache ; the inf
inite variety of po ible compo ite lever allow mo t ituation to be catered
for.
EXERCISES FOR DEVELOPING TECHNICAL SKILL
12
Any p ychomotor kill can be improved and developed by uitable practice. It i
not po ible to practi e every technique or method ufficiently to become kille
d enough o that the proce i automatic without a con iderable quantity of tim
e and effort. However, there are ome exerci e or drill that may help to hort
en the learning time for tudent and I have included a few here. It i unfortun
ate that it i extremely difficult to find activitie that are imilar to the k
ill needed for o teopathic technique. There are many thing that can be done to
improve mechanical handling kill for dealing with inanimate object . The e do
little in helping the tudent to appreciate the ubtle difference in ti ue t
ate that exi t in patient . We are dealing with living tructure in real peopl
e and the p ychology, age, weight, ize and nature of the problem all ubtly alt
er the approach and force nece ary. With thi in mind it mu t be aid that the
be t exerci e i con tant practice with a willing fellow tudent who i empower
ed to give con tructive feedback a to handling, comfort and effectivene . Not
all exerci e or drill will be of equal benefit to all individual . We all have
our own per onal model of learning and ome y tem of kill development will f
eel more natural than other . O teopathic kill can be divided into palpatory
kill and technique kill although one i not really po ible without the other.
From a general viewpoint, anything that improve hand and eye coordination houl
d be u eful. Mo t people, at ome time in their life, undertake port of ome o
rt or another
if they are phy ically able. A experience and practice develop , they gradually
improve their kill to a certain level dependent on the amount of time and effo
rt u ed. If a tudent i going to be an o teopath, their whole life work i go
ing to be concerned with performing o teopathic treatment. It would eem to make
en e therefore to practi e the kill until they are efficient. For many rea o
n ome are unwilling to do thi . Although life place many demand on our time,
without ufficient practice it i fair to ay that a tudent i unlikely to bec
ome an efficient technician. The practice mu t, neverthele , be of the correct
method . Bad habit , particularly regarding po ture, can be very difficult to co
rrect later. The be t port are going to be tho e that the tudent enjoy and w
ill, therefore, participate in regularly. Racquet port help in coordination, b
ut dance and contact port are probably better for po ture, balance and the p y
chomotor development nece ary for o teopathic technique. Phy ical trength i n
ot a requirement for an o teopath. However, a rea onable level of tamina and fi
tne i u eful. A high proportion of tudent who become good technician have
done ome training in martial art . It eem that the di cipline, fitne and bo
dy awarene have many u e in their ub equent o teopathic career. Team game
eem to have le to offer in term of the future need of an o teopath, but that
i not to ay that they do not have their u e in general fitne . Here are a fe
w pecific exerci e :
Exerci e for developing technical kill 1. Face a partner and take up a po ture
with one leg forward of the other. Place a football between one of your out tre
tched hand and that of your partner o that it i andwiched between you. Take
turn in pu hing and pulling. If you coordinate with your partner, the football
will remain between your hand . If you do not coordinate, you will drop it. Re p
onding to the ubtle cue of your partner movement will help develop coordina
tion and re pon e to motion. It will al o help in developing the feel for  etti
ng the hand in light i ometric ten ion which i nece ary in many technique .

Rever e hand . 2. Pull a toilet roll gently until the ti ue i taut. Apply a h
arp tug, aiming the force at the perforation of the heet. Avoid imply unravel
ling the roll. Practi e moving the force to the next row of perforation while m
i ing the nearer one. Thi drill improve the ability to en e ten ion and to d
irect force beyond one point to another. 3. Stand facing a partner and place one
leg in front of the other. Extend one arm lightly and place the back of the ha
nd again t the back of your partner imilarly extended hand. Decide who i to
be the mover and who i to be the follower. Take turn in moving the hand at dif
ferent rate , and in different plane and direction . The follower mu t try to r
emain in contact at all time . Thi i imilar to exerci e 1, but u e direct co
ntact rather than via the football. Alternate hand and leg po ition . 4. Stand f
acing a partner with one leg in front of the other. Grip each other  wri t and
gently a ume a rhythm of pu h and pull. Attempt to pull or pu h your partner f
rom hi firm tance, but be aware that he i doing the ame with you. Force will
not work; you mu t u e balance and tran mi ion of energy and rapid change of d
irection to be effective. Thi will improve balance, coordination
67
and en e of ti ue re i tance. Alternate leg . 5. Acquire ome bubble wrap a u
ed in protective packing. Place it on a table and u e your heel of hand or pi i
form to bur t one bubble at a time. Draw an ink line around one bubble and try t
o bur t the bubble within the line and no other . Examine your hand and ee if y
ou have a neat ink circle around your pi iform or only an arc of a circle. Progr
e to doing the ame thing on your thigh o that there i normal ti ue underne
ath the bubble and not the firmne of the table. Progre to performing the a
me ta k on a partner back to ee how little force you can apply and yet till
achieve the objective. Thi will develop kill and accuracy in minimum force tec
hnique. 6. Have a partner place a ingle hair underneath a ingle page of a moo
th telephone book without you knowing where it i . U e light pre ure and canni
ng of the urface with your finger to locate the hair. See if you can do the a
me with the heel of the hand. Try with eye hut or open and tanding or itting
to ee which i your per onal be t method. When you become adept at thi , have
your partner place the hair under everal page of the book rather than ju t one
. See how deep you can till feel it. Thi improve palpatory perception and tra
in you in which part of the hand i be t to u e in palpation. 7. Perform a imi
lar exerci e to 6, but u e a coin in tead of a hair. Have your partner place the
coin at varied depth and try to judge the depth. Have him vary the ize of the
coin and try to e timate the ize by palpation. Thi palpatory exerci e i imi
lar to the previou one but it develop different a pect of your kill in e tim
ating ize and bulk. 8. Perform the magician trick of natching the tablecloth
from under cutlery or crockery. U e unbreakable crockery at fir t! Once you hav
e acquired the kill to pull
68
Exerci e for developing technical kill movement a if you are a mirror. Gradua
lly make the movement more complex and ee if you can follow hi mime. Thi wil
l improve your ability to learn technique by watching and copying. 13. Sit at a
table with your forearm horizontal. Relax and then try to contract your brachio
-radiali mu cle alone on one ide. You may find that thi i difficult, but wit
h practice it hould be po ible to i olate thi one mu cle alone. When you have
achieved thi , move on to pecifically contracting part of your forearm exten o
r group. To do thi , lift one finger and watch and feel the mu cle contract. Try
different finger . Progre to contracting pecific part of the mu cle group w
ithout moving the finger . Try to contract your bicep mu cle without vi ibly co
ntracting brachio-radiali . The e exerci e give you the ability to u e your mu
cle in a controlled fa hion. Thi i a very u eful facility when performing thr
u t technique that require accurate u e of defined part of your tructure for
pecificity. With a little ingenuity it hould be po ible to develop mo t daily

activitie into uitable practice for ome a pect of technique kill. When vac
uuming, wivel from foot to foot to practi e weight tran fer nece ary in many a
rticulation technique . When wa hing a car try different direction of circling
the hand to find the variety po ible in addition to the mo t natural direction
. Try placing a few piece of fruit or vegetable in a bag and identifying each
one by feel. Trace the outline of the bone of your own wri t; ee how much pr
e ure i comfortable and how you can deflect the tendon out of the way to get
to the deeper tructure . Until it i po ible to develop ome form of thru t me
ter or pre ure- en ing device to objectively mea ure force and direction , the
e imple drill hould help in a mall way to aid under tanding, palpation and
kill.
away the tablecloth without di rupting the article on the table, try the ame e
xerci e itting, or with varied leg po ition . Try it bent over or traight to f
ind the optimum po ture nece ary for the harp pulling action. Thi exerci e h
ould improve your fa t movement and acceleration kill . 9. Play red hand  wit
h a partner. Have your partner extend hi hand in front with palm facing down.
Place your hand , palm upward, lightly under hi o that they are ju t touchin
g. Keep very light movement going and try to extract your hand fa t enough to
gently lap the back of your partner hand before he ha had a chance to nat
ch them away. If you manage to lap him, you repeat the move until he manage to
anticipate your movement and natch hi hand away o that you lap the air. Wh
en thi happen , you rever e role . Try not to natch the hand away at every mo
ve; wait until the lapper i committed. Thi improve re pon e to motion and re
action time, but will give ore hand if continued for too long! 10. Have a part
ner u pend a cri p banknote between finger and thumb. Place your finger and thu
mb on either ide of it near the bottom, without touching. Have him drop the not
e and try to catch it before it pa e through your hand. A you improve, gradua
lly widen the gap between your finger and thumb and rai e your hand nearer the t
op o that there i le time to react. Thi i another exerci e in re pon e to
motion and improve your ability to move only at the right peed when nece ary.
11. Learn to juggle with uitable juggling ball . Thi teache you to relax and
yet remain alert and re pon ive to moving object . Thi i extremely good for c
oordination and hand-eye reaction . 12. Have your partner mime a imple movement
. Stand in front of him and copy thi
TECHNIQUES FOR THE LUMBAR AREA
13
Accurate and efficient technique for the lumbar area are extremely important. T
he highe t percentage of patient pre enting to o teopathic practitioner are u
ffering with low back pain, and technique for the lumbar pine will probably be
u ed more often than any other . The practical difficultie for a mall operato
r in reaching a facet joint no bigger than the thumb nail, in the back of a pati
ent weighing anything up to around 200 pound , can be con iderable. The heer de
pth of the joint , the difficulty of controlling the area in many ubject , and
the common complicating factor of an underlying acute or chronic di c le ion add
to the problem . For treatment to be more pecific, accurate mechanical diagno
i i e ential. There i , therefore, a requirement for technique de igned to b
e efficient and fea ible for the operator to perform without undue tre on hi
own tructure. It can be a problem to localize a force to a egment or egment
while attempting to protect adjacent area that may be hypermobile. It i oft
en difficult to manipulate a pecific egment, but in ome ca e unle thi i
achieved, the relief from a particular pain and dy function yndrome will not
occur. General mobilizing can be extremely helpful, but the nature of relea e ac
hieved with a well-timed, accurate and pecific thru t can be not only a hort c
ut, but the only way to get full re toration of function. Although ize of opera
tor hould not be a critical factor, it mu t be aid that in ome ca e a larger

operator will have a di tinct advantage. The u e of exce ive force rather than
accuracy, however, conceal a weakne in approach. Ca e of po ible di c inju
ry require particular care to avoid wor ening the ituation. An educated, aware
practitioner hould be conver ant with neurological ymptom and ign . Extreme
antalgic po ture i pre ent for a rea on and con ideration hould be given to th
at rea on before attempt to correct the po ture are made. Many pathological
tate in the body can manife t a low back pain and the reader i referred to li
terature on pathology and diagno tic creening a an e ential prerequi ite to t
reatment in thi area. A patient hould be fully inve tigated when they are clea
rly ill, have lo t weight for no apparent rea on, or have intractable pain that
doe not ub ide on re t.
70
Technique for the lumbar area
13.1 Kneading oft ti ue lumbar area prone The operator i working on the mu c
le on the ide furthe t from him. The near hand i holding back on the area to
apply a mall compre ive force toward the table and to prevent rotation of the
pine and to en e the be t re i tance from the ti ue . Pu h with the active ha
nd into the mu cle belly until a en e of re i tance i felt, and then maintain
thi po ition for a few moment until a en e of activation i felt. There houl
d be a en e of melting a the mu cle relax, and then you can follow thi en
e until the mu cle i gently but fully ea ed. The direction of the pathway may
be in a curve, and it i important to allow the ti ue ufficient time to guide
the force direction rather than to impo e on them. The pre ure i then lowly
relea ed until the cycle can be repeated on an adjacent area. The whole cycle ca
n take up to about ix or even econd . Mo t operator will not u e uch a long
cycle, but paradoxically it i often found that the lower one work , the quick
er and more effective the re ult . Tip : Lea t u eful in ca e where prone lying
i a problem and in acute ca e where there may be a tendency for the mu cle t
o go into a greater pa m when the patient ub equently move . Extra con iderati
on : Note that a pillow i placed under the patient abdomen to reduce exce iv
e hyperexten ion of the lumbar pine.
13.2 Kneading oft ti ue lumbar area prone Perform the technique with the near
hand while the other tabilize . There i no particular advantage in which hand
i u ed. However, it may be ea ier to reach right down to the acral attachment
of the erector pinae u ing the caudal hand rather than the cephalic one, a i
n technique photograph 13.1. Tip : Lea t u eful in ca e where lying prone may b
e a problem. Extra con ideration : Try a king the patient to turn the head to on
e and then the other ide to a e which produce the mo t u eful ten ion. Comb
ining pha e of breathing with the technique may be u eful. The pillow i option
al to increa e patient comfort if nece ary. Mo t patient find that a pillow un
derneath the abdomen i preferred when prone lying.
Technique for the lumbar area
71
13.3 Kneading oft ti ue lumbar area idelying The patient ha the knee and h
ip flexed to increa e tability on the table. Stabilize the patient body with
your cephalic hand and apply the kneading force to the mu cle neare t the tabl
e. Tip : Mo t u eful in ca e where prone lying may be a problem. Lea t u eful i
n large patient where the reach for the operator may be too great. Extra con id
eration : In acute ca e thi may be the only way to work on the lumbar mu cle
a prone lying may be impo ible due to pa m. Try varied angle of hip and knee
flexion. Try working on the mu cle by pulling up in tead of pu hing down. Try
performing the ame procedure when tanding behind the patient.

13.5 Articulation into ide hffting lumbar area prone Grip the tran ver e proce
e of the vertebra concerned with the pad , not the tip , of the finger and th
umb and u e a direct ide hifting force from ide to ide. The o cillation can b
e quite firm when u ed carefully, and thi will act a a u eful te t a well a
a mobilizing force. A the hand are applied to adjacent vertebrae, difference
in mobility can ea ily be felt, and thi hold can be u ed to mobilize, or other
technique can be applied to deal with the re triction in movement. Tip : Thi i
a movement of the whole of the patient body around the vertebra, and need q
uite a long amplitude to be effective. Lea t u eful in ca e of antalgic ideben
t po ture a thi movement will cau e pain when working again t the curve.
13.4 Stretching uperficial fa cia lumbar area prone Gather kin and uperficial
fa cia over upper lumbar area, and then apply a lifting force o that a gapping
i produced. In ome ca e thi can be made into a thru t, and the fa cia will
eparate with a vacuum gapping ound. Tip : Mo t u eful in ca e of tightne in
the uperficial fa cia which can be a factor in maintenance of pain yndrome .
Lea t u eful where the kin i very tender.
72
Technique for the lumbar area
13.6 Harmonic technique lumbar area prone Fix the acrum in ome traction toward
the patient feet and fix the thoraco-lumbar area toward the head. The harmo
nic technique i performed by a rhythmic o cillation of the patient whole body
in a caudal and cephalic direction. See earlier ection relating to harmonic te
chnique. Tip : The range of mobility available in a longitudinal plane i going
to be maller than in a rotary plane, but i , neverthele , often u eful in ree
tabli hing rhythm and mobility. Lea t u eful in ca e where prone lying i a pro
blem. Extra con ideration : It i al o po ible to perform rotary harmonic techn
ique if the ten ion i maintained between the hand , and the whole body o cillat
ed into rotation. The pillow under the abdomen often make the po ition more com
fortable for the patient.
13.7 Sacral pringing and traction prone Flex the acrum a far a i comfortabl
e, and fix the thoraco-lumbar area with the other hand. Aid the flexion of the
acrum with the elbow of the one hand by pre ing the other hand into the table.
Tip : Try adding variou amount of rotary movement of the pelvi or the body a
a preliminary before the acral flexion. Thi can enable force to be directed
to either ide of the lumbo- acral joint rather than imply flexing the acrum.
Mo t u eful in ca e of very tight lumbar fa cia where thi po ition remove om
e of the oft ti ue ten ion and allow the force to reach the facet joint . Lea
t u eful in ca e where prone lying i a problem. 13.8 Harmonic technique pelvi
and lumbar pine upine Take up any lack in the ti ue of the pelvi by pre
ing firmly into the table with both hand . Perform the o cillation with alterna
ting pre ure toward the table with the heel of each hand on the anterior uper
ior pine . Thi induce a rotatory movement in the lumbar pine. See earlier e
ction relating to harmonic technique. Tip : Try u ing varied amount of traction
a an additional movement. Mo t u eful in fairly mall patient where the tret
ch to reach i not too far. Lea t u eful in large patient , or for mall operato
r where the reach i a problem.
Technique for the lumbar area
73
13.9 Harmonic technique lumbar pine and pelvi prone Apply pre ure above the c
re t of the ilia and a variable traction force toward the patient feet. A ha
rmonic o cillation i induced into rotation, idebending and traction. See earli
er ection relating to harmonic technique. Tip : Lea t u eful where prone lying

would be a problem.
13.11 Functional technique typical hold The operator i controlling all po ible
vector of patient movement while monitoring the en e of ea e and bind at a pa
rticular egment. The controlling hand i directing the body toward accumulatin
g ea e at the target egment. See earlier ection relating to functional techniq
ue.
13.10 Traction upine Fix the patient leg again t your che t and pu h gently
downward o that the feet are fixed to the table. Lean back to produce a tracti
on force in the lumbar pine. Thi hold can al o be u ed in a harmonic fa hion.
Tip : Mo t u eful where a gentle traction force i required. Extra con ideration
: Try varying the angle of hip and knee flexion to focu the force to differen
t area .
74
Technique for the lumbar area
3.12 Articulation into flexion idelying Flex the patient hip until a en e o
f gapping i felt at the target egment. Rock from foot to foot and turn thi di
agno tic procedure into an articulation with an increa e of pre ure with either
hand to localize the force. Tip : Mo t u eful in almo t all ca e of lumbar ver
tebral dy function. Lea t u eful in extremely large patient or in the pre ence
of any di order preventing hip flexion. Extra con ideration : Try u ing one hand
to pull the pelvi into more flexion or the other hand to hold back above the t
arget vertebra or both.
13.13 Articulation into reinforced flexion Pull the acrum directly toward you w
hile bracing the rib toward the table. The upper hand fixe above the target
egment while you pull the acrum into flexion. Tip : Try varied amount of compr
e ion of the thigh toward the table and adding element of idebending or rota
tion to the flexion, to focu on particular part of the egment. Mo t u eful wh
ere a trong localized force i required. Lea t u eful where the patient i very
large and the reach around them would be too great.
13.14 Articulation into flexion upine Sit on the table and hold over one or mor
e pinou or tran ver e proce e . Flex the hip with the other hand. Hold the p
atient leg again t your che t o that a you rock back and forth, the force c
au e a flexion movement of the lumbar area. Tip : Lea t u eful if there i any
hip di order preventing flexion. Extra con ideration : Try u ing varied degree
of idebending or rotation at the ame time to enhance localization.
Technique for the lumbar area
75
13.15 Articulation upine Stand at the ide of the table and fix over one or mor
e of the pinou or tran ver e proce e . Cro the patient thigh and apply a
flexion and idebending force a you lean forward. Thi hold make it ea y to i
ntroduce ome element of idebending to the primary movement of flexion. Tip : L
ea t u eful in any patient with hip mobility re triction. Mo t u eful in very fl
exible ubject where the po ition automatically ab orb ome of the exce move
ment. Extra con ideration : Try u ing a fi ted hand to form a tronger fulcrum i
n uitable ca e .
13.17 Articulation into idebending idelying Compre toward your elf, and down
into the table. Sidebend the pelvi away from the table with the caudal hand a
the other hand pu he down on the pinou proce e . Tip : Mo t u eful where ve
ry localized articulation i nece ary, and in ca e where a firm lever through

the hip may be unde irable.

13.16 Articulation upine (alternative view) The hold i a in photograph 13.15,
but how hand placement from the other ide.
76
Technique for the lumbar area
13.18 Articulation into exten ion idelying Fix the patient houlder girdle to
the table with a compre ive force and apply a direct po terior to anterior for
ce over the lumbar pine. Tip : Mo t u eful where a very localized force i requ
ired. Lea t u eful in large ubject where a mall operator may find it difficul
t to develop ufficient leverage. Extra con ideration : Try varying the initial
patient po ition on the table to find the optimum in each ca e.
13.19 Articulation into exten ion idelying Fix the patient knee again t your
thigh, and fix the patient feet to the table with your caudal hand. Pull agai
n t the lumbar area to produce a direct force into exten ion. Tip : Lea t u eful
in very tiff ubject who would be very difficult to move with thi hold a it
would require great trength in the pulling hand to overcome the tiffne . Ext
ra con ideration : A exten ion i elicited from above, try a king the patient t
o extend the head and upper back to enhance the action of the technique.
13.20 Articulation into exten ion upine Pull upward on the tran ver e proce e
on both ide . The patient ha interlaced the hand behind her head to enhance
the exten ion force. Tip : Mo t u eful in mall ubject and children, and when
directing the force into the upper lumbar area. Extra con ideration : Try extend
ing and then adding an o cillatory rotation force a an additional vector.
Technique for the lumbar area
77
13.21 Articulation into exten ion idelying Overlap the hand and pull directly
on the area to introduce exten ion. The patient knee are directly fixed by yo
ur thigh to form a counter-force. Keep the patient hip at 90. Tip : Mo t u efu
l where a fairly trong force i de ired. Extra con ideration : Try introducing
an element of idebending or rotation to a i t the primary movement.
13.22 Articulation into exten ion itting Take up the lack of the area with cro
ed hand and a the patient i rocked back and forth an exten ion force i pro
duced. By placing the cephalic hand on either ide of the area, a rotation or i
debending force can al o be introduced. The patient i a tride the table to tab
ilize the pelvi . Tip : Lea t u eful in elderly patient or tho e where the a tr
ide po ition may be a problem. Extra con ideration : Try a king the patient to m
ove the hand further forward to change the angle of approach to the area.
13.23 A Articulation into exten ion itting Apply thumb pre ure to the paravert
ebral area of the egment to be mobilized. The patient i a ked to hold the edge
of the table and then you can pu h again t the area rhythmically to introduce t
he exten ion force. The natural recoil of the ti ue will bring the pine back
to neutral. Tip : Lea t u eful in ca e where a tride itting can be a problem.
Extra con ideration : Try taggering the thumb on either ide of the pine to i
ntroduce a rotation element to the movement.
78
Technique for the lumbar area 13.24 Articulation into idebending idelying Pu
h down with one hand on the leg while pulling up on the pinou proce e to in

duce idebending. Increa e the angle of hip flexion to direct the force to highe
r egment a de ired. Tip : Lea t u eful in patient who have any hip di order
a thi po ition may be a problem. Where the table ha a hard edge the pre ure
may be uncomfortable for the thigh on the table. Extra con ideration : Try intro
ducing an element of exten ion to the technique to change the effect of the pure
idebending if nece ary.
13.25 Articulation into flexion/ idebending idelying Pull up with one hand on t
he pinou proce e while the patient pelvi i andwiched between your body
and arm and lean away from the pinal hand to produce idebending. Varied degree
of flexion are applied by rocking again t the patient knee . Tip : Mo t u ef
ul where very localized articulation i de ired.
13.26 Articulation into idebending idelying Lift the patient feet until ten
ion i felt to accumulate under the other hand which pu he toward the table to
produce idebending. Tip : Mo t u eful where a trong generalized force i requi
red. Lea t u eful in the pre ence of any hip joint dy function. Extra con iderat
ion : Try varied degree of hip flexion.
Technique for the lumbar area
79
13.27 Articulation into idebending idelying Take the patient feet in the cro
ok of your arm and fix again t the upper thigh with your wri t. Your other hand
pu he again t the pinou proce e toward the table. Varied degree of flexion
of the hip are applied to focu the force to the egment de ired. Tip : Lea t
u eful in the pre ence of any hip di order. Mo t u eful where a very trong tr
etch i de ired.
13.28 Articulation into idebending idelying The patient i idelying in a neut
ral po ition of light hip flexion and you can pu h on the rib cage toward her h
ead, on the pelvi toward her feet and on her pine toward your elf. The articul
ation movement i either a fixation with one hand and a pulling with the other o
r a movement of both. Varied degree of rotation or flexion can be introduced a
part of the overall leverage. Tip : Mo t u eful where any hip di order prevent
u e of the hip a a lever and where trong localized force are required. Extra
con ideration : A high compre ive force toward the table help to focu the te
chnique.
13.29 Articulation into idebending prone Apply the finger to the ide of the
pinou proce e and pu h away from your elf to produce a idebending and rotati
on force. Thi i mo t applicable in the upper lumbar area. Tip : Lea t u eful w
here the patient may find the prone po ition a problem. Extra con ideration : Tr
y placing the patient in ome idebending before the initiation of the technique
.
80
Technique for the lumbar area 13.30 Articulation into idebending idelying The
patient i lying in a neutral po ition with hip only lightly flexed. Apply a
compre ive force to the pelvi with your body and caudal hand to produce idebe
nding away from the table. With the other hand apply a downward force toward the
table to localize the idebending. Tip : Mo t u eful where a localized force i
required and it i nece ary to avoid u ing the hip in the leverage. Extra con
ideration : Try u ing varied amount of compre ion through either arm and rota
tion to enhance the idebending force.
13.31 Generalized rotation mobilization upine Pull the flexed hip of the patien
t toward you while holding the thorax of the patient toward the table and away f

rom you. Thi will produce a rotational mobilization focu ed primarily in the th
oraco-lumbar region. Varying the angle of hip flexion will change the localizati
on to ome extent. Tip : Lea t u eful where pecific localization i required.
13.32 Generalized articulation into rotation upine Apply a force with your lowe
r hand through the patient hip u ing your wri t a a fulcrum. U e the other ha
nd to hold the patient folded arm away from you. Tip : Lea t u eful where pe
cific localization i required. Extra con ideration : Try placing the patient in
ome idebending before the on et of the technique to enhance the effect.
Technique for the lumbar area
81
13.33, 13.34 and 13.35 Thru t idelying, building of upper lever component. Ther
e are many way of introducing the upper component to the lumbar roll po ition f
or applying a thru t. Each ha it advantage and di advantage . Individual wil
l have to experiment to find their own preferred method. Photograph 13.33 how
the operator cla ping the patient forearm under hi arm. While pu hing the oth
er houlder toward the table he i pulling the lower capula out of the way. The
pull can be either into pure rotation or idebending either way according to th
e direction he take the arm. In photograph 13.34 the patient arm are folded
and the operator i lifting on the lower elbow while holding down on the upper
houlder. It i le ea y to introduce a idebending component with thi hold. In
photograph 13.35 the operator i liding the lower capula forward while holdin
g down on the upper houlder. Thi hold allow idebending to either ide to be
introduced if nece ary and doe not involve any train on the patient houlde
r.
13.36 Thru t u ing lumbar roll idelying rear view The operator i applying a cl
a ical combined lever and thru t technique. Thi rear view how operator po tu
re and how both feet are pointing to the head of the table with the rear heel ju
t off the floor. Note that the weight i applied to the patient pelvi with t
he thru ting hand. The other arm i only lightly fixing on the lateral a pect of
the thorax and NOT the anterior a pect of the houlder. The patient body i r
olled toward the operator who i , therefore, only lightly flexed and i able to
apply the rotary force of the technique imply by a flexing of hi knee .
13.37 Thru t u ing minimal leverage idelying The patient i po itioned in a bal
anced idelying po ition and a very mall element of rotation ha been introduce
d from above. Apply the hand to the patient houlder to produce a compre ive
force toward the table and your other arm i then flexed to 90 and held clo e to
your ide. Gather ome kin from your forearm and the patient buttock by gripp
ing the buttock with the forearm and pulling it into you before applying the dow
nward compre ion force. The force i applied a a combination of (a) compre io
n to the table, (b) localized compre ion over the egment, (c) rotation of the
pelvi by you flexing your knee and (d) a idebending of the pelvi away from t
he houlder. In minimal leverage thru t the amplitude i very hort and the velo
city i high. Tip : Mo t u eful where it i de ired to produce facet eparation
with minimal di tortion of the pinal area or tor ion through the rib cage. Extr
a con ideration : The final vector of force direction will be lightly differen
t in each ubject. Although the direction given will apply in mo t ca e , it mu
t be remembered that an increa e of one force direction will automatically redu
ce the quantity nece ary for other . The order in which they are applied may al
o be changed to uit the circum tance . Con iderable experimentation will be ne
ce ary to find the optimum for each operator particular kill, the patient
morphology and the facet orientation. Thi depend to a large extent on palpator
y awarene .
13.38 Thru t, upper hand hold, combined leverage and thru t, idelying The opera

tor ha placed the patient in po ition for the thru t, and thi photograph how
the rotation of the upper lever. Note that the patient upper houlder i behi
nd the lower one, but only by a mall amount. The operator head i vertically
over the lumbar pine although the abducted arm i going to apply the thru t. A
compre ion force i applied to the houlder with only enough backward pre ure
to act a an equal and oppo ite force to the thru ting hand. It doe NOT enter i
nto the thru t other than a a tabilizer.
Technique for the lumbar area
83
13.39 Thru t, lower hand hold, combined leverage and thru t, idelying Roll the
patient body toward you and apply the heel of your hand to the ilium o that i
t i po ible to produce a rotation and exten ion force a nece ary. Your pelvi
and ide mu t be applied to the patient thigh, and you mu t fix the patient
thigh to the table with your thigh. Tip : Mo t u eful for larger operator , an
d where an exten ion force i needed. Thi hold can al o be u ed for a direct a
cral thru t. Extra con ideration : Some of the thru t force come from a downwar
d movement of the operator body along with the hand force.
13.40 Thru t u ing minimal leverage, alternative houlder hold Apply a downward
pre ure through the pelvi , after gathering ome ti ue under the applied forea
rm to induce compre ion and ome rotation. Place your hand in direct contact wi
th the vertebra at the apex of the force. Your other arm compre e the patient
thorax through the houlder toward the table. The final thru t direction i a
combination of compre ion, rotation and ome idebending a nece ary. Tip : Mo
re u eful in larger patient where it may be too far to reach up to the axilla w
ith the tabilizing hand. Le u eful for operator with long arm , who may find
it difficult to apply the hand to the pine at the ame time a the u ually app
lied part of the forearm to the ilium. Thi action will cau e hyperflexion of th
e elbow and if repeated many time will make the ri k of injury to it extremely
high!
13.41 Thru t into flexion u ing combined lever and thru t idelying The initial
patient po itioning involve u ing ome flexion and afterward you apply a compr
e ive, rotary and flexion force to the pelvi . Note that the patient lower le
g i off the edge of the table and that you mu t be quare to the table rather t
han in the u ual po ition of facing toward the oppo ite top corner. The combined
effect of thi i to produce a flexion gapping force that may be very u eful in
hyperextended patient . Tip : The final flexion i the gapping force and uffic
ient free play mu t be left to allow thi to operate.
84
Technique for the lumbar area 13.42 Thru t into rotation combined lever and thr
u t idelying The hold u ed here i pecific for the lumbo- acral and L4/5 level
. The difference in thi hold i that your lower hand i not in contact with th
e patient. The upper hand i threaded through and i palpating the relevant pin
al level. The upper hand i u ed to apply a compre ion force at the target egm
ent. If the lower hand were to be in contact with the pine the thru ting elbow
would become exce ively flexed and liable to injury. Tip : The optimum plane fo
r the thru t will be ea ier to find if the patient, a a whole, i rolled gently
back and forth on the table within the thru t po ition. Thi doe not mean that
the lever alter at all, but that a momentum force i being u ed in rolling whi
le keeping the lever the ame. It i al o ea ier to apply the thru t from a dyn
amic rather than a tatic po ition.
13.43 Thru t into rotation combined lever and thru t idelying Thi view how t
he lower arm hold u ed in photograph 13.42. The medial a pect of the elbow i ap

plied to the mall plateau on the lateral a pect of the ilium between gluteu ma
ximu and gluteu mediu . The oft ti ue have been gathered fir t and the fore
arm mu cle have been rolled to form a cu hion between your ulna and the patient
 pelvi . Tip : It i worth pending ome time experimenting to find the mo t c
omfortable way of applying the lower hand hold. It i often u eful to apply the
arm, and then, keeping contact with the patient, adduct it o that your forearm
mu cle are rolled between your ulna and the patient. Avoid u ing the back of th
e ulna or the point of the elbow. The forearm will be at approximately 60 to the
long axi of the table. If it i at 90 thi i likely to be very uncomfortable fo
r the patient.
13.44 Thru t into rotation combined lever and thru t idelying Thi view how t
he upper arm hold u ed in photograph 13.42. Notice that the operator i tanding
fairly upright and that hi arm pre ure i not on the houlder but rather the
antero-lateral a pect of the thorax. He i u ing ulnar deviation of the wri t to
bring the ulnar border of the hand again t the lower rib . To avoid the force d
i ipating when the thru t i applied, en ure your houlder are pulled down by
active contraction of your lati imu dor i mu cle on both ide . A u eful rule
i that if you can ee your houlder , except out of the corner of your eye , t
hey are too high.
Technique for the lumbar area
85
13.45 Thru t into rotation combined lever and thru t idelying The hold hown he
re i for a pure rotary thru t where the operator i focu ing the force and the
n will imply flex hi knee to introduce the rotation. Thi direction of force
i mo t u eful in patient who have very agittal lower lumbar facet plane . Tip
: The optimum plane and timing for the thru t will be found if the patient bo
dy i kept lowly rolling back and forth.
13.47 Thru t into rotation combined lever and thru t idelying Hold directly on
the pinou proce of L4 or L5 to help make the force pecific. Apply the thru
ting arm to produce a direct compre ion and rotation force through a very mall
amplitude. Tip : The tabilizing hand i not applied to the anterior of the tho
rax in thi ca e but more to the lateral ide of the rib a the force hould no
t reach much above the egment concerned. Mo t u eful where exce ive rotation o
f the thoracic pine i be t avoided.
13.46 Thru t into idebending combined lever and thru t The normal po ition of t
he patient leg for HVT (high velocity thru t) technique i rever ed in that
the upper leg i traight. Thi will introduce a idebending toward the table an
d you can apply force to empha ize thi fact. The final thru t u e rotation an
d flexion or exten ion a nece ary but will be primarily into rotation. Tip : M
o t u eful in ca e where the idebending force i de ired to open an inter-vert
ebral foramen and po ibly decompre a nerve root.
86
Technique for the lumbar area
13.48 Thru t into exten ion combined lever and thru t idelying Pull the lumbar
pine into exten ion with your tabilizing hand and then while maintaining thi
vector perform the thru t into rotation and compre ion, avoiding flexion. If it
i de ired to gap the facet nearer the table, thi i po ible with the followi
ng vector . Exten ion i maintained and a idebending force i applied away from
the table and then a compre ion thru t i u ed. Tip : Mo t u eful in patient
who have very tight fa cia, a their uperficial po terior ti ue will come on
ten ion too early in flexion and prevent the technique reaching the facet joint

. Paradoxically thi i al o effective in ome very flexible patient where exte

n ion will produce an ea ier localization than flexion. Thi i becau e they aut
omatically fall into exten ion and, rather than trying to fight thi , the exten
ion can be u ed a one of the component of the technique. The ability to gap th
e lower facet i very u eful where there i nerve root impingement, a rotation,
with the patient lying with the painful ide uppermo t, may be impo ible.
13.49 Thru t u ing combined lever and thru t with patient a i tance Focu force
in the u ual way to the lumbo- acral facet and then a k the patient to turn he
r head to look over her houlder. Thi will often cau e the barrier en e in the
joint to be enhanced which may aid the thru t. Tip : Mo t u eful in patient wh
o are very flexible where it i difficult to produce an accurate focu . It i al
o a u eful di traction for patient who find it very difficult to relax. Extra
con ideration : Try a king the patient to look toward the table - thi will have
the effect of lackening the lever .
13.50 Thru t combined lever and thru t idelying Fix behind the patient knee a
nd again t the upper houlder and apply a rotary force to the lumbar pine. The
patient body i rocked back and forth until a en e of focu ing of force i p
roduced. The thru t i applied with a hort amplitude, high velocity force towar
d the floor with the knee hand. Although thi hold can be a very generalized man
ipulation, ome operator find it i po ible to be very pecific to a particula
r egment. The tendency, however, may be for any relatively hypermobile egment
to gap leaving the re tricted one unaffected.
Technique for the lumbar area
87
13.51 Thru t into idebending combined lever and thru t idelying Place a pillow
under the patient ide which will produce idebending toward the table. Accum
ulate the force at the target egment and apply the final thru t directly into
idebending by pulling up on the pinou proce with the fixing hand and thru t
ing toward the feet with the other. Tip : Mo t u eful where there may be nerve r
oot pre ure or foraminal encroachment and it i de ired to open the foramen dur
ing the technique. Some treatment table have moveable ection capable of produ
cing the initial po itioning rather than needing to u e a pillow.
13.53 Thru t u ing ciatic tretch idelying Thi hold u e imilar principle t
o photograph 13.52 except that you hook your leg around the patient foot. At t
he time of thru t you can traighten your leg to increa e the traction component
through the patient leg. Thi clearly ha di advantage , a you are tanding
on one leg, but i ometime a u eful technique. It i often po ible to produce
a force pecific to the acro-iliac with thi hold.
13.52 Thru t u ing ciatic tretch idelying Sandwich the patient leg between
your thigh and after accumulating ten ion at the facet joint flex the thigh unt
il ciatic tretch begin . Apply the thru t into rotation while maintaining the
ciatic tretch. Thi can be u eful in ca e of long-term ciatica where it may
be po ible to break ome adhe ion around the nerve root. Great care mu t be ta
ken not to over tretch the nerve and traumatize it. Some adhe ion will be tron
ger than the normal ti ue , and if thi i the ca e, damage can occur if exce
force i u ed.
13.54 Thru t into flexion u ing minimal lever and thru t idelying Compre the
lateral wall of the thorax into the table with the tabilizing hand. Apply a com
pre ive force directly over the acrum with the other and then the thru t i pe
rformed a a hort, harp flick into flexion of the acrum to pecifically gap t
he lumbo- acral facet . Tip : Mo t u eful in ca e of acute pa m where any tor
ion i be t avoided. Extra con ideration : Thi i an extremely difficult techni

que to perform effectively. However, it i well worth the effort of practi ing i
t, a it i then po ible to manipulate ca e where mo t other technique would
be too painful due to the leverage nece ary.
88
Technique for the lumbar area
13.55 Thru t lumbo- acral facet prone Apply a three-pha e force to the acrum.
The fir t pha e i pre ure toward the table. Maintain the pre ure and apply th
e econd pha e that carrie the whole acrum toward the head. The third pha e i
to flex the acrum until re i tance i felt and then harply apply a mall forc
e into further flexion while maintaining the other vector . Tip : Mo t u eful wh
ere pecific lumbo- acral gapping i required without pinal tor ion. Lea t u ef
ul in patient where prone lying i a problem for any rea on. Try adding a ideb
ending or rotation vector to direct the force more pecifically to one ide or
the other. 13.56 Thru t lumbo- acral upine Hold the pelvi firmly down onto you
r hand, cupping the acrum, and apply a traction force to the acrum until ome
en e of re i tance i achieved. Flex the di tal interphalangeal joint of your
acral hand to pull effectively toward the feet with the finger . Then u e a ho
rt, harp tug on the acrum to pecifically gap the lumbo- acral facet . Tip : M
o t u eful in heavy or pregnant patient where tor ional manipulation would be a
problem. Extra con ideration : Try varying the hip flexion and initial idebend
ing po ition of the patient a well a the pha e of re piration.
Technique for the lumbar area 13.57 ( ee facing page, top right) Thru t into ro
tation combined lever and thru t itting The patient it a tride the table to h
elp tabilize the pelvi and place her folded arm over the operator padded
houlder. Keep the patient head vertically over her pelvi throughout. Introduc
e idebending of the pine away from you and then, while maintaining thi , rotat
e the pine until ten ion accumulate under your hand applied to the pinou pro
ce e . You and the patient turn a a unit and the thru t i performed during th
i turn by the pinal hand accelerating lightly into rotation. Tip : Mo t u efu
l in large heavy patient where their weight in compre ion on the pine a i t
the technique. Lea t u eful where the itting a tride po ition may be a problem
. Extra con ideration : Try varying the compre ive force forward toward you to
minimize the rotation element. Do not lo e the idebending when applying the rot
ation or the focu of ten ion will be lo t, and train can occur at the acro-il
iac joint .
89
13.58 Thru t into rotation combined lever and thru t itting Thi how an alter
native hold for the ame technique a photograph 13.57. It may be more u eful in
large ubject where you may prefer to avoid taking the weight through your ho
ulder. Thi technique may be u eful where no treatment table i available a it
can be performed on a chair with the patient itting a tride the chair. Tip : Ke
ep the patient head vertically over the acrum throughout the technique.
13.59 Thru t into rotation combined lever and thru t tanding Fix the acrum wit
h your hip on the forward leg and rotate the patient to that ide with your hand
interlaced between the patient cla ped hand . Your other hand hold back on t
he ilium and then you idebend the patient toward your fixing hand. When ten ion
accumulate , harply increa e the pull with both hand . Tip : Mo t u eful in fl
exible ubject where the light pull of p oa in thi po ition will help limit
pinal movement and aid focu ing the force . Thi technique may be u eful where
no treatment table i available.
90

Technique for the lumbar area 13.60 Vertical adju tment po ition tanding Thi
how the mo t common hold u ed where the operator i cupping hi hand to cla p
the patient folded arm by the elbow . Note that one foot of the operator i
in front of the other and that although he i flexed from the hip hi pine i
relatively traight.
13.61 Vertical adju tment tanding The hold hown in photograph 13.60 i applied
and the patient i lifted o that your acrum fit into her lumbar pine. Maint
ain firm compre ion of the patient back again t your . You ri e onto your toe
and the adju tment i performed by dropping to your heel and firming your gri
p at the ame moment. Your knee hould never fully extend. Tip : En ure that th
e patient can extend her lumbar pine before performing thi technique or it i
likely to produce a lot of pain, a he will be in quite a con iderable amount o
f exten ion at completion. Mo t u eful where there i a vertical compre ive com
ponent to any dy function uch a di c herniation or overriding of facet . It i
al o u eful in heavy patient where ome element of traction can be very benefi
cial. Thi technique can often undo fixation that rotary technique will leave
partly unre olved and i u ually be t applied after rotary technique . Lea t u e
ful when the operator i horter than the patient unle he tand on a uitable
platform or tep. Extra con ideration : It i important that the operator pull
firmly through the elbow toward him elf to add a compre ive element. Thi hel
p to limit upper lumbar movement and increa e friction o that the lifting for
ce i le of a train. The movement in the technique i ynonymou with haking
the feather down in a pillow. Thi technique would appear to be quite a train
on the operator but if performed properly the weight i taken mo tly on hi ac
rum. There i not nece arily a big vertical compre ive force on the operator
pine. With practice mall amount of idebending and rotation can be u ed to f
ocu the force to particular location within the lumbar pine.
Technique for the lumbar area
91
13.62 Vertical adju tment itting Lift the tor o of the patient through the fold
ed arm , po ibly u ing a pillow in the lumbar lordo i a a fulcrum. The adju t
ive force i a hort harp lift at the end of the accumulation of ten ion. There
are everal other hand hold for thi procedure that may be tried. You could ha
ve the patient cla p the hand behind her neck. The patient could grip oppo ite
houlder , or he could be cla ped around the lower thorax. Tip : Mo t u eful in
mall, light patient and where a narrow table i available o that the operato
r can bring the patient clo e to him. Extra con ideration : Small element of i
debending and rotation can be introduced by pulling differentially on the elbow
or by the patient cro ing the ankle or knee .
13.63 Adju tive traction upine Cla p above the patient wri t and apply a te
ady pull until force i felt to accumulate at the lumbo- acral joint. Thi i co
nfirmed by watching the pelvi tilt. Keep the arm at approximately 30 from the h
orizontal. Apply a hort, harp tug through the patient arm without relea ing
any of the ten ion produced. Tip : Mo t u eful in very tall ubject where a t
anding adju tment may be difficult. Lea t u eful in the pre ence of any houlder
dy function that may be irritated by the traction force. It i e ential to pre
-load with the initial traction or the force will be di ipated before it reache
the lumbo- acral.
92
Technique for the lumbar area
13.64 Thru t idelying u ing econd operator Apply force in the normal way to f
ocu to a particular joint. The econd operator applie a lowly increa ing trac

tion force through the ankle until you tell him to top. You will be able to do
thi a you hould feel an enhancement of localization during thi traction comp
onent. The thru t can then be performed while the traction i maintained. Tip :
Mo t u eful in very flexible ubject where it i difficult to accumulate ten io
n unaided. Thi i al o a u eful method where there i ome nerve root impingeme
nt a the traction may allow a rotary force to reach the facet while the foramen
i being opened lightly. Extra con ideration : The econd operator, with a min
imum of practice, will al o be able to feel ten ion accumulating a the lever a
re applied.
13.65 Thru t idelying u ing econd operator Thi technique u e exactly the am
e principle a tho e in photograph 13.64, except the econd operator i applyin
g traction to the neck until the required ten ion i felt by the fir t operator.
Tip : With a third operator thi technique can be combined with the previou te
chnique in photograph 13.64 if even more traction i required.
Technique for the lumbar area 13.66 Thru t idelying two operator technique The
patient i in a emi-Simm po ition and the fir t operator ha lifted the patie
nt flexed knee while pu hing toward the floor with the heel of hi hand appli
ed to the pinou proce e . The econd operator re t hi thorax on the patient
 capula and while applying traction through the patient wri t, i fixing un
der the pinou proce e with hi other hand. The force are accumulated by the
contra-rotation of the upper part of the patient body toward the table and th
e lower part away. The thru t i performed by the econd operator fixing while t
he fir t operator pre e down on the pinou proce e and harply lift the pa
tient knee toward the ceiling. Varied angle of hip flexion in the patient wi
ll direct the force higher or lower in the lumbar pine. It i critical to keep
the patient hip flexed throughout or the ten ion i very ea ily lo t. Tip : S
ee photograph 13.67 for clarification of hand po ition . Mo t u eful where conve
ntional rotary technique are ineffective a thi technique work on the princip
le of backward rotation of the lower component that will ometime break fixatio
n in a way not previou ly achieved. Lea t u eful in very heavy patient where th
e train on the operator lifting the leg may be too great. Extra con ideration
: Thi technique need ome practice to achieve appropriate coordination between
operator .
93
13.67 Thru t idelying two operator technique The rear view hown here of photog
raph 13.66 clarifie the hand hold and patient po ition.
TECHNIQUES FOR THE SACROILIAC AREA
14
There are probably more difference of opinion among t o teopath a to how to d
eal with dy function of the acro-iliac articulation than for any other area of
the body. There are multiple theorie of movement direction and type of le ion
ing. I have attempted to implify the i ue into the two main type of le ioning
po ition of anterior and po terior rotation. Thi i not to ay that other typ
e of dy function do not occur, but to ay that they eem to be fairly rare. Tre
ating the acro-iliac joint a a rotary articulation will deal with the va t pro
portion of joint problem of a mechanical nature without exce ive complication
and therefore uncertainty. It i po ible for the acrum it elf to become di tor
ted, a in life it i a omewhat flexible tructure. It can produce apparent ac
ro-iliac dy function which i , in fact, due to acral tor ion. Some technique f
or addre ing thi problem are included in thi ection. Other are more appropr
iate when con idered with the lumbar pine, a the acrum can ometime be cla
ed a a vertebral, midline tructure. The

principle of treatment, therefore, are tho e of pinal joint . The acro-iliac,

or to be more accurate the ilio- acral articulation have a very limited range
of mobility. However, that mobility i often very ea ily induced, and therefore
it i extremely ea y to overlock when trying to produce a pecific force. Then
, a no movement can be felt, even more force i u ed and pain, trauma and ineff
ective technique i the re ult. The acro-iliac articulation re pond far better
to delicate, careful application of force in very pecific direction . Special
precaution include the elimination of pathological tate in the bone them el
ve , and the po ibility of inflammatory di order . If the acro-iliac dy functi
on i recurrent the po ibility of it being part of a po tural compen ation mu t
be con idered. Exce ive recurrence and a en e of precariou ne hould alert
the practitioner to the po ibility of hypermobility. Thi , neverthele , may ma
nife t a recurrent locking.
Technique for the acro-iliac area
95
14.1 Articulation of acro-iliac further ide prone Find the approximate angle o
f the joint plane u ing your cephalic hand, and apply gentle pre ure. The other
hand take the leg through a circumduction movement until a en e of potential
for mobility i perceived through the applied hand. At thi point of tre ing t
he joint, a pre ure along the line of joint movement i applied. The arc of mov
ement in the circumducting leg when a en e of re i tance from the acro-iliac i
reached will vary lightly from per on to per on. It i ometime difficult to
i olate the acro-iliac movement from hip movement in thi hold. Tip : Mo t u e
ful in patient who are not too acute and can lie prone. Lea t u eful in acute c
a e , or in the pre ence of any lumbar di c yndrome where the po ition may be a
problem. May be difficult in very large patient to reach acro thi far for
mall operator . Extra con ideration : It i very ea y to apply too much pre ure
with the cephalic hand, feel nothing, and therefore, pre even harder. If a e
n e of movement cannot be felt, change angle or reduce pre ure; do not increa e
force. U e of a pillow under the abdomen can aid comfort and finding the be t a
ngle for the joint.
14.2 Sacro-iliac articulation nearer ide prone Operator applie gentle pre ure
along perceived plane of joint with cephalic hand and then perform a circumduc
tion movement with other hand controlling leg. A en e of ten ion accumulate i
n acro-iliac joint, firmer pre ure i applied to ilium along joint plane to mo
bilize joint. The arc of movement within the circumduction where the joint play
will be felt varie lightly with different patient . Tip : Mo t u eful in large
r patient where reaching acro may be a problem when working on the other ide
. Lea t u eful if the table i not adju table, and it i not po ible to get the
angle of the articulating arm vertical. Not comfortable in acute patient where
lying prone i a problem, or in the pre ence of lumbar di c le ion where exten
ion may be painful. Extra con ideration : U e of a pillow under the abdomen wil
l often make the correct joint plane acce ible. 14.3 Sacro-iliac articulation a
nteriorly idelying The patient i put into ome rotation of the pine, and the
upper leg flexed to a comfortable po ition. The operator firmly gra p the ilium
between hi own forearm, abdomen and other hand. A rocking back and forward alo
ng the plane of the joint can be applied. Sufficient rotation mu t be applied to
obliterate mo t of the lumbar movement, a otherwi e the rocking action will i
mply produce flexion and exten ion of the lumbar area. Tip : Mo t u eful in pati
ent who cannot lie prone uch a pregnant women, and acute lumbar pain yndrome
. Lea t u eful in very flexible ubject where the lumbar movement tend to ab o
rb the force . Extra con ideration : Harmonic technique can be performed in thi
po ition if the upper part of the body i made to o cillate, and if the ilium i
held very firmly, an anterior gapping can be applied if the operator flexe hi
own knee and hold the pelvi in the ame plane. Thi hold can al o be u ed f
or lumbar idebending and everal other direction of articulation with a little

thought.
96
Technique for the acro-iliac area
14.4 Sacro-iliac pringing prone Thi i a much a te ting hold a it i a treat
ment technique. The operator i palpating in the ulcu of the acroiliac joint
on the oppo ite ide, and the other hand i applying a force vertically toward
the table. If movement i felt in the acro-iliac it mu t be hypermobile, a the
plane of the joint i far more medial to lateral than vertical. Rotary movement
of the lumbar pine i en ed with thi pre ure al o, and if exce ive give a
clue a to po ible cau e of dy function tate . Tip : The pre ure mu t be app
lied carefully over the po terior uperior iliac pine a otherwi e only oft ti
ue mobility will be en ed. Lea t u eful in acute ubject where lying prone i
a problem. Extra con ideration : Thi hold develop into the next one illu tra
ted a a mean of en ing from where movement i being induced.
14.5 Sacro-iliac articulation and pringing prone Thi illu tration directly fol
low on from the previou one. The pringing hand ha gradually changed directio
n until the plane of the joint ha been en ed, and the combined en e of propri
oceptive awarene with the pringing hand and tactile en ing with the other ha
nd allow the optimum direction of movement to be u ed. If the be t direction fo
r joint play cannot be en ed, u e le force not more a the applied weight of
the hand may have obliterated the free play, and more pre ure will only rotate
the lumbar pine and not have any more effect on the acro-iliac joint. Tip : G
radually moving the houlder of the pringing hand through a circle will allow t
he optimum direction of force to be a e ed. A pillow under the abdomen may hel
p to.reduce the lumbar lordo i , and make the acro-iliac joint more acce ible.
Lea t u eful in acute lumbar pain yndrome where lying prone may be a problem.
14.6 Harmonic technique pelvi upine Operator cup anterior uperior iliac pin
e in the palm of both hand and initiate an o cillatory movement into rotatio
n of the pelvi . Thi can be diagno tic a well a therapeutic, a difference i
n rotary capability of the pelvi on the lumbar pine can be en ed. Tip : A pil
low behind the knee may change the angle of the pelvi and make movement ea ier.
Lea t u eful in ca e of acro-iliac hypermobility a thi pre ure may induce
pain in the joint concerned. Thi i in it elf diagno tic.
Technique for the acro-iliac area
97
14.7 Sacro-iliac articulation upine The finger of the palpating hand are place
d o that the tip are in the ulcu of the joint and while gentle downward pre
ure i applied toward the table the knee i taken through a circular movement t
o mobilize the joint. Tip : Exce ive pre ure toward the table hould be avoide
d a thi will obliterate joint movement. Within the arc of circumduction of the
knee, a point of re i tance will be felt and thi can be empha ized with increa
ed downward pre ure at that time to optimize the mobilizing force. Mo t u eful
where patient cannot lie prone for any rea on. Lea t u eful in the pre ence of
o teoarthro i in the hip or knee.
14.8 Sacro-iliac thru t into anterior direction upine The optimum direction of
the joint i found and then a downward pre ure i applied with ome internal ro
tation of the hip to put the hip cap ule on ome ten ion. The knee will be appro
ximately over the midline of the body in mo t ca e . Small adju tment of flexio
n and exten ion of the hip will be nece ary to find the optimum, and then a ho
rt harp downward force i applied to direct the ilium into an anterior rotation
direction. At fir t thi may eem to be doing the oppo ite of what i intended,

but it hould be remembered that the acro-iliac joint i uperior to the hip,
and therefore when a pre ure i applied toward the table, the ilium will be ro
tated forward, not backward. If, however, the hip i taken further into flexion,
the ilium will be rotated backward. Tip : Very accurate joint plane en e i re
quired for thi technique, and it may be nece ary to feel thi with finger in
the ulcu of the joint fir t, and then remove the hand and apply it to the knee
. Mo t u eful in imple uncomplicated acro-iliac dy function in fairly tiff u
bject where the force will not be di ipated into the lumbar pine. Lea t u efu
l in ca e where there i o teo-arthro i of the hip and thi pre ure will be u
ncomfortable or impo ible. Extra con ideration : May be repeated everal time
a a pringing rather than a pecific thru t if correct barrier en e cannot be
found.
98
Technique for the acro-iliac area 14.9 Sacro-iliac thru t and articulation ant
eriorly prone The plane of the joint ha been found with the caudal hand and whi
le maintaining thi direction of force, the operator applie the other hand to t
he acrum near to the other acro-iliac joint. The acral hand become a fixing
hand, and the acro-iliac can be mobilized with pringing into an anterior direc
tion. If a cri p barrier i en ed, a thru t can be applied, but, owing to the
mall range of movement of the joint, thi will be a very hort amplitude. Tip :
The acral hand can be placed in uch a way to tip the acrum into flexion, rota
tion or idebending to optimize the ten ion at the acroiliac joint. Different p
ha e of breathing can be u ed a felt appropriate. If the thru t i applied whe
n the patient i holding a full breath in, the pelvi will become firmer, and th
e thru t may be ea ier. In a very tight ubject, exhalation may be more u eful.
Mo t u eful in uncomplicated ubacute or chronic ca e . Lea t u eful in acute ca
e or where the patient may find lying prone a problem.
14.10 Thru t acro-iliac anteriorly prone The medial a pect of the elbow i appl
ied over the po terior uperior iliac pine and along the cre t of the ilium. Th
e other hand lever again t the table and the other thigh, to produce an anterio
r rotation movement of the ilium. Adju tment will be nece ary of the adduction
and exten ion of the thigh to produce the optimum ten ion in the joint. At the
point of accumulation of force , the thru t i applied along the cre t of the il
ium. Tip : Mo t u eful in mall patient , and where there i no problem lying pr
one. Lea t u eful in acute ca e where the prone po ition may be a problem. Extr
a con ideration : Care mu t be taken not to hyperextend the lumbar pine and thu
cau e pain. In thi re pect keeping the anterior of the pelvi on the table i
a help. If the correct ten ion en e doe not accumulate, reduce ten ion rather
than increa e it, a it i very ea y to over-lock. Different pha e of re pirat
ion may help to produce the be t barrier.
Technique for the acro-iliac area
99
14.11 Thru t acro-iliac anteriorly prone Pre ure i applied to the po terior
uperior iliac pine of the oppo ite ilium in the direction of the joint. The oth
er hand lift the thigh into exten ion and ome adduction of the hip until ten i
on accumulate at the acro-iliac. Varied rotation of the hip will aid the build
-up of ten ion. The thru t i applied with the hand on the ilium, not the liftin
g hand. Tip : Mo t u eful in chronic ca e where fixation i liable to be more c
ri p and, therefore, more ea y to relea e. Lea t u eful in acute ca e where lyi
ng prone may be a problem or where the patient i very heavy, making lifting of
the leg a problem. Extra con ideration : Keep the pelvi firmly applied to the t
able a otherwi e hyperexten ion of the lumbar pine can occur. A pillow under t
he abdomen may a i t patient comfort. Different pha e of breathing will often
a i t in accumulation of optimum ten ion.

14.12 Thru t acro-iliac anteriorly prone The thru ting hand i applied behind t
he po terior uperior iliac pine, and the finger of the other hand interlock w
ith the thru ting hand o that the leg can be lifted until ten ion i felt to ac
cumulate. Adduction of the leg will help the build-up of ten ion. Tip : Mo t u e
ful in patient who are fairly tight a ten ion will accumulate more ea ily. Lea
t u eful where there i any hip dy function or where exten ion of the lumbar p
ine i going to be a problem. Extra con ideration : If the operator i mall, th
e po ition may be difficult, a the leg-lifting hand i drawing the operator tow
ard the feet of the patient, and thu reducing the force available at the acroi
liac. U e of a pillow under the abdomen can be u eful to avoid hyperexten ion of
the lumbar pine. 14.13 < Thru t/articulation acro-iliac anteriorly upine The
patient ha firmly cla ped the other thigh into flexion to lock the lumbar pin
e. The operator ha fixed the patient leg between hi thigh and i a i ting
the patient in holding the other leg in flexion. He applie a downward force to
the knee o that the acro-iliac i tor ioned forward on the acrum. If ten ion
i ufficient, a thru t can be applied toward the table. Tip : Mo t u eful in yo
ung and fairly fit patient a the po ition can be rather extreme. The po ition
of extreme flexion of the other hip put the lumbar pine in a flexed po ition w
hich can help to obliterate movement which may be u eful. Lea t u eful if there
i any hip di order. Extra con ideration : A u eful fulcrum can be made if the p
atient i capable of lying with the acrum on the edge of the table. Thi po iti
on i al o a differential te t of lumbar and acro-iliac dy function. If pain i
reproduced in thi po ition, a the lumbar pine i not involved in the movemen
t, it can be rea onably a umed that the acro-iliac i the ource of the ympto
m . If pain get much wor e a the patient relea e the other knee, there i a g
ood chance that it i movement of the lumbar pine which i implicated in the pa
in yndrome.
100
Technique for the acro-iliac area 14.14 Thru t/articulation acro-iliac anteri
orly idelying Thi po ition i fundamentally the ame a photograph 14.13 excep
t that the leg ha been flexed at the knee. The movement i now much tronger a
the quadricep i put on ten ion earlier. Tip : Varying the flexion of the knee
can focu the ten ion more efficiently. Extra con ideration : The ame implicat
ion of the te ting nature of thi po ition apply a for photograph 13.15.
14.15 Thru t u ing leg tug acro-iliac anteriorly upine The hand hold i hown
before being applied to the foot. Note that one hand i upinated and the other
pronated. The thumb of the pronated hand i interpo ed between the third and fou
rth finger of the other hand. Thi ha the advantage that a the operator lean
back, without actively gripping, the hold become tighter automatically. Tip :
Experimentation will reveal which i the mo t comfortable way to interlock the h
and .
14.16 Thru t anteriorly acro-iliac u ing leg tug upine The hold hown in photo
graph 14.15 i applied and the lower extremity i taken into ome flexion to cle
ar the other leg a adduction i applied. Apply adduction and internal rotation
of the hip until the fa cia lata and hip cap ule, re pectively, are on ten ion.
A preliminary traction force i u ed until ten ion i felt to accumulate in the
acro-iliac and then without relea ing the ten ion a harp longitudinal tug comp
lete the thru t. Tip : Lea t u eful where there i any knee or hip dy function
or in very lax ubject where the force will be di ipated. Extra con ideration
: Greater efficiency i ometime achieved if the patient i a ked to hold their
breath or to cough coincident with the thru t. Try bracing the patient other
leg on the table again t the operator thigh.
Technique for the acro-iliac area

101
14.17 Harmonic technique for acro-iliac Thi technique how longitudinal harmo
nic technique to the pelvi and particularly the acro-iliac. Tip : Refer to ear
lier ection relating to harmonic technique.
14.18 Thru t acro-iliac anteriorly prone Operator focu e force applied to po
terior uperior iliac pine along the plane of the joint and fixe pelvi to the
table. Patient perform a one-handed pu h up and a ten ion accumulate at the
joint, operator applie a very hort amplitude thru t. Tip : Mo t u eful in very
mobile ubject where the u e of active mu cle contraction in the patient help
reduce pinal mobility. Extra con ideration : Try adju ting initial idebending
to find the optimum ten ion.
102
Technique for the acro-iliac area
14.19 Thru t to acro-iliac anteriorly idelying Thi i a modified lumbar roll
po ition. The cephalic hand of the operator i pu hing toward him elf on the po
terior uperior iliac pine of the ilium on the table. The other hand i applyi
ng a rotary force on the pelvi o that the lower acro-iliac i gapped. Firm co
mpre ion with both hand i nece ary to focu the force at the target joint.
Exce ive rotation of the pine mu t be avoided. Tip : Mo t u eful where there i
a di c yndrome making rotation to the other ide difficult. Lea t u eful in v
ery large ubject where it may not be po ible to achieve ufficient compre iv
e force. Extra con ideration : With compre ion maintained, roll the pelvi forw
ard and backward to find the optimum barrier en e.
14.20 Thru t to acro-iliac anteriorly idelying hown on keleton Thi photogra
ph may clarify the hand po ition of photograph 14.19.
14.21 Thru t to acro-iliac anteriorly itting The operator knee pu he firmly
forward again t the po terior uperior iliac pine. The patient i flexed lig
htly and rotated down until ten ion accumulate at the operator knee. The thru
t i a combination of a light increa e of rotation of the patient body and a
forward movement of the knee again t the ilium. Tip : Thi i a very long lever
technique and rarely u ed; however, there may be ome ca e where it can be a m
ethod of choice, particularly tho e where it i de ired to have the pine vertic
al, thereby lightly driving the acrum down between the ilia.
Technique for the acro-iliac area
103
14.22 Thru t to acro-iliac anteriorly part tanding Patient lie acro the tab
le keeping the other foot on the floor. The operator applie the heel of hi thr
u ting hand to the po terior uperior iliac pine and hi knee in the popliteal
pace of the fexed knee. He pu he down with hi knee while pulling up with the
hand holding the ankle until ten ion accumulate at the acro-iliac. The thru t
i mo tly a force with hi hand but the knee and other hand a i t lightly. Ti
p : Thi will be a rarely u ed manoeuvre but wa taught by Andrew Taylor Still.
Mo t u eful where lumbar tor ion i to be avoided. Lea t u eful where there i a
ny knee dy function. Extra con ideration : The barrier en e will accumulate mor
e ea ily, in mo t ca e , if the patient hold the breath for the thru t.
14.23 Thru t acro-iliac anteriorly part tanding The patient lie acro the ta
ble which ha been lifted to the height of the patient pelvi . She flexe her
knee lightly o that the anterior uperior iliac pine upport her weight. Pr
e ure i applied along the plane of the joint with one hand and the other tabi

lize the acrum. The thru t i applied forward on the po terior uperior iliac
pine to break fixation of the acro-iliac furthe t from the operator. Tip : Mo
t u eful where it i nece ary to avoid tor ion of the lumbar pine. Lea t u ef
ul where the patient i elderly and the po ition may be difficult to attain. Ext
ra con ideration : The acrum- tabilizing hand can hold the bone in a variety of
direction . It will be nece ary to experiment to find the optimum direction wh
ich focu e the force at the acro-iliac. It will u ually be ea ier to accumula
te ten ion if the patient hold the breath for the thru t.
104
Technique for the acro-iliac area
14.24 Thru t acro-iliac po teriorly upine Thi photograph how the hand hold
and patient po itioning for thi technique. The palm of the cephalic hand will b
e applied with the anterior uperior iliac pine. The other hand will be placed
under the i chial tubero ity and the flexed knee and hip are po itioned acro t
he operator abdomen.
Technique for the acro-iliac area 14.25 ( ee facing page, top right) Thru t/ a
rticulation acro-iliac po teriorly upine The hold hown in the previou photog
raph i applied and the operator i pu hing the thigh into abduction with hi el
bow. The thru t i performed a a combination of pu hing back with one hand, pul
ling up with the other and a bending of the knee to further abduct and flex the
hip. Thi will drive the ilium back on the acrum. Tip : Mo t u eful in patient
with a fairly rigid lumbar pine where the force will focu more ea ily in the
acro-iliac. Lea t u eful in the pre ence of any hip dy function. Extra con ide
ration : Barrier en e may accumulate more ea ily if the patient hold the breat
h at the time of the thru t.
105
14.26 ( ee facing page) Thru t po ition acroiliac po teriorly upine The patien
t i po itioned into ufficient initial idebending o that when the operator ap
plie the other component , the idebending may reduce, but will not be complete
ly lo t. 14.27 < ( ee facing page) Thru t acro-iliac po teriorly upine The ocalled Chicago technique ha been applied with the operator cephalic hand ma
intaining idebending and producing a rotation of the patient tor o toward hi
m. Hi other hand hold the ilium again t the table, and a ten ion accumulate
in the acro-iliac a hort amplitude thru t i applied to the anterior uperior
iliac pine. Tip : Initial po itioning of the patient i critical with thi tech
nique. The idebending mu t not be lo t or the force will di ipate higher in th
e pine. The direction of force applied to the ilium will govern whether the tec
hnique focu e at the acroiliac or the lumbo- acral. If the thru t i applied w
ith the cephalic hand, it will tend to focu force on the thoraco-lumbar juncti
on. It may be nece ary to experiment to find the optimum. Mo t operator find t
hat ten ion accumulate be t if the patient leg on the operator ide i cro
ed over the other one, but ometime the oppo ite i true. Tip : Lea t u eful f
or mall operator working on large patient where it may be impo ible to reach
ufficiently well to accumulate the correct ten ion. 14.28 A Thru t acro-iliac
po teriorly itting The patient ha folded her arm and the operator ha rotate
d her whole tor o down to the acrum. He hold back on the ilium with index fing
er and thumb. He applie a rotary force away from the ilium with the other hand
and a ten ion accumulate he thru t backward on the ilium. Sidebending toward
the thru t ide i maintained at all time to help focu the force a otherwi
e they will di ipate through the lumbar pine. Tip : Mo t u eful in tight ubje
ct who have no major pinal dy function. Lea t u eful in very tall ubject whe
re it will be difficult to control the lever . Extra con ideration : It may ome
time aid the technique if the patient it a tride the table.

106
Technique for the acro-iliac area 14.29 Thru t/articulation acro-iliac po ter
iorly prone The operator ha abducted and flexed the patient hip and knee and
the tibia i re ting on hi own flexed thigh . He fixe the whole lower extremit
y between hi forearm and abdomen and cup the anterior uperior iliac pine in
the palm of hi thru ting hand. The wri t of hi other hand ha applied pre ure
behind the i chial tubero ity and the two hand together pull the ilium backwar
d . The thru t i applied with a combination of operator hand and body. Tip :
Lea t u eful where the patient may find prone lying a problem. Extra con iderat
ion : It i important to hold the ilium and pelvi again t the table a abductio
n i applied to the hip, a otherwi e rotation occur into the lumbar pine and
the force will not accumulate at the acro-iliac.
14.30 Thru t acro-iliac po teriorly idelying Thi i a modified lumbar roll po
ition. The operator ha applied only a mall amount of rotation to the thorax a
nd lumbar pine but ha ub tituted compre ion toward the table, through the h
oulder. The thru ting forearm i placed behind the ilium and the elbow pu he th
e i chial tubero ity toward him elf to rotate the ilium backward . The thru t i
applied with a compre ion force from the operator body at the ame time a
an adduction and external rotation of hi arm applied to the ilium. Tip : Exce
ive rotation of the lumbar pine will di ipate the force up to the thoraco-lumb
ar junction; hence the u e of compre ion. A gentle o cillatory rolling of the w
hole patient will enhance the ability to find the optimum thru t plane. Lea t u
eful in very flexible ubject where the thru t will merely produce lumbar flexi
on.
14.31 Thru t to acrum prone Thi how a recoil technique where the operator ha
applied a light compre ive force to the acrum toward the table and then qu
eeze the acrum between both hand to lightly buckle it. The technique i perf
ormed with the relea e of ten ion allowing the natural recoil of the bone to act
a the mobilizing force. Thi may need to be repeated two or three time . Tip :
Mo t u eful where de pite ilio- acral gapping, ome dy function remain which m
ay be due to intra- acral di tortion. Extra con ideration : It may be found that
compre ing the acrum more on one ide than the other will produce a more pec
ific re ult.
Technique for the acro-iliac area
107
14.32 Thru t to acrum idelying Thi i a modified lumbar roll po ition. The op
erator ha applied compre ion to the pelvi and the thorax to reduce the range
of rotation nece ary to reach the acrum. The thru t i a combination of an inc
rea ed compre ive force on the ilium with the operator che t at the ame time
a the heel of hi hand drive the acrum forward . Tip : Mo t u eful where tor
ion of the acrum rather than the ilium i the prime element in the dy function
. Lea t u eful in very flexible or very large ubject . Extra con ideration : Th
e direction of the acral thru t can vary according to the optimum en e of barr
ier accumulation.
14.33 Thru t to ymphy i pubi upine Thi i a combined technique where the op
erator hold the patient knee apart a he attempt to draw them together. Th
e patient doe not u e full power but allow him to gradually work the knee fur
ther apart until ten ion accumulate at the joint. He applie a hort amplitude
thru t into abduction of the thigh while the patient maintain mu cle tone. Tip
: Vary the range of hip flexion and knee flexion to find the optimum. En ure th
e amplitude of the thru t i very hort. Lea t u eful when the patient i very
trong and the operator i mall.

14.34 Mu cle energy technique ymphy i pubi upine See earlier ection on prin
ciple of mu cle energy technique. Tip : Vary the angle of hip flexion and knee
flexion to achieve the optimum. Thi hold only allow an i ometric mu cle energy
technique a the patient pre e again t the operator flexed hand and elbow b
etween the knee .
TECHNIQUES FOR THE GLUTEAL REGION AND COCCYX
15
A the glutei are uch trong mu cle and are in clo e proximity to the ciatic
nerve they may play an important role in production and maintenance of ciatic p
ain yndrome . Hypertonic glutei can affect po ture and prevent normal mechanica
l relation hip of the hip to the re t of the body. There may be a tendency for
the hip to rotate outward and thu up et locomotion, itting and tanding po t
ure . Lumbar origin pain yndrome will often pre ent a ten e painful area in
the glutei, piriformi and gamelli, and work on the e can be helpful in treatmen
t of uch ca e . Particular caution i rarely nece ary in working on the glutei
except that a Ewing tumour in the bone or the ilium it elf can pre ent a glu
teal pain. Due to the en itive nature of the area, particular care need to be
taken with the treatment here o that no accu ation of improper handling can oc
cur. Informing the patient of the purpo e or a particular procedure hould elimi
nate thi problem. The coccyx i commonly a ite of pain although many ca e are
due to referred pain from the lumbar pine. It i , however, po ible to have a
dy functional junction between the acrum and the coccyx. There are technique t
hat approach the joint through the rectum, but external technique are generally
preferred by patient and operator alike! Particular precaution include the p
o ibility of fracture if there ha been direct trauma, and rare ca e of referr
ed pain from the rectum in the pre ence of a pace-occupying le ion.
Technique for the gluteal region and coccyx
109
15.1 Kneading prone U e your thumb to apply cro -fibre kneading to the i chial
tubero ity and lower part of gluteu maximu . The other hand applie a counter-f
orce, partly to limit the force to a pecific part of the mu cle , and partly t
o reduce the di comfort of direct pre ure. Tip : Mo t u eful in ca e of i chia
l tubero ity bur iti , mu cle train and re idual ciatica. Lea t u eful where
prone lying might be a problem. Extra con ideration : The pillow under the abdom
en i for patient comfort. Try u ing varied degree of abduction in the thigh, o
r a pillow under the tibia to flex the knee and reduce tretch on the po terior
thigh mu cle .
15.2 Kneading prone Apply a kneading force to the glutei, gamelli and piriformi
on the oppo ite ide of the patient. The other hand monitor the tretch produc
ed, and pread the effect of the hold to reduce any di comfort. Tip : Lea t u e
ful where prone lying may be a problem. Exqui ite tender area will often be fou
nd in the glutei in lumbar dy function yndrome , and although the e are often d
ue to referred pain, they can be maintaining factor . Recovery can be enhanced i
f the e mu cle are relaxed to allow freer pelvic movement. Extra con ideration
: The pillow under the abdomen will u ually aid patient comfort. Try abducting t
he leg by varied amount to find the optimum.
110
Technique for the gluteal region and coccyx 15.3 Kneading prone Work on the lowe
r part of the glutei and the piriformi while holding back on the belly of the m
u cle with the other hand, to localize the force. The lateral mu cle of the thi

gh can al o be addre ed in thi po ition. Tip : Mo t u eful in ca e of re idua

l ciatica where there i a mu cular component a a maintaining factor. Lea t u
eful where prone lying may be a problem. Extra con ideration : The pillow under
the abdomen will u ually aid patient comfort. Try varying the abduction of the t
high to find the optimum for the technique. 15.4 Inhibition prone Apply a direct
pre ure over the piriformi with your thumb. Maintain a teady pre ure and in
ternally rotate the hip until ten ion i felt. Inhibition implie a teady pre
ure, u ually while a lever i applied, and a finite time mu t elap e until a en
e of relea e i attained. Tip : Mo t u eful in ca e of per i tent piriformi t
en ion a in ome ciatica . Lea t u eful where prone lying i a problem. Extra
con ideration : Try varying the hip abduction in the initial et-up for the tech
nique. Thi may be a very uncomfortable procedure, but within about 10-15 econd
the mu cle will be felt to relax, often accompanied by an immediate relief of
ymptom .
15.5 Inhibition prone Carefully apply the tip of your elbow to the piriformi wh
ile maintaining the internal rotation of the hip with the other hand applied to
the ankle. Tip : Mo t u eful in ca e of evere, long-term pa m of piriformi .
It i al o the method of choice when the operator thumb are not trong enough
. Lea t u eful where prone lying may be a problem. Extra con ideration : The pil
low under the abdomen i to increa e patient comfort. Thi i clearly a powerful
technique, and need ympathetic care in handling to avoid exce ive pain; howe
ver, it can be extremely u eful.
Technique for the gluteal region and coccyx
111
15.6 Inhibition prone Apply a double thumb pre ure to the piriformi with the p
ad of the thumb overlaid. Thi ha the advantage over the elbow hold of being
a little more gentle, yet very pecific. Tip : Mo t u eful in ca e of ciatic n
erve irritation where the piriformi i involved, and inhibition alone i requir
ed, without tretch. The previou hold for working on piriformi u e ome longi
tudinal tretch. Lea t u eful where prone lying i a problem.
15.7 Articulation of acro-coccygeal joint prone Abduct and internally rotate th
e hip carefully, a thi i potentially a very trong hold. Hold back on the coc
cyx with your thumb. The technique can be performed a a force with either hand,
or both imultaneou ly. Tip : Lea t u eful where prone lying i a problem. Extr
a con ideration : Try a king the patient to temporarily hold the breath to a i
t in firming up the pelvic floor.
15.8 Articulation of acro-coccygeal joint prone Internally rotate the hip with
varied degree of knee flexion a uitable for the ca e. Hold back on the coccyx
with the other thumb. The technique i performed with either a pre ure on the
coccyx, a circumduction of the leg, or both imultaneou ly. Tip : Lea t u eful w
here prone lying i a problem. Thi hold i more uitable than the one in photog
raph 15.7 if a gentler procedure i required a it avoid tre ing the hip.
112
Technique for the gluteal region and coccyx
15.9 Articulation of acro-coccygeal joint prone Spring alternately on the acru
m and the coccyx. Different lateral pre ure can be introduced if required to f
ind the optimum barrier for the articulation. Tip : Mo t u eful where it i de i
red to avoid u ing the hip a a lever. Lea t u eful where the prone lying po iti
on may be a problem. Extra con ideration : To make the technique more effective
try u ing patient breath-holding to drive the pelvic floor down and firm up the
area.

15.10 Articulation of acro-coccygeal joint prone Hold the coccyx toward your el
f while internally rotating the oppo ite leg. Tip : Mo t u eful where one hip ha
a dy function making it unu able a a lever, o the contralateral ide i u ed
. Lea t u eful where prone lying may be a problem or where the reach may be too
great for a mall operator. Extra con ideration : Try u ing varied degree of hi
p abduction to amplify the technique.
TECHNIQUES FOR THE THORACOLUMBAR JUNCTION AREA
16
When referring to the thoraco-lumbar region we are encompa ing the area from ab
out the tenth thoracic to the econd lumbar vertebrae, not ju t the twelfth thor
acic to fir t lumbar articulation . The term refer to an area rather than a pe
cific egment. Like all the junctional area of the pine, there are difference
from adjacent area . A the curve are changing and the tability of one area m
eet the relative mobility of the other, difficultie occur. From a technique an
d treatment viewpoint the pre ence of the autonomic outflow to the coeliac plexu
, and the diaphragmatic attachment , -further complicate thi area. O teochondr
o i i extremely common and often cau e the characteri tic flexion deformity,
premature arthro i , and tiffening which make the application and choice of tec
hnique difficult. A the pro tate and uteru drain through their vein into thi
area, the po ibility of econdary meta-

tatic depo it from the e ite mu t alway be con idered in hi tory, examinati
on and diagno i . Exce ive tor ion of thi area in treatment can produce nau ea
. Poor application of technique and exce ive leverage into rotation can lead to
acro-iliac joint train. Careful con ideration of appropriate modifying factor
, and greater u e of compre ion rather than rotation can help to avoid thi pr
oblem. Technique for the area can be difficult in extremely mobile young ubjec
t , a it i not ea y to i olate the area, but high force hould not be u ed in
tead of kill. The area can be con idered a part of the lumbar pine, the thora
cic pine, or an area in it own right. It hould be po ible to employ techniqu
e that reach the area without exce ive force, but owing to the length of the l
ever nece ary, uitable protection of adjacent area i important.
114
Technique for the thoraco-lumbar junction area
16.1 Articulation into idebending itting The patient it acro the table at
one end with one arm over the operator houlder. Apply a idebending force by
pulling with one hand while giving a counter-force with the other. Rock from you
r front to your back foot to produce the de ired movement. Tip : Control of the
compre ion through the fixing hand on the patient houlder i critical in thi
hold. Note that the patient head remain over her pelvi . The pine i being
buckled pecifically at the thoraco-lumbar area.
16.2 Articulation prone Stand below the patient pelvi and pull up again t the
anterior uperior iliac pine. Fix with the other hand over the tran ver e proc
e e at the thoraco-lumbar area. If a uitable barrier accumulate , thi can be
made into a rotation and exten ion thru t although to avoid pain the amplitude
mu t be kept very mall. Tip : Try placing the patient in ome idebending fir t
which will have the effect of making the technique reach the deeper or more up
erficial ti ue according to the direction of the idebending. Try a king the p
atient to turn her head to one ide or the other. Try having her arm by her id
e , under her houlder , under her forehead or over the ide of the table. Each
change will make a difference to the technique. Try applying the technique at v
aried pha e of breathing.

16.3 Articulation prone Lift the thigh ju t above the knee of the prone patient.
Fix with the other hand over the tran ver e proce e of the thoracolumbar area
. Adduct the thigh and extend it. Force will be tran mitted through to the upper
lumbar area. The pull on p oa i very powerful and in mo t ubject it will di
rect force to the area without exce ively tre ing the lumbar area. Tip : Lea
t u eful in very heavy ubject where lifting the leg would be a problem. Mo t
u eful where trong articulation i nece ary.
Technique for the thoraco-lumbar junction area
115
16.4 Thru t prone The patient lie prone in a  phinx po ition. Cla p her ankle
between thumb and index finger and index and middle finger of the pronated han
d. Apply the other hand over the pinal level de ired and initiate a rocking mot
ion of the patient from end to end of the table. A you are rocking, increa e th
e traction force with both hand until ten ion i felt to accumulate under the
pinal hand. Apply a mall thru t again t the pinou proce into exten ion, or
again t a tran ver e proce into rotation. Tip : Try a king the patient to tag
ger her elbow lightly which will introduce a preliminary idebending or rotati
on to the area. It may be nece ary to have the patient drop her feet over the e
nd of the table if they are uncomfortable. Try u ing varied pha e of re piratio
n to find the optimum barrier.
16.5 Thru t ideiying The patient i placed idelying with her lower arm behind
the thorax. Pu h backward again t the anterior a pect of the pelvi to tabiliz
e it. Pu h the pine backward from below to introduce rotation. Pull forward ag
ain t the tran ver e proce e of the vertebrae above with the other hand and u
e the forearm to tabilize the capula. Rock the whole patient into a mall ampl
itude of rotation to find the optimum point of ten ion and then apply the thru t
with the upper hand while maintaining the lever po ition with the lower. Tip :
Thi technique require quite firm compre ion with both hand into the table a
well a into rotation. It i critical not to relea e the fixation produced by t
he lower hand at the moment of the thru t a the focu i liable to be lo t. Try
varied pha e of breathing.
16.6 Thru t idelying The patient i idelying in a cla ical lumbar roll po iti
on. Apply the lever in the u ual way for the thru t with ome mall variation .
U e a ub tantial element of compre ion through the pelvi toward the table. U
e a compre ive force through the houlder toward the table and lightly toward
the patient head. The thru ting hand i pu hing into compre ion again t the
lamina of the vertebral egment de ired. A the force accumulate, the thru t i
applied with the lower hand pulling the vertebra and the pelvi toward you a a
unit. Thi en ure that the force i not di ipated in rotation of the lumbar
pine. Thi i primarily a compre ion thru t with a mall local rotary force at
the contact point at the moment of full compre ion.
TECHNIQUES FOR THE THORACIC SPINE
17
The term thoracic will be u ed in preference to the term dor al, which ha now
been largely uper eded. If the reader i accu tomed to dor al, he will need
to make a mental tran lation at each reference. The thoracic pinal area i more
acce ible than the lumbar in that the embryological curve i maintained. The f
acet joint are liable to be involved in mechanical dy function yndrome a the
y are in appo ition during normal po ture, not ju t in flexion of the pine. De
pite the tor ional nature of movement in the thoracic area, di c le ion are muc
h le common than in the other area . However, when they do occur, they produce

equally eriou problem . The flexible nature of the thoracic area in rotation
mean that it i often nece ary to u e a large element of contra-rotation to
produce locking in manipulative technique. Thi in it elf, if exce ive or poorl
y controlled, will be painful and re ult in over-locking. There can be a tendenc
y to apply more leverage when uitable re i tance i difficult to feel, which le
ad to even more di comfort. Special precaution for the area mu t include the p
o ibility of bony weakne uch a o teoporo i and econdary depo it . There i
potential for damage in ca e of advanced o teoarthro i with ligamentou tif
fening, particularly if exce ive force i u ed.
The autonomic chain i very clo e to the thoracic pine and thi mean that appl
ying any phy ical therapy can produce unde ired or unexpected autonomic change
in the body. The e may include weating, en e of coldne , fatigue, yawning and
breathing and dige tive change . While adver e reaction of thi ort are u ual
ly temporary, and not too alarming, it i be t to be aware of thi po ibility
o that the practitioner can advi e the patient accordingly. Progno tic factor f
or a poor re ult include the patient with very  tringy mu cle ti ue in the ar
ea. Thi awarene can allow the practitioner to be more accurate in progno i e
arly on in a treatment erie . Stringy mu cle ha undergone ome partial fibro
tic change , which are by their very nature only partly rever ible. Thi i not
uncommon in the thoracic area and it hould direct treatment to the cau e of the
dy function, rather than ju t the painful area. It i often u eful to con ider
pha e of breathing when performing technique in the thoracic area. Mo t thru t
technique i ea ier if performed a the patient exhale . There are time when th
i i not the ca e. If it i nece ary to tabilize a very mobile ubject, a kin
g them to hold the breath may be of more help. Experimentation i nece ary to f
ind the be t method for each patient.
Technique for the thoracic pine
117
17.1 Harmonic technique prone U e your upper hand to fix or focu the harmonic r
ocking of the pelvi induced by your lower hand. The amplitude of the harmonic m
ovement will increa e depending how far up the thoracic pine you fix a the lev
er lengthen . See earlier ection relating to harmonic technique. Tip : Although
thi i a therapeutic procedure, it perform a u eful diagno tic te t that will
rapidly find area of dimini hed flexibility. 17.3 Articulation in idebending
and exten ion itting The patient re t her folded arm on the operator hould
er and upper arm and he i reaching around to cla p the paravertebral region on
each ide. Rock into idebending and, therefore, induce idebending in the pati
ent. Pull your hand toward your elf again t the fulcrum of the houlder and ind
uce exten ion into the patient pine. Tip : Try adding rotation to either ide
before or after the other movement to help focu them. Extra con ideration : F
ix the patient knee , padded if nece ary, again t you.
17.2 Articulation into rotation itting Fix again t the pinou proce at a cho
en level and rotate the re t of the body back again t thi level by pulling the
houlder backward . The natural recoil of the body will take it forward again
o that you can move up or down to the next egment and repeat the exerci e. Tip
: Thi can be a diagno tic exerci e or a mild therapeutic technique.
118
Technique for the thoracic pine
17.4 Articulation into idebending itting Sit or tand clo e to the patient
ide and hook your abducted arm over her houlder. Apply your thumb or thenar emi
nence again t the pinou proce and buckle the body into idebending while u i
ng your thumb a a fulcrum. Tip : Mo t u eful in fairly tiff ubject where onl

y mall movement i required. Extra con ideration : Try u ing circumduction move
ment of the upper body around the thumb to induce other range of movement. 17.5
(top right) Articulation into idebending itting The patient it with hand c
la ped behind the neck. Cla p the far houlder of the patient. Hold her near ho
ulder firmly again t your che t and keeping her head directly above her pelvi ,
buckle the pine over your thumb or thenar eminence applied to a pinou proce
. Firm compre ion of the patient again t the operator i nece ary and the move
ment come from a light flexing of the knee . Tip : Try adding other range of
movement to the idebending to help focu it. Try circumduction. 17.6 Articulati
on into idebending itting The patient it with folded arm . Lift the further
elbow while pre ing down on the near houlder with your axilla. Buckle the pin
e around your thumb or thenar eminence applied to the near ide of a pinou pro
ce . It i nece ary to have firm compre ion of the patient into the operator
and the idebending movement occur a you flex your knee . Tip : Mo t u eful wh
ere it would be difficult for the patient to cla p hand behind the neck.
Technique for the thoracic pine
119
17.7 Articulation into rotation itting The patient fold her arm and the opera
tor reache either acro the folded arm , or through them, to cla p the further
houlder. Compre the patient into your che t and apply a thumb or thenar emin
ence to one or more pinou proce e . Induce a mall amount of idebending away
from your elf and rotate the patient and your body together to focu the forc
e at the contact point. Tip : Try circumduction in tead of imple rotation to ad
dre different part of the dy function. 17.8 (bottom left) Articulation into r
otation itting The patient cla p her hand behind the neck. Reach around to cl
a p her far houlder and apply your thumb or thenar eminence to one or more pin
ou proce e . Compre the patient into your elf and induce rotation down to yo
ur applied thumb by twi ting your body and the patient a a unit. Tip : The th
umb applied to the pinou proce can be on the near ide to pu h further rotati
on or on the far ide to block rotation below that point. Extra con ideration :
If the patient i very flexible, an a tride itting po ition may be more efficie
nt a it limit thoraco-lumbar mobility with the leg abducted in thi way. 17.9
Articulation into exten ion upper thoracic area itting The patient fold her a
rm and re t them on the operator upper che t. Thread your forearm through h
er folded arm and u ing your hand a a fulcrum lever the pine into exten ion.
Tip : Try making thi into a circumduction movement. Brace the operator thigh
again t the patient knee - padded, if nece ary.
120
Technique for the thoracic pine
17.10 Articulation into flexion itting The patient fold her arm and drop her
head forward onto the operator che t. Fix the top of her head with your chin
and apply a lateral compre ion force, to the thorax, through your wri t and fo
rearm . Pull up under the angle of a cho en pair of rib while bending your kne
e to induce a localized flexion. Tip : Try inducing ome idebending movement o
r circumduction. Fix the patient knee again t the operator thigh , if nece
ary.
17.11 Traction and idebending articulation itting The operator thread hi arm
under the patient arm . Lift under the patient ma toid proce e with the
heel of your hand . The lift al o take place through the axillae. Lean the pat
ient again t your che t and while maintaining the lift, introduce circumduction
to reach the mid thoracic area. Tip : Mo t u eful in maller patient and childr
en a the lift can prove hard work if applied to larger adult .

Technique for the thoracic pine

121
17.14 Articulation into exten ion prone The patient lie prone with her arm fol
ded under the forehead. Lift the folded arm , head and thoracic pine into exten
ion while counter-fixing with your other hand. Tip : Lea t u eful where the pat
ient i very large a con iderable effort would be required to perform thi tech
nique. Extra con ideration : Try introducing idebending or rotation to help foc
u the force .
17.12 ( ee facing page, bottom left) Articulation into rotation prone The patient
lie prone with her arm folded under the forehead. Fix a thoracic pinou proc
e toward your elf and with your other hand under the patient folded arm int
roduce rotation down to the fixing hand. Note that the patient i not being lift
ed a the operator upper hand act a a fulcrum and re t on the table. Tip :
Mo t u eful where it i de ired to limit rotation down to a pecific egment a
the thumb will act a a block to movement below thi point. Extra con ideration
: Try adding different combined force of idebending or exten ion to the rotati
on. 17.13 ( ee facing page, bottom right) Articulation into exten ion prone The
patient lie prone with her arm folded under the forehead. Lift the folded arm
, head and thoracic pine into exten ion while counter-fixing with your other ha
nd. Tip : Lea t u eful where the patient i very large a con iderable effort wo
uld be required to perform thi technique. Extra con ideration : Try introducing
idebending or rotation to help focu the force . 17.15 Springing prone Apply a
cla icalpu h and pull technique by bracing everal pinou proce e with th
e heel of one hand to rotate them away from your elf. Brace everal adjacent one
above to produce a rotation force between the hand . Naturally, the technique
can be u ed with the hand rever ed. Tip : Try fixing with one hand and mobilizi
ng with the other and vary the patient head rotation to find the optimum.
122
Technique for the thoracic pine 17.16 Articulation into rotation prone The ope
rator i working pecifically on one egment, taking hold of it a if it were a
wing nut. Pu h toward the table to ab orb the pring. Apply the thumb and index
finger on oppo ite ide of adjacent pinou proce e . They introduce the rotar
y force a you pronate your forearm. Tip : Lea t u eful where the pine i very
tender. The force needed to make thi technique effective would be too great in
ca e of evere re triction. 17.17 (bottom left) Springing po tero-anterior pron
e The patient i lying prone, in thi ca e with her hand folded under her head.
Stand at the head of the table and u e the pad of your thumb to pring the ch
o en level directly anteriorly. Pre with the thumb over the laminae of the ve
rtebrae, not the pinou proce e a they can tend to be en itive. Tip : Mo t
u eful where pecific egmental dy function i pre ent. Extra con ideration : Tr
y pringing on one ide only, or taggering the thumb to work on adjacent egme
nt . Try having the patient arm by her ide or over the edge of the table. 17
.18 Thru t tarting po ition upine The patient gra p oppo ite houlder withou
t cro ing her forearm . The operator ha rolled the patient toward him elf and
i liding hi hand under the pine. He i u ing a flat hand, but can apply a va
riety of po ible hold to the target egment. Note that the patient head i l
eft on the pillow and that he i only rolled far enough to allow the hand to be
lipped underneath. Thi ha become known a the dog technique after Fryette a
w thi being done badly and commented, I wouldnt do that to a dog! If he had
een it done well, he would not have made that comment, but the name ha tuck
ince then!
Technique for the thoracic pine
123

17.19 Thru t to mid thoracic pine upine The hold hown in photograph 17.18 i
adopted and the patient i rolled over onto the hand. Te t for free play in the
primary lever direction of traction. Focu all other component of flexion, rota
tion away from your elf, idebending toward your elf and light compre ion and
ide hifting away. If thi eem exce ively complex, you hould imply concentr
ate on bringing your elbow in toward your ide ; thi will automatically produc
e the correct component . The underneath hand perform a mall pronation and tra
ction toward the pelvi to help tighten up the lever . At the point of barrier a
ccumulation, empha ize the pronation of the lower hand and apply the thru t thro
ugh your upper hand and che t into traction pu hing her elbow toward her hould
er . It i u ually po ible to reach from about the third to the tenth thoracic
vertebra with thi hold. Tip : Thi i not a flexion thru t. It i not a compre
ion thru t. The u e of all the econdary lever i de igned purely to help mini
mize the amplitude of the primary lever of traction. Extra con ideration : Many
varietie of hand hold underneath are po ible. A flat hand pronated lightly i
mo t comfortable for the patient. The hand can be applied with the finger loo
ely cla ped. It can be applied u ing a fi t o that the pinou proce e fit in
to the pace between flexed finger and thenar eminence. The underneath hand i
a much a part of the technique a the upper hand. If the hold cau e pain to th
e operator in the lower hand, try pu hing the hand firmly into the thoracic pin
e, rather than imply re ting it on the table. Leave a mall pace between the b
ack of the hand and the table o that the wri t i a much part of the fulcrum a
the hand. Try u ing varied quantitie of patient head rotation to help focu t
he technique. Generally rotation away from the operator will tighten the lever .
17.20 Thru t to mid thoracic pine upine The patient i only cro ing one arm o
ver her che t. The operator i u ing a pad between hi che t and the patient e
lbow. Hi upper hand i applied to the pad to direct the force more accurately.
It i u ually po ible to reach from about the third to the tenth thoracic level
with thi hold. Tip : Mo t u eful for the patient with a houlder dy function a
the painful houlder can be left out of the hold. All other factor are a the
detail in photograph 17.19.
124
Technique for the thoracic pine 17.21 Thru t mid thoracic area upine The patie
nt cla p her hand behind her neck. Lift her upper body with your upper hand an
d lip your lower hand under the patient to apply it to the target vertebra. Fle
x the patient down to the egment and while holding her teady, apply a thru t w
ith your thorax to the patient elbow . In thi variation of the ba ic techniqu
e it i difficult to reach much above the fifth thoracic level in mo t ubject .
Tip : Thi i a mobile technique and it i not u ually po ible to tay in the
thru t po ition for more than a few moment . It i much more difficult to u e va
ried econdary lever with thi hold and it tend to become a flexion and compre
ion thru t. A the econdary lever are not u ed to any great extent, thi can
become a rather forceful variation. It i more difficult to be pecific with th
i technique and, therefore, it can become a technique for gapping everal facet
at once. Thi mean that a re tricted egment in the mid t of a mobile area ma
y not be mobilized effectively.
17.22 and 17.23 Thru t upper thoracic area upine The patient cla p her hand b
ehind her neck. The operator make a loo e fi t and lip it under the patient t
o fit the pinou proce e into the palm. Flex the patient elbow and apply a
ll the u ual component . The thru t i performed into traction and ome compre
ion when the lever have focu ed. Tip : Mo t u eful for upper thoracic area from
econd to fifth level .
Technique for the thoracic pine

125
17.24 and 17.25 Thru t mid thoracic upine ingle arm lever The operator i u in
g one or other arm of the patient, cro ed over the che t. It i perfectly po i
ble to make the technique work with one arm only, but thi will be a little more
difficult. All the u ual component are available although more compre ion wil
l probably be required. Tip : Mo t u eful in ca e where any problem with one h
oulder or the other make the normal hold impo ible. 17.26 and 17.27 Thru t mid
thoracic area from ame ide The patient cro e her che t with her arm in the
u ual way avoiding cro ing the forearm . Roll the patient away from you to li
de your hand under the pine from the near ide. Roll the patient onto your hand
and apply your forearm to her folded arm . (Note that the completed hold i how
n from the other ide for clarity.) Te t for free play in the primary lever dire
ction of traction while adding the econdary component to bring the target join
t to the optimum thru t focu . Tip : Mo t u eful in very large ubject where it
may be impo ible to reach all the way around. Thi hold can al o be u ed for c
o to-vertebral and co to-tran ver e joint , it i al o a u eful method when an o
perator, for any rea on, find u e of one particular hand a problem, a it mean
that the ame hand can be u ed from both ide , wherea with the conventional h
old he would need to change hand to reach the other ide.
126
Technique for the thoracic pine
17.28 Thru t u ing cro ed hand mid thoracic area prone The patient lie prone
with her arm over the ide of the table to pread the capulae. Apply your cro
ed hand to oppo ite ide of the pine. The far hand i lightly upinated to
bring the pi iform into contact with a tran ver e proce . The near hand i pro
nated to bring the hypothenar eminence into contact with a tran ver e proce of
the ame or adjacent vertebra. The thru t i applied after the lack ha been t
aken out of the ti ue with a downward pre ure and a mall element of idebend
ing. Tip : Mo t u eful in fairly flexible ubject . Extra con ideration : Try ch
anging the hand to make the far hand pu h toward the head and the near one pu h
toward the feet. It may be nece ary to change the applicator to u e the thenar
eminence for ome operator . The thru t i u ually coincident with exhalation
to avoid rib damage. Try varying the patient head rotation to find the optimum
.
17.29 Thru t mid thoracic area idelying The operator i u ing hi lower arm to
tabilize the pelvi in thi modified lumbar roll po ition. The upper, or fixing
, hand i maintaining a downward pre ure to produce a compre ion force. The ac
tual thru t i applied with a direct compre ion force into the table and again
t the tran ver e proce of the level de ired. Tip : Mo t u eful in ca e where
compre ive force on the che t might be unde irable for any rea on. Extra con i
deration : Although thi po ition might eem difficult, or even impo ible for t
he purpo e intended, it need remarkably little practice to be made into an effi
cient technique. The control of the two very long lever i critical. Careful t
udy of the photograph will reveal the compre ive and tor ional force being app
lied. Try varying the pha e of breathing to find the optimum ten ion.
Technique for the thoracic pine
127
17.30 Thru t mid thoracic area idelying, hown on keleton The hold hown in ph
otograph 17.29 i applied here to the keleton for clarity. Note that the lower
hand i applied to the tran ver e proce e and the rib . The lower elbow i app
lied to the lateral a pect of the pelvi . The upper hand i pu hing the houlder
back lightly, but mo tly down into the table.

17.31 Thru t mid thoracic area itting The patient cla p round her che t grippi
ng oppo ite houlder . She ha not cro ed her forearm . Pull in toward your elf
on her elbow u ing a pad, if nece ary, a a fulcrum between your che t and th
e de ired level of the patient pine. You need to pull in on the elbow to pro
duce a compre ion while engaging the barrier in a traction, flexion, idebendin
g and oppo ite rotation direction. The thru t i an accentuation of the primary
lever of traction. Extra con ideration : Lateral compre ion i available imply
by the operator bringing hi elbow toward hi ide . If the elbow are tagger
ed lightly one above the other, an automatic ide hift will be introduced. Note
that the patient i itting a tride the table in thi illu tration. Thi i not
an e ential part of the technique but may help fix the pelvi in a very mobile
ubject. Try varying the pha e of breathing to find the optimum.
128
Technique for the thoracic pine
17.32 Thru t mid thoracic area itting (taking up the hand hold) The operator i
cla ping the patient wri t . A k her to interlace her hand behind her neck.
Taking up the hold in thi way avoid having to thread your hand through her el
bow , and make the grip much ea ier to acce .
17.33 Thru t mid thoracic area itting The operator ha taken up the hold in the
way hown in photograph 17.32. Place a pad if nece ary between your che t and
the patient pine. Pull her into flexion down to the egment de ired, compre
toward your che t and induce ome idebending and oppo ite rotation. The thru t
i applied with a combination of a lift of your body from the knee , and an inc
rea e of compre ion of the patient again t your che t. The force are produced
by pulling in on her arm at the ame time a the lift. Tip : Lea t u eful where
a houlder problem might make the hold painful or difficult. Try a lateral comp
re ion of the che t in mobile ubject to ab orb the free play in the thorax. T
ry making the thru t after a circumduction of the patient body o that the tec
hnique become a movable procedure rather than ju t a lifting exerci e. Extra co
n ideration : Note that the patient i a tride the table in thi illu tration. T
hi i not e ential to the technique. Neverthele , in mobile ubject the a tr
ide po ition will help to ab orb exce ive free play in the lumbar pine. Note t
hat the patient elbow remain pointing forward; if they are allowed to play t
o the ide the patient houlder may be trained. Try varying the pha e of bre
athing to find the optimum ten ion for the thru t.
Technique for the thoracic pine
129
17.34 Thru t mid thoracic area itting, knee fulcrum The patient i itting with
hand cla ped behind her neck. Thread your hand through the arm and grip the
patient wri t to pull her into flexion down to the level de ired. Apply your
padded knee to the pinou or tran ver e proce . The thru t i performed with a
mall lifting force through the patient arm , again t a very mall increa e o
f pre ure with your knee. Tip : Note that the patient elbow remain pointing
to the front; they are not allowed to play out a thi can train the houlder
. Thi i a very powerful technique, and if performed with exce ive force could
very ea ily become traumatic. Try varying the pha e of breathing to find the op
timum ten ion for the thru t.
17.35 Thru t mid thoracic area itting patient arm acro che t The patient c
la p oppo ite houlder without cro ing her forearm . Grip her elbow and pull
her into your che t over a uitable pad, if nece ary, applied to the de ired
pinal level. Apply a lateral compre ive force to ab orb the free play in the th

orax. Stagger your arm lightly to produce a mall component of ide hift a yo
u queeze the thorax. The patient lean back again t you and you flex her pine
until the barrier accumulate . The thru t i performed with a mall increa e of
the compre ion coinciding with a lift directly into traction. Small vector of
idebending and rotation can be introduced a nece ary. Tip : Try varying the p
ha e of breathing to find the optimum barrier. Note that in thi photograph the
patient i itting acro the table. If the table i very wide, thi will mean t
hat he i too far from the operator to make the technique efficient. Either a n
arrower table would be nece ary or he could it a tride the table. Another alt
ernative might be to u e a tool.
130
Technique for the thoracic pine
17.36 Thru t mid thoracic area tanding The patient cla p oppo ite houlder wi
thout cro ing her arm . Pull her into your che t with a uitable pad interpo ed
if nece ary. Grip her elbow and pull her into compre ion, ome idebending a
nd oppo ite rotation. Flex down to the egment de ired and then lean her back ag
ain t you a you drop your weight onto your back leg. The patient ha been told
what to expect. At the optimum ten ion, apply a mall increa e of compre ion at
the ame time a a mall lift by hrugging your houlder . Tip : At all time t
he patient remain in ome flexion. Thi i not o much a lifting force a a re
i tance to dropping of the egment above the fulcrum. The primary lever i a tr
action force.
17.37 Thru t mid thoracic area tanding, hand cla ped behind neck The patient c
la p her hand behind her neck. Pull her again t your che t with a uitable pad
interpo ed if nece ary. Warn her that he i to be pulled off balance. Apply a
compre ion, idebending and oppo ite rotation force while pulling her into fle
xion down to the egment de ired. At the point of optimum ten ion, perform the t
hru t with a mall hrug of your houlder . Tip : Note that the patient elbow
remain pointing to the front. If they are allowed to play, there i a po ibil
ity of training her houlder .
TECHNIQUES FOR THE THORACIC CAGE AND RIBS
18
The rib and thorax pre ent certain of their own particular problem from the po
int of view of efficient performance of technique. The e problem mainly relate
to the variation in hape of the thoracic curve in different individual and th
e need to modify approache accordingly. There are everal pathological conditio
n that need particular care. Although thi i not the place to be detailing all
the e, the reader i encouraged to think in particular about o teochondro i , o
teoporo i , colio i , and myeloma. All the e are example of the type of condi
tion where pecial con ideration a to type of approach i needed a there will
be deficiency of bone trength. The rib articulation are rarely dy functional
by them elve , and it ha been tated that in a given ca e, the thoracic pine
hould receive attention fir t and ub equently the rib . If rib articulation ar
e addre ed fir t, they may be di turbed again when any thoracic pinal techniqu
e i performed thereby rendering the rib work ineffective. Although there are u
ually twelve pair of rib , they vary progre ively from above down, in hape an
d movement po ibility; technique will inevitably vary according to the area bei
ng worked. The technique , therefore, are divided broadly into tho e applicable
on the lower, middle and upper rib . Naturally the e demarcation are artificial
, and there will be ome overlap. Technique cla ification have often referred
to the nature of movement of rib . They demarcate the bucket-handle type of mo
vement from the pump-handle type. Thi cla ification i not u ed here a rib
dy function i

con idered imply in re pect of good function or lack of it. The choice of techn
ique i by the quality and quantity of the dy function found rather than by any
pre-conceived notion of pecific functional movement po ibilitie . Although thi
doe not accord with traditional thinking when working on rib , it i no le
effective in actual practice, and con iderably impler to apply. Due to the pri
ngy nature of the rib in normal ubject , it i u ually nece ary to ab orb om
e of that pring in performing technique . Thi i done by u ing everal vector
of force of compre ion in more than one direction. Thi can be either along or
acro a particular rib o that the amplitude of a force applied need not be to
o great. Tor ion with thi method of approach i le nece ary, and di comfort
i reduced with the e carefully applied compre ion . The control of rhythm i c
learly nece ary, particularly in rhythmic technique that have to be repeated
everal time . Pha e of breathing al o need to be taken into con ideration. Many
technique require either exhalation to induce relaxation or, alternatively, in
halation to create a firming up of the part and improving the acce to the opti
mum motion barrier. The choice of po ition to perform the technique, uch a up
ine, idelying, etc., i going to be governed by the mo t comfortable po ition f
or the patient at the time, and the po ition mo t effective for the requirement
of the treatment and technique being given. A a rule, idelying i more comfor
table than upine, and upine i more comfortable than
132
Technique for the thoracic cage and rib and articulation will be more u eful.
Thru t technique have a particular u e when there ha been a traumatic on et to
a particular yndrome. They play a maller part in more chronic condition , exc
ept at the out et, to break fixation and pave the way for more rhythmic approach
e later. Care hould alway be taken of the ae thetic nature of the procedure b
eing u ed. Thi i particularly true when working on female patient o a not t
o put pre ure on brea t ti ue which could be embarra ing and po ibly painful
.
prone. Sitting i better where larger movement are nece ary, but thi require
more cooperation from the patient and control by the operator. It i preferable
to avoid the nece ity of changing the patient po ition exce ively during tr
eatment and the relative ize of operator and patient may al o determine the opt
imum po ition in which mo t technique can be performed. Becau e of the deep nat
ure of the interco tal mu cle , direct oft ti ue work i le effective here t
han in ome region , and tretching
Technique for the thoracic cage and rib
133
18.1 Articulation of mid rib upine Hold the patient arm in exten ion, and u
e internal rotation to put the houlder cap ule on ten ion. Apply the applicator
thumb and thenar eminence to the co tal inter pace . Rock from one foot to the
other and apply a careful pre ure to the lower of a pair of rib o it i po i
ble to increa e their pacing and mobility. Thi hold can be u ed from twelfth t
o third rib. Tip : Mo t u eful in kyphotic patient where the rib will be in cl
o e appo ition. Lea t u eful in large-brea ted women where it may be impo ible
to get to the rib without intruding on the brea t . Extra con ideration : It ma
y be u eful to u e different pha e of re piration.
18.2 Articulation of upper rib Stand at the head of the table with the patient
arm extended and internally rotated. Apply a force through the houlder and ri
b by rotating your body. Keep the patient arm held firmly into your ide o t
hat the rotation movement cau e rib articulation force to develop. Tip : Mo t
u eful where the fir t three or four rib are involved in a dy function yndrome

. Lea t u eful where there i any houlder problem making the arm movement diffi
cult in thi plane. Extra con ideration : With ten ion maintained try u ing a ha
rmonic o cillation in thi po ition.
18.3 Articulation of lower rib Apply a light compre ive force to the thoracic
cage and u e the thumb to carefully hold the rib being worked toward the pel
vi while you rhythmically lean back. The patient here i holding a towel betwee
n her hand a the tretch round the operator to cla p her hand may be too grea
t. Tip : Lea t u eful in patient where houlder movement of thi amplitude may
be a problem. Extra con ideration : Try u ing a harmonic o cillation in thi po
ition.
134
Technique for the thoracic cage and rib
18.4 Articulation of mid rib Apply a light lateral compre ion force to the up
per thorax to limit movement there, then, with the back of the hand re ting on
the table, apply an anterior force to the haft of the rib . At the ame time a
pply a traction by leaning back and, thereby, produce a tretch from the patient
 linked hand . Tip : Mo t u eful in kyphotic patient where rib  preading i
an important element of treatment. Lea t u eful where the houlder cannot adop
t the po ition. Extra con ideration : Try u ing different pha e of re piration.
18.5 Articulation of lower rib The patient link her hand behind her head and
then you apply a re i tive force to her elbow. You can lift the lower rib by tw
i ting your own body with relatively fixed arm . Tip : Mo t u eful where there i
a need to elevate and pread the lower rib trongly. Lea t u eful where the
houlder cannot be u ed in thi range of movement. Extra con ideration : Try u in
g varied pha e of re piration.
18.6 Articulation of mid rib With the patient hand linked behind her neck yo
u apply a lifting and preading movement to the mid rib on the oppo ite ide. T
hi i particularly effective in patient who have a very flexed thoracic pine,
for example from age, o teoporo i or o teochondro i . It will only be effectiv
e a far a the ixth or eventh rib, a the capula intervene . Tip : Lea t u e
ful in large ubject where the reach may be too great for maller operator . Ex
tra con ideration : Try u ing varied pha e of re piration and varied amount of
preliminary idebending of the patient body.
Technique for the thoracic cage and rib
135
18.7 Articulation of mid and upper rib Thi hold u e initial patient po itioni
ng a the oppo ite leg i flexed at the hip and adducted to produce ome rotatio
n of the pelvi toward the operator. The arm i placed under the ide of the pat
ient. Apply the thenar eminence to the angle of the rib. With the other hand, ho
ld down the houlder and pronate the applicator forearm to apply the mobilizing
force. Stabilize the patient folded arm with a traction force applied with yo
ur own che t if required. Tip : Mo t u eful in very tight ubject where the pr
inging force developed in thi way will help mobilize the individual joint effe
ctively. Lea t u eful if the rib head are very tender to pre ure. Extra con id
eration : Try u ing varied pha e of re piration and head rotation to optimize t
he ten ion.
18.8 Articulation of mid to lower rib The applicator here i the border of inde
x finger and econd metacarpal. They apply an exten ion and eparating force, wh
ile the patient houlder i held down with the other hand and the elbow of you
r applicator hand rotate the patient pelvi toward you. Tip : Mo t u eful wh

ere a trong mobilizing force i required. Lea t u eful where rotation in the th
orax may be a problem. Extra con ideration : Try u ing different pha e of re pi
ration and varying the patient initial po ition to help localize the force.
136
Technique for the thoracic cage and rib
18.9 Stretching mid rib itting Hold down on the rib being worked while trai
ghtening your knee and idebending the patient with your other hand. Apply rib
tretching or articulation. Note that the patient head i kept in the midline
above the pelvi o that the maximum tretch can be applied to the rib. If the p
atient body i taken out of the midline there will tend to be more of a compre
ive force on the other ide. Thi will be uncomfortable and tend to di ipate
the force into other ti ue than the area intended. Tip : Lea t u eful in very
flexible ubject where it would be difficult to localize the force. Extra con
ideration : Try u ing different pha e of re piration and try circumducting the
patient body around the fixed applicator hand a an alternative approach.
18.10 Articulation of mid to lower rib itting Thi hold can be u ed where ther
e i a colio i , a the rib can be worked on the convexity. It require a firm
compre ion between the bodie of the operator and the patient, and whil t the
hand are held relatively fixed the o cillation of body movement will perform th
e articulation. Extra con ideration : Try u ing varied pha e of re piration and
different amount of active flexion and exten ion in the patient.
Technique for the thoracic cage and rib
137
18.11 Articulation of upper rib itting Apply the hand to the co to-chondral ju
nction and keep it relatively fixed while you abduct and externally rotate the
houlder to a comfortable limit. Then, hold the houlder and perform the articula
tory force by pre ing forward again t the capula to induce a force localized
to the rib. Sometime a light increa e of traction through the arm a i t the
technique. Extra con ideration : Try u ing varied pha e of re piration. 18.12 (
bottom left) Articulation of co tochondral joint Hold back on the ternum and a
pply a traction, external rotation and abduction force to the houlder. Then lea
n forward again t the patient capula to produce a gapping force. Lower down t
he rib cage, more initial rotation need to be induced in the patient body to
e tabli h localization. Tip : Lea t u eful in ca e where houlder dy function w
ould cau e pain in thi po ition. Extra con ideration : Try u ing different pha
e of re piration. 18.13 Articulation of mid to lower rib Sidebend the patient
over your padded knee and compre her firmly again t your ide. The mid rib wi
ll require more exten ion of the patient body than the lower. Tip : Lea t u ef
ul if the patient i very large and the operator very mall. Extra con ideration
: Try u ing varied pha e of re piration.
138
Technique for the thoracic cage and rib
18.14 Articulation of mid rib idelying U e your upper hand to apply a po terio
r to anterior force while your elbow maintain the patient houlder in exten i
on, abduction and external rotation. Thi tran mit the force to ju t below your
fingertip where the other hand applie a light compre ive force and hold do
wn on the rib toward the pelvi . It i po ible to reach up to about the fourth
or fifth rib except in very mobile ubject , where the capula get in the way.
Extra con ideration : Try u ing varied pha e of re piration.

18.15 Articulation of mid to lower rib idelying Apply a downward force through
both forearm on the lateral border of the capula and the pelvi . U e a mall
component of po tero-anterior force and then, while maintaining the e, eparate
your hand lightly to produce the required direction of force. Extra con iderat
ion : Thi po ition can be u ed for harmonic technique where the focu i made w
ith the hand and then the whole patient body rocked around them. Try u ing va
ried pha e of re piration.
Technique for the thoracic cage and rib
139
18.16 Articulation of mid rib prone Extend and abduct the houlder and then, wh
ile the rib i held toward the pelvi with the fingertip and thumb, further abd
uct the houlder to reach the fixation point o that articulation can be perform
ed. Tip : Lea t u eful in patient where prone lying i a problem and where ther
e i houlder dy function. Extra con ideration : Try u ing varied pha e of re p
iration.
18.17 Thru t fir t rib prone Sidebend the neck toward the rib and rotate it away
. Produce a component of ide hifting with the tabilizing hand. Form an apex by
pre ure of your metacarpophalangeal joint a clo e to the head of the rib a p
o ible. When you have taken up the lack, the direction of force will be approx
imately toward the oppo ite axilla. The hand holding the head i a tabilizer in
thi technique and doe not enter into the thru t. A the technique i mo tly u
ed on rib that have been fixated relatively uperiorly by the pull of the cal
eni, it i normally performed a the patient exhale . Tip : Mo t u eful where a
treatment table headpiece can drop below horizontal to take the whole neck into
ome flexion. Lea t u eful where the patient i over the age of 40, where neck e
xten ion and rotation may be limited. Extra con ideration : Avoid thi technique
where there i a brachial nerve compre ion yndrome on the ame ide, a it ma
y aggravate nerve root pre ure.
140
Technique for the thoracic cage and rib
18.18 Thru t hand po ition fir t, econd and third rib
18.19 Thru t fir t, econd or third rib upine U e the hand hold hown in the ph
otograph. Apply your hand to the head of the rib, keeping it in ome pronation
o that the thenar eminence i applied to the angle of the rib. Apply the other h
and ju t lateral to the co to-chondral junction, and u e your thorax to pu h dow
nward on the folded arm and toward the head, to produce traction and ome comp
re ion. A the patient lift her head from the pillow, barrier en e will accum
ulate on the thenar eminence. It i then nece ary to make a mall increa e of p
re ure toward the table with the upper hand, and a further harp pronation with
the table hand to complete the thru t. Tip : Mo t u eful in very tight ubject
where neck lever are be t avoided. Extra con ideration : If exce ive pre ure
i applied to the rib head too early, the patient will be unable to lift the he
ad. Try u ing varied pha e of re piration. 18.20 Thru t mid rib itting Pull th
e patient body into exten ion and apply a localized force u ing your thumb dir
ected to the angle of a rib. Sidebend the pine over that thumb and then rotate
it away to produce locking of the pinal column. Then apply a hort amplitude th
ru t in an upward and forward direction which will break fixation in the co to-v
ertebral or co to-tran ver e articulation , depending on the amount of prelimina
ry rotation. More rotation direct the force laterally to the co to-tran ver e a
rticulation. Tip : Lea t u eful in very flexible ubject where it i difficult
to produce localization. Thi technique may be impo ible in large patient a t
he reach for the operator may be too great. Extra con ideration : The patient ma

y be more comfortable with a pillow between her arm and the operator houlder
. Try u ing different pha e of re piration.
Technique for the thoracic cage and rib
141
18.21 Thru t tarting po ition for mid rib itting Apply the hypothenar eminenc
e to the angle of the rib and tretch the other arm acro the thorax to compre
the rib lightly from front to back. Thi compre ion of the che t make the
thru t point more acce ible. Then rotate your and the patient body, pu hing t
he rib forward. At the la t moment introduce a idebending toward the rib with e
nough exten ion to focu the force . The exten ion i produced partly by pre ur
e on the rib angle, and partly by a king the patient to extend her head and neck
. Mo t technique do not require a pecific order of component . However, in thi
hold it i far too ea y to over-lock if the balance between the component i
wrong. If the order de cribed i u ed there i a greater probability of balance
between comfort and effectivene .
18.23 Thru t for middle rib itting Thi how e entially the ame technique a
photograph 18.22 but an alternative hand hold acro the thorax ha been taken
and the patient i itting acro the table rather than a tride. Tip : Thi han
d hold may be better a it avoid the brea t area in female patient .
18.22 Thru t for mid rib The operator ha applied the rotation away from the ri
b and the ide bending down to it until the force accumulate a in photograph 1
8.21. The patient then extend her head. Apply a compre ive force between the t
wo hand and perform the thru t with the pi iform vertically on the angle of the
rib. If the thru t force i more anterior than vertical, pain will be induced a
nd the technique will be le effective. Tip : Lea t u eful where the operator i
maller than the patient and where the reach around may be a problem. Extra co
n ideration : Thi i one of the few thru t technique where generally it i bet
ter not to apply very much in the way of oft ti ue procedure fir t. It i oft
en difficult to accumulate u eful ten ion if the tructure have been previou ly
relaxed. Try u ing varied pha e of re piration. (The itting a tride po ition
hown here i not an e ential part of the technique.)
18.24 Thru t for mid rib idelying Thi i a modified lumbar roll po ition with
hand applied directly over the co to-tran ver e articulation on the ide of the
thorax clo e t to the table. U e the other hand to compre the pectoral girdle
directly toward the table and prevent forward rotation of the body. Apply the fo
rce with the whole of the thru ting arm and hand and u e a direct compre ive fo
rce anteriorly and lightly uperiorly to produce gapping of the joint. Tip : Mo
t u eful in tight ubject a their tiffne help to produce a localization m
ore ea ily. Lea t u eful where the operator i mall and it may be difficult to
apply ufficient compre ive force for localization. Extra con ideration : Try d
ifferent pha e of re piration.
18.25 Thru t to mid rib idelying hown on keleton The exact po ition of the
finger of the thru ting hand in the previou illu tration are een more clearly
here. Although thi may eem a rather unu ual way of reaching the mid rib , it
i particularly u eful where exce tor ion would be a problem. The controlled c
ompre ive force allow the target rib to be reached. If a pure rotation force w
ere to be u ed in tead of the compre ion, the whole lumbar pine would be at ri
k of train.
18.26 Thru t to lower rib idelying hown on keleton A modified lumbar roll po
ition ha been applied and the finger tip of the thru ting hand have been dire
cted toward the table. Thi will reduce pinal movement. A force i applied then
toward the operator to gap the rib articulation while the other hand hold bac

k on the pinou proce e . The critical element in thi technique i the contro
l of compre ive force to focu the force accurately. Tip : Mo t u eful where t
or ion of the whole thorax i be t avoided a compre ion ub titute for rotati
on. Lea t u eful in the pre ence of lumbar or acro-iliac dy function a ome t
re inevitably occur at the e region .
TECHNIQUES FOR THE SCAPULOTHORACIC AREA
19
The oft ti ue articulation of the capulothoracic junction i frequently invol
ved in ten ion tate and po tural control of the pectoral girdle. Fibrotic mu c
le band are frequently found under the capula, and the e can limit mobility an
d be a ource of ymptom in them elve . Re tricted mobility of the capula can
put undue train on the houlder joint it elf. In ca e of houlder dy function
thi can make the difference between comfort, rea onable u age, and mechanical p
roblem . From a practical viewpoint the difficultie lie mo tly in finding way
to acce the area underneath the capula. Technique mu t be u ed which allow t
he capula to be partly

eparated from the thoracic wall to allow the operator finger or hand acce
to the mu cle . Very tight fa cial tate will make thi difficult, but the e pa
tient are the ubject mo t likely to benefit from uch approache . There are f
ew pecial precaution when working on thi area except to be aware of the force
being put through the houlder joint. It i nece ary to avoid exce ive tre
on the gleno-humeral joint in the proce of reaching the capulo-thoracic jun
ction. Deep emotional ten ion often manife t here, and relea e of the mu cle c
an produce emotional relea e, o the thinking practitioner hould be prepared fo
r thi po ibility.
144
Technique for the capulo-thoracic area 19.1 Kneading of oft ti ue on uperi
or and medial part of capulo-thoracic junction idelying Fix the capula light
ly down toward the table with the tabilizing hand while the other applie knead
ing to the horizontal fibre of trapeziu , and the rhomboid . A the e mu cle a
re involved in re piration, it may be be t to u e the exhalation pha e of breath
ing to enhance the relaxation re pon e. Extra con ideration : Try holding the mu
cle on ten ion and moving the whole body again t the ten ion in very tight ub
ject rather than moving the mu cle again t the body.
19.2 Kneading and tretching rhomboid and ub capulari idelying Pu h the pati
ent houlder and upper arm toward the table with your thorax and arm to wing t
he capula. Then pu h the finger tip up into the pace formed o that they can
work directly on the mu cle . Kneading can be performed directly or the whole c
apula can be lifted while being held again t your che t to perform tretching. I
f you idebend your body it i po ible to work the upper or lower part of the
area. Extra con ideration : Can be u ed a an inhibition technique with u taine
d pre ure.
19.3 Articulation of capulo-thoracic junction idelying Fix the capula and ho
ulder between your thorax and finger tip and rhythmically roll the patient up
per body into rotation. Thi will have the effect of mobilizing the capula on t
he thorax. Extra con ideration : Try taking the whole capula uperiorly and int
eriorly.
Technique for the capulo-thoracic area
145

19.4 Articulation of capulo-thoracic junction idelying Thi lightly complex h

old will reach the capulo-thoracic junction in a number of way depending on th
e pre ure u ed. Pre again t the lateral border of the capula with your lowe
r arm while moving your body in a circle. Thi will produce a completely differe
nt effect than if you compre your upper arm hand again t the lower arm elbow a
nd then perform rotary movement . With ome experimentation of different directi
on of pre ure of the arm , hand and body it i po ible to vary thi techniqu
e in many ubtle way and, therefore, it effect on the ti ue . Tip : Mo t u ef
ul where tretch rather than kneading i the preferred method. Lea t u eful wher
e there i houlder dy function making thi po ition difficult for the patient.
19.5 Kneading and articulation of capulothoracic junction prone Abduct the pati
ent arm to an appropriate point and rotate it a nece ary with your lateral h
and. You can reach the deep inter capular mu cle and pull the capula into diff
erent degree of rotary movement. Tip : Lea t u eful where prone lying i a prob
lem for any rea on. Extra con ideration : Try changing the patient head po iti
on to vary the ten ion on the capular mu cle .
146
Technique for the capulo-thoracic area 19.6 Kneading capular mu cle prone Pu
ll the houlder toward you and backward to wing the capula and apply the thumb
of the other hand to work on the mu cle that have been taken off ten ion. Tip
: Mo t u eful in very tight ubject and where it i required to work on the po
terior thoracic wall. Lea t u eful where prone lying may be a problem and where
operator thumb trength may be in ufficient for the technique. 19.8 Kneading and
friction of capulo-thoracic junction prone Perform the work with fingertip ap
plied to the oft ti ue . Thi allow a wider pread of pre ure that may be le
uncomfortable than thumb pre ure. Some operator will find thi ea ier to pe
rform if their thumb are too mobile for the other hold .
19.7 Kneading and articulation capula prone Place the patient arm in trong i
nternal rotation and pull the houlder toward you and backward to make the ub
capular mu cle more acce ible. The thumb i performing the kneading work. Tip
: A photograph 19.6.
TECHNIQUES FOR THE CERVICOTHORACIC JUNCTION AREA
20
The cervico-thoracic, or cervico-dor al, region can be a ource of more fru trat
ion in attempting to achieve ucce ful technique re ult than almo t any other
area. There are many rea on for thi . The area i junctional which mean that t
here will be a change in orientation of anatomical curve . There i a mobile are
a of the neck meeting a le mobile area of the thoracic pine and rib . There a
re emotional factor which manife t in the area relating to tre and ten ion.
The u e of the arm put mechanical train on the area, requiring it to be tab
le, and thi very tability mean that the range of movement of the joint i o
mewhat limited. Thi can lead to a greater u ceptibility to mechanical train i
n the e joint if they are taken out of their normal range in an uncontrolled fa
hion. Particular care hould be taken with regard to the po ibility of patholo
gical condition in the area affecting bone trength, uch a econdary depo it
from primary carcinoma in the lung and thyroid. Severe brachial nerve entrapmen
t yndrome require particular caution if the neck i going to be u ed a a leve
r. The pre ence of extreme hyperten ion can manife t a a very hypertonic tate
in the mu cle of the cervico-thoracic area. Inability to
get adequate relaxation in the area hould alert the practitioner to thi po ib
ility. A a precur or to incipient cardiac infarction there can al o be a charac
teri tic feel to thi area, pre umably due to aberrant vi cero- omatic reflex pa

ttern . Thi manife t a an increa ing tate of ten ion with a  heet like nat
ure in that there i no pecific point of ten ion. There i , rather, a generaliz
ed tate that partly re olve with manual treatment only to recur very rapidly.
Thi i difficult to de cribe, but once it ha been felt, will never be forgotte
n. Thi mu cular tate di appear almo t immediately after a myocardial infarcti
on, pre umably due to the change in the reflex pattern . There may be other fac
tor in operation here uch a change in life- tyle, the hock to the y tem, a
nd many other , but it i , neverthele , quite characteri tic. Some of the techn
ique are modified ver ion of cervical hold , ome modified thoracic one , and
ome are pecific to the area it elf. There i no inherent benefit in any one ov
er another, except there may be time when it i nece ary to avoid the neck or
the thorax for ome rea on. It i , therefore, nece ary to have a range of techn
ique available to affect the area by a variety of mean .
148
Technique for the cervico-thoracic junction area
20.1 Kneading idelying The patient i idelying nearer you than the back of the
table. Fix the upper capula with your forearm and hand and apply the kneading
to the trapeziu and rhomboid toward the pine with the thenar eminence. Tip :
More u eful for patient who might find prone lying a problem. Better for tight
-mu cled ubject where the action of pu hing the mu cle toward the pine may b
e ea ier than pulling away u ing the more traditional method .
20.2 Kneading prone The patient i prone with her arm over the ide of the tab
le and her face in a breathing hole in the table if available. Fix the nearer i
de of the pine with the lower hand into a light rotation toward you. U e the t
henar eminence of the kneading hand to pu h again t the mu cle on the oppo ite
ide toward the table and away from the pine. Tip : Try u ing the expiration ph
a e of breathing to give the be t relaxation effect.
Technique for the cervico-thoracic junction area
149
20.3 Kneading prone The patient i prone with her face in a breathing hole in th
e table if available. Her arm are over the ide of the table. Fix the pine wi
th the upper hand to prevent too much rolling of the pine into rotation and app
ly the kneading with the other hand. Take up ome kin lack and u e the thumb o
r thenar eminence to perform the kneading to trapeziu and rhomboid . Tip : Lea
t u eful if the operator ha any problem of thumb in tability.
20.4 Articulation itting Stand behind the eated patient and have her place one
hand behind her neck. Fix with your thumb again t a pinou proce directed to
ward her other axilla o that the thumb form a fulcrum for a idebending moveme
nt. Reach under her flexed arm with your arm and fix again t the ide of her hea
d. Keep the vertex in the midline and introduce the idebending movement with bo
th hand imultaneou ly. Tip : Try circumducting the patient body around the f
ixed vertebra to make a tronger and more variable force. Avoid continuing thi
movement for a long duration a there will be a tendency for ulnar nerve irritat
ion from your arm pre ure under her flexed arm.
150
Technique for the cervico-thoracic junction area 20.5 Articulation idelying Th
e patient cla p her hand behind her neck in the idelying po ition. Fix on the
pinou proce of the target vertebra with the finger of one hand. Clamp the
patient flexed arm between the ide of your thigh, hand and forearm. Introduc
e flexion and exten ion with mall amount of any other movement by making a m

all rotation movement of your body. Tip : Lea t u eful where there i any hould
er condition which may make thi po ition difficult for the patient. Try u ing o
nly one arm flexed and gripping the neck rather than both. 20.7 Articulation id
elying Fix the patient flexed arm between your body and abducted upper arm. G
rip around her hand with your upper hand o that the movement of flexion, exte
n ion and mall amount of rotation and idebending can be introduced. Fix the
pinou proce of the target vertebra with the other hand to act a a counter-fo
rce to the movement induced. Tip : Lea t u eful in nervou patient a thi po i
tion i omewhat clau trophobic. Not very u eful if there i any houlder condit
ion which may make the po ition difficult to attain. Try u ing only one flexed a
rm in tead of two. Mo t u eful where it i de ired to block out cervical movemen
t.
20.6 Articulation idelying Fix the under- urface of the patient flexed upper
arm again t your ide and hold her elbow . U e a firm grip on the pinou proce
of the target vertebra with the other hand. Introduce flexion and exten ion b
y rocking your body into rotation, thereby carrying her arm back and forth. Tip
: Lea t u eful where the patient ha any condition of her houlder making the
flexed po ition difficult. Try u ing one flexed arm only. Mo t u eful where it i
de ired to eliminate neck movement from the equation a her hand will help do
thi .
Technique for the cervico-thoracic junction area
151
20.8 Articulation idelying Flex both of the patient arm to pull the capulae
apart. Support her head and mid neck with your upper hand. U e the fingertip o
f the other hand to pu h again t the pinou proce e of the area to introduce
a idebending articulation with ome po ible rotation. Lift the head u ing the
tip of the finger under the neck to limit the movement to the lower cervical a
nd upper thoracic area. Tip : The lower down the neck you fix with the upper han
d, the more localized the force become , and the more the upper neck i protecte
d from train. Avoid having your upper arm bicep again t the patient eye ; ra
ther, fix again t the forehead. U e ome downward compre ion through the upper
capula to tabilize the body. Lea t u eful where there may be any neck in tabil
ity, a however much protection i introduced, there will be ome train induced
.
20.9 Articulation idelying Flex the patient neck and grip a far down a po
ible with the upper hand to avoid neck train. U e the finger of the other hand
to pull the pinou proce e again t the movement of the upper hand. Mo t move
ment direction are po ible with thi hold. Tip : The fixing hand protect the
neck and allow rea onable localization of the articulation. Avoid crowding the
patient face with your upper arm or che t.
152
Technique for the cervico-thoracic junction area
20.10 Articulation itting The patient it acro the table at one end. Stand a
t the end of the table and reach around her cla ped hand to hold her far hould
er and pull her again t you. U e the thumb of the rear hand to pull the pinou
proce of the target vertebra toward you. Keep the head in the midline and rock
the patient into idebending, making thi vertebra a focu and fulcrum for the
movement. Tip : Firm compre ion toward you will allow induction of movement to
be produced by a rocking back of your body. Try introducing circumduction moveme
nt a well a imple idebending. Lea t u eful if the operator ha an un table
thumb. In thi ca e try ub tituting a thenar hold.

20.11 Articulation itting The patient it acro the table at one end. Stand a
t the end of the table and reach around her cla ped hand to hold her far hould
er and pull her again t you. U e the heel of the rear hand to pu h the pinou p
roce of the target vertebra toward you. Keep the head in the midline and rock
the patient into idebending making the vertebra a focu and fulcrum for the mov
ement. Tip : Firm compre ion toward you will allow the induction of movement by
a rocking back of your body. Try introducing circumduction movement a well a
imple idebending. Lea t u eful if the movement need to be very pecific to o
nly one egment a thi pu hing movement will inevitably involve everal vertebr
ae.
Technique for the cervico-thoracic junction area
153
20.12 Thru t prone Sidebend the neck and head of the prone patient at the ame t
ime a you induce a light rotation to the other ide. Fix a low down on the ne
ck a po ible with the head hand to avoid exce ive neck train. Apply a compre
ion force again t the tran ver e proce of the target vertebra toward the tab
le and axilla with the thru ting hand. Gently rock the head on the chin to find
the optimum barrier accumulation under the thru ting hand. At the point of optim
um barrier, apply a very hort amplitude thru t to the tran ver e proce to gap
the joint on the ame ide. Tip : Lea t u eful for patient who find the prone
po ition a problem. Lea t u eful for mo t patient over 45 year of age, a norm
al degenerative change in the neck will make the lever uncomfortable. If a tabl
e with a dropping head leaf i available, it i po ible to perform thi techniq
ue on a wider range of patient a the lightly flexed po ition will remove ome
of the train. Avoid pulling the head harder when the thru t i performed a th
i i often painful. Try varied balance between rotation and idebending to find
the be t mix of effectivene and comfort. Try moving the thru ting hand latera
lly to the angle of the rib to make thi into a rib gapping technique.
20.13 Thru t prone Stand at the ide of the table with the prone patient head
in rotation away from you. Fix the capula with your lower hand and gently fix t
he head into rotation and a mall amount of idebending. Apply the heel of the t
hru ting hand again t the ide of the pinou proce of the target vertebra tow
ard the patient axilla. Balance the force o that the head hand doe not move
when the thru t i applied with the pinal hand to gap the facet on the far id
e of the patient pine. Tip : The direction of thru t govern which ide will
be gapped. If the force i toward the axilla, the other ide will gap a the fac
et on the near ide are in appo ition. If the force i predominantly into the ta
ble, there will be a tendency for the ame ide to gap. Avoid thi po ition in p
atient over about 45 year of age a the train on the neck may be unacceptable
. Avoid the tendency to introduce exce ive exten ion a thi can be traumatic a
nd, moreover, render the technique le effective. Move the thru ting hand more
laterally onto the angle of the rib to make thi into a rib gapping technique.
154
Technique for the cervico-thoracic junction area
20.14 Thru t prone Stand at the head and lightly to one corner of the table wit
h the patient head turned away from you. Carefully fix the neck a low down a
you can reach with the upper hand. Fix the pinou proce of C7 with the thumb
of thi hand to limit movement to the upper thoracic area. Apply the thenar emi
nence of the thru ting hand to the tran ver e proce of the target vertebra, pu
hing it toward the axilla. Stabilize the rotation with the head hand, and incre
a e the pre ure with the thru ting hand until a uitable barrier accumulate . A
pply the thru t with the lower hand only to gap the facet on the thru ting ide.
Tip : Apply the thru t to the angle of the rib to make thi into a rib gapping

technique. Avoid thi po ition in patient over about 45 year of age a the nec
k exten ion may be uncomfortable. It i po ible to perform thi at a more advan
ced age if a drop leaf table i available.
20.15 Thru t prone ( hown on keleton) The technique hown in photograph 20.14 i
demon trated here on the keleton to how the po ition of the hand more accur
ately.
Technique for the cervico-thoracic junction area
155
20.16 Thru t prone Stand at the ide of the prone patient and apply the hypothen
ar eminence of both hand to oppo ite ide of the pine at adjacent egmental
level . Apply ome pre ure onto the tran ver e proce e to induce ome compre
ion and idebending. Rotate your body to empha ize thi movement and vary the p
re ure with the hand to introduce ome rotation to the movement until a uitab
le barrier i found. The thru t can be applied with one hand, the other hand, or
both. Tip : Try rever ing the hand po ition to find the optimum for each ca e.
A each vertebra ha multiple joint , every change in angle or pre ure will var
y which will be the target joint. A idebending thru t rather than a compre ion
thru t will be more comfortable, and le potentially traumatic in ca e of po
ible rib fragility.
20.17 Thru t prone Fix the capula with the forearm and the head into rotation a
way from you with the lower hand. Introduce a very mall element of exten ion an
d idebending with the head hand. Pu h again t the pinou proce of the target
vertebra with the thumb of the thru ting hand into idebending and light compr
e ion into ide hifting. The thru t i applied into idebending with the thru t
ing hand while maintaining the head po ition with the other hand. Thi will gap
the facet on the far ide. If the force i directed more toward the table with t
he thru ting thumb, it will tend to gap the facet on the near ide. Tip : Lea t u
eful where the operator ha problem with thumb tability. The fixation of the
capula and head give ome protection to the neck providing they are held tead
y during the thru t.
156
Technique for the cervico-thoracic junction area
20.18 Thru t prone Re t the patient head on her chin and gently upport it wit
h one hand avoiding throat pre ure. Cup one pinou proce with the area betwe
en the thenar and hypothenar eminence of the other hand and apply a pre ure to
ward the ternum. Carefully o cillate between the hand until a uitable barrier
accumulate under the thru ting hand. The thru t i a very hort amplitude forc
e while maintaining the head teady. Avoid exce ive neck exten ion that can bec
ome traumatic. Tip : Thi i a technique that i potentially dangerou , and mu t
only be u ed with great care in uitable ubject . 20.19 Thru t itting Stand b
ehind and lightly to one ide of the eated patient. Apply the thumb again t th
e ide of a pinou proce of the target vertebra to induce idebending and rot
ation. Support the patient by pulling her again t your abdomen with the hand, an
d introduce light rotation by pulling back again t the clavicle. Flex your othe
r arm and apply the in ide of the forearm again t the ide of the head and face
to produce a direct ide hifting force. Form a fulcrum of the vertebra and thumb
while performing light circumduction to the body to accumulate the be t thru t
barrier. When the ten ion i optimum, apply the thru t with the thumb toward th
e oppo ite axilla while the neck i fixed by the other hand. Tip : Lea t u eful
for operator who might have in tability in the thumb. Try u ing the thenar or h
ypothenar eminence in tead. Vary the flexion and exten ion of the head, and try
antero-po terior hifting to aid the focu of force . If the thru t i applied a

gain t the pinou proce toward the oppo ite axilla, it will gap the facet on
the oppo ite ide. If the thru t i applied more in a forward direction it will
tend to gap the facet on the ame ide. In thi ca e there will need to be a vec
tor of downward pre ure introduced a well.
20.20 Thru t itting Thi hold i ub tantially imilar to photograph 20.19 exce
pt that the patient ha placed her arm over a uitable pad on the operator kne
e to act a a fulcrum. It i ea ier to induce ide hifting with thi hold, and
ome patient feel more table and ecure. It i , however, more difficult for the
operator to balance the force a he i tanding on one leg.
Technique for the cervico-thoracic junction area
157
20.21, 20.22, 20.23 and 20.24 Thru t itting Thi hold i very pecific for the
C7 to T1 joint . The equence of photograph how the order of application of t
he hold. Have the patient place one hand behind her neck and reach under the oth
er arm with your thru ting hand. When you have reached a far a po ible, flex
your elbow to bring the heel of your hand again t the fingertip of the patient
that are flexed over the lower neck. Maintain ome pre ure there, and reach aro
und her che t to gra p her wri t with your other hand. Apply ome idebending to
ward your thru ting hand, and rotate lightly away from thi hand by pulling on
the wri t. Pull the patient firmly again t your che t to produce a direct antero
-po terior compre ion. Thi will better help focu the other vector of force.
Apply the thru t vertically through the patient finger to gap the facet on th
e ame ide. (Note the a tride po ition i hown for clarity only; it i not an
e ential part of the technique.) Tip : Thi i a complex technique, and everal
thing can cau e it to fail. The thru t mu t be vertical not anterior. The pull
on the wri t mu t be quite firm to maintain the idebending. Some exten ion i
nece ary, but too much will block the technique.
158
Technique for the cervico-thoracic junction area
20.25, 20.26 and 20.27 Thru t idelying Fix again t the ide of the pinou proc
e with the thumb, heel of the hand or thenar eminence according to your prefer
ence. Lift the head into idebending away from the table. Abduct the arm holding
the head o that the patient face i not ob tructed and o it i po ible to
reach down to upport the neck with the ulnar border of the hand. Introduce ome
ide hifting away from the table a well a a light anterior hift of the neck
. Fix again t the upper capula with your che t. Accumulate the barrier with a c
ombination of ide hifting and idebending away from the table, light rotation
toward the table, and compre ion again t the houlder. Apply the thru t with th
e cho en applicator to produce a rotation and idebending of the target vertebra
. Tip : Note that the direction of the thru t can be varied to gap the lower or
upper facet. Force directed toward the table will tend to gap the lower one, whi
le force toward your elf will tend to gap the upper one. The lower down the neck
you grip, the more the neck protection, and the le uncomfortable for the pati
ent. The election of applicator i dependent on operator thumb trength and abi
lity to focu with the other part of the hand.
TECHNIQUES FOR THE CERVICAL AREA
21
The cervical pine po e everal problem for the manipulative practitioner. Pat
ient are often nervou of having their neck handled. Thi i either through nat
ural fear of damage to the area from it apparent fragility, or due to having ha

d a previou bad experience. Some operator are al o afraid of harming the patie
nt with technique in the neck and uncon ciou ly tran mit thi to the patient. T
he patient then find relaxation impo ible, thu perpetuating the viciou circl
e. There are clearly particular rea on for extreme caution in the neck, and it
i wi e to u e a gentle technique a i po ible con i tent with a good re ult.
Particular care hould be taken in ca e of hyperten ion, po tural hypoten ion
and ve tibular origin dizzine . Each of the e condition can be irritated by ex
ce ive force or rapid movement. If a particular po ition irritate a brachial
yndrome, it hould al o be topped immediately. Nowhere i it more important to
make technique low, deliberate, and non-threatening than in the neck. Di comfort and pai
n will often occur if not enough care i taken to keep the head ub tantially in
the midline. Mo t of the thru t technique require the vertex to remain almo t
central and the neck to be pu hed or buckled into idebending and ide hifting t
o achieve locking and localization. It may apparently be ea ier to produce an ef
fective barrier if the head i taken out of the midline, but pain, di tre and
over tretch are the common unde irable re ult . Some patient feel threatened if
they cannot ee the practitioner, and in the e ca e technique hould be perfo
rmed from the ide or front of the patient. Some patient inten ely di like chin
hold technique , and in the e ca e cradle hold method are preferable. The u e
of rhythm i e ential to produce a dynamic tate of relaxation, and it i al o
ea ier to accelerate in thru t technique from a lightly moving pathway.
160
Technique for the cervical area
21.1 Kneading of oft ti ue upine Stand at the ide of the patient and tabil
ize the head with the upper hand. Pull up on the po terior and lateral ti ue w
ith the pad and fingertip of the active hand. Depending which finger are u ed
, the lower, mid or upper part of the neck mu cle are kneaded. Tip : Try addin
g a idebending movement with the head hand to empha ize the effect. Try not to
lide over the kin exce ively a thi can be uncomfortable.
21.2 Kneading of oft ti ue upine Thi operator viewpoint photograph how th
e hold illu trated in photograph 21.1. Note that even though the head ha been
idebent, the vertex remain in the midline. Thi avoid exce ive facet compre
ion on one ide, and over tretch on the other.
21.3 Kneading of oft ti ue upine Thi photograph i of a imilar procedure t
o that hown in photograph 21.1. However, it can be een that by changing the ha
nd hold lightly, you can pull into a combined exten ion and rotation at the am
e time a the kneading. Thi will have the effect of making the technique much
tronger. Tip : Try extending the neck with the kneading hand or fixing with thi
hand and extending with the other. Try u ing both hand together.
Technique for the cervical area
161
21.4 Kneading of oft ti ue upine Fix the head into the pillow with one hand
applied to the forehead. Apply the fingertip of the kneading hand parallel to t
he pinou proce e . Pu h the finger upward into the neck and pull laterally
while maintaining thi upward pu h. The effect of thi i to tretch the mu cle
ti ue away from the pine while the neck remain tatic. Tip : U e the back of
the hand on the pillow a a fulcrum. Extra con ideration : Try rolling the head
lightly to enhance the effect of the kneading force.
21.5 Kneading of oft ti ue upine Fix the head into the pillow with the tabi

lizing hand. U e the finger and thumb of the kneading hand to work on the po te
rior ti ue of the neck on both ide at once. Tip : With both finger and thum
b applied underneath it i ea ier to combine an articulation force at the ame t
ime rather than when ju t u ing the finger . Extra con ideration : Try adding a
traction force.
21.6 Kneading of oft ti ue prone Stand to the ide of the table and tabilize
the head with the upper hand. Apply the kneading hand to the ti ue on the oth
er ide of the neck and pull toward your elf. Tip : Thi i not very comfortable
for the patient unle a table with a breathing hole i u ed a exce ive exten
ion can re ult if in ufficient care i taken.
162
Technique for the cervical area
21.7 Kneading of lower oft ti ue prone Stand at the ide of the table and
bilize the head with the upper hand. U e the heel of the kneading hand to pu h
he trapeziu and lower po terior cervical mu cle toward the table. Tip : Thi
not very comfortable for the patient unle a table with a breathing hole i
ed a exce ive exten ion can re ult.

ta
t
i
u

21.9 Kneading of oft ti ue idelying Stand in front of the patient and tabil
ize the head with the upper hand. U e the heel of the kneading hand to work on t
he lateral and po terior ti ue uppermo t to pu h them toward the pine. Tip :
Mo t u eful in ubject with very tight fa cial tate a working toward the pi
ne i often ea ier than working away. Try reaching round to the lower ide and p
ulling up with the fingertip to work on the ti ue nearer the table.
21.8 Kneading of lateral oft ti ue idelying Stand at the head of the table a
nd tabilize the patient head with the front hand. U e the thumb and thenar em
inence of the kneading hand to work on the ti ue on the ide of the neck to pr
oduce the kneading force. Tip : Thi hold allow the anterior ti ue to be work
ed a well a the lateral one . Try varying the rotation of the head with the t
abilizing hand to direct the effect of the technique to different layer and par
t of the mu cle .
21.10 Kneading of oft ti ue idelying Stand at the corner of the table light
ly behind the patient and tabilize the head with the upper hand. U e the thumb
and thenar eminence of the lower hand to work on the oft ti ue of the neck. T
ip : Try u ing a rotation force at the ame time with the head hand to empha ize
the effect of the force applied with the kneading hand.
Technique for the cervical area
163
21.11 Kneading of oft ti ue itting The patient it with her leg over the
ide of the table. Stand in front of her and tabilize her head on your che t; u
e a uitable pad if nece ary. Reach round her neck and u e the fingertip to pu
ll the oft ti ue away from the pine on both ide imultaneou ly. Tip : Try
adding a idebending movement at the ame time to aid the mu cle tretch effect.
21.12 Kneading of oft ti ue itting The patient it with her leg over the
ide of the table and her head i tabilized again t your che t, uitably padded
if nece ary. Control her head with one hand and u e the fingertip of the other
to knead the oft ti ue on the lateral and po terior part of the neck. Tip :
Thi hold allow movement to be combined with the kneading a the head hand ca
n control the direction more ea ily than if two hand are performing the kneadi
ng.

21.13 Kneading of anterior ti ue upine Stand at the head of the table and fix
the head into the pillow with one hand. Reach carefully round the anterior part
of the neck to the caleni mu cle group and pu h toward the table with the thum
b and thenar eminence. When the barrier ha been engaged, keep the thumb till a
nd roll the head away with the other hand to bow the mu cle over the thumb. Tip
: If kneading i applied directly to the caleni, it i extremely uncomfortable
. Working on the mu cle u ing thi method allow quite trong tretch with far
fewer problem .
164
Technique for the cervical area
21.14 Kneading of anterior ti ue upine Stand to the ide of the table and ta
bilize the head on the pillow with the upper hand. U e the heel of the lower han
d to pu h carefully into the caleni until re i tance i felt. Hold the mu cle
on thi light ten ion and roll the head away to bow the mu cle over the thenar
eminence. Tip : A the caleni are o en itive to pre ure, thi allow quite
trong tretch and kneading effect by u ing a lever rather than pre ure.
21.15 Kneading of anterior oft ti ue upine Stand at the ide of the patient
and tabilize the head with the upper hand. Reach round the throat with the lowe
r hand and pull the ti ue toward the midline. When a mall amount of ten ion h
a been achieved, pu h the head away to focu the force in the throat and anter
ior ti ue . Tip : A direct tretch and kneading on the e ti ue can be very un
comfortable o thi technique allow them to be worked with far le di comfort.
21.16 Stretching of lateral ti ue upine Stand to the ide of the patient and
cup the houlder in the lower hand. Hook the upper hand around the occiput with
the thenar eminence re ting again t the mandible. Stabilize the houlder and rot
ate the head away from the houlder until ten ion i felt to accumulate in the l
ateral ti ue . Tip : Try varied degree of rotation to reach different depth o
f ti ue . Try varied degree of idebending, either before or after the rotatio
n to alter the ti ue to be affected. Try reaching down the neck with the head h
and to fix around the tran ver e proce of a mid or even lower cervical egment
. Thi will focu the force below the finger to empha ize the tretch in the lo
wer neck in tead of the whole neck. Try varied direction of pre ure on the ho
ulder, toward the table or toward the axilla for example. Try changing the order
of application with each hand. Every minor change in order, amplitude or direct
ion will vary the technique and only by experimenting in thi way can you find t
he optimum for each ca e.
Technique for the cervical area
165
166
Technique for the cervical area
21.17 ( ee previou page, top left) Stretching of lateral ti ue upine Stand a
t the head of the table and cup the occiput and upper neck in one hand. Fix on t
he di tal part of the houlder with the other hand. Apply a idebending to the n
eck while rotating it away from the houlder until a tretch i felt to accumula
te in the ti ue . Tip : Try varied degree of ide hift with the head hand to f
ocu the force at the area de ired. See tip for photograph 21.16, a the ame
parameter apply. 21.18 ( ee previou page, top right) Stretching of lateral po
terior ti ue upine Stand lightly to the ide of the head of the table and u
pport the head in the palm of the upper hand. Fix the tip of the houlder with t

he other hand. Apply the tretch with the head hand pulling into a combination o
f flexion, idebending and rotation until the ten ion i felt to accumulate in t
he area de ired. Note that the head hand ha pu hed up into the neck in thi vie
w to focu the force more pecifically to the lower neck. Tip : See tip for pho
tograph 21.16 a the ame variation apply in thi hold. 21.19 ( ee previou pag
e, bottom left) Stretching of po terior ti ue upine (hand hold) Cro your fo
rearm with the palm facing the floor. When they are placed under the head and
the hand are again t the houlder it will be po ible to lever downward with
the hand , and upward with the forearm to pu h the head into flexion. Thi will
, therefore, tretch the po terior ti ue . 21.20 ( ee previou page, bottom rig
ht) Stretching of po terior ti ue upine The hand hold hown in photograph 21.
19 i applied. Induce the flexion movement by hrugging your houlder , or by l
ightly traightening your arm . Tip : Try idebending your body to produce a rot
ation of the patient head which will tend to focu the force on one ide more
than the other. Try changing which arm i on top to ee which reache the target
tructure and ti ue be t in each ca e.
21.21 Functional technique or train and counter- train hold upine The patient
lie with the head over the end of the table. Support the head in your palm wit
h the fingertip of both hand contacting the articular pillar of the relevant
egment. With thi hold it i po ible to introduce all po ible movement parame
ter of the neck to find the optimum pathway of ea e for the functional techniqu
e. See earlier ection relating to functional technique .
Technique for the cervical area
167
21.22 Articulation into ide hifting, idelying Stand in front of the patient an
d tabilize the head into the pillow with the upper hand. Reach around the artic
ular pillar with the lower hand and pull directly up toward the ceiling to indu
ce a ide hifting movement. Focu the force by empha izing mo t of the lifting
force with one finger while maintaining ome pre ure with the other finger to
reduce di comfort. Tip : Thi hold allow an accurate te t of individual egment
al mobility a well a being a u eful treatment technique. Re tricted mobility w
ill manife t a a di tinct lack of ide hift capability.
21.23 Articulation into idebending upine The patient head re t on the pillo
w. Pu h with the pad of the finger of one hand over the tran ver e proce e w
hile pu hing the head with the other hand in the ame direction. Note that it i
important to keep the vertex ub tantially in the central plane o that you do
not induce exce ive train on the other ide. Tip : Lea t u eful where the tran
ver e proce e are very tender for any rea on. In thi ca e, u e the pad of t
he finger and pri e the head over them. Thi will be much le uncomfortable th
an pu hing into the neck with the fingertip . Extra con ideration : Paradoxicall
y the operator perform rotation of hi body to perform idebending of the patie
nt neck.
21.24 Articulation into exten ion upine Leave the head re ting on the pillow an
d pri e up toward the ceiling behind the articular pillar to induce exten ion.
The back of the hand remain on the pillow to act a a fulcrum for the movement
. Tip : Lea t u eful in ca e where exten ion movement may be a problem, uch a
po tural hypoten ion or brachial neuriti yndrome .
168
Technique for the cervical area 21.25 Articulation into flexion upine Lift the
head with the palm of the hand and re t it on your upper abdomen or lower che
t. Pull up again t the de ired level with the pad of the finger to induce fle
xion. U e a mall traightening of the knee to aid the movement which will al o

help avoid exce ive fatigue in the hand . Tip : Note that the elbow remain cl
o e to the ide throughout o that the movement i performed by the operator
body, not the arm .
21.26 Articulation into localized exten ion upine Leave the head on the pillow
and u e the forearm a a fulcrum. Pri e the target level up with the pad of the
finger into localized exten ion. Tip : Avoid pre ing too far laterally a the
tip of the tran ver e proce e are u ually very tender. Extra con ideration :
Try adding idebending to the exten ion movement to localize it even more peci
fically.
21.27 Articulation into rotation upine Leave the head on the pillow and tand
lightly to the ide of the table. Rotate the head to the ame ide. Keep the ver
tex in the midline o that a pure rotation can be made. Support the head with on
e hand and allow it to lide under the head a the other hand pull behind the t
ran ver e proce e into rotation. Note that the index finger i applied at a ta
rget level to focu the force principally to one point. Tip : Try tanding in va
ried po ition to find the be t per onal method, at the end of the table, to one
ide or the other. Extra con ideration : Paradoxically the operator perform i
debending of hi body to produce rotation in the patient neck.
Technique for the cervical area
169
21.28 Articulation into rotation upine Keep the head on the pillow and turn it
to one ide, allowing the underneath hand to lide under the head. Apply the oth
er hand to the temporal and maxilla area of the upper ide and pull into rotatio
n. Tip : It i ea y to introduce different element of idebending along with ro
tation in thi hold. Try rotating and then adding idebending, or idebending an
d later adding the rotation. The um of the movement i imilar, but the effect
i completely different according to which ti ue i put on ten ion fir t.
21.29 Articulation into idebending upine For thi alternative hold it at the
head of the table and u e the fingertip of one hand to act a a fulcrum for the
idebending. Pu h with the other hand again t the parietal area on the other i
de to induce the idebending. Tip : The itting po ture form a u eful variation
for the operator who might find tanding tiring, inconvenient or impo ible.
21.30 Springing into exten ion prone The patient lie prone, preferably with the
face in a breathing hole in the table. Apply your pad of thumb directly over
the po terior a pect of the tran ver e proce e of the target egment. Apply a
direct po terior to anterior pringing movement to produce localized exten ion.
Tip : Lea t u eful where the patient may have a problem with prone lying. Lea t
u eful in ca e where exten ion i be t avoided due to any circulatory in uffici
ency cau ing potential ob truction of vertebral arterie .
170
Technique for the cervical area
21.31 Traction upine Thi operator viewpoint photograph how the hand applied
to perform traction. The underneath hand ha carefully gripped round the occipu
t with the head re ting on the palm. The other hand i gripping around the chin
and the heel of the hand i applied to the mandible. The wri t and forearm of th
e chin hand pu h lightly toward the oppo ite ide to induce a light ide hift.
The occiput hand i introducing a variable quantity of ide hifting depending o
n the target egment. More ide hifting will drive the force lower in the neck.
The wri t of the chin hand balance the pre ure of the other hand. Once the id
e hift i applied, the traction force can be brought into play. Remarkably littl

e range of traction i available if the ide hift i maintained, which make it

very pecific to a mall number of egment .
21.32 Traction unilaterally upine Sit at the head of the table and fix the head
to the pillow with the wri t, forearm and lateral border of the hand. Slip the
other hand under the head and hook the finger under the occiput or around the
pinou proce of a vertebra. Maintain the pre ure with both hand and pull gen
tly in the long axi of the pine. Tip : Note that ome rotation and light ide
bending ha been introduced to focu the movement to a pecific area on the lowe
r ide.
Technique for the cervical area
171
172
Technique for the cervical area
21.33 ( ee previou page, top left) Traction and idebending articulation combin
ed Stand at the head of the table and fix the head again t your upper abdomen. A
pply a light lifting force with the index finger to produce a mall anterior
hifting movement. Keep the arm fairly rigid and pivot your body into idebendin
g from ide to ide while leaning back lightly. The um effect i to produce a
idebending and traction force that will vary in level according to how far down
the neck you fix with the hand . Tip : Thi can be rather hard work, but i cap
able of producing rapid change in mobility, making it an extremely u eful techn
ique. Mo t u eful in ca e of brachial neuriti where the idebending force will
be introduced primarily in one direction to open the foramen on the convexity.
21.34 ( ee previou page, bottom left) Thru t u ing cradle hold upine (hand po
ition) Stand lightly to the corner of the table on the ide that the thru t i
to be applied. Supinate the lower hand and pronate the upper one o that they fo
rm a mirror image of each other. Note that the finger are lightly played to
pread the force over a larger area. Note al o that the elbow remain fairly clo
e to the operator ide . 21.35 ( ee previou page, top right) Thru t into rota
tion, cradle hold upine U e the hand po ition hown in photograph 21.34 and tur
n the head gently into the primary lever direction of rotation. There i unlikel
y to be a en e of barrier with thi mall amount of rotation, o add other comp
onent either individually or in combination to accumulate the mo t effective po
tential barrier. You need to con tantly te t the primary lever direction while a
dding the other component until the optimum barrier i en ed. Firm contact poi
nt pre ure i important a it help to further reduce the other component . Mai
ntain all the econdary lever of idebending, ide hifting, compre ion, anteri
or hifting and light exten ion and apply the thru t into a mall amplitude of
rotation. Tip : Note that the elbow are clo e to the ide , and the technique w
ill be more efficient if you tand a upright a po ible o that the arm move
with the body. Thi technique i de igned to lide the facet on the ame ide a
the thru t i applied. 21.36 ( ee previou page, bottom right) Thru t into rota
tion, cradle hold upine Thi operator viewpoint photograph how the underneath
hand acting in compre ion again t the thru ting hand. The vertex i maintained
in the midline. Note that the wri t of the thru ting hand i in a neutral po it
ion. At the end of the thru t the hand accentuate their po ition of upination
and pronation re pectively. Thi hould lide the facet on the ame ide a the
thru t.
21.37 Thru t u ing cradle hold into idebending, upine Stand at the head of the
table lightly to the corner behind the head. Take up the hold with the underne
ath hand upinated to upport the head. Apply the thru ting hand with the arm cl
o e to your body and the lateral a pect of the middle interphalangeal joint of t
he index finger lightly behind and to the ide of the tran ver e proce of the

target egment. Keep gently te ting the primary lever of idebending while addi
ng all the other available component until a en e of barrier accumulate . When
the optimum barrier ha been found, maintain the econdary component and apply
a low amplitude thru t with the index finger directed toward the oppo ite houl
der. The econdary component mo t often u eful in thi technique are contra-rot
ation, ide hifting, compre ion of the head between the hand , light exten ion
and localized pre ure over the contact point with the finger. It i important
that the underneath hand doe not allow the head to move away from the barrier w
hen the thru t i performed, a otherwi e exce ive tor ion can re ult. Tip : Mo
t u eful in ca e of brachial neuriti on the ide to which the neck i being r
otated a the foramina on that ide will be opened by thi thru t. Thi thru t i
de igned to gap the facet on the oppo ite ide to the thru ting hand.
Technique for the cervical area
173
21.38 Thru t u ing cradle hold into idebending, upine Thi operator viewpoint
photograph how the hold de cribed in photograph 21.37. Note the buckling of th
e neck over the operator thru ting hand and that the vertex i almo t in the m
idline. The hand are applying a compre ion toward each other to minimize the q
uantitie of the other econdary lever . Thi hould gap the facet on the oppo i
te ide to the thru t hand.
21.39 Thru t u ing cradle hold into idebending Thi illu tration i of the ide
bending thru t applied to a lower cervical egment. The hold i taken up in the
u ual way, but in tead of applying more lever , the head i being compre ed bet
ween the hand o that the effect of the idebending force i amplified. Note th
at the thru ting forearm i almo t horizontal o that the neck i not being forc
ed into a painful direction, and that the thumb i applied to the mandible to he
lp block out upper cervical movement. Thi i de igned to gap the facet on the o
ppo ite ide to the thru ting hand.
174
Technique for the cervical area
21.40 Thru t into rotation u ing chin hold Stand at the head of the table, ligh
tly at the corner behind the head. Gently rotate the head to one ide and lide
the chin hand under the ide of the head to take up the hold. Take a mall tep
to the back corner of the table and lide the thru ting hand into place with the
metacarpo-phalangeal joint of the index finger applied behind the articular pro
ce . Keep te ting the primary lever direction of rotation while adding the cont
ra- idebending and other component until a uitable barrier ha been accumulate
d. Thru t with the hand behind the head while tabilizing the head and chin with
the other hand. Note that the thru ting hand wri t hould be in line with the f
orearm, and both elbow clo e to the ide . Note al o that the forearm of the th
ru ting arm i almo t horizontal. The mo t u eful econdary component to focu
thi technique are compre ion between the hand , light ide hifting in the opp
o ite direction to the idebending, and a light anterior hift of the whole a
embly. Thi i de igned to lide the facet on the ame ide a the thru ting han
d.
21.41 Thru t u ing chin hold into rotation, upine Thi operator viewpoint photo
graph how the hold illu trated in photograph 21.40. Note the midline po ition
of the head, and the traight po ition of the wri t of the thru ting arm. Note t
he po ition of the upporting forearm under the head i in front of the patient
ear. Thi technique i de igned to lide forward the facet on the ide being t
hru t.

Technique for the cervical area

175
21.42 Thru t u ing chin hold into idebending Thi photograph how the hold app
lied to a lower cervical egment from a different viewing angle. Take up the hol
d in the way previou ly de cribed. Apply the lateral border of part of the index
finger or metacarpal to the po terior part of the tran ver e proce of the eg
ment. Buckle the neck over the thru ting hand o that the idebending and ide h
ifting occur at the ame time. Introduce a mall amount of controlled rotation w
ith the other hand, and then balance all the econdary component again t the pr
imary lever of idebending. At the optimum moment apply the thru t with one hand
while maintaining the head po ition with the other. Note that the thru ting for
earm i almo t horizontal and that the vertex remain in the midline. Thi i de
igned to gap the facet on the oppo ite ide to the thru t hand.
21.43 Thru t u ing chin hold into rotation of atlanto-axial joint Take up the ch
in hold in the manner previou ly de cribed fixing behind the tran ver e proce
of the atla with the thru ting hand. A the principal movement po ible of thi
joint i rotation, only light idebending will be required. Introduce a very
mall exten ion to take the po terior ti ue off ten ion, and apply the thru t i
n pure rotation through a mall amplitude to gap the facet on the ame ide. Tip
: Mo t region of the body can be treated in any order, but if it i nece ary
to thru t the atlantoaxial joint, it i often found to be be t left to the la t
manipulation of a treatment e ion. Thi i not an ab olute rule, but ha ound
rea oning. Thi joint i very u ceptible to becoming hypermobile and therefore
uffering recurrent le ioning. If it i manipulated fir t, it i liable to be
tre ed when any other thru t are done in the area, thereby irritating it furth
er.
21.44 Thru t u ing chin hold into rotation or idebending Thi photograph i to
how the overall po ture u ually adopted for the e type of technique . Note tha
t although the operator i bent from the wai t, he ha not flexed hi pine. Not
e that the leg on the thru ting ide i behind the other o that the operator
body i brought into the thru t. Note that the forearm of the thru ting hand i
horizontal and that the patient head ha been left on the pillow.
176
Technique for the cervical area
21.45 Thru t u ing pulling hold upine Stand to the ide of the table and grip a
round the tran ver e proce of the target egment with the middle phalange of t
he index or middle finger of the thru ting hand. Balance the force with the oth
er hand applied to the ide of the forehead clo e t to you. Introduce part of th
e primary lever of rotation and lowly o cillate in the primary lever direction
while adding the econdary lever until a ati factory barrier accumulate . Main
tain the econdary lever , and apply the thru t with a hort harp pulling actio
n with the thru ting hand into rotation. Tip : Some operator find that pulling
technique are ea ier for them than pu hing one . Thi technique i mo t u eful
in the e ca e . It i al o u eful where the operator ha a problem in u ing one
particular hand for any rea on. The left ide of the neck i u ually thru t with
the left hand of the operator, but with thi technique the right hand i thru t
ing the left ide of the patient neck.
21.46 Thru t into rotation prone Stand to the ide of the prone patient and tab
ilize the head with the upper hand. U e the thru ting hand a a fulcrum to ideb
end the neck. Maintain the head in the midline and carefully pu h the lateral i
de of the index finger into the neck again t the po terior a pect of the articul
ar proce of the target egment. The primary lever of rotation i going to be a

force directed in the line of the forearm a hown. Maintain the po ition with
the tabilizing hand and apply the thru t behind the articular proce through a
very mall amplitude. Tip : Note that there i a tendency to introduce exten io
n, and that thi i potentially dangerou and mu t be avoided. Although a idebe
nding thru t could be applied with thi hold, it i even more likely to produce
an unwanted hyperexten ion. If a table with a breathing hole i being u ed, eith
er plug it up for thi technique, or have the patient move lightly to one ide
o that the chin can act a a pivot directly on the table. Lea t u eful for pati
ent over 40 year of age.
Technique for the cervical area
177
21.47 Thru t into rotation itting Have the patient it acro the table toward
one end. Stabilize the head with the front hand, and apply the pad of the finge
r behind the articular proce of the target egment with the other. Introduce
idebending toward the thru ting hand, and rotation away. Keep te ting the prima
ry lever of rotation while adding the econdary component of the compo ite leve
r until a uitable barrier accumulate . Maintain the econdary lever and apply
a hort harp thru t into rotation with the hand applied to the neck. Tip : Thi
i a rather difficult technique to control a the neck tend to try to e cape f
rom the ten ion induced unle it i very carefully managed. Many operator may
find it difficult to generate the harpne needed to break facet fixation with
thi hold. It i , neverthele , u eful a ome patient will not relax well in a
upine po ition, and may find thi hold more comfortable.
178
Technique for the cervical area
21.48 ( ee previou page, top right) Thru t into rotation itting (hand hold) The
hand reaching in front of the patient i upinated, and either the index or the
middle finger i flexed to make it more prominent and ready to apply behind the
articular proce . The other hand i pread lightly and i applied to the near
ide of the head to act a a tabilizer for the thru ting hand. Note that the op
erator elbow are clo e to hi ide . 21.50 Thru t into rotation itting (hand
hold) The patient it acro the table near the end. Stand at the end of the t
able and reach around the neck with the front hand o that the lateral border of
the hand can pull forward on the far ide articular proce . Place the thumb of
the pronated po terior hand on the far ide of the pinou proce of the next
lower vertebra. Maintain a mall amount of idebending away from you by firm pre
ure again t the ide of the pinou proce . The thru t will be applied with b
oth hand . One i pulling forward from behind the articular proce , and the oth
er pu hing the pinou proce toward you. Thi will produce a contra-rotation f
orce between the adjacent vertebrae. 21.49 ( ee previou page, bottom right) Thr
u t into rotation itting The hold de cribed in photograph 21.48 i applied. Rot
ate the head u ing both hand until it i approximately in mid rotation. Apply
ome idebending to the oppo ite ide. Maintain the rotation and oppo ite ideben
ding and circumduct the patient body through a mall arc keeping the vertex di
rectly over the acrum. When ten ion accumulate in the target egment, apply th
e thru t with a hort harp pulling action with the front hand while aiding it w
ith the other. Note that firm compre ion between the hand i a critical part o
f thi technique. Tip : It ometime help to a k the patient to exhale ju t bef
ore the thru t to produce an optimum tate of relaxation. Another u eful relaxat
ion aid i to a k the patient to let the houlder drop; a they do thi , there
will be an interval of 2 or 3 econd when the thru t i more ea ily performed.
21.51 (bottom right) Thru t into rotation itting Take up the hold with the grip
de cribed in photograph 21.50. Produce a buckling into idebending at the targe
t egment, and pull back lightly on the clavicle with the po terior hand while

pri ing the pinou proce toward your elf. The econdary component of ide hi
fting, antero-po terior hifting and compre ion are balanced. At the optimum ba
rrier, the thru t i applied by a tabilizing force with the po terior hand and
a rotation force with the anterior hand.
TECHNIQUES FOR THE OCCIPITAL AREA
22
The occipital and upper cervical region are area where extreme caution i nece
ary in preliminary a e ment before technique are applied. Care i required
in all area , but in the occipital region thi i even more critical. There i t
he po ibility of damage to the vertebro-ba ilar y tem if in tability i pre en
t. Precaution need be undertaken before performing any thru t technique that i
in any way liable to put the area under mechanical tre . A in the re t of th
e neck, the patient i often fearful of manipulation, and a light a force a wi
ll achieve the required re ult i e ential. It i often difficult for tudent
to achieve the required acceleration and ub equent braking force nece ary in t
hru t technique in thi area, and there i no ub titute for practice and practi
cal experience. The reader i referred pecifically to the ection on contra-ind
ication . However, uffice it to ay that anything that can compromi e the integ
rity of the ligamentou tructure of the area require extreme caution in the c
hoice of technique. Some will ay that all thru t technique hould be avoided in
the area if there i o much potential danger. However, thi would be to deny t
he po ibility of aid in many ca e where almo t nothing el e will work a well.
Occipital thru t technique performed without exce ive force require a well-de
veloped barrier en e. The angle through which any given technique will ucceed
are very mall. Slightly too much leverage will block the technique and more f
orce i then often
mi takenly applied to counteract the lack of ability to focu accurately. Maximu
m afety i paramount and too much force jeopardize thi . If a given technique
i not working, try reducing lever and force rather than increa ing them. It m
ay eem paradoxical, but thi often work much better. It i often nece ary to
make many ubtle change in direction or plane to find the optimum for each tech
nique. Every patient will differ, and change in table height, operator po ition
in relation to the patient, and quantitie of the varied component of a compo
ite lever vary. Many ca e will re pond well to general mobilizing and articulat
ion. Some, however, will only achieve full and la ting relief from treatment whe
n a pecific thru t i ucce fully performed to fully liberate a facet fixation
. Although pecific po ition of le ion fixation are often de cribed, the techni
que hown here are imply de igned to break fixation. If po itional correction
i deemed nece ary, the fixation i dealt with fir t, and then the joint i coa
xed into the direction required afterward with articulatory technique. If a bar
rier doe not eem to accumulate in one direction, another i ought. Thi remov
e ome of the fear of many tudent that they may be performing the technique i
n the wrong direction. If thi doe not ati fy the puri t in relation to le i
on direction , then everal of the technique hown can be applied in a correct
ion direction if required. I have included the detail of the e under the Extr
a con ideration  heading .
180
Technique for
the occipital area
22.1 A r t i c u l a t i o n into flexion upine Pu hthe head into flexion with
your thumb on the mandible while you pull under the occiput with your fingertip
. Tip : Try adding traction to the movement. Try adding idebending to the move

ment. En ure the patient ha time to breathe between repetition a the flexed p
o ition may ob truct the ability to breathe.
22.2 Articulation upine (hand hold) Form a pivot with the pad of thumb and inde
x or middle finger. Re t the back of the hand on the pillow. Apply the other han
d to the forehead of the patient to be able to tip the head into all the directi
on that the technique allow .
Technique for the occipital area 22.3 Articulation into exten ion upine Apply
the hand hold hown in photograph 22.2 and tip the head into exten ion with the
forehead hand while pivoting it over the occiput hand. Once the head i in exten
ion it can be rolled into idebending to either ide to focu the force to each
condyle of the occiput. 22.4 (bottom left) Articulation into flexion upine Thi
operator viewpoint photograph how the hold from photograph 22.3 in u e for f
lexion. Note that the pillow i retained, partly to allow it to be u ed a a ful
crum, and partly a mo t patient eem to prefer the ecurity and familiarity of
a pillow. Tip : Allow the patient time to breathe between pre ure a the trac
hea can be ob tructed in extreme pa ive flexion! 22.5 (bottom right) Articulati
on into exten ion upine In thi operator viewpoint photograph the head i pu he
d up with the lower hand while tipping it into exten ion with the frontal hand.
Note that thi i an exten ion focu ed to the occiput rather than an exten ion o
f the whole neck. Tip : Try alternating thi movement with flexion and idebendi
ng to focu force to one pecific condyle of the occiput. Try varying the paci
ng of the lower hand to find the optimum for efficiency and comfort.
181
182
Technique for
the occipital area
22.7 Traction to o c c i p u t upine Apply the hand hold hown in photograph 22
.6. Lower your elbow over the end of the table to form a fulcrum, and fix your
hooked finger under the occiput. A you lower your elbow , the traction force a
ccumulate automatically. Although thi would not eem to be very powerful, it d
oe reach very pecifically to the occiput and can be very u eful if localized t
raction i de ired.
22.6 T r a c t i o n to o c c i p u t (hand hold) The operator ha hi finger c
lo e together and i hooking them o that they can pull cephalically under the o
cciput into localized traction. The back of the forearm will be applied to the
pillow with the elbow over the end of the table. Thi will allow him to produc
e traction partly by pulling, and partly by levering hi forearm again t the en
d of the table.
Technique for
the occipital area
183
22.8 Thru t to occipito-atlantal j o i n t itting (hand hold) Thi hold i u ed
for a pecific thru t technique to be applied to the occipito-atlantal joint on
the far ide from the operator. The upper hand will be applied around the front
al bone, temporal bone and maxilla of the patient. The lower hand i to be appli
ed with the index or middle finger placed gently in front of the tran ver e proc
e of the atla . The palm will be cupping the occipital bone. 22.9 (top right)
Thru t to o c c i p i t o - a t l a n t a l joint itting The hold illu trated i

n photograph 22.8 ha been applied. Stand to the ide and lightly behind the e
ated patient. Fix the frontal bone with your bicep avoiding the eye . The forea
rm of the upper arm lie around the temporal bone and the in ide of the elbow i
in contact with the maxilla. The technique will not work effectively if the han
d of the upper arm i in contact with the patient. The grip i only performed wi
th the medial a pect of the forearm. Your lower hand grip gently around the ant
erior a pect of the tran ver e proce of the atla with the pad of the index or
middle finger. Pull carefully backward on the atla while performing a bowing
movement with your
body. A the head i in contact with your che t thi bowing action will form a
idebending action of the neck o that the atla i driven lightly toward you. Y
ou mu t imultaneou ly upinate your upper forearm. The patient body i now in
idebending away from you, and you can rock into circumduction from your ankle
. A the barrier accumulate , increa e the compre ion of the head toward you, a
nd momentarily grip the atla firmly a you adduct both arm . The cumulative eff
ect of thi i to produce a rotation of the head toward you, and of the atla aw
ay. Tip : Mo t u eful in ca e where it i be t to avoid exce rotation of the
head on the neck. Thi technique work with the head in any degree of rotation
o it i hardly nece ary to tor ion the neck at all. It i probably the afe t o
f the thru t technique that can be applied to thi joint. Lea t u eful if the a
tla i extremely en itive to touch a the pre ure may be unacceptable. Extra
c o n i d e r a t i o n : En ure that firm compre ion of the head into your
body i maintained throughout. Only pull back on the tran ver e proce of the
atla for a hort a time a po ible to avoid exce di comfort.
184
Technique for
the occipital area
22.10 Thru t to o c c i p i t o - a t l a n t a l j o i n t itting (rear view)
The technique de cribed in photograph 22.9 i et up. Note that the patient i
idebent toward the operator at the pelvi , and away at the neck. Note the upina
ted upper arm that i maintaining the compre ion. Note that the upper arm hand
i not in contact with the patient. Note that the patient head i in a po itio
n of almo t no rotation but a contra-rotation force i , neverthele , being appl
ied to the occipito-atlantal joint.
Technique for the occipital area 22.11 Thru t u ing minimal leverage to gap o c
c i p i t o - a t l a n t a l j o i n t upine Thi technique i de igned to ga
p the occipito-atlantal joint on the ide where the thru t i applied. It i a m
inimal leverage technique a the neck i not taken to full rotation, but merely
placed in an available po ition o that the contact point i available for the t
hru t. Some mall quantitie of lever are u ed, but the empha i i mo tly on c
arefully applied compre ion, and high velocity with very hort amplitude. Keep
the vertex of the head in the midline and take up the chin hold with one hand. K
eep the head re ting on the forearm applied anterior to the ear. En ure that the
head i again t your upper arm and thorax o that it i firmly controlled, crad
led and upported. Place the applicator, the fir t metacarpophalangeal joint of
your thru ting hand, on the po terior a pect of the arch of the atla . Keep the
vertex midline and pre firmly into the atla to take up the lack in the oft
ti ue . Apply the thru t with a rapid force toward the patient oppo ite eye a
nd a imultaneou force of the other forearm again t the ide of the head. The t
otal of the e force hould keep the head till while the atla i driven forwar
d underneath the occiput. Thi i not a tor ional force of the head on the neck
, but a force of the atla under the occiput to break fixation on the ide of th
e thru ting hand. If exce ive head movement i allowed, the occipito-atlantal j
oint on the other ide will be trained, with a idebending force. There will al

 o be a tendency to train the atlanto-axial joint into exce rotation. Note: t

hi i an extremely difficult technique to perform well a there i no en e of
accumulating barrier. The control of acceleration and braking and accurate direc
tion of force are the governing factor . If it i performed a de cribed here t
here i little chance of trauma but if tor ion i added in tead of the method de
cribed it i then no longer a minimal lever technique and ti ue tre i more
likely. Tip : Mo t u eful where tor ion i be t avoided and fixation i not too
evere. Lea t u eful if the operator i not able to develop the kill to apply
the nece ary ultra-high velocity, and the trict control of the braking force.
Extra c o n i d e r a t i o n : Try a king the patient to look over the hou
lder of the ide to which the head i being rotated. Thi will increa e the effe
ct of the mall amount of lever applied a the eye tor ion ten e the cervical
mu culature. Conver ely, try a king the patient to turn the eye to the other i
de if the ten ion accumulate too fa t a thi will have the effect of reducing
neck ten ion.
185
186
Technique for the occipital area
22.12 ( ee previou page, bottom right) Thru t atlanto-axial joint cradle hold
upine A the main movement of the atlanto-axial joint i into rotation, thru t t
echnique normally u ed are rotatory type . Thi technique i de igned to break
fixation on the ide of the thru ting hand. Apply the proximal metacarpo-phalang
eal joint of the thru ting hand behind the arch of the atla . Support the head i
n the palm of the underneath hand and apply a compre ion force toward the thru
ting hand to ab orb the lack into compre ion to avoid exce tor ion. Keep the
vertex in the midline and gently rotate the head to about 5 0 % of it availabl
e range. Apply a mall amount of idebending oppo ite to the rotation and a very
mall exten ion of the head on the neck to take the po terior ti ue off ten i
on. Thi will make room for the applicator to reach the atla . Slowly increa e t
he compre ion while gently o cillating the head into rotation until a en e of
barrier accumulate under the thru ting hand. Apply a hort amplitude and high v
elocity force to the atla to break fixation in a forward or rotation direction
of the atla on the axi . Tip : Mo t u eful in older neck a the compre ion fo
rce help to minimize the tor ion nece ary. Lea t u eful if the operator i not
able to develop the peed nece ary to break fixation in thi type of technique
. Extra c o n i d e r a t i o n : Thi hold make everal variation of the
technique available. It i po ible to thru t into idebending to gap the other
ide of the occipito-atlantal joint. It i po ible to thru t on the occiput it
elf to gap the occipitoatlantal joint on the ame ide. It i po ible to thru t
on the po terior a pect of the atla while holding the head till to gap the oc
cipito-atlantal joint of the ame ide.
22.13 Thru t to occipito-atlantal joint upine Thi hold i u eful if a traction
component i found to be nece ary to gap the joint. It i al o u eful if a id
ebending component i nece ary. Pronate the arm of the thru ting hand and apply
it behind the occiput on one ide of the head. Rotate the head to the other id
e and fix the head again t your che t and the pillow with the underneath hand. R
otate the head to about 5 0 % of the neck range and apply a traction force with
the underneath hand. Compre the head between the hand and pu h firmly up into
the occiput with the pi iform of the thru ting hand. Apply very light exten io
n and idebending toward the thru ting hand to take the ti ue off ten ion and
allow the thru ting hand to reach the target ti ue . Perform the thru t a a tr
action with the underneath hand and a rapid adduction with the pi iform. Tip : M
o t u eful in ca e where there i a idebending component in the le ion complex
. Lea t u eful where operator arm trength may not be ufficient or where the pa
tient may have an extremely large head. Extra con ideration : A idebending, rot

ation or traction force can be u ed according to which give the optimum barrier
.
Technique for the occipital area 22.14 Thru t to occipito-atlantal joint upine
Thi technique i u eful for breaking fixation in a idebending or rotation dir
ection. Leave the head on the pillow and take a mall tep to the corner of the
table. Slip your upper hand under the occiput o that the fingertip ju t curl u
nder the ba e of the kull. Pull gently upward under the occiput on the lower
ide a you apply the other hand to the maxilla and mandible on the upper ide. U
e a mall compre ion between the hand to hold the head firmly, but avoid exce
ive pre ure on the mandible a otherwi e the temporo-mandibular joint can be
trained. Tip the head into very light exten ion to take the po terior ti ue
off ten ion, but not enough to reach the end of range of movement in the occipit
o-atlantal joint . Increa e the rotation to about 5 0 % of full range and then b
alance all the component together until ten ion accumulate on the occipital co
ndyle nearer the table. Apply a hort amplitude thru t into idebending with the
maxilla hand while tugging upward with the occiput hand. Note that thi thru t
can al o become a rotation thru t with the maxilla hand around the fixed axi o
f the underneath hand. Thi will have the effect of gapping the upper condyle ra
ther than the lower a in the idebending thru t. Tip : Mo t u eful in ca e of
idebending re triction. Lea t u eful in patient who have a very mobile mid cer
vical pine a the force will too ea ily di ipate there and not accumulate at t
he occipitoatlantal joint. Extra c o n i d e r a t i o n : Try u ing thi po
ition a a firm articulation procedure, but do not carry it on for very long a
the force generated are very trong and will provoke di comfort.
187
22.15 Thru t to o c c i p i t o - a t l a n t a l joint flywheel t e c h n i q
u e (hand hold) Thi technique i a complex one where both hand work in oppo i
tion to hold the axi and rotate the occiput on it. The axi hand i to be place
d with the thumb gently applied in front of the tran ver e proce and the reinf
orced middle finger po terior to the tran ver e proce on the other ide. The c
hin hand will be rotating the head to one ide a the atla i held to the other
. Even though momentum i u ed, the head hould not reach full rotation even at
the end of the technique. It i the effect of the one hand pulling back on the a
tla a the head i rotated which cau e the joint to gap, not full rotation of
the head on the neck.
188
Technique for
the occipital area
22.16 Thru t to occipito-atlantal joint flyw h e e l  technique Stand to one c
orner at the head of the table. Take up the chin hold with one hand while u ing
the other to take a hold on the atla . The atla hand i applied with the thumb
placed ju t in front of the tran ver e proce . The reinforced middle finger i
placed on the other ide ju t behind the tran ver e proce of the atla . A you
drop the elbow of the atla hand toward the table while pulling carefully back
on the tran ver e proce you will rotate the atla lightly back toward the neu
tral po ition. A you rotate the head toward the pillow, you will tend to carry
the atla with the head. Make a few gentle rotation movement with both hand mo
ving in the ame direction of movement of the chin toward the pillow. At the opt
imum moment accelerate the chin hand to rotate the head harply toward the pillo
w. At the ame time grip for a hort a time a po ible on the atla to hold it
from joining the head rotation. Relea e the atla immediately the technique i
completed a the tran ver e proce i u ually very tender to pre ure. Tip : Mo
t u eful in fairly flexible neck where the mobility make normal locking techn

ique difficult. Lea t u eful in tiff neck with degenerative change a the to
r ional force , however carefully applied, can be potentially problematical. Ext
ra con ideration : Try tipping the head into very light exten ion to take the p
o terior ti ue off ten ion and make the atla more acce ible. At the moment o
f thru t try pu hing the head into the pillow to add a compre ion component tha
t may help in localization.
TECHNIQUES FOR THE SINUSES AND TEMPORO-MANDIBULAR JOINTS
23
The na al inu e are a common ite of conge tion, infection and inflammation an
d o teopathic intervention u ing vibration and frictional technique can play a
part in treatment in ome ca e . Rarely will the e be a complete an wer, and oth
er method from hot or cold pack through to antibiotic are all appropriate in
certain ca e . The o teopathic input can, however, add a dimen ion to treatment
which may not have been con idered by other practitioner . Some apparently impl
e manoeuvre can have a very profound effect on drainage, and produce benefit ou
t of all proportion to the time and effort u ed. Some of the e technique can al
o be taught to the patient for their own u e if appropriate. Special precaution
are mo tly tho e relating to the po ibility of preading infection when the 
ma k area of the face i being worked. The lack of valve in the vein draining
the face, and the chance of infection
backtracking into the kull and brain mu t alway be borne in mind. The temporomandibular joint are a ite of ymptom and dy function in ome patient , and c
an be omewhat improved in their function by o teopathic intervention. Hypermobi
lity i clearly not going to re pond well to mobilizing technique ; however, whe
n that hypermobility i due to over- tre produced by the oppo ite ide being t
oo tight, uitable mobilization of the re tricted ide can be u eful. The dental
urgeon ha a role in a e ing the occlu ion of the teeth and making any uita
ble adju tment here, but gentle tretching of over-tight mu cle and cap ule ca
n be valuable. There are few pecial precaution here except to con ider the ind
ication a much a the contra-indication , and to be aware that poor re ult in
treatment are often due to the wrong choice of treatment a much a the wrong c
hoice of technique.
190
Technique for
the

inu e
and
temporo-mandibular joint
23.1 Friction or vibration frontal inu upine Direct friction over the emi io
n of the upra-orbital nerve a it emit from the ridge of the frontal bone i b
eing u ed here. The operator finger are directly applied to the notch, and ei
ther circular friction or vibration can be performed. Tip : Mo t u eful in conju
nction with other friction and vibration point and particularly when the fronta
l inu i involved. Lea t u eful where acute inu iti make the friction point
too tender. Extra c o n i d e r a t i o n : Start lowly and uperficially
and gradually work deeper, repeating the technique intermittently during treatm
ent rather than keeping up the contact for a long period.
23.2 F r i c t i o n or v i b r a t i o n over na al b o n e u p i n e The n

a al bone can be gently queezed and ma aged in thi hold where the operator i
teadying the head with one hand and applying the technique with the other. T
i p : Mo t u eful in conjunction with other inu friction and vibration techn
ique . Extra c o n i d e r a t i o n : Start gently and gradually increa e p
re ure and perform the technique for hort period during the treatment e ion
, repeating thi everal time .
Technique for
the

inu e
and
temporo-mandibular joint
191
23.3 F r i c t i o n a n d v i b r a t i o n m a x i l l a r y i n u u p i
n e The operator i itting at the head of the table and ha applied the tip o
f middle finger to the maxilla o that direct pre ure can be u ed over the emi
ion of the nerve. Vibration can be applied over the maxillary inu in thi po
ition al o. The patient head i tabilized by the heel of the operator han
d . Tip : Mo t u eful where maxillary inu i involved. Lea t u eful where acut
e inu iti make pre ure extremely uncomfortable. Extra con ideration : Start
gently and gradually increa e pre ure, perform technique for hort period and
return everal time during treatment e ion. 23.4 (bottom left) Springing temp
oro-mandibular j o i n t upine While itting at the head of the table, the oper
ator i holding back on the temporal bone with one hand and applying a repetitiv
e force along the ramu of the jaw with the other. It i po ible to make mall
adju tment to the angle of that force o that the articulation can be tre ed
in different way according to the re triction of motion encountered. It i nece
ary for the patient to let the jaw ag to make the joint acce ible; when the
ma eter i ten e, the technique will not produce any effect. Tip : Mo t u eful
in ca e of unilateral temporomandibular joint dy function and intra-articular d
i c derangement . Lea t u eful in ca e of bruxi m where it i more important to
work on the mu cle and occlu ion than the joint. Extra con ideration : While u
nder tretch try antero-po terior movement and lateral movement to find the op
timum. 23.5 ( ee next page, bottom left) Articulation temporo-mandibular joint
upine From the head of the table the operator can gently grip both ide of the
mandible between finger and thumb and apply a direct di traction force to the
joint on one or both ide . It i nece ary for the patient to relax the jaw
o a to take the mu cle off ten ion to make thi effective. The whole mandible
can al o be taken into a forward or backward direction a required, and if the
patient lowly open and clo e the jaw while the di traction force i applied,
adju tment of the tracking during thi action can be brought to their attenti
on a thi often i an e ential part of any treatment to the e joint . Tip : Mo
t u eful in ca e of mal-occlu ion and bilateral joint dy function where the tr
action c o m ponent i helpful. Lea t u eful where mu cle ti ue i very tight a
nd prevent effective traction. Extra c o n i d e r a t i o n : Extreme tend
erne at the angle of the jaw can be an indication of occipito-atlanto dy funct
ion.
192
Technique for
the

 inu e
and
temporo-mandibular joint 23.6 T r a c t i o n a n d a r t i c u l a t i o n t e
m p o r o mandibular joint upine The operator i holding directly over the joi
nt and palpating for en e of ten ion a the other hand pull on the mandible to
di tract the joint in a variety of direction a nece ary. It i al o po ible
to hold over the articular di c, and en ing when the joint i on optimum ten i
on, apply a force directly over it which may give an opportunity for the di c to
relocate if it i malaligned in the joint. A in mo t temporo-mandibular joint
technique it i nece ary for the patient to relax the jaw mu cle during the e
procedure . Tip : Mo t u eful where mu cle tretch i needed and intra-articula
r di c dy function exi t . Extra c o n i d e r a t i o n : Add antero-po ter
ior and lateral movement for optimum ten ion. 23.7 Traction temporo-mandibular
joint upine A uitable wooden rod i interpo ed between the teeth a far back a
po ible to form a fulcrum, and the operator i carefully clo ing the jaw a d
i tally a po ible. Thi cau e a direct traction force to be generated in the
temporo-mandibular articulation on the ide of the fulcrum. Traction can be a u
eful force a it i an acce ory movement to the joint and can thu produce mech
anical change that primary movement under the control of the patient own mu c
le cannot achieve. Tip : Mo t u eful where mu cle tretch rather than articular
work i required and where patient ha own teeth. Lea t u eful with denture . E
xtra c o n i d e r a t i o n : Pad wooden rod if nece ary and vary the thic
kne to find the optimum ten ion.
TECHNIQUES FOR THE CLAVICLE AREA
24
The clavicle i mentioned often in early o teopathic literature, yet relatively
little attention eem to have been given to it in modern time . Although the no
menclature given to le ion po ition wa attractive, and gave pecific direction
of force to be applied to rever e the po ition , it al o limited thinking into
certain direction only. Some pecific direction of fixation are common, but m
odern thinking i much more directed toward function, and technique de igned in
uch a way that they can re tore perceived deficit of function. The clavicle c
an be thought of in i olation, or a a tructure influenced by trauma, po ture a
nd occupational di order . It can be con idered a a bone with a pair of joint
attaching it to the ternum and the capula. Whatever the type of thinking, the
main object of o teopathic technique i to re tore normal mobility where it ha
been lo t. Thi aim to re-introduce integrity of function to the
pectoral girdle that may have dimini hed if the clavicle i not working properly
. Precaution are minimal, except that hypermobility of the joint can occur, an
d then technique applied will be irrelevant and indeed may wor en the ituation
. Some technique u e the arm a a lever, but po ible houlder condition may n
eed protection, or alternative technique ought in the e ca e . The clavicle ac
t a a prop to join the arm to the body and to protect the ve el and nerve u
nderneath it. It i e entially a table unit, but when the tability i lo t, d
i proportionate ymptom and dy function of the arm may occur. Thi in turn can
make re toration of function of the clavicle an important part of any yndrome a
ffecting the upper thoracic or houlder area. Many difficult upper thoracic ca
e will re pond well if clavicular dy function i attended to a a major part o
f the treatment approach.
194
Technique for the clavicle area

24.1 Articulation of clavicle idelying Stabilize the capula with your body and
while you hold the clavicle down circumduct the houlder. Varied degree of abd
uction will influence the ternoclavicular or acromio-clavicular joint or impl
y place the main empha i on the haft of the bone. Tip : Mo t u eful when the m
u cle attaching uperiorly to the clavicle are involved. Lea t u eful when the
u e of the houlder a a lever would be a problem. Extra c o n i d e r a t i o
n : Try u ing different pha e of re piration.
24.3 Articulation of clavicle upine Mold the patient wri t and forearm firmly
again t you o that a your hand applied to the clavicle fixe toward the table
, a mall traightening of your knee will perform the articulation. The oppo it
e can be applied if you gently lift the clavicle with your fingertip and apply
a downward pre ure through the patient arm. Extra c o n i d e r a t i o n
: Try u ing varied angle of rotation of the houlder.
24.2 Articulation of terno-clavicular joint itting Stabilize the capula with
your body and hold down on the medial end of the clavicle. Abduct the patient
arm and extend and externally rotate it to apply a pull along the length of the
clavicle. Due to the hape of the joint, the mo t common dy function i a riding
upward and medially of the ternal end of the clavicle. In a true ubluxation,
thi technique may produce a realignment of the joint but it will not la t, a
the cap ule and ligament will have been di rupted, probably permanently. Tip :
Mo t u eful where for any rea on the recumbent patient po ition i a problem. Le
a t u eful in very mobile ubject where it would be difficult to achieve a loca
lization. Extra con ideration : Try u ing different pha e of re piration.
Technique for the clavicle area
195
24.4 Thru t to a c r o m i o c l a v i c u l a r j o i n t upine Fixation in th
e acromio-clavicular joint i mo t u ual on the anterior a pect and can be addre
ed well with thi technique. To avoid exce ive ten ion on the brachial plexu
when the tug i applied, the patient head i idebent and rotated to the ame
ide. Pull teadily on the di tal part of the forearm and above the elbow with
the other hand. Make mall circumduction movement and vary the angle of flexio
n and abduction of the houlder until ten ion i felt to accumulate in the acrom
io-clavicular joint. Apply the thru t without relea ing the previou ly applied t
en ion a otherwi e a whipping action will occur which make the technique ineff
ective. Tip : Lea t u eful when gleno-humeral dy function i pre ent. 24.5 Thru
t to a cromio-clavicular joint itting The final po ition for thi technique i
hown here. The operator ha applied an external rotation, flexion and traction
to the patient houlder while firmly holding back on the clavicle o that forc
e accumulate at the acromio-clavicular joint. A a preliminary the patient ar
m i taken from a po ition with the back of her hand facing forward at, or behi
nd her wai t and then thru t out harply forward while upinating her forearm.
Thi put a gapping force on the acromio-clavicular joint. Tip : Lea t u eful in
very flexible ubject a the force will tend to di ipate. Extra c o n i d e
r a t i o n : Try u ing different pha e of re piration and varying the flexi
on angle of the houlder.
196
Technique for the clavicle area
24.6 T h r u t to a c r o m i o - c l a v i c u l a r j o i n t u p i n e St
art with the patient arm by the ide and firmly fix the di tal third of the cl
avicle with the pad of your thumb. Bring the patient arm into elevation and ro
tate it gently until ten ion accumulate at the acromio-clavicular joint. An art
iculatory force can be u ed, or if ten ion i uitable, a mall thru t can aid i

n breaking fixation in the joint. Care mu t be taken to apply the thumb to the p
lateau on the top of the clavicle to keep the di comfort of the pre ure to a mi
nimum. Tip : Lea t u eful where there i gleno-humeral dy function making thi r
ange of movement impo ible. Extra c o n i d e r a t i o n : Try andwiching
the patient wri t between your hand and forearm and then moving your whole bo
dy and arm together.
24.7 Articulation of terno-clavicular joint itting Hold ten ion with both hand
in oppo ite direction and fix the patient capula with your thorax, then tw
i t into rotation both way to produce a mobilizing force on the terno-clavicul
ar articulation and fir t rib anteriorly.
Technique for the clavicle area 24.8 Thru t to terno-clavicular joint upine U
e cro ed hand to put a longitudinal force through the houlder and, therefore
, the clavicle while holding back again t the ternum with the ulnar border of t
he ternal hand. When ten ion accumulate , the ternal hand maintain the pre u
re and the pi iform pu he toward the table and along the clavicle by mean of t
he hand deviating into an ulnar direction. It may be nece ary to rotate the pat
ient head to one ide or the other to optimize the accumulation of ten ion in
the joint. Extra con ideration : Try varying the range of abduction and adductio
n in the houlder to optimize the ten ion.
197
TECHNIQUES FOR THE SHOULDER AREA
25
Patient often pre ent in o teopathic practice with dy function and di order o
f the glenohumeral articulation and the houlder area generally. The houlder ca
n be de cribed a a tructure that i lung from the occiput, and tethered to th
e pelvi , o, like all other area of the body, cannot be con idered in i olatio
n. The joint it elf can manife t mechanical derangement a well a pathological
condition , particularly tho e involving the cap ule and the urrounding mu cle
and tendon . Special precaution that mu t be con idered when working on the h
oulder with manual technique include the po ibility of boneweakening condition
uch a o teoporo i , a ome of the lever u ed will be long and
potentially trong, and exce force could po ibly compromi e bone trength. In
practice the amount of force u ed hould never be enough to cau e bone damage e
ven in a di ea ed tate. However, it may not alway be realized how much leverag
e i being applied. From the point of view of indication , accurate diagno i of
the cau e of any particular yndrome i not alway ea y. Pain due to inflammato
ry di order and econdary protection of hypermobility may not be re pon ive to
phy ical treatment and much wa ted time, di comfort and expen e can be avoided b
y recognizing thi . A the joint i a ball and ocket, thru t technique directl
y applied to the houlder do not play a large part in the treatment of dy functi
on .
Technique for the houlder area
199
25.1 Kneading houlder mu cle upine Thi i an operator view of the technique.
It how the hand applied with the u e of a wringing action to the oft ti
ue to work either the anterior, uperior or even the po terior a pect of the j
oint. Varying degree of abduction, compre ion and rotation can be u ed to aim
the force to the ti ue or area de ired. The technique can be applied with the
medial hand while the other one remain till, or the medial hand can remain ti
ll while the operator body and other hand move in varied direction together.

There are many more variation of thi hold than would appear at fir t glance, a
nd a little experimentation will reveal ome intere ting change in target ti u
e with only mall adju tment of hold and pre ure . Tip : Mo t u eful in ca e
where houlder dy function permit a rea onable degree of abduction. Lea t u efu
l in acute cap ular condition where abduction beyond a mall range i impo ibl
e. Extra con ideration : A with all technique that are de igned to work on of
t ti ue , the duration of the hold i an important element. A finite quantity o
f time of everal econd with the ti ue under light u tained pre ure i ne
ce ary to produce a ignificant change. Simply tretching and immediately relea
ing the ti ue will produce a le efficient re ult a fluid interchange will
not have had time to take place.
25.2 Kneading upper a r m m u c l e upine Thi operator viewpoint photogr
aph how cro fibre kneading being applied to the tricep . If the hand were ch
anged over, the bicep and brachiali could be worked in the ame way. Similar w
ork on the mu cle can be performed with the arm in a neutral po ition. In the p
o ition hown the mu cle and fa cia are on ome ten ion that can allow more eff
icient combined technique with tretch a well a cro -fibre work. Tip : Mo t u
eful in ca e where the belly of the upper arm mu cle rather than the tendon
i the target. Lea t u eful in ca e of articular dy function where the mu cle
may be le important. Extra c o n i d e r a t i o n : Varying the degree of
abduction or adduction will change the initial ten ion on the mu cle to make t
he technique a combined kneading and tretch procedure.
200
Technique for the houlder area
25.3 K n e a d i n g h o u l d e r p r o n e The po terior part of the hould
er and mu cle are more acce ible in thi po ition. Control the arm with your h
and o that you can vary the angle at which the houlder i held to reach the pa
rticular ti ue de ired. Tip : Mo t u eful where the po terior mu cle and the
capula are involved. Lea t u eful where the patient find prone lying a problem
for any rea on. Extra c o n i d e r a t i o n : Varying the po ition of head
rotation can change the effect of the technique.
25.4 Articulation of houlder itting Induce antero-po terior and upero-inferio
r movement in thi po ition. The long head of the bicep i available for fricti
on in ca e of tendoniti , a i the rotator cuff tendon behind the houlder tha
t you can work with your thumb. Tip : Mo t u eful in any patient who find a rec
umbent po ition difficult. Lea t u eful where houlder movement of any amplitude
mu t be introduced to make the technique reach the target ti ue. Extra c o n
i d e r a t i o n : Introduce a traction component with the patient holding a
mall weight a the technique i being performed.
Technique for the houlder area
201
25.5 Harmonic t e c h n i q u e hold f o r houlder upine Cla p the patient h
and between your hand and induce a harmonic o cillation into a pendulum-like mo
vement of the arm into abduction, adduction and internal and external rotation.
Flexion or exten ion can be introduced a well, or combination of everal compo
nent are po ible. An appropriate rhythm for thi technique would be lightly d
ifferent in each ubject but would vary between fifty and a hundred o cillation
per minute. The correct rhythm i determined by finding the rate that can be ma
intained with lea t effort by the operator. Tip : Refer to earlier ection peci
fically relating to harmonic technique.
25.6 T r a c t i o n of h o u l d e r u p i n e Fix the patient arm betwee

n your upper arm and hand while you apply the other hand behind the head of the
humeru . Lean back and allow the fingertip to fall into the pace produced by t
he traction. The e finger monitor the force nece ary and can add a mall tuggi
ng motion to the joint. Tip : Mo t u eful in mild inflammatory di order where t
hi gentle o cillation allow circulatory interchange. Lea t u eful in ca e of
pecific individual mu cle di order where thi technique i non- pecific. Extra
c o n i d e r a t i o n : Try varying the angle of rotation of the arm and
the abduction and flexion angle a well for optimum effect.
202
Technique for the houlder area 25.7 Traction of houlder idelying Fix the pat
ient forearm between your flexed wri t and bicep and keep the elbow well flex
ed o that a you lean back, the forearm and upper arm i carried into traction.
Apply the other hand ju t below the acromium to produce a traction and di tract
ion force at the houlder. Varied degree of abduction and rotation and flexion,
exten ion can be induced to maximize the effectivene and reach different part
of the cap ule. Tip : Mo t u eful where rotation in traction i indicated rath
er than pure rotation. Lea t u eful where there i any dy function in the elbow
or wri t. Extra c o n i d e r a t i o n : Try applying the traction with alt
ernating hand . 25.8 (bottom left) A r t i c u l a t i o n of houlder i t t i
n g The itting po ition allow you to treat the houlder by your body movement
while the houlder i held till rather than the houlder being moved on the bo
dy. Fix the capula to your ternum and rotate or idebend your elf, thereby inf
luencing different part of the houlder joint complex. You mu t maintain a firm
grip o that you and the patient move a a unit. Tip : Mo t u eful where the lo
ng mu cle affecting the houlder interiorly are involved. Lea t u eful in very
mobile ubject . Extra con ideration : Take care to avoid the patient trachea.
Technique for
the houlder area
203
25.10 Traction to houlder i t t i n g Place your interlocked finger ju t bel
ow the acromium and apply a traction and di traction force. While thi force i
maintained take the houlder through a variety of movement while the body i to
rqued into the oppo ite direction . Thi will have the effect of influencing the
houlder directly and the long mu cle acting on it. Tip : Mo t u eful where fo
r any rea on the recumbent po ition may be a problem and where good abduction ra
nge i po ible. Lea t u eful in very flexible ubject where pinal movement wi
ll ab orb the lever . Extra c o n i d e r a t i o n : An initial force appli
ed diagonally toward the oppo ite hip fir t, before the traction, i u eful.
25.11 T r a c t i o n and articulation to houlder p r o n e Cla p the patient
forearm between your thigh and grip the houlder between your hand . Re t the
back of your finger on the table and while you hold the houlder firmly in po i
tion traighten your knee to produce the traction. You can move the hand into
a variety of direction . Tip : Mo t u eful where acce ory movement of antero-p
o terior and upero-inferior movement are required. Lea t u eful where lying pro
ne for any rea on i a problem. Extra c o n i d e r a t i o n : An adju tabl
e table i e ential to allow the patient to be lowered o that the arm i horiz
ontal.
25.9 ( ee facing page, bottom right) T r a c t i o n and articulation to houlde
r idelying Place your interlocked finger ju t under the acromium and apply a f
orce toward the axilla o that you produce a di traction and traction to the joi
nt. Some variation of angle of rotation can be introduced to affect different p
art of the cap ule. Tip : Mo t u eful where it i nece ary to avoid putting fo

rce through the elbow or wri t. Extra c o n i d e r a t i o n : Firm pre u

re toward the table i an e ential component.
204
Technique for the houlder area
Technique for the houlder area 25.12 ( ee facing page, top left) A r t i c u l
a t i o n of houlder upine Hold the acromion and the clavicle into the table
and pull them lightly caudally while your other hand elevate the arm until re
i tance i felt. Different degree of rotation and more or le abduction can be
introduced to focu to particular part of the joint. Tip : Mo t u eful where i
t i de ired to i olate the clavicle from houlder movement. Lea t u eful in ver
y kyphotic ubject a thi range of movement can be very limited a the anterio
r ti ue will be rather hortened. 25.13 ( ee facing page, bottom left) Articul
ation of houlder into external rotation idelying U e your cephalic forearm to
act a a fulcrum while that hand fixe the acromion and clavicle, and palpate t
he houlder joint. The other hand introduce external rotation with varying degr
ee of flexion while your body a i t the movement. The greater the degree of f
lexion in the houlder the tronger thi articulation will be. Tip : Mo t u eful
where a trong mobilizing force i required. Lea t u eful where there i any u
picion of lack of bone trength a thi i a long lever on the humeru . Extra c
o n i d e r a t i o n : Try holding the joint on ten ion and rocking the wh
ole body backward and forward into rotation. 25.14 ( ee facing page, top right)
A r t i c u l a t i o n into external rotation of houlder idelying In thi hol
d, both of your hand are applied to the houlder and your caudal elbow induce
the rotary movement. Thi ha the advantage that c o m pre ive force can be app
lied more firmly to the houlder. Thi limit the range of motion from the joint
it elf which will therefore have a greater influence on the oft ti ue . Tip :
A photograph 25.13. 25.15 ( ee facing page, bottom right) Articulation into ex
ternal rotation of houlder idelying Fix the lateral border of the capula with
your caudal hand and u e your cephalic hand to hold the capula firmly again t
the po terior wall of the thorax. Apply the external rotation force with your up
per abdomen pre ing down on the patient elbow. Tip : A photograph 25.13 and
25.14 but mo t u eful in very flexible ubject to avoid capulothoracic moveme
nt and focu into the houlder it elf.
205
25.16 A r t i c u l a t i o n into external r o t a t i o n of houlder upine T
hi very trong long lever articulation need great care, but with your caudal h
and you apply a po tero-anterior force o that the houlder can be externally ro
tated. The other hand i u ed rhythmically, to increa e range of motion. Tip : M
o t u eful where a trong lever i required. Lea t u eful where there i any fea
r of lack of bone trength. Extra c o n i d e r a t i o n : Vary the angle o
f houlder abduction to find the optimum.
206
Technique for the houlder area
25.17 A r t i c u l a t i o n into external r o t a t i o n and t r a c t i o n
p r o n e Face the head of the table and u e your elbow to fix the capula with
your hand firmly applied in the cubital fo a. The other hand applie a force to
ward the patient head, thereby levering the head of the humeru away from the
glenoid. Varied range of exten ion and abduction and external rotation can be i
ntroduced. Tip : Lea t u eful in patient where lying prone i a problem for any
rea on.
25.18 Traction and external rotation articulat i o n to houlder upine Face the

head of the table and apply traction by placing your hand in the cubital fo a
while the other hand applie the external rotation force. Tip : Mo t u eful a t
he capula i held by the table and trong traction and external rotation can be
applied. Lea t u eful where the bicep tendon are en itive a pre ure on the
m will be a problem. Extra c o n i d e r a t i o n : Vary the angle of abduc
tion and flexion to find the optimum.
Technique for the houlder area
207
25.19 Articulation into internal r o t a t i o n of houlder idelying From in f
ront of the patient you fix the houlder with your cephalic hand while the cauda
l hand applie the internal rotation force. Tip : Mo t u eful where a trong for
ce i required. Lea t u eful in ca e of evere movement re triction a the po i
tion will be difficult to attain. Extra c o n i d e r a t i o n : Try applyi
ng a traction force at the ame time. 25.20 (top right) A r t i c u l a t i o n
i n t o i n t e r n a l rotation of houlder idelying Abduct the houlder ju t
ufficiently o that the patient wri t i fixed behind the lower rib . The int
ernal rotation force will be much tronger in thi po ition. The cephalic hand c
an either hold back on the capula and clavicle or a i t the rotary movement by
pulling on the head of the humeru . Tip : A photograph 25.19. 25.21 Articulati
on into internal r o t a t i o n of t h e houlder idelying Fix the capula fro
m behind with your tor o and introduce the rotary movement by holding firmly wit
h your cephalic hand while carefully pu hing your caudal hand anteriorly. Tip :
Mo t u eful where the capula tend to wing exce ively. Lea t u eful in ca e o
f evere movement re triction where the po ition may be difficult to attain. Ext
ra c o n i d e r a t i o n : Try u ing traction in addition to rotation.
208
Technique for the houlder area
25.22 (top left) Articulation into internal rotat i o n of houlder prone In thi
operator viewpoint photograph you face the foot of the table and while fixing
the capula with your elbow, you apply a traction force through the patient cu
bital fo a. Your other hand carefully rai e the patient hand toward the ceil
ing while maintaining pre ure on the di tal end of the forearm toward the table
to induce traction. Tip : Lea t u eful where the patient may find prone lying a
problem for any rea on. 25.23 A r t i c u l a t i o n into internal rotation an
d t r a c t i o n of houlder upine In thi operator viewpoint photograph you f
ace the foot of the table and fix the houlder to the table with your elbow whil
e the hand in the cubital fo a act a a fulcrum for the other hand. Pu h, at t
he ame time, toward the table for the traction and toward the floor for the int
ernal rotation. Tip : Mo t u eful where a trong force i required. Lea t u eful
where there i any elbow dy function. Extra con ideration : Vary the angle of
abduction and flexion for optimum re ult . 25.24 (top right) Articulation of ho
ulder upine Fix the lateral border of the capula with one hand while firmly gr
ipping the lower end of the humeru with the other hand which can induce rotatio
n movement , elevation and traction a required. The fixation of the capula cau
e the movement to be localized to the houlder joint it elf rather than the c
apulo-thoracic articulation and naturally le range of motion will be achieved.
Tip : Mo t u eful where there are adhe ion in the inferior a pect of the cap u
le. Lea t u eful in acute ca e where thi much abduction would be a problem.
TECHNIQUES FOR THE ELBOW AREA
26
Mechanical dy function in the joint of the elbow are not uncommon. Their impor

tance in painful yndrome and di turbance of u age varie , but i often a fact
or that can be addre ed by o teopathic treatment. From a mechanical viewpoint t
he elbow po e variou problem . There are rather mall but important range of
acce ory movement and it i nece ary to u e the e in treatment technique . The
y may be difficult to control. Other problem are po ed by the fact that the joi
nt will tend to e cape from corrective force if the grip i not firm enough. H
owever, an exce ively firm grip will produce pain and even more re i tance. A b
alance between force, direction and amplitude i critical. The joint complex mu
t be con idered a a whole; the elbow, radio-humeral articulation, and di tal ar
ticulation at the wri t all work
together. Often individual joint dy function will not be corrected until a uita
ble balance i obtained between all the relevant joint and oft ti ue . The fa
cial tate i al o important a poor circulation, healing and myofa cial tone m
ay be part of the equation. Special precaution need to be con idered to exclude
the po ibility of myo iti o ifican , which ometime affect the brachiali
mu cle in the pre ence of a haematoma after trauma. The ulnar nerve i vulnerabl
e to damage on the medial ide of the ulna. An ulnar neuriti take a very long
time to heal when traumatically induced. It would be very hard to do thi in tre
atment, but an exi ting condition could be irritated by injudiciou technique. I
t i e ential to avoid exce ive force, particularly into exten ion a there i
a po ibility of damage to the floor of the olecranon fo a.
210
Technique for the elbow area
26.1 Kneading of lateral ti ue upine Cla p
the arm between your body and forearm and knead the exten or mu cle on the late
ral ide of the arm. The hand hare the pre ure in oppo ite direction to redu
ce the amplitude of movement nece ary in each. Tip : Try varying the angle of
houlder abduction and rotation; thi will change the initial ten ion in the mu c
le before they are kneaded. If there i extreme tenderne , try holding the mu
cle on ome ten ion and moving the arm back and forth rather than the mu cle t
hem elve . 26.2 (bottom left) Kneading of medial and
anterior ti ue upine Fix the patient forearm
to your ide with your forearm and then apply a kneading and tretching to the m
edial compartment mu cle with both thumb . It i po ible to reach deep into th
e cubital fo a with thi hold. Tip : Take care to protect the brachiali and br
achial artery from exce ive pre ure.
26.3 (bottom right) Kneading of lateral compartment mu cle upine Sit on the ed
ge of the
table. Put ome ten ion on the mu cle with the proximal hand and then, while ma
intaining thi pre ure, pronate the forearm with your other hand to generate th
e kneading force. Tip : Try varying the angle of elbow flexion and adding compon
ent of traction or circumduction to the hold. A an alternative, the operator c
an perform thi technique tanding.
Technique for the elbow area
211
26.4 Stretching of medial compartment upine
Fix the arm to the table with your proximal hand and apply ome compre ion to t
he medial ide of the elbow. Apply a tretching force with the other hand by pul
ling the forearm into your ide. Keep your arm again t your ide and rotate aw
ay from the table. Tip : Try varying the pronation and upination during the tec
hnique to reach different part of the mu cle . 26.5 (bottom left) Stretching of

intra-o eou membrane upine Grip the forearm between your hand and apply opp
o ite movement with each hand of traction and compre ion a well a antero-po
terior hearing. Tip : Try varying the angle of flexion before applying the othe
r lever . 26.6 (bottom right) Traction upine Fix in the ante-cubital fo a with
your pronated proximal hand and grip the forearm with the other. Compre the f
orearm again t your abdomen and turn away from the table to pivot round the fixi
ng hand to apply traction. Tip : Mo t u eful where there ha been a longitudinal
compre ion force injury and it i nece ary to di engage the joint .
212
Technique for the elbow area
26.7 Traction to ulna upine Fix the upper
arm to the table with the pronated proximal hand. Grip the ulna with the other h
and and compre the forearm again t your abdomen. Pivot around your fixing hand
to apply a pecific traction to the ulna. Tip : Try adding a mall circumductio
n force to the traction.
26.8 Traction to radiu upine Fix the upper
arm to the table with your pronated hand and reach around the forearm with the d
i tal hand to grip the radiu . Apply traction and mobilize in pronation and upi
nation. Tip : Mo t u eful in ca e of tenni elbow where there tend to be an
element of compre ion in the radio-humeral joint. Lea t u eful where the forear
m i very large and it would be difficult to reach around the ulna to the radiu
.
Technique for the elbow area
213
26.11 Thru t to radio-humeral joint upine The patient i lying upine but ligh
tly turned toward the affected arm. Apply your hand to the lightly flexed arm.
Contact the haft of the radiu and ulna and the upper part of the humeru . Co
mpre with both hand toward the table and apply a gentle rocking force from i
de to ide until ten ion accumulate in the radio-humeral joint. The thru t i a
pplied without relea ing thi preliminary ten ion by dropping ome weight onto b
oth hand . Tip : If preferred, u e cro ed arm to apply the hold.
214
Technique for the elbow area ( ee previou page, top left and
26.9 and 26.10
top right) Thru t to radio-humeral joint upine
Fix the patient forearm to your ide and cla p around the lightly flexed elbo
w, applying the web of your thumb to the medial a pect of the joint. Compre th
e elbow between your hand and circumduct through a mall range to find the opti
mum point of ten ion. The tip of the index finger of either hand palpate for th
i ten ion and applie a local compre ion to the radio-humeral joint. The thru
t i performed with an increa e of compre ion between the hand , an increa e of
fixing of the arm to your ide and a hort, harp rotation of your body away fr
om the table. Tip : Note the angle of the thru ting arm i not directly acro t
he joint but i directed lightly anteriorly, a the radio-humeral joint i ante
rior to the humero-ulnar joint. Thi technique may require everal priming force
of gradually increa ing amplitude until the optimum ten ion accumulate .
26.12 and 26.13 Thru t to radio-humeral joint upine The e operator viewpoint ph
otograph how a complex technique for gapping the radio-humeral joint. Sit on t

he edge of the table and place the internally rotated arm acro your lower thig
h. Fix the medial epicondyle again t your thigh and hold back on the di tal end
of the humeru . Your other hand grip the di tal end of the radiu and applie a
force directly toward the floor. Vary the pronation and upination until ten io
n i felt to accumulate. The thru t i performed by an accentuation of all three
force , the operator thigh and upper and lower hand imultaneou ly. Tip : Try
placing the fifth finger in the palm of the patient hand to allow better cont
rol of the pronation and upination. Thi al o allow control of the flexion and
exten ion of the wri t to help focu the force in the elbow. If a tronger eff
ect i needed, in tead of u ing more force, try introducing ulnar deviation of t
he wri t with the di tal hand. If an even tronger effect i needed, place the p
atient thumb in the palm of their hand to place the exten or mu cle on tretc
h, and then apply ulnar deviation. Note: The internal rotation of the arm i e
ential if thi i not to become an exten ion thru t. It i often very difficult
to maintain thi internal rotation. En ure that the medial and lateral epicondyl
e are vertical before performing the technique.
Technique for the elbow area
215
26.14 and 26.15 Thru t to radial head upine Place your thumb behind the radial
head and hold the di tal end of the forearm again t your abdomen. Maintain ligh
t flexion of the elbow throughout the whole technique. Sharply pronate the forea
rm, flex the wri t and extend the elbow to lap the forearm again t your abdomen
. Thi Mill procedure i often performed with hyperexten ion, which i not on
ly painful, but can be potentially dangerou a the proximal end of the ulna can
be driven through the floor of the cubital fo a. Hyperexten ion al o mean tha
t the force will be unlikely to reach the radial head o the technique will be i
neffective for the purpo e intended! Tip : Try compre ing the radial head betwe
en thumb and finger of the proximal hand to help localize the lever .
216
Technique for the elbow area
26.16 Thru t to humero-ulnar joint into adduction upine Thi operator viewpoint
photograph how the patient forearm gripped firmly again t the operator ab
domen. Hi hand are gripping around the elbow and the metacarpo-phalangeal join
t of the index finger i applied to the medial a pect of the ulna. While grippin
g firmly again t the barrier of joint re i tance he rotate hi body away from t
he table to apply the thru t. The direction of force i from medial to lateral t
o reach the humeroulnar joint, not the radio-humeral joint which would require a
more anterior force. A few degree of flexion mu t be maintained throughout the
technique. Tip : Thi technique would be u ed where there i an increa ed carry
ing angle or a perception of an inability for the head of the ulna to locate in
the fo a on full exten ion.
26.17 Thru t to humero-ulnar joint into adduction upine Stand out ide the ligh
tly flexed arm and pull with your proximal hand on the medial a pect of the ulna
. Pu h the di tal end of the forearm in the oppo ite direction to produce a forc
e de igned to reduce the carrying angle. Tip : Try adding varied amount of wri
t deviation to help focu the technique. Thi hold can be u ed to perform the op
po ite function of increa ing the carrying angle where nece ary, by rever ing t
he hand direction .
Technique for the elbow area
217

26.18 Thru t to humero-ulnar joint into adduction upine Fix the forearm again t
your ide and grip firmly around the elbow with both hand . Apply the metacarpo
-phalangeal joint of your index finger of the proximal hand to the lateral a pec
t of the upper end of the ulna. Maintain light flexion in the elbow and apply t
he thru t with your proximal hand and a mall rotation of your body.
26.19 Thru t for pulled radiu upine Shake
hand with the patient and fix behind the elbow with your other hand. Apply a m
all compre ion force between the hand and gently pronate and upinate the elbo
w until the radial head i felt to relocate within the annular ligament. Tip : T
hi technique hould only be nece ary in children and any u picion of a pulle
d radiu in an adult hould alert the operator to the po ibility of a fracture
or di location. Di location of the ulna mu t NEVER be reduced u ing pronation
and upination a there i a danger of the coronoid proce damaging the brachia
l artery!
TECHNIQUES FOR THE FOREARM AREA
27
The forearm tran mit torque between the elbow and hand by way of the fa cia, bo
ne and mu cle . Bone in life are omewhat malleable, and torque on the e ti u
e can cau e mechanical problem . The tate of the fa cia that enclo e the mu c
le and ve el i particularly important here, and myofa cial technique are ex
tremely u eful in contributing to improvement in function. Ten ion tate in the
mu cle of the forearm can have an adver e influence on wri t and elbow functio
n and may be maintaining factor in dy function at tho e ite . Mu cle energy te
chnique are u eful here and are ba ed on anatomical knowledge of mu cle plane
and attachment . Deep frictional cro -fibre ma age ha a part to play in pecific oft ti ue tate of
adhe ion and local irritability. Thi can be of particular u e over the medial
or lateral epicondyle of the humeru in tate of golfer elbow and tenni el
bow, re pectively. Rehabilitation of po t fracture ca e can al o often be help
ed by direct work on the mu cle and fa cia of the forearm. Particular care hou
ld be applied where there i a true neuriti a thi can be aggravated by phy ic
al treatment applied over the nerve it elf. No technique hold have been illu tr
ated for thi ection a the principle are imilar to tho e in the upper arm or
the calf hold hown in the relevant ection .
TECHNIQUES FOR THE WRIST AND HAND
28
The wri t i an extremely delicate tructure that require mobility and yet tab
ility. Sometime thi balance goe wrong, and then o teopathic intervention i a
ppropriate. Technique can be general or pecific, and although general techniqu
e can often be effective in re toration of function, pecific manipulative kil
l i ometime e ential. Some apparently pathological condition can be influen
ced by o teopathic treatment uch a carpal tunnel yndrome, and o teopathic man
agement including direct work on the wri t and hand can be valuable.
Special precaution need be con idered in relation to the true cau e of ymptom
. In many ca e there i hypermobility, and although thi will manife t a ubje
ctive tiffne , accurate motion te ting hould reveal the real nature of the pr
oblem. Undiagno ed fracture of the caphoid, for example, i well documented in
book on fracture and traumatology and hould never be overlooked. Inflammatory
di order uch a rheumatoid arthriti need pecial care, and although gentle
hort treatment can be helpful, exce ive mobilization i not wi e.

220
Technique for the wri t and hand 28.1 General mobilization The patient i lying
upine and the operator i performing alternating ulnar and radial deviation wi
th hi hand , gripping around the wri t. Thi will produce a hearing force that
will reach many of the carpal articulation . Tip : Try fixing with one hand and
moving the other or u ing both hand . Thi hold can be applied to the palmar or
dor al urface of the wri t.
28.2 (bottom left) Shearing of carpal row The
operator i tanding by the ide of the upine patient and i introducing a
ct dor al and palmar force with the wri t maintained in a neutral po ition.
: Try moving both hand di tally to focu on the inter-carpal articulation
er than the true wri t joint. Try queezing or preading the patient hand
the di tal operator hand or applying traction between the operator two
.

dire
Tip
rath
with
hand

28.3 (bottom right) Kneading thenar eminence

In thi operator viewpoint photograph, the lateral border of the hand i being h
eld table while the operator thumb i applying kneading to the thenar eminenc
e. Tip : U e care and a gradual introduction of force a the e mu cle are often
extremely tender initially. Try adding circumduction articulation imultaneou l
y.
Technique for the wri t and hand 28.4 Stretching palmar urface In thi operato
r viewpoint photograph the hand i being tretched laterally between the operato
r hand . Tip : Mo t u eful in ca e of carpal tunnel yndrome where hortening
of the flexor retinaculum may be a maintaining factor. Extra con ideration : Tr
y inducing an ulnar and radial deviation of the operator hand when ten ion ha
been applied to reach different part of the flexor tructure . 28.5 (bottom l
eft) Stretching proximal part palmar urface The operator i gripping over the h
ammate and caphoid to produce a trong tretch on the flexor retinaculum. Tip :
Mo t u eful in ca e of carpal tunnel yndrome. Extra con ideration : Try u ta
ined tretch for everal econd and then adding a diagonal tor ion to reach the
proximal and di tal part of the retinaculum. 28.6 (bottom right) Stretching pa
lmar urface Thi operator viewpoint photograph how the hand held again t th
e operator abdomen and hi hand applying a diagonal tretch to the ulnar bord
er of the patient hand. Tip : Mo t u eful in carpal tunnel yndrome and Dupetr
en contracture if lowly applied and maintained for everal econd .
221
222
Technique for the wri t and hand 28.7 General mobilization The operator i inte
rlocking finger with the patient and then while hi other hand tabilize the f
orearm he introduce a variety of force to the wri t and hand. Tip : Try each r
ange of po ible movement including combination , not forgetting traction and co
mpre ion. 28.8 (bottom left) Specific articulation carpometarcarpal joint Thi
operator viewpoint photograph how the di tal hand pulling on a metacarpal bon
e while the proximal hand fixe on the appropriate part of the wri t to introduc
e a traction articulation. Other vector can be u ed, uch a rotation, circumdu
ction and abduction/adduction, a the re triction demand . Tip : Mo t u eful in
ca e of carpo-metacarpal dy function rather than the actual wri t it elf. 28.9
(bottom right) Specific articulation carpometarcarpal joint Thi operator viewp
oint photograph how the patient pronated hand being fixed again t the table
while the operator di tal hand grip one of the metacarpal . Traction, with or
without other range of movement, can be introduced. Tip : Mo t u eful in impac
tion injurie where the metacarpal may be driven into the related carpal bone.

Technique for the wri t and hand

223
224 28.10
Technique for the wri t and hand ( ee previou page, top left) Articulation
carpo-metarcarpal joint lateral border The operator cla p the wri t of the up
ine patient to hi abdomen by turning hi back to the table. He hold firmly on
the relevant metacarpal and carpal bone and then turn hi body to produce the t
raction and mobilizing effect. Many different vector of force can be introduced
while the traction i applied, to effectively addre the re triction of moveme
nt. 28.11 ( ee previou page, bottom left) Circumduction articulation/thru t The
operator i very firmly cla ping the patient hand between the heel of hi ha
nd . He applie a circumduction motion and, within the movement, an arc of effec
tive re i tance will be felt. If thi i a oft re i tance, increa ed pre ure b
etween hi hand may create a uitable thru t barrier, where he can perform a h
ort, harp force again t the re triction. Tip : Try varying the part of the appl
icator performing the compre ion, thu directing the force to different part o
f the wri t ( ee photograph 28.12). Extra con ideration : Thi technique require
fairly high compre ion force that mu t be varied to reduce operator train an
d patient di comfort. 28.12 ( ee previou page, top right) Circumduction articulation/thru t See de cription for
photograph 28.11, but note that in thi hold the operator i applying the force
with hi hypothenar eminence to the proximal part of the patient wri t. He i
al o in a different arc of the circumduction cycle. 28.13 ( ee previou page, b
ottom right) Thru t mid carpu pronated Thi operator viewpoint photograph how
the patient hand pronated and the operator i pre ing firmly with hi pi ifo
rm directly over a cho en articulation. Hi other hand i reinforcing the pre u
re and he maintain thi pre ure while lightly deviating the thru ting hand in
to ulnar or radial deviation to find the optimum ten ion. Without relea ing the
ten ion a very mall amplitude thru t i applied. Tip : Mo t u eful where it i
nece ary to reduce the angle of the curve of the wri t and tretch the anterior
tructure while pecifically manipulating any re tricted joint.
28.14 Thru t mid carpu upinated Thi operator viewpoint photograph how the p
atient hand upine and the operator i applying a direct force between the ca
phoid and the hammate to open out the wri t curve, The other hand i reinforcing
. He lowly twi t hi applied hand until ten ion accumulate in the wri t and t
hen a very mall amplitude thru t i introduced. Note: for clarity, thi illu tr
ation how the initial contact; at the point of thru t the finger will be in t
he ame direction a tho e of the patient.
Technique for the wri t and hand
225
28.15 Thru t pecific articulation dor al
urface Thi operator viewpoint photograph how the patient pronated hand hel
d between the operator hand . He cro e hi thumb over the dy functional art
iculation on the dor um of the wri t. He circumduct the wri t while introducing
mall change in ulnar and radial deviation and if ten ion accumulate efficien
tly he can apply a very hort amplitude thru t with the thumb . Tip : Thi can b
e a very uncomfortable technique unle the amplitude i kept extremely mall. T
ry making the thru t more of an increa e of compre ion rather than dor i-flexio
n. 28.16 (bottom left) Thru t metacarpophalangeal joint Thi operator viewpoint
photograph how the operator i fixing and pulling back the metacarpal while a
pplying a traction and flexion force to the phalange. Tip : Lea t u eful in infl

ammatory di order uch a rheumatoid arthriti . 28.17 (bottom right) Thru t int
erphalangeal joint Thi operator viewpoint photograph how the patient prona
ted hand gripped at the wri t. The operator finger and thumb of the other hand
are applying a medial and lateral gapping force to the interphalangeal joint. T
ip : Keep the interphalangeal joint lightly flexed to reduce train on the cap
ule and to take the lateral ligament off ten ion. Thi technique may require e
veral priming movement until the ten ion accumulate efficiently.
226
Technique for the wri t and hand
28.18 Thru t interphalangeal joint of thumb
Thi operator viewpoint photograph how the index finger of the thru ting hand
i applied on the flexor urface of the di tal phalange while the thumb i on th
e dor al urface. The other hand i holding back on the ba e of the patient th
umb. The thru t i applied a a hort, harp flexion and traction movement to th
e di tal phalange while the other hand applie a mall oppo ite force. Tip : Not
all interphalangeal joint can be gapped due to their inherent joint configurat
ion and exce ive attempt to do o are unwi e.
28.19 Thru t/articulation interphalangeal joint
Thi operator viewpoint photograph how an articulatory force into direct media
l and lateral gliding being performed. If a thru t i nece ary a mall componen
t of flexion will be required to produce the optimum barrier en e in the joint.
Tip : Lea t u eful in inflammatory di order where the joint tructure may be w
eakened. Extra con ideration : Several priming movement are often nece ary bef
ore applying a thru t to the e joint .
TECHNIQUES FOR THE HIP AREA
29
The hip joint can be a ource and ite of ymptom in it elf although in mo t ca
e pain originating in the hip will be referred along the third lumbar nerve ro
ot down the medial ide of the thigh to the knee. Hip di ea e, whether of an acu
te or chronic nature, can produce a characteri tic di comfort deep in the groin,
but not u ually where the patient perceive the hip to be. Re toration of even
a mall part of lo t mobility can be very ucce ful in relieving many of the y
mptom of hip di order , even if the progre of the degenerative tate ha not
been changed at all. Technical problem occur mo tly due to the deep nature of t
he joint, and the need to u e quite long lever to reach it. There i ,
therefore, a po ibility of inducing a train of the knee in the proce . Harmon
ic technique are particularly u eful here in re-e tabli hing normal movement pa
ttern . Precaution relate particularly to the po ibility of undiagno ed fractu
re of the neck of the femur, which even though till allowing the patient to hav
e movement, how on Xray a evere damage. Unexpected hip fracture in an inappr
opriate age group can be due to a econdary depo it from a cancerou growth el e
where, and clearly the indication for o teopathic treatment mu t be carefully c
on idered. The full implication of the po ible ill-effect of treatment on po
ible di ea ed bone are con iderable.
228
Technique for the hip area
29.1 Harmonic technique into rotation upine Roll the lower extremity harmonical
ly into rotation a a unit. See earlier ection referring to harmonic technique.
Tip : Mo t u eful in ca e where there i con iderable tiffne and lo of d

ynami m in the limb. Extra con ideration : Try u ing varied degree of prelimin
ary abduction to produce the mo t u eful angle of the hip.
29.2 Harmonic technique pelvic hold upine Choo e whether to fix on the pelvi a
nd roll the leg, or fix on the leg and rock the pelvi . Thi hold will allow the
peri-articular ti ue of the hip to be reached. See earlier ection relating t
o harmonic technique. Tip : Harmonic technique i rarely going to be a complete
treatment by it elf, but i often a u eful preliminary to other procedure to lo
o en the ti ue .
Technique for the hip area
229
29.3 Harmonic technique both leg upine Roll both leg into rotation and apply
alternating traction and abduction/adduction a required. See earlier ection re
lating to harmonic technique. Tip : Thi hold i u eful in ca e of bilateral dy
function. Alternating traction and compre ion will al o addre a lumbar dy fu
nction that i re tricting idebending.
29.4 Articulation into internal rotation upine
Interlock your arm and hold over the knee to avoid training it. Apply a rhythm
ic internal rotation movement to the hip a you vary the range of flexion and ab
duction/adduction according to the need of the ca e. Tip : Mo t u eful where t
rong mobilizing i required. Lea t u eful in ca e of extreme movement limitatio
n a the po ition may not be attainable. Thi hold may be unacceptable in ca e
of evere knee di order a the train may be
230
Technique for the hip area
29.5 Articulation into internal rotation upine
Grip around the knee and you can partly protect the knee joint while you apply a
n internal rotation force to the hip. Tip : Mo t u eful where the knee joint may
require ome protection. Lea t u eful where trong mobilizing i nece ary a t
he lever i not very powerful. Extra con ideration : Try u ing varied angle of
flexion or abduction/adduction a nece ary to reach the part of the hip cap ule
de ired. 29.6 (bottom left) Articulation into abduction and adduction idelying
Fix firmly above the greater trochanter to limit the movement to the hip joint
and prevent movement into the lumbar pine. Hold the lower medial ide of the th
igh with the other hand to produce the abduction force. Varied degree of flexio
n, exten ion and rotation can be introduced a required. Tip : Thi po ition can
al o be u ed for mu cle energy technique hold . To reduce any di comfort in thi
technique try varying the angle of flexion of the lower leg in the initial et
-up of the technique. Thi will allow the lever to change the effect on the pelv
i and lumbar pine. 29.7 (bottom right) Articulation in external rotation and a
bduction idelying Thi hold i imilar to that in photograph 29.6 except that y
ou can hold further round the thigh and it i ea ier to introduce the trong ext
ernal rotation po ible with thi technique.
Technique for the hip area
231
29.8 Traction to hip upine Pull laterally again t the padded upper, inner thigh
at the ame time a you create a fulcrum with your che t again t the lateral a
pect of the knee. The um of the e two force will be to produce a true traction
force that will tend to eparate the head of the femur from the acetabulum. Tip
: Mo t u eful in ca e of degenerative hip di ea e where traction can allow gre

ater circulatory interchange. The actual tretch on mu cle i very mall, but t
hi technique can produce con iderable ymptomatic improvement. Extra con iderat
ion : Try varying the angle of flexion of the hip to find the optimum for the ca
e.
29.9 Traction idelying U e a pillow over your anterior thigh to act a a fulcru
m over which you place the patient upper thigh. Fix down toward the table with
the cephalic hand, and pu h toward the table with the caudal hand to produce a
true traction effect on the hip. Note that the other leg ha been flexed well ou
t of the way. Tip : Mo t u eful in ca e of degenerative di ea e where fairly t
rong traction may be needed. Extra con ideration : Exten ion movement may be a p
roblem in many ca e of hip degeneration o that ome flexion may be nece ary t
o make the technique fea ible.
29.10 Traction and di traction upine Fold your arm and interlace your forearm
under the patient knee. The traction i produced imply by leaning back. Tip :
Mo t u eful in ca e of evere degenerative di ea e a the knee and hip are in
con iderable flexion, reducing the train on them, but till allowing the effect
to reach the hip. Extra con ideration : Try varying the abduction and flexion r
ange to find the optimum.
232
Technique for the hip area
29.11 Low velocity tre into internal rotation
technique upine Place the patient foot lateral to the other knee. Hold the hi
p into firm internal rotation for everal econd until a en e of give i fel
t. Thi can be a multiple tage technique, a there may be more than one en e o
f relea e a different part of the dy function relea e. Tip : Mo t u eful in al
mo t all ca e of hip dy function providing the patient can achieve the po ition
; if not, place the foot medial rather than lateral to the other knee. Thi will
, however, reduce the lever omewhat. Extra con ideration : Try al o varying the
angle of hip flexion to find the optimum.
29.12 Low velocity tre technique into
external rotation upine Place the foot of the affected ide on the thigh of the
other leg. Hold down on the knee with the hip well abducted, while fixing on th
e other ide of the pelvi to prevent lumbar rotation. After a few econd there
hould be a en e of relea e in the hip. Thi may be a multiple tage technique
. Relea e may be only partial if it i not repeated in lightly varied angle . T
ip : Mo t u eful in almo t all ca e of hip joint dy function a tre techniqu
e do not put undue train on the articulation, but allow it to adopt it own pa
th of free movement. Extra con ideration : If the po ition i impo ible due to
evere limitation of movement, try placing the foot medial to the other knee. Tr
y varied degree of hip flexion to find the optimum for the ca e.
TECHNIQUES FOR THE THIGH AREA
30
The thigh contain ome of the tronge t mu cle in the body and can be very inf
luential in po tural problem . It i al o the ite of many port injurie , and
although not a very common pre enting ite of ymptom in mo t o teopathic pract
ice , may well be involved in hip, knee and lower back problem . Although it i
po ible to work on mo t of the mu cle with conventional tructural tretching
and cro -fibre technique , mu cle energy and inhibitory pre ure are often u ef
ul here.
Special precaution include the obviou one of the nece ity to work ometime i

n en itive area near the groin, but with uitable choice of technique po ition
, and care in handling, thi hould not pre ent too much of a problem. There i
al o the chance of interfering with an organizing haematoma, and the po ibility
of myo iti o ifican , particularly in gracilli and to a le er extent in the
quadricep , mu t be con idered.
234
Technique for the thigh area
30.2 Stretching lateral a pect of thigh idelying A it i impo ible to adduct
the thigh more than a mall amount to tretch the lateral ti ue , direct tretc
h with pre ure i ometime the only way to get to the e ti ue . Here the oper
ator ha adducted the patient thigh a much a po ible, and then while holdin
g back on the uperior part, i applying a longitudinal tretch with the other h
and. A the ilio-tibial band i largely non-exten ible, very little tretch can
be produced, and thi procedure can be very uncomfortable. Thi i one of the fe
w o teopathic technique where the u e of a lubricant oil or cream may make the
technique more effective. Tip : Mo t u eful in evere ca e of o teoarthro i of
the hip where it i not po ible to mobilize the hip directly. Lea t u eful whe
n there are fibrou band in the fa cia lata which are very tender. Extra con id
eration : Change the angle of the hip flexion to find the optimum in each ca e.
30.1 Kneading anterior thigh upine From
the oppo ite or ame ide of the table the operator can apply a cro -fibre knea
ding to the quadricep by concentrating either on the hand pu hing away from him
elf, the hand pulling toward him elf, or a bit of both according to the need
of the ti ue. The more the work i hared by both hand , the le the amplitude
of movement with each hand, and therefore the le di comfort for the patient.
Tip : Mo t u eful where the mu cle are not very well developed a it i po ibl
e to get into the belly of the mu cle well. Lea t u eful with large trong mu cl
e and very tight fa cia. Extra con ideration : Deep and low produce the be t
re ult a the mu cle ha time to relax, and fluid interchange take place.
Technique for the thigh area
235
30.3 Kneading lateral and anterior mu cle
of thigh From behind the patient the operator ha gra ped the mu cle on the lat
eral ide of the thigh and, while applying a pu hing force with the thumb , i p
ulling with the finger to produce a cro -fibre kneading to the area. There are
many variation of hand hold that can be u ed here. Tip : Mo t u eful where the
patient can take direct kneading on the mu cle and where the fa cia i mobile
enough to accept tretch in thi way. Lea t u eful where the patient ha any pro
blem with the other ide making idelying difficult. Extra con ideration : Vary
the angle of the hip for optimum ten ion, fix hand and u e body movement rathe
r than hand movement alone.
30.4 Kneading medial or po terior mu cle
of thigh upine With the patient knee and hip flexed and firmly held again t t
he operator abdomen, a cro -fibre kneading action i being performed. The mor
e the work i hared by both hand , the le the di comfort of the movement, and
the deeper the technique can be applied to produce a better and quicker re ult.
With the hip and knee flexed, the mu cle are off ten ion and therefore ea ier
to work in mo t ca e . Tip : Mo t u eful in ca e where the patient cannot exten
d the hip fully. Lea t u eful when the patient i very tickli h. Extra con idera
tion : It may be nece ary to experiment to find whether it i better in a parti
cular ca e to fix with one hand and move with the other or to work both hand .

30.5 Kneading thigh upine Here the operator

ha fixed the anterior part of the thigh to him elf, and i holding the patient
foot into the table at varied angle of flexion of the knee to optimize the te
chnique. The other hand i applying the cro fibre kneading to the adductor mu c
le . In thi po ition it i po ible to reach well up to the origin of the mu cl
e . Tip : Mo t u eful where there are tight band in the adductor mu cle , parti
cularly clo e to the pubi . Lea t u eful when the patient may feel threatened by
thi po ition. Extra con ideration : Sometime the kneading hand can be held t
ill and the operator and hi other hand moved again t it.
236
Technique for the thigh area
30.6 Kneading thigh idelying The operator
i in front of the patient and i working on the medial a pect of the leg on the
table. Both hand are working in oppo ite direction to minimize the amplitude
of force in each. In thi po ition the upper thigh protect  the groin area and
it i po ible to reach very high up into the origin of the mu cle ; thi al o
allow the pubic ramu to be reached without exce ive embarra ment to the pat
ient. Tip : Mo t u eful where it i nece ary to apply the technique high in the
adductor . Lea t u eful where the patient ha difficulty idelying. Extra con i
deration : Change in the angle of flexion of the knee and hip can make it po i
ble to reach different part of the mu cle .
30.7 Kneading thigh medial a pect idelying
The operator i tanding behind the patient and u ing the fingertip to work dee
p into the mu cle . It i po ible to get behind the quadricep in thi po ition
, and right up to the adductor mu cle origin to the pubi a the patient othe
r thigh protect  the groin area. Tip : Mo t u eful where it i nece ary to re
ach deep into the mu cle . Lea t u eful where the mu cle are extremely tender.
Extra con ideration : Find which work be t in each ca e, either pu hing or pull
ing.
Technique for the thigh area
237
30.9 Kneading thigh prone Thi po ition en30.8 Kneading thigh prone The operator
i
tanding on the ame ide a the leg being worked on. The medial and po terior a
pect of the thigh can be reached in thi po ition. According to the need of t
he ca e and the preference of the operator thi can be made into a pulling techn
ique or a pu hing technique a de ired. A in all uch technique , the more the
work i hared between the hand , the le amplitude need be applied with each o
ne a the conjoint effort produce the re ult. Tip : Mo t u eful where mu cle a
re fairly lack a it i po ible to work deeply into them. Lea t u eful where m
u cle are extremely tight, a the technique will be very uncomfortable. Extra c
on ideration : Vary the angle of knee flexion if nece ary, with a pillow. able
the operator to work on the po terior and lateral a pect of the thigh on the op
po ite ide from where he i tanding. A u ual with thi ort of technique, the
hand are performing oppo ite action o that the work i hared and thi reduc
e the amplitude of the force and the di tortion of the ti ue accordingly. Ti
p : Mo t u eful in maller patient where leaning acro the table i not too mu
ch of a tretch. Lea t u eful in large ubject or where the patient find lying
prone difficult. Extra con ideration : Vary the angle of flexion at the knee an
d abduction at the hip for be t re ult .
238

Technique for the thigh area

30.10 Mu cle energy po terior thigh The
lower extremity i taken to a point of ten ion in the po terior mu cle and held
there. The patient i a ked to pu h the leg again t the houlder for about ix
econd , and then after relaxing, a new motion barrier i found at a greater ang
le of flexion of the hip. After a few econd thi i repeated, u ually three ti
me to achieve a tate of enhanced tretch of the mu cle and a new barrier to m
otion at a greater range of joint motion. A with all mu cle energy procedure ,
the re i tance need not be more than about 2 5 % of mu cle force. Tip : Mo t u e
ful in chronic contracture where direct work on the mu cle may be too uncomforta
ble. Lea t u eful in acute mu cle train a it may
be too painful. Extra con ideration : See the
earlier ection on principle of mu cle energy technique.
30.11 Mu cle energy technique thigh upine The operator i holding the patient
foot again t the table and the patient i a ked to pu h again t the hand either
into flexion of the knee or exten ion a nece ary for the ca e. The other hand
i tabilizing the knee. A in mo t mu cle energy technique , the force applied
by the patient i about 2 5 % of mu cle action, and the po ition i held for ab
out 6 econd , repeated u ually three time with hort re t period between. Tip
: Mo t u eful in mall, light ubject where the operator can re i t ea ily. Le
a t u eful in large or trong ubject where it may be difficult to keep a true
i ometric contraction controlled. Extra con ideration : See earlier ection on m
u cle energy technique.
TECHNIQUES FOR THE KNEE AREA
31
The knee i both a imple and a very complex joint. O teopathic thinking embrace
the concept of mechanical dy function with or without po itional di placement.
The bone making up the joint can be working in one of everal different di tur
bed po ition . Thi i not o much a po itional finding a a lack of function in
variou po ible direction . Technique are de igned to re tore function genera
lly, or to mobilize in a particular direction to re tore normal relation hip . F
unction i een to be more nece ary than ymmetry and po ition. Neverthele , i
n ome ca e , direction of force are cho en for pecific rea on . The e u ually
relate to perception
of mechanical dy function manife ting a apparent malpo itioning. Precaution in
clude the need to avoid exce ive force and pain, a in all other area , but al
o include the need to avoid exce ive pre ure over any varico e vein . Some kne
e yndrome are manife tation of hypermobility ma ked by mu cle protection. If
thi i the ca e, mobilizing technique are not indicated, o careful a e ment
hould precede treatment. If a knee i degenerative it will generally tend to h
ave lo t ome of it normal hyperexten ion, and exce ive attempt to re tore th
i are doomed to failure and will only provoke more pain and problem .
240
Technique for the knee area
31.1 and 31.2 Harmonic technique into circumduction itting The patient it wit
h her leg over the ide of the table. Hold the uperior a pect of the tibia with
one hand and rock the leg from ide to ide with the other. At the ame time ci
rcumduct the foot to induce a rhythmic o cillation in the knee. See earlier ect
ion relating to harmonic technique. Tip : Mo t u eful in ca e where rhythm and
pring ha been lo t in normal knee movement. Harmonic technique i particularly

u eful a a preliminary procedure to tructural mobilizing to loo en the ti ue

and help re-e tabli h circulatory function. Extra con ideration : Light patien
t re i tance can be introduced into thi movement a a mu cle re-training proc
edure.
Technique for the knee area
241
31.3 and 31.4 Harmonic technique into flexion
and exten ion itting The patient it with her leg over the ide of the table.
In truct her to re i t your movement with a light pre ure only. Rock the leg
forward and back to flex and extend the knee. Vary the tart and fini h point o
that the flexion and exten ion movement will be through a larger and maller ar
c. Thi ha the effect of retraining the proprioceptive feedback from the join
t. See earlier ection relating to harmonic technique. 31.5 Articulation patello
-femoral j o i n t upine Thi hold allow mobilization of the patella through a
ll po ible range . The acce ory range of diagonal movement in particular can
be addre ed a relea e of the e often improve overall function. Tip : Mo t u e
ful in the majority of ca e of knee dy function a the patello-femoral joint ma
y be involved and can often be a neglected element. To avoid di comfort, prelimi
nary work on the urrounding oft ti ue i helpful to relea e the area prior t
o thi technique.
242
Technique for the knee area
31.6 Articulation flexion upine Flex the knee
over your hand placed in the popliteal pace. U e your other hand to monitor the
ti ue ten ion to avoid exce ive train. Hold the leg firmly between your fore
arm and body. Circumduction movement can be added o that the flexion become o
ne part of the arc of movement. Tip : Avoid exce ive flexion in the pre ence of
any effu ion unle it i very light.
31.8 Articulation exten ion upine Grip the
knee firmly and hold it again t the table while your other hand brace the foot
and lift the heel. Thi hold ha the advantage over the one hown in photograph
31.7, in that the ga trocnemiu i placed on ome ten ion and will be tretched
if de ired. Tip : Try varying the amount of dor iflexion in the foot to a e
at what point the limitation of exten ion manife t . The earlier thi occur , th
e more the ga trocnemiu will be involved in the lack of exten ion.
31.7 Articulation exten ion upine Grip the
knee firmly and hold it again t the table while your other hand lift the lower
end of the tibia. Tip : Many knee dy function manife t a a lack of hyperexten
ion, particularly internal derangement . Thi movement i a much an a e ment
of ucce ful re toration of function a it i a treatment.
Technique for the knee area
243
31.9 Traction upine Hold back on the femur with your fully pronated hand avoidi
ng the patella. The other hand applie the traction and can vary rotation, abduc
tion and adduction of the tibia a nece ary. Tip : Many internal derangement o
f the knee eem to have ome ort of internal entrapment involved. Traction can
be very helpful in the e in tance .
31.11 Articulation rotation and flexion upine

Hold directly over the joint line with your upper hand and introduce internal an
d external rotation with varied degree of flexion with the other. Tip : Mo t u
eful in ca e of meni cal dy function where there i often a limitation of full
flexion and rotation combined, at one pecific point in the range of movement. E
xtra con ideration : In many ca e of meni cal dy function it i u eful to break
fixation in flexion before trying to re-e tabli h full exten ion.
31.10 Traction prone Fix the femur into the table. Lift the
ankle, with the other hand while introducing different range
ation. Thi can be applied to the near or far leg. It ha the
ine method in that it can be performed in flexion and can be
duction movement at the hip to be more rhythmic.

leg, from above the

of flexion and rot
advantage over up
made into a circum

244
Technique for the knee area
31.12 Thru t meni cu upine Fix the femur
between your hand, the table and the lateral a pect of your thigh. You can then
circumduct the tibia to alternately apply medial and lateral gapping to the knee
. Thi form a very efficient articulation technique and if the fixing hand pre
e directly over the joint line, a firm abduction and exten ion force can often
allow a medial meni cu fixation to be relea ed. If the lateral meni cu i the
target, adduction combined with exten ion i nece ary. Tip : A the hand pre
ure increa e over the joint the available range of abduction and adduction wil
l reduce to a pecific mall arc. Within thi arc will be found the appropriate
barrier to apply the final very hort amplitude force.
31.13 Thru t medial meni cu upine Firmly
cla p the ankle between your body and forearm. Grip around the leg ju t below th
e knee and introduce a circumduction movement. Within thi circumduction there w
ill be a point of ten ion. Thi point u ually occur with a combination of abduc
tion and external rotation of the tibia. At thi point apply a force combining f
urther abduction, external rotation and exten ion. It i more of a flick than a
thru t. Tip : Vi ualize a piece of tring with a weight attached tied around the
knee and hanging down on the lateral ide. Try to flick the weighted tring ove
r to the medial ide harply. Thi give an image of the direction and type of f
orce nece ary.
Technique for the knee area
245
31.14, 31.15 and 31.16 Thru t to medial meni cu
upine Thi erie of photograph how the equence of move normally u ed in t
hi technique. Flex and externally rotate the knee to break fixation in the medi
al meni cu . Gently repeat thi a few time until nearly full flexion i attaine
d. Maintaining the external rotation and ome abduction of the knee, extend it u
ntil the po ition hown in photograph 15. At thi point the final part of the te
chnique take over which i an exten ion, traction and internal rotation movemen
t. At the end of the technique it i important to hold the knee firmly into exte
n ion, to avoid reflex mu cle contraction into flexion that may di lodge the men
i cu again. Tip : Lea t u eful where there i a u pected tear in the meni cu
and the exten ion movement would be very painful. Extra con ideration : It may t
ake everal attempt to produce ufficient flexion to unlock the joint. It may b
e very uncomfortable at the po ition hown in photograph 31.15 where the patient
lo e active control of the joint. It i important to pa through thi point f
airly quickly.
246

Technique for the knee area

31.18 Thru t lateral hift of tibia upine Fix 31.17 Thru t for lateral hift of
tibia upine
Internally rotate the patient thigh lightly and place your infra-patella tend
on again t the mall plateau on the antero-lateral part of the tibia. Your upper
hand i placed under the knee maintaining a mall amount of flexion and holding
the femur over the medial condyle. The lower hand cla p the heel. Focu the fo
rce by pulling the femur toward the fulcrum of your applied knee while circumdu
cting the ankle and tibia. The thru t i a combination of a mall pu h with the
knee and a pull toward you with both hand . Tip : Becau e the operator i pivoti
ng on the middle part of hi tibia again t the ide of the table, it i importan
t to adju t the patient po ition accordingly. If the patient leg i not exte
rnally rotated ufficiently, painful pre ure occur on the head of the fibula.
Have the patient clo e enough to the edge of the table o that your tibia can be
nearly vertical at the tart of the technique. the patient leg between your c
ro ed thigh . Apply your taggered hand to the medial a pect of the femur and
the lateral a pect of the tibia with firm compre ion. Circumduct the knee u ing
both hand and a the point of ten ion build , harply increa e the pre ure wi
th both hand while traightening your knee to produce a traction effect. Tip :
A thi po ition i inherently un table for the operator it may help to lean ag
ain t the ide of the table. Keep the patient knee lightly flexed throughout.
Technique for the knee area
247
31.19 Thru t for medial hift of tibia upine
Fix the patient leg between your cro ed thigh . Apply your taggered hand to
the medial a pect of the tibia and the lateral a pect of the femur. U e firm co
mpre ion and circumduct your hand until ten ion accumulate and then apply the
thru t with both hand at the ame time a traightening your knee to produce
the traction. Tip : Maintain a mall amount of flexion to prevent the patient
knee from locking.
31.20 Thru t for medial hift of tibia and lateral meni cu Fix the abducted leg
between your body
and forearm and grip the knee firmly ju t below the joint. Maintain a mall amou
nt of flexion and firm compre ion and apply a rapid circumduction and lateral g
apping force by rotating your body away from the table. A direct lateral force w
ill tend to correct the tibial hift wherea an abduction force will reach the l
ateral meni cu . Tip : Vi ualize a piece of tring with a weight attached tied a
round the knee, with the weight hanging down medially. The movement of thi tech
nique i to flick the weight from the medial ide over to the lateral ide.
248
Technique for the knee area
31.21 Thru t for lateral hift of tibia and medial
meni cu Place the leg between your body and forearm and cla p the knee firmly b
etween your hand . U e everal mall o cillating movement to prime the joint an
d then apply a mall rotation of your body toward the table to open the medial
ide of the joint. Thi can be a circumduction movement or a medial gapping movem
ent. Tip : Thi technique can be uitable for correcting an internal rotation re
triction of the tibia if the tibia i held firmly with both hand and externall
y rotated at the ame time a the gapping force i applied.
31.23 Thru t for internal rotation fixation of

tibia Firmly cla p the upper end of the tibia between your hand and hold the lo
wer end between your forearm and body. Adduct the thigh to produce a tor ional f
orce into external rotation of the knee. Apply a mall exten ion force to break
fixation into an external rotation direction.
31.22 Thru t for external rotation fixation of
tibia Gra p the tibia firmly between your hand and again t your body with your
forearm. Abduct the thigh and flex the knee lightly and then apply an internal
rotation force by u ing your grip and by idebending your body toward the table.
A mall final exten ion force may be nece ary. Tip : U e the weight of a more
abducted thigh a a re i tance to the internal rotation force.
Technique for the knee area
249
31.24 Thru t uperior tibio-fibula joint upine
Place the metacarpo-phalangeal joint of your index finger behind the fibular hea
d. Adduct the thigh and externally rotate and flex the leg until you feel the fi
bular head gripping your hand. Apply a mall flexion force to the leg while pri
ing the fibular head toward you. Tip : Lea t u eful in the pre ence of degenerat
ive di ea e in the knee that may make extreme flexion painful. Extra con iderati
on : Try varying the di tal hold to grip under the foot if preferred.
31.26 (Thru t uperior tibio-fibular joint idelying Dor iflex the foot lightly
with your thigh while holding the inferior end of the fibula back. Your other h
and applie a compre ion and forward movement to the upper end of the fibula. T
he thru t i applied to the upper end while the lower part i held table or the
lower lever i lightly increa ed. Tip : Lea t u eful where the operator may fi
nd the thru t with the abducted arm difficult.
31.25 Thru t uperior tibio-fibular joint idelying Stand behind the patient and
apply a forward force again t the head of the fibula of the flexed knee. Hold b
ack on the di tal end of the fibula with varied degree of dor iflexion of the f
oot to find the optimum ten ion. Hold the foot firmly and then apply the thru t
again t the fibular head. Tip : Mo t u eful where full flexion of the knee may b
e a problem.
TECHNIQUES FOR THE CALF AREA
32
The calf area i often involved in condition of dy function in the foot and the
knee. Condition of the calf mu cle them elve uch a mu cle tear and direct
trauma ometime need addre ing. Contracture due to po tural condition and oc
cupational di order can al o occur. Particular care hould be taken in phlebiti
or varico e vein ca e where there i the po ibility of thrombo i , haemorrha
ge and haematoma formation. If a clot i pre ent, there i a di tinct po ibilit
y of it being hifted and ub equently lodging in heart, lung or brain with pote
ntially di a trou re ult . Technique to be u ed on the oft ti ue can be cro
-fibre, tretching or mu cle energy.
It i performed with the knee extended to reach the ga trocnemiu , and flexed to
take the ga trocnemiu off tretch and make it ea ier to reach the deeper mu cl
e . Calf mu cle are clo ely concerned with po ture, which mean that there may
be only partly rever ible contracture and fibro i pre ent. Technique hould u
ually be low and deep to produce the be t re ult. Control of rhythm i al o imp
ortant. There may be remote cau e of calf mu cle hypertonicity, uch a nerve r
oot pre ure in the lumbar pine or circulatory deficit in the arterial upply.
Initial attention to the e extraneou cau e hould be con idered to get the be

t re ult from any later work on the calf.

Technique for the calf area
251
32.1 Kneading of lateral calf upine Sit on the end of the table, tabilizing th
e patient foot by re ting your thigh on it. Hold the ankle to prevent movement
of the leg. Apply deep cro -fibre kneading to the calf mu cle with the pad a
nd tip of the finger . Tip : Mo t u eful where it i nece ary to reach the lat
eral mu cle of the calf. Extra con ideration : Try varying the angle of knee fl
exion and rotation. 32.3 Kneading of medial part of calf upine Sit on the table
tabilizing the patient foot with your thigh. Hold again t the knee with your
tabilizing hand. The medial a pect of the calf i available to the fingertip
of the kneading hand. Tip : Mo t u eful when working on the mu culotendinou jun
ction deep in the calf. Try varying the angle of knee flexion and rotation to
achieve the optimum for the technique.
32.2 Kneading of lateral calf upine Sit at the end of the table. Stabilize the
patient foot with your thigh. Fix the knee with one hand and knead the mu cle
around the fibular head with the other. Tip : Mo t u eful when it i nece ary
to tabilize the knee when working on the upper part of the calf. Extra con ider
ation : Try varying the angle of flexion and rotation of the knee to vary the t
en ion on the mu cle before the technique i applied.
252
Technique for the calf area
32.4 Kneading and tretching calf prone Stretch the mu cle by applying a dor if
lexion force to the foot. U e a pulling or pu hing force on the bellie of the m
u cle with the other hand. Tip : Mo t u eful where it i nece ary to get very
deep into the mu cle ti ue. Try circumducting the knee to reach different part
of the mu cle. Try changing the order of the tretch and kneading to produce th
e be t combination of comfort and effectivene .
32.5 Kneading of calf prone Support the tibia on a mall pillow and then apply a
cro -fibre kneading to the calf mu cle with both hand working in oppo ite di
rection . Tip : Try u ing varied ize of pillow or no pillow at all, if it i d
e ired to make the principal effect on the ga trocnemiu .
32.6 Kneading of calf idelying Stand behind the patient and apply oppo ite forc
e with both hand , u ing the thumb to knead the mu cle of the calf. Tip : In
thi hold it i po ible to work the whole length of the calf of either leg with
the thumb . If u e of the fingertip i preferred to u e of the thumb , work fr
om the other ide of the table.
TECHNIQUES FOR THE FOOT
33
Some o teopath neglect foot technique in patient care a they find the techniqu
e difficult to ma ter, and ometime uncomfortable on the hand . Many ca e of
foot dy function receive benefit from general mobilization and ome practitioner
are happy to u e uch method rather than pecific technique . However, pecif
ic technique can often be a hort cut to good re ult , and ave not only time a
nd effort, but can produce a better and longer-la ting relief from mechanical dy
function problem . The foot i the foundation of po ture, and to neglect it i
to leave out a very important part of patient care. Clearly a poor foundation i
not going to help in re olution of mechanic in other part of the body if a pr

actitioner i trying to re tore good function. There are al o local problem of

pain and di turbed mechanic in the foot that can often be uitably addre ed by
o teopathic treatment. A in all area of the body, particular care mu t be tak
en in ca e of potential bone weakne a technique may induce quite trong eff
ect on bone. However, the chance of damage i quite mall, a the tre e u ed
are liable to be le than tho e in normal locomotion. Infection, celluliti an
d inflammatory di order are common in the foot, and will need careful pre-treat
ment a e ment. By it very nature o teopathic treatment i de igned to increa
e mobility. Many ca e of di turbed foot mechanic are due to exce mobility or
hypermobility and, therefore, further mobilization may be unwi e. In

ome ca e , relative hypomobility in one part of the foot will cau e relative hy
permobility in another. Thi i where very pecific treatment to the hypomobile
ection will help to unload the exce ively mobile articulation . Hypomobility
al o eem to have reflex effect in maintenance of hypermobility in adjacent a
rea . If a hypomobile joint i ati factorily mobilized, there i often an immed
iate re toration of ligamentou tone in adjacent, previou ly diagno ed, hypermob
ile joint . Thi i too rapid to be imply a balancing of mechanic , and eem t
o be due to fa cial ten ion alteration and ome proprioceptive feedback mechani
m. Mo t po ition u ed traditionally for thru t technique are perfectly uitabl
e for articulation procedure . Thi i not mentioned each time in the de criptio
n appended to the photograph to avoid repetition. Where a technique i declare
d a thru t, a repetitive articulation i u ually performed a a preliminary, and
if ten ion accumulate to a uitable en e of barrier, the thru t can be perfor
med. If it doe not, repeated articulation may deal with the dy function adequat
ely. A uitable thru t barrier ha a potential for the hort amplitude movement
that implie a quite characteri tic cri pne . Without thi the thru t i not
liable to ucceed a a pecific technique and i be t avoided, a the ti ue ma
y become traumatized. A ucce ful hort amplitude thru t performed well i rare
ly traumatic in the foot. An un ucce ful one may be uncomfortable, although rar
ely damaging.
254
Technique for the foot 33.1 Kneading of ole upine Stabilize the foot with the
cephalic hand and u e your thumb to work on the oft ti ue of the ole. Tip :
Many patient are tickli h and the grip hould be firm but not painful. Kneadin
g i more likely to be of benefit than tretching, a a normal tanding po ture
tretche the ti ue more than any technique i able to do. Try varied degree
of plantar flexion to reach different layer of ti ue. 33.3 Thru t to tibio-tal
ar joint upine Cup the calcaneum and pull the foot into a neutral point between
plantar and dor iflexion. Pu h the tibia with the cephalic hand toward the tabl
e and allow the natural recoil of the ti ue to cau e it to pring back. The pl
ane of the joint i not directly antero-po terior and it will be nece ary to l
ightly internally rotate the hip to produce the optimum re i tance for a thru t.
If there i a barrier, everal priming movement will be nece ary before the t
hru t can be executed. Tip : Mo t u eful where the patient mention the ymptom
that they are unable to achieve comfort in the ankle in any po ition and feel t
hat the joint need to crack. Extra con ideration : When a ati factory thru t
i attained with thi technique, the amplitude of movement within the thru t wi
ll often be greater than expected and po ibly alarming to the practitioner!
33.2 Articulation to di tal tibio-fibular joint upine Stand at the foot of the
table and gra p the tibia and fibula between the index finger and thumb of each
hand. Introduce antero-po terior and light upero-inferior movement . Tip : Mo
t u eful in ca e where fibular mobility i particularly relevant, that i mo t
ankle dy function and ome knee condition . Extra con ideration : Try dor i-fle
xing the foot with the operator thigh to produce the optimum ten ion in the jo
int .

Technique for the foot

255
33.4 Thru t to tibio-talar joint upine Fix the calcaneum with your cro ed thum
b , and grip firmly around the front of the ankle. Thi produce a dor iflexion
component and an antero-po terior component. A you lower the foot toward the ta
ble increa e the firmne of the grip. You can then amplify the hold to apply a
force in an anteropo terior direction to the tibia to eparate the joint. Tip :
Lea t u eful where the operator may have any weakne in the thumb . It i al o
difficult in large feet to maintain the dor iflexion nece ary and to grip aroun
d the foot at the ame time. Extra con ideration : The thru t i performed by th
e operator adducting hi arm at the ame moment a the grip tighten .
33.5 Thru t to tibio-talar joint prone Apply ome pre-ten ion to the joint by gr
ipping around the anterior a pect of the ankle while pulling forward on the calc
aneum. Maintain a degree of dor iflexion of the foot with your forearm and when
the force accumulate, execute a harp adduction of your arm to cau e a gapping
at the joint. Tip : Lea t u eful for maller operator , and for tho e where the
nece ary trength may be a problem. Extra con ideration : Try circumducting th
e leg in the build-up to the technique, o that the perception of continuou ten
ion for the patient i not too uncomfortable.
33.6 Thru t to tibio-talar joint prone Stand to the ide of the prone patient fa
cing toward the foot of the table. Grip round the front of the ankle a you pu h
forward with your cro ed thumb behind the calcaneum. Rock the patient leg f
orward and back. When ten ion accumulate empha ize the imultaneou pull and pu
h to gap the joint. Tip : The thumb can al o vary the dor iflexion to focu th
e technique more accurately.
256
Technique for the foot 33.7 Thru t to tibio-talar joint prone Put the patient
foot over the end of the table and fix the tibia with your cephalic hand while
taking up the lack with the other hand pu hing down on the calcaneum. You can v
ary the dor iflexion to achieve the optimum ten ion and then apply the thru t to
ward the floor. Tip : Mo t u eful for mall operator a thi technique relie o
n application of weight rather than u e of lever and force. Extra con ideration
: Try u ing the operator inner thigh to produce the relevant amount of dor if
lexion.
33.8 Traction to tibio-talar joint upine Brace your elbow again t your flexed k
nee on the table under the patient flexed knee. U e your lower forearm a a fi
xed lever while your upper hand pull the foot into dor iflexion and drive it
traight down over the edge of the table. The larger the pace between your elbow
and the patient knee the tronger the lever will be to open the tibio-talar a
rticulation. Tip : Try gently mobilizing the foot in circumduction while it i u
nder traction.
Technique for the foot
257
33.9 Traction to tibio-talar joint upine Brace your elbow again t the back of t
he patient flexed thigh. Cup the calcaneum in the ame hand and fix the talu
and the re t of the foot with the other hand. Traction i produced by maintainin
g the e hold and imply leaning back. The pu h of the patient flexing thigh a
gain t your elbow perform the technique. Tip : Lea t u eful where the operator
arm i very hort and the patient leg i very long. Extra con ideration : Tr
y abducting the hip further a nece ary or placing a pad between elbow and thig

h. 33.10, 33.11 and 33.12 ( ee al o next page, top left and bottom left) Articul
ation to ub-talar joint upine Thi erie how the overlapping finger hand
hold in the hape of a letter  W ; then the hand applied to the foot can produ
ce inver ion, ever ion and circumduction to influence the ub-talar joint . The
operator viewpoint photograph how the pre ure being applied with one thenar e
minence and the oppo ite hypothenar eminence. Thi i then rever ed to gap the m
edial and lateral ide of the joint re pectively. Tip : Try varying the angle o
f dor iflexion u ing the operator abdomen again t the ole of the foot. Extra
con ideration : Thi hold require a firmer grip than may be apparent; it al o r
equire a good control of rhythm.
258
Technique for the foot
33.13 Articulation to ub-talar joint prone Brace the foot again t your lower c
he t. Fix the finger of both hand over the medial ide of the foot while gripp
ing firmly under the lateral maleolu . Rock forward over the foot to put ten ion
on the medial a pect of the joint. Then rotate your body to alternate the tre
applied to the anterior and po terior part of the joint. Tip : Lea t u eful f
or the maller operator who may find the reach a problem. Extra con ideration :
A firm grip i nece ary if thi technique i to be effective.
Technique for the foot
259
33.14 Articulation to ub-talar joint idelying The patient lie on the affected
ide and the operator fixe the dor iflexed foot again t hi inner thigh. Hold
it firmly again t the table with the fixing hand and rock the calcaneum into inv
er ion and ever ion. Dy functional area of the ub-talar joint are reached by v
arying the angle of movement. Tip : Thi technique will only gap the medial a p
ect of the joint. Extra con ideration : If the pre ure on the table i uncomfor
table, try interpo ing a pillow between foot and table. 33.15 (bottom left) Arti
culation to ub-talar joint idelying In thi operator viewpoint photograph the
patient i lying on the unaffected ide and the operator i gripping medially on
the di tal part of the calcaneum. Place your thumb ju t under the lateral male
olu . Maintain a firm grip and lean forward until ten ion i felt to build in th
e joint. The medial ide of the joint i being tretched. Tip : Try varying the
angle of dor iflexion to reach different urface of the joint. 33.16 ( ee next
page, top left) Thru t to ubtalar joint idelying The patient lie on the affec
ted ide with the knee flexed. Fix the foot into dor iflexion to tabilize the a
nkle. Fix the calcaneum to the table, and with your other hand invert the foot b
y fixing on the navicula and pu hing up along the long axi of the tibia. Ten io
n hould accumulate in the ub-talar joint. Rock the foot between the hand unti
l the optimum barrier i en ed. Maintain the pre ure and thru t the whole foot
toward the table thereby applying a gapping force to the target joint. Tip : If
the ten ion i correct the foot will rock back and forth like a aucer rocking
from edge to edge. According to whether the anterior or po terior of the joint i
dy functional, the ten ion will be felt better with the proximal or the di tal
hand. Extra con ideration : If thi po ition i very painful, try working from
the lateral ide of the foot a in next technique illu tration.
260
Technique for the foot 33.17 and 33.18 Thru t to ub-talar joint idelying The
patient lie on the unaffected ide. Pull the medial ide of the dor iflexed foo
t into inver ion with your di tal hand. Maintain pre ure toward the table with
your other hand cupping the lateral maleolu . The calcaneum i , therefore, a ful
crum. Thi ha the effect of putting a tre on the ubtalar joint on the media

l a pect of the foot. Vary the degree of abduction with your di tal hand until a
uitable barrier i felt, and then apply a thru t with your proximal hand to ga
p the joint. Tip : Thi may be u eful when the oppo ite idelying po ition i a
problem. Note that the econd photograph how the di tal hand in the final po i
tion. The wri t i now traight and the foot inverted and abducted. Firm compre
ion i nece ary if the force i not to be di ipated in the ti ue generally.
The ub-talar joint i extremely trong, and if the compre ion i not firm eno
ugh, the technique will be ineffective.
Technique for the foot
261
33.19 and 33.20 Articulation mid tar u upine The e operator viewpoint photogra
ph how a hold for applying general mobilization to the mid foot. The thumb ar
e aligned along the haft of one or more of the metatar al . A tor ional force
i introduced to direct the articulation force to the joint to be mobilized. Ti
p : Try varying the pacing between the thumb or making them more proximal or d
i tal to reach different part of the foot.
33.21, 33.22, 33.23 and 33.24 ( ee next page) Articulation mid tar u upine Thi
erie of photograph how the o-called figure of eight technique in it va
riou pha e . Take the foot through an imaginary figure of eight, in a variety o
f plane , either vertically, horizontally or diagonally. Ten ion accumulate at
the cro -over in the middle or at the outer edge of the figure a direction c
hange. Vi ualize the eight a having a omewhat flat top and bottom. Tip : Mo t
u eful in almo t all ca e of dy function cau ing re tricted mobility in the foo
t. Extra con ideration : Quite firm compre ion i u ually nece ary to the meta
tar al head by the operator lower abdomen to help focu the technique.
262
Technique for the foot
Technique for the foot
263
33.25 Articulation mid tar u and fore foot upine The hand gra p firmly around
the medial and lateral a pect of the foot. The foot i dor iflexed to tabiliz
e the ankle. One hand can tabilize while the other mobilize , or they can both
drive the metatar al up in the centre of the foot by fingertip pre ure. At the
ame time the heel of the hand drive down the border of the foot. Tip : Try
introducing twi ting force to direct the lever to different part of the foot.
With traction and firm moulding of the foot it i po ible to reach mo t of the
mid foot articulation .
33.26 Thru t to mid tar u upine Fix over the target joint with the centre of y
our interlocked hand . Apply ome traction to put the area on ten ion. Then pre
ad the foot with the thumb to allow ome pace for the bone you wi h to manipul
ate. The mid tar al bone are omewhat wedge haped and need a pace to fall in
to. The thru t i performed with a hort tug in the long axi of the tibia. Tip
: Try u ing light variation of abduction and adduction while accumulating the
optimum force .
264
Technique for the foot
33.27 Thru t to middle or lateral cuneiform upine Shake hand with the foot, th

at i right hand to right foot, or left to left. Maintain the foot in a neutral
po ition between dor iflexion and plantarflexion by levering up with the back of
your hand on the table. The other hand applie a firm pre ure down on the cune
iform u ing your pi iform a the applicator. En ure that the tendon are pu hed
to one ide to avoid exce di comfort. Balance the ten ion between the hand an
d adju t the inver ion, ever ion, traction and abduction component with the und
erneath hand. The upper hand maintain the firm pre ure, and at the optimum mom
ent apply a thru t force to the cuneiform toward the heel. Tip : Try varying th
e knee flexion to find the optimum en e of ten ion in the foot. It generally he
lp to have the foot clo er to the ide of the table than the hip o that the le
g doe not fall into external rotation.
33.28 Thru t to middle and lateral cuneiform upine Thi technique u e a imil
ar principle to the one hown in photograph 33.27. The difference i that the op
erator i u ing a reinforced pi iform while a pad i performing the dor iflexion
re i tance. Tip : Mo t u eful where the operator may not be able to develop the
force ufficiently with the ingle-handed grip. The di advantage i that part o
f the control of the inver ion, ever ion and traction i lo t.
Technique for the foot
265
33.29, 33.30 and 33.31 Thru t to middle or lateral cuneiform upine The e three
equence photograph how the foot pulled into dor iflexion to lock the ankle. T
he upper hand then fixe the tibia to the table. The thru ting hand i then very
rapidly pronated, and before the foot can drop into plantarflexion, it applie
a force to the cuneiform with the thenar eminence. Note: Thi require very fa t
movement by the operator to reach the cuneiform before the foot drop . Very few
operator can achieve the amount of peed nece ary, but if it can be developed
, thi i an extremely effective technique. If the thru t i performed on the cu
neiform after the foot ha dropped into plantarflexion, it may be very uncomfort
able on the ti ue on the front of the ankle.
266
Technique for the foot 33.32 and 33.33 Articulation of medial border prone The
foot i gra ped with the target joint between the index finger of one hand and t
he fifth finger of the other. Fix the lateral border of the foot into your che t
. Apply a dor iflexion force with your di tal hand, while holding a plantarflexi
on force with the other. Then rever e the movement. Tip : Try u ing abduction or
adduction, a well a traction, to amplify the primary lever . Extra con iderat
ion : If a uitable barrier accumulate , thi hold can develop ufficient ten io
n to perform a thru t.
33.34 Articulation of lateral border Sit on the table and re t the patient foo
t on your thigh. Fix the ankle with the in ide of your wri t and cla p around th
e lateral border of the foot with the ame hand. The other hand reache under th
e foot and pull the fourth and fifth metatar al into plantarflexion. The direc
tion of force of both hand can be rever ed to produce the articulating force.
Tip : The itting po ition cradling the foot can be quite a u eful one for gener
al mobilization a an alternative to tanding.
Technique for the foot
267
33.35 and 33.36 Thru t to medial border Dor iflex the foot to tabilize the ankl
e and fix the foot into the table with the tabilizing hand. Place the index fin
ger of the tabilizing hand under either the talu , navicula or cuneiform accord

ing to which joint i being mobilized. Apply a buckling force toward the table w
ith the thru ting hand and a combination of plantarflexion, ever ion and abducti
on to focu at the target joint. Tip : Several priming movement are often nece
ary to produce the optimum potential for the thru t.
268
Technique for the foot
33.39 Articulation of medial border Sit on the table o that you can apply a ver
y pecific force to articulate the joint . The hand applied to the ole pu he t
he forefoot into dor iflexion and varied degree of inver ion, ever ion, abducti
on and adduction while the other hand fixe pecifically, proximal to the target
joint. Tip : Although thi hold doe not allow a very powerful articulation it
can be extremely pecific. The itting po ition can be a u eful variation for th
e operator.
33.40 Articulation of lateral border Sit and tabilize the calcaneum or cuboid w
ith the proximal hand. The di tal hand articulate either the cuboid, or the fou
rth and fifth metatar al on the cuboid. Form a fulcrum with the thumb under th
e foot or the fingertip on the dor um. Tip : Try introducing traction and direc
t dor al to plantar movement.
33.37 and 33.38 ( ee previou page, top right and bottom right) Thru t to medial
border Stabilize the foot again t the table holding the hind foot in dor iflexi
on to lock the ankle. Supinate the other hand and pull the medial border of the
foot into plantarflexion, abduction and ever ion. Rever e the direction of force
and then, when a uitable ten ion i developed, execute the thru t with both h
and . The tabilizing hand fixe on either the talu , navicula or cuneiform acco
rding to the target joint. Tip : It may be ea ier to develop the ever ion requir
ed with thi hold than with ome other , a the heel of the thru ting hand can b
e u ed.
Technique for the foot
269
33.41 Articulation of mid tar u upine Sit on the table and reach around the fo
ot with both hand to fix the target joint between the cla ping finger and thum
b . Traction, circumduction, inver ion, ever ion, abduction and adduction are po
ible in thi hold. Although thi i not a very trong hold it i highly pecif
ic and can have a u eful influence to re-introduce mobility in the e otherwi e i
nacce ible joint . 33.43 Articulation of lateral border upine Form a fulcrum e
ither under the foot with the thumb or on the dor um with the fingertip of the
proximal hand. Apply traction, compre ion, abduction, adduction or circumductio
n to the third, fourth or fifth metatar al with the di tal hand. Thi will arti
culate the lateral cuneiform, cuboid and ome of the mid tar al joint . Tip : Le
a t u eful with large feet a the reach may be a problem.
33.42 Articulation of cuboid upine Sit and cla p either the calcaneum or the cu
boid to apply traction and adduction with different degree of dor iflexion and
plantarflexion. Fix the foot again t your abdomen to help block movement in the
re t of the foot. Tip : Mo t u eful in very mobile feet where it may be difficul
t to i olate the cuboid.
270
Technique for the foot
33.44 and 33.45 Thru t to cuboid upine Pull up on the fourth and fifth metatar

al with your fingertip . Apply the pad of the other thumb under the medial bord
er of the cuboid. The thumb become a fulcrum over which you can plantarflex, in
vert and lightly adduct the foot. A ten ion accumulate you can amplify the e
component and add a compre ion force toward the table. The thumb forming the f
ulcrum hould remain in a little abduction a po ible to avoid training it. A
lthough the thumb applie a light increa e of force the thru t come principall
y from the other hand. At the end of the thru t both elbow hould be clo e to t
he operator ide . Tip : Lea t u eful where operator thumb trength may be u
pect. The technique could be modified o that the thenar or hypothenar eminence
could be ub tituted. Extra con ideration : If it prove impo ible to build a
uitable barrier, try lifting the whole leg off the table then bring it down ha
rply and, a the heel hit the table, execute the thru t. The momentum component
may allow the barrier to be acce ed more effectively.
Technique for the foot
271
33.46 Thru t to cuboid prone Pull the foot into dor iflexion with the di tal han
d and fix the other thumb toward the table on the medial border of the cuboid. K
eep the arm clo e to the ide, hrug the houlder of the di tal hand to produce
an inver ion of the lateral border of the foot with the heel of your hand. Tip :
Lea t u eful where thumb trength i a problem but try ub tituting the thenar
or hypothenar eminence . Extra con ideration : Try circumducting the operator
body and the patient lower extremity until the optimum thru t point i en ed.
33.47 (bottom left) Thru t to cuboid prone Cro the thumb under the cuboid an
d dor iflex the foot. Maintain the thumb pre ure and plantarflex, invert and ad
duct the foot with ome c o m pre ion. Thi hold can be applied to mo t of the
mid tar al joint by changing the po ition of the cro ed thumb . Tip : En ure t
he ankle i maintained in dor iflexion throughout a otherwi e the force will di
ipate through the ankle joint and the mid tar al joint will tend to lock in p
lantarflexion.
33.48 and 33.49 ( ee next page, top left and bottom left) Thru t to cuboid prone
leg over ide Cro the thumb and apply them under the cuboid. Overlap the fin
ger on the dor um of the foot and pu h with both thenar eminence again t the
ole to maintain dor iflexion at the ankle. Keep dor iflexion con tant, and flex
and extend the patient hip and knee. Maintain the tibia in an almo t horizonta
l po ition and pu h the leg away and allow it to recoil. Midway through one of t
he e recoil movement drive the thumb again t the cuboid while the finger prod
uce inver ion, adduction and compre ion. Tip : Lea t u eful in patient who may
find the po ition a problem. Extra con ideration : The coordination nece ary f
or thi technique i rather difficult. Note that it i applied a the tibia come
toward the operator, not away. If it i applied a the tibia move away it be
come an unde irable plantarflexion thru t. It i po ible to perform thi thru
t in imple plantarflexion but there are everal di advantage in thi ; greater
force will be required, it will not be a pecific, and the ankle will come unde
r ome train.
272
Technique for the foot
33.50 Articulation to metatar o-phalangeal joint of hallux upine Sit and re t t
he foot acro your thigh. Fix the foot, particularly the fir t metatar al, with
your proximal hand and apply a traction and circumduction force to the fir t me
tatar o-phalangeal joint. Tip : By gripping the di tal end of the fir t metatar
al rather than the phalange the traction force will reach into the medial border
of the foot.

Technique for the foot

273
33.51 Traction to hallux upine Grip the fir t toe between your flexed finger a
nd hold back on the re t of the foot. Thi hold permit the u e of traction, cir
cumduction and rotation. Tip : Thi grip allow a more comfortable hold than a
imple cla ping of the toe with finger and thumb.
33.52 Thru t to metatar o-phalangeal joint upine Fix on the body of the foot a
nd apply a traction, then plantarflexion force to the toe. Small amount of circ
umduction and rotation help localize the force. Tip : Try u ing the finger under
the toe a a fulcrum rather than ju t u ing traction or flexion.

Adjuvant: 2mo greater urvival, 40% reop for necro i (BTCG 8701; 94) Lapierre 1
997: RPT. No change in urvival but u ed ingle catheter. 4 pha e II trial did
how benefit (18-23mo). Chemotherapy No agent hown to increa e MS ignificantly
. Metaanaly i for all agent howed 2mo increa e in MS, increa ed 6mo urvival.
(StewartLA Lancet 02) Temodar: o EORTC: RPT. Addition of Temodar to XRT ( tarti
ng imultaneou ly and continuing 6mo po t-XRT) increa ed MS from 12.1 to 14.6mo
and 2YS from 10% to 26%. o Metaanaly i : No change in MS; longer PFS, improved
QOL (Dinne BJC02). o Only RPT howed no advantage over procarbazine (Yung, BJC
02). o Retro pective tudie : Improved TTP, PFS, MS in pt >65yo (Brande Cancer
03). Neoadjuvant 70% re pon e or table (Gilbert NO02). Given concurrent and po
t-XRT 31% 2YS (Stupp03) o 3% Leukopenia. BCNU: o No increa e in MS; 1 year urvi
val increa ed by 15%; o 40% re pon e rate ha never been urpa ed; however all
urvivor >3yr have received BCNU. o Re pon e better <40yo. o Progre ion durin
g XRT predictive of BCNU failure. o Adjuvant Tx for re idual tumor, <60yo, tabl
e during XRT; if no re idual may wait for recurrence. May be u ed a alvage tx
>60yo. Glidel: Exclu ion: bilateral tumor. Initial and recurrent: increa e MS b
y 2mo ( ignificant for GBM only when adju ted for other factor .), KPS decline
delayed, 11 of 13 long-term urvivor , CSF leak 5% (v 1%) (Brem Lancet 95, Valt
onenS N97, We tphalM NO03, N4/03). May mimic ab ce on po top MRI . Reimplantat
ion at ub equent urgery po ible. No contraindication to y temic chemotherap
y after Gliadel. Communication with CSF i not a contraindication. Only approved
for up to 8 wafer . May exclude patient from clinincal trial . PCV: Initial rep
ort howed benefit in AA (LevinVA 90), not confirmed by RPT (MRCBTWP JCO 01) Rec
urrence Re-re ection done if fea ible and KPS >70; MS 9mo . High quality urviva
l after reop: AA 21mo , GBM 2.5mo . Progno i AA: MS 2-3yr Factor : Age, KPS, E
xtent of re ection. Survival (GR 3 & 4): XRT only=9.5mo; XRT+BCNU=11mo (avg. BTS
G RTOG-ETOG) Glioma Outcome Project (Law 2003): 41wk . Survival of primary v
econdary GBM controver ial. FDG-PET: inc glu 5mo, dec glu 19mo urvival PET bet
ter indicator than hi tology? CBF not ignificant P53 effect equivocal. MIB-1 le
clear than in LGA . RTOG 93: Age (<>50yo), hi tology, KPS, mental tatu , dur
ation of ymptom (<> 3mo), extent of re ection, neurologic function, RT do e (<
>54Gy) 10% have LOH 1p better progno i 10-25% incidence CSF eeding 10yr urviv
al 0.5% (Salford 88), 3yr 2% (Scott 98). Children 5y 25%, 10y 10% Long-term u
rvivor had higher p53, lower EGFR and MIB-1
Giant cell Gliobla toma
Giant cell : not dividing, are nonmalignant Slightly better progno i . Report o
f cure exi t with lobectomy & XRT.

Glio arcoma
2% of GBM. AKA Feigin tumor Superficial, dural inva ion Firm, circum cribed Fa i

cle of pindle cell arcoma 30% meta ta ize Sarcoma come from ve el , meninge
al fibrobla t
Flotte Outline of Neuro urgery Pilocytic A trocytoma
9
Hi tology Bipha ic compo ed of 2 cell type : o 1) compact bipolar cell with Ro
enthal fiber (intracytopla mic compo ed of cry talin, bright blue on Luxol fa t b
lue, may be ab ent ) o 2) loo e multipolar cell with microcy t and eo inophili
c granular bodie (al o een in PXA ) Hyalinized Ve el . Occa ional oligodendro
glial-like cell , plump protopla mic cell . Often calcify. Necro i , va cular prol
iferation, occa ional mito e are not indicative of malignancy - hould be con i
dered anapla tic pilocytic a trocytoma Rarely undergo malignant tran formation (mo
t had undergone previou XRT) but progno i better Can invade ubarachnoid pac
e - no progno tic ignificance, although ome di eminate No identifying marker
64% infiltrate urrounding brain Unclear if there i a diffu e variant Win ton A:
microcy t , Ro enthal, oligodendro- glioma foci (94% 10y ) = juvenile variant Win
ton B: p eudoro ette , inc cellularity, mito i , calcification, no cy t (29% 1
0y ) = Adult variant Pilomyxoid A trocytoma : Hypothalamic/chia matic, 2mo to 7yo
(mean 18mo), monomorphou piloid cell in myxoid background, angiocentric (periv
a cular clu tering) not bipha ic, no Ro enthal fiber , more mito e , myxoid, mor
e likely to recur or CSF eed Genetic : do not have ame genetic change a diff
u e LGA (i.e. p53 mutation are rare) Clinical Age: cerebellar 10yr , cerebral 2
2yr Can occur in any age (70 yo ) Seizure uncommon (compared to LGA) Radiology
Cy t wall: nonenhancing 10% had tumor by Bx, enhancing 70% had tumor 46% cy tic
non-enhanced, 21% cy tic enhanced, 17% olid, 16% fal e cy t (v Lee 89 - all e
nhance; KL - 90% enhance) Location Cerebellar: 70% vermi , 30% hemi pheric Cere
bral hemi phere : May dedifferentiate, more malignant, adult more common, uncir
cum cribed, wor e progno i Hypothalamic/optic: Propen ity for CSF eeding; po t
erior = more malignant. 20% will have NF-1, mu t rule out Brain tem Treatment Pr
imary treatment i urgical GTR: No XRT; erial MRI for 10 yr STR: XRT controv
er ial (mo t tudie how no benefit). Variou chemo regimen u ed (mo t tudie
mixed with LGA) SRS: U ed in mall erie , all pt tabilized Optic nerve: re e
ction; Chia mal: Biop y + XRT Progno i 100% urvival after GTR. STR 80% 20y . S
tability may be maintained for decade - natural hi tory unclear >8 reported ca
e of regre ion, can alternate between progre ion and regre ion. Tumor may r
egre after partial re ection. Recurrence Cerebellar: 5% GTR, 30% STR All locat
ion : 25% GTR, 80% STR Recurrence can occur 4mo to 45yr after GTR (mean 4yr)
Pleomorphic Xanthoa trocytoma
Clinical Peak 7-25yr (range 3-62yr ) Hi tology WHO Grade II Pleomorphic lipidiz
ed GFAP (+) cell with reticulin ba ement membrane, lymphocytic infiltrate , mul
tinucleated cell & eo inophilic granular bodie PXA with anapla tic feature (Gra
de III): Rare. Mito e (>5/10HPF) and/or necro i (Note PXA with necro i i not
con idered GBM )
Flotte Outline of Neuro urgery
Po tulated that they ari e from ubpial a trocyte Some ynaptophy in (+) and o
me are GFAP (-); 15 reported ca e of compo ite PXA/Ganglioglioma Epithelial and
angiomatou variant MIB-1 u ually <1% p53 mutation in only 25% (v LGA) no co
n i tent mutation Imaging U ually uperficial; temporal/ parietal, but reported
in thalamu , cerebellum (2%) Cy tic with mural nodule, trongly enhancing Treat
ment/Progno i GTR may cure Con ider XRT for STR (no evidence) Chemo u ually not
given Survival: 81% 5y , 70% 10y . 10-40yr tability common. 15-20% behave aggr
e ively Mo t important progno tic factor i extent of re ection. Other : mito e
, necro i
10

Subependymal Giant Cell A trocytoma

Alway a ociated with Tuberou Sclero i No hi tologic grading y tem Neuronal
& Glial Antigen : S100+; 50% GFAP+ Ari e from immature germinal matrix cell Tr
eatment GTR cure STR may tabilize
Brain Stem Glioma
Ep tein cla ification: Pontine/Diffu e = 100% = GBM; Cervicomedullary/Medulla =
91% LGA; Dor al exophytic = 75% LGA Treatment Focal/exophytic/cervicomedullary
= urgery Diffu e pontine = focal XRT >50Gy; need for biop y i controver ial Bi
op y: 50% nondiagno tic (Stroink 86); 3-6% M/M. 20% are nonneopla tic (MS, AVM)
SRS: 14-35Gy; 7pt - all alive at 6 yr Chemo: Adjuvant - no benefit; recurrence
: mall trial , LC 0-33%, Tamoxifen, etopo ide mo t promi ing Progno i : 5y (w/
XRT) 30% CSF pread 20%
Oligodendroglioma
Epidemiology Peak: 40yo 6% occur in childhood Seizure in 87% Hi tology Fried-eg
g cytopla m: artifact of formalin-fixation/paraffin-embedding, not een in frozen
ection Moderately celluar with homogeneou nuclei and clear wollen cytopla m
Al o: microcalcification , cy tic degeneration, den e capillary network Mini-gem
i tocyte : GFAP+ (tran itional between a trocyte and oligodendrocyte?); Do not c
orrelate with urvival Nuclear atypia and occa ional mi to e not indicative of
progno i (Anapla tic oligo: Significant mito e , microva cular proliferation, n
ecro i ) Differential: Clear cell ependymoma, central neurocytoma, DNET 8% hemor
rhage (v 2% LGA, 6% GBM) No good immunohi tochemical marker LOH 1p, 19q. Each
occur in 65%, both occur in 40%. Indicative of re pon e to PCV. o 30% have a tro
cytic mutation - ie p53 mutually exclu ive with 1p/19q Imaging 50-60% in frontal
lobe . <10% infratentorial
Flotte Outline of Neuro urgery
11
Calcification occur in 70% 50% how minimal enhancement Treatment Surgery Role
of urgery i controver ial: Shaw 92: GTR better urvival than STR (12.6 v 4.9
yr ); upported by Mork 85, Lindegaard 87, Whitton 90, Celli 94; not by: Sun 88
Celli 94: upport ob ervation if pre enting with eizure only and calcified, l
ocalized, nonenhancing le ion After GTR can ob erve Radiation Remain controver
ial: 9 tudie beneficial, 4 tudie no benefit, 1 wait until recurrence o No pr
o pective, randomized tudie . Metaanaly i : 14% increa e 5y o Shaw 92: STR + X
RT. <50Gy: 4.5yr MS; >50Gy: 7.9 yr MS General practice: STR or biop y with bad h
i tologic feature give 50-60Gy Chemotherapy Only mall erie and anecdotal eve
idence upport low-grade oligo re pon e to PCV (a oppo ed to anapla tic oligo w
ith LOH 1p,19q) Progno i Median urvival 10yr (Shaw 92) Factor : young age, fr
ontal location, KPS, no enhancement, complete removal
Anapla tic Oligodendroglioma
Hi tology: necro i , mito e , cellularity, nuclear atypia, microvacular prolifer
ation Enhance more frequently Treatment: o Surgery + focal XRT >50Gy + PCV o PCV
: With LOH 1p or 1p+19q mean urvival 10yr (v 2 yr without). 50% complete re
pon e to PCV (rare without). 19q alone wa not a ociated with re pon e (Cairncr
o 98) o RTOG 94-02: XRT v XRT + PCV for AO, AOA Survival 35mo ; 19% 5y

Flotte Outline of Neuro urgery Mixed Glioma Oligoa trocytoma

>20% of neopla tic cell are a trocytic Only 14% calcify (v 70% of oligodendrog
lioma ) Survival 6 yr Progno i lightly wor e than for pure oligo Survival 3y
r Treatment ame a anapla tic oligodendroglioma

12
Anapla tic Oligoa trocytoma
Age: bimodal, peak at 1-5yr and 35 yr 66% infratentorial Can occur anywhere i
n ventricle , central canal of pinal cord Hi tology Ro ette : Dont affect progno
i . o True ependymal: lumen with blepharopla t . o P eudoro ette : around blood
ve el. (+) PTAH, GFAP Grading unclear. Necro i doe nt effect progno i . Rarely
tran form into GBM Ultra tructure (EM): Blepharopla t (apical cytopla m), int
ercellular junctional complexe , intracytopla mic lumina, microvilli, cilia Imag
ing Calcification in 50%. V medullobla toma: calcified, inhomogeneou , exophyt
ic 5-25% drop met Hi tologic Type Cellular Papillary Clear Cell: re emble oli
go, lack ro ette Tanycytic Giant-cell: mimic PXA, SEGA Treatment Obtain pina
l MRI and CSF in all patient Surgery: GTR XRT: o Focal to 50-55Gy o Cranio pina
l if CSF (+) or anapla tic infratentorial or upratentorial near ventricle (que
tionable) o WBRT for anapla tic upratentorial not near ventricle o <3yo defer X
RT Chemotherapy for re idual o Chemo ha not increa ed urvival Progno i Infrat
entorial: 33% 5y urgery alone, 58% 5y GTR + XRT; Supratentorial 15% 5y Negat
ive factor : Age <4yo, calcification
Ependymoma
Anapla tic Ependymoma

Myxopapillary Ependymoma
Mucinou material around hyalinized ve el More benign; hi tology not ignifica
nt in progno i XRT effective Age: 40-60 yo Location: 4th ventricle, eptum pell
ucidum Hi tology: Ependymal & a trocytic feature . Hypocellular, re t of cell .
May have p eudo ro ette o V ependymoma: no ro ette , no eeding. Do not give
XRT
Subependymoma
Flotte Outline of Neuro urgery
13

o No grading o May be combined ependymoma / ubependymoma: progno i a umed to d
epend upon ependymoma part (grade 2) Treatment: Surgery No recurrence if complet
e re ection
Choroid Plexu Papilloma
Median age: 12mo. Can occur in adult Mo t common location: Children: lateral ve
ntricle (L atrium); adult : 4th ventricle Hi tology: Columnar/cuboidal cell wit
h va cular troma papillae. o Rare ectopic ti ue (bone, cartilage) o (+) tran t
hyretin o Hi tology not ignificant for progno i /grading o May invade brain, e
ed CSF Imaging: Strongly enhancing, 25% calcify Treatment: Surgery o Con ider pr
eoperative angiography/embolization, prepare for blood lo o GTR may cure Frequ
ent recurrence

Choroid Plexu Carcinoma
Mean age 2 yr Hi tology: Lo of papillae; mito e Treatment: Surgery (GTR), XR
T, +- chemo o STR: <3yo: multiagent chemo; >3yo: XRT (con ider cranio pinal), co
n ider chemo o Con ider urgery for recurrence

Glial Tumor of Uncertain Origin

Chordoid Glioma of the 3rd Ventricle De cribed in 1998. 20+ reported ca e Mean
Age 46yo (12-70yo) Hi tology: Cord of epithelioid cell in mucinou troma. Mit
o e rare. Lymphocytic infiltrate. Strongly (+) for GFAP and vimentin Radiology:
Solid enhancing 3rd ventricular ma . Occa ionally cy tic Progno i : Appear to
be low-growing. A trobla toma Occur in young adult occa ionally in infant
and children Hi tology: Periva cular GFAP (+) cell forming p eudoro ette throu
ghout o High grade variant: increa ed cellularity, atypia, mito e , endothelial
proliferation o Necro i may occur in low or high grade Radiology: U ually hemi
pheric, but can occur anywhere. Well circum cribed, may be cy tic Treatment: Sur
gery o GTR may cure (0/13 at 2yr recurred) o XRT, chemotherapy benefit unclear
in low-grade, probably ju tified in high-grade tumor Progno i : Behavior i var
iable. Low grade may convert to GBM o Anapla tic: higher recurrence, urvival ab
out 2 yr Gliomato i Cerebri Age: peak 40-50yo (0-83yo) Involvement of more tha
n 2 lobe with (60%) or without a defined ma . May be Grade 2, 3, or 4 a trocyt
oma. Mito e variable; endothelial proliferation u ually ab ent Hi togene i con
trover ial Appear to be monoclonal. Chromo omal change are unlike other a troc
ytoma Radiology: 50% enhance. Be t een on T2 or FLAIR
Flotte Outline of Neuro urgery

14
Treatment: Biop y and XRT. May re ect dominant ma . No demon trated benefit to
chemo but may u e BCNU, temozolomide, PCV. Con ider hi tologic type. Progno i :
9mo median. 54% 12month urvival; 8yr longe t reported urvival (all other <4yr
) KPS, enhancement, and grade ignificant? (not age, pre ence of dominant ma )
(N8/03)
Neuronal Tumor
Commonly pre ent with chronic eizure
Gangliocytoma
Occur all age , all location . Can occur in pinal cord Treatment: GTR if ympt
omatic
Dy pla tic Gangliocytoma of the Cerebellum

De mopla tic Infantile A trocytoma/Ganglioglioma
Age: <18 mo Ma ive, frontal, cy tic, adherent to dura Enhance V meningioma:
GFAP(+), EMA (-) Treatment: Cure with GTR o XRT, chemo after STR not defined

De embryopla tic Neuroepithelial Tumor (DNET)
Age: median 7yr (range 1-19 yr ) Symptom : Intractable complex partial eizurez
without deficit mo t common Imaging: Temporal, cy tic, cortical Pathology: Ma
ture neuron , abnormal oligo & a tro . Specific neuroglial element: bundle of ax
on attached to GFAP(-) oligodendroglial like cell in column o Simple & comple
x form o No lympocyte cuffing (v ganglioglioma) Grade I behavior Imaging: Hypo
inten e on T1, hyperinten e on T2. Variable enhancement (multiple ring ) Treatme
nt: GTR u ually cure . No recurrence w/STR. Operate for eizure control.


Ganglioglioma

Age: <30 yo (mean 11yo), but can occur in any age Location: 70% temporal, 10% fr
ontal, but may occur anywhere. Symptom : Seizure common (75-100%). Pathology: B
inucleate neuron ((+) neurofilament, ynaptophy in, ilver tain) and neopla ti
c a trocyte (GFAP (+)); lymphocyte cuffing. o May be a ociated with cortical d
y pla ia, may cau e per i tent eizure after le ionectomy Grading: by a trocyti
c component: u ually Grade II, occa ionally grade III (Anapla tic Ganglioglioma)
, grade IV = GBM Imaging: Cy tic, calcified nodule, +- enhancement Treatment: o
Surgery: GTR. Wide re ection of involved gyru i recommended to decrea e recurr
ence and improve eizure control (becau e of a ociated dy pla ia ). Utility of
ECoG not firmly e tabli hed. o XRT: U ed by ome for STR, adjuvant or at recurre
nce (controver ial) o Chemotherapy: con ider for grade III Mo t common intravent
ricular tumor Near foramen of Monro (al o in cervical pine) Median age 25yr (1
5-38 yr ) Calcified, enhance in 50% Pathology imilar to oligo, but (+) ynapto
phy in, NSE Treatment: Surgery. GTR may cure Rare malignant variant: con ider XR
T, chemo


Central Neurocytoma
Flotte Outline of Neuro urgery Cerebellar Liponeurocytoma
Le than 20 reported ca e . 40-50yo. Benign. Lipomatou . Neuronal.  GFAP.  de min
(myogenic). Low MIB1. Treatment: Surgery o Recurrence: urgery  XRT. (N12/03)
15

From paraganglion cell Contain ecretory granule ; <4% ecrete catecholamine ;
check 24hr urine VMA Location Carotid Body: (=Chemodectoma) painle low growi
ng ma in neck; may cau e ICA teno i Glomu jugulare: uperior vagal ganglion
. May grow into inner/middle ear Glomu Tympanicum: vagu auricular branch Treat
ment Treatment: Surgery. Radiation, preop embolization o Radio urgery ha been u
ed with 100% 7yr LC, 75% 10yr LC
Paraganglioma
Neurobla tic Tumor Olfactory Neurobla toma (E the ioneurobla toma) Olfactory Ne
uroepithelioma Neurobla toma of Adrenal & SNS Pineocytoma
Peak at 30 yr . Pediatric pineocytoma are more aggre ive Symptom : headache ,
Parinaud yndrome, vi ion lo Path: Recapitulate normal pituitary. Homer-Wrig
ht ro ette . MRI: enhancement, calcification, well-defined margin Treatment Opt
ion : re ection, tereotactic or endo copic biop y ( third ventriculo tomy), radi
o urgery Spetzler recommend gro total re ection for ymptomatic le ion with
tectal plate compre ion or hydrocephalu ; radio urgery for mall, re idual, or
recurrent le ion . (N8/04) Some (We tphal) feel that tereotactic biop y of the
pineal i un afe due to vein in the area. Al o ampling error may mi a focu
of pineobla toma (with re ection end a much ti ue for path a po ible). Radi
o urgery: Kondziolka reported 100% local control with SRS for biop y or STR. 10%
permanent gaze pal y after SRS. Type of PNET Age: <20 yo Invade brain, eed C
SF, di tant met Trilateral retinobla toma: bilateral retinobla toma w/ pineobla
toma
Pineobla toma
Pineal Parenchymal Tumor of Intermediate Differentiation
Mixed pineocytoma-pineobla toma

Embryonal Tumor
Age: <15 yr , econd peak @ 28 yr . More commonly located off in cerebellar hemi
phere in adult A ociated with ba al cell nevu yndrome Hi tology
Medullobla toma
Flotte Outline of Neuro urgery

16
Numerou uniform mall blue cell . Homer Wright ro ette . (+)GFAP, neurofilament
, ynaptophy in (glial & neuronal) Staging: Need pinal MRI, CSF cytology (10d a
fter urgery and at recurrence). CSF cytology detect an additional 15% after MR
I. More dedifferentiated = better progno i Chang taging y tem: o T Stage: T1
< 3 cm, T2 > 3 cm, T3a > 3 cm invading adjacent tructure , T3b ari ing from flo
or of IVth ventricle, T4 through aqueduct or foramen magnum o M Stage: M0 No gro
meta ta i , M1 Tumour cell in CSF, M3 Gro nodular eeding, M4 Extraneural
meta ta i Low-ri k: <3yo, no re idual, no met . 5y 70% o High-ri k: >3yo, re i
dual, met . 5y 40% Imaging: Hyperden e on CT 33% CSF di emination, 5% di tant
meta ta e (90% of which are bony) Hi tologic Type De mopla tic Medullobla toma
: Reticulin (+) area . Slightly better progno i Large Cell Medullobla toma Medu
llomyobla toma Melanotic Medullobla toma Treatment Surgery: extent of re ection
correlate with urvival in ome tudie , not in other o Po top muti m occur i
n 20%, la t up to 1 year o Need <1.5cm2 re idual to change progno i XRT: o Cra
nio pinal (23.4 Gy for GTR, 35Gy for STR) with focal boo t (50-55Gy over 5-7 wk
). o Younger than 3yr : defer XRT, u e chemo Chemo: o U ed for patient <3 yo an
d high-ri k patient >3yo (role in good ri k patient unknown) o Ifo famide, etop
ide, ci platin o Recurrence - multiple regimen u ed including bone marrow re cu
e Progno i Po itive factor : extent of re ection?, extent of local di ea e, met
a ta i Age <4yo, GFAP (+) Recommended urveillance imaging: q3mo for 1 year the
n q6-8mo for 8yr ; Brain  contra t & ingle contra ted agittal entire pine Pack
er 94: 5y 85% (67% with met , 90% no met )
Medulloepithelioma
From ventricular matrix cell
Ependymobla toma
More common upratentorially
Supratentorial PNET (Central) Neurobla toma
Age: <5 yr
Ganglioneurobla toma Atypical Teratoid/Rhabdoid Tumor Nerve Sheath Tumor
Schwannoma
AKA neurilemmoma, neurinoma Hi tology Spindle cell . Verocay bodie , Antoni A (d
en e) & B (loo e), ba ement membrane. Encap ulated, cy tic, hemorrhage, not infi
ltrative. No grading, necro i unreliable. Mito e dont change progno i . S100+.
Rarely calcify.
Flotte Outline of Neuro urgery

17
Growth unpredictable (1-10mm/yr). Occur at DREZ. Al o: media tinal/ retroperito
neal, head/ neck, flexor urface of extremitie . Rarely intraparenchymal (on per

iva cular nerve ). Lo Chr 22 Hi tologic Type : Cellular Plexiform Melanotic Cl

inical Hearing lo /tinnitu . CN5 (numbne ) early. Facial weakne late (v men
ingioma - early). Audiogram: ab cence of loudne recruitment, poor peech di cr
imination, U eful hearing: peech reception thre hold <50dB, peech di crimination
core >50%. Gardner-Robert on Scale: Pure Tone Average (PTA), Speech Di crimina
tion (SD): I = <30, >70%; II = 31-50, 69-50%; III = 5190; 49-40%; IV = 91-max; 3
9-1%; V = no re pon e. Serviceable: Grade I-II, PTA thre hold <50dB, SDS > 50% Hou
e-Brackmann: facial trength Radiology MRI: enhance trongly, CSF cleft. CT tem
poral bone: how po ition of labyrinth Size: Intracanilicular > Small (<1cm in
CPA) > Medium (1-2.5cm) > Large (2.5-4.5cm) > Giant (>4.5cm) Meningioma cau e h
ypero to i , le widening of IAC. Purely intracanilicular meningioma can occur
. Treatment Ob ervation: 23% decrea ed, 42% table, 35% grew. (Steiner JN 00?) S
urgery 98% local control. 14-46% hearing pre ervation. (50% if <2cm, 25% if 2-4c
m). Approache : o Middle fo a: Small (<1cm) or intracanalicular o Tran labyrint
hine acrifice hearing. U e only if Gardner-Robert on cla III or IV. o Subocc
ipital: large, medial tumor . Facial n. location: anterior > uperior > inferior
. Con ider intraoperative facial nerve monitoring, BAER . STR: XRT can recurrenc
e. Radio urgery Marginal do e 12-13Gy (12Gy if hearing intact, 13Gy if not, 11Gy
in NF2). 4mm collimator u ed for intracanilicular portion. 90-98% local contro
l. Tumor may enlarge before hrinking. 60-80% (<12Gy) hearing pre ervation. Hea
ring lo occur at 6-24mo . Hearing pre ervation may be 85% with SRT (2Gy/d) co
ntrover ial. Delayed facial weakne 5% (0-8%) at 6-28 mo (none if <15Gy) (LINA
C 24-32%). Facial pa m 2-8%. Facial numbne 3%. Over 50% of po t-SRS cranial n
europathie re olve in 3-6 mo . Schwannoma ver u Neurofibroma: Schwannoma: enca
p ulated, cy tic/hemorrhage, not infiltrative, degeneration rare. Neurofibroma:
not encap ulated, not cyctic, infiltrative, 10% arcomatou . No verocay bodie .
Neurofibroma
1. Dermal: NF1 2. Plexiform: neck, media tinum 3. Vi ceral
Perineurinoma
Intraneural Perineurinoma Soft-Ti ue Perineurinoma
Malignant Peripheral Nerve Sheath Tumor (MPNST)
Aka neurofibro arcoma Location : Brachial & Sacral Plexu , Pleural, Retroperiton
eal, media tinum Hi tologic Subtype Epithelioid MPNST w/divergent me enchymal a
nd/or epithelial Differentiation Melanotic
Flotte Outline of Neuro urgery
Melanotic P ammomatou
18
Meningioma
Epidemiology Female 2:1. Peak 45yr o 20 year recurrence: male 60%, female 100%
Increa ed ri k: HRT (premenopau al RR 2.5, po tmenopau al RR1.9), brea t CA, pre
gnancy, men e , ?obe ity (not OCP ) Location: Para agittal > convexity > tubercu
lum ella > phenoid wing >olfactory groove > falx > intraventricular (Children
28%, Adult 4%, u ually on left) > tentorial > po terior fo a > orbital > pina
l. Rarely parencymal, Sylvian fi ure, kull, extracranial (0.3%). Radiation-ind
uced meningioma : 100% recurrence, 38% atypical or malignant Genetic 70% have C
hr22 abnormality (95% of fibrobla tic, 33% of meningothelial; 40% of benign, 80%
of atypical, 90% of anapla tic) Merlin: NF2 gene, tumor uppre or, Chr22, anch
or cyto keleton to membrane protein . Mutated in 60% of poradic meningioma . P
roge terone receptor pre ent in 66% (more in female), e trogen in 10%, androgen
in 66%; omato tatin receptor in 100%, ignificance unknown. (Al o glucocortic
oid, dopamine receptor ) Hi tology P ammoma bodie , whorl , hole in nuclei. (+)
vimentin, EMA. MIB1predict recurrence. Necro i unreliable predictor. Hypothe
ized to ari e from arachnoid cap cell En plaque meningioma grow longitudinally

, rather then pherically Edema prominent in ome (paracrine effect?) Blood upp
lied by ECA meningeal branche (except olfactory groove meningioma - upplied b
y ophthalmic a.) Grading/Hi tologic Type Jaa kelainen grading: lo of architec
ture, hypercellularity, nuclear pleomorphi m, mito e , necro i , brain inva ion.
Inva ion of dura, kull, kin do not increa e grade. 15% are atypical, 5% are a
napla tic. Grade I: Meningothelial: ( yncytial) whorl , no p ammoma; Fibrou (Fi
brobla tic); Tran itional (Mixed): whorl , p ammoma common; Secretory: CEA+, PAS
+. Edema. P ammomatou ; Angiomatou ; Microcy tic; Lymphopla macyte-rich; Metapla
tic GradeII: Clear Cell Chordoid Atypical: >4 Mito e per 10HPF (high powered f
ield), or 3 or more: hypercellularity, high nuclear:cytopla m ratio, prominent n
ucleoli, heet-like growth, focal necro i (not brain inva ion or labeling index
v Jaa kelainen criteria). 5% meta ta ize. Grade III: Papillary: young pt .; br
ain inva ion, 20% di tant meta ta e . Rhabdoid Anapla tic: >20 mito e per 10HPF
. Invade brain or meta ta ize . Criteria = more anapla ia Al o: Meningioma of
any ubtype with high proliferation index and/or brain inva ion o Brain inva ion
debated. 23% hi tologically benign, 61% atypical and 17% anapla tic. Tho e that
were hi tologically benign acted benign. WHO 2000 note brain inva ion may occu
r in benign meningioma, where it increa e recurrence rate. Radiology CT: calcif
ication, hypero to i , enhancement (75%), dural tail Correlation of radiology to
grade i poor Angiography: ome recommend routine on all patient to look for p
ial blood upply Venou inu patency by MRV (may not prove total occlu ion) or
angio Treatment Embolization Gelfoam powder, PVA foam, NBCA, micro phere , fibri
n, coil u ed ICA feeder (ophthalmic) not u ually embolized
Flotte Outline of Neuro urgery
19
Ri k: 2.5% permanent deficit Particularly for: firm (hypointen e on T2), va cula
r, kull-ba e, large, inacce ible blood upply. Done day before urgery. Can ca
u e wor ened edema: hydration, teroid , ICU (may do urgery immediately if larg
e) Some feel that it i not u eful Surgery Favored if growing, ymptomatic, or
ignificant edema. Dementia may improve after re ection with ignificant edema (C
heeCP 85) Simp on grade: extent of re ection: (I-V) Dural tail hould be re ecte
d. Pial inva ion correlate with ize, edema, pial va cularization Patent inu e
hould not be re ected (controver ial but not recommended by Al-Mefty or DiazDay); recurrence rate i about 20% with or without inu recon truction (N7/04).
Radiation 50-55Gy. Retro pective tudie : effective both after STR and at recur
rence, with or without reoperation. STR: recurrence 32% (v 60% no XRT), longer
urvival. Le conclu ive evidence for efficacy a primary treatment in inoperat
ive meningioma Malignant meningioma: little evidence but routinely u ed. Decrea
ed recurrence (GTR: 58% to 36%; STR: 90% to 41%). Radio urgery <3cm only. 93% 5
y CR. 11-18 Gy margin do e. U e with tandard XRT in malignant meningioma, con i
der for atypical. Chemotherapy Mixed re ult . Chemotherapy (Hydroxyurea, CDV, in
terferon -2B) nd hormone ther py (T moxifen, Mifeprestone/RU486) show minim l
e
nefit in sm ll series Specific Loc tions Optic nerve meningiom resection lmost
lw ys c uses
lindness; only resect if lre dy
lind, otherwise use XRT C vern
ous sinus: Mor
idity 10-45%. Resection of tumor in the medi l c vernous comp rtm
ent lmost lw ys c uses cr ni l nerve mor
idity. Some recommend only resecting
tumor in the l ter l comp rtment nd using r diosurgery on the residu l (N6/04)
Petrocliv l: Mor
idity 30-40%. Tentori l: Y s rgil cl ssific tion (N7/04) CPA: C
uses hyperostosis, dur l t il, inv sion of petrous
one, less IAC erosion th n
coustic neurom . Su
occipit l ppro ch. M y
e purely intr c nillicul r (N7/04)
Tu
erculum Sell e: M y
e removed
y tr nssphenoid l ppro ch. Petrous: JN6/04.
P r s gitt l: Kondziolk nd others suggest using r diosurgery for residu l tum
or in the superior s gitt l sinus (2-6mo fter resection), inste d of sinus reco
nstruction (N9/98) Prognosis N tur l History: M y h ve sudden incre se in growth
r te. Growth r tes higher in young pts, lower with c lcific tion or T2. No corre
l tion with size. MS: m lign nt = 7yr, typic l = 9yr Recurrence: 10yr: 25% GTR,
61% STR. F ctors: Necrosis, mitoses,
r in inv sion, in ccessi
ility, l
eling

index. o 20% of
enign meningiom s will
e typic l or n pl stic on recurrence.

30% of typic l meningiom s will
ecome n pl stic.
Meninge l mel nocytom
Most common in spin l cord nd posterior foss (mel nocyte remn nts in perimedul
l ry dur ). Tumor is
l ck with superfici l siderosis, HMB-45 (+). Enc psul ted.
C n
e tot lly excised.
Hem ngiopericytom
Sep r te entity from meningiom s. Histology (vs meningiom ): no ps momm
odies,
l ck desmosomes & g p junctions, st ghorn vessels, vimentin+, EMA-, reticulin+
Arises from pericyte cells round c pill ries Gr ding unreli
le 25% met st size
Blood supply from ICA

Flotte Outline of Neurosurgery
Tre tment Surgery: preferred tre tment R diosurgery: M y
e more responsive to S
RS th n meningiom .
20

Hem ngio
l stom
Uncert in histogenesis Age: 20-40yrs 20% h ve VonHipple-Lind u Loc tion: cere
el
l r hemisphere most common. Supr tentori l hem ngio
l stom s r re (<100 reported
c ses) o Spin l cord: usu lly dors l, come to pi l surf ce (m y
e hidden under
vessels c n use ultr sound to loc te). Resected circumferenti lly like n AVM.
Syrinx resolves with remov l of tumor P thology: V cuol ted strom l cells. F t s
t ins with Oil-red-O, NSE+, m st cells; 10-20% h ve vHL o Resem
le Ren l cell me
ts histologic lly,
ut (+) vimentin, (-) EMA. C n secrete erythropoetin Tre tmen
t: surgic l resection.


Chordom
20-60yrs; m le 40% clivus, 60% s crum P inful C n ffect ll cr ni l n. on one s
ide (G rcin syndrome) 30% met st size, m y ch nge to s rcom Histology: Phys lip
horous cells o Echordosis psyph lliphorous:
enign rests of notochord, seen in 1
% of utopsies Tre tment: Surgery. R dioresist nt Surviv l 2 yrs

Loc tion: Pine l or supr sell r (50% sell r h ve pine l) Supr sell r: tri d = DI
, visu l loss, hypopituit rism Tre tment: R diosensitive (
iopsy) Germinom 10-3
0yrs; m le T-cell follicles M les: precocious pu
erty XRT neur xis Em
ryon l C r
cinom AFP & BHCG Yolk S c Tumor AFP; Schiller-Duv l
odies Chorioc rinom B-HCG
; hemorrh ges Ter tom 2nd most common M les CEA Mixed Germ Cell Tumors
Germ Cell Tumors
Cr nioph ryngiom s
Bimod l ge distri
ution: 5-10yrs nd 30-50yrs Usu lly supr - nd intr sell r. M
y
e entirely intr sell r, p rti lly or entirely 3rd ventricul r. R rely: CPA,
pine l, n soph rynge l 3rd ventricul r tumors lw ys c use hydroceph lus, extr v
entricul r tumors r rely do. Acom indic tes position of the optic chi sm on MRI.
From R thkes cleft cells Str tified squ mous epithelium. Cont in m chine-oil flu

id

Flotte Outline of Neurosurgery
21
90% c lcify, usu lly enh nces. Infundi
ul r more often c lcified, intr ventricul
r more often solid. Histology Ad m ntinom tous: Rests of epithelium surrounded

y l yer column r cells w/ myxoid strom of whorls & ker tin nodules. M y cont i
n teeth P pill ry: Adults. 3rd ventricle, solid, no c lcific tion. Better progno
sis Tre tment Surgery Pre- & postop endocrine ev lu tion needed. Appro ches: Int
r sell r: Tr nssphenoid l ( nd extended TSRP). Supr sell r extr ventricul r: Pte
rion l, su
front l, or
itozygom tic, r rely su
tempor l (middle foss extension)
. Intr ventricul r: tr nsc llos l, tr nsl min termin lis. Some reports of TSRP
for supr sell r tumors. 44% STR, 19% CSF le k. Less cognitive nd visu l deficit
s. (JN3/04) DI prominent postop. Hypoth l mic insufficiency more common with int
r ventricul r tumors. Tot l resection vs. STR le ving tumor on hypoth l mus is c
ontroversi l. XRT Controversi l Prognosis Recurrence: most occur within 1-3yrs.
0-50% with GTR, 30-100% with STR. L ws: repe t resection does not c use ddition
hypoth l mic d m ge.
Pituit ry Adenom
Epidemiology Incident l on 10% of routine MRIs. M les: GH. Fem les: PR, ACTH. El
derly: FSH/LH. Pedi tric: ACTH prepu
erty, Prol ctin during & fter pu
erty. Ass
oci tions: MEN type I. (check C , glucose). Aneurysms up to 7 times more common
(5%). Histology P th: sheets of uniform cells w/round nuclei, dense core gr nule
s, loss of nesting & reticulin. Sheds on touch prep. Micro denom < 1cm, M cro den
om >1cm. Occurrence: Prol ctin > Null cell > GH > ACTH (> TSH > FSH/LH very r r
e) Acidophilic: GH (50%), FSH/LH. B sophilic: ACTH, TSH. Chromopho
ic: PR, null
cell. L ter l: PR, GH. Medi l: ACTH Higher recurrence: cidophilic stem cell, si
lent corticotroph, oncocytom , plurihormon l, high MIB-1 ACTH: PAS+. GH: Chromop
ho
ic more ggressive. Null-Cell: nonsecretory. Oncocytom : eosinophilic due to
mitochondri . Acidophilic stem cell denom : prol ctin, perinucle r v cuoles (mi
tochondri ), r pidly inv sive,
romocriptine doesnt work. Silent corticotroph (no
nfunctioning, ACTH+): r re, ggressive, consider postop XRT (N11/03) Clinic l M
ss effect: o Visu l loss (
itempor l hemi nopsi from chi sm l compression. o Ot
her CN p lsies. Hypopituit rism: o Nonfunctioning: 75% hypogon d l, 36% hypo dre
n l, 18% hypothyroid. o Pedi tric: Growth f ilure, menorrhe common with ll ty
pes. Hormone hypersecretion: o Prol ctin: fem les: menorrhe / g l ctorrhe , m l
es: impotence. Apoplexy: hypotension, VA, HA, CN p lsy, Addisons. Due to hemorrh
ge or necrosis (perip rtum = Sheeh ns syndrome). C n occur cutely during r dios
urgery. Tx: steroids, urgent surgery. (If mild check prol ctin first nd tre t w
ith
romocriptine). L
or tory GH: Serum GH, IGF-1. ACTH: Serum cortisol (loss o
f diurn l v ri tion), serum ACTH, 24hr urine cortisol & 17 OHCS. o Low dose dex
meth sone suppression (DST): o High dose DST.
Flotte Outline of Neurosurgery
o o
22

Insulin or Cortosyn (tetr cos ctide) stimul tion test: incre se of cortisol to 5
50 nmol/L fter dministr tion is norm l. Inferior petros l sinus s mpling: for
confirm tion of pituit ry source & l ter liz tion. Do not give pre/post-procedur
e steroids. TSH: Serum TSH, free T4. o TRH stimul tion test: TRH given, TSH s mp
led t 0, 20, nd 60min. FSH/LH: Serum FSH, LH, testosterone. o Hormon l cure: GH:

norm l IGF-1, OGTT suppresses GH <1

R diology Micro denom s: hypointense, enh nces less th n norm l pituit ry (enh n
ces on del yed im ges). M cro denom s enh nce more th n norm l pituit ry Tre tme
nt Prol ctinom : Dop mine gonists, surgery for f ilure or cute loss of vision.
GH: Surgery, if nonoper tive SRS or octreotide, then
romocriptine. IGF-1 drops
4wks postop. GH >40 difficult to cure. ACTH: Surgery then SRS. o Medic l: ketoc
on zole, or metyr pone (or Cyprohep t dine). o Adren lectomies s l st resort
(le ds to Nelsons syndrome in 30%). Nonfunctioning: Surgery, then SRS. TH: Surger
y (octreotide) Medic l Dop mine gonists: o Bromocriptine: dop mine gonist ; pr
olonged = reduced ch nce surgic l cure; m y enl rge on d/c. 2.5mg po tid. SE: em
esis, postur l hypotension, (
oth resolve). S fe with OCPs, pregn ncy. o Dostine
x: longer- cting. Octreotide: Som tost tin n logue; SQ. Expensive. Surgery Tr n
ssphenoid l: endon s l, su
l
i l (l rge, supr sell r tumors), tr nsn s l. Consi
der stereot xy for redo. Consider Xr y/flouro, endoscope. Hormone levels norm li
zed in: 75% of Cushings (50% if second surgery or m cro denom ), thyroid in 14%,
gon d l in 11%. Signific nt complic tions 1-2% (c rotid or hypoth l mic injury,
visu l loss, meningitis). C rotid injury: get ngio postop to r/o neurysm. o A
lcohol instill tion reported to c use
lindness (JN6/04) Ch ndler o
t ins postop
MRI t 9mos, then ye rly for 5 ye rs, then every 2 ye rs. Weiss o
t ins first s
c n t 3mos. For residu l or recurrence: Weiss tre ts fter 1st postop sc n if p
tient is hypopituit ry, o
serves if hormon lly int ct. XRT 50Gy conform l. Nonf
unctioning: Postop XRT decre ses recurrence t 10yrs from 50% to 2% (N7/04). O
s
erv tion until recurrence ppe rs prefer
le to st nd rd postop XRT s long s g
ood followup c n
e o
t ined. Complic tions: Hypopituit rism 40% (GH > FSH/LH >
ACTH). Cognitive imp irment. Optic neurop thy(1-3%). Second ry neopl sms (3%). R
diosurgery Prim rily for nonoper tive p tients, unresect
ility (ie c vernous s
inus). 12-24mo l tency. M rgin dose: 12-16Gy for nonfunctioning, 16-30Gy for fun
ctioning,
ut limited
y 8-10Gy to optic chi sm. Stop medic l tre tment 1-2mos

efore SRS (3-4mos for Dostinex). Tumor control: 97%. 40% reduced size. (N7/03).
Nonfunctioning: 88% 5yr LC. (JN3/04) Hormone norm liz tion: 80% ACTH, 60% GH, 40
% prol ctin t 60mos. L tency: 12mos for ACTH, 24mos for GH & prol ctin. Complic
tions: Optic neurop thy 0-1%. None if <10Gy. (JN98). 1% for 12Gy. Dose limits r
nge 9-12Gy. Some limit ICA cover ge to <50% of lumen to void ICA stenosis (con
troversi l) 3 reports of second ry neopl sms. Hypopituit rism: 50% if >17Gy, 10%
if <17Gy. Use <16Gy to the st lk. Chemo: Glidel used for recurrent, ggressive
tumors (N8/03) Recurrent/residu l: Timing controversi l immedi te vs progression
. Nonfunctioning: 2y &5y Control 97%. 50% reduced size. 28% new hormone deficits
(N11/03) Differenti l Di gnosis Prol ctin cell hyperpl si : pregn ncy, l ct tio
n, estrogen tre tment, end org n f ilure (hypothyroidism, etc), extr pituit ry G
RH producing tumors, hep tic or ren l dise se, phenothi zines, ver p mil, cimeti
dine Nelsons syndrome: Pituit ry enl rgement nd incre sed ACTH (c uses hyperpig
ment tion) fter dren lectomy. Crookes hy line ch nge occurs from extr pituit ry
source of ACTH.
Flotte Outline of Neurosurgery Gr nul r Cell Tumor
From posterior pituit ry pituicytes. Clinic lly resem
les nonsecretory denom .
No c lcific tion (vs cr nio). Strongly enh nces. Densely p cked, l rge PAS+ eosi
nophilic cells. Pituicytom (GFAP+) pro

ly s me tumor. (per WHO).
23
R thkes cleft cyst
Symptom tic in 30-40yo women. Found in 33% of utopsies. C uses he d ches,
l
norm lities, endocrinop thies Usu lly intr sell r 50% rim enh ncement; no c
lcific tion P th: Cili ted cu
oid l/column r epithelium with mucin-producing go

let cells (vs cr nioph ryngiom str tified squ mous) Cholesterol cyst (not motor
-oil fluid like cr nioph ryngiom ) Tre tment: surgic l dr in ge nd p rti l exci
sion of cyst w ll. Recurrence is r re. 20-40 yo (r re in kids). P th: Single l y
er column r cells w/ cili & go
let cells, mucin (PAS+). Endoderm l origin (p r

physis vs dienceph lic recess of postvel r rch). Loc tion: For men of monroe/ 3
rd ventricle (r rely outside ventricles). Sxs: He d che (#1), dementi , seizures
, drop tt cks, sudden de th. Im ging: Hyperdense on CT; hyperintense on T1, hyp
o on T2, w ll m y enh nce, no c lcific tion.
Colloid Cyst

Neuroepitheli l cyst Ependym l, choroid plexus, or choroid l fissure. Neurenteri
c cyst AKA Enterogenous cyst. M les. 1st dec de. P th: Endoderm, GI/Resp mucos ,
mucin+. Loc tion: Spin l. Intr cr ni l (CPA): <10 reported c ses. Cervic l/ tho
r cic most common. Usu lly nterior, c n
e posterior. C n
e intr - or extr dur
l, or intr medull ry. Symptoms: P in, septic meningitis. Not ssoc. with cute
neous stigm t . C n
e contiguous with GI or respir tory tr ct. Verte
r e m y
e
norm l or split. C n h ve derm l sinus. Tre tment: Dr in cyst, c uterize w ll.
Epidermoid Cyst
30-50yo P th: Line r growth r te. R rely ruptures. Frequently recurs. Insinu tes
long
s l cisterns Moll rets meningitis: recurrent chemic l meningitis from re
pe ted rupture; l rge cells in CSF Loc tion: Off-Midline. CPA > suprsell r > int
rventricul r > th l mic. 10% intr diploic Im ging: simil r to CSF (T1 v ri
le d
epending on f t content), no enh ncement. Diffusion-weighted MRI differenti tes
from r chnoid cyst. Tre tment: GTR of entire cyst w ll. Seldom h ve cle n pl ne
of dissection Both epidermoid nd dermoid c used
y incomplete cle v ge of neur
l from cut neous ectoderm;
oth lined
y squ mous epithelium.

Dermoid Cyst
10-20yo P th: Pilose
ceous units, occ sion l teeth. Frequently ruptures. R
undergo ch nge to squ mous cell CA B cteri l meningitis occurs due to cut neous
tr ct. Loc tion: Midline. P r sell r, 4th vent, interhemispheric Im ging: Simil
r to f t except hyperintense on T1 nd T2, c licific tion, r re enh ncement Tre
tment: GTR. Midline: corpus c llosum, qu drigemin l, 3rd vent, CPA, Sylvi n fis
sure 50% h ve ssoci ted
r in m lform tions. Tu
eronodul r or curviline r
Lipom
Flotte Outline of Neurosurgery
24
Prim ry CNS Lymphom
Epidemiology Affects ll ges, including children Pe k 50-60yo 50% multiple Etiol
ogy No univers lly ccepted cl ssific tion scheme (WF, Kiel, REAL) 98% B-cell, 2
% T-cell (usu lly immuno-competent, in cere
ellum) Intr cere
r l pl sm cytom ,
ngiotropic, Hodgkins lymphom occur
ut re r re Histogenesis: Unknown (the
r i
n l cks lymph tic system). Three theories: B-cells tr nsform outside CNS then
develop dhesion molecules specific for cere
r l endotheli . Lymphom cells re
immunologic lly protected in the CNS B-cells tr nsform in preexising polyclon
l infl mm tory re ction in the CNS Predisposing F ctors: Immunosuppresion, Autoi
mmune dise se (RA, Sjorgens, SLE) EBV: present in tumor cells in 95% of immuno-c
ompromised p tients, 0-20% of immuno-competent p tients P thology Prim ry & Seco
nd ry lymphom s re p thologic lly identic l. Periv scul r cuffing ch r cteristi
c Growth is diffuse, not follicul r Some well-defined m ss, others infiltr tive
M y lso
e ocul r, spin l, leptomeninge l Second ry lymphom is usu lly meninge
l MIB-1 l
eling up to 90% Di gnosis Systemic workup (CT
domen/pelvis 
one m
rrow Bx) controversi l 3% of p tients will h ve extr neur l dise se Seizures les
s common (10%) th n with mets or gliom s 1) Im ging: MRI
r in  spine (if clinic l
ly indic ted) Diffuse enh ncement. R rely non-enh ncing or ring-enh ncing (more c
ommmon w/AIDS) Edem m y
e less th n with other lesions 40% h ve leptomeninge l

dissemin tion t di gnosis, ll p tients t utopsy PET or SPECT helpful in dif

ferenti l di gnosis of AIDS p tients (toxo, etc) 2) CSF: Cytology helpful in 30%,
especi lly if IHC shows monoclon lity PCR to detect EBV DNA 100% specific, 80%
sensitive in AIDS p tients 3) Slit l mp ex m: ocul r involvement seen in 18% 4) H
IV test Tre tment L rge ph se III tri ls h ve not
een possi
le due to r rity Su
rgery Stereot ctic Biopsy. Resection only when urgent decompression is necess ry
. XRT 40-50Gy WBRT st nd rd. Cr niospin l XRT shows no
enefit. With ocul r lymp
hom 40Gy to posterior 2/3 of glo
e Some re withholding WBRT until fter Methot
rex te f ilure Steroids 30-40% response r te (15% complete, 25% p rti l) 9 repor
ted c ses of long-term remissions (6mo-6yrs) with steroids lone,
ut eventu lly
ll reoccur No uthors recommend di gnostic tri l of steroids If p tient presents
on steroids most uthors recommend stopping steroids (for 5d ys), reim ging, th
en
iopsy if there is suit
le t rget Mech nism: Induction of poptosis Chemot
her py High-dose methotrex te (>1g/m2). o Systemic lymphom tre tment (CHOP) is
ineffective o Intr thec l MTX: ppe rs to incre se surviv l reg rdless of CSF cy
tology o Complic tion: Leukoenceph lop thy. Toxicity of MTX nd XRT dditive. Oc
curs prim rily in p tients >60 yo (70% @ 2yrs, 100% @ 4yrs). Giving MTX
efore X
RT (neo djuv nt) m y decre se toxicity
Flotte Outline of Neurosurgery
o
25
Chemother py lone (esp. >60 yo) m y
e option currently
eing studied. XRT timi
ng nd dose m y
e decided on response to MTX
Prognosis Supportive c re only: 2 months XRT only: 10 - 18 mo XRT + high-dose MT
X: 33- 45 mo (AIDS: 14mo) Positive predictive f ctors: ge < 60, KPS > 70, singl
e lesion, no periventricul r lesions, immunocompetence Predictive v lue not cle
r: histologic su
type, prolifer tion m rkers. Spont neous remission m y occur (gh
ost tumors)

Met st sis
Lung>
re st>mel nom >(colorect l<>ren l). Men: lung #1; Women: Bre st. (Lun
no>sm ll cell>l rge cell>squ mous) Peds: neuro
l stom , rh
domyos rcom , Wilms
Develop mets: Mel nom 50%, lung 33%,
re st 20%, ren l 10%. 20% unknown prim ry
(most end up
eing lung) Hemorrh gic: ren l cell, mel nom , chorioc rcinom Sin
gle
r in m ss in p tients with h/o CA 93% ch nce it is met (7% other); in p t
ient without CA < 15% will
e met Must use MRI (not CT) to determine # of mets.
Consider dou
le- or triple-dose contr st. Met st tic work-up: CT chest/
domen/p
elvis, serum PSA nd CEA,
one sc n
Tre tment Consider tions: P tient ge, symptoms, KPS; Tumor size, loc tion, m ss
effect, histology, num
er, prim ry dise se control Question
le mets c n
e w t
ched up to 1cm
efore SRS or surgery (N8/03) Surgery Single mets or 1 l rge lesi
on & multiple mets. KPS >70. Controlled 1. Not r di tion sensitive (sm ll cell lu
ng). Surgery + WBRT vs WBRT: 2 RPT showed surgic l
enefit in MS: P tchell 90 (1
0 vs 4mo), Vecht 93/ Noordjik 94 (10 vs 6mo). Not confirmed
y Mintz 96 (h d mor
e p tients with ctive extr cr ni l dise se & poor KPS). Multiple mets: Bind l 9
3. Retrospective. <=3 mets incre sed MS when ll resected (14mo vs 6mo), especi
lly with controlled prim ry dise se, high KPS, r dioresist nt tumors. XRT Resist
nt: mel nom , ren l cell. Sensitive: Sm ll cell lung, germ-cell, lymphom , leuk
emi , multiple myelom , Dose: P lli tive: WBRT 30Gy (10 3Gy fr ctions over 2 wks
). Cur tive ( fter GTR): 40-45Gy (2Gy fr ctions over 4wks). No difference with fr
ction tion. Extends surviv l 4mos. 25% CR, 33% PR on CT. (Mel nom 0% response,
>1cm 3% CR) Prim ry tx: preferred for multiple (>4) mets; r diosensitive tumors,
uncontrolled prim ry, medic l pro
lems Adjunctive: controversi l; Some not usin
g XRT if single met; preferred for multiple mets. Lundsford feels only lung CA

enefits from WBRT fter SRS. Surgery vs surgery + WBRT: o P tchell 98: RPT, 70%

vs 18% recurrence, fewer de ths due to neurologic c uses,
ut no difference in K

PS, MS. o Retrospective: no ch nge in MS, decre sed recurrence, Dosoretz 80, DeA
ngelis 89, H gen 90 SRS vs SRS + WBRT: o All retrospective: Sneed 99, IJROBP 02:
MS, loc l control s me, remote control worse for SRS lone (28 vs 69%). Chidel
2000: MS
etter for SRS lone (10mo vs 6mo). Pirzk ll 98: With no extr cr ni l d
ise se, WBRT improves MS fter SRS (15mo vs 8mo). o R ndomized tri l underw y wi
th ACOSOG. Prophyl ctic XRT used for SCCL in complete remission R diosurgery <3c
m di meter. Del yed results. M rgin dose 16Gy with WBRT, 20Gy for SRS lone or i
f WBRT >1yr previous. MS 8-10mo. Loc l control 85%. LC s me for SRS lone, WBRT
+ SRS
oost, or WBRT + SRS t recurrence. R dioresist nt tumors (mel nom , ren l
cell, s rcom ) re responsive. Histology m kes no difference? SE: edem 5-10%,
hemorrh ge 1-8% (higher with mel nom ), necrosis 1-5%. SRS Vs Surgery: No RPT. R
etrospective: Auchter 96 f vors SRS (14 vs 10mo); Bind l 96 f vors surgery (16 v
s 7 mos); Cho 98, Mu cevic JN 99: no difference.
Flotte Outline of Neurosurgery

26
WBRT vs WBRT + SRS
oost: o Flickinger 94 RPT. Kondziolk 99: RPT. 2-4mets. MS i
mproved (11 vs 7.5mo), decre sed loc l recurrence (8% vs 100%). Sperduto 00 (RTO
G 9508): RPT. No difference in MS for multiple mets,
ut incre sed MS for single
mets nd RPA I (high KPS, young),
etter loc l control, improves QOL, KPS. S ng
h vi 01: Met - n lysis. Improved MS in ll RPA cl sses. o RTOG 9508: Andrews DW,
L ncet 5/04. Chemother py Gli del h s
een used (Ph se III tri l in progress) P
rognosis RTOG RPA cl sses (G sp r 97): ge, KPS, st te of prim ry dise se, extr
cr ni l mets. o Cl ss I <65yo, KPS >70, controlled 1 = 7mos. o Cl ss II others =
4 mos. o Cl ss III KPS <70 = 2mos. (RPA I 18mo, II 22mo, III 10mo N7/03) H seg w
N6/03: MS 8mos., <60yo 12mo, >60yo 5mo, KPS >90 9mo, <90 3mo, st
le prim ry 1
1mos, progressive prim ry 5mos, RPA cl ss I 28mos. Mel nom 7 mos, ren l 7mos, l
ung 11mo,
re st 13mos, denoCA 15mos. Follow-up MRI: 3mos for 1st ye r, then 46mos.
Miscell neous Lesions Hypoth l mic h m rtom
Age <3yo C uses gel stic seizures initi lly, then complex-p rti l or gener lized
. Responds well to r diosurgery. o Gel stic seizures: l ughing fits. Other c use
s: hypoth l mic gliom , 3rd ventricle tumor, tempor l lo
e epilepsy, inf ntile s
p sms, etc. Also c uses precocious pu
erty Associ ted with P llister-H ll (polyd
ctyly nd imperfor te nus) nd other congenit l syndromes. Up to 4cm. Nonenh n
cing, isodense on T1, some hyperintense on T2. Two types: peduncul ted supr sell
r m ss rising from tu
er cinereum (c uses precocious pu
erty), or entirely int
r hypoth l mic (c uses gel stic seizures) Do not grow on seri l im ging

Ph kom toses

Neurofi
rom tosis-1 (NF1)
Chr 17, 50% spor dic. Neurofi
romin: tumor suppressor, inhi
its r s oncogene
y
R s-GTP se ( lso ? EGFR, p53) Skin: >6 c f u l it spots, xill ry freckling, Eye
: Lisch nodules (iris),
uphth lmos (cow-eye, due to lid neurofi
rom ), retin l
ph kom s Spine Xr y: enl rged for men, scoliosis, verte
r l sc lloping, dur l ec
t si , l ter l thor cic meningocoele. CNS lesions: o meningo ngiom tosis (coll r
s of meningotheli l cells round vessels) o sphenoid dyspl si (empty or
it) o m
oy -moy , neurysms, ect si o white m tter nonneopl stic h m rtomtous lesions: dy
spl stic gli , diminish w/ ge, T2, c n enh nce o
t in seri l MR o BG lesions: T2
, nonenh ncing CNS tumors: optic gliom s, spin l strocytom , neurofi
rom s Othe
r tumors: plexiform neurom s common in V1, viscer l, endocrine tumors


Neurofi
rom tosis-2 (NF2)
Chr 22, Protein: Merlin ( k Neurofi
romin2, schw nnomin). Links mem
r ne to ct
in cytoskeleton. Tumor suppressor. (Also in regul r schw nnom s & meningiom s) N
o lisch nodules or cere
rov scul r
norm lities (C f- u-l it spots, cut neous or
plexiform neurofi
rom s r re). Skin pl ques (rough, r ised, h iry re s), juven
ile c t r cts, c lcified choroid plexus, meningiom tosis
Flotte Outline of Neurosurgery

27
Tumors: Bil ter l coustic sch wnnom s (or 1 w/f mily history), other CN schw nn
om s, spin l ependymom , meningiom s (multiple, kids), strocytom s,

Tu
erous sclerosis (TS)
Ak Bournvilles dise se Usu lly spor dic, Chr 9/11/16, v ri
le penetr nce Genes:
TSC1 (chr9, h m rtin), TSC2 (chr16, tu
erin). Tumor suppressors, functions unkn
own Tri d: denom se
ceum, MR, seizures (inf ntile sp sms). Seen in <50% CNS l
esions: o Cortic l tu
ers: <5% enh nce, no tr nsform tion, thick gyri o Su
epend
ym l nodules: c ndle gutterings, 1/3 enh nce, c lcify (c n see on Xr y), no tr n
sform tion o Su
ependym l gi nt cell strocytom o H m rtom tous white m tter le
sions o Aneurysms, stenoses Skin: sh le f spots, su
ung l fi
rom s, sh green p
tch; Tumors: c rdi c rh
domyom s, retin l ph kom s/ ngiomyolipom , viscer l cys
ts, p ncre tic & liver denom (m lign nt >
enign) tumors

Sturge-We
er
Nonheredit ry Port wine st in in V1 f ci l distri
ution Foc l seizures, hemip re
sis, hemi nesthesi , tr mtr ck c lcific tion of cortex (not cortic l vessels), l
rge enh ncing choroid ipsil ter l, leptomeningi l ngiom (enh nces), hemispher
ic trophy, gl ucom in kids
Von Hippel-Lind u (vHL)
Chr3, AD, v ri
le penetr nce pVHL (vHL protein) regul tes proteosom l degred ti
on of proteins, including hypoxi induci
le f ctor-1 (HIF-1). Hem ngio
l stom (C
NS + retin l), ren l cell c rcinom , pheochromocytom , ren l nd p ncre tic cyst
s, epididym l cyst denom s. Endolymph tic s c tumors (p pill ry cyst denom s): c
use de fness, f ci l p r lysis. Erosive cystic tumor in EAC. Tre tment is tot l
resection (vi com
ined tr nsm stoid-su
occipit l ppro ch) (JN3/04) Erythrocyt
osis; 25% hem ngio
l stom s h ve VhL. No skin lesions.
Differenti l Di gnosis
Pine l: GCT (germinom ) > pineocytom > pineo
l stom > pine l cyst > strocytom
, meningiom , met Front l horn/For men: Kids: SEGA. Adults: Neurocytom , su
epen
dymom L ter l vents: Kids: PNET, strocytom (
ody), CPP, ependymom ( trium) A
dults: strocytom , su
ependymom (
ody), meningiom , lymphom ( trium) 3rd vent
/supr sell r: Kids: strocytom , histiocytosis, germinom , cr nio Adults: Pituit
ry denom > meningiom > cr nio > gliom > r chnoid cyst, R thkes cleft cyst,
s rcoid, LH Pure Sell r: Adenom , hyperpl si , R thkes cyst, cr nio, dermoid, ep
idermoid, p r medi n c rotid rteries, neurysm (Posterior/St lk: lymphocytic hy
pophysitis, s rcoid, histiocytosis, germinom s) Supr sell r hot spot on T1: Cr n
io, R thkes cyst, lipom , dermoid,
lood, ectopic neurohypophysis, s rcoid, hist
iocytosis, tr nsected st lk Infundi
ulum: Histiocytosis, germinom , s rcoid, men

ingitis, met CPA: Schw nnom (85%) > meningiom (10%, c lcific tion) > dermoid (
5%) In tempor l
one: Gr denigos, Choleste tom ( T1 & T2, chronic otitis), glomus
tymp nicum, m lign nt extern l otitis (DM), jugul r
ul
Bells & R ms y-Hunt enh
nce. Posterior foss : Kids: Pilocytic strocytom > medullo
l stom (homogeneou
s enh ncement) > ependymom (c lcified, hemorhh gic, cystic, extend thru Luschk
). Adult: Met, hem ngio
l stom , strocytom IAC: neuritis (Bells, R ms y-Hunt)
Or
it l: c vernous hem ngiom > mel nom > retino
l stom (kids) Optic n.: Menin
giom , pseudotumor, gliom Anterior skull
se: meningiom , met, mucocoele, oste
om , polyposis, inverted p pillom , esthesioneuro
l stom , coc ine gr nulom tosi
s. Middle skull
se: mets, juvenile n soph rynge l ngiofi
rom ( dolescent m l
es, origin tes t sphenop l tine for men), meningiom , chordom
Flotte Outline of Neurosurgery
28
N soph rynge l: squ mous cell CA, denoCA, denoid cystic CA, chondros rcom , es
thesioneuro
l stom . Dur l (meningiom mimics): hem ngiopericytom , dur l c vern
ous m lform tion, s rcom s, met, extr medull ry hem topoesis (foc l enh ncing m
ss), mel nocytom , s rcoid, Tempor l lo
e: gliom s, HSV, lim
ic enceph litis Pit
uit ry Lesions Lymphocytic Hypophysitis: More common in fem les, perip rtum/preg
n ncy (20%), utoimmune dise se (30%) Present tion: he d che (75%), hypopituit r
ism (DI 30%; leth rgy, loss of li
ido), m ss effect Im ging: Enh ncing sell r m
ss, 80% supr sell r extension, 30% thickened infundi
ulu r st lk. Other types of
infl mm tory hypophysitis: gr nulom tous, x nthogr nulom tous, x nthom tous, ne
crotizing o Infundi
uloneurohypophysitis: Involves st lk only. Resolves with con
serv tive tre tment. Tre tment: o Steroids: L ws recommends tri l of high-dose s
teroids with clinic l suspicion (pregn nt p tients with hypopituit rism) except
with progressive visu l loss,
ut response is inconsistent (60% response) nd hy
pophysitis m y recur upon discontinuing steroids o TSRP: He d che nd visu l fie
lds improve, hypopituit rism doesnt. o SRS. Gr nul r Cell Tumor (Choristom ): Fro
m posterior pituit ry pituicytes. Clinic lly resem
les nonsecretory denom . No
c lcific tion (vs cr nio). Strongly enh nces. Densely p cked, l rge PAS+ eosinop
hilic cells. Pituicytom (GFAP+) pro

ly s me tumor. (per WHO).
Skull lesions: Fi
rous dyspl si : Thick, sclerotic, enh nces. Young. Lionlike f
cies. 70% monostotic. Woven
one: imm ture. No s rcom tous degener tion. Visu l lo
ss/ CN deficits. Al
rights syndrome: unil ter l polyostotic fi
rous dyspl si , p
recocious pu
erty, skin lesions, fem les Eosinophilic Gr nulom : No sclerotic ri
m, p inful, gi nt cells, excision or XRT. <30 yo Epidermoid cyst: sclerotic rim,
sc lloped Hem ngiom : Sun
urst. P inful. Aneurysm l Bone Cyst: r re, tender, oc
cipit l. No endothelium. Osteom : Dense lesion in cortex. P gets: Lytic > sclero
tic ph ses. CN compression, osteos rcom tr nsform tion. Men >40yo. Hyperostosis
front lis intern , p riet l for min : norm l v ri nts (HFI elderly women) Idiop
thic hypertrophic p chymeningitis: diffuse dur l enh ncement ( lso occurs w/ s
rcoid, histiocytosis, intr cr ni l hypotension)
Flotte Outline of Neurosurgery
29
Epilepsy
Intr ct
le: f iled t le st 3 meds. Occurs in 30% Antiepileptics Non-enzyme ind
ucing: Kepr (po only), L mict l (po only), Dep kote (c uses
leeding?) Tegretol
: c uses leucopeni , hep titis Hippoc mp l Sclerosis AKA Ammons Horn Sclerosis (A
HS) Correl ted with Mesi l Tempor l Sclerosis (MTS) on MRI ( FLAIR/T2). Hypomet

olism on PET Correl ted with fe
rile seizures Seen with Timm st in CA1 & CA3 los
s Hypoth l mic h m rtom C uses gel stic seizures initi lly, then complex-p rti
l or gener lized. Responds well to r diosurgery. o Gel stic seizures: l ughing f
its. Other c uses: hypoth l mic gliom , 3rd ventricle tumor, tempor l lo
e epile

psy, inf ntile sp sms, etc. Tumors Seizure focus is usu lly in the surrounding

r in, not in the tumor Single or controlled seizures do lesionectomy. Intr ct
l
e seizures lesionectomy plus intr oper tive ECOG nd resection of seizure focus
Cortic l Dyspl si With complete resection of epileptogenic foc l cortic l dyspl
si , 87% h ve good seizure outcome
Gener l
Preoper tive Ev lu tion
Ph se I: Im ging MRI (FLAIR) If nonlesion l then proceed with: PET o Ict l PET s
hows hypermet
olism in focus. Interict l PET shows hypomet
olism in focus in 7
0%. o Sensitvity in TLE 60-90% o Does not correl te with histop thologic l ch ng
es (eg trophy) SPECT o Su
tr ction Ict l-Interict l SPECT shows hypermet
olism
in focus C n coregister T1 MRI with PET, SPECT, grid xr y for im ge guid nce (J
N3/04) Sc lp EEG/Video-EEG Neuropsychologic l Testing Ph se II: Inv sive monitor
ing Su
dur l Grids & Strips: Sc lp EEG f ils to loc lize focus in 30%. St nd rd:
2 pl ced perpendicul r to tempor l lo
e (consider 1 medi l & p r llel) Electroc
orticogr phy m y ssist in grid pl cement Low- mplitude high-frequency ctivity
t onset correl tes with good surgic l outcome. Electrodes recording higher-freq
uency spikes re closer to focus Depth Electrodes WADA test: Injection of sodium
mo
r
it l into ICA. Used to predict l ngu ge & memory loc liz tion. Speech:
rrest not enough, must h ve n ming errors. Memory: Uses: o 1) Memory loc liz tio
n: less reli
le th n speech. (Note hippoc mp l
lood supply m inly from PCA) o
2) Focus loc liz tion:
le to detect side of eplieptogenic focus in 40-80%, inc
orrect in <10%, indetermin te 20-50%. Memory improves when given on side of focu
s.
Surgery
Flotte Outline of Neurosurgery
Lesionectomy MRI lesion with concord nt EEG: 80-90% seizure-free t 12 months No
nlesion l postop seizure-free 30-50%
30
Tempor l Lo
ectomy Complic tions: Superior qu dr ntopsi most common (higher if
resection >5.5cm posterior) o Anterior choroid l . c n loop into choroids plexu
s void co gul ting choroid plexus Anterior tempor l lo
ectomy: o 70-94% Engle I
-II. o Diplopi 19% (due to CN4 p lsy, resolves). o Extent: 4-5cm in nondomin nt
lo
e, 3.5-4cm domin nt lo
e. o Open choroid l fissure
etween hippoc mpus nd c
horoids plexus (vessels run
etween choroids plexus & th l mus). Be w re of
s
l tempor l l ngu ge re (N5/04) o Tempor l lo
otomy: See N6/04 Amygd lohippoc mp
ectomy: Tr nssylvi n ppro ch descri
ed
y Y s rgil. 74% Engle cl ss I-II in kid
s. Versus ATL: No difference in dults, worse control in peds. Corpus C llosotom
y: Indic tions: Second ry gener lized, tonic, inf ntile hemiplegi , R smussens,
Lennox-g st ut. Contr indic ted in crossed domin nce (left-h ndedness with left
-sided speech) o
t in WADA in ll left-h nded p tients. Complic tions: o Anterio
r: spont neous speech (SMA), nondomin nt leg p resis & gr sping, urge incontinen
ce ( ll usu lly tempor ry). Perm nent speech deficit with mixed cere
r l domin n
ce (speech & motor on opposite sides). o Posterior: interhemispheric disconnecti
on syndrome ( t ctile sens tion & vision on nondomin nt side,
r dyphreni , incon
tinence o Complete: s
ove + nondomin nt h nd doesnt perform comm nds (c n perf
orm nt gonistic ctions) Hemispherectomy Indic tions: Hemiplegi with intr ct

le seizures: R smussens dise se, cortic l dyspl si , hemispheric inf rct. An tomi
c: Complete resection of hemisphere. Complic tions: hydroceph lus, superfici l s
iderosis. Function l: Remov l of centr l cortex nd tempor l lo
e,
s l g ngli
left int ct
ut disconnected from cortex, disconnection of contr l ter l hemisp
here. 25% reoper tion for recurrent seizures. Other techniques: Hemisphereotomy
(JN:P2/04). V g l Nerve Stimul tor: Effective on left side only (re son unknown)
. Effic cy: All seizure types equ lly reduced. 42% reduction in szs @ 18mo, very

few seizure free. Equiv lent to dding nother medicine. Complic tions: Coughin
g, voice ch nges, drooling, l ughing, torticollis, urin ry retention. (No c rdi
c ch nges reported.) Multiple Su
pi l Tr nsections Used to m ke disconnections i
n eloquent cortex Used lone: 15% seizure-free, 35% improved, 50% unch nged. Com

ined with resection: 40% seizure-free, 40% improved, 20% unch nged. Used in L n
d u-Kleffner Syndrome in which epileptic ph si develops in previously norm l c
hild Intr oper tive Electrocorticogr phy Done either with strips or the Hellr ise
r Under gener l n esthesi void
enzodi zepines nd

itu tes. Under loc l use
only n rcotics (fent nyl) nd droperidol. Perioper tive T per nd discontinue A
EDs 24hrs preop. Continue AEDs 1-2 ye rs.
R diosurgery
Dose limited
y optic chi sm nd
r instem. T kes >9mos to work; ur s or seizur
es m y incre se
efore decre sing. M y t ke up to 3yrs
efore considering retre
tment. One report using 20Gy (N6/04) showed 0/5 improvement with MTS nd possi
l
y worsened cognitive testing. M rseilles group (Regis) report 82% seizure-free
nd nother 12% signific ntly improved using 25Gy. T2 sign l pe ks t 1yr. Multic
enter tri l (led
y UCSF) currently ongoing.
Deep
r in stimul tion: of nterior th l mic nucleus nd other re s (centromedi
n nucleus, STN, hippoc mpus)
eing investig ted
Flotte Outline of Neurosurgery
31
Infectious
Cere
r l
scess Most common org nisms: nero
ic/ micro erophilic Strep. Immunoc
ompromised: Noc rdi , Toxo. Neon tes: Proteus, Citro
cter. Predisposing f ctors
: Sinus/dent l infection, pulmon ry
scess/empyem , cy notic c rdi c dise se (T
etr logy, kids), pulmon ry AVF (Osler-We
er-Rendu, heredit ry hemorrh gic tel ng
ect si ), endoc rditis (r re), AIDS Symptoms: 50% h ve low-gr de fever. L
: CXR
, ESR/CRP, CBC (WBC >10K), BCx (10% positive), HIV, Toxo testing. LP contr indic
ted. Im ging o Cere
ritis vs enc psul ted
scess: On del yed CT c psule enh nc
ement dec ys, cere
ritis doesnt. Cere
ritis usu lly thicker o MR-Spect: l ct te,
cet te, pyruv te. M y use to follow response to tre tment. Also WBC-t gged sc n
s. Tre tment o Medic l: nti
iotics  steroids o Indic tions: Cere
ritis, <3cm
s
cess, multiple, deep, eloquent cortex, <2 weeks of symptoms. o Steroids controve
rsi l: m y del y enc psul tion,
ut most evidence rgues g inst routine use. Us
u lly used only if signific nt edem present. o Anti
iotics: V nc/Rocephin/Fl gy
l (empiric). Noc rdi = B ctrim. Toxo = Pyrimeth mine + Sulf di zine. An ero
ic
GNR (B cteroidies) = Fl gyl. Strep = PCN. St ph epi = V nc  Rif mpin. St phj ure
us = n fcillin. Fung l = Ampho B. o Surgic l: o Indic tions: Enc psul ted, periv
entricul r (>80% mort lity w/rupture), m ss effect, di gnosis uncle r, difficult
follow-up, medic l f ilure (incre sed fter 2wks or no ch nge fter 4wks) o Asp
ir tion: Stereot tic, CT or MR guided, or open w/ultr sound.  irrig tion (s line,
nti
iotic). Possi
le lower incidence or seizures & other sequel e. o Excision:
Aspir te first then corticectomy & c psule excision (in noneloquent re s). Pre
ferred for penetr ting tr um , fung l, multilocul ted, f ilure of repe ted spir
tions, posterior foss , g s-cont ining. o Follow-up: Continue nti
iotics for 6
-8wks (12wks if empiric) then m y d/c even if CT
norm lities persist (m y t ke
36mos to resolve). CT q2-4 wks until CT resolution, then q2-4mos for 1yr. Su
du
r l empyem Usu lly second ry to sinusitis/otitis in young people. Strep & St ph
ureus re most common org nisms. C uses cortic l vein throm
osis. Tre tment: e
mergent cr niotomy. Do not remove mem
r ne dherent to cortex. AIDS Foc l lesion
s: most common re o Toxopl smosis: most common (75%). L rge hypodense re with
edem nd ring-enh ncement. Common in
s l g ngli . Tre tment: pyrimeth mine
nd sulf di zine. o Progressive Multifoc l Leukoenceph lop thy (PML): due to JC p
pov virus. Demyelim tion occurs. Hypodense, hyperintense on T2, no edem or enh
ncement. Inv ri
ly f t l no effective tre tment. o Prim ry CNS Lymphom . Enh n

ce strongly (m y rim enh nce or re t rget lesions in AIDS lymphom ). Tre tment: W
BRT. M n gement: PML c n
e differenti ted
y l ck of enh ncement. O
t in CSF fo
r cytology (lymphom ) nd serum toxo titers. If lesions enh nces nd p tients tox
o titers re positive (will
e positive in 90% of norm l popul tion), then tre t
empiric lly for toxo. If no response fter 3wks or titers re (-), then conside
r
iopsy. Single lesions re more likely lymphom th n toxo. Cryptococcus c uses
meningitis Neurocystercercosis Infection of l rv e of pork t peworm T eni soli
um. Most common CNS p r sitic infection. Endemic in Mexico. Incu
tion: months t
o >10ye rs. T peworm (intestin l) infection results from e ting undercooked pork
. Cystercercosis results from e ting t peworm eggs (ie through fec l cont min ti
on of food or utoinocul tion). M y
e meninge l, p renchym l, ventricul r. Usu
lly ring-enh ncing lesions with minim l edem . M y h ve su
cut neous nodules. CS
F m y show eosinophili . Serum nd CSF nti
ody titers c n
e checked. Tre tment
: Pr ziqu ntel nd steroids. R rer p r sities: echinococcus (hyd tid cysts) dog
t peworm. (m y grow to
e l rge,
e c reful not to rupture cyst); me
i sis Gr d
enigos Syndrome: osteomyelitis of petrous pex. CN6 p lsy & retroor
it l p in, f
rom otitis
Flotte Outline of Neurosurgery
32
Whipple dise se: C used
y
cteri Tropherym whippelii. Symptoms: g strointest
in l symptoms nd migr tory rthr lgi s. P th: periv scul r m croph ges with di
st se-resist nt PAS (+) gr nules. Involves CNS in 25%: dementi , multifoc l grey
-m tter lesions, especi lly tempor l cortex, th l mus, etc.
Flotte Outline of Neurosurgery
33
Peripher l Nerve
Neuropr xi > Axonotmesis (perineurium nd epineurium int ct) > Neurotmesis. Sun
derl nd cl ssific tion I-V. Spont nteous recovery: 40% C5/6, 18% C5-7, 5% C5-T1
(fl il rm) Motor recovery occurs within 18mos (no limit on sensory) Tre tment L
cer tion: Rep ir within 72hrs if sh rp. 2-4 weeks if
lunt (to llow deline tio
n of injury) Penetr ting: explore when 1 wound he led. GSW: 2-5 mos for GSW with
complete or severe lesions-in-continuity with intr oper tive o E rly explor tion
is dvoc ted
y some Tr ction/Blunt : 3-6mos. Seri l EMG/NCVs (& SSEP?) q3mos,
rep ir if no improvement on EMG or clinic lly. Rep ir possi
le for upper element
s only. Also for p in, pseudo neurysm. Most effective if p tient is <50yo. Surgi
c l Rep ir Consider tourniquet (remove during intr op nerve testing). Expose nor
m l nerve proxim l & dist l 1st. Nerve Action Potenti ls (CNAP): Stimul tion pp
lied cross neurom or
lunt/strech injury site (proxim l stimul tion, dist l re
cording). o Kline: If conduction occurs then only perform neurolysis (others per
form neurolysis nd dist l tr nsfer, or no neurolysis
ut dist l tr nsfer only).
If no conduction then perform gr ft or nerve tr nsfer. o Others perform gr ft o
r tr nsfer reg rdless of CNAP. Techniques Neurom : Determine need for resection

y p lp tion (firm = worse), CNAP (see
ove). Immo
ilize joint x 6wks. Neurorrh
phy: end-to-end rep ir. Must h ve no tension. Use 10-0 suture. Suture epineuriu
m (not perineurium/f sicles) Neurolysis: If nerve is in continuity nd shows evi
dence of regener tion
y positive NAP dist lly or muscle contr ction in response
to stimul tion Neurotiz tion (nerve tr nsfer): Used for root vulsion. o Avoid
intervening gr fts, use direct tr nsfer when possi
le. Impl nt s close s possi

le to the site where function is to
e restored Interposition nerve gr ft: o Do
nors: sur l, medi l nte
r chi l cut neous, superfici l r di l. o C
le gr ft: sev
er l sm ller nerves used to rep ir l rge n. Burying into muscle: equivoc l, repo
rted to
e effic cous
y some
Nerve injury

Entr pment Neurop thies

P in is usu lly t the entr pment site, not in the distri
ution of the ffected
nerve. P r sthesi s more common th n num
ness. Clumsiness more common th n discr
eet we kness. NCV shows conduction del y cross the site of entr pment Tinel sig
n: p in/p resthesi s reproduced on mech nic lly stimul ting (eg t pping) the ner
ve

Upper extremity
Root C5 C6 C7 C8 T1 Clinic lly Relev nt Gross Motor Function Shoulder
duction;
 el
ow flexion El
ow flexion, pron tion/supin tion,  wrist extension Diffuse loss
of function in the extremity without complete p r lysis of specific muscle gr
oup Finger extensors, finger flexors, wrist flexors, h nd intrinsics H nd intrin
sics
Br chi l Plexus
Flotte Outline of Neurosurgery

34
Supr sc pul r n.: Shoulder
duction (first 15). Supr spin tus/ infr spin tus. Mo
st often rep ired
y spin l ccessory n. tr nsfer. Spin l ccessory n.: injured
in surgery of posterior cervic l tri ngle (lymph node
iopsy). We k shoulder
d
uction, drooping shoulder, winging of sc pul , shoulder p in. No SCM p r lysis (
tr pezius only). SAN is ceph l d to gre t uricul r n. t l ter l
order of SCM.
(N11/03) Roots (myotomes & derm tomes) show consider
le overl p, peripher l ne
rves h ve sh rp
ound ries Cords: o L ter l = muscles to fore rm nd sens tion o
f medi n n. o Medi l = ll medi n nd uln r intrinsic h nd muscles Injuries Er
s
p lsy: C5 & 6,
ellhops h nd o Er
s
irth injury: Controversi l whether to oper
te or o
serve Klumpkes p lsy: C8 & T1, cl w h nd Uln r n. vs C8 root injury: Sen
s tion: uln r n. splits 4th digit, C8 covers entire finger. Motor: C8 root lesio
n c uses loss of ll intrinsic h nd muscles (uln r nd medi n) P nco st tumor: H
orners + C8/T1 loss Supr sc pul r entr pment: in
ility to
duct shoulder first
15 Winged sc pul : o long thor cic n. or serr tus nterior injury (lymph node
i
opsy): winging when rm extended (eg g inst w ll) o spin l ccessory n. injury:
winging when el
ow flexed ( cross chest)
Br chi l Plexus Rep ir
Priorities for function l improvement: el
ow flexion, shoulder
duction, finger
flexion, wrist extension, shoulder rot tion Dist l recovery is much more diffic
ult to chieve Preoper tive workup MRI, CT-myelogr m: Kline feels CT-myelogr m
re usu lly necess ry unless MRI is conclusive for vulsion o Meningocoeles roun
d nerve root imply root vulsion. o A
sence of meningocoele does not rule out
n vulsion EMG: documents the degree nd p ttern of dennerv tion, nd ny signs
of reinnerv tion o Kline feels th t SSEPs do not dd ny inform tion Differenti
te root vulsion from postg nglionic injury: Horners syndrome, p r lysis of serr
tus nterior (winged sc pul ) or rhom
oids, di phr gm p r lysis on CXR (for C35),
sent Tinel sign, e rly neurop thic p in, EMG shows p r spin l deinnerv tio
n (w it >3wks), norm l SNAP (lost in postg nglionic lesions. More sensitive th n
n
sent SSEP), MRI/CT shows meningocoele. Root vulsion No spont neous recove
ry. Gr fting not yet fe si
le (experiment l). Most tre tment is
y nerve tr nsfe
r. Intr oper tively some dissect the nerve root into the for min to prove vuls
ion Br chi l Plexus Explor tion Trunks t l ter l
order of nterior sc lene. Co
rds t level of 1st ri
. Supr cl vicul r exposure: Supine, shoulder roll, he d t
urned w y. Incision posterior
order SCM, long cl vicle. Pl tysm divided. EJ

divided. Tr nsverse cervic l . & v. lig ted only if su
cl vi n . p tent. Cl vi

cul r he d SCM det ched. CN11 under SCM preserved. Omohyoid divided. Phrenic n.
identified on nterior side of nterior sc lene & mo
ilized medi lly. Br chi l p
lexus runs
etween nterior & middle sc lenes. 1-2cm of nterior sc lene resecte
d. Thyrocervic l trunk preserved if possi
le. Infr cl vicul r: Incision long cl
vicle to deltopector l groove. Ceph lic v. exposed. Cl vicul r he d of pector l
is m. divided. Pector lis minor det ched from cor coid process. Su
cl vius m. de
t ched from cl vicle. L ter l cord identified. Avoid sectioning cl vicle he ls p
oorly. Axill ry: Extend infr cl vicul r incision long pector lis to humer l ins
ertion (m y
e det ched). Musculocut neous n.
elow pector lis. Posterior ppro
ch: useful fter nterior ppro ch f ilure or TOS. P tient prone, rm
ducted &
flex, he d turned contr l ter l. Incision
etween medi l
order of sc pul & sp
ine. Tr pezius, lev tor sc pul e & rhom
oids divided. 2nd ri
divided medi lly,
1st ri
& T1 tr nsverse process resected. Sc lenus posterior & medius tr nsected
. Trunks visi
le Neurotiz tion (Nerve Tr nsfer) Spin l ccessory n. (CN11) works

est for supr sc pul r nerve, less for xill ry or musculot neous nn. (where in
terposed gr fts re necess ry) Intercost l-musculot neous n. restores
iceps in
70%
Flotte Outline of Neurosurgery

35
Medi l pector l-musculocut neous n. works if these
r nches re su
st nti l O
er
lin procedure: f scicle of uln r n. co pted to dist l musculocut neous n. o Pref
erred
y some to restore
iceps in p tients presenting over 8mos fter injury C7
: ipsil ter l or contr l ter l (requires lengthy gr ft). C rries sm ll risk of w
e kness. Cervic l plexus or C3-4 provide some we k motor function Phrenic n.: Kl
ine voids using it. Hypogloss l n. not effective per Kline Ex mples (see JN 9/0
4) C5-T1 vulsions: Accessory-supr sc pul r, intercost ls-musculocut neous/ xill
ry/medi n (or medi l pector l, or phrenic). Thor cic Outlet Syndrome/Cervic l r
i
From C7 m y
e incomplete ri
or fi
rous
nd. Elev tes nd stretches the
lower
r chi l plexus, su
cl vi n . & v. Affects lower trunk: we kness in ll
h nd muscles (uln r nd medi n), uln r num
ness No neck p in. M y h ve mild chi
ng p in of uln r fore rm/h nd. H nd we kness/clumsiness is prominent. Atrophy (gu
ttering) of the l ter l then r eminence (APB) is ch r cteristic. Bil ter l in 50%
,
ut less ffected side is usu lly su
clinic l, di gnosed
y EMG Chest or (o
li
que) c-spine Xr ys or CT usu lly show cervic l ri
NCV shows low APB mplitudes
(1st dors l interosseous uln r is norm l or slightly low), low uln r sensory pot
enti ls with norm l medi n sensory potenti ls Adsons test: turn he d
ck & to
ffected side & lose r di l pulse Other Br chi l Plexop thies Acute Br chi l Neur
itis (P rson ge-Turner Syndrome): Sudden onset of very severe p in (p tient pres
ents to ER) in shoulder girdle. P in persists for hours or weeks then
ecomes du
ll che. As p in su
sides r pid proxim l rm we kness
ecomes prominent (deltoid
, supr /infr spin tus,
iceps). We kness usu lly recovers (90% t 3 ye rs),
ut
degree nd dur tion of recovery re v ri
le. Sensory ch nges re mild. 33%
il
ter l. Occurs t ny ge,
ut pe ks in 20s nd 60s. M les f vored 4:1. 25% follo
w vir l illnesses or v ccin tions. M y occur postop, even with dist nt surgery.
No fever. WBC nd ESR re norm l. No tre tment. Steroids ineffective.
Uln r nerve Entr pment o Signs: Interosseous w sting. W rten
ergs sign (we kness

duction 5th digit); Froments sign (grips p per
/t thum
& fingers w/tip only
dductor policis we kness) o Cu
it l tunnel: Medi l el
ow
etween 2 he ds of FC
U. Surgic l options include simple decompression, medi l epicondylectomy, or tr
nsposition (su
cut neous, su
muscul r or intr muscul r) controversi l. o Guyons
c n l: Wrist sensory to dorsum of h nd sp red,
Flotte Outline of Neurosurgery

36
o Arc de of Struthers: 8cm proxim l to el
ow, r re Exposure t el
ow: Incision
t medi l epicondyle. Follow dist lly thru cu
it l tunnel & FCU. Tr nsposition: m
oving uln r n. out of uln r foss (
/t medi l epicondyle & olecr non process) Ex
posure t wrist: Incision over uln r rtery
etween pisiform & h m te, followed
proxim lly.
Medi n entr pment 1) C rp l tunnel Sxs: w kens @ night, num
ness/ tingling, m y
h ve p in in 5th digit & up rm (re son uncle r), then r trophy, Ph lens (61%
sens, 83% spec) /Tinels (74% sens, 91% spec). Dou
le-crush: CTS + cervic l r dic
ulop thy DDx: DeQuerv ins syndrome: tendonitis of APL, tenderness t
se of thu
m
, ssoc. with pregn ncy. Dx: NCV/EMG: medi n sensory (> motor) l tency (norm l
in 20-30%). Tre tment: Wrist splint, steroid (no nesthetic) injection (uln r t
o p lm ris longus). C rp l Tunnel Rele se: P lm r cut neous
r nch is superfici
l to flexor retin culum on r di l side. Recurrent motor
r ch usu lly exits dist
l to lig ment
ut m y pierce it. 2) Pron tor teres syndrome More prominent p lm
r p in (medi n p lm r cut neous
r nch rises proxim l to TCL). Aching fore rm
fter use, we k h nd. No night sxs. Dx: P in on resisted pron tion. NCV not usef
ul (episodic) C use: repe t pron tion. Tre tment: rest fore rm. 3) Anterior inte
rosseous syndrome No sensory loss. Loss of Pron tor, FPL, index FDP. NCV not hel
pful. Usu lly resolves. R di l nerve Supin tor tunnel syndrome: At el
ow. Mimics
refr ctory tennis el
ow. Posterior interosseous syndrome: finger drop without w
rist drop (ECR sp red). No sensory loss (?). Tr pped t rc de of Frohse. Tre tm
ent: conserv tive Mer gli P resthetic : L ter l femor l cut neous n. Sensory on
ly, l ter l thigh. Dx: Loc l
lock just medi l to ASIS. Electrodi gnostic studie
s only helpful to rule out other c uses. Tre tment: Neurectomy more effective th
n decompression. Incision medi l to ASIS (longitudin l or tr nsverse), Section
f sci l t over nterior
order of s rtorius. Section inguin l lig ment. Lower
Extremity O
tur tor n.: Adductor we kness Femor l n.: JN 6/04. Injured in thigh
fter herni or hip oper tions. From lum
r plexus (L1-L4). Supplies iliopso s,
qu driceps Sci tic n.: L4-S2. H mstrings nd ll mm
elow the knees Common Peron
e l n.: Deep n.: dorsiflexion ( nterior ti
i lis), toe extension. Superfici l n.
: foot evertors. Lesion c uses foot drop. G nglion cysts: resection recommended.
Surgic l exposure: prone. S-sh ped incision. See N6/04. Ti
i l n.: pl nt r flex
ion, toe flexion, inversion, sens tion to sole of foot. G strocnemius, soleus mm
. T rs l tunnel syndrome: ti
i l n. t medi l m lleolus. p resthesi s sole of fo
ot no motor loss. Tinels. NCV. 90% improved. (only 50% in reops) (N11/03)
Flotte Outline of Neurosurgery
37
Function l
P rkinsons Dise se
Etiology unknown. Decre sed neurons in SNpc, DMN v gus & locus ceruleus. P th: L
ewy
odies (eosinophilic intr cytopl smic inclusions with h lo). Symptoms: resti
ng (pill-rolling) tremor,
r dykinesi , cogwheel rigidity. Also: microgr phi , d
ecre sed
link, m sked f cies, festin ting g in, p in in 50%, GI pro
lems, dys u
tonomi , weight loss. Symptoms usu lly symmetric. 20% h ve dementi . Di gnosis
is clinic l. P rkinsons Plus syndromes: DBS is ineffective, even when responsive
to levodop . o P rkinsonism: 80% due to P rkinsons dise se, 10% P rkinsons-Plus
(MSA, PSP, CBGD, diffuse lewy
ody dz), 10% second ry. (drugs: neuroleptics, res
erpine, C -ch nnel
lockers, lithium) o Multisystem trophy (MSA): Younger. P th
: -synuclein positive gli l cytopl smic inclusions, no Lewy
odies. Poor response
to dop mine. Includes: Stri tonigr l degener tion: syncope, stridor. Put men t
rophy Olivopontocere
ell r trophy: AD, Chr 6, 15yo, LE t xi , trophy middle c
ere
r l peduncle Shy-Dr ger: utonomic pro
s, impotence, no lewy
odies(?); loss
in put men, SN, & interomediol ter l horn cells o Progressive Supr nucle r P ls

y (PSP): downg ze p lsy, pseudo
ul
r, g it p lsy, eyelid freezing, no tremor, s

ymmetric. MR: trophy of mid
r in & tectum. 70yos. No tre tment. o Cortic l
s l
Degener tion (CBD): P rkinsonism, cortic l signs ( pr xi , myoclonus) nd lien
lim
. No tre tment. Tre tment: Medic l:
enztropin (Cogentin)/ Art ne: ntichol
inergics. Am nt dine: Rele ses dop mine. Loses effect with time. Sinemet: Dop mi
ne + c r
idop ( dop dec r
oxyl se inhi
itor). 2nd tier. SE: N/V, orthost tis,
rrythmi s, on-off periods, dyskinesi s (Tx B6). Contr indic ted with Mon mine
Oxid se Inhi
itors (MAOIs). Bromocriptine/ pergolide: Stimul tes D2 receptors. S
E: v soconstriction, fi
rosis (
romocriptine is used for prol ctinom , pergolide
isnt). Selegiline/ Eldypryl: MAOI. Slows progression. deprenyl: MAOBI. Surgery M
od lities Lesioning: GPi, VIM th l mus, STN Deep Br in Stimul tion (DBS): of STN
, ( lso GPi, th l mus.). Up to 35% of p tients develop toler nce (for tremor) R
diosurgery: only Vim Th l mus. 130Gy m rgin dose. One 4mm collim tor. Success: 8
2% good response. Onset 2mos. SE: Dys rthri . Experiment l surgeries: Fet l mese
nceph lic tr nspl nt tion into SN c used dyskinesi s, some improvement <60yo. Ad
ren l medull ry tr nspl nt
ndoned. Loc tions 1. P llidotomy: GPi (6mm lesion,
posteromedi l GPi, 2mm
ove optic tr ct, 3-4mm nterior to intern l c psule.)
Usu lly done unil ter lly only due to cognitive risk. Indic tions: refr ctory to
meds, dop -induced dyskinesi (90% success), rigidity (75%),
r dykinesi (85%)
, on-off, dystoni (tremor only 57% success). SE: field cut 3%, dys rthri 8%, h
emip resis, cognitive pro
s if
il ter l. CI: dementi ( cognitive pro
s), ipsil
ter l hemi nopsi , >85yo, 2 p rkinsonism. Withold meds d y of surgery. Avoid IC,
optic tr ct. T rget: midpoint of AC-PC line: 2mm nt, 21mm l ter l, 5mm inferior
. C n l st >5yrs. 2. Th l motomy: VL: tremor/rigidity. VIM/VOP: tremor. Complic
tions: confusion, speech
norm lity ( w/
il ter l). 3. STN stimul tion: M y
e
done
il ter lly. Effective for
r dykinesi , tremor, rigidity. Medic tion reduc
ed or elimin ted (r rely with p llidotomy over ll results
etter, however more e
xpensive $36K vs $12K). Improved g it usu lly requires
il ter l stimul tion. FD
A pproved J n. 2003. Appe rs to
e 10% more effective th n GPi stimul tion. STN
c n
e seen on T2 low
ndwidth. Position v ri
le enough th t microelectrode r
ecording is needed. (JN3/04) SE: cognitive decline, depression, hypophoni . Effe
ct declines with time. Mech nism unknown. Also effective in essenti l tremor. Es
senti l Tremor Occurs in elderly. F mili l: Autosom l domin nt with v ri
le pen
etr nce, 60% of c ses. Spor dic in 40%. Bil ter l ction (not rest) tremor of h
nd/ rms or he d. No other neurologic signs. Tre tment: Prop nolol, prim done, ST
N stimul tion (or th l motomy). (Neurologist 9/04) Torticollis
Flotte Outline of Neurosurgery

38
Tre tment: CN11 neurectomy & upper cervic l ventr l rhizotomies (97% success), t
h l motomy (66% success), MVD (70% success), DCS, Botox
Hemif ci l sp sm Intermittent, p inless. Begins ocul r, goes c ud lly. Persists
during sleep. Fem les. MRI (no ngio). o Atypic l sp sm
egins in
ucc l muscles
, usu lly dors l-rostr l surf ce, h rder to tre t or preserve he ring. Tre tment
: o Botox injections q3-4mos for life (Tegretol, B clofen). o MVD: 34% AICA, 31%
PICA, 31%
oth, 4%
sil r. Vessel usu lly cont cts ventroc ud l surf ce of CN
VII. Dissect DREZ only, not dist l. Intr op BAERS (Pe k V l tency del y: 0.6ms =
w rning, 1.0ms = critic l). SE: de fness (3-10%) > f ci l we kness (1%). M y pe
rsist >3d postop. Success 94%, 10% recurrence, 86% t 1mo. Some use p p verine i
ntr op. Idiop thic Intr cr ni l Hypertension (IIH, Pseudotumor Cere
ri): D ndy c
riteri : symptom tic, no loc lizing findings (except CN6,7 p lsy), lert, norm l
CT/MRI, ICP >25. O
ese women, 20-45yo. Usu lly self-limited, tr nsient during p
regn ncy. Sxs: He d ches in ne rly ll worse in m, with V ls lv . Also tr nsien
t visu l ch nges. P pilledem in lmost ll (r rely
sent not necess ry for di
gnosis). Ventricles m y
e norm l or slit. Empty sell nd enl rged optic n. she
th in 50%. LP: Some symptom tic o
ese women m y h ve ICP > 25. CSF norm l. Ass

oci ted with vit min A & retinoids, nti
iotics (tetr cycline), hormones, steroi
d withdr w l, lithium. Stop meds if possi
le (stopping OCPs not necess ry). Also
ssoci ted with lupus, uremi , etc. Blindness most signific nt sequel e occurs
in 25%. C n
e r pid. Follow visu l fields. Tre tment: Weight loss. Di mox (SR 5
00mg BID, ter togenic)  Dec dron (for cute
lindness). Progressive visu l loss d
espite medic l tre tment: 1. Seri l LPs: cum
ersome 2. Su
tempor l decompression
: historic l 3. Optic nerve fenestr tion: 90% successful. Unil ter l fenestr ti
on ppe rs to lower ICP, improves vision
il ter lly nd CN deficits, help HAs (
de
ted). 2% risk of
lindness. 4. LP shunt: Use horizont l (high pressure) vert
ic l (medium) v lve. Complic tions: f ilure 55% in 1 ye r, overdr in ge 15%, lum

r r diculop thy 5%, infection 1%, cquired Chi ri nd syringomyeli . 5. VP shu
nt: for repe ted LP f ilure. o De
ted wether ONSF or LP shunt is
est. H ve equ
iv lent effic cy. Empty Sell syndrome 1 (incompetent di phr gm) or 2 (surgery, st
roke, etc). Sxs: HA, CSF rhinorrhe , visu l loss, menorrhe -g l ctorrhe . Surge
ry only for CSF rhinorrhe tr nsphenoid l rep ir  lum
r dr in. Shunt m y c use p
neumoceph lus Sheeh ns syndrome: ischemic necrosis 2 to intr p rtum shock Intr cr
ni l hypotension: C uses dur l enh ncement on MRI/CT. C uses include shunts, CS
F le ks. Spont neous cr ni l CSF le k: Due to genesis of the nterior foss , em
pty sell , sinus infection, tumor. Spont neous spin l CSF le ks M y
e due to os
teophytes, dur l te rs,
sent nerve root she ths. Connective tissue disorders m
y predispose. C uses postur l he d ches. MRI: dur l enh ncement, downw rd displ
cement of cere
rum. Dx: Myelogr m. (Low opening pressure on LP). Tre tment: Mos
t resolve spont neously. Bedrest,
lood p tch, surgic l rep ir. (JN11/03) Norm l
Pressure Hydroceph lus (NPH): Term coined
y H kim 1964. Idiop thic c ses m y

e c used
y unrecognized SAH, meningitis, etc. Symptoms: tri d of dementi , g it
inst
ility, nd incontinence. Usu lly occurs in the elderly. Di gnosis: o High
-volume LP: remove 30cc nd look for clinic l improvement. Consider m
ul tory l
um
r dr in tri l. o CT: Communic ting hydroceph lus. o Cisterogr phy: positive
nd neg tive predictive v lue only 50% - not recommended
y most. Activity persi
sting >48hrs implies good response to shunting. Tre tment: Shunt: Medium-pressur
e v lve. 66% improve. Incontinence improves first. Endoscopic third ventriculost
omy h s
een used (N7/04) o Better outcome seen with minim l ch nge in ventricul
r size th n in p tients with m rked decre se
Flotte Outline of Neurosurgery
Sp sticity: Tre tment: o Botox loc l only; l sts 3mo. o B clofen pump. Overdose:
stop pump, IV physostigmine, remove 30ml from side-port or
y LP
39
Dystoni Sust ined muscle contr ctions c using twisting, repetitive movements. C
n
e loc l or gener lized, prim ry (idiop thic) or second ry to
r in insult. P
rim ry: 30% h ve utosom l domin nt DYT1 mut tion Tre tment: o Medic l, IT
clo
fen: often ineffective. o P llidotomy or GPi DBS equ lly effective in prim ry n
d cervic l dystoni . Neither effective in second ry dystoni or p tients with n
y MRI
s l g ngli
norm lity. Selective Dors l Rhizotomy: intr oper tive EMG
to preserve useful sp sticity. Preserves m
ul tion. F ci l P lsy: c n n st mose
CN12, 11, or 9 to CN7 OCD (O
sessive-Compulsive disorder): Tre tment: Bil ter l
nterior cingulotomy. Only 6-30% perm nently respond (MGH reports 50% success).
M ny rel pse within 1yr m y need repe t procedure. Also used for m jor depressio
n. T rget: 2.5cm posterior to tip of front l horn. Bil ter l nterior c psulotom
y ( nterior lim
of intern l c psule) Lim
ic leucotomy. A
ove m y
e done stereo
t ctic lly or with SRS.
P in
Deep Br in Stimul tion for P in: Peri cqueduct l grey (PAG). SE: diplopi , nxie
ty. Periventricul r grey (PVG): S fest. VPL/VPM Cingulotomy: must
e
il ter l;
recurs 3mo; SE: 10-30% fl t ffect Medi l Th l motomy: he d/neck p in. SE: 20-70
% cognitive pro
s, ph si Mesenceph lotomy: he d/neck p in Cordotomy Unil ter l
p in
elow nipple, termin l pt, ching, de fferenti tion p in Open: C1/2 l mino

tomies; Cut contr l ter l spinoth l mic tr., st rt nterior to dent te, 5mm deep
; Check PFTs, di phr gm fxn preop Percut neous: Aw ke, use stimul tion Success:
94% initi l; 60% 1yr; 40% 2yr. SE: p resis, incontinence Commisur l Myelotomy:

il ter l p in, thor cic &
elow, l minectomy 3 levels
ove; 60% success Spin l
Cord Stimul tor AKA dors l column stimul tor Pl ced epidur lly. Works when pl ce
d ventr lly lso (for unknown re sons). Le d tips C3-C6 for UE, T8-T12 for LE. D
one w ke w/propofol & loc l nesthesi . M y
e done percut neously of vi l min
ectomy, with RF-controlled receiver or IPG. Results: Few RCTs. Met n lysis (n=3
679), success r tes: SCI, f iled-
ck, ph ntom lim
= 60%, peripher l neurop thy
= 70%, ischemic lim
p in, postherpetic neur lgi , CPRS = 80%. Poor for SCI, ro
ot vulsion (
norm l Centr l Conduction Time (CCT) on SSEPs, hypesthesi ), or m
lign ncy. In RSD response m y
e predicted
y symp thetic
lock de. Used common
ly for ngin in Europe (off-l
el in US) MRI with DCS h s
een reported (Sh RV
04)
Flotte Outline of Neurosurgery
DREZ rhizotomy: good for nerve root/
r chi l plexus vulsion Intr thec l pumps:
For nociceptive c ncer p in
ove C5. Suggest IT tri l 1st
40
Complex Region l P in Syndrome (CPRS) Type I = Reflex Symp thetic Dystrophy (RSD
), no nerve injury. Type II = C us lgi , due to incomplete m jor nerve injury (i
e GSW). Both types c n
e symp thetic lly m int ined p in (SMP) or symp thetic l
ly independent p in (SIP). Sxs: Hyperesthesi (not hypesthesi ) Tri d:
urning p
in, utonomic dysfxn ( or swe ting & h ir, v soconstriction or dil tion), trophi
c ch nges. Sudeks trophy (skin,
one, etc not nerve). Etiology unknown (epiph t
ic tr nsmission discredited). Most h ve onset <24hrs, usu lly within 1mo. Dx: Sy
mp thetic
lock: Stell te for UE, lum
r for LE. Confirmed
y 1C incre sed temper
ture. Question
le f iled RCT. Tre tment: TCA, reserpine, gu nethidine symp the
tic
lock (18-25% success, repe t
le), symp thectomy, DCS Symp thectomy Used fo
r hyperhidrosis, R yn uds, ngin , RSD/CRPS, peripher l v scul r dise se. Resect
2nd ( 3rd or 4th) thor cic g ngli for UE (le ve T1 to prevent Horners), 2nd & 3
rd (L1) for LE Appro ches: supr cl vicul r, tr ns xill ry, posterior. C n
e done
endoscopic lly. Success: hyperhidrosis >90%. Complic tions: Thor cic:
r dyc rd
i (usu lly symptom tic). Lum
r: retrogr de ej cul tion. Ot lgi M y come from
CN 5,7,9, or 10. Give tri l of TGN meds. Intr ct
le: explore CNs MVD vs sectio
ning (nervus intermedius, 9, upper 2 roots of 10)
Trigemin l neur lgi
Unil ter l, no deficits, sensory trigger. Due to epiph tic tr nsmission. More co
mmon on right. V2&V3> V2> V3> V1&2> V1. R>L. Bil ter l or V1 think MS. <1% due t
o tumor (not responsive to meds). Onset >50yo. Di gnosis: MRI if typic l or con
sidering surgery. DDx: zoster (continuous p in). Tre tment Medic l: Tegretol > d
il ntin >
clofen. Microv scul r Decompression (MVD): o 80% SCA ( lso PPTA, AIC
A,). In 5% of c ses compression is prim rily venous (tr nsverse pontine & trigem
in l veins (N8/04) o Indic tions: >5yr surviv l, <65yo, f iled PTR, V1 (less ris
k of ker titis th n PTR). o 85-95% initi l success, 70% t 10yrs. o Pexy stitch
on petros l surf ce m y
e used to keep vessel off nerve with 8-0 nylon, or Tefl
on nd fi
rin glue. o J nett stresses voiding cere
elll r retr ction, use CSF
dr in ge. 2mm retr ctor used, r chnoid dissected on cere
ell r side only to pro
tect CN. Dissect vessels off DREZ only (not dist l? Others s y whole nerve must

e dissected (vs HFS) N12/03). Pl ce insul ting sponge on vessel. o Superior pet
ros l veins m y
e cut nd divided when multiple veins exist nd it is not the s
ingle m in dr iner. o No nesthesi doloros . Mort lity 0.3%, neurologic mor
idi
ty 2% (higher in redos, verte
ro
sil r dolichoect si ). Less risk of f ci l num

ness th n PTR. Sm ll pontine inf rctions occur on MRI in 24% o Also used for he
mif ci l sp sm & glossoph rynge l neur lgi . Percut neous Trigemin l Rhizotomy (
PTR): o R diofrequency,
llon, glycerol. 3yr: RFT 62%, Glycerol 54%,
lloon 69
%. Br dyc rdi , HTN m y occur (consider tropine preop). Str ight or curved elec

tode (5mm 1div, 7.5mm 2 div, 10mm 3div). Pl ce needle 3cm l ter l to or l commis
sure. Aim 3cm nterior to EAM, medi l pupill ry line. P lp te intr or lly. P tie
nt winces when entering for men ov le. O
t in CSF (in trigemin l cistern). Stimu
l te to reproduce p in
efore lesioning. SE: P resthesi s/ dysesthesi s 20%, n
esthesi doloros 4%, m sseter we kness, he ring (tensor tymp ni), EOM p resis,
neurop r lytic ker titis (2%). (N4/04) R diosurgery: o Success: 70% h ve >50% im
provement, 55% no p in with meds, 40% no p in. (55% t 3yrs). 58% for second ry
TN, 0% for typic l. 13% rel pse ( t vg 15mos). Not s good s initi l MVD,
ut
s good for second procedure. o For repe t SRS: 50-60Gy, nterior to 1st t rget
. o 25% new num
ness, 12% other complic tions. T kes 3 months to work. All impro
vement occurs
y 1 ye r, most
y 6 mos. o 80 (70-90) Gy, one 4mm collim tor. Br
instem surf ce t 30% isodose line, center t DREZ. Also: peripher l neurectomy
(supr or
it l, infror
it l, inf. dent l nerves only; nonoper tive c ndid tes); I
ntr dur l neurectomy (f iled PTR w/ preexisting CN5 n esthesi . V1 superior, V3
inferior. Cut lower )
Flotte Outline of Neurosurgery

41
R eders syndrome: V1 & V2 p in with oculosymp thetic p r lysis ptosis nd miosis
(no nhydrosis-pseudoHorners); due to ICA dise se Tic convulsive: TGN w/hemif c
i l sp sm
Glossoph rynge l neur lgi P in in e r, tongue, tonsil, nd ngle of j w. 10% h
ve v g l
r dyc rdi / systole. Dx: coc niz tion of tonsill r foss relieves the
p in. Tre tment: MVD (PICA) or rhizotomy (9 & top 2 roots of 10). 11% risk of h
o rseness/dysph gi h ( lso f ci l p resis). C rdi c inst
ility m y occur intr o
p, give tropine
efore m nipul ting nerve. Monitoring CN9-10 not helpful. (N4/0
4) MVD Used for trigemin l neur lgi , hemif ci l sp sm, glossoph rynge l neur lg
i . Also reported for torticollis, Refr ctory essenti l hypertension: decompress
ion of the left rostr l nteromedi l medull . J nett reported 75% success. Neur
ogenic hypertension: B sil r rtery on left rostr l ventrol ter l medull (RVLM)
, responds to MVD; m y lso
e due to
sil r impression & responsive to odontoi
dectomy. Genicul te neur lgi : ot lgi , prosop lgi (deep f ci l structures). Tr
e tment: symptom tic Postherpetic neur lgi : Persists >3mos fter zoster. Lidoc
ine p tch ( lso IT lidoc ine, Zostrix, El vil.) No surgery. Occipit l neur lgi :
Block, TENS. Surgery poor. Neurop thic p in: Neurontin, TCAs, other AEDS, lidoc
ine gel/ p tch, c ps cin
Flotte Outline of Neurosurgery
42
Spine
Lines McR e: for men m ngum di meter, >35mm, ny protrusion of odontoid
ove is

norm l; Ch m
erl in: p l te to for men m gnum, odontoid not >1/3 or 6mm
ove
; W ckenheim: clivus, tip
ehind; McGregor: p l te to occiput, tip not >4.5mm

ove; Fishgolds dig stric: tip not
ove; Pl ty
si : >145. B sion-Dens interv l:
<12mm. Atl ntodent l interv l (ADI):
norm l >3mm dults, >4mm kids. >4mm = tr
nsverse lig ment disruption possi
le, >6mm likely. Preverte
r l sh dow: <7mm t
C2, <22mm t C6 (kids 14mm) An tomy PICA c n rise from VA extr dur lly, c n
e
injured during dissection of C1. VA is 3mm from l ter l uncoverte
r l joint, m y
run through verte
r l
odies. VA enters tr nsverse for men
ove C6 or t C7 in
13%. 15% h ve hypopl stic VA. 2% of VA do not enter BA. L ndm rks: hyoid (C3)
, thyroid c rtil ge nd
ifurc tion of the common c rotid rtery (C4), cricoid c
rtil ge (C6) Conus lesion: Vs c ud equin : less p in, symmetric, e rly utonom
ic signs

Cervic l Spine
Congenit l A
norm lities
Im ging: Xr ys (pl in nd dyn mic), CT (3D), MRI (dyn mic), ngiogr phy (MR/CT)
See JN:S 9/04
Os Odontoidium Odontoid ossific tion centers: 2 prim ry t
se, 1 second ry t
tip. Believed to
e tr um tic. Associ ted with Downs syndrome. Indic tion for st

iliz tion: >1cm inst
ility on flexion/extension, progression, deficits. Reduce
preop. Posterior C1/2 fusion. Klippel-Feil syndrome Congenit l fusion of cervic
l verte
r e Associ ted with Sprengel deformity (elev tion of sc pul ), Chi ri I
. Workup required for ssoci ted c rdi c, ren l
norm lities. Follow with flexi
on/extension xr ys for inst
ility. B sil r Impression Motor/ sensory symptoms m
ore prominent th n cere
ell r/ verte
r l symptoms (opposite of Chi ri). Short ne
ck, torticollis, & verte
r l . nomolies common. Tre tment: Reduce 7>15l
s over
week; Reduci
le: fusion  C1 l minectomy; Nonreduci
le: odontoidectiomy.
Degener tive Dise se
Neck p in Differenti l di gnosis of neck/shoulder p in with no neurologic signs:
rot tor cuff te r, su
cromi l
ursitis, dhesive c psulitis (frozen shoulder),
glenohumer l impingement, l ter l epicondylitis (tennis el
ow) Differenti l di
gnosis with neurologic signs: r diculop thy, neurop thy, plexop thy (ie P rson g
e-Turner syndrome), Thor cic outlet syndrome Cervic l R diculop thy/Herni ted Di
sc Symptoms nd signs: o R dicul r p in: r di tes in distri
ution of nerve o Der
m tom l num
eness/p r sthesi s o Myotom l we kness: usu lly mild. ( trophy/f sic
ul tions re r re) o Decre sed reflexes in root distri
ution
Flotte Outline of Neurosurgery
o
43

Almost ll pts h ve decre sed neck motion nd neck p in init lly. Chronic lly p
in
ecomes dull, ching, tingling. Sc pul r p in m y develop. o Spurlings sign: r
dicul r p in reproduced on tilting he d tow rd ipsil ter l side. Hyperextension
with or without vertex compression lso reproduces p in. o C7: 15% su
sc pul r
or
re st/chest p in. C6: intr sc pul r p in. EMG: Fi
rill tions, xon l loss. P
r spin l muscle involvement confirms root involvement. NCV not useful Im ging:
MRI, CT-myelogr m (if MRI is equivoc l) Tre tment o 95% recover with conserv tiv
e tre tment (lum
r 85%). Conserv tive tre tment includes: NSAIDS, cervic l tr c
tion, physic l ther py o Surgic l options: Anterior cervic l discectomy & fusion
(ACDF) Anterior for minotomy (Jhos procedure) Posterior for minotomy
Cervic l stenosis Cross-section l re of cord m y
e more ccur te predictor of
risk of myelop thy th n c n l di meter. DDx: ALS: (+) j wjerk, tongue f sicul t
ions, dys rthri , no sensory s. Radiology: T2 signal controversial: reversi
le (e
dema) or not (cystic necrosis). Snake-Eye Appearance: poor recovery. Treatment: AC
DF; laminectomies  lateral sta
ilization, laminoplasty. If needs anterior & poste
rior decompression do ACDF
efore laminectomy. Canal <12mm with myelopathy needs
surgery.
DISH: Diffuse Idiopathic Skeletal Hyperostosis. Usually asymptomatic,
ut may ca
use esophageal compression (Forestiers Disease) OPLL: Ossification of Posterior L
ongitudinal Ligament. Su
type of DISH. More common in Japan. Risk of developing
myelopathy near 100% if >60% canal compromise. If no myelopathy at presentation
then patient has 30% risk of developing myelopathy. If myelopathic than chance o
f
ecoming wheelchair dependent/
ed
ound is 10% with surgery, 90% with conserva
tive treatment. Surgery ineffective if already wheelchair dependent. Surgery: An
terior if it involves 2 levels, otherwise posterior. Ossification of Ligamentum

Flavum: very rare, occurs in Japan Rheumatoid Arthritis AA su
luxation, Basilar

Invagination common. Flex/extend all patients
efore intu
ation. Fuse if symptom
atic or >8mm su
lux.
Thoracic Spine
Thoracic Herniated Disc Back pain most common symptom. Treatment: 1) laminectomy
: highest risk of injury 2) transpedicular, 3) costotransversectomy (risk: Adamk
iewicz A. T9-12 on Left); 4) transthoracic / thoracoscopic 5) lateral extracavit
ary
Lum
ar Spine
Low-Back Pain Pain may
e axial (low-
ack) or radicular. May also
e referred to

uttock, hip, or thigh Pain generators include nerve, disc, facet joint, and pa
raspinal muscles Pain may
e mechanical (exacer
ated
y movement) or neuropathic
(constant, usually radicular) Differential Diagnosis o Sacroilitis
Flotte Outline of Neurosurgery
Workup o Xrays: L-spine series, flexion/extension o CT scan for previous surgica
l instrumentation o MRI L-spine o Injections/
locks: nerve root, facet, trigger
point in paraspinal muscles Treatment o Injections/
locks: may
e done as a seri
es of 2-3
locks, repeated every 6mos as indicated
44

Lum
ar Herniated Disc

Symptoms o Straight Leg Raise (SLR): tests L4 to S1. Postive if it produces scia
tic pain, NOT
ack pain. Reverse SLR positive for L3. o L1-3 and facet pain refe
rred to
uttocks/thigh thru superior cluneal nn (not
elow the knee). o HNP with
out sciatica: 0.1% o Great toe weakness: L5/S1 60%, L4/5 30% o L2-4: weakness wa
lking up stairs Differential Diagnosis: Hip pathology perform Patricks test (hip
rotation) Treatment Conservative treatment o Intradiscal steroid injection fail
ed in RCT Lum
ar Discectomy o Pain
etter @ 1 year, no difference @ 4yrs. o L3/4
: 94% improvement for leg pain, 87% for
ack pain. L2/3 and a
ove: only 50% impr
ove. o Arthroplasty: Artifical disc. Far Lateral Disc: males >50yo, DM, upper lu
m
ar levels (N4/04) Lum
ar stenosis Neurogenic claudication (vs vascular): varia

le distance, slow relief, requires sitting persists while standing. Canal Diame
ter: Avg. 22mm, Lower limit normal 15mm, severe stenosis <11mm. Treatment: Lamin
ectomies  lateral decompression (facetectomy) Lateral recess stenosis: caused
y
superior facet hypertrophy Spondylolisthesis L4/5 = 66%, L5/S1= 30%. Grades: I <
25%, II 25-50%, III 50-75%. IV >75%. Insta
ility: >4mm movement. Types: 1) Isthm
ic: due to spondylolysis. If sclerosis do nothing, if not then Boston
race. 2)
degenerative, 3) dysplastic (congenital), 4) traumatic, 5) pathologic. Treatment
: o Decompression: for foraminal or spinal stenosis without insta
ility. o Poste
rolateral fusion: with or without reduction & decompression o PLIF: with postero
lateral fusion. Distracts disc space, strengthens posterior construct. o Reducti
on necessary in Grades III & IV, controversial in I & II. Easier if younger, L45
(harder if L5S1). o No clinical difference in outcome
etween PLIF & posterolat
eral fusion,
ut PLIF mechanically stronger (less translation, disc space decrea
se. (JN:S9/03) o Benzel feels Disc space distraction may
e unnecessary after de
compression, and compression may make posterolateral fusion stronger. Failed Bac
k Syndrome Causes: Arachnoiditis (Surgery only improves 10-20%), epidural fi
ros
is. Diagnosis: o L-spine xrays, flexion/extension, o
liques (look for postlamine
ctomy fractures of the pars interarticularis) o MRI with contrast: Fi
rosis enha
nces, recurrent disc doesnt. o Consider CT scan  myelography Treatment: medical, s
pinal cord stimulator (poor for axial or
ilateral leg pain), intrathecal morphi
ne pumps (palliative) See Neurologist 9/04 Coccydynia Treated with NSAIDS and lo
cal steroid injections Ankylosing Spondylitis 90% HLA-B27, onset <40yo

Flotte Outline of Neurosurgery

Initially in SI joints, uveitis/conjunctivitis, prostatitis, Back pain relieved

y exercise, not relieved when supine, AM stiffness. Xray:
am
oo spine.
45
Pagets Disease Progresses from lytic to sclerotic phases. 15-30% familial. Spine
, skull, pelvis, long
ones most common sites. La
s: alk phos, Ca WNL, urine hyd
roxyproline. Causes
ack pain, foraminal & spinal stenosis, pathologic fractures
, neurologic deficit from vascular steal. Myelopathy rare. Sarcomatous transform
ation occurs. Treatment:
isphosphonates. Surgery is
loody. Tethered cord Dx: C
onus
elow L2 or filum >2mm diameter. Occurs in kids or adults. Adults: pain mor
e common (perineal). Kids: foot deformities, scoliosis, cutaneous stigmata (80-1
00% vs adults <50%). Both: urinary sxs. Aggravated
y growth spurts. In myelomen
ingocoele patients must diagnose tethering clinically (worse gait, spasticity, u
rodynamics, scoliosis) as all will have radiographic tethering. If painful suspe
ct tethered cord, painless syringomyelia. Incontinence: SCI = spastic
ladder, T
reatment: oxy
utinin (Ditropan); LMN injury = atonic
ladder (overflow), Treatme
nt:
ethanecol
Spinal tumors
Intramedullary: ependymoma (adults, symmetric, well-delineated, cervical, hemorr
hage), astrocytoma (kids, assymmetric, infiltrative, syrinx), hemangio
lastoma (
3%). Intradural-extramedullary: schwannoma (enlarge neural foramen) > meningioma
(thoracic,
one erosion rare). Extradural: metastases. (Rare: spinal angiolipom
a: <10yo, female, dorsal thoracic; arachnoid cyst: thoracic). Filum: myxopapilla
ry ependymoma, schwannoma, paraganglioma. Sacral: chordoma, aneurismal
one cyst
, Giant cell tumor. Children: teratoma most common.
Hemangio
lastoma: Usually dorsolateral (early sensory symptoms), come to pial su
rface. Enhances. Treatment: Timing controversial, some recommend surgery if symp
tomatic or tumor or cyst enlarging (N12/03). Surgery: Resected like AVM Use lowpower
ipolar. Some recommend preop em
olization (Spetzler), SSEPs/MEPs, CO2 las
er, SRS. May
e hidden under vessels can use ultrasound to locate. Most feel pre
op angio not helpful. Syrinx (pseudocyst) resolves w/removal of tumor, does not
require resection. Edema = higher surgical mor
idity. Only 30% improve.
Thoracic (70%), Lum
ar (20%), cervical (10%). Treatment XRT: 30Gy Surgical indic
ations: pain, cord compression, sta
ilization. Cervical: Use of
one graft recom
mended for patients with over 6mo survival; disadvantages include pseudarthosis
(due to XRT, malnutrition), recurrent tumor may invade graft. Alternatives: poly
methylmethacrylate (PMMA) with Steinmann pins, screws, contoured plate, or coaxi
al dou
le-lumen (Chest-tu
e) for scaffold (Gelfoam or fat over dura to prevent h
eat damage), cages (with chest tu
e around it). Aspirin has unique effectiveness
in pain from
ony mets Metastatic Epidural Spinal Cord Compression May
e from
epidural or verte
ral
ody mets Steroids: Patchell uses Decadron 100mg IV then 2
4mg IV q6hrs Patchell 04: RCT. Surgery + XRT led to improved am
ulation vs XRT a
lone as initial therapy (within 24hrs of symptom onset) (surgery was not as good
for salvage therapy after XRT), especially in am
ulatory patients. Also prolong
ed continence (142 vs 12 days) Spinal cysts: arachnoid = posterior, neurenteric
= anterior
Spinal mets
Flotte Outline of Neurosurgery
46
Synovial cysts Aka juxtafacet cysts, lined
y synovium; ganglion cyst = no linin
g. Usually cause claudication. May hemorrhage (acute radiculopathy, cauda equina

), may contain air. Treatment: Surgery vs aspiration vs facet injections of stro

ids to rupture cyst. (tend to recur with last 2). May regress. Check for insta
i
lity. Tarlov cyst: Perineurial cyst, nerve root sleeve at dorsal root ganglion.
Between peri- and endoneurium. Delayed filling on myelogram may or may not commu
nicate w/ CSF. Usually sacral. Spinal meningeal cysts: extradural, extradural +
nerve roots, or intradural (arachnoid cyst) Conjoined nerve roots: can mimic lum

ar mass, cause sciatic. Present in 6% of patients. Hemangioma Most commom in 46th decade. Neoplastic. Occur in verte
ral
ody. Most are asymptomatic
ut can c
ause myelopathy CT: trae
eculations (polka-dot), T1 & T2. Treatment: XRT Aneurys
mal Bone Cyst: No endothelium. Nonneoplastic. Most common <20yo. Occur in poster
ior elements (60%) or
ody (40%). Painful, recurrent, expansile/lytic. Osteoid O
steoma: <2cm, young, lum
ar. Osteo
lastoma: >2cm, older (20yo), cervical. Both:
neural arch, pain relieved w/aspirin, lytic lesion w/central nidus, males. Osteo
chondroma: C2 spinous process or transverse processes, pedunculated cauliflower, s
olid. Occur in <30yo. Giant Cell Tumor: Benign, lytic, expansile. Have fluid-flu
id levels. 20-40yo. Females. Sacral. Osteosarcoma: rare in spine Epidural lipoma
tosis: Risk factors: o
esity, Cushings, steroids (may resolve after stoping ster
oids) Extramedullary hematopoesis: can cause epidural compression. Treatment: XR
T. Spinal AVM Signs: Bruit 3%, skin angioma 3-25%. Type I: most common, middle-a
ged, acquired. Single feeder (radicular artery), nidus on nerve roots in foramen
(dural AVF). Natural history: progressive myelopathy. 50% disa
led at 3 years.
98% o
literated with surgery, 45% with em
olization (Meta-analysis N7/04). Reaso
na
le to attempt em
olization at time of diagnostic angiogram,
ut
e aware of h
igh recurrence. Mor
idity with
oth is minimal. With surgery 55% improve, 10% wo
rse. Coil placed in feeding artery during angiography may
e used for intraopera
tive localization of fistula on Xray. Type II: glomus, intramedullary, young, ce
rvicomedullary. Type III: juvenile, intra- & extramedullary Type IV: extramedull
ary/ intradural, anterior, fed
y ASA. Types II & III: true AVMs, congenital, he
morrhage common (Coup de Poignard) Types I & IV: venous HTN, progressive myelopa
thy (Foix-Alajouannine). Osteomyelitis Staph aureus most common organism. Cultur
es usually o
tained
y CT-guided needle
iopsy. Osteomyelitis involves the disc
space (discitis), spinal mets do not (Cancer = C-shape around the disc) Can use
ESR, CRP to follow treatment Potts disease: Tu
erculosis verte
ral osteomyelitis.
Psoas a
scess common. Treatment usually medical Discitis may occur without oste
omyelitis Discitis is usually postoperative,
ut may occur spontaneously in adul
ts (with same risk factors as spinal epidural a
scess) or juveniles (average 2-3
years-old due to persistence of nutrient feeding arteries). In children present
s as refusal to walk. Spinal epidural a
scess Symptoms: Similar to osteo:
ack p
ain, fever, tenderness (with neurologic deficit). Risk factors: immunocompromise
, IV drug use, dia
etes, alcoholism. Most common organisms: Staph aureus.
Flotte Outline of Neurosurgery

47
Treatment: o No deficits or complete deficits for >3days: immo
ilization (TLSO)
and anti
iotics (4wks IV then 4wks oral). Evacuation controversial. o Deficits:
emergent evacuation.
Radiation myelopathy Causes painless paresthesias. Occurs12-15mo post-XRT. Tx: S
teroids Verte
ral
ody changes: fatty marrow replacement (T1 vs tumor T1) Radiati
on plexopathy: painless (vs cancer invasion painful) Syringomyelia Syringomyelia
= no ependyma. Hydromyelia = ependymal lining. Communicating: filled with CSF (
eg Chiari). Noncommunicating: filled with proteinaceous fluid (trauma etc). Sept
ae in syrinx are called haustrae. Sxs: Cape sensory loss. Charcot joints. O
tain
contrasted MRI to r/o tumor. Pain less likely to improve in syrinxes deviated i
nto the dorsal horn. Treatment: o Chiari: Decompress. If no improvement then shu
nt. o Tumor: resect lesion and follow for resolution. o Idiopathic: CT cisternog
ram. If reflux into 4th ventricle then pleural shunt; if no reflux then decompre

ssion. o Shunting: Pleural for traumatic, arachnoiditis. Su
arachnoid for occult

dysraphism, Chiari.
Spinal Procedures
Grafts
Woolfs Law: Fusion potential is improved if graft is placed under compression Typ
es Autograft: local, iliac crest o Anterior iliac crest harvesting: Keep at leas
t 1cm posterior to ASIS (anterior superior iliac spine) to avoid damaging the la
teral femoral cutaneous n. Allograft: cadaveric iliac crest or fi
ula. No AIDS t
ransmission since 1985. Osteogenic Su
stances rhBMP2 (recom
inant human BMP2): f
usion rates of 95% in LIF Occipitocervical Fusion Occipital plate, C2/3 screws,
rods. Historic: 1. CD horseshoe with either su
laminar or interspinous wiring. 2
. Luque rectangle. 3. Contoured Steinman pin. C1/2 (Atlantoaxial) sta
ilization:
C1 lateral mass-C2 pedicle screws: Easier with thoracic kyphosis. Screws may
e
placed
efore reduction. C1/C2 transarticular screws: 80-100% success. 4% verte

ral a. injury identify VA on imaging. Difficult with thoracic kyphosis. Reducti
on required
efore screw placement. May
e done anterior or posterior. A
ove two
techniques are
iomechanically equal. Su
laminar/Intraspinous Wiring: 60-100% s
uccess. Pro
lems: risk of passing su
-laminar wires, 25% Pseudoarthrosis o Brook
s: o Gallie: o Sonntag: Halifax interlaminar clamps: C1/2 Anterior Harms Plate:
Less
iomechanically secure. Requires posterior wiring in addition. Anterior Cer
vical Discectomy (Fusion): C3-C7/T1 only. Look for sternal notch in relation to
verte
rae on imaging for lower limit. Fusion rate unchanged with graft,
ut kyph
osis dropped (62.5 to 42%) no RCT. Plating improves fusion rate 90% to 96% for s
ingle-level and 72% to 90% for 2-level Arthroplasty: see NF9/04
Flotte Outline of Neurosurgery

48
Complications: o Hoarseness: Usually temporary (paratracheal swelling). Recurren
t laryngeal n. loops around su
clavian a. on right and aortic arch on left, runs

etween esophagus & trachea, more varia
le on right. o Singers & speakers use p
osterior approach to avoid recurrent laryngeal n. injury. o Thoracic duct may as
cend to C6 on left
ehind the carotid sheath. Ligation carries 50-80% mortality.
o Durotomy: place fascial graft on top of dura
ehind graft, HOB elevated, cons
ider LD. o Horners syndrome: sympathetic plexus runs in longus coli laterally. o
Graft extrusion usually doesnt require reoperation. o 20% develop adjacent level
disease requiring operation
y 10 years (higher at C5/6, C6/7)
Cervical Corpectomy Use iliac crest, fi
ula, or cage + plate (with or without en
dplate drilling or cage end caps) Cervical Laminectomy Consider concurrent sta
i
lization with lateral mass screws & rods or plate, especially if a
normal motion
on flexion/extension. C5 root injured in 10% due to stretch. Laminoplasty: Inte
rspinous ligaments at C2/3 and C7/T1 removed. Laminotomies at C2 and C7. Open si
de completely drilled, hinge side scored with drill. 10-15mm Allograft ri
space
rs placed. Collar x 3mos. Lower incidence of kyphosis. Preop kyphosis, a
normal
motion on dynamic xrays, or OPLL >60% of canal diameter are contraindications. (
N1/04). Meta-analysis showed no
enefit over laminoplasty (NQ2004) Posterior Cer
vical Foraminotomy For unilateral osterophyte, facet hypertrophy, extruded disc
causing unilateral radiculopathy (no myelopathy) Some do sitting position. Scovi
lle retractor or endoscopic (JN:S7/04). 1/3 of lamina and facet drilled. Posteri
or Cervical Sta
ilization: Lateral mass screws: Center of lateral mass (or 1mm i
nferomedial), aim towards superior-lateral corner of facet. Cervical pedicle scr
ews advocated
y some,
ut are technically difficult. C7/T1 pedicle screws stron
ger than C7 lateral mass/ T1 pedicle screws. A
ove C6 makes no difference. Lamin
ar hooks have risk of neurologic deficfit. Verte
ral artery injury: If possi
le,
primarily repair. If not ligate or clip VA proximally & distally (12% mortality
esp. w/contralateral VA hypoplasia). Transthoracic: Upper thoracic = sternal sp

litting; midthoracic (T4-6) = right (heart); low-thoracic = left (aorta easier t

o mo
ilize than SVC); thoracolum
ar = left (liver) Costotransversectomy: Lum
ar
discectomy Consider 250mg solumedrol, Marcaine epidurally Arthroplasty: SB Chari
te artificial disc FDA approved 6/04. Complications: o Great vessel injury: 0.01
-0.1%. Sxs: Refractory hypotension,
ack/leg pain, rarely su
cutaneous hematoma.
Usually presents within hours. Late complications include pseudoaneurysm, AVF.
Left common iliac a. at L4/5 most commonly injured. Repair via transperitoneal a
pproach, o
taining vessel control first. Lum
ar Corpectomy:
Lum
ar Fusion
Data on effectiveness of lum
ar fusion and instrumentation is mixed (NEJM 2/04)
Lum
ar Pedicle Screws Percutaneous placement: Sextant (Medtronic) Posterolateral
Lum
ar Fusion 50-92% fusion rates
Inter
ody Fusion
96% fusion rates
Flotte Outline of Neurosurgery

49
Advantages over posterolateral fusion: more compression, more surface area, more
vascularity, retores coronal & sagittal
alance, easier to diagnose pseudarthro
sis Ta
les from JN:S 7/04 Anterior Lum
ar Inter
ody Fusion: Can only
e performe
d at L4/5 or L5/S1 Haid uses standalone ALIF for L5/S1 DDD ( anterior plate). Als
o uses for L5/S1 low-grade spondylolisthesis with posterior instrumentation. Avo
id in young men due to retrograde ejaculation Anterior plate may
e used at L5/S
1 if it doesnt lie under iliac vessels Class II evidence: 80% have improved pain.
Iliac vessels must
e retracted (especially at L4/5) Complications: retrograde
ejaculation in up to 45% (due to hypogastric plexus injury), a
dominal hernia Ma
y
e com
ined with pedicle screws May
e done
y laparoscopic or mini-open techn
iques. Retrograde ejaculation: 45% laparoscopic, 0% mini-open Posterior Lum
ar I
nter
ody Fusion (PLIF): Bilateral exposure. Requires significant dural and nerve
root retraction Performed L3/4 and
elow (avoid conus retraction) Transforamina
l LIF (TLIF): Any level
elow L1. Pedicle screws placed first. Unilateral lamine
ctomy/ facetectomy on side of radiculopathy. Discectomy performed with pedicle s
crew distraction. Posterior osteophytes removed with osteotome. Pedicle screws d
istracted on opposite side. Bone graft placed anteriorly in disc space. Two cage
s placed. Compression applied. (N2/04, JN:S7/04)
Flotte Outline of Neurosurgery
50
Flotte Outline of Neurosurgery
51
Vascular
Blood on MRI: (T1/T2) <24h (oxyhemoglo
in): iso/iso. 1-3d (DeoxyHg
): iso/hypo.
3-14d (intracellular metHg
): hyper/hypo. >14d (extracellular metHg
): hyper/hyp
er. hemosiderin: hypo/hypo Angiogram: 0.5% risk of stroke, 7% w/atherosclerosis
Berry aneurysms: False aneurysms (lack media, defects in internal elastic lamina
). Other types of aneurysms: o Oncotic aneurysms: most common with left atrial m
yxoma, choriocarcinoma Prevalence 2-6%. 20-30% multiple. Pcom (35%) > Acom (30%)
> MCA (20%) >
asilar tip (5%). Risk factors: age, smoking (3-10x), heavy alcoh
ol use, HTN (3x), ?hormones Rupture risk: Large size (no critical size), high do
me/neck ratio, high aspect (aneurysm depth:neck width). Smaller aneurysms produc

e more extensive SAH (JN8/03). Slow flow increases rupture risk. If two or more
family mem
ers have aneurysms, others should
e screened
y MRA or CTA (9% posit
ive). Frequency is de
ated (every 6mos to 5yrs) (N8/03) Predisposing factors: Ao
rtic coarctation, AD polycystic kidney disease, fi
romuscular dysplasia, Marfans
, Ehlers-Danlos, homocystinuria, NF1, AVMs Complications: o Rerupture: 20% 2wks,
50% 6mos, then 3%/yr. Unruptured 1-2% (<10mm 0.5%). o Vasospasm: See
elow. o H
ydrocephalus: 10% require shunting. No difference in shunt placement
etween gra
dual and rapid EVD weaning (JN2/04) Effect of increased ICP on outcome unproven
(JN9/04) Diagnosis: o CT. If CT is negative then LP. If CT and LP negative then
consider angio (LP may
e negative with loculated
lood) o Cere
ral angiogram: g
old standard. Risk of hemorrhage 3% with SAH. o MR angio: False-positive and fal
se-negative rates 10%. o CT Angio: Sensitivity/specificity >95% for aneurysms >7
mm. Look for neck calcification or plaques. Especially
eneficial for emergent I
CH evacuation. With SAH: If aneurysm seen it is relia
le. Perform angio to confi
rm (-) CTA. No SAH: If (+) consider angio to confirm small aneurysms. If (-) the
n pro
a
ly relia
le. o Angiogram-negative (or Occult) SAH 10% of SAH. Most common ca
use is non-visualized aneurysm due to aneurysm throm
osis or inadequate study. O
ther causes: Perimesencephalic Benign SAH, spinal AVM, Spontaneous throm
osis of
aneurysms may occur (10% of autopsy series); however they may reappear and rupt
ure years later. Acom is most common location. Hemorrhage rate is 0.5%/year (low
er than angiogram (+) SAH). Other SAH complications occur (vasospasm may
e less
likely). Consider repeat angiogram at 10-14 days. Overall 2-25% positive yield
on repeat angiogram,
ut up to 70% with interhemispheric SAH. Do not repeat for
perimesencephalic SAH. Surgical exploration has
een advocated
y some, especial
ly if re
leeding occurs or if the SAH is in a typical aneurysm location (interhe
mispheric, Sylvian fissure) Benign Perimesencephalic SAH: aka Perimesenchephalic
Nonaneurysmal SAH. May
e due to rupture of small perimesenchephalic vessels. M
ay have similar presentation to SAH (headache, meningismus),
ut Hunt-Hess is 12. All are angiogram-negative. CT/MRI shows SAH only in interpeduncular/am
ient/
prepotine cisterms. 3% of
asilar tip aneurysms mimic BPS, so angiogram is manda
tory. Repeat angiography is controversial. Re
leeding and vasospasm in true BPS
have not
een reported. Nimodipine not recommended. Treatment o Unruptured: ISUI
A (International Study of Unruptured Intracranial Aneurysms, NEJM, 1998): <10mm
unruptured
leed rate 0.05%/yr does not justify surgery, advocated
y AHA. Other
studies refute this. Most aneurysms with SAH are <10mm. Heros feels <5mm should
rarely
e treated (only if very young). 5-9mm controversial. o With ICH: Higher
re
leed rates than pure SAH. Clot evacuation alone mortality 75-100%. Must secu
re aneurysm. Consider coiling then evacuation.

Cere
ral Aneurysms
Flotte Outline of Neurosurgery
o
52

E-ACA (-aminocaproic acid, Amicar): R

bl

ds d
cr
as
d from 20% to 12%, but
pasm incr
as
d from 23% to 32%, mortality unchang
d. Consid
r in nonsurgical
i
nts. Surg
ry o Us
mannitol, CSF drainag
, mild hypoth
rmia (33-36). K

p
140 until clip plac
d. o Hypoth
rmia: no RPT. Must b
normoth
rmic by
nd of

vasos
pat
SBP <
sur

g
ry to pr
v
nt: long
r ana
sth
sia r
cov
ry, postop bl

ding, inf
ctions and mo
rbidity. o Som
routin
ly f
n
strat
th
lamina t
rminalis to pr
v
nt hydroc
pha
lus o Consid
r microdoppl
r, MEPs (mor
s
nsitiv
than SSEPs). o Pr
viously coil

d an
urysms: do not r
mov
coils if plac
d >3mos pr
viously. <6mos r
moval is c
ontrov
rsial. o Intraop
rativ
angiogram: Controv
rsial. Us
d routin
ly by som
.
Estimat
d to av
rt s
rious complications in 2-10% of pati
nts (JN2/04). o T
mpo
rary occlusion: normot
nsion, Etomidat
to burst suppr
ssion (Barbs caus
hypot

nsion). L
ss lik
ly to b
tol
rat
d w/MCA an
urysm (26% infarction, v
rsus 9% wi
th ICA, 16% with Acom) (l
ss risk distal to l
nticulostriat
s). Ov
rall about 10
% strok
rat
. o N
ck avulsion: wrap with cotton th
n apply clip (JN11/03) Coili
ng o Cranial n
rv
s d
ficits usually improv
post-coiling. o Timing of coiling d
o
s not aff
ct proc
dural morbidity or outcom
. o M
asur
dom
-to-n
ck ratio o C
oil typ
s: 3-D, compl
x fill, biologically activ
. o Balloon r
mod
ling for wid

-n
cks. St
nting for wid
-n
ck & fusiform. o 5-10% morbidity, 2% mortality. Isch

mia 9%, h
morrhag
3%. Coiling vs Clipping o Johnston (Strok
2001): California
databas
. Mortality CE 0.5%, SC 3.5%. Poor outcom
CE 9%, SC 22%. o Raftopoulos
(N6/03): Unruptur
d. Occlusion rat
s: Total: CE 56%, SC 93%. Subtotal: CE 15%,
SC 2%. Fail: CE 29% (60% w
r
MCA), SC 5%. Complications: t
mporary CE 10% SC 16
% p
rman
nt CE 8% SC 2%. R
ccom
ndation: coil only for DNR >2.5. o ISAT (Int
rna
tional Subarachnoid An
urysm Trial - Lanc
t 2002): 1yr n
urologic outcom
b
tt
r
for CE. 7% risk r
duction CE vs SC. (Only 2 pts from US). Only 2100 of 9300 pat
i
nts randomiz
d mo t of re t were clipped. o No difference in hunt-dependent h
ydrocephalu between the two group except with IVH higher hunt rate with coil
ing (2 tudie , 1 howed higher rate with coiling) (JN 9/04) Specific aneury m
o Proximal ICA/Paraclinoidal: Aka carotidophthalmic. May cau e blindne . Expo
e cervical ICA. U ually require drilling anterior clinoid. No radiographic way
to determine if they are intradural or extradural (JN8/03). If anterior wall (ak
a uperior hypophy eal) or true ophthalmic (off C2) con ider urgery. If carotid
cave (C3) or pointing inferiorly, con ider coiling. o Po terior Communicating:
Ari e di tal to pcom. If no CN3 pal y more likely to be adherent to temporal lob
e. Pupil paring CN3 pal y (10-20% involve pupil, may be painful): DM, HTN. Comp
lete CN3 pal y (or pupil only, often painful): pcom OR ba ilar apex aneury m . L
ook for fetal PCA on angio. Clipping: En ure patency of anterior choroidal & tha
lamoperforator off pcom. Pcom may be acrificed if nece ary if not fetal. CN3
pal y: evacuate fundu , do not di ect off CN3. Small: clip perpendicular to ICA
. Large: fene trated Sundt encircling clip around & parallel to ICA (prevent ki
nking). o Anterior Choroidal: Supplie internal cap ule, optic tract, lentiform
nucleu , amygdale. Injury: Hemiplegia, hemihype the ia, homonymou hemianop ia (
cognition unimpaired). o ICA bifurcation: May cau e ICH (re embling putaminal IC
H). Know whether acom fill from A1 (can acrifice A1). Open medial ylvian fi
ure di tal to proximal after expo ing ICA. Watch for anterior choroidal & lentic
ulo triate (off ACA & MCA). o Ba ilar apex: Approache : o Pterional (Ya argil):
wide di ection of Sylvian Fi ure. Generally go between ICA & CN3. Al o COZ fo
r high bifurcation. Advantage : Le temporal re traction, le CN3 manipulation
. Di advantage : Deep field, poor proximal control. See N7/04. o Subtemporal (Dr
ake): Better for low bifurcation, po terior projection. Advantage : Short workin
g di tance, lateral view of aneury m and perforator . Di advantage : Temporal r
etraction, CN3 & CN4 injury. o Combined (Hero ): Uncu mobilized uperolaterally
out of inci ura. o Temporopolar (Sano) o MCA bifurcation: Cau e ICH, SDH. Highe
r ri k with temporary occlu ion. More often multiple (up to 45%). Treatment: cli
pping (coiling too ri ky). Approache : o 1) Tran ylvian. Expo e ICA for proxim
al control, then follow to MCA bifurcation.
Flotte Outline of Neuro urgery
o
53

o 2) Superior Temporal Gyru . Better if ICH pre ent. Acom: Look for ACA filling
from each ide. Surgery: Pterional. Con ider re ecting gyru rectu . Identify bo
th A1 & A2 . Avoid perforator from po terior Acom. o Di tal ACA: Note if aneur
y m i upracallo al (above genu) or infracallo al (may require removal of na io
n or anterior corpu callo otomy), azygou ACA (7-16%). Pterional/ ubfrontal u
ed for low A2, interhemi pheric for other o Vertebroba ilar junction: Far later
al (tran condylar), retrolabyrinthine. Between CN7/9 & CN9. o PICA: Occur at ver
tebral-PICA junction. Di tal PICA aneury m rare. Occa ional 4th ventricle IVH.
Surgery: Clipping or proximal occlu ion  bypa (contalatertal PICA or occipital
a., indication unclear). Proximal: Lateral, vertex tilted lightly toward floo
r, chin rotated 10 to floor. Can include CN12 fiber in clip. Spare brain tem per
forator . Far-lateral approach N7/04. Subarachoid Hemorrhage grading o Hunt-He
: 1=a ymptomatic; 2= CN pal y, evere HA, nuchal rigidity; 3=focal deficit, conf
u ion, 4= tupor, hemipare i , early po turing; 5=decerebrate,coma; +1 for y tem
ic di ea e, va o pa m. Grade 1/2 operated, >2 managed until <3 o WFNS: GCS 15=1;
13-14=2; 13-14+deficit = 3; 7-12=4; 3-6=5. 40% of Grade 4/5 patient independen
t at 3mo argue for aggre ive treatment. o Fi her: CT grading. 1=none; 2 = <1m
m thick; 3 = >1mm, clot; 4=ICH, IVH. All pt with va o pa m were > grade 3 Va o
pa m o Incidence: Angiographic 50%, ymptomatic 30%. o Peak 7-10d. #1 cau e of m
orbidity. Cigarette moking i a ri k factor. o ACA pa m give frontal lobe yn
drome. o Lovenox howed no benefit in prevention. o TCD : Mild: 120-200cm/ , MCA
:ICA ratio 3-6; Severe: >200 cm/ , ratio >6. o HHH: IVF + colloid, HCT 30-35 pro
phylactically. For deficit rai e SBP >200. (unclipped CVP & PCWP 6-10, SBP <160)
; No RPT. Increa ing CO, MAP effective (not CVP) (N11/03) o Nimodipine: improve
outcome by 40%, reduce troke by 34% and clinical va o pa m from 30% to 20%, n
o change in mortality or angiographic va o pa m. 60mg po q4hr for 21 day o Angi
opla ty: Repeat CT fir t to rule out ICH. o Papavarine: Intraarterial papavarine
efficacy i inconclu ive. Half-life i le than 24hr , va o pa m recur in 2448hr . Can increa e ICP, cau e tran ient deficit , blindne , eizure , thromboc
ytopenia,a nd paradoxical exacerbation of va o pa m. o Lumbar drainage reported
in retro pective trial to decrea e va o pa m from 50% to 20% (JN2/04). (Randomi
ed trial underway). o Ci ternal thrombolytic therapy with inflow/outflow catheth
er or head haking have had mixed re ult . RCT howed no overall difference in va
o pa m with intraoperative ci ternal injection of rt-PA, but did how 56% decre
a e in patient with thick ubarachnoid clot. Mycotic aneury m o 18% have SAH, 1
0% w/SBE develop. Mo t common in di tal MCA. Staph aureu , hemolytic strep most c
ommon. Resolves w/in 6 wks w/ nti
iotics.
AVM
Hemorrh ge: 4%/yr. Rehemorrh ge: 6% @ 6-12mos then 3%/yr. Risk of de th from rup
tured AVM: 1%/yr. Mort lity from hemorrh ge: 15%. Lifetime risk: 1-0.3life expec
t ncy, or 105- ge. For deep (
s l g ngli , th l mus,
r instem) risk is 10%/ye
r. Higher rupture r te: previous hemorrh ge, sm ll size, deep dr in ge. No incre
sed risk if symptom tic (HA, seizures, etc). 15% h ve ssoci ted neurysms. V s
osp sm occurs in 1%. Present tion: hemorrh ge 50%, seizure 25%, deficit (2 to ste
l) 25%, he d ches (migr ine-like, more common w/occipit l AVM). Younger present
tion th n neurysms. Often wedge-sh ped, pex t ventricle. Spetzler-M rtin Gr
de: (1) Size: <3cm = 1, 3-6cm = 2, >6cm = 3. (2) Venous dr in ge: superfici l =
0, deep = 1. (3) Eloquent re : no = 0, yes = 1. Tre tment o B sed on ge & loc
tion, gr de, not present tion. o 55yo cutoff. o Ruptured AVMs usu lly tre ted el
ectively fter 2-4weeks. Emergent ICH ev cu tion c n
e done with or without AVM
resection. Surgery o Em
oliz tion should
e done 24-48 hrs preop. o Wide exposu
re. Circumferenti l dissection, preserving dr ining veins. C n use controlled hy
potension. Alw ys get postop ngio. o Medi l tempor l AVMs: c n use pterion l if
nterior, su
tempor l-tr nscortic l (thru inferior tempor l or fusiform gyrus),
or COZ.

Flotte Outline of Neurosurgery

o
54

Complic tions: Postop ICH or severe edem in 3-12%. Norm l Perfusion Pressure Br
e kthrough (NPPB): Hyperemi detected with Perfusion MRI. Em
oliz tion: o NBCA (
N-
utyl cy no cryl te), polyvinyl lchohol p rticles, coils. o Almost lw ys dj
unctive, not prim ry tre tment (cure r tes 5-40%). o C n reduce NPPB. o Some rec
ommend 24-72 hrs ICU monitoring. o Hopkins recommends not em
olizing >50% t one
time if multiple feeders, use loc l/propofol, pedicles tested with mo
r
it l.
o Risks: ICH 1-4%. AVM m y rec n lize fter successful em
oliz tion. R diosurge
ry: o Bleeding risk unch nged for 2-3yr l tency (controversi l some studies repo
rt lower or higher r tes). o M rgin doses: Sm ll AVMs 23Gy (no
enefit from high
er doses). L rge, risky loc tion: 16-18Gy. (<15Gy ineffective). Use MRI (Or CTA)
with (or without) ngio for t rgeting. o 2yr o
liter tion: >4cm3 = 40-60%, <4cm
3 = 85-100%. (N11/03, N6/03). Over ll 75%. o F ctors reducing success: size, loc
tion, ge, em
oliz tion. Em
oliz tion only helps if it decre ses volume periphe
r lly. o >15cm3 consider st ged SRS t 6mo interv ls. o Hem tom m y compress ve
ssels, m y t rgeting difficult. Perform SRS 2-3mos fter hemorrh ge. o Complic t
ions: cyst form tion, edem (10% symptom tic, 4% need surgery), necrosis 3-6%. o
Repe t ngio not performed until 3yrs, then consider retre tment, repe t SRS. I
f e rly dr ining vein
ut no nidus, consider it tre ted. o Follow-up MRI h s 100
% positive nd 85% neg tive predictive v lues some get only MRI only for followup. o Br instem AVMs: 66% success, 10% perm nent deficits. Risk higher in tectum
. (JN2/04,3/04)
Dur l AVFs Acquired, due to venous sinus throm
osis, fed from ECA. Cortic l/ lep
tomeninge l venous dr in ge is #1 SAH risk 10% mort lity. Tr nsverse/ sigmoid: m
ost common. Symptoms: Puls tile tinnitus ( lso c used
y
err nt ICA, glomus ty
mp nicum.), occipit l
ruit. Less SAH risk (higher with or
it l/ nterior f lx, t
entori l) C vernous sinus (CCF) Tre tment: If only retrogr de or cortic l dr in
ge requires urgent tre tment. If h s nterogr de dr in ge c n tre t symptom tic
lly. o Em
oliz tion: tr ns rteri l (higher recurrence) or tr nsvenous (riskier)
o SRS o Surgery (excision nd p cking of sinus). Single leptomeninge l dr ining
vein c n
e tre ted with vein lig tion. C rotid-c vernous fistul : Types: o 1) P
osttr um tic. o 2) Spont neous ) Dur l AVF: women, 50% throm
ose.
) C vernous
neurysm rupture. Symptoms: Proptosis, chemosis, or
it l
ruit. M y c use epist
xis, visu l loss (emergency), intr cere
r l ICH (through tempor l veins). Tre tm
ent: B lloon occlusion (tr ns rteri l or tr nsvenous thru ophth lmic veins), CCA
lig tion  intr cr ni l tr pping C vernous m lform tion Hemorrh ge r te 0.5%/yr.
50% multiple (80% in f mili l c ses). F mili l: Hisp nics. 3 loci identified (CC
M1= Krit1) MRI: popcorn ppe r nce. (Look for ddition l lesions w/ gr dient ech
o). Angio norm l. Tre tment: o Surgery: Most recommend resection only for recurr
ent hemorrh ge (except in
r instem higher hemorrh ge r te) or intr ct
le seizu
res. o R diosurgery controversi l. Some m int in its ineffective. Venous ngiom
: Medus s he d on MRI, ngio. Hemorrh ge r re. 33% h ve ssoci ted c vernous m lfor
m tion. Vein of G len M lform tion: Type I: true AVF from posterior choroid l, p
resents in neon tes s CHF, inf nts s hydroceph lus or seizures. Type II: Mid
r
in or th l mic AVM th t dr ins into VOG. Presents in dults s SAH, ICH, dement
i . Tre tment: Tr ns rteri l or tr nsvenous (tr nstorcul r) em
oliz tion.
Flotte Outline of Neurosurgery
C rotid lig tion Used for c vernous or ophth lmic neurysms, or intr cr ni l c r
otid dissection. A
solute CI: V sosp sm on gr m. Rel tive CI:
il ter l neurys

ms, extr cr ni l therosclerosis.

55
C rotid stenosis
Asymptom tic
ruit: CVA risk 2%/ye r Angio: me sure ICA dist l to pl que for nor
m l; U/S & MRA overestim te. U/S: me sure norm l dist lly. Higher velocity = red
. Systolic velocity is
est velocity p r meter. Medic l Tre tment: de
t
le. AS
A (325 qD)  Ticlid (more expensive, neutropeni , use only of ASA intoler nt). Onl
y lowers risk in symptom tic pts. o Intr cr ni l: ASA, if f il EC-IC
yp ss. End
ov scul r ngiopl sty & stenting: 5-7% M&M. Higher restenosis th n CEA
ut equiv
lent stroke prevention (CAVATAS r ndomized tri l, L ncet 2001). 2 single center
tri ls for symptom tic & symptom tic p tients showed equiv lent effic cy. Rest
enosis occurs w/in 1 ye r due to myointim l hyperpl si ,
ut m y remodel & reope
n w/in 3yrs. SAPPHIRE (high-risk) tri l: Stroke, de th & MI: ngiopl sty + stent
6% vs CEA 12%. Stroke & De th: 4% vs 7% (NS). CREST (low-risk) tri l in progres
s. C rotid end rterectomy (CEA) Indic tions: 1. Symptom tic >70%. NASCET: lowers
risk of CVA 26 to 9% t 2yrs (NEJM 91) 2. Asymptom tic >50%. ACAS: lowers CVA r
isk 11% to 5% (JAMA 95) 3. Ulcer ted pl que; 4. Symptom tic refr ctory to medic
l tre tment 5. Crescendo TIAs (urgent); 6. <1mm di meter or fresh throm
us (emer
gent) 7. CVA during ngio w/stenosis (emergent) 8. CVA w/loss of previous
ruit.
Complic tion r te must
e <3% nd >5yr life expect ncy Completely occluded: CEA
, EC-IC
yp ss. Emergent should not
e performed >2hrs for cute deficit. Resolv
ing deficit oper te ASAP Recommend: w it 4-6wks fter CVA? CI: (Sundt) Completed
CVA w/in 1 wk, MI w/in 6mos, ngin , HTN (>180/110), contr l ter l ICA occlusio
n, siphon stenosis, pl que >3cm in ICA Preop: c rdi c workup; Preop CT; ASA x 25d (continue DOS) If
il ter l oper te on symptom tic side 1st Angio: look t le
vel of
ifurc tion vs m ndi
le, extent of ICA pl que, contr l ter l dise se (for
shunt) Intr op: 0.1%-0.3% lidoc ine to c rotid
ul
to prevent
r dyc rdi & hy
potension; v gus m y
e nterior; rteriotomy 5000U hep rin (revers l not needed
), SBP 110-150 (Stump pressure < 40-50 > shunt). Postop: check pron tor, speech,
pupil, tongue, ho rseness, lip depressor (m ndi
ul r
r. of f ci l n.) Complic
tions: TIAs: emergent CT, then ngio CVA: in PACU: reexplore, others s
ove; c
heck
ckflow from ICA if none use #4 Fog rty; fluids, pressors for SBP 180, O2,
hep rin? Hyperperfusion Syndrome: (Norm l Perfusion Pressure Bre kthrough): 1-3
%. Foc l edem or ICH. Unil ter l he d che, f ce/eye p in, seizures, foc l defic
it. Occurs d3-8. Risks: severe or
il ter l stenosis, previous CVA, perioper tiv
e hypertension. TCDs felt to
e unreli
le. Tre tment: BP control. Hem tom : ree
xplore immedi tely if stridorous, intu
te, use De
key Verte
ro
sli r Stenosis
/Insufficency Usu lly occurs t verte
r l origin. Em
oli less common th n in c r
otid stenosis. 11%/yr stroke r te (30% 5yr fter TIA). Requires 2 of:
il ter l
motor/sensory, diplopi , dys rthri , homonymous hemi nopi . Suggested
y positio
n l syncope. Dx: ngio. Tre tment: o Intr cr ni l: ntico gul tion (ASA unproven

ut consider if mild). EC-IC
yp ss for f ilure. Endov scul r
lloon ngiopl s
ty  stenting. Proxim l occlusion. o Extr cr ni l: Verte
r l . tr nsposition (ver
t to CCA) or end rterectomy. Endov scul r
lloon ngiopl sty  stenting (43% rest
enosis,
ut most rem ined clinic lly improved, low complic tions). V scul r Diss
ection: Between intim & medi (dissecting neurysm = medi & dventiti . Pseudo
neurysm = enc psul ted hem tom ).
Flotte Outline of Neurosurgery
Extr cr ni l: su
intim l, rupture r re. Intr cr ni l: l cks extern l el stic mem

r ne, rupture more common.
56
Extr cr ni l Dissection: C rotid: C n occur spont neously (OCPs, FMD, coll gen d
z, migr ines, HTN), during delivery, ph rynge l infections. Usu lly 2cm
ove
i
furc tion. Sxs: F ci l/eye p in, pseudo-Horners (no nhydrosis), c rtotidyni , c
ervic l
ruit, c n ffect CN10,11,12. HA preceeds deficit
y d ys/weeks. If SAH

gre test risk of rerupture 1st 24hrs, unlikely fter 1mo. Verte
r l: Between sku
ll
se & C2. Occipit l HA. Dx: ngio/MRA: string sign, pe rl nd string sign (h
igher re
leed r te?), dou
le lumen, fusiform dil tion. Tre tment: Antico gul tio
n 6-12 wks or until >50% rec n lized on MRA/ ngio then ntipl telets. If persist
ent em
oli: vein gr ft, EC-IC
yp ss, lig tion. Fusiform (Dissecting) Aneurysms:
SAH: 30% re
leed r te, highest in first 24hrs. Commonly verte
ro
sil r. Tre tm
ent: Bec use of high re
leed r te e rly tre tment is w rr nted Options: 1) Proxi
m l (Hunteri n) lig tion or tr ppping: F vored tre tment. Done with GDC coils or
clips. B lloon test occlusion (BTO) necess ry first (20 minutes). B sed on loc
tion: VA Involving PICA t keoff: clip/coil proxim l only VA Proxim l to PICA: tr
p (clip proxim l nd dist l) VA Dist l to PICA: clip/coil dist l to PICA (
ut p
roxim l to neurysm). B sil r: st ged
il ter l verte
r l occlusion if pcoms de
qu te. (N8/03) If p tient does not toler te BTO com
ine with PICA
yp ss, VA-PCA
n st mosis (with r di l rtery gr ft), or
oth. Others feel th t clipping prox
im lly only does not gu r ntee g inst re-
leeding nd ll VA neurysms must
e
tr pped with PICA rev scul riz tion if p tient doesnt toler te BTO or neurysm in
volves PICA (JN 12/03) Risk of perm nent deficits: 14%
sil r, higher with hypo
pl stic pcoms. Note if vert is domin nt, or contr l ter l v sosp sm exists. M y
not c use resolution, occ sion l re
leed. 2) Wr pping: Unproven effic cy. 3) Cli
pping/Reconstruction 4) Endov scul r
lloon occlusion  coils  stenting. MCA fusif
orm neurysms: Occur in children, young pts (<40yo). Lower
leed r te th n VA &
ICA fusiform neurysms. Sm ll neurysms present with SAH, l rge ones with ischem
i or m ss effect. M y
e due to therosclerosis or dissection. M y
e stenotic
or dil ted on ngio,
ut MRI/MRA shows extern l dil tion. Tre tment: Tre t with
SAH, when very l rge, or symptom tic from chronic ischemis or m ss effect ( cute
ischemi tre t s for stroke). Proxim l occlusion nd dist l
yp ss (s phenous
or rteri l interposition if M1, otherwise c n use STA). Note c nnot tr p nd ex
cise M1 due to lenticulostri tes (will throm
ose with proxim l occlusion). P rti
l clipping ineffective (JN8/03). Dur l Sinus Throm
osis Risks: infection, pregn
ncy (w/in 2wks postp rtum), OCPs. Deep veins usu lly thromo
ose in children. Sx
s: HA, seizures, p pilledem , deficits. CT: hyperdense. Contr st: empty delt sign
(dur enh nces SAH gives pseudodelt sign). MRV, ngio. Tre tment: Hep rin, AEDs,
ICP Tre tment (use M nnitol l st), void steroids. Coum din x 3-6mos. If ICP un
controlled consider throm
olytics (systemic or direct), surgery r rely. Visu l l
oss: optic she th decompression. Mort lity 30%
Cere
r l Inf rct

Stroke Risk f ctors: ge > HTN (#1 modifi
le) > DM > c rdi c dise se ( ll types
) > smoking. OCP in smokers >35yo risk (due to estrogen content), Postmenop us l
HRT no effect Sm ll vessel therosclerosis = lipohy linosis TIA: 20-30% risk of
CVA in 5 yrs. Higher risk of MI needs c rdi c workup. Im ging
Flotte Outline of Neurosurgery
57

o CT: 1-3d: wedge, hyperdense vessel, loss of insul r ri

on. 4-7d: gyr l enh nc
ement. o MRI: 2h: intr v scul r enh ncement. 12h: gyr l/ meninge l enh ncement,
edem . o Diffusion-weighted MRI most sensitive for e rly ischemi . Tre tment: o
t-PA: give within 3hrs from symptom onset if stroke h s non-c rdi c origin. 30%
incre se in excellent outcome. 6% incidence of ICH. Hold ASA/ hep rin x 24hrs, k
eep BP <185/100. Contr indic tions: ICH, l rge stroke on CT, CVA or TBI within 3
mos, h/o ICH, m jor surgery within 2wks,
leeding, severe HTN, throm
ocytopeni /
co gulop thy, pregn ncy, peric rditis 2 to MI. MCA strokes: Intr rteri l tPA if

<6hrs from onset. o Antico gul tion: not
enefici l unless p tient h s -fi
or
mech nic l v lve. C n use 2-4mo ASA/Ticlid long term. o Decompressive cr niecto
my: for MCA inf rcts improves surviv l from 25% to 75%, 40% h ve severe dis
ili
ty. After ge 60 recovery to independent function l st tus us very unlikely.(JN8
/04) Cere
ell r Inf rct: Tre tment options: m nnitol/steroids (not recommended

y AHA Stroke Guidelines), EVD, cr niotomy nd de
ridement for deterior tion,
r
instem signs, or tight posterior foss (others rgue for e rly cr ni) (N11/03)
Cere
r l rev scul riz tion (EC-IC
yp ss)
Indic tions: In dequ te cere
r l perfusion: intr cr ni l c rotid stenosis, moy moy . Requires persistent ischemic symptoms despite m xim l medic l ther py nd
exh usted cere
rov scul r reserve. P rent vessel s crifice: gi nt neurysm,
r i
n tumors involving m jor rteries For MCA neurysm: STA-MCA
yp ss within sylvi
n fissure For neurysms/tumors requiring ICA s crifice: S phenous vein or r di l
rtery gr ft from cervic l c rotid to M2 B lloon occlusion tests re not reli

le ccording to Spetzler; he uses
yp sses in ll c ses of c rotid s crifice Cer
e
rov scul r reserve m y
e determined
y CT-perfusion sc n with Di mox ch lleng
e, Xenon-CT, PET Recommended w it 2mos w it fter cute inf rct to perform surge
ry. RCT showed no reduction in ischemic stroke (NEJM 85). C rotid Occlusion Surg
ery Tri l currently underw y. STA-MCA
yp ss: STA n st mosed to M2, M3, or M4.
Reverse s phenous vein gr ft used if STA is in dequ te (ie prior surgery). From
STA trunk to M2/M3. Preoper tive testing: Tempor ry
lloon occlusion lso used.
ECA ngiogr m. Mild hypothermi . Norm l pCO2. Slightly high MAP. Procedure: See
N6/04 nd N8/04. Postop gr ft p tency check with
edside Doppler qhr in ICU. PO
D1: ngiogr m,
egin ASA. Verte
ro
sil r: Donors: Occipit l ., STA Recipients:
PICA/AICA/SCA/PCA.

Intr cere
r l Hemorrh ge (ICH)
Loc tion: Put men> lo
r> th l mus> pons> cere
ellum. L ter l to intern l c psul
e:
etter prognosis, surgery f vored. Hypertensive enceph lop thy/ Ecl mpsi : oc
cipit l hemorrh ges Ocul r findings: Th l mic: persistent downg ze. Put min l: d
evi tion tow rd. Cere
ell r: devi tion w y & ocul r
o

ing. Pontine: pinpoint.
Fluid-fluid levels common w/ co gulop thy. 40% enl rge >33%
y 24hrs. Angio yie
ld: lo
r, >45yo = 10%. IVH = 65%. Volume = (AxBxC)/2 Antico gul tion: with mech
nic l he rt v lves w it 1-2wks
efore rest rting ntico gul tion Medic l: o Rec
om
in nt F ctor VII: Ph se II tri l, (GCS >6, within 3hrs of onset). Throm
oem
o
lic complic tions twice s common. Mort lity reduced
ut not st tistic lly signi
fic nt. (N9/04) Surgery: o Cr niotomy 20-60cc
est (>85cc: 0% surviv l reg rdles
s of tre tment). Lo
r, cere
ell r, extern l c psule. Young. <24hrs. Cere
ell r:
surgery for GCS <14 OR >4cm 4 r ndomized tri ls, 3 show no
enefit, 1 showed en
doscopic ev cu tion
etter th n medic l. 3 r ndomized tri ls inconclusive. Surgi
c l Tre tment for Intr cere
r l Hemorrh ge (STITCH) tri l: no
enefit to surgery
<72hrs, possi
ly except those <1cm from surf ce. Consider ultr sound for loc li
z tion. o Stereot ctic spir tion: fr me-
sed, CT- or MRI-guided. M y use tPA,
urokin se. C n le ve ventricul r c theter. Improved ADLs only if opening eyes to
p in on dmission in single-center RCT (JN 9/04)
Flotte Outline of Neurosurgery

58
Intr cr ni l Hemorrh ge of Pregn ncy: M y
e ssoci ted with ecl mpsi ( lso inc
re sed rupture of AVMS, neurysms). O
t in shielded CT/ ngio. MRI is s fe. Avoid
m nnitol, Nipride (hydr l zine is OK). V gin l delivery vs c-section for ll le
sions is controversi l. Intr ventricul r Hemorrh ge (IVH): 4mg intr thec l tPA i
njected once- -d y, EVD cl mped for 30min-1hr. IT urokin se h stened clot resolu
tion
y 44% in sm ll (n=12) RCT. (N2/04). t-PA speeds ventricul r cle r nce, kee

ps EVD p tent, reduces ICP. No ch nge in shunt pl cement(N9/04) Moy -Moy Dise s
e Idiop thic occlusion of ICAs with form tion of coll ter ls (puff-of-smoke on ng
iogr m). More common in Asi (J p n). Associ ted with NF-1, cr ni l irr di tion,
Downs syndrome, sickle cell More ggressive, more pre- & postop inf rcts in chi
ldren <3yo Symptoms: He d ches (difficult to tre t), r rely chore . Inf rctions
in young children, SAH/ ICH in older p tients Tre tment: o Enceph lodur lsyn ngi
osis (EDAS): STA nd g le l tissue sutured into dur l opening. M y
e done 2wks
fter stroke. If sm ll inf rct do th t side first, if l rge inf rct do norm l si
de. W it 1-2mos to do contr l ter l hemisphere or do
oth concurrently o Enceph
lomyosyn ngiosis (EMS): tempor lis muscle sutured to dur l opening through wide
cr niotomy o Pi l syn ngiosis: Int ct STA tr nsposed onto pi l surf ce. Intr ope
r tive EEG used to monitor for intr oper tive cere
r l ischemi . 7% risk of stro
ke 30 d ys postop. o He d ches resist nt to surgery Su
cl vi n ste l: Occlusion
of su
cl vi l . proxim l to verte
r l . C uses vertigo, syncope, etc. Dx: Dopp
ler U/S, ngio. Tre tment: ngiopl sty, c rotid-su
cl vi n
yp ss.
Flotte Outline of Neurosurgery
59
Pedi tric
He d Circumference: Neon te: 1cm/wk; 1-3m: 2cm/wk; 4-6mo: 1cm/mo; 6-12mo: 0.5cm/
wk Cr niosynostosis S gitt l = sc phoceph ly (most common). Metopic = trigonocep
h ly. Oxyceph ly, turriceph ly, croceph ly = l m
doid nd coron l (cone he d, o
xycep ly = pointed, turriceph ly = fl t forehe d). Kle

l tsch del (Clover-Le f)
= ll sutures except squ mos l. Nonsynostotic sc phoceph ly = sticky s gitt l su
ture (JN:P 8/04) C uses incre sed ICP in 11%. Enceph locoele Occipit l = whites,
Front l (sincipit l, frontoethmoid l) = si ns. Also: p riet l, tr nsphenoid l,
n s l ( ssoc. with n s l gliom s dyspl stic tissue). 50% develop HCP w/in 1mo. M
eckels syndrome: enceph locoele + dyspl stic kidneys, c rdi c pro
s, f ci l clef
ts. Myelomeningocoele Risk f ctors: M tern l low fol te, v lpro te, Tegretol. Pr
evention: Fol te supplement tion (0.4mg/d y) from 1 month
efore pregn ncy to en
d of 1st trimester. Di gnosis: m tern l serum AFP > mniocentesis AFP & cetylch
olinester se (pe ks 12-14wks). Complic tions: Tethered cord, Chi ri II, syrinx,
HCP. With neurologic deterior tion lw ys check for shunt m lfunction first. Tre
tment: If ruptured st rt nti
iotics. Close MM within 24hrs whether open or not
. Decre ses infection, doesnt ch nge function. Controversi l whether to w it >3d
fter closure
efore shunting or perform it simult neously. Occult spin l dysr p
hisms (OSD) Meningocoele, lipomyelomeningocoele, di stem tomyeli , f tty filum,
etc. Defects of second ry neurul tion. Cut neous
norm lity: F wns t il, lipom , d
erm l sinus tr ct, dimple, etc. M y not correspond to level of dysr phism. F wns
t il very suggestive of di stem tomyeli . C pill ry hem ngiom s re norm l ne r
occiput, 10% OSD if thor colum
r. In gener l not ssoci ted with
r in
norm l
ities. M tern l AFP not elev ted. Dx: MRI +- xr ys. Consider intr op monitoring
(cystometrogr m,
l dder/ rect l EMG, nerve stimul tion, EPs). If dur is dhere
nt posteriorly, excise dur circumferenti lly. Meningocoele Usu lly neurologic l
ly norm l, no ssoci ted
norm lities. Rule out hydroceph lus. M y cont in
er
r nt nerve roots/ gliotic tissue. M y occur off-midline. Tre tment: elective rep
ir. Usu lly ends in n rrow neck. Myelocystocoele = meningocoele + hydromyeli .
Occurs with omph locoele,
l dder exstrophy, imperfor te nus. Lipomyelomeningoc
oele AKA spin l lipom of the conus (SLC). Dors l insertion: Nerve roots lie ven
tr l. Usu lly contiguous with su
cut neous f t. C ud l insertion: Lipom either
repl ces filum or runs sep r tely. Nerve roots m y run in lipom . M y
e entirel
y intr spin l or from su
cut neous f t. C n cont in ter tom s (i.e.
owel). Usu
lly h ve cut neous stigm t . Not ssoci ted with Chi ri II. M y
e off midline (
ipsil ter l LE more ffected), cont in CSF. Symptoms: Usu lly norm l in inf ncy.
Develop leg p in, LE neurologic deficit. Tre tment: Most recommend prophyl ctic
surgery
ut is controversi l. Kulk rni (N4/04) showed deterior tion is higher f
or surgic lly tre ted p tients th n o
served ones. Midline incision (even if off
midline). L minectomies, durotomy 1-2 levels
ove. Untether cord, remove lipom

 , reconstruct dur . Remove norm l l min
elow to find norm l dur . Dors l: C n
use l ser to remove f t, close pi . C ud lly inserting: divided fter t ke-off
of l st norm l root unnecess ry to remove ll gross lipom . Keeping the p trient
prone postop m y reduce retethering? Only in the event of progressive neurologic
deterior tion should one reuntether McCom
.
Flotte Outline of Neurosurgery
60
Di stem tomyeli Lum
r > thor cic. Cut neous
norm lity (75%) m y not correspo
nd to di stem. F wns t il most common. Clinic lly presents s tethered cord. Asso
ci ted with scoliosis, myelomeningocoele, verte
r l n molies. M y consist of 1
or 2 dur l tu
es; m y h ve duplic tion of spin l cord (diplomyeli ). Di stem m y
incompletely sp n c n l, m y
e dors l or ventr l; m y not h ve
ony di stem. T
re tment: Symptom tic: oper te. Asymptom tic: controversi l. Some dvoc te oper
ting
efore 2yo (
ony di stem or not). Surgery mostly prophyl ctic, lthough som
e improve. Surgery: M y h ve lipom t mous filum - Remove septum
efore detetheri
ng. Closing nterior dur not necess ry. Elliptic l dur l incision m de round s
eptum - cuff of dur left on seputm
se. M y
e
err nt nerve roots in midline
ne r septum, c n s crifice. Butterfly verte
r e/ Hemiverte
r e: m y mimic fr ct
ure Occult spin
ifid : Norm l v ri nt in up to 30% S1 > L5. M y
e symptom tic

ove L5. No studies needed if t L5/S1 nd no cut neous
norm lities. Tethere
d cord Occurs in kids or dults. Conus
elow L2 (L2 t term, L1/2 t 6mos). M y

e due to lipom tous filum, fi
rous
nd from conus to dur (dors l or ventr l),
epidur l sc rring. Lipom tous (F tty) filum: >2mm di meter Found in 5% of popul
tion. P tients m y h ve symptoms with norm l conus level, nd p tients with thi
ck filum m y rem in symptom tic. Adults: p in more common (LBP/ perine l). Tr u
m c n produce cute deficits. Kids: foot deformities, scoliosis, cut neous stig
m t (80-100% vs dults <50%). Both h ve urin ry symptoms. Aggr v ted
y growth
spurts. F t within 13mm of conus h s higher risk of deficits. In myelos must det
ect clinic lly (worse g it, sp sticity, urodyn mics, scoliosis) s ll will h ve
r diogr phic tethering. If p inful suspect tethered cord, p inless syringomyeli
. Tre tment: Section filum if symptom tic. Anterior s cr l meningocoele M y con
t in neur l elements or not. M y present s pelvic m ss, HA with defec tion. M y
complic te l
or. Asymptom tic m y
e followed if not enl rging, no ch nce of p
regn ncy. Symptom tic p tients: surgery. L5-S4 l minectomy performed. Ostium ove
rsewn, filum divided, pelvic m ss not removed. If dur l defect is wide c n perfo
rm digit l decompression thru rectum or f sci l gr ft. Aspir tion thru rectum or
v gin should not
e performed. Derm l sinus: M y end extr du lly or extend int
r dur lly. In cervic l or thor cic spine it c n end in centr l c n l. Lum
r c n
termin te in filum or intr dur l dermoid cyst (m y
e in filum or intr medull r
y). M y
e ssoci ted with lipom . M y h ve purulent dr in ge. C n c use recur
rent meningitis. S cr l or coccyge l sinuses dont need to
e explored, lum
r do.
Pro
ing nd contr st injection re worthless. If extends to dur , open dur . C
n require l minectomies to T12. If dermoid cyst h s ruptured nd sc rred, or is
em
edded within conus, void tot l remov l do intr c psul r decompression. S cr
l Agenesis 15% ssoci ted with m tern l di
etes. Motor deficit = lowest level w
ith int ct pedicles. V ries from coccyge l to thor cic genesis. C n
e symmetr
ic. Neuro loss c n
e from minim l to complete. Tre tment: fusion C ud l regress
ion syndrome: S cr l genesis, imperfor te nus, ren l dyspl si , sirenomyeli .
Also ssoci ted with VATER syndrome. Sinus pericr nii: Presents s sc lp m ss. L
rge su
cut neous venous sinus. Growing skull fr cture: 75% of p tients re <1yo
. R re >3yo. Requires >3mm di st sis to occur. Ceph lohem tom : su
perioste l, d
oes not cross sutures, C put succed neum in su
cut neous f t, crosses sutures; S
u
g le l hem tom ; ll m y c use signific nt
lood loss, check Hct. Ar chnoid Cy
st C n occur nywhere. Most common in middle foss (seizures), supr sell r (visu
l loss, precocious pu
erty,
o

le-he ded doll).
Flotte Outline of Neurosurgery


61
Symptoms: seizures, deficits, he d che, visu l deficits, development l del y, en
docrinop thies. C n rupture, c use hyperostosis or hydroceph lus. Associ ted wit
h intr cystic nd su
dur l hemorrh ge. N tur l history is v ri
le: m y enl rge,
regress, or rem in st tic Tre tment .Most recommend tre ting only if symptom ti
c (intr ct
le he d ches, seizures, foc l deficits) or incre sing size o Needle/

urrhole most recur o Cr niotomy & fenestr tion: most effective if fenestr ted i
nto
sil r cisterns (2cm Microcr niotomy: N11/03). Improves seizure control nd
foc l deficits; visu l distur
nces, development l del y, nd endocrinop thies
persist. o Cystoperitone l shunt: usu lly if fenestr tion f ils. Shunt dependenc
y m y develop. o All c ses presenting with hydroceph lus required VP shunting re
g rdless wether fenestr tion w s performed. o Endoscopic fenestr tion o Supr sel
l r: tr nsc llos l, ventriculocystostomy. HCP m y incre se fter tre tment. Conc
omit nt hydroceph lus usu lly requires VP shunt. Outcome: Improvement (keyhole c
r ni) hemip resis & CN6 p lsy 100%, he d ches 66%, seizures 50%. Chi ri I 3-5mm
tonsill r herni tion. Controversi l whether degree of herni tion correl tes with
symptoms or postop improvement. He d ches: cl ssic lly occipit l, worsen w/V ls
lv . Also cr ni l nerve,
r instem & spin l cord syndromes. Down
e t nyst gmus.
60% h ve syrinx (m y
e thor cic or lum
r); 25% hydroceph lus; 25% cr niocervi
c l junction
norm ilities; 20% scoliosis; 5% Klippel-Fiel, 5% GH deficency. No
t ssoci ted with other development l
r in
norm lities; No recommend tion for
p rticip ting in thletics. Di gnosis: MRI. O
t in preop flexion/extension c-sp
ine xr ys. Cine-MRI not of m jor di gnostic v lue Tre tment o Asymptom tic: Decomp
ress only for syrinx; if no improvement then shunt. (But - Nishiz w (N01) 8/9 w
/ syrinx rem ined symptom tic.) o Symptom tic: If HCP: shunt. No HCP: decompres
sion, if f ils then syringosu
r chnoid shunt. o Anterior compression (VBSC): tr
nsor l odontoidectomy BEFORE decompression. o Scoliosis: improves with decompre
ssion; perform spin l fusion only for Co

ngle >50. (JN8/03) o Chi ri Decompres
sion: 3x3cm su
occipit l cr niectomy, C1 (or lower) l minectomy, dur pl sty,  shr
inking tonsils. If extensive sc rring c n use ultr sound to find 4th ventricle.
Stenting worsens outcome. Postop: w tch for sleep pne . 50-85% success. If h s
occipit lcervic l inst
ility, c n do concomit nt OC st
iliz tion (N6/04). Best
if done within 2yrs of onset. SE: cere
ell r s g: requires cr niopl sty. Chi ri I
I 100% ssoci ted with myelomeningocoele. 90% h ve hydroceph lus. Decompress for
pne , stridor, dysph gi , progressive sp sticity or t xi , opisthotonus, recu
rrent spir tion pneumoni . Check shunt function first. D ndy-W lker Syndrome As
soci ted with c rdi c
norm lities, polyd ctyly, genesis of the corpus c llosu
m, 80% hydroceph lus. Tre tment: 4th ventricul r shunt (low pressure v lve) with
or without l ter l ventricle shunt (medium pressure). 3rd & l ter l ventricles
communic te with the cyst in 50% - requires iohexol CT to identify. D ndy-W lker
V ri nt: vermi n hypopl si , norm l posterior foss , no HCP Meg cistern m gn
: norm l vermis, cere
ellum IVH Source: Neon te = germin l m trix; full-term = c
horoid plexus. Tre tment: seri l LPs 7-15cc/d. If un
le to remove enough CSF to
norm lize ICP then ventriculostomy or Omm y . Shunt: >1500g, protein <200 (or 2
g, 500). Achondropl si : Tre t HCP only if symptom tic (consider jugul r for men
decompression inste d) Hydroceph lus Aqueduct l stenosis: some re X-linked. Co
nsider endoscopic 3rd ventriculostomy Tre tments o Ventriculoperitone l (VP) Shu
nt Common shunt v lves: PS Medic l, Cordis (H kim), Holter v lve (with Rickh m r
eservoir) During pregn ncy: 1st two trimesters pl ce VP shunt (no troc r), 3rd t
rimester VA shunt. Prophyl ctic nti
iotics during delivery. o Ventriculo tri l
(VA) Shunt: C uses SBE, shunt nephritis (immune). O
t in CXR q ye r. Revise when
tip @ T4.
Flotte Outline of Neurosurgery
62

o Ventriculopleur l Shunt: Pl ce
/t 2nd & 3rd ri
s o Ventriculo-s gitt l sinus
Shunt: Inf nts, thru nterior font nelle o Ventriculo-su
g le l shunts o Endosco
pic 3rd ventriculostomy: Consider pl cement of Omm y reservoir for emergency (N
7/03) Complic tions o Shunt infection: 66% St ph epi, 25% St ph ureus. St ph: I
V + IT V nc. H flu usu lly cle r w/in 48hrs
y IV lone. Risks: <1yo, preop hosp
it liz tion, surgery dur tion Use prophyl ctic nti
iotics
efore dent l procedu
res,
l dder instrument tion o Slit ventricle syndrome: Intr cr ni l hypertensio
n with decre sed ventricul r compli nce. Symptoms resem
le shunt m lfunction (
often intermittent). Must
e differenti ted from intr cr ni l hypotension from o
vershunting (spin l he d che relieved with recum
ency). Tre tment: r ise v lve p
ressure > ntisiphon device> su
tempor l cr niectomies. P tients with shunts sho
uld not
e restricted from pl ying sports (N5/04)
Isol ted 4th Ventricle Occurs due to shunting for communic ting hydroceph lus (I
VH, meningitis) C uses he d ches, t xi , qu drip resis, pne ,
r dyc rdi . Tre
tment: 4th ventricul r shunt, endoscopic queductopl sty  stent, endoscopic inte
rventriculostomy Benign su
dur l collection of inf ncy: Usu lly resolves
y 9mo
Su
g le l/su
perioste l hem tom : void spir ting, follow Hct
Flotte Outline of Neurosurgery
63
Tr um
He d Tr um
Intr cr ni l pressure (ICP)
Cere
r l Perfusion Pressure (CPP) = ICP MAP (Me n Arteri l Pressure) ICP monitor
ing: o ICP W veform: P1 = percussion w ve, systolic contr ction, decre sed with I
CP, compli nce; P2 = tid l w ve; P3 = ortic v lve closure. o Lund
erg W ves: A
(Pl te u) = > 50mmHg rise for > 20min. B = >20mmHg, l sts 1-2min. C = 4-8Hz. o M
onitors: Intr ventricul r C theter Intr p renchym l Monitor Su
r chnoid Bolt: l
ess ccur te Su
dur l & Epidur l monitors: less ccur te Signs of incre sed ICP:
o Pupil ry dil tion/ CN3 p lsy: 90% ipsil ter l m ss. Hemip resis (cere
r l ped
uncle compression): 70% contr l ter l m ss. Jugul r Venous Monitoring o Necess r
y during
r
itur te com . Norm l SjO2 >50%. ICP Tre tment me sures HOB 20-30 Sed
tion/P r lysis Ventricul r dr in ge M nnitol o Contr indic tions: hypotension,
ren l f ilure. o Common dose: 1mg/kg initi lly, then 0.25-0.5 mg/kg q6hrs. Check
Serum N & Osm
efore giving. Hold if N > 150-160, or Osm > 320 (limits c n v
ry depending on the situ tion). o High-dose (1.4g/kg) given wide open in p tient
s GCS3 & fixed pupils h d 33% more f vor
le outcome. (JN3/04) o Altern tives or
djuncts include L six, hypertonic s line (ie 9%). Hyperventil tion: Keep CO2 3
0-35. Use cutely (CO2 to 25
y m nu l
gging) only for cute pl te us. Decompr
essive cr niectomy o 14cm di meter ppe rs optim l. o Reconstruction: Timing con
troversi l B r
itu te Com o To
urst-suppresion on EEG. Serum levels re used,

ut h ve poor correl tion to clinic l
enefit. o Pento
r
it l: Lo ding: 10 mg/k
g over 60min, then 5mg/kg/hr x3hrs, then 1mg/kg/hr (Thiopent l m y
e su
stitute
d). o Side-effects: hypotension Hypothermi : to 95 is ccepted. <95 is controversi
l. o NABIS:Hypothermi study showed reduced ICP
ut no difference in 6 month ou
tcomes Minor He d Injury LOC <1min, norm l ment l st tus, no deficits on initi l
ex m, no skull fr cture Guidelines for children with mild he d injury pu
lished
(Pedi trics, 1999) For LOC (more th n seconds), mnesi , vomiting, leth rgy, GC
S >13, no foc l deficits or seizures, skull fr cture (except cross MMA, venous
sinuses, or depressed), otherwise norm l CT: o
served for 2hrs, if GCS 15, no de
terior tion,
le to hold down liquids, nd reli
le c ret ker, c n disch rge (J
N:P 8/04) 0.3% incidence of deterior tion with norm l CT (del yed EDH, diffuse

r in swelling) Sports-concussion: Return fter 1 week if mnestic, 2 weeks if (+

)LOC Tr um tic Br in Injury (TBI) Keeping ICP<20 improves outcome. Keep CPP >70.
Norm lize BP, temper ture, oxygen. Keep mildly hypervolemic (CVP >8) Nutrition:
p r lyzed 100% BME, non-p r lyzed 140% BME Steroids re not indic ted in TBI. P
edi tric TBI B r
com , CSF dr in ge more effective th n in dults. Hyperventil
tion, m nnitol m y lower ICP without lowering CBF. Incre sed risk of seizures.
Flotte Outline of Neurosurgery
Diffuse Cere
r l Swelling: Due to venous congestion nd hyperemi (not cytotoxic
/ v sogenic edem ). 50% mort lity. Denny-Brown syndrome: Br dyc rdi , git tion,
HA due to v g l syncope, mimics EDH
64
Child
use (Sh ken B
y) Common findings: Interhemispheric SDH, skull fr ctures
, multiple long-
one fr ctures of different ges. Diffuse Axon l Injury (DAI): P
th: Axon l retr ction
lls. Loc tions: White m tter, corpus c llosum, dors l

r instem Tr um tic Cr ni l Nerve injury Indirect optic nerve injury: No prospect
ive tri l showing decompression
etter th n steroids except for del yed onset
l
indness. Surgery: Done within 1-3weeks, tr nsethmoid l route Tr nsient cortic l

lindness: Children m y develop l sting 1-2d fter he d injury. CN7 injury: foll
ow ENOG, decompress if no improvement on steroids. Posttr um tic Seizures Antiep
ileptics prevent e rly (with 7 d ys), not l te ( fter 7 d ys) seizures Begin AED
s for: GCS <11, ny hem tom or contusion, seizures, penetr ting injury, history
of lcoholism, open depressed skull fr cture If no seizures fter 1 week discon
tinue ntiepileptics except for: penetr ting injury, prior seizure history, cr n
iotomy. M int in 612mos nd o
t in EEG
efore discontinuing. Skull Fr ctures Di
st tic fr ctures sep r te cr ni l sutures M ndi
ul r fr cture: think c rotid dis
section. Depressed fr ctures o Criteri to elev te: >1cm or thickness of the sku
ll, neurologic deficit, CSF le k, open fr cture. o Elev tion m y improve deficit
s, not seizures o Fr ctures over m jor venous sinuses: controversi l criteri . H
ve Fog rty c theter re dy. Prep out s phenous vein. B sil r skull fr cture: o G
ive Pneumov x & Tdt. o Get CT with thin cuts. o In the
sence of n open fr ctu
re, pneumoceph lus is di gnostic of
sil r skull fr cture. o Require tre tment:
tr um tic neurysm, C-C Fistul , persistent CSF rhinorrhe , meningitis/
scess
(m y occur ye rs l ter) o Prophyl ctic nti
iotics controversi l. Most tre t wit
h
ro d-spectrum nti
iotics (Cipro) 7-10 d ys. Tempor l fr cture: o Tr nsverse:
Perpendicul r to IAC. Higher risk of CN 7,8 tr nsection. o Longitudin l: P r ll
el to IAC. Del yed f ci l p lsy is usu lly due to edem , resolves. o F ci l p ls
y: Tre t with steroids. Immedi te onset: if no improvement on steroids, consider
expor tion (timing controversi l). Del yed onset: Follow with ENOG (f ci l EMG)
. Consider explor tion for continuous deterior tion on steroids nd <10% functio
n comp red to norm l side on ENOG. Controversi l. Front l Sinus fr cture: o Ante
rior w ll fr cture: o
serve. o Posterior w ll: Controversi l. Intr dur l pneumoc
eph lus implies dur l l cer tion. o Front l Sinus Cr ni liz tion: Bicoron l inci
sion. Perserve pericr nium. Remove posterior w ll. Mucos removed (exenter ted)
nd p cked into fronton s l duct. Rem ining w lls of sinus drilled to remove muc
os l crypts (preventing mucocoele form tion). Fronton s l duct then p cked with
muscle or f sci . Perioste l fl p is then pl ced over sinus nd floor. o If dur
l te r is suspected (pneumoceph lus, CSF le k) then do intr dur l explor tion n
d rep ir with gr ft sutured down nd fi
rin glue. Tension pneumoceph lus: c n ev
cu te with spin l needle thru
urr hole. Avoid N2O. Tr um tic CSF le k: Loc t
e: 1) CT  contr st (look for p renchym l enh ncement) 2) CT cisterogr phy. Iohexo
l, metrizimide (w ter solu
le contr st) inject
y LP, requires ctive clinic l l
e k 3) MR Cisternogr phy. Tre tment: Conserv tive (
edrest, Di mox) x 5-7d then
seri l LPs (30-50cc) or lum
r dr in. Surgery if persists > 2wks. Front l: Intr
dur l explor tion preferred over extr dur l. Epidur l Hem tom (EDH) / Su
dur l
Hem tom (SDH) Del yed enl rgement in 10-30%.
Flotte Outline of Neurosurgery
Chronic SDH Get intermedi te CT windows. Bl ck
nd on intern l mem
r ne on T2-MRI

m y predispose to enl rgement Penetr tiong Br in Injury (PBI)/ Gunshot Wound (G

SW) to the He d Tdt, nti
iotics (not proven). Consider ngio for: del yed hemor
rh ge, tr jectory involving vessels, l rge hemorrh ge in s lv ge
le pt (on d2-3
). Only remove fr gments which come out with gentle irrig tion.
65
Spine Tr um
Spin l Cord Injury Level: motor 3/5 AND p in/temper ture sens tion int ct. Sens
tion or s cr l sp ring ( n l motor/sensory) = incomplete. Complete: 3% h ve reco
very w/in 24hrs. After 24hrs no recovery Conversion disorder (hysteric l p r lys
is): preserv tion of norm l reflex p ttern, norm l rect l sens tion, nd norm
l
l dder nd
owel functions. Flex p tients knees nd rele se conversion disord
er will m int in knees in flexed position. Some uthors recommend motor or som t
osensory evoked potenti ls. Tre tment: Solumedrol (methylprednisolone) 30mg/kg x
1hr then 5.4mg/kg/hr x 23 hr only if <8hrs out. MAP 85-90 for 1st 7 d ys. DVT p
rophyl xis for 3 months. Anterior cord syndrome: p r plegi + dissoci ted sensor
y loss. Must differenti te cord inf rct from surgic l lesion ( nterior fr gment)
Spin l shock: Hypotension &
r dyc rdi . L sts 3d-3wk. Bul
oc vernosus 1st refl
ex to return (pinching gl ns penis/ clitoris > n l contr ction, lowest v il
l
e reflex). SCIWORA (Spin l Cord Injury Without R diogr phic A
norm lity) MRI, CT
, Xr ys (including flexion/extension) neg tive with neurologic deficit. Most com
mon 1-16yo. Tre tment: Bedrest, C-Coll r until norm l Flex/ext. Guilford
r ce f
or 3mos or h lo for 1-3wks recommended
y some. Discontinue if flexion/extension
xr ys norm l t 3mo. No sport p rticip tion for 3mos. Whipl sh: Gr de I = p in/
stiffness, Gr de II = limited ROM, point tenderness. Both: ROM exercises, II = c
-coll r < 72hrs (h rd only) Centr l cord: no evidence e rly surgery
enefits or
hurts. With myelop thy pts f re
etter w/fusion

Spine fr ctures
Cervic l Spine Xr ys: A/P, l ter l, odontoid. Must see to C7/T1 disc. If no f
tures on Xr ys
ut h s neck p in or tenderness, then get flexion/extension xr ys
. If cervic l muscle sp sm is present then keep in c-coll r nd repe t flexion/e
xtension films in 1 week. O
t in CT for non-visu lized re s on Xr y, level of n
eurologic deficit D/C coll r fter norm l flexion/extextension xr ys (under flou
ro if o
tunded) OR norm l MRI (within 48hrs) Bone sc n to delini te old versus n
ew fr ctures (rem in hot for 24-48hrs up to ye r). White-Punj
i guidelines for
inst
ility: 4mm su
lux tion, 11o ngul tion (inferior endpl tes). Dont get flexi
on/extension xr ys. If <4mm su
lux tion get flexion/extension. Reduction: if not
w ke during reduction o
t in MRI first to rule out herni ted disc (requires de
compression
efore reduction). MRI recommended if closed reduction f ils. Emgerg
ent decompression for: incomplete SCI nd progression, complete CSF
lock, decom
pression of vit l cervic l root, compound/penetr ting fr cture, cute nterior c
ord syndrome, nonreduci
le fr cture
Cervic l fr cures
Cervic l tr ction o Pl ce G rder-Wells tongs 1cm
ove pinn . Pl ce nterior to
EAC for extension, posterior for flexion. MRI-comp ti
le G rdner-Wells tongs m y
not h ve the weight c p city for lower cervic l disloc tions. o Begin t 3-5l
s
per level of weight. 10l
s/level m ximum. Stop if ny disc height >1cm. Use V l
ium/Demerol. o Do not use tr ction with AO disloc tion or type IIA, III h ngm ns
fr ctures. Extern l st
iliz tion o Cervic l coll r (C-coll r). Soft: for comfo
rt only, no st
ility. H rd. o Cervic l-thor cic orthoses (CTO): Guilford, SOMI

r ces o H lo vest AO disloc tion: immedi tely pply h lo, no tr ction. (surgery
vs h lo) AA disloc tion: MRI to look t tr nsverse lig ment. If int ct, h lo. I
f disrupted, surgery.
Flotte Outline of Neurosurgery
66

Jeffersons Fr cture: Anterior nd posterior ring fr ctures of C1 Rule of Spence:

>7mm overh ng needs h lo, <7mm CTO. Need thin cut CT C1-C3. C1 fr ctures: Poste
rior rch: coll r. L ter l m ss: >2mm displ cement = h lo, <2mm = coll r. Atl nt
o xi l Rot tory Su
lux tion: Most resolve spont neously. If not, use tr ction. K
ids: st rt 8l
s, incre se to 15l
s over few d ys. Adults: st rt 15l
s incre se t
o 20l
s. If present <3mos, h lo x 3mos. If chronic or recurs 2-3 wks tr ction th
en ORIF  fusion. Crock rd: ORIF vi extreme l ter l ppro ch, h lo x 3mo or if h
lo not w nted then midline ppro ch nd C1/2 tr ns rticul r screws. H ngm ns Fr
cture: 3mm su
lux tion: c-coll r. 4mm su
lux tion: reduce with tr ction, then h
lo x 8-12wks. Check cspine xr y in h lo to verify reduction. If in dequ te then
surgery (C2 to C3 +- C1) then coll r x 34mos. Locked f cets: do not reduce, surge
ry Odontoid fx: Type I: very r re. Type II: depends on displ cement, ge. Gener
lly: >7yo with >6mm su
lux tion (or inst
ility in h lo) needs surgery. Others 1
012 wks in h lo. Type III: h lo. C2 fr cture: L min : coll r. F cets,
ody, l te
r l m ss: CTO or h lo. Cl y shovelers: Coll r prn p in. Anterior wedge: CTO if m
ild, h lo if severe Te rdrop: True Tre rdrop ssoci ted with: s gitt l
ody fr ctu
re, retrolisthesis, nterior wedge, f cet disruption, preverte
r l swelling, los
s of disc height inferiorly, neurologic deficit. Requires st
iliz tion. Simple t
e rdrop: If none of the
ove re present then o
t in flexion/extension xr ys. If
norm l then keep in c-coll r nd repe t in 4-7d. Qu dr ngul r fr cture: O
lique
fr cture through
ody ( nterior-superior to inferior), retrolisthesis, nterior
wedge, disruption of disc & lig ments. Tre tment: Anterior & Posterior st
iliz
tion. C3-C7 fr ctures: Follow White-Punj
i guidelines. Anterior st
iliz tion
(ACDF) m y need to
e supplemented
y posterior st
iliz tion if posterior tensi
on
nd is disrupted. Locked f cets: M nu l reduction in tr ction: Unil ter l: t
orque tow rds locked side. Bil ter l: xi l distr ction nd flexion Once reduced
le ve in 5-10l
s for st
iliz tion. H lo x 3mos m y
e tried if f cet fr cture
fr gments re present. O
t in Flexion/Extension Xr ys in h lo
efore disch rge.
If no f cet fr cture surgery. Posterior ppro ch preferred. Mech nisms: Flexion:

il ter l locked f cets (neck flexed w/ flexing force), wedge, cl y-shoveler, t
e rdrop (neck flexed w/ compression). Flexion-Rot tion: unil ter l locked f cet
Extension-Rot tion: pill r fr cture (neck flexed, compressing force) Axi l compr
ession: Jeffersons,
urst (
oth neck neutr l) Extension: h ngm ns (distr cting fo
rce, neck extended), posterior fr cture-disloc tion, l min r fr cture Pedi tric
Fr ctures C23 pseudosu
lux tion: C2 posterior spin l line should
e less th n 2m
m posterior to line
etween C1 & C3 posterior spin l lines Odontoid synchondro
sis (fuses @ 7yrs) ADI interv l wider th n dults <9yo odontoid xr y not useful
<7yo C2 epiphysiolysis (synchodrosis injury @ odontoid
se): closed reduction &
h lo Thor cic fr ctures: Decompression nd st
iliz tion prim rily
y pedicle s
crews nd l minectomy. Anterior ppro ch (sternotomy or m nu
riotomy) limited to
T3
y ortic rch. Thor colum
r fr ctures: Wedge fr cture: TLSO if severe. M y
require surgery if >50% loss of height with ngul tion, ngul tion >40, progress
ive kyphosis, or 3+ fr ctures in row. Burst fr cture:
Flotte Outline of Neurosurgery
o
67

If deficit, ngul tion >20, nterior height <50% of posterior height, or >50% c n
l compromise: surgery. If not void e rly m
ul tion, TLSO, seri l xr ys to loo
k for progression. o Surgic l Options: o Posterior: Pedicle screws 1 level
ove
&
elow w/distr ction  tr nspedicul r decompression (push fr gments out of c n l
w/ ngled curette), NOT l minectomy. Some report 20-50% screw f ilure, worsening
kyphosis. Some recommend 2 levels
ove &
elow. Consider djunctive verte
ropl
sty o Anterior corpectomy. L4 nd
ove only. o Com
ined nterior/posterior. o
L5 (r re): 2wks
edrest, TLSO w/thigh cuff x 4-6mos, seri l xr ys Se t-
elt/Ch n

ce fr cture: o Fr cture through nterior nd posterior
ony or lig mentous eleme

nts with no su
lux tion nd no deficit. o Tre tment: TLSO or surgery (Pedicle sc
rews 1 level
ove &
elow with compression) Fr cture-disloc tion: Surgery (Pedi
cle screws 2 levels
ove &
elow) Osteroporotic: Bedrest x 7-10d then PT, TLSO
GSW Spine Surgery for: deterior tion, c ud equin injury w/compression, nerve c
ompression, CSF le k, inst
ility, de
ridement, v scul r injuries, migr tion, pl
um
ism.
Flotte Outline of Neurosurgery
68
Miscell neous
Critic l C re
Anesthesi Sed tives: Versed, Thiopent l N rcotics Nitrous oxide: void with pne
umoceph lus P r lytics o Succinylcholine: 1mg/kg 3.5 to 5cc, l sts 5-10min. do n
ot use with spin l cord injury, hyperk lemi o P vulon revers l: Neostigmine (2.
5-5mg IV) + tropine (0.5mg/mg neostigmine) or Ro
inul (0.2mg/mg neo). T kes 20
min M lign nt hyperthermi : D ntrolene (2.5mg/kg, up to 10mg/kg), 100% O2, D/C
nesthesi & ch nge tu
ing. Occurs w/ inh l tion l + Sux. ETCO2. 50% previous nor
m l nesthesi . Air Em
olism Occurs when venous pressure is lower th n tmospher
ic pressure nd the venous system is open to the tmosphere. Most likely to occu
r in the sitting position Detection: precordi l doppler (most sensitive), EtCO2
(e rliest), FEN2, CO, PAP, pulmon ry v scul r resist nce, ventil tion-perfusion
mism tch. Tre tment: Lower the he d-of-
ed, cover wound with wet l ps, spir te
ir through centr l line. Ventil tion Intu
tion/Extu
tion: 100mg lidoc ine IVP
, 100% O2 x 5min. Tu
e: 20-22cm t gum line, tip 5cm
ove c rin PCWP <12, PA 1
5-30/4-12, CI 2.8-4.2 Arrythmi s Afi
/Aflutter: Ver p mil (5mgx2), Dilti zem (0.
25mg/kg 10-20mg). ( denosine, proc inimide; digoxin for flutter) If unst
le c r
diovert 100J. Other SVT: V g l m neuver, Adenosine (6mg-12mg-12mg), ver p mil, d
ilti zem Vt ch, Vfi
: Pulseless: Epi (1-3-5mg); Other: Lidoc ine (1mg/kg q5m x 3
), proc in mide (20-30mg/min to 12mg/kg),
retylium (10mg/kg q5m x 3) Asystole:
Epi, tropine (0.5mg x 4) Br dyc rdi : tropine Myoc rdi l inf rction Clopidogre
l with spirin is recommended for unst
le ngin or minor myoc rdi l inf rction
; ticlopidine is not recommended Antihypertensives Nipride: onset seconds, Follo
w thiocy n te levels if used >24hrs. 0.3-10g/kg/min. Avoid in pregn ncy Nitroglyc
erin: 10-20 g/min, 0.4mg SL q5m x 3 Both r ise ICP. Hydr l zine: onset 3-5min, du
r tion 2-4hrs. OK in pregn ncy. SE: t chyc rdi . IM 10mg, IV 20-40mg prn. L
et
lol: onset 5m, dur tion 3-3hrs; 20-40-60-80mg IV, 200mg po
id Esmolol. V sotec:
1.25-5mg q6hrs prn Shock Dop mine: 2-20 g/kg/min, >. Do
ut mine: 2.5-10g/kg/min,
y (inotrope, BP unch nged). Use for c rdi c f ilure if normotensive. P lp
le pu
lses: r di l 80, femor l 70, c rotid 60 Steroids C use p ncre titis Addisoni n c
risis: hydrocortisone (Solucortef) 100mg IVP then 50mg q6h (not Solumedrol) An p
hyl xis Tre tment: Epinephrine 1:1000 5ml SQ, Ben dryl 50mg IM, Dec dron 10mg IV
Urtic ri : Ben dryl 50mg PO/IM + Cimet dine 300mg PO/IV V sov g l re ction: hyp
otension,
r dyc rdi . Tx: Atropine 0.75mg IV, q 15min to 3mg DVT Incidence in n
eurosurgic l p tients: 15-20% M y
e incre sed in cr niotomies due to rele se of

r in throm
opl stin C lf vein throm
osis h s <1% risk of PE, however they m y
progress to DVT Prophyl xis: Hep rin 5000 U SQ BID, Lovenox 30mg SQ BID. TEDs/SC
Ds (do not use if DVT is present)
Flotte Outline of Neurosurgery
o
69
ACCP guidelines: fond p rinux is ltern tive to low-molecul r-weight hep rin (
LMWH),
ec use it is equ lly s fe nd effective
ut h s longer h lf-life, mo
re predict
le response, nd fewer dverse effects o Moder te-risk surgic l p ti

ents: Hep rin 5000mg SQ BID or LMWH (less th n 3,400 U once d ily) o High-risk s
urgery p tients: Hep rin 5,000 U SQ TID or LMWH more th n 3,400 U d ily o Aspiri
n is not recommended Di gnosis: o Doppler ultr sound: St nd rd o Clinic l di gno
sis (c lf tenderness, w rmth) is unreli
le. o The fi
rinogen upt ke test nd im
ped nce plethysmogr phy h ve low ccur cy nd re not recommended o Contr st ven
ogr phy h s high sensitivity
ut limited v il
ility nd question
le use for s
m ll dist l throm
i nd high p tient discomfort. Use is limited to rese rch Tre
tment: Bedrest x 10d ys, then c reful m
ul tion. 3-6 months full ntico gul tio
n then low-dose coum din (INR 1.5-2). o Three r ndomized tri ls of ntico gul nt
s vs no ntico gul nts in DVT showed no
enefit with hep rin nd vit min K nt g
onists (com
ined ll-c use mort lity: ntico gul nts = 6/66, un- ntico gul ted c
ontrols = 1/60, P = .07). No pl ce
o-controlled tri ls of low-molecul r-weight h
ep rins or throm
olytic drugs h ve
een done; therefore, their effic cy in VTE d
epends entirely on r ndomized comp risons with unfr ction ted hep rin. They h ve
not
een proven s fer or more effic cious th n unfr ction ted hep rin. Throm
ol
ysis c uses more m jor nd f t l
leeds th n hep rin nd is no more effective in
preventing PE (CundiffDK 9/04). Pulmon ry Em
olism Di gnosis: o V/Q sc n: Norm
l sc n rules out PE. High pro

ility (88% true positive) then tre t. Low or mod
er te pro

ility then o
t in leg dopplers nd if positive then ngiogr m to con
firm. o Spir l CT o Angiogr m Tre tment: ntico gul tion or IVC (Greenfield) fil
ter. M ssive PE c using hemodyn mic compromise should
e tre ted with ntico gul
tion reg rdless of intr cr ni l risk. F t em
olism Occurs 12-48hrs post-injury
Symptoms: dyspne , petechi e over thor x, t chyc rdi , t chypne . L
s: serum li
p se in 50%. Look for f t in
lood, urine. No specific test. Cere
r l em
olism (
c using confusion, somnolence, seizures) does not occur without lung symtoms unl
ess PFO or ASD exists. Tre tment: O2, PEEP. Steroids controversi l. Antico gul
nts Preoper tive m n gement o Mech nic l He rt V lve: stop coum din 2d preop &
dmit on hep rin. o A-fi
: Stop coum din 5d preop, c n rest rt 5d postop Anti
io
tics Aminoglycosides/Gent mycin: Poor CSF penetr tion. SE: nephrotoxic (ATN), ot
otoxic, vesti
ulitis, worsens my sthenic crises,. Cover ge: Gr m (-) (no strep).
Sinus entry: Gent mycin, Clind mycin Electrolytes AG = N (Cl + HCO3) Osm = 2(N
+K) + BUN/2.8 + Glu/18 Hypon tremi : 1.0-1.5 meq/L/hr, 25meq/L/d, 3% N 25-50cc
/hr + L six SIADH: Dx: N <134, Osm<280, UN >18. Tx: fluid restrict <1L/d. Chron
ic: demeclocycline DI: 1/2NS + v sopressin Hyperk lemi : 10% C Glucon te 5-10cc
over 2m; 1 mp HCO3; 5-10U regul r insulin + 1 mp D50; K yex l te Hep rin: Repo
rtedly no higher risk in p tients with
r in tumors Coum din: lw ys pre-hep rin
ize Hem tology Pl telets: 1 units r ises pl telet counts
y 5-10K. Do not use wi
th utoimmune destruction (eg ITP). Fresh Frozen Pl sm : Vit min K: 10mg IM. PT
revers l requires 6-12 hrs. (Do not give IV) Prothrom
in complex concentr te rev
erses coum din 5x more quickly th n FFP DIC L
s: Incre sed PT, PTT & Bleeding T
ime. d-dimer, fi
rin degr d tion products (FDPs). fi
rogen =
est correl tion ()
. Tre tment: FFP  Hep rin (throm
otic) (cryo if fi
rinogen is low, pl telets if l
ow)
Flotte Outline of Neurosurgery
70
Antico gul tion/Antipl telets: Preop o Mech nic l v lves: stop coum din 2-3d pre
op, hep rinize. A-fi
: stop 4-5d preop. o Bleeding time not predictive of intr o
per tive
leeding o Br in tumors reportedly c rry no higher risk of hemorrh ge w
ith ntico gul tion o Stop coum din, hep rinize, nd stop hep rin 6hrs prior to
ngiogr phy, myelogr py, or surgery Postop o Cr niotomy: w it t le st 3-5d
efo
re rest rting ntico gul tion. o St rting LMWH <24hr postop clinic lly signific
nt hemorrh ge: 1.5% for m jor procedures, 0.07% for minor procedures (vs 4.3% wi
th SQ hep rin) (N11/03) Coum din o Alw ys prehep rinize
efore st rting coum din
(decre ses proteins C&S initi lly c using hyperco gu
ility). o INR: Mech nic l
He rt v lve: 3-4. All other (DVT, TIA, fi
, PE): 2-3. Hep rin o Incre ses Anti
throm
in III. o IV: 5000U
olus then 1000U/hr. SQ: 5000U Q8h. o C uses throm
ocy
topeni (use lepirudin/Reflud n). o Prot mine: 1mg reverses 100U hep rin. Low mo

lecul r weight hep rin (LMWH) o AKA fr ction ted hep rin o enox p rin (Lovenox):
30mg SQ BID. n drop rin (Fr xip rin) 0.3ml SQ. d ltep rin. o Versus SQ hep rin:
gre ter
io v il
ility, more predict
le ntico gul tion, lower risk Antipl te
lets: Clopidogrel o ntithrom
otic ther py for coron ry rtery dise se, stroke,
etc:
ec use of m ny dverse effects which re sometimes f t l, ticlopidine is n
o longer recommended for coron ry interventions when other tre tments re v il

le. Clopidogrel plus spirin is now recommended for most p tients with unst
le
ngin or minor myoc rdi l inf rction GP II
-III inhi
itors: Integrellin (epti
fi
tide), m xic
(Reopro), tirofi
n (Aggr st t) Fluids Het st rch: Che per t
h n l
umin. Possi
le ntico gul nt effect t high dos ge (>500 cm3/d) Alcohol W
ernickes syndrome o Symptoms: Tri d: g it t xi , nyst gmus/opth lmoplegi , confu
sion. ( lso hypothermi ). Due to thi mine deficiency. Ocul r pro
lems recover 1s
t. Usu lly reversi
le. IV glucose worsens lw ys give thi mine (
n n
g)
efo
re IV glucose. Kos koffs syndrome: memory deficits, usu lly perm nent
R diology
Iodin ted Contr st Allergy: If minor, c n prep with prednisone 32mg PO 12hrs nd
2hrs
efore; Ben dryl 50mg either IM 1hr
efore, or IV 5min
efore. Use non-ion
ic cont st (Iohexol) if possi
le. With history of n phyl xis do not give even
with prep. IV Iodin ted contr st nd Glucoph ge (metformin) c n c use ren l f i
lure.\ MR l ndm rks for precentr l gyrus: on most rostr l xi l cuts, look for L
-sh pe. On mids gitt l cuts it is just nterior to the termin tion of the cingul
ted sulcus. On l ter l s gitt l cuts it is
isected
y perpendicul r line em
n ting from the posterior corner of the insul r tri ngle. Myelogr phy Only intr
thec l contr sts gents: iohexol (Omnip que), metriz mide (suppl nted
y Iohexol
, not usu lly v il
le), or P ntop que (non-w ter solu
le). Others m y c use se
izures, etc. Lum
r puncture performed, dye injected. For cervic l myelogr m he
d of t
le lowered. Pl in films usu lly com
ined with CT (CT-myelogr m) Spin l

lock p tterns: Fe thering = extr dur l; meniscus = intr dur l, extr medull ry MR S
pectroscopy Me sures met
olites in 1cm2 voxel o Choline: indic tive of cell m
em
r ne turnover (eg tumors) o L ct te: indic tive of necrosis o NAA: N- cetyl
sp rt te, found in neurons (norm l
r in) Tumor: Choline (& choline:cre tinine r
tio), NAA, l ct te. o Cho:Cr r tio c n predict surviv l & guide
iopsy in gliom
s. MR tr ctogr phy (Diffusion Tensor Im ging) M ps su
cortic l fi
er tr cts (ie
corticospin l p thw ys) using diffusion tensor im ging. Limited in re s of tum
or or edem .
Flotte Outline of Neurosurgery Function l im ging
71
Function l MRI (fMRI) Detects ch nges in deoxyhemoglo
in. Useful for motor m ppi
ng, not sensitive enough for speech. Motor p r digms: 1) thum
-index opposition,
2) toe flexion, 3) tongue movement. Speech: 1) visu lizing presented ver
s, 2)
decipher complex noun. Positron Emission Tomogr phy (PET) Me sures met
olism. R
dioisotopes (emit positrons, eg 18F) conjug ted to met
olic lly ctive su
st n
ce (eg glucose). o Requires cyclotron (for r dioisotopes). Positron = s me m ss
of electron
ut + ch rge. R diotr cers used: o 18F flouro-deoxyglucose (FDG): me
sures glucose met
olism. o 11C-methionine (Met): Amino cid, me sures protein
synthesis. Hot in low-gr de tumors (unlike FDG). M y
e
etter for stereot ctic
t rgeting (see JN9/04) Resolution 8mm. Findings: o Hot: GBM/ high gr de tumor, i
ct l seizure foci o Cold: Low gr de tumor, r di tion necrosis, cortic l dyspl si
, interict l seizure foci, mesi l tempor l sclerosis. Single Proton Emission To
mogr phy (SPECT) Me sures
lood flow. 99Tc (technetium, HMPAO) or 133Xe used. Re
solution 10mm. Findings simil r to PET.
T lr ich or Sch lten
r nd tl ses used AC-PC line: m y
e used to sc le other me
surements Stereot ctic Biopsy 4% mor
idity, 1% mort lity
Stereot xy

Stereot ctic R diosurgery

Given s single dose, s opposed to convention l r diother py o St nd rd XRT k
ills r pidly dividing cells, sp res norm l tissue nd hpoxic tissue resist nt. S
RS kills tissue reg rdless of mitotic ctivity, oxygen tion, or inherent r diose
nsitivity. o Stereot ctic R diother py: Fr ction ted r diosurgery. Optic n. c n
toler te 50Gy. o St ged-r diosurgery: tre ting portions of l rge lesions 3-12 mos
p rt Systems: o LINAC: Line r cceler tor cceler tes electrons into he vy met
l (tungsten) which emits photons. 1-5 isocenters used. o Proton Be m: Requires c
yclotron, Uses Br gg-pe k effect. Currently only t Lom Lind nd MGH. o Cy
erK
nife: Fr meless, multiple-fr ction tion possi
le, c n tre t extr cr ni l lesions
, re l-time im ging. o G mm Knife: 5-15 isocenters usu lly used. 201 Co
lt60 s
ources, emit photons. 4, 8, 14, 18mm collim tors. Im ging: MR: spoiled-GRASS seq
uence, 1-2mm slices, 512x256 m trix, 2 excit tions. F t suppression for previous
TSRP with f t gr ft. MR ppe rs to
e s ccur te s CT (shift is minim l). Use
short posts to void rtif ct. o MRSpect d t c n
e used for pl nning Dose tol
er tion o M x. dose to optic n.: Tishler 93: 8Gy. Le
er 98: 10Gy h d 0% optic ne
urop thy (h d
etter dosing). St fford 01: up to 12-16 Gy if < 9-12mm exposed (1
% if <12Gy). 2mm m rgin from optic p thw y is de
te
le. o Br instem 15Gy m x.
Side-effects o R di tion- ssoci ted neopl sms: 6 reported c ses of, 6-19yr l ten
cy. Estim ted risk 1:1000. o Deficits m y ppe r over 2yrs postop. Complic tions
or tumor progression r rely occur fter 3yrs with
enign lesions. (N03) o Tumor
s m y enl rge
efore contr cting. De
te
le whether SRS m kes su
sequent surger
y more difficult. o C vernous sinus 2% risk of CN4 or CN6 injury. (37% of pre-tr
e tment cr ni l nerve deficits improve). Specific Conditions o C vernous sinus t
umors: 98% loc l control. 37% improved cr ni l nerve deficits, 2% new deficits.

Aw ke cr niotomy
Anesthesi
Flotte Outline of Neurosurgery
o
72

Some do not intu
te, use IV propofol during opening nd closing, with versed or
fent nyl while w ke. In this c se do not open dur until the p tient is fully
w ke, to prevent swelling o Loc l nesthesi (lidoc ine+m rc ine) is used on in
cision. Addition l loc l nesthetic is infused round the supr or
it l rim, the
zygom , the posterior uricul r region, nd the tempor lis insertion to produce
field
lock. Pin sites re injected if he dholder is used. The dur is lso
injected. o Asleep- w ke- sleep technique: Intu
tion/Gener l nesthesi > openi
ng with hyperventil tion > extu
tion > m pping > intu
tion (fi
eroptic l ryngo
scope or tu
e ch nger) > gener l nesthesi o Inh l tion l (isoflu ne, nitrous o
xide) nd remifent nyl fter verifying muscle rel x nts re worn off
y tr in of
four. o Berger feels propofol m y dversely ffect cortic l depol riz tion. Som
e use he d-pins, others use doughnut.
Slow-growing lesions m y shift eloquent re s, even contr l ter l Stopping resec
tion 1-2cm from eloquent cortex gre tly reduces postop deficits Cortic l stimul

tion Done with Ojem nn
ipol r stimul tor (60Hz, 1msec, single-ph se, 2-6mA w k
e, 4-16mA gener l nesthesi ). Incre se current
y 2mA until response is o
t ine
d. Hold tips on cortex for 2-3 secs. P tient temper ture >36. EMG recording m y i
mprove sensitivity. M y
e done sleep or w ke. Alw ys h ve cold irrig tion re
dy to irrig te cortex in the event of stimul tion-induced seizure Some v ry cu
rrent t different sites (while monitoring fterdisch rges), others dont (JN9/04)
Afterdisch rges monitored
y electrocorticogr phy i.e. 5-grid strip l id next t
o stimul ted re . This prevents seizures nd ensures th t stimul tion effects
re loc l only (ie specific) Seizures stopped
y cold irrig tion of cortex,
ezod
i z pines, nd Dil ntin Neg tive results m y not ensure s fe resection, positi
ve result (ie speech rrest) is necess ry to
e sure of the loc tion of essenti
l l ngu ge sites M y
e done t
edside with impl nted electrode grids. L ngu ge
M pping Hemispheric domin nce: Left hemisphere for 99% of right-h nders. Over l
l: 85% left, 9%
il ter l, 6% right. In left-h nders use WADA test to determine
l ngu ge domin nce. O
t in
seline l ngu ge function. Test the p tient preoper
tively nd elimin te o
jects the p tient c nt identify. N ming errors must
e les
s th n 25%. If p tient is un
le to p rticip te preoper tively tri l of higher
-dose steroids c n
e ttempted to see if enough improvement occurs th n l ngu g
e testing c n
e used. Aw ke cr niotomy with cortic l stimul tion O
ject n ming
is the most reli
le test. Re ding m y lso
e tested (posterior tempor l) E ch
site is tested 3 times, never twice in succession Essenti l l ngu ge sites exist
prim rily on the surf ce of the gyri nd not in the depths. Prim ry l ngu ge si
tes re vertic lly org nized with respect to su
cortic l fi
ers, nd surf ce sti
mul tion c n
e used to predict the results su
cortic l resection. But su
cortic
l m pping c n
e used to identify l ngu ge fi
ers ne r the insul Motor M pping
Resection m y
e continued into the nterior
nk of the precentr l gyrus witho
ut c using n dverse event Resection m y
e performed in
oth the domin nt nd
nondomin nt supplement ry motor re s without c using perm nent sequel e, s lon
g s the prim ry motor cortex is not viol ted Prim ry som tosensory cortex resec
tion will produce tempor ry hypesthesi nd proprioceptive deficit. When they
ffect the domin nt h nd, such deficits re pro
lem tic to the p tient nd the p
tient should
e counseled preoper tively reg rding this Cortic l stimul tion: M
otor re identified in 94%. o M y
e done under gener l nesthesi or w ke. Mo
tor stimul tion is elicited in 50% under gener l nesthesi nd 100% of consciou
s sed tion c ses in sm ll series, with electrogr phic seizures in 30% nd 10%
respectively (NF7/03) Gener l nesthesi : inh l tion gents com
ined with Versed
nd fent nyl without p r lytics. o Children under ge 5 often c nnot
e m pped
with stimul tion
ec use of cortic l inexcit
ility use SSEPs inste d o After re
section, motor p thw ys should
e stimul ted g in to ensure their function. If
motor p thw ys respond to stimul tion fter resection, ny motor deficit o
serve
d fter the oper tion will
e tempor ry Su
cortic l stimul tion: Identifies su
c
ortic l motor tr cts in 50%. If motor cortex is identified then tr cts re m ppe
d from there. If not, then white m tter is stimul ted t 10-16mA. Stimul tion of
corpus c llosum does not elicit clinic l response. 8% of p tients h ve su
corti
c l p thw ys within gross tumor Revers l of SSEP w ve
Cortic l M pping
Evoked Potenti ls
Flotte Outline of Neurosurgery

73

Avoid inh l tion gents, BZDs,
r
s; use nitrous or n rcotics, short- cting mus
cle rel x nts (not for MEPs) Amplitude 50% or l tency 10% is signific nt Motor E
voked Potenti ls (MEPs) o Used for intr medull ry spin l cord tumors, spin l AVM

s, m pping motor cortex, neurysm clipping. o Stiuml tion m y
e done tr nscr ni

lly or
y cortic l grids. o Twitch rtif ct of erector spin e muscles m y limit
. Leg MEPs m y require too strong stimul tion. (Supr tentori l MEPs N5/04) o MEP
s superior to SSEPs in detecing motor imp irment during neurysm clipping. Som t
osensory Evoked Potenti ls (SSEPs) o Stimul te medi n n. (unless oper ting on AC
A use posterior ti
i l n.) o Squ re w ve, 300sec, 25-30mA m x, 5.3Hz. Br instem
uditory evoked responses (BAERS): o I cochle r n. > II cochle r nuc > III Superior
olive > IV l ter l lemniscus > V Inf. Colliculus > VI MGB > VII Cortex o Intr oper t
ive: microphone pl ced in e r, electrodes on sc lp
Surgic l An tomy
Extern l L ndm rks Rol ndic sulcus: 30 line from or
it l rim midpoint
etween EAC
nd s gitt l suture For men of Monroe Coron l suture Cere
r l cortex Supplement
ry Motor Are Syndrome: Contr l ter l kinesi with mutism (when on domin nt si
de very r rely on nondomin nt side) fter d m ge to SMA. SMA: re 8 - mesi l po
sterior front l lo
e, role in init ting speech nd motor function. Recovery of s
peech occurs in 4-12 d ys nd motor
y 2-6mos. M y not
e evident during w ke c
r ni (m y ppe r postop). Stimul tion reuires high volt ge to elicit responses (
speech nd motor rrest, complex postur l movements, sensory & utonomic phenome
non.) Corpus C llosum: o Anterior: Slow ide tion o Middle: SMA syndrome o Poster
ior: Sensorimotor deficits. o Hemispheric Disconnection Syndrome Middle foss tr
i ngles: Gl sscocks: For men spinosum to rcu te eminence to GSPN. Exposes petrou
s ICA. Posterior Foss Rhotons rule of 3: 1) Meckles c ve, CN5, SCA, tentori l sur
f ce. 2) IAC, CN7/8, AICA, petros l surf ce. 3) Jugul r for men, CN9/10/11, PICA
, su
occipt l surf ce. L ndm rks: Tr nsverse-Sigmoid junction t sterion. M sto
id emiss ry vein/for men t posterior edge of sigmoid sinus. Posterior end of in
cisur m stoide is t level of IAC. Veins L

e: enters t tr nsverse-sigmoid j
unction.
Procedures
Gener l
Dur l gr fts: pericr nium, f sci l t , c d veric f sci ,
ovine peric rdium (Du
r -Gu rd) L ser: NdYAG (not CO2) in
loody tumors; focused
e m for gross remov
l, defocused for m rgins
Cr niopl sty M teri ls: o Autologous
one (cr nium, ri
, ilium): lower infection
risk th n rtifici l m teri l, m y resor
o Methylmeth cryl te
one cement: hightensile strength, infection 23%, fr ctures occur o Hydroxy p tite or c lcium pho
sph te (Bonesource):
iocomp ti
le nd osteoconductive, in dequ te setting, sett
ling,
rittle, less s tisf ctory for l rge defects o Prem de llopl stic impl nt
s (polyethylene - Porex) o Tit nium mesh Bone fl p impl nted in p tients
domen
or stored in freezer. Autocl ving not recommended due to
one protein den tur ti
on. 50%
one resporption in children when repl ced; no correl tion with time to
reimpl nt tion (JN:P2/04)
Loc tions
Flotte Outline of Neurosurgery
74
3rd Ventricle No hydoceph lus tr nsc llos l Inferior pterion l, su
front l, su
t
empor l, tr nsphenoid l, COZ B sil r rtery ccess: Or
itozygom tic: Top 2/5, SC
A, CN3/4, mid
r in Tr nscochle r: Middle 1/5, AICA, pons, CN5-8 F r l ter l: Low
er 2/5, PICA, CN9-12 (Henn, Spetzler, Clin NSurg 49) Perimesenceph lic Cisterns
Tr nssylvi n pretempor l, su
tempor l, occipit l tr nstentori l, supr cere
ell r
infr tentori l, tr nstempor l tr nschoroid l. See N6/04 C vernous Sinus Some re
commend resecting tumors (meningiom s) from l ter l comp rtment only, tre ting t
he residu l with SRS. If tumor touches
ut doesnt enc se ICA (C4) it c n
e tot l
ly resected. Excision of medi l c vernous sinus lesion c rries very high r te

of cr ni l nerve mor
idity. (N6/04)

Positions
Sitting o Risks: Air em
olism, tension pneumoceph lus, remote ICH, spin l cord i
njury/inf ction. o Avoid with p tent for men ov le, c rdiov scul r dise se, seve
re hypertension, cervic l stenosis. Consider preop cspine MRI to rule out cervic
l stenosis (c n l should
e >12mm)
Anterior Appro ches
Tr nsor l Clivus, cr niocervic l junction (Dorsum sell e to C2-3). Extr dur l le
sions prim rily (Intr dur l used for
sil r neurysms.) Supine, he d extended.
Retr ctor h s groove for ET tu
e. Posterior ph rynge l w ll incised,  soft p l te
(l ter l to uvul ),  h rd p l te. Use Dingm nn mouth g g. (N1/04) Tr nsphenoid l
P tient supine, on doughnut, forehe d p r llel to floor. Prep
domen for f t g
r ft. Use intr n s l coc ine for hemost sis. Endon s l: Middle conch followed t
o sphenoid osti , n s l mucos reflected medi l/inferiorly, sphenoid sinus opene
d thru osti , sphenoid mucos removed, posterior sphenoid/ nterior sell w ll op
ened (with chisel if necess ry). Dur opened in cruci te f shion. Sell explored
with ringed curettes. Altern tive: Su
l
i l, tr nsn s l (su
mucos l) CSF le k:
Consider f t p cking, fi
rin glue, etc. Consider pl cing lum
r dr in in the O.
R. C rotid te r: Hemorrh ge controlled wth h rvested f sci . Immedi te postop n
gio to rule out pseudo- neurysm (repe t fter 7-10 d ys if neg tive). Endoscopic
ppro ch dvoc ted
y P. C pp
i nc Extended tr nsphenoid l: o Presell r: pl n
um sphenoid le removed o Tr nssell r-Tr nsdi phr gm tic: Gl nd incised, di phr g
m opened to resect supr sell r m ss. o Intr -c vernous sinus: consider preop
l
lon occlusion test, c rotid exposure for proxim l control. o Sell r-Cliv l o See
N9/04 Bifront l Bicoron l incision. Pericr nium s ved for dur l gr ft. Burrhole
s t keyholes, str ddling sinus posteriorly ( nteriorly). Tr ns
s l Extr dur l
ccess only. Bifront l cr niotomy. Olf ctory nn. divided, front l sinuses cr ni
lized, ethmoid sinuses removed, sphenoid sinus unroofed, optic n. unroofed, cliv
us drilled (sell to for men m gnum). Extended tr ns
s l:
nde u included. Tr ns
m xill ry
Flotte Outline of Neurosurgery
75

Neurosurgery h s continued to
ene t consider
ly from wide r nge of technologic

l dv nces th t h ve en
led
etter im ging of centr l nervous system dise se,
underst nding of dise se processes nd the consequent development of r tion l tr
e tments. M gnetic reson nce im ging h s now
ecome the st nd rd r diologic l te
chnique to investig te centr l nervous system dise se, nd this h s further demy
sti ed the di gnostic process in neurosurgery. However, it h s ent iled new le r
ning process not only for students,
ut lso for pr ctising clinici ns. M gnetic
reson nce spectroscopy h s
ecome routine di gnostic tool s h s m gnetic res
on nce ngiogr phy. Improved underst nding of the
iology of the centr l nervous
system nd tumour
iology, h s led to the introduction of more r tion l tre tme
nt regimes, with improved outcomes. Molecul r
iology techniques, the introducti
on of
iologic l ther pies including gene ther pies, nd the development of endo
v scul r surgery h ve consider
ly
ro dened the horizon for the m n gement of
wide r nge of neurologic l dise ses. Technologic l dv nces in the oper ting th
e tre h ve incre sed the surgic l possi
ilities, p rticul rly com
ining stereot
ctic techniques with microneurosurgery. Our p tients h ve
ene ted consider
ly fr
om these dv nces, nd over the next two dec des
iologic l nd technic l dv nc
es will continue to provide consider
le
ene t for even more of our p tients. Thi
s third edition of Essenti l Neurosurgery h s essenti lly
een
sed on the rst
nd second

editions,
ut h s incorpor ted m ny of the dv nces descri
ed. Modern neurosurgi

c l pr ctices still differ consider
ly in North Americ nd Europe, nd despite
the glo
l vill ge there continues to
e su
st ntive differences in the philosoph
ic l ppro ch to the m n gement of clinic l pro
lems. The uthor h s descri
ed h
is own pr ctice, which hopefully continues to utilize the
est of
oth systems,
s well s incorpor ting the unique dv nces nd philosophies of the Asi P ci c rim
region. It is not possi
le to list nd cknowledge ll the m ny people who h ve
helped in the prep r tion of this third edition. However, I p rticul rly cknow
ledge my neurologic l nd neurosurgic l colle gues t The Roy l Mel
ourne Hospit
l. Stephen D vis nd Christine Kilp trick h ve g in provided ch pters on their
own re s of expertise. I m very gr teful to Nichol s M rtens for his conside
r
le help with ch pters on He d Injury, Br in Tumours nd Pituit ry Tumours, Jo
hn L idl w for his ssist nce with ch pter on Su
r chnoid H emorrh ge nd Bh
du K v r for his input into the rewriting of the Spin l Injuries ch pter. I woul
d like to especi lly th nk K te L gerewskij for the m ny hours she spent prep ri
ng the m nuscript nd to Helen H rvey t Bl ckwell Pu
lishing for m king this ed
ition possi
le. As lw ys I m especi lly gr teful to the encour gement nd p ti
ence of my wife Judy nd son Ben. Andrew H. K ye, Mel
ourne, 2004
vii
Pref ce to the

rst edition

Clinic l neurosurgery requires n underst nding of the rt of neurology nd of t

he principles of the neurosciences, p rticul rly neurop thology nd neurophysiol
ogy. In the p st the mystique of neurosurgery h s in dvertently prevented
oth m
edic l tr inees nd physici ns from proper ppreci tion of even
sic neurosur
gery nd consequently h s cre ted r ther nihilistic view of neurosurgic l illn
esses. The improvements in medic l technology h ve m rkedly improved the ccur c
y of the di gnosis, the ef c cy of neurosurgic l tre tment nd the r nge of dise s
es th t c n
e di gnosed nd tre ted. In p rticul r, the exciting dv nces in ne
uror diology h ve simpli ed the di gnostic process nd m de neurosurgery more cce
ssi
le. This
ook is intended s n introduction to neurosurgery. It is hoped th
t it will
e useful for physici ns in tr ining, neurosurgic l tr inees nd medi
c l students. The
ook is not intended to
e n exh ustive cover ge of neurosurg
ery
ut r ther concentr tes on the more common neurosurgic l pro
lems nd only

rie y mentions r re entities. The neurologic l principles, p thologic l
sis nd
relev nt investig tions th t form the
sis of the di gnosis re emph sised. The
neurosurgic l m n gement is outlined
ut the surgic l techniques re only
rie y
mentioned, so th t the re der will underst nd the postoper tive pro
lems likely
to
e encountered in the m n gement of the p tient. Modern neurosurgery h s evol
ved princip lly from North Americ n nd Europe n pr ctices nd there re often s
igni c nt differences in the philosophic l ppro ch in the m n gement of clinic l
pro
lems. The uthor h s in
gener l descri
ed his own pr ctice, which hopefully utilises the
est of
oth sy
stems. The references h ve
een chosen for their gener l cover ge of the topics,
e se of ccess, historic l interest nd, in some c ses,
ec use they will provi
de thought provoking ltern tives th t give different perspective to the su
je
ct. It is not possi
le to list nd cknowledge ll the m ny people who h ve help
ed in the prep r tion of this
ook,
oth knowingly nd s result of their in uen
ce on my own neurosurgic l pr ctices. However, the l te John Bry nt Curtis w s t
he m jor initi l in uence not only on my own neurosurgic l educ tion
ut on th t o
f m ny other Austr li n neurosurgeons. I p rticul rly cknowledge the help of my
neurologic l nd neurosurgic l colle gues t the Roy l Mel
ourne Hospit l in th
e prep r tion of this
ook. Stephen D vis nd Christine Kilp trick h ve provided
ch pters on their own re s of expertise. Professor Bri n Tress, Director of R
diology t the Roy l Mel
ourne Hospit l, h s lw ys
een ccessi
le nd helpful
nd I m inde
ted to him for his expert te ching over m ny ye rs nd for ssist

nce with the det ils on m gnetic reson nce im ging. His dep rtment supplied most
of the Xr ys. Dr Meredith Weinstein, neuror diologist t the Clevel nd Clinic,
kindly provided m gnetic reson nce sc ns (Figs 7.9, 12.7, 13.5). Professor Colin
M sters, Dep rtment of P thology, University of Mel
ourne nd Dr Mich el Gonz l
es, neurop thologist t the Roy l Mel
ourne Hospit l, g ve ssist nce with the p
thology det ils nd illustr tions. My residents nd registr rs t the Roy l Mel

ourne Hospit l h ve lw ys provided stimul ting dvice nd criticisms. I p rix
x
PREFACE TO THE FIRST EDITION
ticul rly cknowledge the ssist nce of Drs John L idl w nd Mich el Murphy, reg
istr rs in neurosurgery, who proof re d the m nuscript nd offered constructive
criticism. I th nk Sue D mmery for the m ny hours spent prep ring the m nuscript
nd Rich rd M honey for the illustr tions.
The
ook would not h ve
een possi
le without the guid nce nd stimulus from Pet
er Rich rdson t Churchill Livingstone. I m especi lly gr teful to the encour g
ement nd p tience of my wife Judy nd son Ben. Andrew H. K ye, Mel
ourne, 1990
CHAPTER 1
1
Neurologic l ssessment nd ex min tion
An ccur te neurologic l ssessment is fund ment l for the correct m n gement of
the p tient. The
sic im of the neurologic l ex min tion is to solve the foll
owing four questions: 1 Is there neurologic l pro
lem? 2 Wh t is the site of t
he lesion (or lesions) in the nervous system? 3 Wh t re the p thologic l condit
ions th t c n c use the lesions? 4 H ving scert ined the neuro n tomic l site
nd the p thologic l c use from the history, wh t is the most likely di gnosis? A
nswering these four questions in turn will indic te the type of investig tion ne
cess ry to con rm the di gnosis. The neurologic l ssessment involves: the history
of the illness clinic l ex min tion: ( ) of the nervous system (
) gener l ex m
in tion.
The neurologic l history
As in gener l medicine nd surgery the neurologic l history is the key to the di
gnosis. The history involves not only questioning the p tient
ut lso c reful
o
serv tion. M ny neurologic l illnesses c n
e di gnosed just
y o
serving the
p tient. The p tients gener l m nner, mood, posture, g it, f ci l expression nd
speech re ll vit l clues to the n l di gnosis. In ddition, p tients who do not
h ve n org nic dise se m y present in ch r cteristic m nner, p rticul rly wi
th n ex gger tion of the compl int. The history nd ex min tion commences with
o
serv tion, nd this should
egin when rst
meeting the p tient nd while t king the history. The w y in which the p tient w
lks into the ex min tion room, sits on the ch ir, nswers questions nd clim
s
on to the ex min tion couch will provide vit l clues in the se rch for the di gn
osis. Initi lly it is import nt to llow the p tient dequ te opportunity to exp
l in their symptoms in n unstructured nd unprompted m nner. Direct questioning
should then follow. The questions concerning neurologic l symptoms re in essen
ce ver
l ex min tion of the neurologic l system. It is not just the content o
f the nswer th t is import nt
ut the w y in which the p tient responds to the
questions. The following is gener l cl ssi c tion of neurologic l symptoms. 1 Ge
ner l neurologic l symptoms: ( ) he d che (
) drowsiness (decre sed conscious st
te) (c) vertigo (d) seizures,
l ckouts. 2 Symptoms of meningismus: ( ) he d ch
e (
) photopho
i (c) neck stiffness (d) vomiting. 3 Symptoms rel ted to the spe

ci l senses: ( ) vision (
) he ring (c) t ste (d) smell. 4 Symptoms rel ted to s
peech nd comprehension. 5 Motor symptoms: ( ) power (
) coordin tion. 1
2
CHAPTER 1
6 Sensory symptoms. 7 Cognitive symptoms, e.g. memory. 8 Symptoms of other syste
ms which m y rel te to dise ses of the nervous system. C reful questioning will
scert in the import nt det ils concerning e ch symptom. These include: The time
, mode of onset, progression nd dur tion of the symptom. The mode of onset is
v lu
le clue in discerning the p thologic l process. Sudden onset of neurolo
gic l distur
nce is usu lly due to v scul r or epileptiform c use; sudden s
evere he d che is ch r cteristic of su
r chnoid h emorrh ge where s slowly pr
ogressive he d che is more in keeping with cere
r l tumour. Simil rly, the
r
upt onset of hemiplegi m y result from v scul r c t strophe nd slowly prog
ressive we kness m y
e due to compressive or in ltr tive c use. Wh t f ctors re
sult in llevi tion or ex cer
tion of the symptom? He d che from r ised intr cr
ni l pressure is ch r cteristic lly worse in the morning nd on coughing nd st
r ining. P tients nd the h nd p in ssoci ted with c rp l tunnel syndrome is ofte
n worse t night nd is llevi ted
y sh king the h nd over the side of the
ed.
Is there p st history of ny simil r event? It is often helpful to o
t in det
ils of the history from the p tients rel tives or witness; it is vit l to do t
his if the p tient is child or if there is imp irment of conscious st te or me
mory distur
nce. Det ils of the n ture of epileptic seizures should lw ys
e o

t ined from rel tive or friend who h s witnessed n event. A thorough underst
nding of the n ture of the illness nd symptom tology should h ve
een o
t ined

efore the ex min tion is commenced.
( ) posture (
) w sting (c) tone (d) power (e) re exes (f) sens tion (g) coordin t
ion nd g it.
Ment l st te
Ex min tion of the ment l st te involves n ssessment of: conscious st te orien
t tion in time, pl ce nd person memory emotion l st te presence of delusions or
h llucin tions. A correct ssessment of the ment l st te is essenti l prior to
the ev lu tion of the other neurologic l signs. The rem inder of the neurologic
l ex min tion will
e undert ken within the context of the p tients ment l st te.
The ccur te ssessment of conscious st te is especi lly import nt in neurosurg
ic l disorders nd the ev lu tion of the level of consciousness using the Gl sgo
w com sc le is descri
ed in the ch pter on he d injuries (Ch pter 4). Imprecise
terms such s stuporose should
e voided nd the ex miner should o
jectively ss
ess nd descri
e the p tients response to speci c stimuli. Drowsiness depressed con
scious st te is the most import nt neurologic l sign nd indic tes m jor intr cr
ni l p thology. As with ll neurologic l symptoms nd signs it is essenti l to
o
t in n ssessment of the progression of the drowsiness
y questioning the p t
ients friends or rel tives. A deterior ting conscious st te is neurosurgic l em
ergency. Memory distur
nces should
e tested form lly for
oth short-term nd l
ong-term preserv tion. Short-term memory should
e tested
y listing n me, dd
ress nd type of ower nd sking the p tient to rec ll it fter 5 minutes. Loss o
f short-term memory with rel tive preserv tion of memory for long-p st events is
typic l of dementi , e.g. Alzheimers dise se. In Kors koffs psychosis the distur

nce of recent memory nd disorient tion m y
e so severe th t the p tient
Neurologic l ex min tion
The form l neurologic l ex min tion should
e undert ken in systemic f shion i
n the following order. 1 Ment l st te. 2 Speech. 3 Cr ni l nerves. 4 Ex min tion
of lim
s nd trunk:
NEUROLOGICAL ASSESSMENT AND EXAMINATION

3
will m ke up stories to provide convincing nswer to the questions. This is co
nf
ul tion nd is cl ssic lly ssoci ted with lcoholism, lthough it m y r rel
y
e seen s result of nterior hypoth l mic lesions due to tr um or followin
g su
r chnoid h emorrh ge nd v sosp sm.
Dys rthri m y result from lesions of the lower motor neurones nd the muscles,
such s occur in p l t l p lsies or p r lysis of the tongue. Rigid dys rthri . Thi
s is ch r cteristic of P rkinsons dise se. In severe c ses the phenomenon of p li
l li is seen, in which there is const nt repetition of p rticul r syll
le.
Speech disorders
There re four m in speech disorders: 1 Mutism. 2 Aphoni . 3 Dys rthri . 4 Dysph
si .
Mutism Mutism is ch r cterized
y the p tient
eing lert
ut m king no ttempt
to spe k. It m y result from lesions ffecting the medi l spect of
oth front l
lo
es, cl ssic lly occurring s result of v sosp sm following su
r chnoid h
emorrh ge from ruptured nterior communic ting rtery neurysm. Aphoni Aphoni
is s id to occur when the p tient is
le to spe k
ut is un
le to produce n
y volume of sound. It is due to distur
nce of the voc l cords or l rynx. If t
he p tient is
le to cough norm lly then it is usu lly hysteric l. Dys rthri D
ys rthri is due to imp ired coordin tion of the lips, p l te, tongue nd l rynx
nd m y result from extr pyr mid l,
r instem or cere
ell r lesions. The volume
nd content of the speech will
e norm l
ut the enunci tion will
e distorted.
Sp stic dys rthri . This is due to
il ter l upper motor neurone dise se due to
pseudo
ul
r p lsy, motor neurone dise se or
r instem tumours. At xic dys rthr
i . This is due to incoordin tion of the muscles of speech; the words re often
st cc to or sc nning nd the rhythm is jerky. This type of dys rthri is seen in
cere
ellopontine ngle tumours, cere
ell r lesions, multiple sclerosis nd phen
ytoin toxicity.
Dysph si Dysph si m y
e either expressive or receptive. P tients with express
ive dysph si c n underst nd speech
ut c nnot formul te their own speech. P tie
nts with receptive dysph si c nnot underst nd spoken or written speech. Althoug
h one type of dysph si m y predomin te there is frequently mixture of the two
p tterns of dis
ility. Dysph si results from lesions of the domin nt hemisphe
re, which is the left hemisphere in right-h nded people s well s in high pro
portion of left-h nded people. Expressive dysph si . This is due to lesion ff
ecting either Broc s re in the lower p rt of the precentr l gyrus (Fig. 1.1) or
the left posterior temporop riet l region. If the l tter region is ffected the
p tient m y h ve nomin l dysph si , in which the
ility to n me o
jects is l
ost
ut the
ility to spe k is ret ined. Receptive dysph si . This results from
lesions in Wernickes re , which is the posterior p rt of the superior tempor l
gyrus nd the dj cent p riet l lo
e. Alexi Alexi is the in
ility to underst
nd written speech. Alexi with gr phi (in
ility to write) is due to lesion
in the left ngul r gyrus. The p tient is un
le to re d or write spont neously
nd the condition is often ccomp nied
y nomin l dysph si , c lculi , hemi nop
i nd visu l gnosi . Gerstm nns syndrome consists of nger gnosi for
oth the p
tients own nger nd the ex miners nger, c lculi , right/left disorient tion nd g
r phi without lexi . It is found in lesions of the domin nt hemisphere in the
region of the ngul r gyrus.
4
CHAPTER 1
Motor ctivity Cortex Trunk Hip Shoulder El
ow Knee Wrist Fingers Leg Thum
Neck

Ankle Brow Eyelid Nose Lips Tongue L rynx Toes

Sensory ctivity
Hip Trunk Shoulder El
ow Knee Wrist H nd Fingers Leg Thum
Ankle Neck Brow Toes
Eyelid Nose Lips J w
Prim ry motor re Supplement ry motor re Precentr l gyrus Centr l sulcus Prim
ry som tosensory re Second ry visu l re Broc s motor speech re Prim ry v
isu l re
Fig. 1.1 M jor re s of som totopic org niz tion of the cere
rum.
Ex min tion of the cr ni l nerves
Olf ctory nerve The sense of smell should
e tested
y the p tient snif ng through
e ch nostril s the other is compressed. The common c uses of nosmi re olf c
tory nerve lesions resulting from he d injury, nd tumours involving the oor of t
he nterior cr ni l foss , especi lly olf ctory groove meningiom s. It is import
nt to use non-irrit nt su
st nces when testing olf ction, s irrit ting compoun
ds (e.g. mmoni ) will c use irrit tion of the n s l mucos . The stimulus is the
n perceived
y the gener l sensory
res of the trigemin l nerve. Optic nerve The
optic nerve should
e tested
y: me suring the visu l cuity nd colour vision c
h rting the visu l elds fund l ex min tion with n ophth lmoscope the pupill ry l
ight re ex.
Visu l cuity The visu l cuity should
e tested using the st nd rd Snellen type
ch rts pl ced t 6 m. The cuity is recorded s fr ction, e.g. 6/6 or 6/12, i
n which the numer tor indic tes the dist nce in metres from the ch rt nd the de
nomin tor the line on the ch rt th t c n
e re d. 6/6 is norm l vision. Refr cti
ve errors should
e corrected
y testing with the p tients gl sses or
y sking t
he p tient to view the ch rt through pinhole. Visu l elds The visu l elds c n
e
ch rted
y confront tion, with the p tient f cing the ex miner nd o
jects of v
rying size
eing moved slowly into the visu l eld (Fig. 1.2). Form l testing usi
ng perimetry should
e undert ken in ll c ses of visu l f ilure, pituit ry tumo
ur, p r sell r tumour, other tumours possi
ly involving the visu l p thw ys nd
demyelin ting dise se, or if there re ny dou
ts fter confront tion th t the el
ds m y
e restricted. Perimetry c n
e performed using either t ngent screen,
such s Bjerrum screen (Fig. 1.3), or
NEUROLOGICAL ASSESSMENT AND EXAMINATION
5
Ex miner
tot l visu l loss optic nerve lesion ltitudinous hemi nopi p rti l lesion of t
he optic nerve due to tr um or v scul r ccident homonymous hemi nopi lesions
of the optic tr ct, r di tion or c lc rine cortex
itempor l hemi nopi optic ch
i sm lesions such s pituit ry tumour, cr nioph ryngiom or supr sell r meningio
m .
Test o
ject
P tient
Fig. 1.2 Visu l eld testing
y confront tion.
BJERRUM SCREEN 30 20 10
Fix tion point

Fund l ex min tion The fundus should
e ex mined using the ophth lmoscope with p
rticul r ttention to the: optic disc vessels retin . A p le optic disc is due
to optic trophy which m y
e either prim ry, s result of n optic nerve lesi
on c used
y compression or demyelin tion, or consecutive, which follows severe
swelling of the disc. P pilloedem is due to r ised intr cr ni l pressure nd is
evident
y:
lurring of the disc m rgins lling in of the optic cup swelling nd
engorgement of retin l veins, with loss of norm l puls tion of the veins h emorr
h ges round the disc m rgin (if severe).
Record t rget colour nd di meter/dist nce of eye from fix tion point, e.g. 10/2
000
Fig. 1.3 The Bjerrum screen.

Goldm nn perimeter. The Bjerrum screen records the centr l eld of vision. By en
l rging the centr l re out to 30 it is e sier to detect scotom s nd to me sure
the
lind spot nd, provided sm ll enough t rget is used, the t ngent screen
provides n ccur te represent tion of the peripher l elds. An utom ted perimetr
y m chine will en
le n ccur te nd reproduci
le eld test th t is p rticul rly
useful in cooper tive p tients. The p ttern of visu l eld loss will depend on the
n tomic l site of the lesion in the visu l p thw ys (Fig. 1.4):
Third, fourth nd sixth cr ni l nerves As these cr ni l nerves re ll involved
in innerv tion of the extr ocul r muscles they re usu lly ex mined together. Th
is ex min tion involves ssessment of: the position of the eyelids the pupils ex
tr ocul r movements.
Position of the eyelids Ptosis is due to p r lysis of the lev tor p lpe
r e supe
rioris s result of 3rd cr ni l nerve lesion or due to we kness of the t rs
l muscle due to symp thetic lesion (Horners syndrome). The pupils An ssessment
should
e m de of the pupil size, sh pe nd equ lity. The pupils re ction to lig
ht should
e tested
y shining
e m into the eye nd noting the re ction in th
t eye, s well s the
6
CHAPTER 1
Tempor l field
N s l field
Left eye
Right eye
Left
Right A
B A C
Optic nerve Optic chi sm Optic tr ct B
C
D
L ter l genicul te
ody D Geniculoc lc rine tr ct
Occipit l cortex

Fig. 1.4 Di gr mm tic represent tion of visu l p thw ys, the common sites of les
ions nd the resulting eld defects.
consensu l response in the opposite eye. The re ction to convergence nd ccommo
d tion for ne r vision should
e tested
y sking the p tient to x on dist nt o

ject nd then pl cing pen pproxim tely 12 cm in front of the
ridge of the n
ose. A unil ter l constricted pupil (miosis) often indic tes lesion in the sym
p thetic supply to the pupill ry dil tor muscle. Horners syndrome, in its complet
e st te, consists of miosis, ptosis, enophth lmos nd dryness nd w rmth of h lf
of the f ce. It is due to lesion of the symp thetic supply such s results fr
om n intr c vernous c rotid rtery neurysm, or P nco sts tumour of the pex o
f the lung. A dil ted pupil (mydri sis) results from p r lysis of the p r symp t
hetic
res origin ting from the nucleus of EdingerWestph l in the mid
r in, nd is
therefore seen in 3rd nerve p lsy. The possi
le c uses re n enl rging poste
rior communic ting rtery neurysm c using
pressure on these
res in the 3rd cr ni l nerve (Ch pter 9) nd tentori l herni t
ion resulting from intr cr ni l pressure with the herni ted uncus of the tempor
l lo
e compressing the 3rd nerve (Ch pter 5). The ArgyllRo
ertson pupil is sm l
l, irregul r pupil not re cting to light, re cting to ccommod tion
ut respondi
ng poorly to mydri tics; it is usu lly c used
y syphilis. The myotonic pupil (H
olmesAdie) usu lly occurs in young women nd presents s unil ter l dil t tion
of one pupil with f ilure to re ct to light. The pupil shows slow constriction
occurring on m int ining convergence for prolonged period. In the complete sy
ndrome the knee nd nkle jerks re
sent. Ocul r movement The following re th
e gener l ctions of the extr ocul r muscles.
NEUROLOGICAL ASSESSMENT AND EXAMINATION
7
L ter l rectus (6th nerve) moves the eye horizont lly outw rds. Medi l rectus (3
rd nerve) moves the eye horizont lly inw rds. Superior rectus (3rd nerve) elev t
es the eye when it is turned outw rds. Inferior o
lique (3rd nerve) elev tes the
eye when it is turned inw rds. Inferior rectus (3rd nerve) depresses the eye wh
en it is turned outw rds. Superior o
lique (4th nerve) depresses the eye when it
is turned inw rds. The p tient should
e tested for diplopi , which will indic
te ocul r muscle we kness
efore it is evident on ex min tion. The following rul
es help determine which muscle nd cr ni l nerve re involved. The displ cement
of the f lse im ge m y
e horizont l, vertic l or
oth. The sep r tion of im ges
is gre test in the direction in which the we k muscle h s its purest ction. Th
e f lse im ge is displ ced furthest in the direction in which the we k muscle sh
ould move the eye. Disorders of eye movement m y
e due to imp ired conjug te oc
ul r movement. The centre for the control of conjug te l ter l g ze is situ ted
in the posterior p rt of the front l lo
e, with input from the occipit l region.
The n l common p thw y for controlling conjug te movement is in the
r instem, p
rticul rly the medi n longitudin l
undle. A lesion of the front l lo
e c uses
contr l ter l p r lysis of conjug te g ze (i.e. eyes devi ted tow rds the side o
f the lesion) nd lesion of the
r instem c uses ipsil ter l p r lysis of conj
ug te g ze (i.e. eyes devi ted to side opposite to the lesion). Nyst gmus should

e tested
y sking the p tient to w tch the tip of pointer. This should
e h
eld rst in the midline nd then moved slowly to the right, to the left nd then v
ertic lly upw rds nd downw rds. Jerk nyst gmus is the common type, consisting o
f slow drift in one direction nd f st correcting movement in the other. Horizon
t l jerk nyst gmus is produced
y lesions in the vesti
ul r system which m y occ
ur
peripher lly in the l
yrinth, centr lly t the nuclei, in the
r instem or in t
he cere
ellum. In peripher l lesions the quick ph se is w y from the lesion nd

the mplitude is gre ter in the direction of the quick ph se. In cere
ell r les
ions the quick ph se is in the direction of g ze t th t moment
ut the mplitud
e is gre ter to the side of the lesion. By convention the quick ph se is t ken t
o indic te the direction of the nyst gmus, so th t if the slow ph se is to the r
ight nd the quick ph se to the left the p tient is descri
ed s h ving nyst gmu
s to the left. Vertic l nyst gmus is due to intrinsic
r instem lesions such s
multiple sclerosis,
r instem tumours or phenytoin toxicity. The so-c lled down
e
t nyst gmus, which is ch r cterized
y vertic l nyst gmus ex gger ted
y downg
ze, is p rticul rly evident in low
r instem lesions s c used
y Chi ri syndro
me, where the lower
r instem h s
een compressed
y the descending cere
ell r t
onsils (Ch pter 11).
Trigemin l nerve The 5th cr ni l nerve (trigemin l nerve) is tested
y ssessing
f ci l sens tion over the three divisions of the cr ni l nerve; corne l sens ti
on should
e tested using ne piece of cotton wool. The motor function of the 5t
h nerve c n
e tested
y p lp ting the muscles while the p tient clenches their
j w, testing the power of j w opening nd l ter l devi tion of the j w (Fig. 1.5
). F ci l nerve The f ci l nerve is tested
y ssessing f ci l movement. In n u
pper motor neurone f ci l we kness the we kness of the lower p rt of the
Gre ter occipit l C. 2, 3 Lesser occipit l C. 2 Gre ter uricul r C. 2, 3 Dors l
r mi of C. 3,4,5 Supr cl vicul r C. 3,4
Ophth lmic (V1) M xill ry (V2) M ndi
ul r (V3) Tr nsverse cut neous nerves of ne
ck C. 2,3
Fig. 1.5 Cut neous nerve supply of the f ce, sc lp nd neck.
8
CHAPTER 1
f ce is very much gre ter th n the upper, with the strength of the or
icul ris o
culis
eing rel tively preserved. This is due to lesion
etween the cortex nd
the f ci l nucleus in the pons. Lower motor neurone we kness is evident
y equ
l involvement of the upper nd lower p rts of the f ce nd is due to lesion in
, or dist l to, the f ci l nerve nucleus in the pons. The chord tymp ni c rries
t ste sens tion from the nterior two-thirds of the tongue nd this should
e e
x mined using test vours pl ced c refully on the nterior tongue.
Glossoph rynge l nd v gus nerves The glossoph rynge l nd v gus nerves c n
e m
ost e sily ssessed
y testing p l t l movement nd sens tion from the ph rynx
nd soft p l te. If necess ry the voc l cords (v gus nerve) c n
e ex mined nd t
ste from the posterior one-third of the tongue (glossoph rynge l nerve) c n
e
tested. Accessory nerve The ccessory nerve supplies the motor power to the uppe
r p rt of the tr pezius nd sternocleidom stoid. The l tter muscle c n
e tested

y turning the p tients he d g inst resist nce nd w tching nd p lp ting the o
pposite sternom stoid muscle. The tr pezius muscle is
est tested
y sking the
p tient to shrug the shoulders nd ttempting to depress the shoulders forci
ly.
Hypogloss l nerve The hypogloss l nerve is responsi
le for movements of the ton
gue. The tongue should
e inspected to detect w sting nd movements from side to
side should
e o
served to detect we kness. The tip of the protruded tongue wil
l devi te tow rd the side of we kness.
Vesti
ulocochle r nerve The 8th cr ni l nerve consists of: the cochle r nerve he
ring the vesti
ul r nerve.
The cochle r nerve He ring c n
e ex mined t the
edside
y moving nger in the
me tus on one side, to produce m sking noise, nd repe ting words t st nd
rd volume nd from set dist nce in the other e r. Differenti tion
etween cond
uction nd sensorineur l de fness c n
e ided using tests with tuning fork. T

he Rinnes test involves holding vi
r ting tuning fork in front of the extern l
me tus nd then on the m stoid process. In nerve de fness
oth ir nd
one cond
uction re reduced,
ut ir conduction rem ins the
etter. In conductive de fnes
s
one conduction will
e
etter th n ir conduction. In We
ers test the vi
r tin
g tuning fork is pl ced on the centre of the forehe d. In nerve de fness the sou
nd ppe rs to
e he rd
etter in the norm l e r,
ut in conductive de fness the
sound is conducted to the
norm l e r. Form l udiometry should
e performed if
there re symptoms of imp ired he ring. The vesti
ul r nerve The simplest test
of vesti
ul r function is the c loric test, which is usu lly performed in p tien
ts suspected of h ving cere
ellopontine ngle tumour or s test of
r instem
function in p tients with severe
r in injury. The test is descri
ed in Ch pter
4, p. 44.
Ex min tion of the periphery
Posture nd gener l inspection The p tients posture m y indic te n underlying ne
urologic l dis
ility, or n
norm l posture m y result from p in. A p tient wi
th sci tic will often lie on the opposite side with the ffected leg exed t the
hip nd knee. The decere
r te posture is discussed in Ch pter 4. The lim
s shou
ld
e inspected to comp re size nd sh pe nd to detect deformity; longst nding
neurologic l lesions m y result in imp ired growth or w sting. Lesions of lower
motor neurone in inf ncy, such s
r chi l plexus p lsy or poliomyelitis, will
c use m rked ret rd tion in lim
growth. Upper motor neurone lesions of long st
nding, such s cute inf ntile hemiplegi nd cere
r l
irth tr um , will lso
c use ret rd tion in growth,
ut of lesser degree, with hemiplegic posture
nd ex gger ted re exes.
NEUROLOGICAL ASSESSMENT AND EXAMINATION
9
W sting The lim
s nd shoulder girdles should
e inspected to detect w sting nd
f scicul tion. As well s p lp ting for speci c muscle w sting in e ch lim
the c
ircumference of the lim
s should
e me sured t cle rly identi
le positions, suc
h s 8 cm
ove or
elow the olecr non, 10 cm
ove the p tell nd 8 cm
elow t
he ti
i l tu
erosity. The p ttern of w sting will
e n import nt clue s to the
underlying neurologic l dise se.
W sting of the fore rm nd sm ll muscles of the h nd. This results from lower mo
tor neurone lesions ffecting p rticul rly the C7, C8 nd T1 levels nd m y
e d
ue to lesions of the: spin l cord motor neurone dise se, syringomyeli , cervic l
cord tumours cervic l nerve root cervic l disc prol pse
r chi l plexus tr um ,
cervic l ri
, xill ry tumour peripher l nerve uln r nerve compression t the e
l
ow, c rp l tunnel syndrome (medi n nerve). W sting of the muscles of the lower
leg. This will result from compression of the c ud equin or lum
os cr l nerve
roots c used
y lum
r disc prol pse or tumour. Muscul r dystrophies. These
re genetic lly determined inherited degener tive myop thies nd c use p rticul r
p tterns of muscle w sting. F ciosc pulohumer l dystrophy involves the f ce nd
shoulder girdle. Proxim l lim
girdle dystrophy involves
oth shoulder nd hip
girdles. Dystrophi myotonic involves the f ce, sternom stoids nd qu driceps f
emoris. Myotoni (the f ilure of muscle to rel x fter contr ction) is present,
p rticul rly in the peripher l muscles nd tongue. Perone l muscul r trophy, wi
th predomin nt involvement of the lower lim
s, c uses the inverted
ottle ppe r
nce with simil r
ut less striking ch nges in the upper lim
s. Duchennes muscul r
dystrophy occurs m inly in young
oys nd ffects the rms nd
legs; the muscles h ve pseudohypertrophic ppe r nce.
Tone The tone in the upper lim
s should
e tested using exionextension movement
of the wrist,
y holding the p tients termin l ph l nges nd
y pron tionsupin tio
n of the fore rm. The tone in the lower lim
s should
e tested
y exion of the hi
p, knee nd nkle.

Decre sed tone This is due to: lower motor neurone lesion involving the spin l
roots or nterior horn cell of the spin l cord lesions of the sensory roots of
the re ex rc, e.g. t
es dors lis cere
ell r lesions, which c use ipsil ter l hyp
otoni myop thies spin l shock (the cute ph se of severe spin l lesion usu ll
y due to tr um ). Incre sed tone This will
e produced
y ny upper motor neuron
e lesion involving the corticospin l tr cts
ove the level of the nterior horn
cell in the spin l cord. There re three m jor types of hypertonicity. 1 Cl sp k
nife sp sticity, in which the resist nce is most pronounced when the movement is r
st m de. It is usu lly more m rked in the exor muscles of the upper lim
s nd ext
ensor muscles of the lower lim
s nd is sign of n upper motor neurone lesion.
2 Le d pipe rigidity, in which there is equ l resist nce to ll movements. This i
s ch r cteristic fe ture of lesion of the extr pyr mid l system
ut is lso
seen in severe sp sticity from n upper motor neurone lesion. 3 Cog wheel rigidity
, in which there is n ltern ting jerky resist nce to movement nd which occurs
in degener tive lesions of the extr pyr mid l system, p rticul rly P rkinsons di
se se. Clonus is
est demonstr ted
y rm r pid dorsi exion of the foot nd is indic t
ive of m rked incre sed tone.
10
CHAPTER 1
Power The power should
e tested in ll lim
s, comp ring e ch side. A system tic
ev lu tion will en
le the recognition of p rticul r p ttern of we kness th t
will
e in keeping with either cere
r l, spin l cord, plexus or peripher l ne
rve we kness. The m jor nerve nd m in root supply of the muscles re shown in T

le 1.1. The Medic l Rese rch Council cl ssi es the degree of we kness
y recordi
ng power, r nging from 0 to 5 (T
le 1.2). It is pp rent th t there is consid
er
le r nge of power
etween gr des 4 nd 5 nd some clinici ns m ke their own
further su
cl ssi c tion in this region. We kness due to corticospin l tr ct les
ion is most m rked in the
ductors nd extensors of the upper lim
s nd the exor
s of the lower lim
s. It is norm lly ssoci ted with incre sed tone nd ex gger
ted re exes. We kness due to lower motor neurone lesions is usu lly more severe th
n when the upper motor neurone is involved nd is seen in the distri
ution of t
he nerve ffected. It is ssoci ted with w sting, hypotoni nd diminished re exes
. F scicul tion is n irregul r, non-rhythmic l contr ction of muscle f scicles
which is most e sily seen in the deltoid or c lf muscles. It occurs cl ssic lly
in motor neurone dise se
ut m y lso occur in lower motor neurone lesions, e.g.
in the lower lim
s following long-st nding lum
r root compression. Re exes The d
eep tendon re ex requires the stimulus, sensory p thw y, motor neurone, contr ctin
g muscle nd the syn pses
etween the neurones in order to elicit response.
Reduced or
sent tendon re ex This m y occur due to ny
re ch in the re ex rc: se
nsory nerve polyneuritis sensory root t
es dors lis nterior horn cell poliomye
litis nterior root compression peripher l motor nerve tr um muscle myop thy.
Incre sed deep tendon re exes Due to lesions of the pyr mid l system, incre sed de
ep tendon re exes m y
e excessively prolonged, with l rger mplitude in cere

ell r lesion. In myxoedem the rel x tion ph se of the re ex is ret rded. E ch dee
p tendon re ex is ssoci ted with p rticul r segment l innerv tion nd peripher
l nerve s listed in T
le 1.3. The super ci l
domin l re ex h s segment l inner
v tion extending from T9 in the upper
domin l region to T12 in the lower re .
The re ex m y
e
sent in pyr mid l lesions
ove the level of segment l innerv
tion, p rticul rly in spin l lesions. However, the re ex m y lso
e dif cult to eli
cit when the
domin l muscles h ve
een stretched or d m ged
y surgic l oper t
ions, or in l rge, pendulous, o
ese
domen. Pl nt r re ex This should result in
the gre t toe exing the met t rsoph l nge l joint. The B
inski response consist
s of extension of the gre t toe t the met t rsoph l nge l joint, nd usu lly t
the interph l nge l joint, nd indic tes distur
nce of the pyr mid l tr ct.
Sens tion The mod lities of sens tion which should
e tested re: light touch pi

nprick (p in) temper ture position (proprioception) vi
r tion. Sensory testing i
nvolves n ccur te underst nding of the n tomic l p thw ys of sens tion. All m
od lities of sens tion tr vel
y the peripher l nerve nd sensory root to the sp
in l cord, or vi the cr ni l nerves to the
r instem. The
res for p in nd temp
er ture sens tion enter the posterol ter l spect of the spin l cord, tr vel cr
ni lly for few segments nd then cross to the opposite nterol ter l spinoth l
mic tr ct. This tr ct scends to the
r instem nd is joined
y the quintoth l
mic (trigeminoth l mic) tr ct in the pons. The
res end mostly in the ventrol ter
l nucleus of the th l mus nd from here the
NEUROLOGICAL ASSESSMENT AND EXAMINATION
11
T
le 1.1 Nerve nd m jor root supply of muscles. Spin l roots Upper lim
Spin l
ccessory nerve Tr pezius Br chi l plexus Rhom
oids Serr tus nterior Pector li
s m jor Cl vicul r Stern l Supr spin tus Infr spin tus L tissimus dorsi Teres m
jor Axill ry nerve Deltoid Musculocut neous nerve Biceps Br chi lis R di l nerve
Triceps Long he d L ter l he d Medi l he d Br chior di lis Extensor c rpi r di
lis longus Posterior interosseous nerve Supin tor Extensor c rpi uln ris Extenso
r digitorum A
ductor pollicis longus Extensor pollicis longus Extensor pollicis

revis Extensor indicis Medi n nerve Pron tor teres Flexor c rpi r di lis Flexor
digitorum super ci lis A
ductor pollicis
revis Flexor pollicis
revis* Opponens
pollicis Lum
ric ls I nd II Anterior interosseous nerve Flexor digitorum profun
dus I nd II Flexor pollicis longus Uln r nerve Flexor c rpi uln ris Flexor digi
torum profundus III nd IV Hypothen r muscles Adductor pollicis Flexis pollicis

revis P lm r interossei Dors l interossei Lum
ric ls III nd IV Spin l roots
C3, C4 C4, C5 C5, C6, C7 C5, C6 C6, C7, C8 C5, C6 C5, C6 C6, C7, C8 C5, C6, C7 C
5, C6 C5, C6 C5, C6
C7, C8, T1 C7, C8 C8, T1 C8, T1 C8, T1 C8, T1 C8, T1 C8, T1
Lower lim
Femor l nerve Iliopso s Rectus femoris V stus l ter lis V stus interm
edius V stus medi lis O
tur tor nerve Adductor longus Adductor m gnus
}
L1, L2, L3 Qu driceps femoris L2, L3, L4
} }
L2, L3, L4
C6, C7, C8 C5, C6 C5, C6 C6, C7 C7, C8 C7, C8 C7, C8 C7, C8 C7, C8 C7, C8 C6, C7
C6, C7 C7, C8, T1 C8, T1 C8, T1 C8, T1 C8, T1 C7, C8 C7, C8
Superior glute l nerve Gluteus medius nd minimus Tensor f sci e l t e Inferior
glute l nerve Gluteus m ximus Sci tic nd ti
i l nerves Semitendinosus Biceps Se
mimem
r nosus G strocnemius nd soleus Ti
i lis posterior Flexor digitorum longu
s Flexor h llucis longus Sm ll muscles of foot Sci tic nd common perone l nerve
s Ti
i lis nterior Extensor digitorum longus Extensor h llucis longus Extensor
digitorum
revis Peroneus longus Peroneus
revis
L4, L5, S1
L5, S1, S2 L5, S1, S2 L5, S1, S2 L5, S1, S2 S1, S2 L4, L5 L5, S1, S2 L5, S1, S2
S1, S2 L4, L5 L5, S1 L5, S1 L5, S1 L5, S1 L5, S1
* Flexor pollicis
revis is often supplied wholly or p rti lly
y the uln r nerv

e.
12
CHAPTER 1
sensory impulses p ss through the posterior lim
of the intern l c psule to the
postcentr l sensory cortex (see Ch pter 19, Fig. 19.1). Fi
res c rrying light to
uch, proprioception nd vi
r tion sens tion scend m inly in the ipsil ter l pos
terior columns of the spin l cord on the s me side to the nuclei gr cilis nd cu
ne tus. The
res cross the midline to scend through the
r instem in the medi l
lemniscus, to syn pse in the th l mus nd then on to the sensory cortex. The sen
sory loss involving nocioceptive stimuli (p in nd temper ture) should conform t
o p rticul r p ttern: peripher l nerve derm tome (nerve root) spin l cord resu
lting in sensory level glove nd stocking due to peripher l neurop thy hemi n lg
esi th l mic or upper
r instem
loss of p in nd temper ture on one side of the f ce nd the opposite side of th
e
ody lesion of the medull ffecting the descending root of the 5th nerve nd
the scending spinoth l mic tr ct from the rem inder of the
ody.
Coordin tion Coordin tion should
e tested in the upper nd lower lim
s. In the
upper lim
it is
est ssessed using the ngernose test nd in the lower lim
using t
he heelknee test. It is import nt to determine whether
norm lities of coordin tio
n re due to defects in: cere
ell r function proprioception muscul r we kness. G
it An essenti l p rt of the ex min tion is to o
serve the p tients g it. This is

est done not only s form l p rt of the ex min tion
ut lso when the p tien
t is not w re of o
serv tion. The type of g it is ch r cteristic of the underly
ing neurologic l distur
nce. A hemip resis will c use the p tient to dr g the l
eg nd, if severe, the leg will
e thrown out from the hip, producing the moveme
nt c lled circumduction. A high stepping g it occurs with foot drop (e.g. L5 r
oot lesion due to disc prol pse, l ter l poplite l nerve p lsy, perone l muscul
r trophy). The p tient r ises the foot too high to overcome the foot drop nd t
he toe hits the ground rst. In t
es dors lis the high stepping g it is due to
profound loss of position sense
ut
T
le 1.2 Medic l Rese rch Council cl ssi c tion of power. 0 Tot l p r lysis 1 Fli
cker of contr ction
ut no movement of lim
2 Muscle only
le to m ke norm l mo
vement when lim
is positioned so th t gr vity is elimin ted 3 Norm l movement
g inst gr vity
ut not g inst ddition l resist nce 4 Full movement
ut overcom
e
y resist nce 5 Norm l power
T
le 1.3 Deep tendon re exes, peripher l nerve nd segment l innerv tion. Tendon
re ex Biceps jerk Supin tor jerk Triceps jerk Flexor nger jerk Knee jerk Ankle jerk
M jor segment l innerv tion C5(6) C5/C6 C7(8) C6T1 L3/L4 S1(2) Peripher l nerve
Musculocut neous R di l R di l Medi n nd uln r Femor l Medi l poplite l nd sci
tic
NEUROLOGICAL ASSESSMENT AND EXAMINATION
13

simil r g it, of lesser severity, will result from involvement of the posterio
r column of the spin l cord or severe sensory neurop thy which interferes with p
osition sense. The g it is worse in the d rk nd the heel usu lly strikes the gr
ound rst. In P rkinsons dise se or other extr pyr mid l dise ses the p tient w lks
with stooped, shuf ing g it. The p tient m y h ve dif culty in st rting w lking
nd stopping. A slight push forw rd will c use r pid forw rd movement (protopulsi
on). In the t xic g it, the p tient is unst
le due to cere
ell r distur
nce.
A midline vermis tumour will result in the p tient reeling in ny direction. If

the cere
ell r hemisphere is involved then the p tient will tend to f ll to the
ipsil ter l side. A w ddling g it is ssoci ted with congenit l disloc tion of t
he hips nd muscul r dystrophy. The hysteric l g it is often
iz rre nd is dimi
nished when the p tient is un w re of ny o
serv tion. Following the clinic l s
sessment, presumptive di gnosis is m de nd further investig tions c n
e perf
ormed to con rm the di gnosis. These l
or tory investig tions nd r diologic l pr
ocedures re descri
ed in the following ch pter.
Br in de th
The use of donor org ns for tr nspl nt tion nd the dvent of improved intensive
c re f cilities h ve resulted in the necessity of medic lly nd leg lly ccepte
d criteri of
r in de th. If there is irrecover
le
r instem d m ge nd the te
sts descri
ed
elow show no evidence of
r instem function, then the p tient is
medic lly nd leg lly de d. If rti ci l ventil tion is continued the other org ns
m y continue to function for some time. However, continued prolonged ventil tio
n of the p tient fter the di gnosis of
r in de th is not only undigni ed for the
de d p tient nd distressing to the rel tives,
ut is lso w steful of expensiv
e medic l resources th t re often in short supply. The di gnosis of
r in de th
relies on: preconditions
efore testing c n
e performed
r in de th tests.
The preconditions re th t ll reversi
le c uses of
r instem depression h ve
e
en excluded. These include: depress nt drugs hypothermi (temper ture must
e gr
e ter th n 35C) neuromuscul r
locking drugs met
olic or endocrine distur
nce
s c use of the p tients condition. Br in de th testing must
e del yed until th
ese preconditions re
solutely s tis ed. The tests for
r instem function re: l
ck of pupil response to light l ck of corne l re ex to stimul tion l ck of oculoc
eph lic re ex f ilure of vesti
ulo-ocul r re ex (c loric testing) f ilure of g g o
r cough re ex on
ronchi l stimul tion no motor response in the f ce or muscles su
pplied
y the cr ni l nerves in response to p inful stimulus f ilure of respir t
ory movements when the p tient is disconnected from ventil tor nd the P CO2 i
s llowed to rise to 50 mmHg. The tests should
e repe ted fter n interv l of
30 minutes nd it is essenti l th t they should
e c rried out
y two doctors of
dequ te seniority nd with expertise in the eld.
Further re ding
Conference of Medic l Roy l Colleges nd Their F culties in the UK (1979) Di gno
sis of de th. British Journ l of Medicine 1, 322. H rrington D (1974) The Visu l
Fields, 4th edn. C V Mos
y, St Louis. Jennett B (1981) Br in de th. British Jou
rn l of An esthesi 53, 11111119. Medic l Rese rch Council (1976) Aids to the ex
min tion of the peripher l nervous system. Her M jestys St tionery Of ce, London. P
lum F (1980) Br in de th. L ncet ii, 379. Plum F, Posner JB (1980) Di gnosis of
Stupor nd Com , 3rd edn. F A D vis, Phil delphi . W lton J, ed. (1977) Br in. I
n: Dise ses of the Nervous System. Oxford University Press, Oxford.
CHAPTER 2
2
Neurosurgic l investig tions
Investig tions to determine the ex ct di gnosis re ne rly lw ys necess ry foll
owing the clinic l ex min tion. The following is list of the more common inves
tig tions th t m y need to
e undert ken: cere
rospin l uid (CSF) studies r diolo
gic l investig tions electroenceph logr phy nerve conduction studies evoked pote
nti l studies nucle r medicine investig tions. Some of these investig tions will

e descri
ed in this ch pter. The others will
e de lt with in the ch pters de
ling with the relev nt neurosurgic l pro
lems.
Cere
rospin l uid investig tion
The CSF is produced
y the choroid plexus t r te of pproxim tely 0.4 ml per

minute. The uid circul tes from the l ter l ventricles through the interventricul
r for men (of Monro) into the 3rd ventricle, through the cere
r l queduct of S
ylvius into the 4th ventricle, nd into the su
r chnoid sp ce vi the two l ter
lly pl ced for min of Luschk nd medi l perture in the roof of the 4th ven
tricle the for men of M gendie. The uid circul tes c ud lly into the spin l su
r
chnoid sp ce, throughout the
s l cisterns, up through the tentori l hi tus n
d then over the cere
r l hemispheres. It is
sor
ed
y the r chnoid villi of t
he dur l sinuses, nd especi lly
y the superior s gitt l sinus. Approxim tely 5
00 ml of CSF is produced e ch d y. The tot l CSF volume is 140 ml; the l ter l v
entricles cont in pproxim tely 25 ml, the spin l cord 14
su
r chnoid sp ce 30 ml nd the rem inder of the uid is found in the
s l ciste
rns. T
le 2.1 shows the norm l constituents of CSF. The CSF glucose content is
pproxim tely 65% of the
lood pl sm level in the f sting st te. There is gr
dient for m ny of the constituents of CSF long the cere
rospin l xis (T
le 2.
2). The uid is norm lly cle r nd colourless; it will ppe r tur
id if it cont in
s more th n 400 white
lood cells or 200 red
lood cells per mm3. Yellow discolo
ur tion, x nthochromi , is due to the
re kdown products of red
lood cells; the
se follow h emorrh ge into the CSF. CSF c n
e o
t ined
y: lum
r puncture cist
ern l puncture c nnul tion of the l ter l ventricle. The uid is usu lly o
t ined

y lum
r puncture. Cistern l puncture is performed if the lum
r puncture h s f
iled due to technic l dif culties, if there is loc l skin sepsis or, in some r di
ology investig tions, where it is the preferred route of contr st dministr tion
for myelogr phy. Ventricul r puncture is usu lly only performed s n intr oper
tive procedure or for tempor ry reduction of intr cr ni l pressure in n emerge
ncy.
Lum
r puncture
The most common indic tions for CSF ex min tion
y lum
r puncture re: meningit
is su
r chnoid h emorrh ge neurologic l dise ses such s multiple sclerosis
NEUROSURGICAL INVESTIGATIONS
15
T
le 2.1 CSF st tistics (lum
r). Volume R te of production Pressure (recum
ent
) Cells Protein Glucose IgG Chloride 140 ml 0.4 ml/min 1015 cm of CSF Less th n 34
white cells/mm3 0.150.45 g/l (1545 mg/100 ml) 2.84.2 mmol/l (5075 mg/100 ml) 1012% o
f tot l protein 120130 mmol/l
The v lues re expressed in SI (Systme Intern tion le) units nd the correspondin
g tr dition l units re in p rentheses.
this lies t the L3/4 level. The lum
r puncture c n
e c rried out t this sp c
e or t the sp ces immedi tely
ove or
elow. The re is prep red with ntisep
tic solution nd dr ped. The procedure must
e performed under completely steril
e conditions. The interspinous re is p lp ted nd the skin injected with 12 ml
of 1% lignoc ine loc l n esthetic. The lum
r puncture needle is inserted
etwe
en the two spinous processes, pointing in slightly cr ni l direction. If perfo
rmed c refully it is usu lly possi
le to feel the needle p ss through the inters
pinous lig ment nd then through the dur . The stilette of the lum
r puncture n
eedle is withdr wn nd m nometer tt ched to me sure the pressure. The uid is d
r ined into sterile cont iners nd sent for ex min tion.
T
le 2.2 CSF gr dients long the cere
rospin l xis. Ventricle Protein (g/l) Gl
ucose (mmol/l) 0.1 4.5 Cistern l 0.2 4.0 Lum
r 0.4 3.4
cytologic l ex min tion for neopl stic dise se r diologic l im ging (e.g. myelog
r phy) or r dio-isotope investig tions me surement of intr cr ni l pressure. The
most import nt contr indic tion to lum
r puncture is clinic l evidence of r is

ed intr cr ni l pressure. P pilloedem is n
solute contr indic tion nd lum

r puncture should never
e performed in p tient in whom n intr cr ni l sp c
e-occupying lesion is suspected. If there is ny dou
t CT sc n or MRI must
e
performed prior to lum
r puncture. A lum
r puncture should not
e performed if
there is loc l infection.
Technique of lum
r puncture The p tient should
e positioned on the side, the

ck vertic l on the edge of the
ed nd the knees exed up to the chest. The ili c
crest is p lp ted;
Complic tions of lum
r puncture If performed properly, with the ppropri te ind
ic tions, lum
r puncture is well toler ted nd complic tions should
e minim l.
However, there re sever l potenti l h z rds nd complic tions; these include:
progression of
r in herni tion progression of spin l cord compression injury to
the neur l structures he d che
ck che infection loc l nd meningitis impl nt
tion of epidermoid tumour (r re). The potenti l risk of lum
r puncture worsenin
g
r in herni tion c n
e voided if the procedure is not undert ken in p tients
with r ised intr cr ni l pressure. Neurologic l deterior tion m y follow lum
r
puncture nd myelogr phy in p tients with spin l tumours where there is severe
cord compression. Although the procedure m y occ sion lly
e necess ry to m ke t
he di gnosis, myelogr phy should
e voided s m gnetic reson nce im ging is the
investig tion of choice for spin l tumours. Neurologic l deterior tion requires
prompt surgery; this is discussed in Ch pter 15. Infection should
e voided
y
the use of scrupulous sterile techniques. If the procedure is performed t le
vel th t is too high there is risk of neur l d m ge, p rticul rly to the conus
medull ris. R rely, nerve root m y
e in16
CHAPTER 2
jured
y the improper pl cement of the needle. Injury to spin l r dicul r rte
ry m y occ sion lly give rise to spin l su
dur l or epidur l h em tom ; this r
isk is incre sed if the p tient is t king ntico gul tion ther py. The tr um tic
effects of the lum
r puncture re responsi
le for minor, tr nsient low
ck di
scomfort. Very r rely, fr nk disc herni tion h s
een reported due to d m ge of
the nnulus
rosus of the disc. He d che The most common complic tion of lum
r p
uncture is he d che. In most c ses this is due to low CSF pressure th t results
from persistent le k ge of the uid through hole in the r chnoid nd dur . It i
s gener lly recommended th t p tients should rem in t for 12 hours following l
um
r puncture to minimize the risk of this complic tion. The use of n rrow-g
uge needle (20 g uge or less) nd voiding multiple puncture holes in the mening
es lso decre ses the ch nce of trou
lesome postlum
r puncture he d che. If the
he d che develops following mo
iliz tion the p tient should
e instructed to li
e t for further 24 hours nd encour ged to drink l rge volumes of non- lcoholi
c uids. Some clinici ns dvoc te the use of
lood p tch for the tre tment of persis
tent postspin l he d che. This technique uses the epidur l injection of utologo
us
lood t the site of dur l puncture to form throm
otic t mpon de which se l
s the dur l opening,
ut this is usu lly unnecess ry.
CSF ex min tion
The CSF should
e ex mined immedi tely. If the uid is
lood-st ined it should
e
spun down in centrifuge nd ex mined for evidence of x nthochromi , this
eing
indic tive of h emorrh ge into the CSF. Three m jor pigments derived from red c
ells m y
e detected in CSF: oxyh emoglo
in,
iliru
in nd meth emoglo
in. Oxyh
emoglo
in is red,
ut fter dilution it ppe rs pink or or nge. It is rele sed

y lysis of red cells nd m y
e detected in the CSF within 2
hours of the rele se of
lood into the su
r chnoid sp ce. It re ches m ximum
in the rst 36 hours nd gr du lly dis ppe rs over the next 710 d ys. Biliru
in is

yellow nd is the iron-free deriv tive of h emoglo
in produced in vivo following

the h emolysis of red cells. Biliru
in form tion in the CSF pro

ly depends on
the
ility of m croph ges nd other cells in the leptomeninges to degr de h em
oglo
in. It is rst detected
out 10 hours fter the onset of su
r chnoid
leedi
ng nd re ches m ximum t 48 hours. It m y persist for 24 weeks fter extensive
h emorrh ge. Meth emoglo
in is reduction product of h emoglo
in. It is
row
n pigment th t is d rk yellow in dilution nd it is ch r cteristic lly found in
enc psul ted su
dur l h em tom s. Although it m y
e detected
y spectrophotomet
ry of the spin l uid in p tients with l rge enc psul tions of this sort, the pigm
ent is not usu lly o
served in other x nthochromic spin l uids. X nthochromic spi
n l uid m y lso occur in j undice, such s j undice second ry to liver dise se o
r in h emolytic dise se of the new
orn. The uid should
e sent for micro
iologic
l nd
iochemic l ex min tion nd, if clinic lly indic ted, cytologic l ex min t
ion for m lign nt cells. The common
norm lities re shown in T
le 2.3. Norm l
CSF cont ins no more th n four lymphocytes or mononucle r cells per mm3. Polymo
rphonucle r cells re never found in norm l CSF
ut n isol ted gr nulocyte, pre
sum
ly derived from
lood t the time of lum
r puncture, m y
e seen if the CS
F h s
een cytocentrifuged. A gr nulocyte pleocytosis is the h llm rk of
cteri
l infection; gr nulocytic ph se lso occurs t the onset of vir l meningiti
s, prior to the development of purely mononucle r re ction. Eosinophils re no
t seen in norm l CSF. The most common c uses of prominent eosinophilic re ction
re p r sitic dise ses,
ut eosinophili m y lso occur in in mm tory dise ses n
d in r nge of other dise ses, s shown in T
le 2.3. Ex min tion of the CSF us
ing the polymer se
NEUROSURGICAL INVESTIGATIONS
17
T
le 2.3 CSF
norm lities. CSF
norm lity Polymorphonucle r pleocytosis Monon
ucle r pleocytosis Dise se suspected B cteri l meningitis Vir l meningitis Tu
er
culous meningitis Acute demyelin tion P r sitic infections Trichinell nd Asc r
is Toxopl sm Cysticercosis In mm tory dise ses Tu
erculosis Syphilis Su
cute sc
lerosing p nenceph litis Fung l infections Other dise ses Lymphom Hodgkins dise
se Multiple sclerosis CNS infection Spin l
lock (very high levels Froins syndrom
e) C rcinom tosis of the meninges Spin l neuro
rom s Acoustic neurom s Guill inB r
r syndrome B cteri l meningitis Tu
erculous meningitis
Eosinophils
R ised protein
Low sug r Low chloride (<110 mmol/l)
ch in re ction (PCR) technique is useful in con rming the di gnosis of herpes simp
lex enceph litis (Ch pter 12).
CSF electrophoresis Electrophoresis of the spin l uid is useful in the di gnosis
of p tients suspected of h ving demyelin tion. An IgG of over 15% of the tot l p
rotein is suggestive of dissemin ted sclerosis
ut it m y lso
e r ised in uto
immune st tes, such s Guill inB rr syndrome nd c rcinom tosis. Electrophoresis o
f the CSF m y lso demonstr te myelom protein. In ddition to the
solute incr
e se noted in g mm glo
ulins in in mm tory dise ses of the nervous system, qu li
t tive ch nges in CSF
g mm glo
ulins h ve
een demonstr ted in concentr ted CSF with g rose gel elec
trophoresis nd other gels. This technique demonstr tes discrete
nds in the g
mm glo
ulin p ttern which h ve
een c lled oligoclon l
nds. The term descri
e
s popul tion of proteins, h ving identic l electrophoretic ch r cteristics der
ived from the s me popul tion of immunocompetent cells. A single ntigen is pres

umed to give rise to single
nd. Oligoclon l
nds re reported in
out 90%
of p tients with multiple sclerosis nd re frequently o
served whenever CSF g m
m glo
ulin is incre sed due to v riety of in mm tory disorders of the nervous
system. In p tients with multiple sclerosis the
nd p ttern seems to
e
18
CHAPTER 2
unique for e ch p tient, nd it rem ins st
le over time. Serologic l investig t
ions for neurosyphilis should
e performed on the CSF if suspected.
evidence of met st tic tumour with erosion or sclerosis of the verte
r l
ody, p
edicles or l min enl rgement of neur l for men indic ting spin l schw nnom
congenit l
norm lities such s spin
i d .
R diologic l investig tions
The m jor r diologic l investig tions re: pl in X-r ys CT sc n cere
r l ngiogr
phy myelogr phy MRI.
Computerized tomogr phy sc nning
Computerized tomogr phy (CT) sc nning w s introduced in the 1970s nd t th t ti
me revolutionized the r diologic l investig tion of neurologic l dise se. Since
then consider
le technic l dv nces h ve gre tly improved the qu lity of sc nni
ng which c n now
e performed in
oth the xi l (horizont l) nd coron l pl nes.
S gitt l reconstruction pictures c n
e o
t ined
y computer m nipul tion of th
e d t . The CT sc n is the initi l investig tion of choice in the investig tion
of ne rly ll intr cr ni l dise ses. Figure 2.1 shows the norm l structures seen
in xi l CT sc ns t v rious positions through the cr nium. Intr cr ni l c lci c
tion m y
e seen on the pl in CT sc n. Intr cr ni l lesions th t show c lci c tion
on the pl in CT sc n include: meningiom will lso show hyperostosis of cr ni l
v ult most oligodendrogliom s strocytom 30% of low-gr de tumours
ut infreque
ntly in high-gr de tumours ependymom nd su
ependymom cr nioph ryngiom w ll o
f gi nt neurysm, rteriovenous m lform tions. The pine l gl nd is usu lly c lci e
d nd c lci c tion of the choroid plexus,
s l g ngli nd f lx m y occur in norm
l sc ns. Following pl in CT sc n iodine-
sed contr st medium is dministered
intr venously; this will enh nce re s with incre sed v scul rity or with imp i
rment of the
lood
r in
rrier. The non-ionic iodine gents h ve reduced the ver
y sm ll risk following intr venous dministr tion of contr st, the most serious
side-effect
eing n n phyl ctic re ction. Intr cr ni l lesions th t enh nce fo
llowing contr st dministr tion include: high-gr de cere
r l gliom s
Skull X-r y
The usefulness of the pl in skull X-r y h s
een l rgely superseded
y CT sc nni
ng. However, it is still helpful prelimin ry investig tion in p tients with he
d injuries. The det ils of the use of this investig tion in tr um re discusse
d in Ch pter 4. The m jor
norm lities to look for on skull X-r y re: fr ctu
res hyperostosis, e.g. meningiom
one erosion due to skull v ult tumours midlin
e shift of the pine l gl nd from sp ceoccupying lesion
norm l c lci c tion, e.g.
tumours such s meningiom , oligodendrogliom , cr nioph ryngiom or c lci ed w ll
of n neurysm signs of long-st nding r ised intr cr ni l pressure erosion of t
he dorsum sell e copper
e ting of the skull v ult. Enh nced digit l m rkings re
not uncommon under the ge of 30
ut m y indic te long-st nding r ised intr cr n
i l pressure if present over the whole v ult.
Pl in X-r ys of the spine
These re useful prelimin ry investig tions for p tients presenting with spin l
p in. P rticul r note should
e t ken of: verte
r l lignment presence of degene
r tive dise se with n rrowing of the neur l for min nd spin l c n l

NEUROSURGICAL INVESTIGATIONS
19
Front l lo
e L ter l ventricle Front l horn of l ter l ventricle P riet l lo
e O
ccipit l lo
e Septum pellucidum
Corpus c llosum
Sulci
F lx cere
ri
Occipit l horn of l ter l ventricle Sylvi n fissure
Pine l gl nd
3rd ventricle Front l sinus Chi sm tic cistern Qu drigemin l cistern Or
it l roo
f Tempor l lo
e Pons M stoid ir cells
Fig. 2.1 Norm l intr cere
r l nd cr ni l structures on CT sc n t v rious level
s through the cr nium.
Tempor l lo
e Mid
r in Qu drigemin l cistern
4th ventricle
Cere
ellum

meningiom s coustic neurom s l rge pituit ry tumours met st tic tumours rterio
venous m lform tions. Cere
r l
scesses usu lly enh nce with peripher l ring.
Low-gr de gliom s often h ve sc nty, if ny, enh ncement. An intr cr ni l m ss
will c use distortion of the l ter l ventricles either s result of the lesion
itself or
ec use of the ssoci ted cere
r l oedem , which ppe rs s n re o
f decre sed density round the lesion. CT sc nning of the spine is v lu
le in t
he m n gement of: lum
r disc prol pse degener tive dise se of the lum
r spine
lum
r c n l stenosis cervic l disc prol pse cervic l c n l stenosis
spin l tr um spin l dysr phism. CT sc nning, when com
ined with intr thec l iod
ine contr st, h s
een utilized s useful im ging technique for cervic l disc
prol pse
ut h s
een superceded
y MRI. This is discussed in Ch pter 14.
Cere
r l ngiogr phy
Angiogr phy of the intr - nd extr cr ni l vessels is now usu lly performed usin
g computerized digit l su
tr ction ngiogr phic techniques. The procedure is usu
lly done under loc l n esthesi in the dult p tient. The c theter is inserted
into the femor l rtery nd thre ded up into the c rotid or verte
r l rtery or
igin with the id of n im ge intensi er. Digit l su
tr ction ngiogr phy h s cons
ider
ly reduced the complic tions of st nd rd
20
CHAPTER 2

ngiogr phy, lthough there is still very sm ll risk of cere
r l em
olus from
clot or n therosclerotic pl que
roken off
y the c theter tip. The m jor in
dic tions for ngiogr phy re: investig tion of cere
r l isch emi due to c roti
d rtery dise se nd intr cr ni l therom investig tion of su
r chnoid h emorr

h ge, e.g. cere
r l neurysm, rteriovenous m lform tion investig tion of venous
sinus throm
osis preoper tive em
oliz tion of meningiom . Cere
r l ngiogr phy
is now only infrequently used in the investig tion of intr cr ni l tumours. The
m jor intr cr ni l vessels re shown in Fig. 2.2.
Myelogr phy
Myelogr phy h s
een used in the p st in the
Posterior cere
r l Frontopol r C llosom rgin l
investig tion of spin l dise se c using compression of the dj cent neur l struc
tures. The use of w ter-solu
le contr st gents h s m de the technique s fer nd
produces higher qu lity im ging th n w s chieved with the previously used oil
sed medi . In p rticul r, the dre ded complic tion of postmyelogr phy r chnoi
ditis does not occur with w ter-
sed medi . Complic tions, which re now very u
ncommon, include epileptic seizures, systemic re ctions to the contr st medium
nd the risks of the lum
r puncture itself. The m jor indic tions for myelogr ph
y were: cervic l disc prol pse lum
r disc prol pse spin l tumour cervic l c n l
stenosis c using cervic l myelop thy lum
r c n l stenosis.
Peric llos l
Anterior cere
r l Ophth lmic Middle cere
r l Posterior communic ting Anterior ce
re
r l ( ) Townes view L ter l view Intern l c rotid Middle cere
r l rtery Ant
erior choroid l
Posterior cere
r l rteries Posterior cere
r l rtery
B sil r rtery Superior cere
ell r rtery Anterior inferior cere
ell r rtery
Verte
r l rteries (
)
Posterior inferior cere
ell r rtery
Fig. 2.2 The m jor intr cr ni l vessels seen on cere
r l ngiogr phy.
NEUROSURGICAL INVESTIGATIONS
21
However, the dvent of high-qu lity CT sc nning nd MRI h ve consider
ly reduce
d the indic tions for myelogr phy. Myelogr phy (often com
ined with CT) is now u
sed occ sion lly for p tients with clinic l fe tures of cervic l or lum
r nerve
root compression, such s due to disc prol pse (often recurrent) or perineur l

rosis (th t c n follow previous surgery) nd in whom the MRI ndings re equivoc l
nd not di gnostic.
M gnetic reson nce im ging
M gnetic reson nce im ging (MRI) is di gnostic r diologic l technique which ut
ilizes the m gnetic properties of the
odys hydrogen nuclei to produce cross-sect
ion l im ges in ny pl ne. A moving ch rged p rticle cre tes sm ll m gnetic eld
. At equili
rium the multiple tiny m gnetic elds cre ted
y the r ndomly spinning
hydrogen nuclei (protons) within the
ody c ncel e ch other out. If the
ody is
pl ced within strong extern l m gnetic eld, the protons tend to lign themselv
es within th t eld. If energy, in the form of pulses of electrom gnetic w ves of
precisely the right frequency nd
nd width (usu lly in the FM r dio r nge), is
introduced into the
ody, the protons c n
e induced to spin in unison, or reso
n ntly (Fig. 2.3). When the extern l energy source is removed, the energy from t
he excited protons is emitted in the form of
N

N
S
S
Fig. 2.3 The spinning protons re ligned in m gnetic
netic pulse displ ces the protons (right).

eld (left). An electrom g

r dio sign l, which progressively dies w y. Although f int, the dec ying sign
l c n
e detected
y sensitive ntenn e (receiver coils) pl ced str tegic lly i
n rel tion to the p rt of the
ody
eing sc nned. Initi lly, the strength of the
sign l is proportion l to the distri
ution of the protons within the tissue. Th
e r te of dec y, or rel x tion, is dependent upon three f ctors. The rst is the ef ci
ency with which energy is tr nsferred from the protons to their immedi tely dj
cent molecul r l ttice, or fr mework, which is descri
ed
y n exponenti l curve
with time const nt T1. Although this is commonly n med T1 rel x tion time, other
eponyms used include longitudin l rel x tion time, spin l ttice rel x tion time nd t
herm l rel x tion time. The second f ctor contri
uting to sign l dec y is the des
tructive interference of the protons spins with e ch other. Bec use the protons
re exposed to minute differences in loc l m gnetic eld, their spins
ecome out of
ph se, resulting in loss of synchroniz tion, or reson nce. The r te of sign l d
ec y due to this f ctor is descri
ed
y nother exponenti l curve with time cons
t nt T2, which is commonly c lled T2 rel x tion time. It is lso known s horizont
l rel x tion time nd spinspin rel x tion time. The third f ctor is m gnetic suscepti

ility of tissue. This refers to the e se with which tissue
ecomes m gnetized
when pl ced in strong m gnetic eld. The induction of rel tively strong loc l m
gnetic elds induces m rked ph se dispers l nd sign l loss. This phenomenon is co
mmonly exhi
ited
y h em tom degr d tion products such s deoxyh emoglo
in nd
h emosiderin. M gnetic suscepti
ility is directly proportion l to the squ re of
the m gnetic eld, so th t 1.5-tesl m gnet is 25 times more sensitive to m gnet
ic suscepti
ility th n 0.3-tesl m gnet. The phenomenon is
est exhi
ited t
ll eld strengths when gr dient echo sequences re used. Contr st
etween differen
t tissues in MRI im ges is due to differences in proton concentr tion, T1 nd T2
, m gnetic suscepti
ility nd ow. These differences c n
e m ximized
y v rying t
he r te of the pulses of electrom gnetic energy (TR, or pulse repetition time)
nd the time interv l
22
CHAPTER 2
following the pulses t which the sign l is recorded (TE, or echo time). In MRI
studies of the CNS the T1-weighted sc ns show the n tomic l structures in det i
l (Fig. 2.4) ; the CSF is
l ck. The T2-weighted sc ns show intr cr ni l p tholo
gic l processes, ll of which re ssoci ted with
norm l ccumul tions of w te
r: the CSF is white, nd f st-moving
lood in rteries nd venous sinuses is
l
ck. A signi c nt exception to the rule th t T2-weighted sequences depict the CSF
s white is the sequence known s uid- ttenu ted inversion recovery (FLAIR) which
is he vily T2-weighted sequence,
ut h s pulse timing such th t norm l CSF sig
n l is dulled so th t it ppe rs
l ck. P thologic l ccumul tions of uid still
ppe r white g inst predomin ntly grey
ckground. The differenti l sign l of
moving
lood is utilized
y su
tr cting out the st tic
ckground to depict only

lood vessels in technique known s m gnetic reson nce ngiogr phy (MRA). The
im ges of the
lood vessels re ret ined in the computer s three-dimension l
d t st ck, which llows viewing from ny ngle. The resolution of MRA is still
inferior to th t of digit l su
tr ction ngiogr phy (DSA),
ut intr venous cont
r st-enh nced MRA h s p rti lly
ridged th t g p. M gnetic reson nce spectroscop
y (MRS) exploits the empiric l f ct th t the frequency with which protons spin (
process) in sp ce is directly proportion l to the m gnetic eld to which they re

exposed. Electrons h ve 8001000 times the m gnetic strength of protons. Thus prot
ons in different molecules nd even in different p rts of the s me molecule re
exposed to very slightly different net m gnetic elds nd therefore spin t very s
lightly different frequencies. MRS me sures those differences in spin frequency
nd depicts them s spectrum of pe ks, with sep r tion of the pe ks me sured i
n p rts per million. The result nt proton spectr show l ct te pe ks in re s of
isch emi or n ero
ic met
olism, s seen in inf rcts nd m lign nt tumours. D
ecre sed N- cetyl sp rt te (NAA) levels indic te neuron l loss, nd incre sed c
holine is seen in re s of incre sed mem
r ne tumour. The most sensitive results
re o
t ined

y me suring single voxel,
ut using sp ti lly encoding gr dients s is done w

ith st nd rd MRI, multiple voxels c n
e simult neously ex mined. Individu l pe
ks c n
e selected nd their distri
ution shown s coloured overl y on st nd r
d MRI im ges. This technique is known s chemic l shift spectroscopy. MRI, or nu
cle r m gnetic reson nce, h s consider
le potenti l dv nt ges over CT sc nning
including: no ionizing r di tion no
one rtef ct so th t lesions round the sk
ull
se re cle rly identi ed high resolution. Intr venous contr st medium using
g dolinium compounds consider
ly enh nces the v lue of MRI. These medi re w t
er-solu
le nd cross the
norm l
lood
r in
rrier in m nner simil r to the i
odine-
sed, w ter-solu
le contr st medi used in CT sc nning. The p r m gnetic
compounds function
y ch nging the loc l m gnetic environment. The sign l intens
ity of those hydrogen nuclei th t re in direct cont ct with the p r m gnetic co
mpounds is ltered. The consequent shortening of the T1 rel x tion time results
in n enh ncement or
rightening of the re . Ultr f st im ge cquisition with e
chopl n r im ging (EPI) llows ccur te me surement of
lood ow. The technique ei
ther utilizes
olus of g dolinium contr st medium or detects ch nges in the r
tio of oxyh emoglo
in to deoxyh emoglo
in in stimul ted portions of the
r in (

lood oxygen level-dependent im ging, or BOLD). BOLD llows ccur te loc liz tion
of r nge of motor or sensory functions. Of p rticul r use in neurosurgery is
the ccur te deline tion of the function l motor strip, which is
Fig. 2.4 Norm l MRI. T1, T2 nd m gnetic reson nce spectroscopy. ( ) Axi l T1 MR
I through midpons showing upper 4th ventricle. (
) Axi l T1 MRI through lower mi
d
r in. (c) Axi l T1 MRI showing cere
r l peduncles. (d) Axi l T1 MRI showing sy
mmetric l midline l ter l ventricles. (e) Axi l T1 MRI showing hyperdense CSF in
l ter l ventricles nd su
r chnoid sp ce. (f) Midline s gitt l T1 MRI. Arrow p
oints to 4th ventricle. Arrowhe d shows genu of corpus c llosum.
NEUROSURGICAL INVESTIGATIONS
23
( )
(
)
(c)
(d)
(f)
(e)
24
CHAPTER 2
simply o
t ined
y performing BOLD sequences during repetitive h nd movements. T

his form of im ging is sometimes n med function l MRI (fMRI). EPI o
t ins individu
l im ges in s little s 30 milliseconds. Diffusion-weighted sequences re
le
to im ge ctu l w ter diffusion r te differences t the molecul r level. Cytoto
xic or cellul r oedem results in restricted diffusion, which ppe rs white in d
iffusion-weighted sequences. St nd rd diffusion-weighted sequences me sure diffu
sion in ll directions (isotropic diffusion). By pplying gr dients in t le st
six different directions, direction lly restricted or nisotropic diffusion c n

e me sured. Bec use diffusion in xons is l rgely restricted to their longitudi
n l xes, white m tter m ps c n
e constructed nd superimposed on st nd rd im g
es. The m in dis dv nt ge of MRI t present is its rel tive un v il
ility due t
o insuf cient v il
ility of equipment. It is v lu
le investig tion in the fol
lowing neurosurgic l conditions. Intr cr ni l tumours especi lly meningiom , co
ustic neurom s, pituit ry tumours, skull
se tumours, met st ses, lymphom , men
inge l in ltr tion (with g dolinium contr st), gliom . CNS infection cere
r l
sc
ess, herpes simplex enceph litis. Arteriovenous m lform tions. Venous sinus thro
m
osis. Cr niospin l
norm lities such s the Chi ri m lform tion. Syringomyeli
. Hippoc mp l or mesi l sclerosis. Spin l tumours. Lum
r disc prol pse, lum
r
c n l stenosis. Cervic l cord compression cervic l myelop thy, cervic l centr l
disc prol pse. Cervic l disc prol pse. Thor cic disc prol pse.
r dionecrosis, s the th llium will
e t ken up into the tumour region. R dio-is
otope cisternogr phy is sometimes useful in the detection of the site of CSF le
k ge following fr cture of the skull
se. The technique is lso sometimes use
d to ssess CSF ow in p tients with communic ting hydroceph lus; re ux into the ven
tricul r system, followed
y slow cle r nce, suggests communic ting or norm l-pr
essure hydroceph lus. However, the technique h s
een l rgely superseded
y intr
cr ni l pressure monitoring. Other highly sophistic ted techniques using r dioi
sotopes, such s single photon emission computerized tomogr phy (SPECT) or posit
ron emission tomogr phy (PET), re used to me sure cere
r l
lood ow or cere
r l
met
olism. They re p rticul rly useful in the ev lu tion of p tients for epile
psy surgery (Ch pter 21). SPECT utilizes single photon emitting r dioph rm ceuti
c ls which distri
ute in the
r in ccording to region l
lood ow. Im ging is per
formed using g mm c mer nd computer n lysis. PET utilizes positron-emittin
g isotopes which depend on cyclotron for their production nd, in gener l, the
ir short h lf-life dict tes th t cyclotron should
e re dily v il
le. The sc
nning is of p rticul r use in studying the rel tionship
etween cere
r l
lood o
w, oxygen utiliz tion nd extr ction in foc l isch emi or inf rction. Both tech
niques h ve
een used for the investig tion of epilepsy nd to study the
iologi
c l ctivity of
r in tumours, especi lly gliom s, in order to differenti te low
-gr de tumours from high-gr de nd post-r di tion necrosis from recurrent tumour
.
Electroenceph logr phy
Electroenceph logr phy (EEG) records the spont neous electric l ctivity of the

r in. The det ils re descri
ed in the ch pter on epilepsy (Ch pter 21). The m
jor indic tions for EEG recordings in neurologic l pr ctice re: suspicion of ep
ilepsy in new p tient ssessment of epilepsy in p tient with recurrent seizu
res ssessment of the risk of epilepsy in p tient
R dio-isotope studies
Isotope
r in sc nning for the initi l di gnosis of cere
r l tumour is now o
s
olete. However isotope sc nning using the th llium isotope m y
e helpful in dis
tinguishing recurrent gliom from
NEUROSURGICAL INVESTIGATIONS
25
who h s undergone intr cr ni l surgery or following severe he d injury s n
id in the di gnosis of herpes simplex enceph litis nd CreutzfeldtJ ko
dise se.

Nerve conduction studies/electromyogr phy

The electric l ctivity within p rticul r muscle is recorded
y needle electro
myogr phy. Nerve conduction studies me sure the electric l ctivity occurring wi
thin p rticul r nerve. In electromyogr phy needle is inserted into muscle n
d the electric l ctivity ssessed; norm l muscle is electric lly silent t rest.
As the muscle contr cts motor unit potenti ls ppe r. This ctivity, which is se
en on volunt ry contr ction of the muscle, is known s the interference p ttern.
Neurop thy or myop thy will produce ch r cteristic
norm lities. Spont neous
ctivity t rest Fi
rill tion potenti ls re due to single muscle
re contr ction
nd indic te ctive denerv tion, e.g. neurop thy, motor neurone dise se, some my
op thies. F scicul tion spont neous contr ction of
undle of muscle
res. Slow
neg tive w ves preceded
y sh rp positive spikes known s positive sh rp w ves in
chronic lly denerv ted muscle. Motor unit potenti ls In neurop thy. Where there
is signi c nt denerv tion the surviving motor unit potenti ls re polyph sic with
l rge mplitude nd long dur tion. In myop thy the potenti ls re polyph sic wit
h sm ll mplitude nd short dur tion. Interference p ttern Neurop thy reduced in
terference due to diminished motor units. Myop thy interference p ttern norm l.
Nerve conduction studies me sure the l tency from the stimulus to the recording
electrodes (dist l l tency), mplitude of the evoked response nd conduction vel
ocity. The studies re useful in ssessing:

peripher l nerve injuries (Ch pter 17) peripher l nerve entr pment (Ch pter 17)

r chi l plexus injury (Ch pter 17) neurop thy myop thy (studies re norm l) mus
cul r dystrophy (studies re norm l).
Evoked potenti ls
Visu l, uditory nd som tosensory evoked potenti l monitoring m y
e of v lue i
n the detection of neurologic l nd neurosurgic l dise ses s well s providing
useful intr oper tive monitoring. Stimul tion of the sensory receptor will evoke
sign l in the ppropri te region of the cere
r l cortex. Visu l evoked potent
i l This involves retin l stimul tion using either stro
oscopic sh or n lter
n ting checker
o rd p ttern. The evoked visu l sign l is recorded over the occip
it l cortex. It is p rticul rly useful in the di gnosis of multiple sclerosis. I
ntr oper tive visu l evoked potenti l monitoring h s
een used
y some neurosurg
eons during pituit ry surgery to detect su
tle interference with the optic nerve
s nd chi sm
ut the technique is not suf ciently developed for gener l use t thi
s time. Br instem uditory evoked potenti l This stimul tes the uditory p thw y
s in the vesti
ulocochle r cr ni l nerve nd records the electric l ctivity in
the uditory cortex. The technique h s
een used in the detection of sm ll cous
tic neurom s
ut h s
een l rgely superseded
y high-qu lity MRI. Intr oper tive
recording h s
een performed during coustic tumour surgery nd microv scul r d
ecompression oper tions
ut its use is limited. Som tosensory evoked potenti l T
his involves sensory evoked potenti l recording over the cortex in response to s
timul tion of peripher l nerve nd h s
een used in the detection of lesions w
ithin the sensory p thw ys, p rticul rly the
r chi l plexus, spin l cord or
r
instem. The technique is used in some
26
CHAPTER 2
neurosurgic l units during complic ted spin l nd v scul r surgery s n dditio
n l intr oper tive monitoring technique.
Further re ding
Chien D, Edelm n RR (1992) B sic principles nd clinic l pplic tions of m gneti
c reson nce ngiogr phy (Review). Semin rs in Roentgenology 27, 5362. DuBoul y GH

(1965) Principles of X-R y Di gnosis of the Skull. Butterworth, London. Edelm n

RR, W r ch S (1993) M gnetic reson nce im ging (Review I). New Engl nd Journ l
of Medicine 328, 708716.
Edelm n RR, W r ch S (1993) M gnetic reson nce im ging (Review II). New Engl nd
Journ l of Medicine 328, 785791. Fishm n RA (1980) Cere
rospin l Fluid in Dise se
s of the Nervous System. W B S unders, Phil delphi . McCom
JG (1983) Recent res
e rch into the n ture of cere
rospin l uid form tion nd
sorption. Journ l of N
eurosurgery 59, 369383. Stevens JM, V lentine AR (1987) M gnetic reson nce im gin
g in neurosurgery. British Journ l of Neurosurgery 1, 405426. T ver s JM, Wood EH
(1986) Di gnostic Neuror diology, 2nd edn. Willi ms & Wilkins, B ltimore.
CHAPTER 3
3
R ised intr cr ni l pressure nd hydroceph lus
R ised intr cr ni l pressure
R ised intr cr ni l pressure is m jor clinic l fe ture of m ny neurologic l il
lnesses. It is most import nt neurologic l condition, requiring prompt di gnos
is nd often needing urgent tre tment.
P thophysiology
The mech nisms of r ised intr cr ni l pressure re
est understood
y considerin
g the norm l physiology of pressure within the intr cr ni l c vity. The norm l s
upine intr cr ni l pressure is 1015 mmHg, me sured t position equ l to the lev
el of the for men of Monro. The intr cr ni l pressure is directly rel ted to the
volume of the intr cr ni l contents within the skull. The
sis of the MonroKell
ie doctrine is th t the cr ni l c vity is rigid sphere lled to c p city with no
ncompressi
le contents nd th t n incre se in the volume of one of the constitu
ents will le d to rise in intr cr ni l pressure. In 1783, Alex nder Monro (the
third Monro to
ecome Professor of An tomy in Edin
urgh nd the son of the m n
who descri
ed the connection
etween the l ter l nd 3rd ventricles) pu
lished h
is o
serv tions on the intr cr ni l contents. Forty ye rs l ter, Kellie m de o
s
erv tions th t ppe red to support Monros hypothesis, lthough t th t time neith
er w s w re of CSF. Su
sequently, the MonroKellie doctrine h s
een gener lly c
cepted
ut with the qu li c tion th t the cr niospin l intr dur l sp ce is ne rly
const nt in volume nd its contents re ne rly incompressi
le.
The intr cr ni l contents re:
r in CSF
lood. The rel tive volumes of the cont
ents re shown in Fig. 3.1. R ised intr cr ni l pressure m y
e due to: incre se
d volume of norm l intr cr ni l constituents sp ce-occupying lesion. The incre
se in volume of norm l intr cr ni l contents m y
e due to:
r in cere
r l oede
m
enign intr cr ni l hypertension CSF hydroceph lus
lood volume v sodil t tio
n due to hyperc pni . The incre se in volume of intr cr ni l contents will deter
mine the rise of intr cr ni l pressure. Figure 3.2 shows the intr cr ni l pressu
re volume rel tionship. Initi lly, sm ll incre se in the volume of the intr cr
ni l contents c uses no rise in pressure; sm ll mount of CSF c n move into th
e spin l su
r chnoid sp ce, which is very slightly distensi
le. However, the sk
ull
eing rel tively closed cont iner, critic l volume is soon re ched when
sm ll rise in intr cr ni l volume will result in n exponenti l rise in pressu
re. The rel tionship of the volume to pressure is descri
ed in terms of compli n
ce or el st nce of the intr cr ni l sp ce. Compli nce is expressed s dV/dP nd
is the mount of give v il
le within the intr cr ni l sp ce. El st nce is the in
verse of compli nce nd is the resist nce 27
28
CHAPTER 3

Gli 700-900ml Blood 100-150ml ECF 100-150ml CSF 100-150ml 1 Neurones 500-700ml
Fig. 3.1 The volume of the intr cr ni l contents.
3
2
Intr cr ni l pressure
4
Volume
Fig. 3.2 Intr cr ni l pressure ch nges rel ted to the volume of the intr cr ni l
contents.
offered to exp nsion of m ss or of the
r in itself (Fig. 3.3). A
r in th t h
s sm ll degree of compli nce, i.e. very little give within the intr cr ni l sp
ce, would
e re ected
y sm ll ch nge in volume producing l rge ch nge in intr
cr ni l pressure. This is the situ tion on the vertic l portion of the volume/p
ressure curve, where the compli nce is s id to
e low nd the el st nce is high.
Experiments using Rhesus monkeys, nd involving the gr du l exp nsion of n ext
r dur l
lloon, showed th t the vertic l section of the curve could
e shifted
to the left with either more r pid in tion of the
lloon or p thologic l ch nges
, such s experiment lly produced
r in swelling, th t reduced the mount of dis
pl ce
le CSF
efore the
lloon w s exp nded.
Fig. 3.3 Br in herni tions. A l ter l supr tentori l m ss will c use displ cemen
t of the l ter l ventricles with: (1) su
f lcine herni tion of the cingul te gyr
us
elow the f lx cere
ri; (2) herni tion of the uncus into the tentori l hi tus
; (3) c ud l displ cement of the
r instem. R ised pressure within the posterior
foss m y c use herni tion of the cere
ell r tonsils into the for men m gnum (4
). (Ad pted from Jennett & Te sd le (1981). Reproduced with permission.)
insult such s su
r chnoid h emorrh ge, this
ility is compromised nd the cer
e
r l perfusion pressure (CPP)
ecomes virtu lly dependent on the me n rteri l
pressure. Norm l cere
r l
lood ow is
out 800 ml/min or 20% of the c rdi c outp
ut. The cere
r l
lood ow is function of the CPP nd the cere
r l v scul r resi
st nce (CVR): CBF = CPP/CVR The cere
r l perfusion pressure is function of the
systemic me n rteri l pressure (MAP) nd the intr cr ni l pressure (ICP): CPP
= MAP - ICP Thus in order to m int in cere
r l perfusion in the presence of r is
ed ICP, the systemic
lood pressure needs to
e elev ted.
Cere
r l
lood ow
Between physiologic l r nges in
lood pressure, the
r in is
le to m int in
const nt cere
r l
lood ow. This is chieved
y process c lled utoregul tion w
here
y the
r in djusts the intr cr ni l v scul r resist nce
y ltering vessel
di meter nd tone. Following severe cere
r l
RAISED INTRACRANIAL PRESSURE AND HYDROCEPHALUS
29
Cere
r l herni tion
Depending on the c use of the r ised intr cr ni l pressure or the position of th
e intr cr ni l m ss,
r in herni e m y occur s shown in Fig. 3.3. The three m j
or herni tions of the
r in re descri
ed s: 1 Tr nstentori l. 2 For men m gnum
. 3 Su
f lcine. Tr nstentori l herni tion involves displ cement of the
r in nd
herni tion of the uncus of the tempor l lo
e through the tentori l hi tus, c us

ing compression of the 3rd cr ni l nerve nd mid
r in. The 3rd cr ni l nerve is
ffected, initi lly on the ipsil ter l side, nd in most c ses compression of th
e pyr mid l tr cts in the crus cere
ri c uses contr l ter l hemip resis. However
, l ter l displ cement of the
r instem m y result in the opposite crus cere
ri

eing compressed g inst the sh rp rigid tentori l edge, indenting the crus (Ker
noh ns notch), nd c using n ipsil ter l hemip resis. Simil rly, the posterior c
ere
r l rtery m y
e kinked, c using cere
r l isch emi resulting in hemi nop
i . Compression of the
r instem nd the reticul r ctiv ting system will result
in deterior tion of conscious st te le ding to com , hypertension nd
r dyc
rdi (the Cushing response) nd respir tory f ilure, often
eing initi lly m nif
est
y CheyneStokes periodic
re thing (T
le 3.1). Incre sed pressure within the
posterior foss
will result in herni tion of the cere
ell r tonsils into the for men m gnum nd
compression of the medull . If this is slowly progressive the p tient m y develo
p n
norm l neck posture nd child with posterior foss tumour m y h ve
he d tilt. Neck stiffness results from irrit tion of the dur round the for men
m gnum. Compression of the medull m y c use r pid respir tory f ilure, which i
s m nifest s pnoe or
norm lities of respir tory r te nd rhythm, such s Ch
eyneStokes
re thing. These m y occur without signi c nt imp irment of conscious st
te. The pressure from the herni ted tonsils m y c use
rupt lim
p resis nd s
ensory distur
nce. Progressive r ised intr cr ni l pressure c uses further down
w rd herni tion of the
r instem into the for men m gnum or coning. This results i
n she ring of the perfor tors supplying the
r instem nd h emorrh ge within (Du
ret h emorrh ges). The descent of the intr cr ni l contents with r ised intr cr
ni l pressure c uses tr ction d m ge to the pituit ry st lk, resulting in di
et
es insipidus. With progressive herni tion nd destruction in the
r instem the p
upils ch nge from dil ted nd xed to midsize nd unre ctive. These re inv ri
ly
irreversi
le events le ding to
r instem de th.
Clinic l symptoms nd signs of r ised intr cr ni l pressure
The common c uses of r ised intr cr ni l pressure re: sp ce-occupying lesion ce
re
r l tumour ( nd oedem ),
scess, intr cr ni l h em tom hydroceph lus
enign
intr cr ni l hypertension. The clinic l fe tures will
e determined in l rge p
rt
y the underlying c use of the r ised pressure. However, some of the clinic l
symptoms nd signs will
e the s me, no m tter wh t the c use of the r ised pre
ssure. The m jor fe tures re: he d che n use nd vomiting drowsiness p pilloed
em .
T
le 3.1 Tr nstentori l herni tion. Compression of 3rd cr ni l nerve c using in
iti l dil t tion of the ipsil ter l pupil Compression of the mid
r in Hemip resi
s, usu lly contr l ter l Occ sion l compression of opposite crus cere
ri c uses
ipsil ter l hemip resis Hypertension,
r dyc rdi Cushing response Respir tory f
ilure Compression of posterior cere
r l rtery
30
CHAPTER 3
He d che. The he d che ssoci ted with incre sed intr cr ni l pressure is usu ll
y worse on w king in the morning nd is relieved
y vomiting. Intr cr ni l press
ure incre ses during sleep, pro

ly from v scul r dil t tion due to c r
on diox
ide retention. The c use of the he d che in r ised intr cr ni l pressure is pro


ly tr ction on the p in-sensitive
lood vessels nd compression of the p in-se
nsitive dur t the
se of the cr nium. N use nd vomiting. The n use nd vom
iting is usu lly worse in the morning. Drowsiness. As is often repe ted in this

ook, drowsiness is the most import nt clinic l fe ture of r ised intr cr ni l p
ressure. It is the portent of r pid neurologic l deterior tion nd must never
e

rushed side s simply sleepiness, or dis ster will lmost cert inly occur. P pi
lloedem . The de nitive sign of r ised intr cr ni l pressure, p pilloedem is due

to tr nsmission of the r ised pressure long the su
r chnoid she th of the opti
c nerve. The oedem of the nerve he d, which m y lso
e due to o
struction of
xopl smic ow, results initi lly in lling in of the optic cup nd dil t tion of the r
etin l veins. The experienced o
server will
e
le to note th t there is f ilur
e of the norm l puls tions of the retin l veins nd venous congestion. As the pr
essure rises the nerve he d
ecomes more swollen nd the disc m rgins will
ecom
e
lurred on fundoscopic ex min tion. Fl me-sh ped h emorrh ges develop, p rticu
l rly round the disc m rgins nd longside the vessels. In severe p pilloedem

lo
h emorrh ges nd exud tes ppe r. Long-st nding p pilloedem from prolonged r
ised intr cr ni l pressure will su
sequently develop into second ry optic trop
hy. Cushing re ex. As intr cr ni l pressure rises, in order to m int in const nt
CPP, there h s to
e compens tory rise in the systemic
lood pressure. A hype
rtensive response is therefore elicited which is cl ssic lly ssoci ted with

r dyc rdi . This is termed the Cushing re ex in
honour of the eminent neurosurgeon H rvey Cushing who rst descri
ed it. Sixth ner
ve p lsy, c using diplopi , m y occur in r ised intr cr ni l pressure due to str
etching of the 6th nerve
y c ud l displ cement of the
r instem. This is so c
lled f lse loc lizing sign, s it need not occur on the side of the prim ry lesio
n. In n inf nt, r ised intr cr ni l pressure will c use tense,
ulging font n
elle. Other clinic l m nifest tions of r ised intr cr ni l pressure m y result f
rom
r in herni tion, s descri
ed
ove, nd from the m ss lesion th t h s c us
ed the rise in pressure.
Me surement of intr cr ni l pressure Monitoring nd recording the intr cr ni l p
ressure w s rst descri
ed in the e rly 1960s
y Lund
erg nd L ng tt nd within d
ec de w s
eing extensively used in clinic l pr ctice. The indic tions for monit
oring the intr cr ni l pressure v ry consider
ly in neurosurgic l pr ctice. The
most common indic tions re: He d injury (Ch pter 4). Following m jor intr cr n
i l surgery, when me surement of the intr cr ni l pressure m y help in the m n g
ement of p tients. In p rticul r, fter posterior foss surgery e rly detection
of prolonged rise in intr cr ni l pressure will indic te evolving hydroceph lu
s nd the possi
le need for CSF shunt or ventricul r dr in. In the ssessment
of dementi nd
enign intr cr ni l hypertension, descri
ed l ter in the ch pter
. The m jor
norm lities in the pressure re: elev tion of the
seline intr cr
ni l pressure the development of pressure w ves. Norm l intr cr ni l pressure h
s
seline
etween 10 nd 15 mmHg, with sm ll puls tions due to respir tion
nd the c rdi c pulse. The norm l mplitude of the com
ined c rdi c nd respir to
ry v ri tion is pproxim tely 35 mmHg. As the intr cr ni l pressure incre ses the
pulse pressure will incre se. When the pressure is r ised
norm l pressure w v
es m y occur. Pl te u w ves, descri
ed
y Lund
erg s A w ves, re pressure w ves

ove 50 mmHg nd
RAISED INTRACRANIAL PRESSURE AND HYDROCEPHALUS
31
( )
involve resection of sp ce-occupying lesion, or in the c se of hydroceph lus,
CSF shunt. In n emergency situ tion, when the p tient h s
ecome com tose nd
h s f iling respir tion, it is essenti l th t the p tients ventil tory st te is
urgently m int ined nd this will necessit te the p ss ge of n endotr che l tu

e nd ventil tory support. While the p tient is
eing tr nsferred for de nitive tr
e tment of the r ised pressure it m y
e possi
le to tempor rily lower the intr
cr ni l pressure
y hyperventil tion which will reduce rteri l CO2 nd diminish
v sodil t tion, nd
y the dministr tion of diuretic such s m nnitol or fru
semide (furosemide) (Ch pter 4).
(
)

Hydroceph lus
Hydroceph lus is n
norm l enl rgement of the ventricles due to n excessive
ccumul tion of CSF resulting from distur
nce of its ow,
sorption or, uncommo
nly, secretion. The norm l volume of CSF is 140 ml. CSF is produced
y the choro
id plexus in the ventricles t r te of 0.4 ml per minute (or
out 500 ml in 2
4 hours). The CSF ows from the l ter l ventricles through the for men of Monro in
to the 3rd ventricle, vi the queduct of Sylvius into the 4th ventricle nd the
n through the for min of M gendie nd Luschk into the su
r chnoid sp ce nd

s l cisterns. The CSF circul tes throughout the spin l su
r chnoid sp ce nd t
he
s l cisterns up through the tentori l hi tus. It ows over the cere
r l hemis
pheres nd is l rgely
sor
ed
y the r chnoid villi of the dur l sinuses. Ther
e re num
er of w ys of cl ssifying hydroceph lus
ut the most useful cl ssi c t
ion system is: o
structive hydroceph lus when there is n o
struction to the ow o
f CSF through the ventricul r system communic ting hydroceph lus when there is n
o o
struction to the ow of CSF within the ventricul r system
ut the hydroceph lu
s is due either to o
struction to CSF ow outside the ventricul r system or to f i
lure of
sorption of CSF
y the r chnoid gr nul tions. The most common c uses
of hydroceph lus re: 1 O
structive hydroceph lus:
Fig. 3.4 Intr cr ni l pressure w ves. ( ) A w ves, or pl te u w ves, re elev tion
s of intr cr ni l pressure
ove 50 mmHg l sting 520 minutes. (
) B w ves h ve sm
ller mplitude nd short dur tion.
l st t le st 5 minutes,
ut often up to 20 minutes. They re lw ys p thologic
l nd re pro

ly due to incre sed cere
r l
lood ow nd
lood volume. B w ves re
sm ller in height nd h ve short dur tion (12 minutes) (Fig. 3.4). If they re
infrequent nd of low mplitude they m y
e norm l nding. The intr cr ni l pre
ssure m y
e recorded from the ventricle,
r in su
st nce, su
dur l or extr dur
l sp ce. The intr cr ni l c theters re tt ched
y tr nsducer to continuous
recorder. There re now numerous monitoring devices with v rious degrees of tec
hnic l sophistic tion. Every method h s its own p rticul r dv nt ges nd compli
c tions nd the type of monitoring performed will depend on the clinic l situ ti
on (e.g. size of the ventricles) nd the neurosurgeons preference. The m jor comp
lic tion from intr cr ni l pressure monitoring is infection nd the risk is dire
ctly proportion l to the dur tion of the monitoring.
M n gement of r ised intr cr ni l pressure The tre tment of r ised intr cr ni l
pressure will depend on the underlying c use. The de nitive tre tment involves rem
oving the c use. This m y
32
CHAPTER 3
( ) l ter l ventricle o
struction
y tumours, e.g.
s l g ngli gliom , th l mi
c gliom (
) 3rd ventricul r o
struction, due to colloid cyst of the 3rd ventric
le or gliom of the 3rd ventricle (c) occlusion of the queduct of Sylvius (eith
er prim ry stenosis or second ry to tumour) (d) 4th ventricul r o
struction du
e to posterior foss tumour, e.g. medullo
l stom , ependymom , coustic neurom .
2 Communic ting hydroceph lus: ( ) o
struction to ow of CSF through the
s l ci
sterns (
) f ilure of
sorption of CSF through the r chnoid gr nul tions over
the cere
r l hemispheres. The most common c uses of communic ting hydroceph lus
re infection (especi lly
cteri l nd tu
erculous) nd su
r chnoid h emorrh g
e (either spont neous, tr um tic or postoper tive). Other uncommon c uses re c
rcinom tous meningitis, incre sed CSF viscosity from high protein content nd
excessive secretion of CSF due to choroid plexus p pillom .
surviv l of very low
irth weight prem ture inf nts h s resulted in n incre se
in inf nts with hydroceph lus resulting from perin t l intr cr ni l h emorrh ge.

Hydroceph lus c n present s cute r ised intr cr ni l pressure
ut
ec use of
the rel tive distensi
ility of the inf nt skull the present tion c n
e more su

tle. The m jor clinic l fe tures in inf nts re: f ilure to thrive f ilure to c
hieve milestones incre sed skull circumference (comp red with norm l growth curv
es) tense nterior font nelle cr cked pot sound on skull percussion tr nsillumin t
ion of cr ni l c vity with strong light when severe, imp ired conscious level n
d vomiting setting sun ppe r nce due to lid retr ction nd imp ired upw rd g ze f
rom 3rd ventricul r pressure on the mid
r in tectum thin sc lp with dil ted vein
s.
Presenting fe tures
Hydroceph lus in inf nts The incidence of inf ntile hydroceph lus is pproxim te
ly 34 per 1000
irths nd most c ses re due to congenit l
norm lities. The inc
idence of hydroceph lus occurring s single congenit l disorder is 11.5 per 100
0
irths. Hydroceph lus occurring with spin
i d nd myelomeningocele v ries fro
m 1.5 to 2.9 per 1000
irths,
ut with pren t l screening nd fol te supplement
tion the incidence of spin
i d is decre sing (Ch pter 11). The most common cong
enit l c use is stenosis of the queduct of Sylvius. This is m jor c use of hy
droceph lus in children with spin
i d nd myelomeningocele who lso h ve Chi
ri type II m lform tion (Ch pter 11). Congenit l tresi of the for men of Lusch
k nd M gendie (D ndyW lker cyst) is r re c use. The cquired forms of hydroce
ph lus occur most frequently fter intr cr ni l
leeding, p rticul rly in prem t
ure inf nts, in meningitis nd
ec use of tumours. The m rked improvements in th
e
Adult hydroceph lus (Fig. 3.5) Adult p tients with hydroceph lus m y present wit
h either:
Fig. 3.5 Hydroceph lus of the l ter l nd 3rd ventricles due to queduct stenosi
s.
RAISED INTRACRANIAL PRESSURE AND HYDROCEPHALUS
33
cute onset nd deterior tion or gr du l onset nd slowly deterior tion.
progressive
R diologic l investig tion
The most import nt investig tion is either CT sc n (Fig. 3.5) or MRI of the
r
in (Fig. 3.6) which will show which ventricles re dil ted. If the l ter l vent
ricles nd 3rd ventricle re ll very dil ted, nd the 4th ventricle is sm ll, i
t is likely th t the o
struction is t the level of the queduct of Sylvius. An
enh nced CT sc n or MRI will help determine the c use, s it will
etter de ne the
presence of n o
structing tumour. In communic ting hydroceph lus ll the ven
tricles re dil ted. M gnetic reson nce im ging. In the s gitt l pl ne MRI is p
rticul rly helpful in showing queduct stenosis nd lesions round the 3rd ventr
icle c using o
structive hydroceph lus. Ultr sonogr phy. Ultr sonogr phy through
the open nterior font nelle is useful in ssessing ventricul r size in inf nts
nd m y o
vi te the need for repe ted CT sc ns. Pl in skull X-r y. M y demonstr
te spl yed sutures, erosion of the
ony
uttresses round the tu
erculum sell e
or copper
e ten ppe r nce to the inside of the c lv rium. Records of the he d
circumference nd its comp rison with
ody weight nd length centile ch rts re
n integr l p rt of postn t l follow-up of ny child.
Acute-onset dult hydroceph lus This type of present tion occurs p rticul rly in
p tients with tumours c using o
structive hydroceph lus, lthough it m y occur
with ny of the c uses of hydroceph lus nd n cute r pid neurologic l deterior
tion m y occur in p tients who h ve h d long-st nding chronic hydroceph lus. Th

e m jor presenting fe tures re due to the signs nd symptoms of r ised intr cr

ni l pressure s descri
ed e rlier: he d che vomiting p pilloedem deterior tion
of conscious st te. Upg ze will often
e imp ired due to pressure of the dil te
d 3rd ventricle on the superior colliculus of the tectum. Gr du l-onset dult hy
droceph lus This type of onset occurs less frequently th n the previous type in
p tients with o
structive hydroceph lus due to tumour. The symptoms of r ised
intr cr ni l pressure re only very gr du lly progressive nd l te di gnosis is
common. E rly fe tures in the dolescent involve deterior ting school perform nc
e s result of he d ches, f iling ment l function, memory loss nd
eh viour l
distur
nces. Endocrine
norm lities such s inf ntilism nd precocious pu
ert
y c n occur in ssoci tion with chronic hydroceph lus in older children nd dol
escents due to distur
nce of the hypoth l mus nd possi
le compression of the p
ituit ry gl nd. If the condition is unrecognized progressive visu l f ilure will
occur, second ry to p pilloedem nd optic trophy. As mentioned e rlier, cute
decompens tion m y occur nd the p tient m y suddenly develop r pid deterior
tion of conscious st te. In elderly p tients chronic form of hydroceph lus is
c lled norm l-pressure hydroceph lus; this is descri
ed l ter in the ch pter.
Tre tment
In gener l, the tre tment of hydroceph lus is CSF shunt or 3rd ventriculosto
my. If there h s
een r pid neurologic l deterior tion this will need to
e perf
ormed s n emergency. If the hydroceph lus is due to n o
structing tumour th t
is surgic lly ccessi
le, resection of the m ss m y le d to resolution of the h
ydroceph lus nd shunt might not
e necess ry. Arrested hydroceph lus. This is
st te of chronic hydroceph lus in which the CSF pressure h s returned to norm
l nd there is no pressure gr dient
etween the cere
r l ventricles nd
r in p
renchym . It is uncommon nd is most likely to occur in communic ting hydroceph
lus. The p tients, often children nd dolescents, should
e followed c refully
with neurologic l ex min tions, IQ tests nd c reful ssessment of
34
CHAPTER 3
their development. If there is ny deterior tion of those p r meters shunt wil
l
e necess ry. It is interesting to
rie y review the history of surgic l tre tme
nt of hydroceph lus, s this common neurosurgic l pro
lem h s only rel tively re
cently
een solved. Until e rly this century numerous remedies h d
een employed
, including
lood-letting, he d-wr pping nd repe ted ventricul r t ps. In 1918
W lter D ndy introduced the technique of excising the choroid plexus to decre se
the form tion of CSF in p tients with progressive hydroceph lus. In 1922 he des
cri
ed n oper tion of 3rd ventriculostomy, in which hole w s m de in the thin
ned oor of the 3rd ventricle cre ting stul into the chi sm tic cisterns. Su
seq
uently, the procedure w s performed through the l min termin lis. In 1939 Torki
ldsen introduced the ventriculocisternostomy to tre t hydroceph lus c used
y le
sions o
structing the out ow from the 3rd ventricle. A thin ru

er tu
e w s insert
ed into the l ter l ventricle through n occipit l
urr hole nd p ssed su
cut n
eously into the cistern m gn . All these procedures h d signi c nt limit tions, r
isk of mor
idity nd su
st nti l f ilure r tes. The extr cr ni l shunting proced
ures relied on the development of ow-directed v lves, llowing CSF to ow in one di
rection without re ux through the c theter. This type of v lve w s invented
y n
engineer, Holter, nd w s rst used in 1952. Su
sequently, numerous improvements
nd modi c tions h ve
een m de nd t present there re l rge v riety of reli
l
e shunt devices.
( )
(
)
Oper tive procedure The usu l method of CSF diversion is ventriculoperitone l

shunt, in which c theter is pl ced into the l ter l ventricle nd is connected

to su
cut neous unidirection l pressure-regul ted
(c)
Fig. 3.6 MRI demonstr ting o
structive hydroceph lus. ( ) Axi l T1 MRI showing d
il ted l ter l ventricles. (
) Axi l T2 MRI showing hyperdense CSF in l ter l ve
ntricles. (c) Coron l T1 MRI with rrow pointing to dil ted 3rd ventricle.
RAISED INTRACRANIAL PRESSURE AND HYDROCEPHALUS
35
V lve loc ted in
urr hole
Ventricul r c theter in l ter l ventricle
Su
cut neous c theter
C theter in the peritone l c vity
Fig. 3.7 Di gr m of ventriculoperitone l shunt.
l ter l ventricle, nterior to the choroid plexus. Insertion of the c theter in
this w y minimizes the other m jor complic tion, shunt o
struction. As one of th
e m jor c uses of o
struction of the ventricul r c theter is
lock ge
y the cho
roid plexus it is wise to pl ce the perfor tions of the c theter into the front
l horn. The peritone l c theter c n
e thre ded su
cut neously
etween the
dom
in l nd cr ni l wounds using one of m ny devices. E ch c theter is joined to th
e v lve, which is then sutured in pl ce. After checking th t the system is funct
ioning properly, the peritone l c theter is pl ced within the peritone l c vity.
There re numerous shunt systems nd the type of shunt used, the p rticul r cli
nic l situ tion nd the neurosurgeons own preference result in m ny modi c tions of
this
sic system of impl nting ventriculoperitone l shunt.
v lve which is tt ched to c theter thre ded su
cut neously down to the
dome
n nd inserted into the peritone l c vity. Altern tive dr in ge sites such s th
e trium, pleur l c vity nd ureter h ve now
een l rgely
ndoned, except in e
xception l circumst nces. Modern v lves c n h ve their dr ining pressures djust
ed percut neously nd shunts re
eing developed llowing intr cr ni l pressure
to
e monitored percut neously. Technique of ventriculoperitone l shunt (Fig. 3.
7). The oper tion is performed under gener l n esthesi nd the shunt is usu ll
y inserted on the right side, so s to void interference with the domin nt hemi
sphere. The he d is turned to the left on the neurosurgic l he drest. The he d,
neck, chest nd
domen re sh ved, prep red with ntiseptic solution nd dr ped
. It is
solutely essenti l to m int in the most strict sterile techniques to
void the serious complic tion of infection of the shunt. A sm ll curviline r inc
ision is m de in the p rieto-occipit l re nd sm ll skin p elev ted. The per
itone l c vity is opened, either through tr nsverse rectus splitting incision
in the right hypochondrium or through midline incision. A
urr hole is perform
ed, the l ter l ventricle c nnul ted nd the ventricul r c theter inserted into
the l ter l ventricle so th t its tip lies in the front l horn of the
Postoper tive c re The postoper tive m n gement is simil r for ny intr cr ni l
procedure. Initi lly the p tient is nursed t, to void r pid decompression of th
e ventricul r system. Deterior tion of neurologic l st te or f ilure to improve
will require n urgent CT sc n to con rm th t the c theter h s
een pl ced ccur t
ely into the ventricul r system nd to exclude the possi
ility of intr cr ni l c
omplic tions such s intr cere
r l h em tom . Complic tions of ventriculoperiton
e l shunt The m jor possi
le complic tions re: infection of the shunt o
structi

on of the shunt intr cr ni l h emorrh ge. Shunt infection is dre ded complic t
ion with possi
le dis strous consequences, p rticul rly in p tients who re shun
t dependent. Avoid nce of this complic tion is ided
y: meticulous sterile tech
nique, including the use of no touch technique of the shunt nd tot l void nce
of skin cont ct with the shunt intr oper tive prophyl ctic nti
iotic medic tion
.
Infection The use of intr oper tive prophyl ctic nti
iotics
36
CHAPTER 3
during shunt pl cement is rel tively well su
st nti ted. Although the continu ti
on of the nti
iotics for 2436 hours postoper tively h s not
een proven to
e ef
fective, it is re son
le prec ution. An infected shunt lmost inv ri
ly need
s to
e removed nd repl ced
y new shunt, prefer
ly in different position
nd under ppropri te nti
iotic cover. O
struction The shunt m y f il to perfor
m s tisf ctorily due to
lock ge of the ventricul r c theter, m lfunction or
lo
ck ge of the v lve or o
struction of the peritone l c theter. The p tient will u
su lly present with recurrent symptoms of r ised intr cr ni l pressure nd in so
me c ses there m y
e n l rmingly r pid deterior tion of neurologic l st te. A
useful clinic l sign in the less cute c se of shunt m lfunction is f ilure of
upg ze due to pressure of the distended 3rd ventricle on the superior colliculus
. The di gnosis will usu lly
e con rmed
y CT sc n. Compression of the v lve is o
ften helpful in determining the position of the o
struction. If the ventricul r
c theter is
locked the contents of the pump c n
e expressed
ut the v lve will
re ll slowly. If the
lock lies in the v lve or the peritone l c theter the v lve
is usu lly not compressi
le. The tre tment of m lfunctioning shunt is explor
tion nd revision of the component th t is not functioning dequ tely. Intr cr n
i l h emorrh ge Intr cr ni l h em tom s m y occur following the insertion of v
entriculoperitone l shunt nd m y
e either: intr cere
r l or su
dur l. The intr
cere
r l h em tom will
e due to the tr um of the p ss ge of the ventricul r
c theter. Su
dur l h em tom s re p rticul rly likely to occur in p tients with
long-st nding severe hydroceph lus. If there is sudden decompression of the ve
ntricul r system the cortic l m ntle will f ll w y from the cr ni l v ult; this
m y c use rupture of
ridging vein nd the development of su
dur l h em tom
. Consequently, p tients thought to
e p rticul rly t risk should
initi lly
e nursed lying t nd should
e elev ted gr du lly. Some neurosurgeons
use shunts th t incorpor te n ntisiphon device to decre se the possi
ility of
siphoning effect c using further reduction of intr cr ni l pressure.
Other CSF shunts Also used sometimes re: ventriculo tri l shunts ventriculopleu
r l shunts lum
operitone l shunts. The ventriculo tri l shunt, in which the dist
l end is pl ced through the intern l jugul r vein into the right trium, is occ
sion lly necess ry when there h s
een m rked intr peritone l sepsis or multipl
e
domin l oper tions. It will occ sion lly
e necess ry to pl ce the shunt int
o the pleur l c vity. The lum
operitone l shunt involves dr in ge of the CSF fro
m the lum
r thec r ther th n the ventricle. This type of shunt c n only
e con
sidered in p tients with communic ting hydroceph lus. A c theter is thre ded per
cut neously into the lum
r thec nd then tunnelled su
cut neously to the nter
ior
domin l w ll nd pl ced into the peritone l c vity. The technique h s the
theoretic l dv nt ge th t the
r in is not m nipul ted,
ut the dis dv nt ge is
th t the shunts re not s reli
le nd re more dif cult to ssess if the p tien
t develops symptoms resem
ling m lfunction of the shunt. Third ventriculostomy I
n recent ye rs this old technique, origin lly performed
y W lter D ndy in 1922,
h s
een reintroduced for tre tment of o
structive hydroceph lus using n endos
copic technique. A ventriculoscope is introduced into the l ter l ventricle vi
front l
urr hole nd dv nced through the for men of Monro. The oor of the 3rd
ventricle just nterior to the m mill ry
odies is then fenestr ted, llowing C

SF to
yp ss ny o
struction in the CSF p thw y nd
e re
sor
ed
y the r chno

id villi.
Norm l-pressure hydroceph lus
Norm l-pressure hydroceph lus w s rst deRAISED INTRACRANIAL PRESSURE AND HYDROCEPHALUS
37
scri
ed s clinic l entity
y H kim nd Ad ms in 1965. They descri
ed group
of p tients with symptoms of dementi , t xi nd incontinence, where the r diol
ogic l studies showed hydroceph lus
ut the lum
r CSF pressure w s norm l.
Aetiology
The ex ct c use of the ventricul r dil t tion c nnot
e de ned in every c se. Howe
ver, in l rge percent ge the communic ting hydroceph lus m y h ve resulted fro
m o
liter tion of the su
r chnoid p thw ys in the
s l cisterns following n e
pisode of meningitis or su
r chnoid h emorrh ge, from either rupture of n neu
rysm, rteriovenous m lform tion or following tr um . Although lum
r puncture p
ressure is,
y de nition, within the norm l r nge, continuous monitoring of the in
tr cr ni l pressure in these p tients will frequently reve l
norm l w ve form
tion, especi lly t night.
Other investig tions, including isotope cisternogr phy, neuropsychologic l sses
sment nd CSF infusion studies, h ve
een used. These investig tions h ve high
f ilure r te in predicting which p tients will
ene t from CSF shunt. A
norm l
isotope cisternogr phy, with prolonged ventricul r retention of isotope nd slig
ht or del yed ow over the cere
r l convexity, is ssoci ted with improvement foll
owing shunting in only h lf to two-thirds of c ses of norm lpressure hydroceph
lus. Improvement in symptoms fter lum
r puncture nd remov l of CSF uid m y
e
good prognostic sign
ut f ilure to improve does not exclude the di gnosis. C
ontinuous intr cr ni l pressure monitoring is useful technique s it will excl
ude p tients with low intr cr ni l pressure nd m ke de nite di gnosis in those
p tients with intermittent w ves of r ised intr cr ni l pressure, which occur p
rticul rly t night. However, some p tients with intr cr ni l pressure in the hi
gh norm l r nge will lso
ene t from CSF shunt.
Clinic l present tion
The cl ssic clinic l tri d consists of: 1 Dementi . 2 At xi . 3 Incontinence. The
syndrome is progressive nd the distur
nce of g it, which m y
e the rst nd mos
t prominent symptom, is more of n pr xi th n true g it t xi . Urin ry inco
ntinence is common
ut not univers l. The dementi is simil r to th t seen in Al
zheimers dise se, with profound loss of short-term memory. The p tient does not u
su lly compl in of he d ches.
Tre tment
The m jor dif culty in the tre tment of norm lpressure hydroceph lus is in decidin
g which p tient should
e shunted. Dementi is dev st ting dise se with dis st
rous effects for the p tient nd profound soci l consequences for the p tients f
mily nd the gener l community. It is not surprising th t the neurosurgeon is fr
equently sked to consider p tients for shunt where the di gnosis is f r from
cert in. The slim ch nce th t n oper tive procedure might
e of some
ene t is of
ten considered worthwhile
y the p tient nd their rel tives. The following crit
eri c n
e used to ssess those p tients with the gre test ch nce of improvemen
t following shunt. A clinic l present tion of the cl ssic tri d, p rticul rly
if the fe tures of g it distur
nce predomin te. The CT sc n or MRI showing m rk
ed hydroceph lus with minim l cortic l trophy. A cle rly de ned c use for the hyd
roceph lus, such s p st episode of su
r chnoid h emorrh ge, tr um or mening
itis.

Investig tions
The CT sc n or MRI will show dil ted ventricles without signi c nt cortic l troph
y. The dif culty rises th t norm l-pressure hydroceph lus m y occur in p tients w
ith sc n ppe r nce of cortic l trophy,
ut in these p tients the degree of v
entricul r dil tion should
e more th n would
e expected just to compens te for
the degree of trophic ch nge.
38
CHAPTER 3
A
norm l pressure w ves on continuous intr cr ni l pressure monitoring. N tur ll
y, p tient who h s ll these positive criteri deserves shunt nd should m k
e good recovery following the oper tion. However, the usu l situ tion is p t
ient who presents with only few of these criteri nd the neurosurgeon needs t
o m ke c reful ssessment of whether shunt is truly ppropri te.
throm
osis. Prior to nti
iotic ther py chronic m stoiditis w s c use of pseud
otumour cere
ri s consequence of spre d of in mm tion to the sigmoid nd l ter
l sinuses. This is now rel tively r re occurrence
ut recent studies h ve sho
wn th t n occult venous sinus throm
osis m y pl y role in the development of
e
nign intr cr ni l hypertension.
Oper tive procedure The usu l oper tion is ventriculoperitone l shunt prefer

ly with progr mm
le v lve. If the di gnosis h s
een correct nd the shunt wo
rks s tisf ctorily, the p tient c n m ke striking recovery with lmost complet
e resolution of symptoms.
Presenting fe tures
Most p tients re o
ese fem les who present with: he d ches visu l distur
nce.
The he d ches h ve the fe tures of r ised intr cr ni l pressure in th t they re
worse in the morning nd ex cer
ted
y str ining, stooping nd coughing. The v
isu l pro
lems result from: p pilloedem second ry optic trophy diplopi due to
6th cr ni l nerve p lsy. The p pilloedem m y
e severe nd the visu l elds will
show enl rgement of the
lind spot. O
scur tions of vision m y occur, p rticul
rly on st nding or stooping, nd the swelling of the optic discs m y
e so sever
e s to le d to visu l f ilure nd ssoci ted second ry optic trophy. An unusu
l
ut well-recognized complic tion of
enign intr cr ni l hypertension is spont
neous CSF rhinorrhoe , usu lly ssoci ted with the empty sell syndrome (Ch pter
8).
Benign intr cr ni l hypertension
Benign intr cr ni l hypertension, lso known s pseudotumour cere
ri, is, s its
n me implies, dise se of r ised intr cr ni l pressure which usu lly runs se
lf-limiting course. Although termed
enign, this condition c n c use
lindness due
to severe p pilloedem . The p thogenesis is poorly understood. The condition us
u lly occurs in o
ese fem les.
Aetiology
The etiology is gener lly poorly understood nd the ex ct mech nisms of the r i
sed pressure re not known. The condition is found typic lly in young, o
ese wom
en, often with menstru l irregul rities or t king n or l contr ceptive pill, n
d n endocrine distur
nce h s
een suggested. However, c reful endocrine studie
s h ve f iled to show signi c nt endocrine
norm lities. In minority of p tient
s de nite precipit ting c use is found; these include: hypop r thyroidism vit mi
n A excess (used to tre t cne) pernicious n emi drug re ction tetr cycline, n
lidixic cid, sulf methox zole, indometh cin, d n zole, lithium c r
on te, or l
contr ceptive steroids. A simil r condition results from venous sinus

Investig tions
The CT nd/or MRI sc n will show no c use for the p pilloedem nd the ventricle
s will often
e sm ller th n usu l. Digit l su
tr ction cere
r l ngiogr phy or
m gnetic reson nce venogr phy m y
e performed to exclude throm
osis of venous
sinus s the c use. If the CT sc n or MRI shows no m ss or lesion lum
r punc
ture is usu lly performed; the pressure will
e r ised. CSF ex min tion is norm
l in
enign intr cr ni l hypertension
ut
iochemistry nd cytologic l investig
tions should
e performed to exclude underlying p thology.
RAISED INTRACRANIAL PRESSURE AND HYDROCEPHALUS
39
If there is dou
t s to the di gnosis continuous intr cr ni l pressure monitorin
g is occ sion lly performed in order to ssess the level of intr cr ni l pressur
e.
ing the p pilloedem . Recently endov scul r stenting of the n rrow regions of th
e tr nsverse sinus h s
een suggested s tre tment,
ut its s fety nd ef c cy
re s yet unproven in l rge clinic l tri ls.
Tre tment
Benign intr cr ni l hypertension is usu lly self-limiting dise se nd most c s
es respond to simple conserv tive tre tment. The usu l me sures undert ken re:
weight loss (the p tients re usu lly o
ese) stopping ny medic tion th t m y h
ve led to the dise se, e.g. or l contr ceptives, tetr cycline diuretic ther py
cet zol mide (reduces CSF production). Visu l cuity, visu l eld ex min tion (esp
eci lly size of the
lind spot) nd fund l photogr phy re essenti l to ev lu te
the progress of the dise se. If there is no improvement with the
ove me sures
, tre tment with glycerol or steroids m y
e tried. However,
oth of these medic
tions will tend to incre se o
esity. Some clinici ns recommend seri l lum
r pu
nctures
ut this is of limited v lue s the form tion of CSF quickly repl ces n
y th t is withdr wn. The m jor indic tions for surgic l tre tment re: persisten
t severe p pilloedem despite conserv tive me sures f iling vision intr ct
le h
e d ches despite conserv tive me sures. The surgic l procedures th t c n
e perf
ormed re: optic nerve she th decompression lum
operitone l shunt. If the sympto
ms re prim rily visu l nd he d che is not pro
lem then optic nerve she th de
compression m y
e useful. In this procedure sm ll window of dur is excised f
rom the optic nerve she th to decompress the optic nerve he d. If this procedure
is not successful in improving p pilloedem or reversing the f iling vision, or
if he d ches re m jor component of the dise se, then lum
operitone l shunt
c n
e performed. This oper tion is usu lly highly effective in reversing the s
ymptoms nd in improvFurther re ding
Ad ms RD et l. (1965) Symptom tic occult hydroceph lus with norm l cere
rospin
l uid pressure: tre t
le syndrome. New Engl nd Journ l of Medicine 273, 117126.
Beks JWF, Bosch DA, Brock M (1976) Intr cr ni l Pressure III. Springer Verl g,
Berlin. Bl ck P McL (1980) Idiop thic norm l pressure hydroceph lus. Journ l of
Neurosurgery 52, 371377. Cor
ett JJ et l. (1982) Visu l loss in pseudotumour cer
e
ri. Archives of Neurology 39, 461474. Greer M (1968) M n gement of
enign intr
cr ni l hypertension (pseudotumour cere
ri). Clinic l Neurosurgery 15, 161174. H
kim S, Ad ms RD (1965) The speci l clinic l pro
lem of symptom tic hydroceph lus
with norm l cere
rospin l uid pressure: O
serv tions on cere
rospin l uid hydrody
n mics. Journ l of Neurologic l Science 2, 307372. Jefferson A, Cl rk J (1976) Tr
e tment of
enign intr cr ni l hypertension
y dehydr ting gents with p rticul
r reference to me surement of the
lind spot s me ns for recording improvemen
t. Journ l of Neurology, Neurosurgery nd Psychi try 39, 627639. Jennett B, Te sd
le G (1981) M n gement of He d Injuries. Contempor ry Neurology Series. F A D v
is, Phil delphi . K ye AH, Bl ck P McL (2000) Oper tive Neurosurgery. Churchill

Livingstone, London, New York, Edin
urgh. L ng tt TW et l. (1964) Tr nsmission of

intr cr ni l pressure. I. Within the cr nio-spin l xis. Journ l of Neurosurger
y 21, 989997. L ng tt TW et l. (1964) Tr nsmission of intr cr ni l pressure. II. W
ithin the supr tentori l sp ce. Journ l of Neurosurgery 21, 9981005. Nulsen FE, S
pitz EB (1952) Tre tment of hydroceph lus
y direct shunt from ventricle to jugu
l r vein. Surgic l Forum 2, 339343. Shulm n K et l. (1980) Intr cr ni l Pressure
. IV. Springer Verl g, Berlin. Torkildsen A (1939) A new p lli tive oper tion in
c ses of inoper
le occlusion of the sylvi n queduct. Act Chirurgic Sc ndin
vic 82, 117119.
CHAPTER 4
4
He d injuries
He d injuries re m jor c use of mor
idity nd mort lity in the community. Tr
um is the third most common c use of de th in the United St tes, exceeded only

y c rdiocere
r l v scul r dise se nd c ncer. Tr um is the le ding c use of de
th in youth nd e rly middle ge nd the de th is often ssoci ted with m jor h
e d tr um . He d injury contri
utes signi c ntly to the outcome in over h lf of tr
um -rel ted de ths. There re pproxim tely 2.5 de ths from he d injury per 10
000 popul tion in Austr li nd neurotr um c uses pproxim tely 3.5% of ll de
ths. Ro d tr f c ccidents re responsi
le for
out 65% of ll f t l he d injurie
s in Austr li . There is wide spectrum of he d injury from mild concussion to
severe
r in injury resulting in de th. The m n gement of the p tient following
he d injury requires the identi c tion of the p thologic l processes th t h ve o
ccurred.
events ssoci ted with wh t w s considered irreversi
le
r in injury re potenti
lly prevent
le, or even reversi
le if tre tment is instituted e rly enough. Th
e distinction
etween prim ry nd second ry injury h s
ecome
lurred, nd the t
erms, whilst useful concepts, re now
ecoming o
solete. Most he d injuries resu
lt from
lunt tr um , s distinct from penetr ting wound of the skull nd
r i
n c used
y missiles or sh rp o
jects. The p thologic l processes involved in
he d injury re: direct tr um cere
r l contusion intr cere
r l she ring cere
r
l swelling (oedem ) intr cr ni l h emorrh ge hydroceph lus. Direct tr um . Altho
ugh penetr ting injuries produce most of their d m ge
y direct tr um to the
r
in this is not the c se with
lunt injuries, in which the energy from the imp c
t h s more widespre d effect. Cere
r l contusion. This m y occur loc lly under
the position of the imp ct lthough it usu lly occurs more severely t dist n
ce from the re of imp ct s result of contre-coup injury. As the
r in is mo

ile within the cr ni l c vity the sudden cceler tion/deceler tion force will r
esult in the opposite poles of the
r in
eing j mmed g inst the cr ni l v ult. A
sudden
low to the
ck of the he d will c use the tempor l lo
es to slide cro
ss the oor of the middle cr ni l foss nd the front l lo
es cross the oor of the
P thophysiology of he d injury
The m n gement of he d injury h s
een
sed on the concept of prim ry nd secon
d ry
r in injury. The prim ry
r in injury w s de ned s the irreversi
le p tholo
gy sust ined t the time of the tr um , where s the second ry
r in injury h s

een considered the su
sequent or progressive
r in d m ge th t occurs due to n
evolving p thology following the injury. It h s
een the gener l contention th t
the prim ry injury is irreversi
le, nd m n gement should
e directed t preven
ting or tre ting second ry p thology (such s cere
r l swelling, hydroceph lus
nd intr cere
r l h em tom ). However, it is now cle r th t some of the
iochemic
l 40
HEAD INJURIES

41

nterior cr ni l foss , c using contusion on the undersurf ce of those lo
es nd

to the tempor l nd front l poles of the
r in s they strike the sphenoid ridg
e nd front l
ones, respectively. Cere
r l contusion consists of l cer ted h em
orrh gic
r in, nd
urst tempor l lo
e m y result when the tempor l pole h s
e
en severely injured. She ring forces. Intr cere
r l she ring forces occur s r
esult of the differenti l
r in movement following
lunt tr um , frequently in c
onjunction with contre-coup type of injury. The rot tion l cceler tion follow
ing injury will c use she r forces th t result in petechi l h emorrh ges (p rtic
ul rly in the upper
r instem, cere
rum nd corpus c llosum), nd te ring of xo
ns nd myelin she ths. The e rly p thologic l ch nges consist of retr ction
ll
s or microgli l sc rs, nd if the p tient lives for num
er of months
efore de
th then widespre d degener tion of myelin will
e pp rent t postmortem. Cere

r l swelling. This occurs following tr um , either in foc l p ttern round n
intr cere
r l h em tom or diffusely throughout the cere
rum nd/or cere
ellum.
The n ture of the p thologic l processes re not cle rly understood
ut involve
distur
nce of v somotor tone c using v sodil t tion nd cere
r l oedem . In
ddition, cere
r l contusion nd petechi l h emorrh ges will contri
ute to the
r
in swelling. Intr cr ni l h emorrh ge. Intr cr ni l h emorrh ge following tr um
is discussed in more det il in Ch pter 5 nd m y
e: intr cere
r l su
dur l ex
tr dur l. The intr cr ni l h em tom or cere
r l swelling m y c use the types of
cere
r l herni tion descri
ed in Ch pter 3. The medi l surf ce of the hemispher
e m y
e pushed under the f lx (su
f lcine), the uncus nd hippoc mp l gyrus of
the tempor l lo
e m y herni te through the tentorium c using pressure on the 3rd
nerve nd mid
r in (Fig. 4.1), or there m y
e c ud l disFront l lo
e
Mid
r in distorted Posterior cere
r l rtery
3rd nerve deformed Tempor l lo
e H em tom Tr nstentori l herni tion of uncus of
tempor l lo
e
Fig. 4.1 Herni tion of the uncus nd hippoc mp l gyrus of the tempor l lo
e into
the tentori l notch c using pressure on the 3rd nerve nd mid
r in. (Ad pted fr
om Jennett & Te sd le, 1981. Reproduced with permission.)
pl cement of the
r instem nd/or cere
ellum, herni ting into the for men m gnum
. Hydroceph lus. This occurs infrequently in the e rly st ges fter he d injur
y. It m y
e due to o
struction of the 4th ventricle
y
lood, swelling in the p
osterior foss , or the result of tr um tic su
r chnoid h emorrh ge c using o

struction to the
sorption of CSF nd resulting in communic ting hydroceph lu
s. This l tter type of hydroceph lus is n uncommon,
ut import nt, c use of del
yed neurologic l deterior tion either in the weeks following the he d injury or
some ye rs l ter (see Norm l-pressure hydroceph lus, Ch pter 3).
Concussion
Concussion involves n inst nt neous loss of consciousness s result of tr um
. The term concussion w s introduced
y P re nd is derived from the L tin concuter
e which me ns to sh ke. In 1941 Denny-Brown nd Russell showed th t concussion w
s produced
y
low on the cr nium when it w s free to move nd su
sequent stud
ies showed th t the cceler tion/deceler tion of the he d resulted in she r str
ins, contre-coup injury, petechi l nd punct te h emorrh ges throughout the
r i
nstem, cere
r l hemispheres nd corpus c llosum, nd neuron l injury, the extent
depending on the force of the
42
CHAPTER 4

imp ct. The term concussion is not strictly de ned with respect to the severity of
the injury. However, minimum criterion is th t the p tient will h ve h d pe
riod of mnesi . The retrogr de mnesi of most cere
r l concussion is usu lly s
hort term, l sting less th n 1 d y. The initi l retrogr de mnesi m y extend ov
er much longer period
ut it gr du lly shrinks down. A more reli
le ssessmen
t of the severity of the he d injury is the post-tr um tic mnesi . If the mnes
i following the he d injury l sts more th n 1 d y then the concussion is reg rd
ed s
eing severe.
the nerve is e sily d m ged
y torsion or herni tion of the
r in. The 3rd nerve
. This m y lso
e d m ged
y direct tr um or
y
r in herni tion, the herni te
d uncus of the tempor l lo
e either impinging on the mid
r in or directly stretc
hing the nerve. The optic nerve. This is infrequently injured
y direct tr um .
Associ ted injuries
Cr ni l nerves The cr ni l nerves m y
e injured s result of either direct tr
um
y the skull fr cture, movement of the
r in, or cere
r l swelling.
The olf ctory nerves. These re the most commonly ffected nd this m y
e s
result of either fr cture through the nterior cr ni l foss , directly ffecti
ng the tr cts, or te ring of the delic te nerve rootlets p ssing through the cri

riform pl te c used
y the sudden
r in movement, p rticul rly from
low to t
he
ck of the he d. The 8th nerve. D m ge to this nerve is often ssoci ted wit
h fr cture of the petrous tempor l
one. De fness m y
e conductive, due to
h emotymp num, or sensorineur l s result of injury to the inner e r or to the
nerve itself. Vertigo nd nyst gmus re due to vesti
ul r nerve or end-org n d
m ge nd usu lly resolve within few months of the injury. F ci l p r lysis. Th
is is usu lly ssoci ted with fr cture through the petrous tempor l
one, lth
ough this m y only
e evident on highresolution CT sc n using the
one window. I
t m y
e either immedi te, s result of direct compression of the nerve, or de
l yed, due to
leeding nd/or swelling round the nerve. The 6th cr ni l nerve.
This h s long course from the
r instem to its entry into Dorellos c n l nd
Skull fr ctures Tr um m y result in skull fr ctures which re cl ssi ed s: simpl
e line r fr cture of the v ult depressed when the
one fr gments re depressed

ene th the v ult compound when there is direct communic tion with the extern
l environment. This m y result from either l cer tion over the fr cture or
fr cture of the
se of the skull which will
e compound
ec use there will
e
direct connection outside the v ult, usu lly vi the ir sinuses. Sc lp l cer t
ions The extent of the sc lp l cer tion does not necess rily indic te the degree
of tr um to the underlying
r in. Other injuries The most common ssoci ted in
juries re to the chest, skelet l nd c rdiov scul r systems.
Initi l m n gement of he d injury
The key spects in the m n gement of p tients following he d injury involve: cc
ur te clinic l ssessment of the neurologic l nd other injuries determin tion o
f the p thologic l process involved the concept th t ch nge in the neurologic
l signs indic tes progression or ch nge in the p thologic l processes. Immedi
te tre tment t the site of the injury involves r pid restor tion nd m inten
nce of n dequ te irw y, ventil tion, essenti l circul tory resuscit tion, rst
id tre tment of other
HEAD INJURIES
43
injuries nd the urgent tr nsfer of the p tient to hospit l. It is essenti l to
void hypoxi nd hypotension s these will c use further
r in injury.
Clinic l ssessment

It is fund ment l to the m n gement of the p tient to know of ch nges in the neu
rologic l condition s soon s possi
le. It is essenti l to scert in the type o
f ccident th t c used the he d injury nd the time injury occurred. An ccur te
ssessment of the p tients initi l neurologic l condition, l
eit in non-medic l
terms, c n
e o
t ined from
yst nders t the site of the ccident or from the
m
ul nce of cers.
different me nings to different o
servers. The ssessment is more ccur te nd r
eproduci
le if either the ex ct n ture of the response is descri
ed or, more sim
ply, the Gl sgow com sc le is used. The Gl sgow com sc le (T
le 4.1) gives
numeric l v lue to the three most import nt p r meters of the level of conscious
ness opening of the eyes,
est ver
l response nd
est motor response. The ex c
t response c n
e represented on ch rt (Fig. 4.2) or the level of consciousnes
s given s numeric l score the sum of the three p r meters of the Gl sgow com
sc le. A score of 8 or less indic tes severe injury. Pupill ry size nd re ct
ion C reful ev lu tion of the pupil size nd response to light is essenti l t t
he initi l clinic l ssessment nd during further o
serv tion. R ised intr cr ni
l pressure c using tempor l lo
e herni tion will c use compression of the 3rd n
erve, resulting in pupill ry dil t tion, which ne rly lw ys occurs initi lly on
the side of the r ised pressure. The pupil will t rst rem in re ctive to light

ut su
sequently will
ecome sluggish nd then f il to respond to light t ll.
As the intr cr ni l pressure incre ses this s me process commences on the contr
l ter l side. A tr um tic mydri sis will lso result from direct tr um to the e
ye, nd the dil ted pupil should not
e confused with th t due to 3rd cr ni l
nerve p lsy. Disorders of ocul r movement occur following he d injury s resul
t of injury to n extr ocul r muscle or its nerve supply, or due to distur
nce
of the conjug te g ze centres nd p thw ys. A destructive lesion of either fro
nt l or pontine g ze centre results in tonic over ction of the opposite front lpo
ntine p thw y for horizont l eye movement, c using ipsil ter l devi tion of the
eyes with front l lo
e lesion nd contr l ter l g ze devi tion with pontine le
sions. The oculoceph lic m noeuvre nd c loric stimul tion re import nt tests o
f function l ctivity of the
r instem reticul r form tion. The oculoceph lic re
sponse should only
e performed fter cervic l spine fr cture h s
een exclude
d. The he d is r ised 30 nd rot ted from
Neurologic l ex min tion An ccur te neurologic l ex min tion will help to deter
mine the type nd position of the p thologic l process nd provide
seline fo
r comp rison with su
sequent ex min tions. Although full neurologic l ex min t
ion should
e undert ken, speci l emph sis should
e given to the: conscious st
te pupill ry size nd re ction foc l neurologic l signs in the lim
s.
The conscious st te If the p tient will respond ver
lly n ssessment should
e
m de of the retrogr de mnesi nd post-tr um tic mnesi . There is continuum
of ltered consciousness
etween those p tients who re lert nd respond ppro
pri tely to ver
l comm nd nd those who re deeply unconscious. The rst sign of
depressed conscious st te is drowsiness, t which time the p tient m y
e e si
ly rous
le nd orient ted in time, pl ce nd person. As the level of conscious
ness deterior tes the p tient will
ecome confused nd more drowsy. The term com
is gener lly restricted to p tients who show no response to extern l stimuli, do
not o
ey comm nds, re un
le to utter comprehensi
le words, nd do not open th
eir eyes. However, the use of the words com , semicom or stuporose should
e voided,
s they convey
44
CHAPTER 4
T
le 4.1 The Gl sgow com sc le. P r meter Eye opening Response Spont neous To
speech To p in None Orient ted Confused In ppropri te Incomprehensi
le sounds No
ne O
eys comm nds Loc lize to p in Flexion to p in withdr w l Flexion
norm l E
xtension to p in None Numeric l v lue 4 3 2 1 5 4 3 2 1 6 5 4 3 2 1 315

Best ver
l response

Best motor response to p inful stimulus
TOTAL
side to side in the horizont l pl ne. In the norm l response the eyes m int in t
heir position in sp ce
y moving opposite to the rot tion of the he d. The ffer
ent impulses re from the cervic l nerve roots nd the semicircul r c n ls. C lo
ric testing should lso
e performed with the he d elev ted 30 so th t the horizo
nt l c n ls re positioned in the vertic l pl ne. Irrig tion with ice-cold w ter
c uses ipsil ter l tonic g ze devi tion. Skew devi tion involves divergence of
the eyes in the vertic l pl ne nd is sign of lesion within the
r instem. O
cul r
o

ing occurs only fter very severe he d injury resulting in pontine d
m ge. Foc l neurologic l signs in the lim
s Neurologic l ex min tion of the lim

s will ssess the tone, power nd sens tion. A hemip resis will result from n
injury of the corticospin l tr ct t ny point from the motor cortex to the spin
l cord. Following severe
r in injury the lim
s m y dopt n
norm l posturin
g ttitude. The decere
r te posture consists of the upper lim
s dducted nd inte
rn lly rot ted

g inst the trunk, extended t the el
ow nd exed t the wrist nd ngers, with the
lower lim
s dducted, extended t the hip nd knee with the feet pl nt r exed. T
here is continuum of severity of
r in injury with the decere
r te posturing r
esponse
eing p rti l nd unil ter l nd occurring only s result of p inful
stimulus to severe continuing
il ter l decere
r te rigidity. The posture pro


ly results from n upper
r instem injury. Less frequently, the upper lim
s m y

e exed, pro

ly due to the injury predomin ntly involving the cere
r l white m
tter nd
s l g ngli corresponding to posture of decortic tion. P rticul r
ttention must
e given to the p tients ventil tion,
lood pressure nd pulse. At
ll times it is essenti l th t c re is t ken to ensure the p tients ventil tion
is dequ te. Respir tory pro
lems m y result either s direct m nifest tion of
the severity of the he d injury or due to n ssoci ted chest injury. CheyneStok
es
re thing is due to either intrinsic
r instem d m ge or r ised intr cr ni l
pressure c using pressure nd distortion of the
r instem. Br dyc rdi nd hyper
tension, the Cushing response, re lso
HEAD INJURIES
45
Fig. 4.2 The st nd rd o
serv tion ch rt used t the Roy l Mel
ourne Hospit l nd
t m ny m jor tr um centres. The ch rt incorpor tes the Gl sgow com sc le.
46
CHAPTER 4

oth indic tive of
r instem compression due to r ised intr cr ni l pressure (Ch
pter 3). Pyrexi frequently occurs following he d injury. A temper ture l sti
ng for more th n 2 d ys is usu lly due to tr um tic su
r chnoid h emorrh ge or
m y occur in p tients with severe
r instem injury.
Gener l ex min tion C reful ssessment must
e m de of ny other injuries. Chest
, skelet l, c rdiov scul r or intr
domin l injury must
e di gnosed nd the p
propri te m n gement instituted. Hypotension or hypoxi m y ggr v te the
r in
injury, nd, if severe, will themselves c use
r in d m ge.
Cere
r l ngiogr phy is indic ted if c roticoc vernous

stul is suspected
y th

e presence of
ruit over the or
it or
y puls ting proptosis. C rotid or verte

r l ngiogr phy will
e necess ry if rteri l dissection is considered possi

ility. NB Full r diologic l ssessment of the cervic l spine utilizing pl in X-r
y nd CT sc n is essenti l in p tients who h ve sust ined signi c nt he d injur
y, p rticul rly if there re ssoci ted f ci l injuries.
Further m n gement of he d injury
Following the clinic l nd r diologic l ssessment the su
sequent m n gement wil
l depend on the intr cr ni l p thology nd the extent of ny neurologic l injury
.
R diologic l ssessment
Following the clinic l ev lu tion r diologic l ssessment will
e essenti l unle
ss the injury h s
een minor. The CT sc n will show the m croscopic intr cr ni l
injury nd should
e performed where: there is loss of consciousness (post-tr u
m tic mnesi ) of gre ter th n 10 minutes the p tient is persistently drowsy or
h s more seriously depressed conscious st te there is persisting n use or vom
iting there re l ter lizing neurologic l signs there is neurologic l or foc l d
eterior tion there is skull fr cture there is CSF rhinorrhoe there re ssoci t
ed injuries which will ent il prolonged ventil tion so th t ongoing neurologic l
ssessment is dif cult. The CT sc n will cle rly show the presence of intr cere
r
l or extr cere
r l h em tom , s well s cere
r l contusion, oedem nd inf rct
ion. Sm ll slit ventricles nd
sence of the
s l cisterns will indic te gener l
ized
r in swelling. The indic tions for skull X-r y h ve diminished since the
introduction of the CT sc n, especi lly s the
ony v ult c n
e ssessed
y th
e CT sc n using the
one windows. If CT sc n h s not
een performed, skull X-r
y is o
lig tory if there h s
een ny loss of consciousness or if the mech nism
of injury is suggestive of n underlying fr cture.
Minor he d injury
The p tient would
e ssessed s descri
ed
ove. Any p tient who h s suffered
he d injury must
e o
served for t le st 4 hours. The following re the minim
l criteri for o
lig tory CT sc n nd dmission to hospit l: loss of consciousne
ss (post-tr um tic mnesi ) of gre ter th n 10 minutes persistent drowsiness foc
l neurologic l de cits skull fr cture persisting n use or vomiting fter 4 hours
o
serv tion intr cr ni l p thology noted on CT sc n if the p tient does not h
ve dequ te c re t home. The further m n gement of these p tients will
e c ref
ul o
serv tion; the neurologic l o
serv tions should
e recorded on ch rt disp
l ying the fe tures of the Gl sgow com sc le. If there h s
een period of sig
ni c nt loss of consciousness, or if the p tient is drowsy, then the following me
sures should
e instituted to minimize the development of cere
r l swelling: ele
v tion of the he d of the
ed 20 mild uid restriction to 22.5 l/d y in n dult. Sh
ould the p tients neurologic l st te deterior te n immedi te CT sc n is essenti
l to reHEAD INJURIES
47
ev lu te the intr cr ni l p thology; further tre tment will depend on the outcom
e.
Severe he d injury
The m n gement of p tient following severe he d injury depends on the p tien
ts neurologic l st te nd the intr cr ni l p thology resulting from the tr um . I
n gener l, the following pply. 1 The p tient h s clinic l ssessment nd CT s
c n s descri
ed previously. 2 If the CT sc n shows n intr cr ni l h em tom c
using shift of the underlying
r in structures then this should
e ev cu ted imm
edi tely. 3 If there is no surgic l lesion, or following the oper tion, the m n
gement consists of: ( ) C reful o
serv tion using ch rt with the Gl sgow com

sc le. (
) Me sures to decre se
r in swelling; these include: (i) c reful m n g

ement of the irw y to ensure dequ te oxygen tion nd ventil tion. Hyperc pni
will c use cere
r l v sodil t tion nd so ex cer
te
r in swelling (ii) elev ti
on of the he d of the
ed 20 (iii) uid nd electrolyte
l nce. M inten nce of
tonic uid requirements, voiding dextrose solutions nd following resuscit tion
hould
e dministered until the p tient is
le to commence n sog stric feeding.
Blood loss from other injuries should
e repl ced with colloid or
lood nd not
with cryst lloid solutions. C re should
e t ken to void overhydr tion, s thi
s will incre se cere
r l oedem . Following gener l injury there is retention of
s lt nd w ter nd excretion of pot ssium. The retention of w ter is usu lly gre
ter th n the retention of sodium, resulting in mild hypon tr emi . Following
severe he d injury uid nd electrolyte
norm lities m y occur for v riety of
e sons. Severe hypon tr emi (sodium less th n 130 mmol/l) m y
e due to excessi
ve uid int ke or, occ sion lly,
ec use of in ppropri te excessive secretion of
ntidiuretic hormone (SIADH). The urine is usu lly hypertonic with high sodium
level, pro

ly

iso
s

s result of suppression of ldosterone secretion occurring s response to o

verhydr tion nd exp nsion of the circul ting volume. The term cere
r l s lt w st
ing, which h s
een pplied to this syndrome, is usu lly in ppropri te. Serum sod
ium of less th n 125 mmol/l m y produce neurologic l imp irment with depression
of conscious st te. If due to SIADH the usu l tre tment is to restrict uid int ke
to 800 ml per d y or less. Hypern tr emi is usu lly ssoci ted with hyperosmol
lity nd often results from in dequ te uid int ke. Other c uses re di
etes ins
ipidus, s result of hypoth l mic injury nd excessive use of osmotic gents f
or control of intr cr ni l pressure. Excessive dministr tion of some feeding mi
xtures m y le d to electrolyte
norm lities, p rticul rly when complic ted
y d
i rrhoe . (c) Temper ture control pyrexi m y
e due to hypoth l mic d m ge or t
r um tic su
r chnoid h emorrh ge. However, infection s c use of the fever mu
st
e excluded. The most common sites of infection fter he d injury re the r
espir tory nd urin ry tr cts, p rticul rly if urin ry c theter h s
een inser
ted. If the injury is compound, nd especi lly if there h s
een CSF le k, int
r cr ni l infection should
e suspected. The temper ture c n usu lly
e controll
ed using tepid sponges, nd rect l p r cet mol or spirin. Chlorprom zine, to

olish the shivering response, should
e dministered if cooling
l nket is req
uired. Every ttempt should
e m de to control the temper ture
ec use hyperther
mi c n elev te the intr cr ni l pressure, will incre se
r in nd
ody met
oli
sm nd will predispose to seizure ctivity. Although hypothermi h s
een dvoc
ted in the m n gement of severe he d injury no cle r-cut
ene t h s
een demonst
r ted. (d) Nutrition during the initi l 23 d ys the uid ther py will include 1.52 l
of 45% dextrose, providing 250400 c lories per d y. Proper nutrition l support sh
ould
e commenced fter 34 d ys. Feeding t this st ge is
est done
y intr g str
ic dministr tion,
48
CHAPTER 4
usu lly
y n sog stric tu
e, unless this is precluded
y other injuries. The n
sog stric feeding should supply 25003000 c lories per d y with c lorie : nitro
gen r tio of 180 : 1. The feeding should commence slowly, with dilute mixtures,
nd the stom ch should
e spir ted regul rly to prevent regurgit tion nd pulmo
n ry spir tion. (e) Routine c re of the unconscious p tient including
owel,
l
dder nd pressure c re. More ggressive methods to control intr cr ni l pressur
e re dvis
le if: the p tients neurologic l st te continues to deterior te nd
the CT sc n shows evidence of cere
r l swelling without n intr cr ni l h em tom
there is posturing (decere
r te) response to stimuli the Gl sgow com score
is less th n 8. In these p tients n intr cr ni l pressure monitor should
e ins
erted to ssess the intr cr ni l pressure s ccur tely s possi
le. A tr nsduce
r c n
e pl ced intr p renchym lly vi twist drill cr niostomy. A c theter pl

ced into the ventricle will give n ccur te re ding of the intr cr ni l pressur
e nd CSF c n
e dr ined to help in the control of the pressure. However, the di
s dv nt ges of n intr ventricul r c theter include dif culty of pl cement if the
ventricles re sm ll, possi
le injury to the
r in during pl cement nd infectio
n resulting in ventriculitis following prolonged monitoring. A su
dur l c theter
will lso give n dequ te me surement of the intr cr ni l pressure
ut m y
e
dif cult to insert s tisf ctorily if the
r in is swollen, nd will tend to
lock.
Extr dur l monitors re less ccur te, lthough s tisf ctory recordings re o
t
in
le with meticulous pl cement technique. An intr cr ni l pressure monitor wi
ll lso
e useful in p tients requiring prolonged sed tion nd ventil tion s
result of other injuries. Me surement of the intr cr ni l pressure will provide
nother useful monitoring p r meter nd ny sust ined rise in the pressure will

e n indic tion for c reful re ssessment nd, if necess ry, CT sc n. Following
the insertion of the intr cr ni l
pressure monitor the p tient will
e tr nsferred to the intensive c re dep rtmen
t. The techniques used to control intr cr ni l pressure re s follows. Controll
ed ventil tion, m int ining P CO2 t 3035 mmHg. Reduction of the P CO2 will reduc
e cere
r l v sodil t tion nd consequently decre se the intr cr ni l pressure. I
f the pressure rem ins elev ted despite hyperventil tion CSF c n
e dr ined from
ventricul r c theter if this h s
een inserted. Diuretic ther py utilizing in
termittent dministr tion of m nnitol or frusemide (furosemide) c n
e used if t
he preceding techniques h ve f iled to control the intr cr ni l pressure. M nnit
ol is n osmotic diuretic nd m y lso exert its effect
y incre sing serum osmo
l lity nd dr wing w ter out of the
r in. The usu l dose is 0.51.0 g/kg. The ser
um osmol lity should not exceed 320 mosmol/kg. B r
itur te ther py c n
e consid
ered if the intr cr ni l pressure is resist nt to tre tment with the
ove techn
iques. Pento
r
itone (thiopentone) when given s
olus dose (35 mg/kg) is freq
uently effective in tempor rily reducing the intr cr ni l pressure. There is pro


ly little v lue in using
r
itur te infusion t dose to control
urst supp
ression on EEG, lthough it h s
een postul ted th t this provides
r in protect
ion
y reducing cere
r l met
olism. Steroids. Although steroids dr m tic lly re
duce the oedem round cere
r l tumours they h ve little effect in controlling t
he
r in swelling following he d injury. Steroid medic tion is no longer consi
dered dvis
le s there is no proven
ene t for the p tient nd possi
le complic
tions, such s g strointestin l
leeding, poor wound he ling nd infection, m y
result from their dministr tion. Hypothermi . In some centres hypothermi (to r
educe cere
r l met
olism nd intr cr ni l pressure) h s
een dvoc ted, with co
oling the p tient to 34C. However there is no cle r evidence it is
ene ci l. Hyper

ric oxygen h s
een used in the p st,
ut without proven
ene t. Decompressive c
r niotomy involving remov l of l rge re of cr ni l v ult from the
HEAD INJURIES
49
front l nd tempor l regions
il ter lly h s
een dvoc ted s me ns of contro
lling r ised intr cr ni l pressure due to severe cere
r l swelling following h
e d injury. The technique is controversi l,
ut it needs to
e performed e rly f
ollowing the injury if it is to h ve ch nce of
eing of
ene t to the p tient. A
t present there re no clinic l tri ls proving its ef c cy in he d injury. There i
s some controversy concerning the effectiveness of the more ggressive technique
s to tre t p tients with severe he d injuries. If p tient h s suffered profo
und
r in injury nd the neurologic l ex min tion shows little or no rem ining

r instem function then it is o
vious th t the ggressive techniques will provide
no
ene t nd only del y the inevit
le. Simil rly, there re some p tients who h
ve suffered severe he d injury nd whose intr cr ni l pressure continues to r
ise despite ll the
ove techniques. Other p tients will h ve f t l
r in inj
ury without ny su
st nti l rise in intr cr ni l pressure, usu lly when the
r i
nstem h s
een the prim ry site of injury. However,
out 30% of p tients who h

ve suffered severe
r in injury will o
t in su
st nti l
ene t from control of t
he intr cr ni l pressure. Clinic l studies h ve not yet conclusively proven the
v lue of intr cr ni l pressure control in reducing mor
idity following
r in i
njury s it is thought th t the p tients in the studies th t were performed were
overventil ted nd the cere
r l
lood ow might h ve
een compromised. There is n
ow consensus th t reduction in r ised intr cr ni l pressure will not only decr
e se the mort lity
ut will improve the qu lity of the p tients outcome fter s
evere he d injury. Cere
r l perfusion pressure is vit l physiologic l p r mete
r in the m n gement of severely he d injured p tients. The cere
r l perfusion pr
essure me n rteri l BP minus me n intr cr ni l pressure should
e m int ined

ove 70 mmHg. Consequently he d injury m n gement involves ensuring th t the rte
ri l
lood pressure is m int ined whilst the intr cr ni l pressure is reduced. T
his often involves close cooper tion
etween the neurosurgeon nd the intensive
c re physici n.
M n gement of ssoci ted conditions
Sc lp injury Sc lp injuries m y include:
r sion confusion l cer tion su
g le l
h em tom . A l rge sc lp l cer tion m y result in consider
le
lood loss. When
the p tient rrives in the emergency dep rtment spurting rteries should
e contr
olled with h emost tic clips prior to the pplic tion of sterile
nd ge to th
e he d. The extent of the soft tissue sc lp injury m y not re ect the severity of
the underlying
r in injury. The principles of m n gement re simil r to those o
f soft tissue injury t other sites of the
ody nd the wound should
e closed w
ithout del y. The h ir should
e sh ved widely round the wound, which should
e
meticulously cle ned nd de
rided. The closure should
e performed in two l yer
s if possi
le, with c reful pposition of the g le prior to closing the skin. T
he skin sutures should pproxim te the cut edges of the skin nd c re should
e
t ken to void excessive tension which would c use skin necrosis nd wound
re k
down. Str ightforw rd, cle n sc lp l cer tions c n ne rly lw ys
e closed with
loc l n esthetic in ltr tion. However, if the sc lp wound h s resulted in loss of
soft tissue the wound m y need to
e extended to provide n extr p of he lthy ti
ssue so th t the skin edges c n
e pproxim ted without tension. Skull fr ctures
Simple line r fr cture. There is no speci c m n gement for simple skull fr ctur
e th t is undispl ced without n overlying skin injury. However, the presence of
fr cture is n indic tion th t the tr um w s not trivi l nd it should provi
de w rning th t h em tom m y develop
ene th the fr cture. The p tient shou
ld
e dmitted for o
serv tion nd CT sc n performed.
Compound fr cture. A skull fr cture m y
e compound either
ec use of n overlyi
ng sc lp
50
CHAPTER 4
Fig. 4.4 Depressed skull fr cture with underlying
r in contusion. Fig. 4.3 Depr
essed skull fr cture.
l cer tion or if it involves n ir sinus. The sc lp wound should
e de
rided n
d closed s descri
ed
ove. A short course of prophyl ctic nti
iotics should

e dministered to reduce the risk of infection. Depressed skull fr cture (Fig. 4
.3). A skull v ult fr cture is considered to
e signi c ntly depressed if the inne
r t
le fr gments re depressed
y t le st the thickness of the skull. A
out h
lf the injuries re due to ro d tr um nd most of the rem inder re due either
to o
jects f lling on the he d t work or to ss ult with he vy,
lunt instrum
ent. A depressed fr cture c used
y non-missile injury usu lly c uses only foc
l
r in d m ge, so th t m ny p tients never lose consciousness. If the underlyi
ng injured
r in is n eloquent re the p tient will exhi
it foc l neurologic l
signs. H emorrh ge from the
ony edges, the dur or underlying
r in tr um m y
result in n intr cr ni l h em tom which will c use progressive neurologic l d
eterior tion. If the depressed fr cture is compound nd the dur h s
een l cer

ted there is signi c ntly incre sed risk of intr cr ni l infection. If the depre
ssed skull fr cture is compound, prophyl ctic nti
iotics nd tet nus prophyl xi
s should
e dministered nd surgery, usu lly requiring gener l n esthetic, should
e performed s soon s possi
le. A preope
r tive CT sc n will show not only the position of the depressed skull fr gments

ut lso the presence of ny underlying intr cr ni l p thology (Figs 4.4 nd 4.5
). At oper tion the sc lp wound should
e cle ned nd de
rided, s descri
ed pre
viously, nd the
one fr gments elev ted. If the dur h s
een penetr ted, or if

one fr gments nd extern l foreign m teri l h ve
een driven down into the
r
in, this must
e meticulously de
rided nd h emost sis o
t ined. It is desir
le
th t the dur should
e closed nd this m y require the use of p tch of peric
r nium or f sci l t from the thigh. If the wound nd
one fr gments re he vil
y cont min ted, nd p rticul rly if there h s
een some del y in surgery, the
o
ne should not
e repl ced nd reconstructive cr niopl sty m y
e necess ry l t
er. If the depressed fr cture is closed there is no urgency in elev ting the
on
e fr gments, provided there is no underlying intr cr ni l complic tion. There is
controversy over whether depressed fr gment might le d to epilepsy due to con
tinued pressure on the
r in. In gener l, the depressed fr gments should
e elev
ted if: c reful studies using the
one windows on the CT sc n show th t the dur
might h ve
een penetr ted
HEAD INJURIES
51
Sphenoid sinus
Ethmoid sinus
Front l sinus
Fig. 4.6 Rel tionship of
se of skull to ir sinuses.
( )
(
)
Fig. 4.5 ( ,
) CT showing severe tr um resulting in multiple fr ctures, disrupt
ion of the or
it, intr cr ni l contusions involving the right tempor l lo
e nd
intr cr ni l ir.
there is signi c nt
r in compression the fr cture is compound there re cosmetic
consider tions such s front l fr cture in young child. The risk of epilepsy
following depressed fr cture is 15% for the whole group,
ut the risk r nges
from 3 to 70% depending upon other ssoci ted intr cr ni l p thology resulting f
rom the injury. Prophyl ctic nticonvuls nt medic tion should
e continued for 1
ye r if the dur h s
een penetr ted.
Cere
rospin l uid rhinorrhoe A fr cture involving the
se of the nterior cr ni
l foss m y c use te ring of the
s l dur resulting in stul into the front
l, ethmoid or sphenoid sinuses (Fig. 4.6). This type of stulous connection should
lso
e suspected if the p tient suffers from n episode of meningitis or if th
e r diologic l investig tions show fr cture in the ppropri te site. An intr c
r ni l erocele (Fig. 4.7) is proof of stulous connection. CSF rhinorrhoe m y
lso occur s result of stul through the tegmen tymp ni into the c vity of t
he middle e r, nd m y le k vi the eust chi n tu
e. B se of skull fr ctures re
rel tively frequent s skull fr ctures re often directed into the skull
se

y its
ony
uttresses. They re often occult r diologic lly
ut di gnosed clinic
lly. Anterior foss fr ctures m y open into the front l, sphenoid or ethmoid si
nuses, often running cross the cri
riform pl te. They present with: su
conjunct

iv l h emorrh ges extending to the posterior limits of the scler . Perior
it l h

em tom s or r coon eyes indic te su
g le l h emorrh ge nd not necess rily
se
of skull fr cture nosmi n s l tip p r esthesi e due to nterior ethmoid l ner
ve injury. Middle foss fr ctures involving the petrous tempor l
one present wi
th:
52
CHAPTER 4
Fig. 4.7 CT sc n showing intr cr ni l ir in su
r chnoid sp ce nd within the
l ter l ventricles.
CSF otorrhoe (or rhinorrhoe ) vi the eust chi n tu
e de fness due to 8th nerve
injury or ossicul r disruption h emotymp num B ttles sign
ruising over m stoid

one 7th nerve p lsy often del yed. The di gnosis of CSF rhinorrhoe m y
e dif cu
lt. In the e rly st ges following he d injury involving fr ctures to the f ci
l
ones, CSF needs to
e differenti ted from
loody n s l disch rge. Allergic
rhinitis is the m jor differenti l di gnosis in p tients presenting weeks or mon
ths fter he d injury. Testing for sug r or B2-tr nsferrin in the n s l disch
rge m y help to identify the uid s
eing CSF. CSF isotope sc ns using technetium
-99 injected through the lum
r thec re only likely to
e positive if there is
l rge le k. High-resolution CT sc nning following the dministr tion of intr
cistern l contr st m y help to identify the position of the hole. The m jor conc
ern of dur l stul is the risk of intr cr ni l infection, p rticul rly
cteri
l meningitis. A CSF le k m y not
ecome pp rent for few d ys fter the he d i
njury,
ut s the
r in swelling decre ses the dur l te r
ecomes
unplugged. Altern tively, CSF le k ge m y ce se due to
r in herni plugging the
hole in the dur nd
one. Although the
r in herni might stop the CSF esc pin
g it will not provide protection g inst future intr cr ni l infection, s the d
ur l defect will rem in. There is controversy concerning the indic tions for n
nterior cr ni l foss explor tion nd dur l rep ir,
ut there is gener l greem
ent th t surgery should
e performed if: CSF le k ge continues for more th n 5 d
ys, indic ting the stul is not trivi l there is n intr cr ni l erocele there
h s
een n episode of meningitis in p tient with fr cture of the nterior c
r ni l foss . P tients with possi
le dur l stul should
e pl ced on prophyl ct
ic nti
iotic medic tion. In gener l, penicillin is recommended s Pneumococcus
is the most common org nism; moxycillin is ppropri te in children due to the r
isk of H emophilus infection. Bro d-spectrum nti
iotics m y le d to the develop
ment of resist nt org nisms nd should
e voided. N s l sw
s m y indic te the
need for more individu lized nti
iotic prophyl xis. It is
est to del y surgery
for
out 2 weeks, until the initi l
r in swelling h s resolved. E rly surgery
, using cr niof ci l type of exposure, h s
een dvoc ted
y some neurosurgeon
s if there re ssoci ted m jor f ci l nd nterior v ult fr ctures. However, e
rly surgery m y result in further d m ge to n lre dy swollen front l lo
e duri
ng the retr ction necess ry to o
t in dequ te exposure of the dur l te r. The o
per tive procedure involves front l cr niotomy with rep ir of the dur l defect
using either pericr nium or f sci l t t ken from the thigh.
Cere
rospin l uid otorrhoe CSF otorrhoe m y occur s result of
se of skul
l fr cture involving the petrous tempor l
one. Unlike fr ctures of the nterior
cr ni l foss the le k ge ne rly lw ys settles nd the stul does not usu lly p
rovide route of infection, unless there is evidence of chronic middle e r infe
ction. Occ sion lly, persistent le k m y require surgic l explor tion nd rep
ir.
HEAD INJURIES
53

Cr ni l nerve injuries Injuries to the cr ni l nerves occurring directly s re

sult of the tr um re not helped
y surgery. Steroid medic tion is ppropri te
for p tients with del yed f ci l nerve p lsy following fr cture of the petrous
tempor l
one. Some otologists recommend surgic l decompression of the f ci l n
erve when the p lsy is del yed
ut, s the f ci l function ne rly lw ys recover
s, oper tive intervention is usu lly not justi ed. Post-tr um tic epilepsy The ind
ic tions for prophyl ctic medic tion following he d injury re discussed in Ch p
ter 21.
Missile injuries
Although most liter ture on missile injuries is rel ted to w rf re, these injuri
es re unfortun tely
ecoming more common in civili n con ict, p rticul rly s r
esult of the incre sed v il
ility of re rms. In gener l the cr ni l injury is d
irectly proportion l to the velocity of the missile. The high-velocity injury is d
e ned s resulting from missile tr velling f ster th n the speed of sound (1050
ft/s), nd modern ri e
ullets h ve muzzle velocity gre ter th n 3000 ft/s. Ther
e re three c tegories of missile injury: t ngenti l the missile does not enter
the cr nium
ut c uses depressed skull fr cture, l cer ting the sc lp with n
underlying cortic l contusion, l cer tion or h em tom s penetr ting the missile
enters the cr nium resulting in the deposition of met l,
one fr gments nd de
r
is within the
r in through- nd-through the missile enters nd exits the cr nium
, frequently cre ting more th n one tr ct due to fr gment tion The cr ni l injur
y is directly rel ted to the velocity of the missile. The energy dissip ted
y t
he missile equ ls MV2 where M is the m ss nd V the velocity of the missile. Mod
ern
llistics h s designed missiles to h ve m xim l velocity nd st
ility in ig
ht with m xim l dissip tion of energy upon imp ct. The prim ry missile frequentl
y fr gments nd c n c use further second ry missiles from fr gments of
one or m
et l.
The missile c uses cere
r l d m ge vi three mech nisms: mech nic l l cer tion o
f
r in tissue during tr nsit the shock w ve promulg ted he d of the missile c
vit tion in the w ke of the missile. The p thologic l processes involve sc lp in
jury, depressed skull fr cture, intr cr ni l h emorrh ge nd the intr cr ni l p
thologic l sequel e resulting from closed he d injury, including cere
r l contus
ion, h emorrh ge, swelling nd r ised intr cr ni l pressure. The p ttern of inju
ry will depend on the velocity of the we pon nd the tr jectory of the missile t
hrough the
one nd
r in. A high-velocity wound m y result in r pid incre se
of intr cr ni l pressure of more th n 3000 mmHg due to the tempor ry c vity
ou
t the missile, which might
e 50 times s l rge s the missile itself. The high
intr cr ni l pressure resulting from the c vit tion m y result in de th from f i
lure of the respir tory nd c rdi c centres in the
r instem.
M n gement
R pid tr nsport of the p tient to hospit l nd urgent tre tment is of p r mount
import nce. The e rly de nitive tre tment resulting from prompt tr nsport nd the
introduction of nti
iotics were the m jor f ctors in lowering mort lity from he
d wounds in the Kore n nd Vietn m w rs. The m n gement of the p tient fter tr
nsfer to hospit l is essenti lly the s me s descri
ed for severe he d injuries
previously. Anti
iotics should
e dministered immedi tely, in l rge intr venou
s doses
ec use of the risk of infection; penicillin nd chlor mphenicol were th
e most commonly used during the Vietn m con ict. Optim l nti
iotic dministr tion
should provide
ro d cover of Gr m-positive, Gr m-neg tive nd n ero
ic org
nisms. After neurologic l ssessment, CT sc n should
e performed to scert in
the position of the intr cr ni l h em tom s, depressed
one fr gments nd met l
fr gments. Surgery is not ppropri te if the p tient is
r in
54
CHAPTER 4

de d with no evidence of
r instem re exes. P tients with less severe injuries sho
uld h ve urgent surgic l intervention, p rticul rly s e rly explor tion reduces
the risk of su
sequent infection. The oper tion is performed under gener l n e
sthesi nd intr venous diuretic ther py is dministered to reduce intr cr ni l
pressure. A l rge sc lp p is designed, with excision nd de
ridement of the entr
y nd exit wounds. Meticulous c re is t ken to remove ny ccessi
le
one or met
llic fr gments. H em tom nd necrotic
r in de
ris re excised. A w tertight d
ur l closure should
e performed nd the sc lp should
e closed in two l yers (g
le nd skin). Following surgery, repe t CT sc n will identify ny further re
t ined
one or met llic fr gments. Accessi
le fr gments should
e removed,
ut i
sol ted deep or in ccessi
le
one or met llic fr gments re pro

ly
est left
s further neurologic l d m ge m y occur during n ttempt t excision of these p
rticles. In civili n pr ctice, infection is unlikely if explor tion h s t ken p
l ce within 2 hours of the injury. In gener l it is thought th t ret ined met ll
ic fr gments h ve less potenti l for infection th n other de
ris. Postoper tive
m n gement is simil r to th t descri
ed for severe he d injury, with p rticul r
ttention to controlling intr cr ni l pressure. Prophyl ctic nti
iotics nd nt
iconvuls nt medic tion re dministered.

nd soci l services. MRI now pl ys n import nt role in determining the chronici

ty of cere
r l injuries in inf nts. Collections of different chronicity, or in u
nusu l loc tions, should lert the physici n to the possi
ility of non- ccident
l injury.
Reh
ilit tion
Some form of reh
ilit tion is essenti l following ny signi c nt he d injury. If
the injury h s
een rel tively minor then the necess ry reh
ilit tion m y invol
ve only dvice nd re ssur nce to the p tient nd f mily. However, reh
ilit tio
n following severe he d injury will usu lly involve te m of p r medic l pers
onnel, including physiother pists, occup tion l ther pists, speech ther pists n
d soci l workers. The m jor groups of dis
ilities resulting from he d injury
re: imp irment of motor function hemip resis, qu drip resis, t xi , poor coord
in tion speech distur
nces dysph si , dys rthri imp irment of speci l senses v
ision, he ring cognitive distur
nce memory imp irment, intellectu l dis
ility,
person lity ch nge. The gener l ims of the reh
ilit tion process re: in the
initi l period, to prevent complic tions such s contr ctures of the lim
s nd t
o provide counselling for the f mily to m ximize the neurologic l recovery
y re
storing old skills nd te ching new skills this is usu lly undert ken in reh

ilit tion unit retr ining for future employment, if necess ry nd if possi
le. T
he reh
ilit tion process should commence s soon s possi
le fter the he d inj
ury nd should initi lly concentr te on preventing complic tions. Lim
contr ctu
res nd pressure sores re voided
y frequent p tient turning, physiother py n
d the use of splints. As the neurologic l st te improves the p tients reh
ilit t
ion will norm lly
e undert ken in reh
ilit tion unit. Orthotic devices will
ssist hemiplegic p tients to w lk nd, if they c n follow simple instructions,
most re
le to rele rn the ctivities of d ily living.
Non- ccident l he d injury
The inf ntile chronic su
dur l h em tom or effusion is distinct clinic l enti
ty. Birth tr um is frequent c use
ut in m ny c ses p st history is in dequ
te to est
lish the n ture of the injury with cert inty. Chronic su
dur l h em
tom s occur in pproxim tely 20% of
ttered children. The violent sh king of th
e imm ture
r in might
e suf cient to rupture
ridging veins or c use she ring t
the grey/white interf ce without evidence of extern l tr um . If n in dequ te
history is provided in such setting, it is import nt to screen for co gulop
thy, ex mine the fundi for retin l h emorrh ges, rr nge skelet l survey nd w
hen ppropri te involve p edi trici n
HEAD INJURIES

55
The speech ther pist m y provide v lu
le ssist nce for p tients with dys rthri
nd sw llowing dif culties. Form l speech ther py pro

ly does little to improv
e glo
l ph si
ut it does offer import nt psychologic l support for the p tie
nt with severe communic tion disorder. D m ge to the non-domin nt hemisphere r
esults in perceptu l distur
nces, p rticul rly rel ting to visu l sp ti l t sks
. Although the perceptu l pro
lems m y resolve with time nd reh
ilit tion, the
pro
lems ssoci ted with cognitive distur
nces nd lter tion of person lity m
y persist. F mily counselling nd support is essenti l to help the rel tives un
derst nd nd cope with these long-term dis
ilities.
Further re ding
Becker DP, Miller JD, W rd JD, Green
erg RP, Young HF, S k l s R (1977) The outc
ome from severe he d injury with e rly di gnosis nd intensive m n gement. Journ
l of Neurosurgery 47, 491502. Bl ckwood W, Corsellis JAN, eds (1976) Green elds Neu
rop thology. Edw rd Arnold, London. Cushing H (1908) Surgery of the he d. In: Ke
n WW, ed. Surgery Principles nd Pr ctice. W B S unders, Phil delphi , Vol 3, 2
17276. Cushing H (1918) Notes on penetr ting wounds of the
r in. British Medic l
Journ l 1, 2226. Gurdji n ES, Thom s RS (1964) Surgic l m n gement of p tient
with he d injury. Clinic l Neurosurgery 12, 5674. Hol
ourn AHS (1943) Mech nisms
of
r in injuries. L ncet ii, 438441. J mieson KG, Yell nd JD (1975) Surgic l rep
ir of nterior foss
ec use of rhinorrhoe , erocele or meningitis. Journ l of
Neurosurgery 39, 328331. Jefferson A, Reilly G (1972) Fr ctures of the oor of the

nterior cr ni l foss . The selection of p tients for dur l rep ir. British Jour
n l of Surgery 59, 585592. Jennett B, Miller JD (1972) Infection fter depressed
fr cture of the skull. Implic tions for m n gement of non-missile injuries. Jour
n l of Neurosurgery 36, 333339. Jennett B, Miller J D, Br km n R (1974) Epilepsy
fter non-missile depressed skull fr cture. Journ l of Neurosurgery 41, 208216.
Jennett B, Te sd le G (1981) M n gement of He d Injuries. Contempor ry Neurology
Series. F A D vis, Phil delphi . Johnston IH, Johnston JA, Jennett B (1970) Int
r cr ni l pressure ch nges following he d injury. L ncet ii, 433436. K ye AH, Bl
ck P McL (2000) Oper tive Neurosurgery. Churchill Livingstone, London, New York,
Edin
urgh. L ng tt TW (1978) Me suring the outcome from he d injuries. Journ l of
Neurosurgery 48, 673678. Levy ML, M sri LS, L vine S, Apuzzo M (1994) Outcome pr
ediction fter penetr ting cr niocere
r l injury. Neurosurgery 35, 7785. Plum F,
Posner JB (1972) The Di gnosis of Stupor nd Com , 2nd edn. F A D vis, Phil delp
hi . Rosner MJ, Rosner SD, Johnson AH (1995) Cere
r l perfusion pressure: M n ge
ment protocol nd clinic l results. Journ l of Neurosurgery 83, 949962. Russell W
R, Schiller F (1949) Crushing injuries of the skull: Clinic l nd experiment l o

serv tions. Journ l of Neurology, Neurosurgery nd Psychi try 12, 5260. Stone JL
, Lichtor T, Fitzger ld LF (1995) Gunshot wounds to the he d in civili n neurosu
rgery. Neurosurgery 37, 11041112. Te sd le G, Jennett B (1974) Assessment of com
imp ired consciousness. L ncet ii, 8184. W lsh FB, Hoyt WF (1969) Clinic l Neuro
-ophth lmology, Vol 3. Willi ms & Wilkins, B ltimore.
CHAPTER 5
5
Tr um tic intr cr ni l h em tom s
Intr cr ni l h em tom form tion following he d injury is the m jor c use of f t
l injuries in which de th m y h ve
een potenti lly void
le nd in which m ny
survivors re unnecess rily dis
led following he d injury due to del y in th
e ev cu tion of the h em tom . The incidence of intr cr ni l h em tom s nd the
type of h em tom v ries widely depending on the different dmission policies. I
n gener l hospit ls th t receive n unselected series of p tients, the incidence

v ries
etween 1 nd 5% of ll he d injuries, while the incidence will
e much

higher in speci list neurosurgic l centres.
m y occur in the presence of severe he d injury nd coexist with severe prim
ry
r in injury, the import nt fe ture of n extr dur l h em tom is th t it m
y occur when the injury to the underlying
r in is either trivi l or negligi
le.
Distri
ution of extr dur l h em tom s
The most common sites for extr dur l h em tom re the tempor l region followed

y the front l re . Posterior foss nd p r s gitt l extr dur l h em tom s re
rel tively uncommon. The rel tive proportions in consecutive series of 200 c s
es from The Roy l Mel
ourne Hospit l re shown in Fig. 5.1. In most c ses the h
emorrh ge is from torn middle meninge l rtery or its
r nches
ut h em tom s
m y lso develop from h emorrh ge from extr dur l veins, the superior s gitt l s
inus, tr nsverse sinus or posterior meninge l rtery, the l st two
eing respons
i
le for the posterior foss extr dur l h em tom s. A fr cture overlies the h em
tom in ne rly ll (95%) dults nd most (75%) children.
Cl ssi c tion of tr um tic intr cr ni l h em tom s
The gener l cl ssi c tion depends on the rel tionship of the h em tom to the dur
nd
r in. H em tom s c n
e: extr dur l su
dur l intr cere
r l. However, m ny
h em tom s occupy more th n one of the intr cr ni l sites (T
le 5.1).
Extr dur l h em tom
Extr dur l h em tom s re more likely to occur in the younger ge group s the d
ur is
le to strip more re dily off the underlying
one. In p tients under 20
ye rs of ge, extr dur l h em tom s ccount for
out two-thirds of ll tr um ti
c intr cr ni l h em tom s,
ut represent less th n 5% of h em tom s in p tients
over the ge of 50. Although n extr dur l h em tom 56
Clinic l present tion
As previously mentioned, n extr dur l h em tom m y occur s result of seve
re he d injury nd the h em tom will then
ecome m nifest s further deterior
tion of the neurologic l st te, p rticul rly with l ter lizing fe tures involvi
ng 3rd nerve p lsy (dil t tion of the pupil) nd progressive hemip resis. More
frequently the extr dur l h em tom
TRAUMATIC INTRACRANIAL HAEMATOMAS
57
T
le 5.1 Position of tr um tic intr cere
r l h em tom s. Extr dur l only (%) 16
Extr dur l nd intr dur l (%) 7 Su
dur l only (%) 22 Su
dur l nd intr cere
r l
(%) 34 Intr cere
r l only (%) 20
Series Intern tion l Coll
or tive Study (Gl sgow, Rotterd m, Groningen, Los Ang
eles) Bris
ne (J mieson & Yell nd) Mel
ourne (The Roy l Mel
ourne Hospit l)
13 13
11 9
34 29
36 31
6 18
7

11 8
9
66
Deterior ting conscious st te. This is the most import nt neurologic l sign, p r
ticul rly when it develops fter lucid interv l. It is essenti l th t the drowsi
ness th t occurs in p tient following he d injury is not misinterpreted just
s the p tient wishing to sleep. It is well to remem
er the nursery rhyme: Its r
ining, its pouring, The old m n is snoring, He
umped his he d nd went to
ed,
And couldnt get up in the morning. This is cl ssic description of n extr dur l
h em tom le ding to drowsiness nd de th. Foc l neurologic l signs. These will
depend upon the position of the h em tom . In gener l, tempor l h em tom wil
l produce progressive contr l ter l sp stic hemip resis nd n ipsil ter l dil
ted pupil. Further progression will result in
il ter l sp stic lim
s, decere

r te posture nd
il ter lly dil ted pupils (see Ch pter 4, Fig. 4.1). Occ sion
lly the hemip resis m y initi lly
e ipsil ter l, due to compression of the con
tr l ter l crus cere
ri of the tentori l edge,
ut it is r re for the opposite p
upil to
e involved rst. Ch nge in vit l signs. The ch nge in vit l signs shows t
he cl ssic Cushing response to rise in intr cr ni l pressure
r dyc rdi ccom
p nied
y n incre se in
lood pressure. Distur
nces in respir tion will develo
p into CheyneStokes p ttern of
re thing. Extr dur l h em tom s occurring t ot
her th n
Fig. 5.1 Frequency of sites of extr dur l h em tom s in The Roy l Mel
ourne Hosp
it l series of 200 consecutive c ses.
occurs following he d injury th t h s resulted in only tr nsient loss of con
sciousness nd in pproxim tely one-qu rter of c ses there h s
een no initi l l
oss of consciousness. In these p tients the most import nt symptoms re: he d ch
e deterior ting conscious st te foc l neurologic l signs (dil ting pupil, hemip
resis) ch nge in vit l signs (hypertension,
r dyc rdi ). He d che. This is the
outst nding initi l symptom in p tients who h ve not lost consciousness or who h
ve reg ined consciousness. The he d che incre ses in severity nd is followed

y vomiting.
58
CHAPTER 5
deterior tion m y
e so r pid th t there is not suf cient time for CT sc n nd t
he p tient should
e tr nsferred immedi tely to the oper ting the tre. Infusion
of m nnitol (20% solution, 1 g/kg) or frusemide (20 mg intr venously) m y tempor
rily reduce the intr cr ni l pressure during the tr nsfer to the oper ting the
tre. If unconscious, the p tient must
e intu
ted nd hyperventil ted during th
e tr nsfer. It is essenti l th t there should
e no del y in ev cu ting the h em
tom . An extr dur l h em tom is surgic l emergency which will result in de t
h if not removed promptly.
Fig. 5.2 Extr dur l h em tom with the typic l hyperdense
iconvex ppe r nce.
the tempor l position show modi c tions of this clinic l present tion. Front l h e
m tom s show evidence of l ter lizing signs l te in their evolution, the predomi
n nt fe tures
eing deterior tion of consciousness nd pupil
norm lities. In
the posterior foss the vit l signs tend to
e ffected e rly, followed
y ch
nge in conscious st te. The pupils nd lim
s m y not
e ffected until the p ti
ent
ecomes deeply unconscious. H em tom s in the posterior foss m y c use sudd
en respir tory f ilure.

R diologic l investig tions The CT sc n is the r diologic l investig tion of cho

ice nd must
e performed urgently if n extr dur l h em tom is considered po
ssi
ility. The CT sc n will show the typic l hyperdense (white)
iconvex h em to
m (Fig. 5.2) with compression of the underlying
r in nd distortion of the l t
er l ventricle.
Tre tment
The tre tment of extr dur l h em tom is urgent cr niotomy with ev cu tion of th
e clot. The p tient should h ve n urgent CT sc n s soon s n extr dur l h em
tom is suspected clinic lly. In some c ses the r te of neurologic l
Oper tion for extr dur l h em tom The type of oper tion performed will depend o
n the circumst nces in which the p tient is
eing tre ted. 1 If CT sc n h s
e
en performed nd the position of the h em tom is known, the skin p will
e lift
ed directly over the h em tom . 2 If the p tients neurologic l st te is st
le or
only slowly progressive nd if the surgeon is tr ined in neurosurgic l oper tio
ns, form l cr niotomy c n
e performed over the site of the h em tom . 3 A cr
niectomy, r ther th n cr niotomy, should
e performed: ( ) if the surgeon is i
nexperienced (
) if cr niotomy instruments re not v il
le (c) if the r te of
neurologic l deterior tion h s
een so r pid th t time h s not permitted CT sc
n to
e performed. Explor tory
urr holes should
e inserted rst in the tempor l
region nd then in the front l nd p riet l re s (Fig. 5.3). When the h em tom
is identi ed the
urr hole incision should
e extended nd the
one over the reg
ion of the h em tom should
e r pidly removed. If the h em tom is not found on
the rst side th t is explored
urr holes should
e performed in the s me order o
n the other side. The following re guidelines for the position of the h em tom
if CT sc n h s not
een performed: (i) it underlies the fr cture (th t m y h
ve
een seen on the skull X-r y) (ii) it underlies
oggy swelling on the skull
TRAUMATIC INTRACRANIAL HAEMATOMAS
59
( )
(
)
(c)
Fig. 5.3 Emergency surgery for suspected extr dur l h em tom . ( ) Position of e
xplor tory
urr holes. (
) If h em tom found in the tempor l position the skin
wound is extended. (c) Further
one removed to en
le complete ev cu tion of h e
m tom nd h emost sis.
(iii) it is on the s me side s the pupil th t dil ted rst (iv) in 85% of c ses i
t is on the contr l ter l side to the hemip resis. Following remov l of the
one
of the v ult
y cr niotomy or cr niectomy it is e sy to ev cu te the h em tom .
The origin l source of the h em tom , usu lly the
leeding middle meninge l rt
ery in the tempor l h em tom , is controlled
y di thermy or with h emost tic
clip. The h em tom will h ve stripped w y the dur from the inner t
le of the
v ult, often resulting in consider
le oozing from the dur l surf ce. The dur
should
e opened, if CT sc n h s not previously
een performed, to exclude the
coexistence of su
dur l h em tom . It should then
e closed in w tertight f
shion. It is usu lly dvis
le to insert closed-system, lowpressure extr dur
l dr in to ev cu te ny
lood th t m y continue to ooze. The postoper tive c re
is simil r to th t for ny other intr cr ni l procedure. If the neurologic l st
te f ils to improve following the ev cu tion of the h em tom , or if there is fu
rther deterior tion, nother CT sc n should
e performed to exclude recurrence o
f the h em tom form tion. Prognosis. If the initi l he d injury h s resulted in
only tr nsient loss of consciousness, the p tient should m ke full recovery

following remov l of the extr dur l h em tom , provided the h em tom h s
een
ev cu ted e rly enough to prevent perm nent neurologic l dis
ility. The d m ge
c used
y n extr dur l h em tom is
potenti lly reversi
le, provided the h em tom is ev cu ted
efore pressure from
the
lood clot h s c used second ry intr cr ni l p thologic l effects.
Su
dur l h em tom
Su
dur l h em tom s h ve
een cl ssi ed into cute, su
cute nd chronic, dependin
g on the time they
ecome clinic lly evident following injury: cute su
dur l h
em tom less th n 3 d ys su
cute su
dur l h em tom 421 d ys chronic su
dur l h
em tom more th n 21 d ys. The CT sc n en
les further cl ssi c tion depending o
n the density of the h em tom rel tive to the dj cent
r in. An cute su
dur l
h em tom is hyperdense (white) nd chronic su
dur l h em tom is hypodense.
Between the end of the 1st week nd the 3rd week the su
dur l h em tom will
e
isodense with the dj cent
r in.
Acute su
dur l h em tom
The cute su
dur l h em tom frequently results from severe tr um to the he d
nd commonly rises from cortic l l cer tions. However, n cute su
dur l h em to
m c n result from less severe tr um c used
y rupture of
ridging vein or
foc l te r of cortic l rtery, especi lly if the p tient h s
een ntico gul t
ed for other medic l re sons (e.g. for tri l
rill tion). C ses
60
CHAPTER 5
of spont neous cute su
dur l h em tom h ve
een reported nd in these p tients
it is essenti l to exclude ruptured neurysm or
leeding di thesis s c use
. Acute su
dur l h em tom s re
il ter l in pproxim tely one-third of c ses, i
n comp rison with less th n 3% of extr dur l h em tom s. An cute su
dur l h em
tom often presents in the context of p tient with severe he d injury whose
neurologic l st te is either f iling to improve or deterior ting. The fe tures o
f deterior ting neurologic l st te decre se in conscious st te nd/or incre se i
n l ter lizing signs should r ise the possi
ility of su
dur l h em tom . The C
T sc n will show the ch r cteristic hyperdense h em tom , which is conc ve tow r
ds the
r in, with compression of the underlying
r in nd distortion of the l t
er l ventricles (Fig. 5.4). Over 80% of p tients with cute su
dur l h em tom s
h ve fr cture of either the cr ni l v ult or the
se of the skull, which m y

e evident on the
one windows of the CT sc n. A cr niotomy is ne rly lw ys neces
s ry to ev cu te n cute su
dur l h em tom . If the h em tom is liquid the
lo
od c n sometimes
e
w shed out with gentle irrig tion through
urr holes. However if
leeding persis
ts cr niotomy will
e required for h emost sis.
Chronic su
dur l h em tom in the dult
In 1863, Virchow rst proposed chronic in mm tion of the meninges s
eing the c us
e of chronic su
dur l h em tom . In 1914, Trotter suggested tr um s the eti
ologic l f ctor nd in 1932 G rdner, nd l ter Zollinger nd Gross, proposed th
t n osmotic gr dient occurred from the
re kdown of h emoglo
in. However, it w
s su
sequently shown th t the osmol rity of the h em tom did not ch nge with ti
me nd so this theory w s
ndoned. Chronic su
dur l h em tom c n
e divided i
nto two m jor groups. The rst involves p tients h ving suffered signi c nt, nd o
ften severe he d injury. However, in pproxim tely onethird of p tients there is
no de nite history of preceding he d tr um . The etiology of the su
dur l h em t
om in this non-tr um tic group is pro

ly rel ted to rupture of fr gile
rid
ging vein in rel tively trophic mo
ile
r in. In this group the m jority of p t
ients re over 50 ye rs of ge. Shrink ge of the
r in resulting from trophy l

lows the
r in to
ecome more mo
ile nd incre ses the sp ce tr versed
y the ve
ins
ridging
etween the cortex nd the v ult. A rel tively trivi l injury m y r
esult in movement of the
r in, like w lnut inside its shell, with te ring of
the
ridging vein. P tients who re ntico gul ted re especi lly prone to devel
op su
dur l h em tom following rel tively minor tr um .
Fig. 5.4 Acute su
dur l h em tom c using m rked shift of the l ter l ventricle.
Clinic l present tion The presence of chronic su
dur l h em tom should
e con
sidered if the neurologic l st te of p tient
eing tre ted in hospit l for s
igni c nt he d injury
egins to deterior te. Altern tively, the p tient m y presen
t without the history of signi c nt he d injury in one of three ch r cteristic w
ys. 1 R ised intr cr ni l pressure without signi c nt loc lizing signs. The p tie
nt presents with he d che, vomiting nd drowsiness nd the
TRAUMATIC INTRACRANIAL HAEMATOMAS
61

sence of foc l neurologic l signs, r ising the differenti l di gnosis of cer

e
r l neopl sm or chronic su
dur l h em tom . 2 Fluctu ting drowsiness. The pred
omin nt ch r cteristic is decline in the level of consciousness nd the p tien
t m y
ruptly
ecome deeply unconscious. 3 A progressive dementi , which m y
e
misdi gnosed s Alzheimers dise se. However, the course of the dementi is usu l
ly more r pid nd progressive. Foc l neurologic l signs m y develop, p rticul rl
y hemip resis with n extensor pl nt r response. In up to 20% of c ses the hem
ip resis m y
e ipsil ter l to the side of the h em tom due to shift of the
r
in c using the contr l ter l crus cere
ri to
e compressed
y the tentori l edge
. A chronic su
dur l h em tom will
e di gnosed on the CT sc n s hypodense,
extr cere
r l collection c using compression of the underlying,
r in (Fig. 5.5)
. In 25% of c ses the h em tom is
il ter l (Figs 5.6 nd 5.7).
excise ll the mem
r ne of the h em tom . As these h em tom s m y
e multilocul
ted it is dvis
le to insert more th n one
urr hole nd to visu lize the under
lying
r in t e ch site.
Oper tion The chronic su
dur l h em tom c n
e dr ined through
urr holes or
cr niotomy loc ted over the h em tom . No ttempt should
e m de to
Fig. 5.6 Bil ter l chronic su
dur l h em tom . The collection on the left is not
s hypodense, indic ting th t it is more recent th n the h em tom uid on the ri
ght. It lso h s hyperdense re , indic ting more recent h emorrh ge.
Fig. 5.5 Chronic su
dur l h em tom . The uid is hypodense comp red with the dj c
ent
r in.
Fig. 5.7 T1 MRI showing
il ter l chronic su
dur l h em tom .
62
CHAPTER 5
The h em tom uid should
e w shed out completely nd fter the oper tion it is u
su lly dvis
le to pl ce su
dur l c theter for further dr in ge in closed d
r in ge system. Following the oper tion the p tient is nursed t, or even with th
e he d down initi lly, to encour ge the
r in to exp nd into the h em tom sp ce
. C reful ttention should
e given to the uid int ke nd serum electrolytes. The
norm l d ily uid requirements re given (3 l/d y in dults) provided there is no
clinic l or r diologic l evidence of
r in swelling. The p tient should
e slig
htly more hydr ted fter this type of oper tion th n other intr cr ni l procedur

es, in n ttempt to encour ge the
r in to swell into the previous h em tom sp

ce. However, hypon tr emi is common occurrence,
oth prior to nd following
surgery, nd if the serum sodium decre ses to less th n 130 mmol/l the uid int ke
should
e reduced.
Intr cere
r l h em tom
Tr um tic intr cere
r l h em tom s occur s result of penetr ting injury (su
ch s missile injury) or depressed skull fr cture, or following severe he
d injury. Intr cere
r l h em tom is frequently ssoci ted with su
dur l h em to
m . The size of the h em tom v ries consider
ly nd multiple h em tom s re fr
equently seen on the CT sc n following severe he d injury. The contre-coup inj
ury descri
ed in Ch pter 4 m y
e responsi
le for
urst tempor l lo
e which resu
lts in l rge tempor l h em tom ssoci ted with su
dur l
lood. An intr cere
r
l h em tom should
e suspected in ny p tient with severe he d injury or in
p tient whose neurologic l st te is deterior ting. The CT sc n will show the s
ize nd position of the h em tom s (Fig. 5.8). It should
e noted th t tr um tic
intr cere
r l h em tom s not infrequently evolve more th n 24 hours fter the t
r um . Consequently if is essenti l to repe t CT sc n if the initi l sc n perf
ormed fter the injury w s neg tive
ut the p tients neurologic l st te deterior
tes.
Su
dur l h em tom s in inf ncy
The inf ntile chronic su
dur l h em tom , or effusion, is distinct clinic l en
tity. Birth tr um is frequent c use
ut in m ny c ses p st history is in de
qu te to est
lish the n ture of the injury with cert inty. Chronic su
dur l h e
m tom s occur in 10% of
ttered children nd the violent sh king of n inf nt m y

e suf cient to l cer te
ridging cere
r l veins without evidence of extern l tr
um . Su
dur l
leeding in inf nts occurs
il ter lly in 85% of c ses nd is usu
lly over the dorsol ter l surf ces of the front l nd p riet l lo
es. The e rlie
st nding in inf nts with chronic su
dur l h em tom s is excessive cr ni l enl rge
ment s the sutures re unfused. The symptoms re non-speci c nd usu lly involve
listlessness, irrit
ility nd f ilure to thrive. The di gnosis will
e con rmed

y CT sc n. Tre tment initi lly involves spir tion of the uid
ut if, fter 2 or
3 weeks, repe ted t ps h ve f iled to reduce the volume signi c ntly, shunt m y

e inserted to dr in the uid from the su
dur l sp ce to the peritone l c vity.
Fig. 5.8 Tr um tic front l intr cere
r l h em tom s resulting from contre-coup i
njury.
TRAUMATIC INTRACRANIAL HAEMATOMAS
63
A l rge intr cere
r l h em tom should
e ev cu ted, unless the p tients neurolog
ic l st te is improving. Sm ll intr cere
r l h em tom s, p rticul rly if multipl
e, re not removed
ut the clinici n must
e w re th t they m y exp nd nd requ
ire su
sequent ev cu tion.
Further re ding
G rdner WJ (1932) Tr um tic su
dur l h em tom with p rticul r reference to the
l tent interv l. Archives of Neurology nd Psychi try 27, 847855. Hooper RS (1959
) O
serv tions on extr dur l h emorrh ge. British Journ l of Surgery 47, 7187. J
mieson KG, Yell nd JD (1968) Extr dur l h em tom . Report of 167 c ses. Journ l
of Neurosurgery 29, 1323. J mieson KG, Yell nd JD (1972) Tr um tic intr cere
r l
h em tom . Report of 63 surgic lly tre ted c ses. Journ l of Neurosurgery 37, 52
8532. Jennett B, Murr y A, C rlin J et l. (1979) He d injuries in three neurosur
gic l units, Scottish He d Injury
M n gement Study. British Medic l Journ l 2, 955958. Jennett B, Te sd le G (1981)
Intr cr ni l h em tom . In: Jennett B, Te sd le G, eds. M n gement of He d Inju

ries. Contempor ry Neurology Series. F A D vis, Phil delphi . K ye AH, Bl ck P M

cL (2000) Oper tive Neurosurgery. Churchill Livingstone, London, New York, Edin

urgh. Reilly PJ, Ad ms JH, Gr h m DI et l. (1975) P tients with he d injury who
t lk nd die. L ncet ii, 375381. Te sd le G, G l
r ith S (1981) Acute tr um tic
intr cr ni l h em tom s. In: Te sd le G, G l
r ith S, eds. Progress in Neurologi
c l Surgery 10. K rger, B sel. Trotter W (19141915) Chronic su
dur l h emorrh ge
of tr um tic origin nd its rel tion to p chymeningitis h emorrh gic intern . B
ritish Journ l of Surgery 2, 271291. Weir BKA (1971) The osmol rity of su
dur l h
em tom uids. Journ l of Neurosurgery 34, 528533.
CHAPTER 6
6
Br in tumours
Br in tumours re responsi
le for pproxim tely 2% of ll c ncer de ths. Centr l
nervous system tumours comprise the most common group of solid tumours in young
p tients, ccounting for 20% of ll p edi tric neopl sms. The over ll incidence
of
r in tumours is 810 per 100 000 popul tion per ye r. A study
y the United S
t tes Dep rtment of He lth in 1966 showed the incidence to
e 21 per 100 000 per
ye r t 2 ye rs old nd 1 per 100 000 during the teen ge ye rs. The incidence i
ncre ses fter the 4th dec de of life to re ch m ximum of 16 per 100 000 per y
e r in the 7th dec de. There h s
een n intense de
te concerning the incre sed
incidence of
r in tumours, especi lly in the elderly,
ut this possi
le incre
se could
e expl ined due to the dvent of CT nd MRI le ding to
etter detectio
n of tumours.
Cl ssi c tion
The gener l
r in tumour cl ssi c tion is rel ted to the cell of origin, nd is sh
own in T
le 6.1. T
le 6.2 shows the pproxim te distri
ution of the more commo
n
r in tumours. This ch pter will discuss the tumours derived from the neuroect
oderm nd met st tic tumours. The following ch pters will descri
e the
enign
r
in tumours nd pituit ry tumours.
Aetiology
Epidemiology studies h ve not indic ted ny p rticul r f ctor (vir l, chemic l o
r tr um tic) th t c uses
r in tumours in hum ns, lthough r nge of cere
r l t
umours c n
e induced in nim ls experiment lly. There is no genetic predis64
position
ut chromosome
norm lities h ve
een noted in m ny CNS tumours (T
le
6.3). Neuro
rom tosis type 1 (NF1), previously known s von Recklingh usens dise
se, occurs with n pproxim te frequency of 1 in 4000 live
irths. It is inherit
ed s n utosom l domin nt p ttern nd there is high spont neous mut tion r t
e. NF1 is ssoci ted with v riety of centr l nd peripher l nervous system tum
ours. An optic nerve gliom is the most common CNS tumour ssoci ted with NF1, o
ccurring in
out 15% of those ffected. Less commonly low-gr de gliom of the h
ypoth l mus, cere
ellum,
r instem or spin l cord m y occur. Peripher l neuro
rom
s re the h llm rk of NF1 (Ch pter 17). Neuro
rom s of the spin l roots re co
mmon fe ture of NF1 (Ch pter 15). The gene c using NF1 is loc ted on the long r
m of chromosome 17 (17q 11.2). Neuro
rom tosis type 2 (NF2), previously known s
centr l neuro
rom tosis, is n utosom l domin nt disorder which,
eyond few su
per ci l simil rities, is phenotypic lly nd genetic lly distinct from NF1. It h s
n incidence of pproxim tely 1 in 100 000 live
irths. The h llm rk of NF2 is

il ter l coustic (vesti
ul r) schw nnom s,
ut p tients with NF2 h ve n incre
sed risk of other intr cr ni l schw nnom s, multiple meningiom s (
oth cr ni l
nd spin l) nd gliom s. The NF2 gene locus is sited on the long rm of chromoso
me 22 (22q 11.2) (T
le 6.4). There is no speci c evidence linking CNS tumours to
environment l c rcinogens, lthough m ny chemic ls, especi lly ethyl nd methyl
nitrosoure nd nthr cene deriv tives, show

BRAIN TUMOURS
65
T
le 6.1 Gener l cl ssi c tion of
r in tumours. Neuroepitheli l tumours Gliom s
Astrocytom (including glio
l stom ) Oligodendrocytom Ependymom Choroid plexus
tumour Pine l tumours Neuron l tumours G ngliogliom G ngliocytom Neuro
l stom
Medullo
l stom Nerve she th tumour coustic neurom Meninge l tumours Meningi
om Pituit ry tumours Germ cell tumours Germinom Ter tom Lymphom s Tumour-like
m lform tions Cr nioph ryngiom Epidermoid tumour Dermoid tumour Colloid cyst M
et st tic tumours Loc l extensions from region l tumours e.g. glomus jugul r (i.
e. jugul re), c rcinom of ethmoid
T
le 6.2 Incidence of common cere
r l tumours (%). Neuroepitheli l Astrocytom
( ll gr des including glio
l stom ) Ependymom Oligodendrogliom Medullo
l stom
Met st tic Meningiom Pituit ry Acoustic neurom 52 44 3 2 3 15 15 8 8
T
le 6.3 Chromosom l deletions nd loci of loss of heterozygosity (LOH) in CNS
tumours. Chromosom l deletions/loci of LOH #10, #13, 17p, 17q, 19q, #22 9, #10,
17p, 19q, 22q 1p, 4, 6, 11p, 19q, 22q 17p, 10, 11, 19 13q 1.4 1p, 14, #22, 22q 1
2.3-qter 3p 11q 22q 17q
Tumour Astrocytom Glio
l stom multiforme Oligodendrogliom Medullo
l stom Ret
ino
l stom Meningiom H em ngio
l stom (von HippelLind u) Pituit ry denom (ME
N1) Acoustic nerve tumours (NF2) Neuro
rom s (NF1)
c rcinogenic ctivity in nim ls nd produce CNS tumours. Vir l induction of
r
in tumours h s
een used in nim l models
ut there is no rm evidence for vir l
etiology in hum ns. A hum n polyom JC virus injected into prim tes produces tum
ours simil r to hum n strocytom s fter n 18-month incu
tion period. This typ
e of slow virus effect m y ccount for some of the pro
lems of isol ting viruses f
rom hum n tumours. Although immunosuppression is known to incre se m rkedly the
risk of prim ry lymphom
of the
r in, p rticul rly in tr nspl nt recipients, there is not the correspond
ing incre sed incidence of gliom s. At present there is consider
le conjecture
reg rding the role of other possi
le etiologic l gents, including tr um , elec
trom gnetic r di tion nd org nic solvents
ut, s yet, there is no
66
CHAPTER 6
T
le 6.4 Di gnostic criteri for neuro
rom tosis. NF1: Two or more of the follow
ing 1 Six or more c f u l it m cules, e ch gre ter th n 5 mm in di meter in prep
u
ert l persons nd over 15 mm in di meter in postpu
ert l persons 2 Two or more
neuro
rom s or ny type of one plexiform neuro
rom 3 Freckling in the xill ry o
r inguin l regions 4 Optic gliom 5 Two or more Lisch nodules (iris h m rtom s)
6 A distinctive osseous
norm lity such s sphenoid dyspl si or thinning of lo
ng
one cortex with or without pseudo rthrosis 7 A rst-degree rel tive (p rent, s
i
ling or offspring) with NF1
y the
ove criteri NF2: One of the following 1
Bil ter l 8th nerve tumours seen with m gnetic reson nce im ging or computerized
tomogr phy 2 A rst-degree rel tive with NF2 nd either unil ter l 8th nerve tu
mour or two of the following: neuro
rom meningiom gliom schw nnom juvenile po
sterior su
c psul r lenticul r op city
the identi c tion of v riety of lter tions in the genome of the tumour cell, in
cluding those of
r in tumours. The present concept of oncogenesis involves
oth
the ddition of oncogenes to the genome nd the loss of the norm lly occurring

tumour suppressor genes. Tr nsform tion (spont neous or induced) is multistep

process requiring
oth initi tion nd promotion. Oncogenes encode proteins th t
p rticip te in the sign l tr nsduction nd second messenger systems th t modul t
e cell met
olism nd prolifer tion. These proteins include
oth growth f ctors
nd growth f ctor receptors such s epiderm l growth f ctor receptor, pl telet-d
erived growth f ctor, tryosine-speci c protein kin ses nd gu nine-
inding protein
s. Tumour suppressor genes re norm lly present in the genome nd ct s
r ke o
n cell tr nsform tion. Mut tions in the p53 tumour suppressor gene on chromosome
17 re the most common gene lter tion found to d te in tumours nd h ve
een s
hown to occur in
oth strocytom s nd meningiom s. The LiFr umeni syndrome is du
e to germ line mut tion in the p53 gene with the development of numerous c nce
rs including gliom s, ependymom s nd medullo
l stom s.
Gliom
Neuroectoderm l tumours rise from cells derived from neuroectoderm l origin. Gl
iom s comprise the m jority of cere
r l tumours nd rise from the neurogli l ce
lls. There re four distinct types of gli l cells: strocytes, oligodendrogli ,
ependym l cells nd neurogli l precursors. E ch of these gives rise to tumours w
ith different
iologic l nd n tomic l ch r cteristics. The neuroepitheli l ori
gin of microgli is in question.
convincing evidence to implic te these s
eing involved with the development of

r in tumours in hum ns. The four h llm rks of the development of c ncer cell
re the
ility to prolifer te, with the intr cellul r growth p thw ys constitu
itively ctiv ted, the ev sion of poptosis, with the c ncer cells h ving esc pe
d from cell de th p thw ys, the ttr ction nd induction of new
lood vessels (
ngiogenesis) to supply incre sed met
olic ctivity of tumour cells, nd tissue
inv sion. E ch of these re dependent on lig ndreceptor inter ctions on the cell
surf ce le ding to c sc de of cytopl smic events th t eventu lly result in dif
ferenti l gene expression. Molecul r
iology techniques h ve en
led
Astrocytom
The most common gliom s rise from the strocyte cells which comprise the v st m
jority of intr p renchym l cells of the
r in. Their m in function ppe rs to

e s supporting tissue for the neurones. The tumours rising from stroBRAIN TUMOURS
67
cytes r nge from the rel tively
enign to the highly m lign nt. The term m lign n
t for
r in tumours differs from its us ge for systemic tumours. Intrinsic
r in
tumours very r rely met st size (except for medullo
l stom nd ependymom ), nd
m lign nt refers to ggressive
iologic l ch r cteristics nd poor prognosis.
Cl ssi c tion
There re m ny cl ssi c tion systems of
r in tumours in gener l nd gliom s in p
rticul r. The period of system tic cl ssi c tion of tumours
eg n in 1846, when Vi
rchow descri
ed the neurogli nd rel ted it to
r in tumours. Although Virchow
cre ted the term gliom , these tumours h d lre dy
een descri
ed under other n me
s. In 1926, B iley nd Cushing descri
ed histogenetic cl ssi c tion system which
comp red the predomin nt cell in the tumours with the em
ryon l development of
the neurogli . The comp rison with st ges of cytogenesis w s pro

ly more of
working hypothesis th n n oncologic l theory for the origin of the tumours cells
. The theory th t gliom s origin te from prolifer tion of cells of v rying degre
es of m turity lying dorm nt in the
r in is not gener lly ccepted except in th
e c se of medullo
l stom , which m y rise from primitive l yer in the cere
el
l r cortex.
A v lu
le prognostic system of su
cl ssi c tion of strocytom w s descri
ed
y K

ernoh n in 1949. Astrocytom s were gr ded from I to IV, with Gr de IV
eing the
most m lign nt nd Gr de I cytologic lly,
ut not necess rily
iologic lly,
eni
gn. Ringertz simpli ed the four-gr de cl ssi c tion of Kernoh n into three-tiered
system; the comp rison
etween the two is shown in Fig. 6.1. The glio
l stom mu
ltiforme, equiv lent to the Kernoh n Gr de III nd IV tumours, is the most commo
n dult cere
r l tumour, ccounting for pproxim tely h lf of ll gliom s. The l
ow-gr de gliom s the strocytom , or Gr de I or II Kernoh n strocytom ccount
for only 1015% of strocytom s. The World He lth Org niz tion (WHO) cl ssi c tion r
ecognizes four gr des of strocytom . Gr de I is ssigned to the pilocytic stro
cytom which is
iologic lly distinct from the diffuse strocytom s, which re c
l ssi ed s strocytom (WHO Gr de II), n pl stic strocytom (WHO Gr de III) nd
glio
l stom multiforme (WHO Gr de IV). A gr ding system proposed
y D um sDupo
rt nd lso known s the St AnneM yo System ssessed the tumours ccording to the
presence or
sence of four morphologic l fe tures nucle r typi , mitosis, end
otheli l prolifer tion, nd necrosis nd they re gr ded ccording to the cumul
tive fe tures score. Gr de I tumours h ve none of the fe tures, Gr de
Ringertz
Gr de I (Well differenti ted)
Gr de II (An pl stic strocytom )
Gr de III (Glio
l stom multiforme)
Kernoh n
Gr de I Gr de II Gr des III nd IV
WHO
Gr de I Juvenile pilocytic strocytom
Gr de II Astrocytom v ri nts Fi
rill ry Protopl smic Gemistocytic Gr de II Scor
e: 1
Gr de III An pl stic strocytom
Gr de IV Glio
l stom v ri nts Gi nt cell Glios rcom
St Anne-M yo
Gr de I Score: 0
Gr de III Score: 2
Gr de IV Score: 3 or 4
Fig. 6.1 Rel tionship of the Kernoh n system, the three-tiered cl ssi c tion system,
the WHO system nd the St Anne/M yo (Dum s-Deport) gr ding system for strocyto
m s.
68
CHAPTER 6
II tumours h ve one fe ture, Gr de III tumours h ve two fe tures nd Gr de IV tu
mours h ve three or four fe tures.
P thology

M croscopic ch nges An strocytom m y rise in ny p rt of the
r in, lthough

it usu lly occurs in the cere
rum in dults nd the cere
ellum in children. A lo
w-gr de tumour in the cere
r l hemispheres inv des diffusely into the
r in. The
tumour does not h ve c psule nd there is no distinct tumour m rgin. The lowgr de gliom s re usu lly rel tively v scul r with rm
rous or ru

ery consiste
ncy. Fine deposits of c lcium re present in 15% of strocytom s. Occ sion lly,
low-gr de strocytom m y inv de diffusely throughout the cere
r l hemisphere.
In contr st, the m croscopic ppe r nce of highgr de tumour, the glio
l stom
multiforme, is ch r cterized
y highly v scul r tumour m rgin with necrosis i
n the centre of the tumour. Although in cert in re s the m rgin of the tumour m
y seem to
e m croscopic lly well de ned from the surrounding
r in, there re mi
croscopic nests of tumour cells extending well out into the
r in. Microscopic c
h nges The histologic l ppe r nce of the tumour v ries
with the tumours gr de. The low-gr de strocytom is ch r cterized
y n incre se
d cellul rity, composed entirely of strocytes (Fig. 6.2). Intermedi te-gr de tu
mours show nucle r pleomorphism, mitotic gures re frequent, nd there is incre s
ed v scul rity, s evidenced
y endotheli l nd dventiti l cell prolifer tion.
In the high-gr de strocytom very few strocytes ppe r norm l. There is m rked
cellul r pleomorphism, extensive endotheli l nd dventiti l cell prolifer tion
nd numerous mitotic gures with extensive necrosis (Fig. 6.3). The m jor histolo
gic l fe tures of glio
l stom multiforme re endotheli l prolifer tion nd necr
osis. The n pl stic strocytom is ch r cterized
y nucle r pleomorphism nd mi
toses, which re
sent in the strocytom .
Clinic l present tion
The presenting fe tures c n
e cl ssi ed under: r ised intr cr ni l pressure foc l
neurologic l signs epilepsy. The dur tion of the symptoms nd the progression
nd evolution of the clinic l present tion will depend on the gr de of the tumour
th t is, its r te of growth. A p tient presenting with low-gr de strocytom
(Gr de I or II) m y h ve history of seizures extending over m ny ye rs, nted
ting the development of
Fig. 6.2 Low-gr de strocytom . There is only slight incre se in the cellul ri
ty nd mild nucle r typi . There is distur
ed rchitecture with form tion of
triplets nd qu druplets of strocytes (h em toxylin nd eosin, 40).
BRAIN TUMOURS
69
( )
(
)
Fig. 6.3 Glio
l stom multiforme. ( ) There is m rked incre se in cellul rity
nd v ri tion in nucle r size, sh pe nd st ining density with frequent mitotic g
ures (h em toxylin nd eosin, 40). (
) Low-power view of the s me tumour showing
p llis ded necrosis typic l of glio
l stom multiforme.
progressive neurologic l signs nd r ised intr cr ni l pressure. The tumours m y
evolve histologic lly into the more m lign nt n pl stic strocytom or glio
l
stom multiforme. P tients with the higher-gr de tumours present with shorter
history nd glio
l stom multiforme is ch r cterized
y short illness of weeks
or few months.
st te, is the most import nt symptom nd sign of r ised intr cr ni l pressure. T
he extent of imp irment of conscious st te will
e rel ted to the severity of r
ised intr cr ni l pressure. An lert p tient with severely r ised intr cr ni l p
ressure m y r pidly deterior te nd
ecome deeply unconscious when there is only

 very sm ll further rise in the pressure within the cr ni l c vity.

R ised intr cr ni l pressure R ised intr cr ni l pressure is due to the tumour m
ss, surrounding cere
r l oedem nd hydroceph lus due to
lock ge of the CSF p
thw ys. The fe tures of r ised intr cr ni l pressure re descri
ed in det il in
Ch pter 3. The m jor symptoms re he d che, n use nd vomiting, nd drowsiness.
He d che is the most common symptom in p tients with cere
r l strocytom nd o
ccurs in ne rly three-qu rters of p tients; vomiting occurs in
out one-third.
The he d ches re usu lly gr du lly progressive nd lthough frequently worse on
the side of the tumours, they m y
e
itempor l nd diffuse. Ch r cteristic lly
, the he d che is worse on w king nd improves during the d y. N use nd vomiti
ng occur s the intr cr ni l pressure incre ses, nd the p tient frequently indi
c tes th t vomiting m y tempor rily relieve the severe he d che. Drowsiness, th
t is, deterior tion of conscious
Foc l neurologic l de cits Foc l neurologic l de cits re common in p tients present
ing with cere
r l gliom s; the n ture of the de cit will depend on the position of
the tumour. P tients presenting with tumours involving the front l lo
es freque
ntly h ve pseudopsychi tric pro
lems, person lity ch nge nd mood distur
nce. T
hese ch nges re p rticul rly ch r cteristic of the
utter y gliom , so c lled
ec u
se it involves
oth front l lo
es
y spre ding cross the corpus c llosum, givin
g it ch r cteristic m croscopic (Fig. 6.4) nd CT or MRI ppe r nce. This type
of tumour m y lso occur posteriorly, with spre d cross the splenium of the co
rpus c llosum into
oth p rieto-occipit l lo
es. Lim
p resis results from inter
ference with the pyr mid l tr cts, t either cortic l or su
cortic l level,
nd occurs in just under 50% of p tients. Field defects ssoci ted with tumours
of
70
CHAPTER 6
Fig. 6.5 Low-gr de strocytom . CT sc n shows non-enh ncing low-density lesion w
ith little or no m ss effect. Fig. 6.4 Butter y gliom . Glio
l stom multiforme of c
orpus c llosum spre ding into
oth front l lo
es.
Investig tions
Computerized tomogr phy CT sc n or MRI of the
r in re the essenti l r diologic
l investig tions (Figs 6.5 nd 6.6); n ccur te di gnosis c n
e m de in ne rl
y ll tumours. Low-gr de gliom s show decre sed density on the CT sc n; this doe
s not enh nce with contr st nd there is little or no surrounding oedem . C lci c
tion m y
e present. High-gr de gliom s re usu lly l rge nd enh nce vividly fo
llowing intr venous injection of contr st m teri l (Fig. 6.7). The enh ncement i
s often p tchy nd nonuniform nd frequently occurs in
ro d, irregul r rim r
ound centr l re of lower density. Although tumour cysts m y occur in the hig
hgr de tumours, the centr l re of low density surrounded
y the contr st enh n
cement is usu lly due to tumour necrosis. High-gr de tumours re surrounded
y m
rked cere
r l oedem nd there is frequently consider
le distortion of the l t
er l ventricles. Compression of the l ter l ventricle in one hemisphere, with pr
essure extending cross the midline, m y result in n o
structive hydroceph lus
involving the opposite l ter l ventricle.
the tempor l, occipit l nd p riet l lo
es re common,
ut m y
e evident only o
n c reful testing. Dysph si , either expressive or receptive, is p rticul rly
distressing symptom occurring in p tients with tumours involving the relev nt r
e s of the domin nt hemisphere. The p rticul r ch r cteristics of posterior foss
nd
r instem gliom s will
e discussed in the following section on p edi tric
tumours.
Epileptic seizures Seizures re the most frequent initi l symptom in p tients wi

th cere
r l strocytom nd occur in 5075% of ll p tients. Tumours dj cent to t

he cortex re more likely to
e ssoci ted with epilepsy th n those deep to the
cortex nd tumours involving the occipit l lo
e re less likely to c use epileps
y th n those which re more nteriorly pl ced. Astrocytom s m y produce either g
ener lized or foc l seizures; the foc l ch r cteristics will depend on the posit
ion within the
r in nd the cortic l structures involved.
BRAIN TUMOURS
71
( )
(
)
Fig. 6.6 MRI showing low-gr de gliom in posterior front l region. ( ) T1 sc n,
(
) T2 sc n.
Fig. 6.7 Glio
l stom multiforme. CT shows l rge tumour with contr st enh ncem
ent p rticul rly t the m rgins surrounding necrotic centre. There is m rked s
urrounding oedem with compression of the ventricles.
Fig. 6.8 Glio
l stom multiforme. MRI shows l rge enh ncing tumour inv ding into
corpus c llosum nd ventricle.
M gnetic reson nce im ging When used with g dolinium contr st enh ncement, MRI i
mproves the visu liz tion nd n tomic l loc liz tion of the tumours (Figs 6.8
nd 6.9). MRI h s the dv nt ge of
eing more
sensitive th n CT sc n, en
ling the detection of sm ll tumours nd p rticul rly
low-gr de gliom s th t might
e missed
y CT sc n. MRI provides
etter n tomic
l det il nd is more useful in visu lizing skull
se, posterior foss nd
r i
nstem tumours.
72
CHAPTER 6
( )
(
)
Fig. 6.9 Cystic n pl stic strocytom .
Low-gr de strocytom s m y show up on MRI s
norm l re s of incre sed T2 sign
l nd decre sed T1 sign l, even if the CT sc n w s norm l. High-gr de strocyto
m s ch r cteristic lly h ve low sign l intensity on T1-weighted im ges nd high
sign l intensity on T2-weighted im ges. G dolinium enh ncement is more likely to
occur in the higher-gr de tumour. Perfusion-weighted MRI is used to determine t
he region l cere
r l
lood volume, which is incre sed in high-gr de gliom nd m
y
e of v lue in differenti ting recurrent tumour from r di tion necrosis. M gn
etic reson nce spectroscopy (MRS) is non-inv sive technique th t provides info
rm tion on the composition nd sp ti l distri
ution of cellul r met
olites. On
proton MRS, tumours h ve n incre sed l ct te production, loss of N- cetyl sp r
t te (due to loss of neurones in the tumour re ) nd incre sed choline levels (
due to ctive mem
r ne
iosynthesis). Cere
r l ngiogr phy This w s the st nd rd
study in most p tients with strocytom s prior to the introduction of CT. It pr
ovides helpful inform tion on the v scul r supply of the tumours
ut is now only
r rely indic ted. Pl in X-r ys Pl in X-r ys of the skull do not need to
e

performed s routine. The most common
norm lity is erosion of the sell turc
ic due to long-st nding r ised intr cr ni l pressure. R diologic lly visi
le c
lci c tion is present in
out 8% of p tients with strocyte-derived gliom s.
M n gement
Following the presumptive di gnosis of gliom the m n gement involves: surgery
r diother py other djuv nt tre tments.
Surgery Surgery is performed with three princip l ims. To m ke de nite di gnosi
s. Tumour reduction to llevi te the symptoms of r ised intr cr ni l pressure. R
eduction of tumour m ss s precursor to djuv nt tre tments. The p tient is st
rted on glucocorticoid steroid ther py (e.g. dex meth sone) when presenting wit
h clinic l fe tures of r ised intr cr ni l pressure with the im of decre sing t
he cere
r l oedem prior to surgery. The type of oper tion performed will l rgel
y
e determined
y the position of the tumour nd
y the p tients clinic l presen
t tion. In gener l, the tumour is excised s r dic lly s possi
le,
BRAIN TUMOURS
73
provided the surgery will not result in ny dis
ling neurologic l de cit. Cr niot
omy is performed in the position th t provides the
est ccess to the tumour nd
usu lly with the id of fr meless stereot ctic system to id ccur cy of loc
liz tion. If the tumour h s not grown to involve the cortic l surf ce, sm ll i
ncision is m de in non-eloquent gyrus or sulcus nd the su
cortic l
r in is d
ivided down to the tumour m ss. The tumour is excised, often with the id of n
ultr sonic spir tor. Occ sion lly, the tumour m y involve one of the poles of the
hemisphere nd the excision m y ent il p rti l lo
ectomy. Although cr nioto
my with r dic l tumour excision will llevi te the symptoms of r ised intr cr ni
l pressure, there h s
een controversy s to whether r dic l resection improv
es surviv l. Most high-gr de gliom s weigh pproxim tely 100 g t the time of di
gnosis nd consist of 1011 cells. A r dic l tumour excision is
le to excise t
he m croscopic tumour
ut c nnot remove the tumour cells th t re in ltr ting deep
into the dj cent, often vit l, re s of norm l or oedem tous
r in. Consequent
ly, r dic l excision is unlikely to chieve more th n 9095% reduction in tumo
ur cell num
ers, resulting in 1010 cells rem ining. Whether the 12 logs of tumour
cell reduction re signi c nt reduction in tumour
urden prior to djuv nt ther
py nd whether it improves the effectiveness of su
sequent tre tment is still n
ot completely resolved, lthough recent clinic l studies do seem to show survi
v l
ene t following tumour resection nd this is f voured
y most neurosurgeons p
rovided the tumour excision c n
e performed without c using signi c nt neurologic
l mort lity. Altern tively,
iopsy, which c n
e performed most ccur tely us
ing stereot ctic methods, m y
e undert ken to o
t in the de nite histologic l di
gnosis, without m croscopic tumour excision, if: the tumour is sm ll nd deep se
ted the tumour is diffuse, without m jor fe tures of r ised intr cr ni l pressu
re, nd m croscopic resection is not fe si
le the tumour involves highly eloquen
t re s (e.g. speech centre) without pronounced fe tures of r ised intr cr ni l
pressure.
Fig. 6.10 Schem tic di gr m of stereot ctic
iopsy of cere
r l tumour using the
Cosm nRo
ertsWells (CRW) system.
Stereot ctic
iopsy involves loc liz tion of the tumour with stereot ctic fr m
e pplied to the he d of the p tient using the CT sc n or MRI. The three-dimensi
on l coordin tes of the tumour re scert ined. The surgeon chooses the point of
entry nd the desired p th through the
r in nd computer progr m determines
the necess ry ngles for the
iopsy pro
e nd the depth to the tumour (Fig. 6.10
). Postoper tive c re The postoper tive m n gement of strocytom involves the r
outine c re of p tient following cr niotomy. C reful neurologic l o
serv tio

ns re performed, s prompt intervention is essenti l if the p tients neurologic

l st te deterior tes s result of either incre sing cere
r l oedem or postope
r tive h emorrh ge. A postoper tive h em tom m y occur in the region of the tum
our excision or it m y
e extr cere
r l, either su
dur l or extr dur l. A CT sc
n should
e performed urgently if there is neurologic l deterior tion, to determ
ine the ex ct p thology. Occ sion lly, postoper tive deterior tion m y
e so r p
id s to require urgent re-explor tion
74
CHAPTER 6
of the cr niotomy without prior r diologic l ssessment. In the initi l postoper
tive period it is essenti l to void overhydr tion of the p tient so s not to
precipit te cere
r l oedem . The p tient is nursed with the he d of the
ed elev
ted 20, so s to promote venous return nd reduce intr cr ni l venous pressure.
Steroid medic tion is usu lly required in the initi l postoper tive period nd i
s gr du lly decre sed over the following d ys. The steroids m y need to
e re-in
stituted during the course of r diother py. The p tient is usu lly mo
ilized s
soon s possi
le, if necess ry with the help of physiother pist.
R di tion ther py Postoper tive r di tion ther py is gener lly n effective dju
nct to surgery in the tre tment of higher-gr de gliom s. It h s
een shown to do
u
le the medi n surviv l for high-gr de gliom s to 37 weeks. R di tion tre tment
is pl nned to optimize the homogeneity of the r di tion dose throughout the tum
our volume selected nd to minimize high dose regions in norm l
r in tr nsited

y the r di tion
e m. The size of the d ily r di tion fr ction is rel ted to th
e incidence of complic tions nd m xim l d ily dose is usu lly
etween 1.8 nd
2.0 gr y. The tot l r di tion dose v ries depending on the tumour type, loc tio
n nd size of eld,
ut for gliom s it is usu lly
etween 45 nd 60 gr y. Opinion
v ries reg rding the tissue volume th t should
e tre ted for m lign nt gliom

ut r di tion to the tumour re nd generous volume of surrounding
r in is now
dvoc ted, r ther th n r di tion to the whole
r in. The selection of the proper
r di tion dose for gliom s is s controversi l. Although incre sing the r di ti
on dos ge from 50 to 65 gr y does slightly improve surviv l, the higher dose of
r di tion ther py, especi lly over 65 gr y, signi c ntly incre ses the risk of
r
in necrosis. Prognosis At present there is no s tisf ctory tre tment for the m l
ign nt cere
r l gliom the n pl stic strocytom nd glio
l stom multiforme. T
he
medi n surviv l following surgery is pproxim tely 17 weeks nd when r di tion t
her py is used s n djuv nt the medi n surviv l is pproxim tely 37 weeks. Che
mother py for highgr de gliom s h s
een dis ppointing nd the
est results with
surgery, r di tion ther py nd chemother py consistently show medi n surviv l
time of less th n 1 ye r. The medi n norm l for the low-gr de gliom (
rill ry
strocytom ; WHO Gr de II, D umusDuport Gr des I nd II) is pproxim tely 8 ye rs,
with most tumours progressing to higher gr de. The role of surgery, r di tion
ther py nd other djuv nt ther pies in low-gr de gliom s is even less cert in
th n for the high-gr de gliom . The low-gr de tumour m y rem in rel tively quies
cent for some ye rs
efore it either continues to grow slowly or ch nges to mo
re n pl stic tumour with resulting de
ilit ting neurologic l deterior tion. In
gener l, the s me principles for surgic l excision pply for lowgr de gliom s s
for high-gr de gliom s. However, num
er of clinic l studies h ve shown th t p
tients h ving r dic l excision of tumour h ve longer 5-ye r surviv l th n
those with su
tot l excision. R di tion ther py h s not
een shown to improve
the surviv l in p tients with low-gr de tumours. In gener l, other djuv nt the
r pies re not used for the tre tment of low-gr de strocytom ,
ut m y
e of
e
ne t for oligodendrogliom s.
Other djuv nt ther pies M ny different djuv nt ther pies h ve
een investig te

d for the tre tment of gliom . These include the use of new chemother peutic ge
nts, new methods of dministering cytotoxic chemic ls, immunother py, hypertherm
i , new techniques of r diother py, photodyn mic ther py, nd gene ther py. The
l ck of effectiveness of the present tre tment of gliom s is rel ted to the
iol
ogy of the tumour. The most common position for tumour recurrence following conv
ention l tre tment is loc lly, in the tumour
ed, indic ting th t tre tment h s
f iled in loc l control. Although light microscopy shows high-gr de gliom s to h
ve rel tively well-de ned
order with the dj cent
BRAIN TUMOURS
75

r in, speci l st ining techniques, including monoclon l nti
odies, show m lign

nt cells extending well out into the surrounding
r in. It is the f ilure to co
ntrol the growth of these cells th t is l rgely responsi
le for the loc l tumour
recurrence. As indic ted previously, good surgic l resection with 90% of the
tumour excised would still result in 1010 cells
eing present. Effective r dioth
er py will result in 1 log (90%) or t the most 2 logs (99%) of cell kill nd it
is unlikely th t su
sequent chemother py would reduce rem ining tumour cells
y
more th n 90%. Consequently, the effect of cytoreductive surgery, r diother py
nd chemother py would result in pproxim tely 108 cells rem ining; the immune s
ystem is unlikely to
e
le to cope with tumour
urden of more th n 105 cells
. It follows th t, for ny extr tre tment to
e effective, whether surgery or
djuv nt ther py, it must provide t le st 1 log of cell kill. Chemother py Conve
ntion l chemother py h s
een dis ppointing. M ny of the chemother py gents th
t re ctive in vitro, or in other systemic tumours, h ve reduced ctivity in m
lign nt
r in tumours, either
y exerting n inherently limited cytotoxic potent
i l on
r in tumour cells or
y the in
ility of the chemother peutic gent to r
e ch the cells th t re responsi
le for the tumour recurrence. A study of
r in
tumour cell kinetics of high-gr de gliom s shows only sm ll proportion of the
cells (510%) in n ctive growth ph se; this h s serious consequences for ny cel
l cycle-speci c cytotoxic gent. Until recently the most commonly used single gen
t cytotoxic regime involves dministr tion of nitrosoure compounds. The high li
pid solu
ility nd low ioniz tion of these gents ensures rel tively effective
penetr tion of the cytotoxic compound into the tumour. Com
in tion ther py, uti
lizing m ny different cytotoxic compounds, h s
een used in v rious tri ls
ut n
one of the com
in tions h s
een shown to
e more
ene ci l th n the use of the si
ngle nitrosoure . Temozolomide is n lkyl ting gent th t c n
e t ken or lly,
penetr tes the CNS nd is well toler ted with predict
le myelotoxicity. Clinic
l
studies h ve shown it to
e effective in
out 40% of p tients. It is now the in
iti l chemother py gent of choice,
ut it is usu lly used t the time of tumour
recurrence, r ther th n s n djuv nt to surgery. It h s
een postul ted th t
re son for the l ck of effectiveness of chemother py is the in
ility of the c
ytotoxic compound to re ch the tumour cells which re inv ding the norm l dj ce
nt
r in. This h s resulted in new techniques of delivering the cytotoxic gent.
High-dose chemother py with
one m rrow rescue h s l rgely
een
ndoned
ec u
se of its high mor
idity nd l ck of effectiveness. Techniques of disrupting the

lood
r in
rrier h ve
een used
efore chemother py infusion to improve the de
livery of chemother peutic gents to tumour cells within the environment of no
rm l
lood
r in
rrier. This h s resulted in su
st nti lly incre sed neurotoxici
ty to the norm l
r in without signi c ntly improving surviv l. Intr c rotid chemo
ther py suffers from serious limit tions the perfusion of the tumour m ss is les
s th n expected
ec use most tumours re not supplied entirely
y one c rotid r
tery nd stre ming of the cytotoxic gent results in very high doses of chemother
py to sm ll re s, with rel tive hypoperfusion in other regions. Complic tions s
uch s serious retin l d m ge nd neurotoxicity h ve further reduced the ttr ct
iveness of this technique. R diother py Attempts to enh nce the effect of r diot

her py h ve included the use of r diosensitizers, such s metronid zole nd miso

nid zole, which incre se the r diosensitivity of hypoxic tumour cells without
corresponding incre se in the sensitivity of euoxic cells. However, the clinic l
tri ls h ve shown only m rgin l dv nt ge. The use of interstiti l
r chyther
py, involving stereot ctic lly impl nted r dio ctive sources into the tumour, h
s the dv nt ge of pplying high dose of r diother py to the tumour while sp
ring the surrounding
r in. However, the clinic l tri ls performed so f r h ve r
esulted in high incidence of r dionecrosis to the surrounding
r in nd improv
ements in the technique will need to
76
CHAPTER 6

e devised
efore this method would
ecome ccept
le. Simil rly, stereot ctic r
diosurgery, th t involves r di tion to very highly focused re , is of limite
d use s it does not t rget the in ltr tive tumour cells th t re responsi
le for
tumour recurrence. Hyperthermi This h s inherent
sic limit tions s, lthough
cell de th occurs t pproxim tely 42C, d m ge to the surrounding
r in occurs
t 45C, so there is very n rrow ther peutic index. In ddition, there is m rke
d toler nce of tumour cells to hyperthermi nd the tre tment h s not
een effec
tive. Immunother py The possi
ilities for immunother py s n djuv nt tre tment
h ve
een investig ted for m ny ye rs. Investig tions h ve included the use of
ctive immunother py techniques nd, more recently, the pplic tion of doptive
immunother py. This technique involves stimul ting peripher l
lood lymphocytes
in vitro with hum n recom
in nt interleukin-2 to produce lymphokine ctiv ted ki
ller cells (LAK cells) which c n
e dministered in conjunction with interleukin
-2. However, the LAK cells do not cross the
lood
r in
rrier nd so need to
e
injected in close proximity to the tumour cells. Recent studies using this techn
ique h ve
een dis ppointing. Photodyn mic ther py This is technique th t offe
rs speci l dv nt ges s n djuv nt ther py of m lign nt
r in tumours since it
h s
een shown to
e n effective method of controlling loc l tumours. The tech
nique involves the selective upt ke of sensitizer into the
r in tumour, followe
d
y open or intr oper tive irr di tion of the sensitized tumour cells with ligh
t of n ppropri te w velength to ctiv te the sensitizer nd selectively destro
y the tumour cells. Clinic l studies using this method h ve shown f vour
le t
rend, lthough form l ph se III studies h ve not
een undert ken. Gene ther py E
xperiment l ther pies of gliom t present

eing investig ted involve the use of retrovirus, denoviruses or deno- ssoci t
ed viruses to c rry v riety of gene ther pies to the c ncer cell. It is hoped
th t s these tre tments re re ned over the next dec de they will
e useful in th
e tre tment of cere
r l gliom .
Oligodendrogliom
Oligodendrogliom s re responsi
le for pproxim tely 5% of ll gliom s nd occur
throughout the dult ge group with m xim l incidence in the 5th dec de. The
tumour is r re in children.
P thology
Ne rly ll oligodendrogliom s occur
ove the tentorium; most re loc ted in the
cere
r l hemispheres nd
out h lf of these re in the front l lo
es. Oligoden
drogliom s m y project into either the 3rd or l ter l ventricles. Oligodendrogli
om s h ve the s me spectrum of histologic l ppe r nce s strocytom s, r nging
from very slow growing,
enign tumours to more r pidly growing, m lign nt v ri
ety with
und nt mitotic gures, endotheli l prolifer tion nd foci of necrosis.
C lcium deposits re found
y histologic l ex min tion in up to 90% of oligodend
rogliom s. Unlike the strocyte group, most oligodendrogliom s re well differen
ti ted. Not infrequently tumours h ve mixed histology, with
oth oligodendrogli
l nd strogli l fe tures.

Clinic l present tion

The presenting fe tures re essenti lly the s me s for the strocyte group
ut,
s these tumours re more likely to
e slow growing, epilepsy is common, occurr
ing in 80% of p tients nd seen s n initi l symptom in 50%. The fe tures of r
ised intr cr ni l pressure nd foc l neurologic l de cits re e ch present in ppr
oxim tely one-third of p tients. As for strocyte tumours, MRI with contr st m y

e
ene ci l
ut other investig tions re usu lly unnecess ry.
BRAIN TUMOURS
77
( )
(
)
Fig. 6.11 Oligodendrogliom s. ( ) Non-enh ncing low-gr de c lci ed tumour. (
) Con
tr st-enh ncing high-gr de oligodendrogliom .
R diologic l investig tion
CT sc nning nd MRI re the fund ment l investig tions. They will con rm the di gn
osis of n intr cr ni l tumour nd in m ny c ses the di gnosis of oligodendrogli
om will
e highly pro

le. C lci c tion will
e present in 90% of c ses nd over
h lf show contr st enh ncement (Fig. 6.11).
of histologic l m lign ncy. Five-ye r surviv l r tes re
etween 30 nd 50% with
sm ll num
er of p tients living for m ny ye rs (up to 5% for 20 ye rs). Howev
er, m ny tumours with histologic l fe tures of oligodendrogliom lso h ve com
ponent of strocyte-derived cells, usu lly n pl stic strocytom , nd the tumou
r
eh ves
iologic lly nd clinic lly s n n pl stic strocytom r ther th n
n oligodendrogliom .
Tre tment nd results
Tre tment involves: surgic l resection r diother py other djuv nt tre tments. T
he st nd rd tre tment for oligodendrogliom h s
een n ggressive resection of
the tumour followed
y r di tion ther py, lthough r diother py would now not
e
given to low-gr de tumours, nd utilized only for the intermedi te- or high-gr
de oligodendrogli l tumours. Oligodendrogliom s h ve
een shown to
e more sensi
tive to chemother py th n the strocytom tumours, especi lly if the oligodendro
gliom s
elong to the group with loss of heterozygosity OF chromosome 1p or 19q.
The surviv l of p tients depends on the degree
Recurrent cere
r l gliom
As discussed e rlier, most high-gr de cere
r l gliom s will recur within 1 ye r
of the initi l tre tment with surgery nd r diother py. Lowgr de tumours m y eit
her recur s continuing progression of the slow growth or, ltern tively, the
histologic l ch r cteristics m y lter nd the tumour m y
ecome more n pl stic
nd r pidly growing. The clinic l present tion of recurrent tumour will
e ev
idenced
y either progression of the foc l neurologic l signs or the signs of
n incre se in the intr cr ni l pressure. The di gnosis will
e con rmed
y CT sc
n or MRI in most c ses. The m jor differenti l di gnosis is postr diother py r d
i tion necrosis, which m y develop s e rly s 4 months or s l te s 9 ye rs f
ter r diother py.
78
CHAPTER 6
The r diologic l fe tures of necrosis re n v scul r m ss, nd the di gnosis m

 y
e suspected from the dose of r diother py th t h s
een dministered. Howeve

r, there m y
e consider
le dif culty in differenti ting necrosis from recurrent
gliom , nd sometimes n oper tion is required
oth for de nitive di gnosis nd to
remove the m ss. The initi l deterior tion following di gnosis of recurrent g
liom c n usu lly
e tempor rily h lted
y the use of steroid medic tion. The m
jor decision is whether further surgery nd other djuv nt ther py should
e und
ert ken. In gener l, further oper tion involving de
ulking of the tumour would

e considered if: the p tient is less th n 65 ye rs old there h s
een sympto
m-free interv l of 1 ye r or more since the rst oper tion de
ilit ting irreversi

le neurologic l signs re
sent the tumour is in n ccessi
le position nd rep
e t surgery would not result in ddition l mor
idity.
Adjuv nt ther pies
Adjuv nt ther pies h ve limited
ene t for p tients with recurrent tumour nd,
sidering the mor
idity involved, m y not
e indic ted. Chemother py, utilizing
emozolomide dministered or lly, h s shown to h ve tempor ry
ene t in up to
of p tients. Other chemother py gents, especi lly the nitrosoure compounds,
ve much gre ter toxicity.

con
t
40%
h

Ependymom
Ependymom s re gli l neopl sms rising from the ependym nd constitute pproxi
m tely 5% of ll gliom s. Approxim tely two-thirds of ependymom s occur in the i
nfr tentori l comp rtment nd most of these present in children, dolescents nd
young dults. The supr tentori l ependymom s occur mostly in dults.
P thology
The tumour rises from the ependym of the ventricle nd, lthough predomin ntly intr ventricul r, the tumour often inv des in
to the dj cent cere
ellum,
r instem or cere
r l hemisphere. Fourth ventricul r
tumours usu lly rise from the oor or l ter l recess of the 4th ventricle nd th
ey m y extend into the su
r chnoid sp ce to enc se the medull or upper cervic
l spin l cord. Altern tively, the tumour m y grow l ter lly through the for men
of Luschk nd into the cere
ellopontine ngle. The tumours re well dem rc ted,
nodul r, soft nd p le. C lci c tion is common, especi lly in supr tentori l epen
dymom s. There re numerous histologic l cl ssi c tion systems of ependymom s, nd
the World He lth Org niz tion cl ssi c tion divides these tumours into cellul r,
p pill ry nd cle r cell types of non- n pl stic ependymom nd n pl stic epend
ymom . The myxop pill ry v riety is slow-growing distinct v ri nt of ependymom
th t occurs in the c ud equin nd is discussed in the ch pter on spin l cord
tumours (Ch pter 15). In n dult the su
ependymom v ri nt m y
e encountered
s n incident l utopsy nding discrete nodul r m ss
sed in the
r ins ventric
ul r surf ce, p rticul rly the oor or l ter l recess of the 4th ventricle or the
septum pellucidum or it m y
e l rge enough to present clinic lly. It is usu lly
he vily c lci ed nd is composed of cells with strocytic s well s ependym l fe
tures. The p pill ry nd n pl stic v rieties of ependymom re responsi
le for
the m jority of clinic lly symptom tic ependymom s. The cellul rity nd rchite
cture of the ependymom s v ry
ut di gnostic fe ture is the presence of rosett
es, nd most ependymom s cont in re s in which periv scul r pseudorosettes re
conspicuously developed. In these form tions the
lood vessel is surrounded
y
n eosinophilic h lo composed of the r di ting t pering processes of the cells. B
leph ropl sts frequently occur in ependymom s
ut m y
e dif cult to visu lize. Th
ese re tiny intr cytopl smic spheric l or rod-sh ped structures which represent
the
s l
odies of cili , nd re most frequently encountered in the pic l po
rtion of cells th t form ependym l rosettes.
BRAIN TUMOURS
79

Clinic l present tion

Posterior foss ependymom s Det ils will
e discussed in the section on p edi tr
ic tumours (p. 91). P tients present with fe tures of r ised intr cr ni l pressu
re due to hydroceph lus s result of o
struction of the 4th ventricle, t xi
due to cere
ell r involvement, nd occ sion lly fe tures of
r instem pressure o
r in ltr tion. Supr tentori l tumours Virtu lly ll p tients with supr tentori l e
pendymom s present with fe tures of r ised intr cr ni l pressure, often due to h
ydroceph lus s result of o
struction of the CSF p thw ys. At xi is common n
d foc l neurologic l de cits m y occur due to involvement of the underlying cere
r
l hemisphere.
R diologic l investig tion
The CT sc n nd MRI will show tumour th t rises in the ventricle nd enh nces
fter dministr tion of intr venous contr st. C lci c tion is common in tumours
rising from the l ter l ventricles (Fig. 6.12). There is frequently ssoci ted h
ydroceph lus. In the posterior foss differenti tion from medullo
l stom m y

e dif cult.
Fig. 6.12 C lci ed contr st-enh ncing ependymom involving l ter l ventricles.
p thw ys, sometimes whole neur xis r di tion is recommended. The prognosis is re
l ted to the degree of n pl si of the tumour nd for intr tentori l tumours v
ries from 20% to 50% 5-ye r surviv l. The prognosis for the supr tentori l tumou
rs is
etter, p rticul rly in dults.
Tre tment
The tre tment of ependymom s is initi lly surgic l, with n ttempt to perform
r dic l m croscopic resection of the tumour. Supr tentori l tumours re often v
ery l rge nd m y extend throughout the l ter l nd 3rd ventricles,
ut the sso
ci ted hydroceph lus m kes the excision of the intr ventricul r portion fe si
le
. However, the tumour m y rise from the ventricul r w ll in the region of the

s l g ngli nd
lend impercepti
ly with the underlying cere
r l structures so
th t complete excision is not possi
le. Fourth ventricul r tumours c n
e exci
sed from the ventricle
ut microscopic in ltr tion into the underlying
r instem c
nnot
e removed surgic lly. Postoper tive r di tion ther py is dvis
le nd,
s these tumours m y spre d through the CSF
Pine l tumours
Tumours rising in the region of the pine l gl nd re mostly not of pine l origi
n,
ut re gener lly c lled pine l, s they h ve simil r clinic l present tion.
The tumours re rel tively uncommon, ccounting for 0.5% of ll intr cr ni l tum
ours. However, they occur more frequently in J p n nd Chin , where the incidenc
e is up to 5%. Most tumours m ke their clinic l ppe r nce
etween 10 nd 30 ye
rs of ge.
Cl ssi c tion
Pine l region tumours re cl ssi ed in decre sing frequency s:
80
CHAPTER 6

germinom ter tom pineocytom pineo
l stom miscell neous: gliom cyst. Germino
m is the most common pine l region tumour nd is simil r in histologic l ppe r
nce to germinom of the gon ds nd medi stinum; it occurs predomin ntly in m le
s. These tumours m y lso rise in the supr sell r region, nd synchronous tumou
rs in
oth the pine l nd supr sell r region occur occ sion lly. Ter tom is lik
e germinom ; it lso rises from displ ced em
ryonic tissue. The other tumour ty

pes occur less commonly.

R diologic l investig tions CT sc n nd MRI will show pine l region tumour nd
will often suggest the correct p thologic l di gnosis (Fig. 6.13). On CT sc n,

efore contr st, germinom will
e hyperdense lesion in the region of the pi
ne l gl nd in ltr ting into the surrounding tissue nd there will
e uniform vivid
enh ncement following intr venous contr st. C lci c tion is uncommon. On MRI germ
inom s re rel tively isointense to norm l white m tter on T1-weighted im ges n
d slightly hyperintense on T2-weighted sc ns. G dolinium contr st de nes the tumou
r well s germinom s enh nce m rkedly nd homogeneously.
M n gement
This consists of surgery nd r diother py. A ventriculoperitone l shunt or dr in
ge of CSF
y 3rd ventriculostomy m y
e required if the hydroceph lus is seve
re. There is controversy over whether the de nitive tre tment should
e n ttempt
t surgic l excision or r diother py. As most of the tumours re germinom s, n
d these tumours re very r diosensitive, course of r diother py c n
e given
s the initi l tre tment if the r diologic l ppe r nce is typic l for germinom .
If seri l CT sc ns show the tumour h s f iled to respond to r diother py then s
urgery m y
e necess ry. Altern tively, if the fe tures on the initi l CT nd MR
I sc ns re typic l, nd the lesion does not resem
le germinom , explor tion
of the tumour nd surgic l excision m y
e ppropri te s the initi l procedure.
The preferred surgic l ppro ch is usu lly
y posterior foss cr niotomy,
o
ve the cere
ellum nd
elow the tentorium cere
elli. Altern tive supr tentori l
surgic l exposures include ppro ching the tumour through the corpus c llosum or

y retr cting the occipit l lo
e.
Clinic l present tion
P tients with pine l tumours present with: r ised intr cr ni l pressure neurolog
ic l signs due to foc l compression endocrine distur
nce. R ised intr cr ni l p
ressure. The fe tures of r ised intr cr ni l pressure, such s he d ches, drowsi
ness nd p pilloedem , re due to hydroceph lus, which is result of the tumour
occluding the queduct of Sylvius. Foc l compression. Compression of the effere
nt cere
ell r p thw ys in the superior cere
ell r peduncle results in lim
t xi
nd distortion of the qu drigemin l pl te, produces limit tion of upg ze, conv
ergence p resis with imp irment of re ction of pupils to light nd ccommod tion
(P rin uds syndrome), nd m y result in convergence-retr ction nyst gmus on upg
ze (Koer
erS liusElschnig syndrome). Endocrine distur
nce. Endocrine distur
nces
re uncommon
ut include precocious pu
erty in 10% of p tients, lmost inv ri

ly m le, nd di
etes insipidus in 10%. The endocrine effects c n either
e due
to direct tumour involvement of the hypoth l mus or result from the second ry ef
fects of hydroceph lus.
Met st tic tumours
Met st tic tumours re responsi
le for pproxim tely 15% of
r in tumours in cli
nic l series
ut up to 30% of
r in tumours reported
y p thologists. Approxim t
ely 30% of de ths re due to
BRAIN TUMOURS
81
( )
c ncer nd 1 in 5 of these h ve intr cr ni l met st tic deposits t utopsy. The
met st tic tumours most commonly origin te from: c rcinom of the lung c rcinom
of the
re st met st tic mel nom c rcinom of the kidney g strointestin l c r
cinom . In 15% prim ry origin is never found. Most met st tic tumours re mult
iple nd onethird re solit ry. In
out h lf of these systemic spre d is not p
p rent. The incidence of tumours in the cere
rum rel tive to the cere
ellum is 8

: 1, nd most occur in the distri
ution of the middle cere
r l rtery. The size
of the tumours m y v ry consider
ly if the deposits re multiple. Met st tic t
umours re often surrounded
y intense cere
r l oedem .
Clinic l present tion
The interv l
etween di gnosis of the prim ry c ncer nd cere
r l met st ses v r
ies consider
ly. In gener l, second ry tumours from c rcinom of the lung prese
nt rel tively soon fter the initi l di gnosis, with medi n interv l of 5 mont
hs. Although cere
r l met st ses m y present within few months of the initi l
di gnosis of m lign nt mel nom or c rcinom of the
re st, some p tients m y li
ve m ny ye rs (up to 15 ye rs)
efore n intr cr ni l tumour ppe rs. The presen
ting fe tures re simil r to those descri
ed for other intr cr ni l tumours: r i
sed intr cr ni l pressure foc l neurologic l signs epileptic seizures. He d che
nd vomiting, indic tive of r ised intr cr ni l pressure, occur in most p tients
nd the presenting history is usu lly short, often only few weeks or months.
The incre sed intr cr ni l pressure will
e c used
y either the tumour m ss nd
surrounding oedem or, in posterior foss tumours, o
structive hydroceph lus. T
he p ttern of foc l neurologic l signs will depend on the position of the tumour
deposits nd the p tient m y present with progressive hemip resis or speech d
istur
nce with supr tentori l tumours or g it t xi with cere
ell r tumours.
(
)
(c)
Fig. 6.13 MRI in coron l ( ), s gitt l (
) nd xi l (c) pl ne showing pine l re
gion germinom c using o
structive hydroceph lus.
82
CHAPTER 6
Epileptic seizures occur in pproxim tely 25% of p tients nd m y
e either foc
l or gener lized. Occ sion lly, met st ses, especi lly mel nom or chorioc rcino
m , present following n intr cere
r l h emorrh ge.
Tre tment
Steroid medic tion (e.g. dex meth sone) will control cere
r l oedem nd should

e commenced immedi tely if there is r ised intr cr ni l pressure. Surgery to re
move the met st sis is indic ted if: there is solit ry met st sis in surgic
lly ccessi
le position there is no systemic spre d. Remov l of solit ry secon
d ry is prefer
le only if the prim ry site of origin h s
een, or will
e, cont
rolled. However, excision of single met st sis will provide excellent symptom
tic relief nd consequently m y
e indic ted even if the prim ry site c nnot
e
tre ted s tisf ctorily. Surgery is, of course, m nd tory if the di gnosis is unc
ert in. R diother py, together with steroid medic tion to control cere
r l oedem
, is used to tre t p tients with multiple cere
r l met st ses nd m y
e dvis

le following the excision of single met st sis. The tre tment, up to 45 gr y,
is usu lly given in 2-week course. Over the p st dec de stereot ctic r diosur
gery utilizing highly focused
e m of r di tion h s
een used to tre t single
nd multiple cere
r l met st ses. The ther py does seem to
e effective in some
c ses nd its role rel tive to surgery is
eing ev lu ted. Anticonvuls nt medic
tion is given
oth to p tients who h ve suffered epileptic seizures nd s pro
phyl ctic me sure.
R diologic l investig tions
CT sc n or MRI will di gnose the met st tic tumour nd will show whether the dep
osits re solit ry or multiple (Fig. 6.14). Most met st tic tumours re rel tive
ly isodense on the unenh nced CT sc n nd they enh nce vividly fter intr venous
contr st injection. Tumours th t m y
e hyperdense prior to contr st re mel no

m , chorioc rcinom , mucoid denoc rcinom (e.g. from the g strointestin l tr ct

) nd 50% of lymphom s. There is usu lly consider
le surrounding cere
r l oedem
with distortion of the ventricul r system. MRI following g dolinium contr st w
ill demonstr te sm ll met st tic tumours often not visi
le on the CT sc n (Fig.
6.15).
Prognosis A
out 30% of p tients with solit ry met st tic deposits from c rcinom
of the lung or mel nom nd 50% of p tients with c rcinom of the
re st surviv
e 1 ye r following surgic l excision. In those p tients where the source of the
met st tic tumours is undetermined,
out 50% survive 1 ye r.
Leptomeninge l met st ses
Fig. 6.14 Multiple contr st-enh ncing tumours typic l of met st tic mel nom .
Meninge l c rcinom tosis is widespre d, multifoc l seeding of the leptomeninges

y systemic c ncer. The clinic l present tion includes:
BRAIN TUMOURS
83
hydroceph lus, c using he d ches nd vomiting cr ni l nerve
norm lities due to
direct inv sion
y the tumours spin l root involvement due to loc l in ltr tion.
The CT sc n or MRI ndings m y
e su
tle
ut frequently show excessive enh ncement
of the meninges. Lum
r puncture c n
e performed if there is no evidence of r
ised intr cr ni l pressure. M lign nt cells m y
e seen in the CSF, the protein
concentr tion is incre sed nd the glucose reduced.
Cere
r l lymphom
The term cere
r l lymphom encomp sses num
er of distinct p thologic l nd cli
nic l entities. Current nomencl ture divides cere
r l lymphom into: non-Hodgkins
lymphom or Hodgkins dise se; prim ry or second ry dise se; nd p tients who re
immunocompetent or immunosuppressed. Historic lly, lymphom involving the CNS w
s considered to
e r re,
eing less th n 3% of ll CNS tumours, with
out h lf
of these lymphom s
eing prim ry cere
r l lymphom , th t is, the tumour
eing c
on ned entirely to the CNS. Over the p st dec de there h s
een n unprecedented i
ncre se in the incidence of cere
r l lymphom , which c n
e ttri
uted to t le
st two known f ctors: the cquired immune de ciency syndrome (AIDS) epidemic nd t
he use of immunosuppressive ther py. However, there h s lso
een n unexpl ined
incre se in the incidence of prim ry cere
r l lymphom in nonimmunosuppressed p
tients. Cere
r l lymphom m y
e second ry to systemic lymphom nd in l rge st
udies of p tients with systemic lymphom , up to 30% of p tients develop clinic l
or p thologic l evidence of cere
r l involvement. However, lmost ll c ses re
ssoci ted with either rel psed or progressive systemic dise se nd n isol ted
CNS rel pse is very r re.
( )
(
)
Fig. 6.15 MRI following g dolinium shows multiple sm ll met st tic tumours.
(c)
84
CHAPTER 6
Intr cere
r l Hodgkins dise se is very r re entity.
R diologic l investig tions (Figs 6.16 nd

6.17) The CT sc n ch r cteristic lly shows hypodense or isodense or sometimes

hyperdense tumour, which enh nces following contr st injection with ssoci ted m
ild to moder te oedem . Multifoc l dise se is o
served in
out 40% of CT sc ns.
Most prim ry cere
r l lymphom s rise in the periventricul r region. MRI is now
the investig tion of choice for prim ry cere
r l lymphom . The lesions re usu
lly hypointense to isointense on T1-weighted im ges nd isointense to hyperinten
se on T2-weighted im ges. The tumours enh nce following intr venous injection of
g dolinium. Although CSF ex min tion m y show popul tion of
norm l lymphocy
tes, concern reg rding r ised intr cr ni l pressure usu lly prevents lum
r pu
ncture
eing performed in the m jority of p tients.
Clinic l present tion
The most common site for prim ry cere
r l lymphom is in the front l lo
e, follo
wed
y the tempor l lo
e, p riet l lo
e nd deep nuclei. Tumours m y lso occur
in the cere
ellum nd
r instem. The tumours m y
e either solit ry or multiple
nd prim ry leptomeninge l dise se h s
een reported in up to 10% of prim ry cer
e
r l lymphom . There re no p thognomonic presenting symptoms or signs in prim
ry cere
r l lymphom nd the presenting fe tures re simil r to those descri
ed
for other intr cr ni l tumours: r ised intr cr ni l pressure, foc l neurologic l
signs nd epileptic seizures. The high frequency of front l lo
e involvement re
sults in common mode of present tion s memory loss, forgetfulness nd ltered
ffect. Up to 10% of p tients with prim ry cere
r l lymphom present with sei
zure. In view of the incidence of multiple lesions, it is not surprising th t m
ny p tients present with constell tion of symptoms nd signs. The presenting f
e tures in prim ry cere
r l lymphom rising in immunode cient p tients (including
AIDS-rel ted prim ry cere
r l lymphom ) do not ppe r to
e different from thos
e in immunocompetent p tients with prim ry cere
r l lymphom .
M n gement
The principles concerning the m n gement of prim ry cere
r l lymphom involve: h
istologic l di gnosis ensuring th t the dise se is con ned to the
r in excluding
n underlying, predisposing illness instituting ppropri te ther py. The usu l s
equence of events is CT nd MRI sc n followed
y
iopsy proving intr cere
r
l lymphom . In gener l, it is thought ppropri te
( )
(
)
Fig. 6.16 ( ) MRI nd (
) CT of cere
r l lymphom .
BRAIN TUMOURS
85
Fig. 6.17 Autopsy specimen of cere
r l lymphom .
the ther py of systemic non-Hodgkins lymphom nd include methotrex te, corticost
eroids, nthr cyclines, vinc lk loids, cytosine, r
inoside nd lkyl ting g
ents. Medi n surviv l times of up to 44 months h ve
een reported. However, meth
otrex te given fter r diother py incre ses the risk of leukoenceph lop thy with
consequent serious neuropsychologic l imp irment. The need for postchemother py
r di tion h s
een questioned, in view of the effectiveness of chemother py, es
peci lly in the elderly.
to exclude systemic dise se, lthough concurrent cere
r l nd systemic lymphom
is uncommon. P tients usu lly undergo
one m rrow spir te nd trephine, chest X
-r y, CT sc n of the chest nd
domen nd testing for evidence of HIV infection
.

P r n s l sinus tumours
Tumours of the p r n s l sinuses m y spre d directly to involve the
r in. These
uncommon tumours most frequently rise from the ethmoid or m xill ry sinuses, l
ess frequently from the sphenoid sinus nd r rely from the front l sinus. The tu
mours inv de through the oor of the nterior cr ni l foss in the region of the c
ri
riform pl te nd m y extend through the dur into the front l lo
e (Fig. 6.18
). The tumours re usu lly squ mous cell c rcinom nd less frequently denoc rc
inom or denoid cystic denoc rcinom . The esthesioneuro
l stom is r re n s
l
Surgic l tre tment There is no cle r evidence th t cr niotomy with excision of t
he lymphom is superior to o
t ining n ccur te tissue di gnosis using stereo
t ctic
iopsy. It must
e noted th t the e rly use of corticosteroids, to tre t
cere
r l oedem , m y m ke histologic l ssessment dif cult due to the exquisite se
nsitivity of prim ry cere
r l lymphom to steroids. The tumour m y dis ppe r on
the CT sc n or MRI fter the commencement of corticosteroids nd this h s signi c
nt implic tions for o
t ining stereot ctic
iopsy. R diother py Prim ry cere
r
l lymphom is usu lly r diosensitive with clinic l response r te of up to 80%
. However, cr ni l r diother py lone s tre tment for prim ry cere
r l lympho
m r rely produces long-term survivors, despite the high response r te nd n im
provement in medi n surviv l time to 15 months. Chemother py Numerous chemother
py regimes h ve
een reported, including the use of
oth intr thec l nd intr ve
nous ther pies which c n
e given prior to, synchronous with, or following r dio
ther py. The most commonly used drugs re those used in
Fig. 6.18 C rcinom of the ethmoid extending through the cri
riform pl te into t
he nterior cr ni l foss .
86
CHAPTER 6
tumour rising from the olf ctory epithelium th t m y inv de through the cri
rif
orm pl te. The p tients usu lly present with
loodst ined or purulent n s l di
sch rge nd p in in the involved region. CSF rhinorrhoe m y occur if the dur h
s
een
re ched. Surgic l excision using cr niof ci l resection m y
e the on
ly method of controlling these tumours nd, if the tumour h s spre d into the or

it, n dequ te resection m y involve or
it l exenter tion.
v cuol ted nd some will cont in single l rge v cuole giving signet ring ppe
r nce. The ch r cteristic histologic l ppe r nce is phys liphorous (
u

le-
e r
ing) cells cont ining multiple v cuoles.
Clinic l present tion
The m jority of intr cr ni l chordom s rise
etween 20 nd 60 ye rs of ge. The
clinic l fe tures result from the widespre d tumour extension nd include: r is
ed intr cr ni l pressure, c using he d ches nd vomiting multiple cr ni l nerve
p lsies, often unil ter l n soph rynge l o
struction. The r diologic l ppe r nc
es re of destructive lesion t the
se of the skull or in the verte
r l
odi
es (Fig. 6.19).
Chordom s
Chordom s re r re tumours rising from notochord cell nests. They m y rise thr
oughout the cr niospin l xis
ut occur predomin ntly t the ends of the xi l s
keleton in: the
sioccipit l region the s crococcyge l region. The intr cr ni l
chordom presents s skull
se tumour. It in ltr tes nd erodes the sphenoid
nd
siocciput nd m y spre d into the petrous
ones, the p r n s l sinuses, the
sell turcic nd the c vernous sinuses. The tumour will compress nd distort t
he dj cent
r in nd engulf the cr ni l nerves nd rteries. These tumours do n
ot h ve histologic l fe tures of m lign ncy nd only r rely met st size. However

, it is not usu lly possi
le to excise the cr ni l tumours completely; most p ti

ents die within 10 ye rs of initi l present tion. Spin l chordom s occur predomi
n ntly in the s crococcyge l region, lthough they m y lso rise in the cervic
l re . Like the cr ni l tumours, spin l chordom s inv de nd destroy
one nd c
ompress dj cent neur l structures. Remote met st ses occ sion lly occur. P tien
ts with spin l chordom s present with
ck p in, r dicul r p in nd slowly progr
essive lum
os cr l nerve root involvement resulting in sphincter dif culties nd s
ensory nd motor distur
nces in the legs.
Tre tment
It is r rely possi
le to excise ll the tumour. Postoper tive r diother py is us
u lly dministered
ut is of dou
tful v lue.
P edi tric tumours
Intr cr ni l tumours re the most common form of solid tumours in childhood, wit
h 40% of the
Histologic l ppe r nce
The tumours consist of notochord cells nd mucoid strom . M ny of the cells m y

e co rsely
Fig. 6.19 S cr l chordom . A destructive tumour extending into the verte
r l c n
l.
BRAIN TUMOURS
87
tumours occurring
ove the tentorium cere
elli. The most common supr tentori l
tumours re strocytom s, followed
y n pl stic strocytom s nd glio
l stom m
ultiforme. Cr nioph ryngiom occurs more commonly in children th n in dults nd
is situ ted in the supr sell r region; this tumour is descri
ed in Ch pter 8. O
ther, less common, supr tentori l tumours include primitive neuroectoderm l tumo
urs (PNETs), ependymom s, g ngliogliom nd pine l region tumours.
f lse loc lizing sign. P pilloedem is usu lly present t the time of di gnosis. I
n inf nts, n exp nding he d size is n ddition l sign of r ised intr cr ni l p
ressure. Foc l neurologic l signs These re due to the tumour inv ding or compre
ssing the cere
ellum (nuclei nd tr cts), the
r instem nd cr ni l nerves. Trun
c l nd g it t xi result p rticul rly from midline cere
ell r involvement. Hor
izont l g ze p retic nyst gmus often occurs with tumours round the 4th ventricl
e. Up
e t vertic l nyst gmus is indic tive of
r instem involvement. Distur
nce
s of
ul
r function, such s dif culty in sw llowing with n s l regurgit tion of u
id, dys rthri nd imp ired p l t l nd ph rynge l re exes, result from
r instem
involvement. In ddition, compression or tumour inv sion of the pyr mid l tr cts
m y result in hemip resis nd, if the scending sensory p thw ys re involved,
sensory distur
nce will occur in the trunk nd lim
s. The tumour m y directly e
nvelop the lower cr ni l nerves glossoph rynge l, v g l, spin l ccessory nd hy
pogloss l s well s the 7th cr ni l nerve. Neck stiffness nd he d tilt m y occ
ur in children with posterior foss neopl sms, nd m y
e due to herni tion of
cere
ell r tonsil or tumour tissue resulting in dur l irrit tion.
Posterior foss tumours
Sixty per cent of p edi tric
r in tumours occur in the posterior foss . The rel
tive incidence of the tumours is: 1 cere
ell r strocytom 30% 2 medullo
l stom
(infr tentori l primitive neuroectoderm l tumour) 30% 3 ependymom 20% 4
r in
stem gliom 10% 5 miscell neous 10%: ( ) choroid plexus p pillom (
) h em ngio

l stom (c) epidermoid, dermoid (d) chordom .
Clinic l present tion The presenting clinic l fe tures of posterior foss neopl
sms in children re rel ted to: r ised intr cr ni l pressure foc l neurologic l

signs.
R ised intr cr ni l pressure This is the most common presenting fe ture. It is d
ue to hydroceph lus c used
y o
struction of the 4th ventricle nd is m nifest

y he d ches, vomiting, diplopi nd p pilloedem . The he d ches
egin insidiousl
y, gr du lly
ecoming more severe nd frequent; they re worst in the e rly morn
ing. There is usu lly no speci c he d che loc liz tion. Vomiting is frequently ss
oci ted with the he d ches nd m y tempor rily relieve the he d che. R ised intr
cr ni l pressure m y result in str
ismus c using diplopi due to stretching
of one or
oth of the 6th (
ducens) cr ni l nerves. This is so-c lled
Investig tions CT sc n nd MRI h ve repl ced the need for the previous r diologi
c l investig tions th t included r dio-isotope
r in sc nning, ir ventriculogr
phy nd posterior foss ngiogr phy. The CT sc n nd/or MRI will show the presen
ce of posterior foss tumour, its position nd whether it rises prim rily in
the
r instem, 4th ventricle or the cere
ellum (Figs 6.206.24). M n gement The tr
e tment of posterior foss tumours involves: surgery r diother py chemother py.
88
CHAPTER 6
( )
Fig. 6.21 Cystic cere
ell r strocytom c using o
structive hydroceph lus. There
is contr stenh ncing nodule in l rge cyst.
A prelimin ry CSF shunt m y need to
e performed in child with severely r ised
intr cr ni l pressure due to hydroceph lus. The CSF diversion c n
e chieved

y either n extern l dr in or ventriculoperitone l shunt. An extern l dr in is
tempor ry me sure only,
ec use of the risk of infection. A ventriculoperiton
e l shunt provides immedi te nd controlled relief of intr cr ni l hypertension
nd the su
sequent posterior foss oper tion c n
e performed s pl nned elect
ive procedure, r ther th n n urgent oper tion in su
optim l conditions. A criti
cism of preoper tive ventriculoperitone l shunt is th t it might promote the m
et st tic spre d of tumour. A ltering ch m
er in the shunt system m y lessen this
risk
ut this predisposes to shunt m lfunction.
Fig. 6.20 ( ) CT sc n. Contr st-enh ncing medullo
l stom rising from the vermi
s c using o
structive hydroceph lus. (
) MRI sc n. Medullo
l stom .
(
)
BRAIN TUMOURS
89
( )
Fig. 6.22 Contr st-enh ncing ependymom in the 4th ventricle c using o
structive
hydroceph lus.
Steroid medic tion to control loc l oedem is commenced preoper tively. The oper
tion is performed in either the sitting or prone position through vertic l mi
dline incision. A posterior foss cr niotomy is performed, usu lly with excision
of the
one down to nd round the for men m gnum. Tumour excision is ided
y
the use of m gnifying loupes nd illumin tion with
reoptic he dlight, or
y th
e use of n oper ting microscope. Postoper tive c re involves c reful monitoring
of the neurologic l signs. Postoper tive h emorrh ge or oedem m y result in r
pid deterior tion of the neurologic l st te, nd in respir tory rrest. An urgen
t CT sc n m y indic te the c use nd site of the pro
lem
ut the deterior tion m
y
e so r pid th t the wound m y need to
e reopened without the
ene t of prior

sc nning. If ventriculoperitone l shunt h s not
een inserted prior to tumour

remov l n ex cer
tion of the o
structive hydroceph lus m y occur if the tumour
excision h s f iled to relieve the CSF o
struction. Distur
nces of
ul
r nd
lower cr ni l nerve function m y c use dif culty in
(
)
Fig. 6.23 MRI sc n showing ( ) pontine gliom , (
) cystic
r instem gliom .
90
CHAPTER 6
sw llowing. N sog stric feeding m y
e necess ry until the protective mech nisms
return, nd gre t c re should
e t ken to void spir tion.
Medullo
l stom
Medullo
l stom , lso referred to s n infr tentori l PNET, is m lign nt tumo
ur usu lly rising in the midline from the cere
ell r vermis, lthough it m y oc
cur more l ter lly in cere
ell r hemisphere in older p tients. The tumour exp
nds to inv de the dj cent cere
ellum nd l rge tumours completely ll the 4th ven
tricle (see Fig. 6.19). The tumours rise from the extern l gr nul r l yer of th
e fet l cere
ellum (O
ersteiners l yer). Histologic lly the medullo
l stom is hi
ghly cellul r, with numerous mitoses. True rosettes do not occur
ut the cells
re seen in concentric p tterns round homogeneous m teri l or
lood vessels (pse
udorosettes). Presenting fe tures. The presenting fe tures re rel ted to hydroc
eph lus nd cere
ell r dysfunction. Trunc l t xi is typic lly present in child
ren with medullo
l stom
ut cr ni l nerve de cits, except for 6th nerve p lsy,
re uncommon in the e rly st ges. Surgery. At surgery the cere
ell r vermis is s
plit in the midline nd it is usu lly possi
le to o
t in gross m croscopic exc
ision of the tumour with complete remov l from the 4th ventricle. R di tion ther
py. Medullo
l stom is rel tively r diosensitive nd r di tion ther py is recom
mended to the entire neur xis
ec use of the tendency of the tumours to seed lo
ng the CSF p thw ys. Chemother py. Adjuv nt chemother py is usu lly recommended
nd numerous protocols using v riety of chemother peutic gents h ve
een inve
stig ted. There is no uniformity of opinion s to which drugs or routes of dmin
istr tion re the most effective nd whether chemother py should
e dministered
s p rt of the initi l tre tment pl n or only used t the time of recurrence of
the tumour. Young children re exquisitely sensitive to the neurotoxicity due t
o r di tion ther py, such th t the possi
ility of utilizing chemother py lone
( )
(
)
(c)
Fig. 6.24 MRI sc n showing glio
l stom inv ding through the
r instem of 25-y
e r-old m n. Before intr venous g dolinium contr st: ( ) s gitt l T1. After intr
venous g dolinium contr st: (
) xi l nd (c) coron l.
BRAIN TUMOURS
91

nd postponing the use of r diother py h s
een tri lled. Prognosis. Although th

e com
in tion of r dic l surgery nd irr di tion h s improved the prognosis, the
5-ye r surviv l r te is pproxim tely 40%.
Cere
ell r strocytom

The cere
ell r strocytom is frequently
enign, slowly growing cystic tumour

which is the most f vour
le of ll the intr cr ni l p edi tric neopl sms. The t
umours m y rise in either the hemisphere or vermis nd frequently consist of
l rge tumour cyst with rel tively sm ll solid component in the w ll of the cys
t (see Fig. 6.20). Less frequently the tumour m y
e entirely solid with little
or no cystic component. Histologic lly, the solid portion of the tumour is usu l
ly Gr de 1 or 2 strocytom . Presenting fe tures. The clinic l presenting fe t
ures re simil r to those of medullo
l stom ,
ut s the tumour m y
e loc ted
more l ter lly the presenting fe tures re ccomp nied
y ipsil ter l cere
ell
r distur
nce. The dur tion of symptoms is v ri
le
ut tends to
e longer th n
with medullo
l stom , ver ging 612 months. Surgery. A complete surgic l excision
is usu lly possi
le nd it is only necess ry to excise the solid component from
the cystic tumour. R di tion ther py. Postoper tive r di tion ther py is not us
u lly indic ted if n excision h s
een possi
le. The prognosis is the most f vo
ur
le of ll intr cr ni l childhood tumours with cure r te in excess of 75%.

lly due to hydroceph lus. Involvement of the dors l
r instem results in unil t
er l or
il ter l f ci l we kness. Surgery. The surgic l excision of the ependym
om involves splitting the inferior vermis to o
t in ccess to the 4th ventricle
. It is usu lly possi
le to perform gross m croscopic excision of the tumour f
rom the ventricle nd dj cent cere
ellum
ut, s the tumour often origin tes fr
om the oor of the 4th ventricle, tot l excision is r rely possi
le. R di tion the
r py. Postoper tive r di tion ther py is usu lly dministered to the posterior f
oss nd, s the tumour seeds long the CSF p thw ys, entire neur l xis irr di
tion is often recommended, p rticul rly in the higher-gr de ependymom . There is
no de nite dv nt ge from djuv nt chemother py, lthough it m y
e used t the t
ime of tumour recurrence.
Br instem gliom
The
r instem gliom rises predomin ntly in the pons, less frequently in the me
dull
ut m y in ltr te extensively throughout the
r instem. The tumour in ltr tes

etween the norm l structures with histologic l ppe r nce v rying from the re
l tively
enign strocytom to n pl stic strocytom nd glio
l stom multiform
e. Over 50% of
r instem gliom s ex mined t utopsy will h ve microscopic fe tu
res of glio
l stom multiforme. Clinic l present tion. The clinic l present tion
ch r cteristic lly includes progressive multiple
il ter l cr ni l nerve p lsie
s with involvement of pyr mid l tr cts nd t xi . F ci l we kness nd 6th cr ni
l nerve p lsy re common nd n internucle r ophth lmoplegi is indic tive of
n intrinsic
r instem lesion. The childs person lity often ch nges they
ecome p
thetic. R ised intr cr ni l pressure is less common th n with other p edi tric
posterior foss neopl sms, s o
struction of the 4th ventricle or queduct of Sy
lvius occurs l te in the illness. The CT nd MRI ppe r nce is of n exp nded
r
instem. MRI h s consider
ly improved the ccur cy of the di gnosis (Fig. 6.22)
.
Ependymom
The ependymom of the 4th ventricle rises from the oor of the 4th ventricle nd
is tt ched to, nd m y in ltr te, the underlying
r instem (see Fig. 6.21). P tho
logic l fe tures. The p thologic l fe tures nd histology re descri
ed e rlier
in the ch pter (see p. 78). Presenting fe tures. The presenting fe tures re sim
il r to those descri
ed for medullo
l stom , lthough the initi l symptoms nd
signs re usu92
CHAPTER 6
Surgery. Surgic l tre tment is not usu lly indic ted, lthough either n open or
stereot ctic
iopsy m y
e performed to con rm the di gnosis. R di tion ther py
. P lli tive r di tion ther py is the only tre tment. The tumour usu lly c uses

de th within 24 months of di gnosis, lthough some p tients with low-gr de tumou

rs will live longer. Chemother py h s limited
ene t.
Further re ding
Apuzzo MJ et l. (1984) Ionising nd non-ionising r di tion tre tment of cere
r
l m lign nt gliom s: speci lised ppro ches. Clinic l Neurosurgery 31, 470496. B
iley P, Cushing H (1971) A Cl ssi c tion of Tumours of the Gliom Group on Histo
genetic B sis with Correl ted Study of Prognosis. J B Lippincott, Phil delphi
, 1926. Reprinted Argosy Antiqu ri n, New York. Burger PC, Vogel FS (1982) Surgi
c l P thology of the Nervous System nd its Coverings, 2nd edn. John Wiley, New
York. C irncross JG, Ueki K, Zl tescu MC et l. (1998) Speci c genetic predictors
of chemother peutic response nd surviv l in p tients with n pl stic oligodendr
ogliom s. Journ l of the N tion l C ncer Institute 90 (19), 14731479. Dohrm nn GJ
, F rwell JR, Fl nnery JI (1976) Ependymom s nd ependymo
l stom s in children.
Journ l of Neurosurgery 45, 273283.
Higgin
oth m NL, Phillips RJ, F rr HW (1979) Chordom : thirty ve ye r study t th
e Memori l Hospit l. C ncer 29, 18411850. K ye AH, Bl ck P McL (2000) Oper tive N
eurosurgery. Churchill Livingstone, London, New York, Edin
urgh. K ye AH, L ws E
L (1995) Br in Tumours. Churchill Livingstone, London. K ye AH, Morstyn G, Apuzz
o MJ (1988) Photor di tion ther py nd its potenti l in the m n gement of neurol
ogic l tumours. Journ l of Neurosurgery 69, 114. Kernoh n JW, M y
on RF, Svein HJ
, Adson AW (1949) A simpli ed cl ssi c tion of gliom s. M yo Clinic Proceedings 24,
7175. Korn
lith PL, W lker M (1988) Chemother py of gliom s. Journ l of Neurosurg
ery 68, 117. Lie
el SA, Sheline GE (1987) R di tion ther py for neopl sms of the

r in. Review rticle. Journ l of Neurosurgery 66, 122. Rich TA et l. (1985) Cli
nic l nd p thologic review of 48 c ses of chordom . C ncer 56, 182187. Ringertz
N (1950) Gr ding of gliom s. Act P thologic Micro
iologic Sc ndin vic 27, 516
4. Wold LF, L ws ER Jr (1983) Cr ni l chordom s in children nd young dults. Jo
urn l of Neurosurgery 59, 10431047. Yung WK (2000 June) Temozolomide in m lign nt
gliom s. Semin rs in Oncology 27 (3 Suppl. 6), 2734. Zulch KJ (1986) Br in Tumou
rs, Their Biology nd P thology, 3rd edn. Springer Verl g, Berlin.
CHAPTER 7
7
Benign
r in tumours
The
enign
r in tumours m y
e intim tely ssoci ted with, nd surrounded
y, t
he dj cent
r in,
ut the tumour cells do not inv de the underlying
r in. This
is in contr distinction to the gliom s, which re intrinsic
r in tumours ctiv
ely inv ding the dj cent
r in. This ch pter will discuss the more common
enig
n
r in tumours meningiom nd coustic neurom nd give
rief description of
the less common tumours: h em ngio
l stom , epidermoid nd dermoid cysts nd col
loid cysts.
Meningiom
Meningiom s re the most common of the
enign
r in tumours nd constitute
out
15% of ll intr cr ni l tumours,
eing
out one-third of the num
er of gliom s
. Although they m y occur t ny ge, they re ch their pe k incidence in middle
ge, re very uncommon in children nd occur more frequently in women th n men.
The term meningiom w s introduced
y H rvey Cushing in 1922, lthough the tumou
r h d
een descri
ed in the l te eighteenth century. The tumour rises from the
r chnoid l yer of the meninges, princip lly the r chnoid villi nd gr nul tion
s.
Aetiology
As for other
r in tumours, no de nite etiologic l f ctor h s
een identi ed. Howev
er, the possi
ility th t he d tr um predisposes to the development of meningiom

 h s
een the su
ject of controversy for m ny ye rs. Although epidemiologic l s

tudies do not support tr um s
n etiologic l f ctor, there h ve
een c ses reporting the development of menin
giom s t the site of su
st nti l meninge l tr um . Meningiom s re known to occ
ur following low levels of irr di tion s w s given in the p st for tine c piti
s, nd n n lysis of the N g s ki tomic
om
survivors found high correl tio
n
etween the incidence of meningiom s nd the dist nce from the epicentre of th
e explosion. Meningiom s occur with high frequency in p tients with neuro
rom t
osis type 2 (NF2) (often multiple). This ssoci tion h s prompted cytogenetic st
udies, which h ve shown th t monosomy of chromosome 22 is the most common chromo
som l
norm lity noted in meningiom s, occurring in 5080% of spor dic tumours. I
n ddition, lter tions of m ny other chromosomes (including chromosomes 1, 6, 9
, 10, 11, 13, 14, 18 nd 19) h ve
een noted to
e involved in the form tion nd
progression of meningiom . The import nce of sex hormones nd their receptors i
n meningiom is suggested
y the 24 times incidence in fem les. Oestrogen
inds i
n less th n 30% of meningiom s, with the m jority of those receptors
eing type
II su
type, th t h ve lower f nity nd speci city for oestrogen th n the cl ssic
type I receptor usu lly found in
re st c ncer. Progesterone receptors re much
more commonly ssoci ted with meningiom s, occurring in 50100% of tumours tested.
Position of meningiom s (Fig. 7.1)
Meningiom s rise from the r chnoid l yer of the meninges, especi lly the r ch
noid c p cells. 93
94
CHAPTER 7
B s l Olf ctory groove Sphenoid l wing Tu
erculum sell e Clivus For men m gnum P
osterior foss - cere
ellopontine ngle
P r s gitt l section P r s gitt l/f lcine
Olf ctory groove Tu
erculum sell e Posterior foss (convexity) For men m gnum
Clivus
Fig. 7.1 Typic l positions of intr cr ni l meningiom s.
The most common loc tion is in the p r s gitt l region rising either from the w
ll of the superior s gitt l sinus (p r s gitt l) or from the f lx (f lcine). Le
ss frequently the tumours m y rise from the convexity of the cr ni l v ult, whe
re they re p rticul rly concentr ted in the region of the coron l suture. Sphen
oid l ridge meningiom s re divided into those th t rise from the inner p rt of
the lesser wing of the sphenoid nd the dj cent nterior clinoid process, nd
those rising from the outer sphenoid l ridge, comprising the gre ter wing of th
e sphenoid nd the dj cent pterion (the junction of the tempor l, p riet l nd
front l
ones). Less frequently, the tumours m y rise from the olf ctory groove
, tu
erculum sell (supr sell r), oor of the middle cr ni l foss , c vernous sinu
s or posterior foss (T
le 7.1). Meningiom s usu lly occur s single intr cr
ni l tumour
ut multiple intr cr ni l meningiom s m y present in NF2 (see p. 64)
.
T
le 7.1 Position of intr cr ni l meningiom s (%). P r s gitt l nd f lx Convex
ity Sphenoid l wing Olf ctory groove Supr sell r Posterior foss Ventricle Optic
she th 25 20 20 12 12 9 1.5 0.5
P thology
Unlike gliom s, where the cl ssi c tion system is


sed on the histologic l ppe r nce of the tumour, meningiom s re usu lly cl s
si ed ccording to their position of origin r ther th n their histology. The re so
n for this is th t the
iologic l ctivity of the tumour, the presenting fe ture
s, the tre tment nd prognosis re ll rel ted more to the site of the tumour th
n to the histology. The m jor histologic l types re listed
elow. Syncyti l or
meningotheliom tous sheets of cells with v rying mounts of strom . The tr nsit
ion l type is ch r cterized
y
BENIGN BRAIN TUMOURS
95
whorls of cells which m y undergo hy lin degener tion with su
sequent deposition
of c lcium s lts. These c lci ed concentric ps mmom
odies form the ch r cterist
ic fe ture of m ny tr nsition l meningiom s
ut they m y lso
e present in the
syncyti l or
ro
l stic types. The
ro
l stic type cont ins
und nt reticulin nd
coll gen
res. Angiom tous meningiom s re much less common nd their ch r cteri
stic fe ture is the predomin nce of v scul r ch nnels sep r ted
y sheets of cel
ls. Histologic lly, these tumours resem
le cere
ell r h em ngio
l stom s. M lign
nt meningiom s occur infrequently. The indic tions of m lign ncy include cellul
r pleomorphism, necrosis, incre sed num
ers of mitotic gures nd loc l inv sion
of
r in. Atypic l meningiom s re tumours th t l ck the histologic l fe tures o
f m lign ncy,
ut h ve
iologic l
eh viour intermedi te
etween the typic l
nd m lign nt meningiom . These tumours re most likely to recur.
( )
Clinic l present tion
Meningiom s present with fe tures of: r ised intr cr ni l pressure foc l neurolo
gic l signs epilepsy. The position of the tumour will determine the fe tures of
the clinic l present tion. The tumours grow slowly nd there is frequently lon
g history, often of m ny ye rs, of symptoms prior to the di gnosis.
(
)
P r s gitt l tumours (Fig. 7.2) These tumours most often rise in the middle thi
rd of the v ult nd the p tient m y present with foc l epilepsy nd p resis, usu
lly ffecting the opposite leg nd foot s the motor cortex on the medi l spec
t of the posterior front l lo
e is ffected. Tumours rising nteriorly re ofte
n
il ter l nd p tients present with fe tures of r ised intr cr ni l pressure.
As these tumours involve the front l lo
es, pseudopsychi tric symptoms, s well
s imp irment of memory, intelligence nd person lity, m y occur. Urin ry incont
inence is occ sion lly symptom of l rge
Fig. 7.2 P r s gitt l meningiom . ( ) CT sc n. (
) MRI.
front l tumour, especi lly if it is
il ter l. Tumours rising from the posterio
r f lx m y ffect the p rieto-occipit l region nd produce homonymous hemi nop
i . If the tumour lies
ove the c lc rine ssure the inferior qu dr nt is more f
fected; when the tumour is
elow the ssure the upper qu dr nt is predomin ntly f
fected.
Convexity tumours (Fig. 7.3) Convexity tumours m y grow to l rge size if
96
CHAPTER 7
( )

( )
(
) (
)
Fig. 7.3 Convexity meningiom . ( ) CT sc n. (
) MRI.
Fig. 7.4 ( ) CT sc n. Hyperostosis of the left sphenoid l ring c using unil ter
l proptosis due to sphenoid l ring meningiom . (
) MRI. Inner sphenoid l wing
meningiom .
situ ted in front of the coron l suture. They present with r ised intr cr ni l p
ressure. More posterior tumours will c use foc l neurologic l symptoms nd foc l
epilepsy.
Sphenoid l ridge tumours (Fig. 7.4) Tumours rising from the inner sphenoid l ri
dge c use compression of the dj cent optic nerve nd p tients m y present with
history of uniocul r visu l f ilure. If the tumour is l rge enough to c use r
ised intr cr ni l pressure p pilloedem will develop in the contr l ter l eye.
The presenting fe tures of prim ry optic trophy in one eye nd p pilloedem in
the other is known s the Foster Kennedy syndrome, nd w s descri
ed in 1911. In
ner sphenoid l ridge tumours m y lso c use compression of the olf ctory tr ct,
resulting in nosmi . P tients with tumours involving the outer sphenoid l ridge
present with fe tures of r ised intr cr ni l pressure, often severe p pilloedem
with rel tively inconspicuous loc lizing symptoms or signs. Tumours in this re
gion occur s
BENIGN BRAIN TUMOURS
97
thin sheet, nd re known s en pl que. They m y c use n excessive
ony re ction
(hyperostosis) resulting in proptosis (Fig. 7.4).
Olf ctory groove tumours (Fig. 7.5) Olf ctory groove meningiom s c use nosmi ,
initi lly unil ter l nd l ter
il ter l. The presenting fe tures m y include sy
mptoms of r ised intr cr ni l pressure, nd f iling vision either from chronic p
pilloedem or from direct compression of the optic nerve or chi sm c using visu
l eld defects. These tumours m y lso present with the Foster Kennedy syndrome
nd the intellectu l nd psychi tric pro
lems c used
y front l lo
e compression
descri
ed for inner spheroid l ridge meningiom s.
( )
Supr sell r tumours (Fig. 7.6) Supr sell r meningiom s rising from the tu
ercul
um sell e will c use visu l f ilure nd
itempor l hemi nopi ,
ut the l ck of
endocrine distur
nce will distinguish the clinic l present tion of this tumour
from th t of pituit ry tumour. Ventricul r tumours (Fig. 7.7) Tumours rising
in the l ter l ventricle present with symptoms of r ised intr cr ni l pressure
extending over sever l ye rs nd ssoci ted with mild glo
l distur
nce of fu
nction of one hemisphere nd frequently homonymous hemi nopi . Posterior foss
tumours (Fig. 7.8) Posterior foss tumours m y rise from the cere
ell r convex
ity or from the cere
ellopontine ngle or clivus. The cere
ellopontine ngle tum
ours simul te n coustic neurom with symptoms involving the coustic nerve, tr
igemin l nerve nd f ci l nerve, t xi due to cere
ell r involvement nd r ised
intr cr ni l pressure, often due to hydroceph lus c used
y o
struction of the
4th ventricle. Meningiom s rising from the
Fig. 7.5 ( ) CT sc n. Olf ctory groove meningiom . (
) MRI. Olf ctory groove men
ingiom extending on to tu
erculum sell nd over pituit ry foss .

(
)
98
CHAPTER 7
( )
Fig. 7.7 Intr ventricul r meningiom .
(
)
Fig. 7.6 Tu
erculum sell e meningiom . ( ) CT sc n. (
) MRI.
the tumour tt chment or, s seen with en pl que meningiom , more diffuse scle
rosis. These
one ch nges m y lso
e seen on pl in skull X-r y. M gnetic reson
nce im ging will demonstr te meningiom s following the intr venous injection of
g dolinium contr st (Figs 7.97.11). Meningiom s re usu lly isointense on T1weigh
ted im ges,
ut enh nce intensely nd usu lly homogeneously following dministr
tion of g dolinium. Cere
r l ngiogr phy is no longer necess ry s di gnostic
investig tion
ut m y
e useful preoper tively to scert in the position of the
cere
r l vessels. It will demonstr te extern l c rotid rtery supply to the tumo
ur with ch r cteristic tumour
lush, differenti ting it from gliom or met s
t tic tumour (Fig. 7.12). Angiogr phy lso llows preoper tive em
oliz tion of t
he tumour, if necess ry.
clivus or the for men m gnum region m y compress the
r instem directly.
Preoper tive m n gement
Meningiom s re frequently surrounded
y severe cere
r l oedem nd p tients sho
uld
e tre ted with high-dose steroids (dex meth sone) prior to surgery if possi

le. Preoper tive em
oliz tion of the tumour v scul ture m y
e considered dvis

le in some nterior
s l nd sphenoid l wing tumours where the m jor
R diologic l investig tions
The CT sc n ppe r nce shows tumour of slightly incre sed density prior to con
tr st; it enh nces vividly nd uniformly following intr venous contr st. Hyperos
tosis of the cr ni l v ult m y
e foc l process t the site of
BENIGN BRAIN TUMOURS
99
( )
( )
(
)
Fig. 7.9 MRI of p r s gitt l meningiom . The meningiom m y
e isodense on the p
l in T1 nd T2 sc ns ( )
ut will enh nce vividly fter intr venous g dolinium (

).
(
)
v scul r supply is not re dily ccessi
le in the e rly st ges of the oper tion.
Tre tment
The tre tment of meningiom s is tot l surgic l excision, including o
liter tion
of the dur l tt chment. Although this o
jective is usu lly possi
le there re s
ome situ tions where complete excision is not possi
le
ec use of the position o

f the tumour. Tumours rising from the clivus, in front of the
r instem or thos
e situ ted within the c vernous sinus, re notoriously dif cult to excise without
c using serious mor
idity. R di tion ther py m y
e used to tre t residu l tumou
rs following su
tot l resection, in order to reduce the risk of recurrent growth
.
(c)
Fig. 7.8 ( ) CT sc n. Meningiom rising in the cere
ellopontine ngle nd from
the tentori l edge. (
) MRI. Clivus meningiom . (c) MRI. For men m gnum meningio
m .
100
CHAPTER 7
( ) ( )
(
)
(
)
Fig. 7.11 MRI. Extensive p r s gitt l meningiom . ( ) S gitt l view. (
) Coron l
view.
Fig. 7.10 MRI. F lcine meningiom . ( ) Before contr st tumour is iso- or hypoden
se. (
) Tumour enh nces with intr venous contr st.
( )
(
)
(c)
Fig. 7.12 Cere
r l ngiogr m of olf ctory groove meningiom showing displ cement
of nterior cere
r l rteries ( ,
) nd the ch r cteristic tumour
lush, usu ll
y due to the extern l c rotid rtery supply (c).
BENIGN BRAIN TUMOURS
101
Stereot ctic r diother py h s
een used to tre t sm ll meningiom s (less th n 3
cm in di meter), p rticul rly if the tumours re loc ted in portions not e sily
men
le to surgery, or in the elderly or medic lly in rm p tient. Clinic l studie
s h ve shown short-term control r tes of over 90%,
ut long-term studies will
e
necess ry to prove the ef c cy nd s fety of focused r di tion tre tment.

nd it is slightly more common in m les th n in fem les. The presenting fe tures

re dependent on the tumour loc tion, with symptoms usu lly
eing present for l
ess th n 1 ye r.
Acoustic neurom
Acoustic schw nnom s rise from the 8th cr ni l nerve nd ccount for 8% of intr
cr ni l tumours. Schw nnom s occur less frequently on the 5th cr ni l nerve nd
r rely involve other cr ni l nerves. The coustic schw nnom t kes origin from
the vesti
ul r component of the 8th cr ni l nerve ne r the intern l uditory me
tus, t the tr nsition zone where the Schw nn cells repl ce the oligodendrogli .
As such it should more correctly
e c lled vesti
ul r schw nnom , lthough th
e term coustic neurom or schw nnom is more commonly used. M croscopic lly, th

e coustic schw nnom is lo
ul ted with c psule th t sep r tes it from the sur
rounding neur l structures. Sm ll tumours usu lly rise from within the intern l
uditory c n l nd occupy the porus of the intern l uditory c n l nd, s the
tumour grows, the 8th nerve is destroyed nd the dj cent cr ni l nerves
ecome
stretched round the tumour. The 7th nerve is typic lly displ ced on the ventr l
nd nterior surf ce of the tumour nd the trigemin l nerve is c rried upw rds
nd forw rds
y the upper pole. The 6th nerve lies ventr l nd usu lly medi l to
the m jor m ss nd the lower cr ni l nerves re displ ced round the inferior p
ole of the tumour. As the tumour grows medi lly it compresses nd displ ces the
cere
ellum nd distorts the
r instem. L rge tumours will result in o
struction
of the 4th ventricle nd hydroceph lus. Bil ter l coustic neurom s re the h ll
m rk of neuro
rom tosis type 2 (NF2), inherited s n utosom l domin nt conditio
n (see Ch pter 6).
Postoper tive m n gement The postoper tive c re of p tients following excision o
f meningiom involves the routine m n gement of p tients following cr niotom
y
ut with p rticul r ttention to the minimiz tion of cere
r l oedem . Steroid
ther py is continued initi lly nd gr du lly t pered. C re is t ken to void exc
essive hydr tion nd the p tient is nursed with the he d of the
ed elev ted to
promote venous return. Neurologic l deterior tion requires urgent ssessment nd
CT sc n will determine the p thologic l c use, either postoper tive h emorrh
ge or cere
r l oedem .
Tumour recurrence
The risk of tumour recurrence depends on the extent of tumour excision. When the
tumour nd its dur l origins re completely excised, the risk of recurrence is
remote. The most common source of recurrence is from tumour th t h s inv ded
venous sinus nd which w s not resected (e.g. superior s gitt l sinus or c vern
ous sinus). Recurrence is more common if the tumour h s histologic l fe tures of
m lign ncy.
Meninge l h em ngiopericytom
Meninge l h em ngiopericytom is m lign nt neopl sm with s rcom -like
eh viou
r. It w s origin lly cl ssi ed
y Cushing nd Eisenh rdt in 1938 s n ngio
l sti
c v ri nt of meningiom . The tumours r diologic l nd m croscopic ppe r nce rese
m
les v scul r meningiom ,
ut it rises from the meninge l c pill ry pericyte
nd typic lly cont ins su
popul tion of cells th t express f ctor VIII . The
tumour incidence is 24% of meningiom
Clinic l present tion
The presenting fe tures will depend on the size of the tumour t the time of di
gnosis. The e rlier symptoms re ssoci ted with 8th nerve involve102
CHAPTER 7
ment. Tinnitus nd unil ter l p rti l or complete sensorineur l he ring loss re
the e rliest fe tures. Episodes of vertigo m y occur
ut these m y
e dif cult to
distinguish from Menires dise se. Although the tumour c uses compression of the f
ci l nerve, the growth of the tumour is so slow th t f ci l p resis is not evid
ent until the tumour is l rge. At th t st ge 5th nerve compression m y
e eviden
t, with diminished f ci l sens tion nd depressed corne l re ex. Cere
ell r invo
lvement will result in t xi , nd compression of the pyr mid l tr cts from ve
ry l rge tumour c using
r instem compression will c use contr l ter l hemip r
esis. If l rge tumour h s c used o
structive hydroceph lus the p tient will l
so present with fe tures of r ised intr cr ni l pressure.
Fig. 7.13 Acoustic neurom . A contr st-enh ncing tumour in the cere
ellopontine
ngle rising from the 8th cr ni l nerve in the intern l uditory c n l.

R diologic l investig tions

The CT sc n or MRI will show n enh ncing tumour extending from the intern l ud
itory c n l into the cere
ellopontine ngle (Fig. 7.13). The intern l uditory m
e tus will
e widened indic ting th t the tumour h s risen from the 8th cr ni l
nerve (Fig. 7.14). While there is no dif culty in di gnosing tumour l rge enoug
h to
e evident on the CT sc n, very sm ll coustic neurom s, which re predomin
ntly within the intern l uditory c n l, m y
e more dif cult to di gnose. These
tumours m y
e seen on highqu lity CT sc n
ut re p rticul rly evident using MR
I, especi lly following g dolinium contr st (Fig. 7.15), which is now the invest
ig tion of choice.
Fig. 7.14 Widened intern l uditory me tus, indic tive of n coustic neurom .
Other investig tions
Pure tone udiometry,
y
oth ir nd
one conduction, is n essenti l p rt of t
he investig tion of p tient with n coustic neurom , the most common nding
ei
ng high-frequency he ring loss. Other speci l uditory tests include the use of

r instem uditory evoked responses which re p rticul rly sensitive for ch nges
in the retrocochle r uditory system; these re helpful in di gnosing sm ll i
ntr c n licul r tumour. Vesti
ul r function is imp ired e rly in
p tients with coustic neurom . The H llpike c loric test is c rried out with th
e p tient supine on couch nd the he d r ised to 30C
ove horizont l,
ringing
the horizont l c n ls into the vertic l pl ne with the position of m ximum sens
itivity to therm l stimuli. W rm nd cool w ter is irrig ted nd the nyst gmus r
e ction o
served. The c loric response on the side of the coustic nerve tumour
is depressed or
sent.
Differenti l di gnosis
The m jor differenti l di gnoses for cere
elloBENIGN BRAIN TUMOURS
103
pontine ngle tumour, in decre sing frequency, re: meningiom met st tic tumour
exophytic
r instem gliom epidermoid tumour.
Tre tment
The tot l excision of l rge coustic neurom rem ins one of the m jor oper tiv
e ch llenges in wh t Cushing h s descri
ed s the gloomy corner of neurologic sur
gery. The im of the oper tion is complete resection of the tumour while sp ring
the dj cent neur l structures. If the p tient presents with l rge tumour c us
ing severe hydroceph lus nd r ised intr cr ni l pressure, prelimin ry ventric
uloperitone l shunt or ventricul r dr in m y
e considered. Steroid dministr ti
on prior to surgery m y
e dvis
le if the tumour is l rge. There re three
s
ic ppro ches to the cere
ellopontine ngle:
y excision of the l
yrinth (tr ns
l
yrinthine); through posterior foss cr niectomy (su
occipit l/retrosigmoid)
; or vi the middle cr ni l foss . No cle r consensus h s emerged from the liter
ture s to which is the procedure of choice. There re de nite dv nt ges nd dis
dv nt ges ssoci ted with e ch surgic l ppro ch. The route chosen is governed

y tumour size, the degree of he ring loss, the he ring level in the contr l ter
l e r, nd the surgic l preference nd expertise of the oper tor. The m jor dv
nt ge of the tr nsl
yrinthine oper tion is th t the f ci l nerve c n
e identi e
d l ter l to the tumour t n e rly st ge in the dissection, nd ccess to the f
undus of the intern l uditory me tus is excellent. Furthermore, retr ction of t
he cere
ellum is minim l nd the risk of postoper tive oedem is consequently le
ss. The m jor dis dv nt ge of this route is th t residu l he ring is irrevoc
ly
destroyed. The ppro ch is unf mili r to neurosurgeons, nd requires the close

cooper tion of neurotologist experienced in dissection of the tempor l
one. A

ccess is con ned,
ut even the l rgest of tumours c n
e removed s fely vi this
ppro ch.
( )
(
)
Fig. 7.15 ( ) MRI showing sm ll intr c n licul r meningiom . (
) MRI showing l r
ge coustic neurom inv ding into tempor l
one nd extending into cere
ellopont
ine ngle with severe compression of the
r instem.
104
CHAPTER 7
As consequence of progressive improvements in oper tive results, p rticul rly
in mort lity nd f ci l nerve outcome, ttention h s turned more recently to the

ility to preserve useful he ring. The su
occipit l oper tion provides good c
cess to the cere
ellopontine ngle
ut, if he ring is to
e conserved, tumour t
the fundus of the intern l uditory me tus m y
e dif cult to expose under direct
vision. This is true p rticul rly when the posterior semicircul r c n l is medi
lly pl ced. Theoretic lly, this m y incre se the risk of su
tot l tumour excisi
on when comp red with the tr nsl
yrinthine oper tion. Recently there h s
een r
enewed interest in the middle foss ppro ch for remov l of intr c n licul r tum
ours or those with sm ll cere
ellopontine ngle component, p rticul rly where
the tumour in the intern l uditory c n l extends to the fundus. Higher r tes of
he ring preserv tion h ve
een reported without ny compromise of f ci l nerve
function,
ut this route provides more limited ccess to the cere
ellopontine n
gle, nd is therefore restricted to the tre tment of sm ll lesions. The question
of he ring conserv tion deserves c reful consider tion when selecting the surgi
c l ppro ch. An tomic l preserv tion of the inner e r nd cochle r nerve does n
ot gu r ntee function, nd it is exception l for he ring to
e improved
eyond i
ts preoper tive level. Whether such he ring is useful depends upon the level of
he ring in the contr l ter l e r. He ring loss need not
e profound
efore it is
soci lly useless when the other e r is norm l. For he ring to
e useful soci ll
y there must
e
oth good speech discrimin tion, nd pure tone udiogr m withi
n 2040 dB of the contr l ter l e r. It is lso essenti l th t the ttempt to pres
erve he ring should not compromise the likelihood of complete tumour remov l. At
The Roy l Mel
ourne Hospit l the tr nsl
yrinthine oper tion is f voured for l
rge tumours, reg rdless of he ring level, nd for medium-sized lesions with poor
he ring. It provides more direct ppro ch to the cere
ellopontine ngle, nd
retr ction of the cere
ellum is negligi
le. For he ring preserv tion the retrosi
gmoid ppro ch for tumours with up to 2 cm cere-

ellopontine ngle extension is preferred. The middle foss ppro ch is preferre

d for intr c n licul r tumours nd for those with up to 1 cm cere
ellopontine n
gle extension where tumour completely lls the intern l uditory c n l. Stereot ct
ic r diosurgery using either 60CO G mm Knife or highly focused line r ccel
er tor h s
een dvoc ted for the tre tment of sm ller tumours, less th n 3 cm i
n di meter. The control r tes re gre ter th n 90% over 5-ye r period,
ut pos
t-r di tion neurologic l complic tions h ve
een reported including del yed f ci
l num
ness nd dys esthesi , f ci l we kness nd he ring loss. To minimize the
complic tions of single-dose stereot ctic r diosurgery m ny centres dvoc te fr
ction ted stereot ctic r diother py. There is continuing de
te s to the rel ti
ve dv nt ges of surgery nd stereot ctic r di tion tre tment. Whilst some clini
ci ns dvoc te r di tion tre tment for sm ller tumours, others would only recomm
end it for elderly or medic lly in rm p tients or if there is residu l tumour or r
egrowth fter su
tot l resection. The m n gement pl n for p tients with
il ter
l coustic neurom s (NF2) is complex, nd must
e t ilored for e ch p tient, wit

h the im of preserving useful he ring for s long s possi
le, whilst minimizin

g the possi
le serious neurologic l complic tions from enl rging tumours c using
cr ni l nerve, cere
ell r nd
r instem compression. Ther peutic options includ
e surgery, r diosurgery nd he ring preserv tions/restor tion utilizing
r inste
m electrode impl nts t the time of tumour resection.
Postoper tive c re The postoper tive m n gement is simil r to th t indic ted for
the posterior foss tumours in the previous ch pter. Any neurologic l deterior
tion must
e investig ted urgently. A postoper tive h emorrh ge in this region m
y
e r pidly f t l. Postoper tive sw llowing dif culties m y occur if there h s

een injury to the lower cr ni l nerves or
r instem. Gre t c re should
e t ken
to void spir tion nd n sog stric feeding m y
e necess ry. F ci l p r lysis w
ill occur if the 7th nerve is not int ct t the end of the oper tion nd
BENIGN BRAIN TUMOURS
105
m y result even if the nerve is in continuity due to neuropr xi of the nerve. A
t rsorrh phy m y
e necess ry to prevent corne l ulcer tion nd will
e essenti
l if there is f ci l p lsy nd corne l sens tion is diminished due to 5th ner
ve d m ge. Altern tively tempor ry closure of the eye c n
e o
t ined
y using

otulinum toxin. The cosmetic ppe r nce of perm nent f ci l p r lysis c n
e i
mproved
y num
er of procedures including: nerve n stomoses, such s hypogl
oss l f ci l n stomosis cross-f ci l nerve gr fts f ci l slings.
( )
H em ngio
l stom
H em ngio
l stom s re uncommon intr cr ni l tumours ccounting for 12% of ll
r
in tumours nd pproxim tely 10% of posterior foss tumours. The h em ngio
l st
om rises from prolifer tion of endotheli l cells. The tumour usu lly occurs in
young dults, lthough it m y occur t ny ge. It usu lly occurs in the poster
ior foss nd often produces l rge cyst. Although h em ngio
l stom m y occur
s component of von HippelLind us dise se, which includes multiple h em ngio
l s
tom s, h em ngio
l stom s of the retin (von Hippel tumour), ren l tumour, ren l
cyst, p ncre tic cyst nd tu
ul r denom t of the epididymis, the m jority of
p tients with the cere
ell r tumour do not h ve von HippelLind us dise se. Incompl
ete forms of the syndrome m y occur nd cere
ell r h em ngio
l stom s occur in

out 20% of p tients with retin l h em ngio
l stom .
(
)
Fig. 7.16 H em ngio
l stom in the vermis of the cere
ellum. ( ) A vividly enh n
cing tumour nodule in the w ll of cyst. (
) Verte
r l ngiogr m shows sm ll tu
mour nidus ( rrowhe d).
R diologic l investig tions
CT sc n or MRI show cere
ell r tumour which m y involve the vermis nd hemisph
eres nd which shows vivid enh ncement following intr venous contr st (Fig. 7.16
). There is usu lly low-density cyst surrounding the tumour nodule (Fig. 7.17)
, lthough h em ngio
l stom s m y sometimes
e solid. If considered necess ry, v
erte
r l ngiogr phy will con rm the highly v scul r m ss. Tot l surgic l excision
through posterior foss cr niotomy is ne rly lw ys possi
le. Gre t c re must

e t ken not to enter the highly v scul r tumour during the dissection nd exci
sion.
Clinic l present tion
The tumour presents s slowly growing posterior foss m ss with fe tures of r
ised intr cr ni l pressure nd cere
ell r involvement. Occ sion lly the p tient
m y
e polycyth emic due to incre sed circul ting erythropoietin.

106
CHAPTER 7
R diologic l investig tions
The usu l CT sc n picture is high-density, rounded tumour in the nterior 3rd
ventricle which enh nces following intr venous contr st (Fig. 7.18), lthough is
odense, hypodense nd non-enh ncing tumours h ve
een reported. MRI helps to de ne
the position of these tumours (Fig. 7.18) nd will
e
le to differenti te
et
ween colloid cyst nd n neurysm of the
sil r tip, which m y occ sion lly

e indistinguish
le on CT sc n.
Tre tment
Fig. 7.17 MRI. Cystic h em ngio
l stom .
Colloid cyst of the 3rd ventricle
The colloid cyst of the 3rd ventricle is situ ted in the nterior p rt of the ve
ntricle nd is tt ched to the roof just
ehind the for men of Monro. Sever l po
ssi
ilities s to the origin of the tumour h ve
een proposed, including the p r
physis, choroid plexus epithelium, ependym or diverticulum of the dienceph l
on. The cyst consists of thin, outer
rous c psule lined
y l yer of epitheli
um; the contents consist of mucoid m teri l, epitheli l de
ris nd mucin. The cy
st m y
e very sm ll nd symptom tic, s w s the c se with H rvey Cushing, wher
e 1-cm colloid cyst w s found t postmortem. As the tumour grows it will c use

il ter l o
struction to the for min of Monro, le ding to r ised intr cr ni l
pressure from hydroceph lus. The he d ches m y uctu te,
eing ggr v ted
y stoop
ing nd relieved
y st nding upright. Episodes of
rupt, sudden leg we kness c
using the p tient to f ll m y occur without ch nge in conscious st te. Altern
tively, n
rupt loss of consciousness m y occur nd this, lthough usu lly tr
nsient, might
e f t l.
Surgic l excision is performed through cr niotomy with sm ll incision in the
nterior corpus c llosum giving ccess to the l ter l ventricle. The tumour is
seen exp nding the for men of Monro nd, using the oper ting microscope, compl
ete excision is usu lly possi
le. Gre t c re must
e t ken during the oper tion
to preserve the venous structures, including the sept l veins, th l mostri te ve
in nd intern l cere
r l veins. D m ge to the columns of the fornix will result
in severe postoper tive memory distur
nce.
Epidermoid nd dermoid cysts
Epidermoid nd dermoid cysts rise from epitheli l cells em
ryologic lly mispl c
ed intr cr ni lly, p rticul rly into the meninges nd ventricles nd, less frequ
ently, into the p renchym of the
r in. R rely, the cells c n
e impl nted s
result of tr um such s lum
r puncture, which c n impl nt skin into the spi
n l c n l c using n epidermoid cyst. Epidermoid cysts m ke up
out 1% of
r in
tumours, lthough their incidence is higher in J p n, where the incidence of de
rmoid cysts is much less. Epidermoid tumours re found princip lly in the r chn
oid sp ces, the cisterns or the diploe of the
one. The most frequent loc liz ti
ons re the cere
ellopontine ngle, the supr sell r nd p r sell r regions, the
l ter l or 4th ventricles, nd the qu drigemin l cistern.
BENIGN BRAIN TUMOURS
107
( )
( )

(
)
(c)
Fig. 7.18 Colloid cyst of the 3rd ventricle. ( ) CT sc n shows hyperdense tumour

efore contr st. (
) MRI. Colloid cyst. (c) MRI. Colloid cyst s gitt l view. Fi
g. 7.19 ( ) Dermoid cyst. A very low density lesion on CT sc n th t does not enh
nce. (
) MRI. Epidermoid cyst dj cent to
r instem.
(
)
108
CHAPTER 7
Dermoid tumours occur mostly in the posterior foss s midline lesion nd
l m y connect the dermoid with the skin.

stu

Tre tment
The tre tment is oper tive, with resection of the cyst. Complete excision m y
e
prevented if the cyst w ll is densely dherent to m jor vessels nd import nt n
eur l structures.
Histology
The epidermoid cyst cont ins desqu m ted epithelium surrounded
y ker tin-produc
ing squ mous epithelium. The dermoid cyst includes derm l elements such s h ir
follicles, se
ceous gl nds nd sometimes swe t gl nds.
Further re ding
Cushing H (1917) Tumours of the Nervus Acusticus nd the Syndrome of the Cere
el
lopontine Angle. W B S unders, Phil delphi . Cushing H, Eisenh rdt L (1938) Meni
ngiom s: their Cl ssi c tion, Region l Beh viour, Life History nd Surgic l End Re
sults. Ch rles C Thom s, Spring eld. Di Tullio MV, R nd RW (1978) The R ndKurze Su

occipit l Tr nsme t l Oper tion. In: R nd RW, eds. Microneurosurgery. C V Mos
y,
St Louis, 206232. K ye AH, L ws ER (2001) Br in Tumours. Churchill Livingstone,
London. K ye AH, Bl ck P McL (2000) Oper tive Neurosurgery. Churchill Livingston
e, London, New York, Edin
urgh. Kennedy F (1911) Retro
ul
r neuritis s n ex c
t di gnostic sign of cert in tumours nd
scesses in the front l lo
es. Americ
n Journ l of Medic l Science 142, 355368. King TT (1982) The tr nsl
yrinthine op
er tion for remov l of coustic nerve tumours. In: Schmidek HH, Sweet WH, eds. O
per tive Surgic l Techniques: Indic tions, Methods nd Results. Grune & Str tton
, New York, 609636. Little JR, McC rty CS (1974) Colloid cysts of the third ventr
icle. Journ l of Neurosurgery 39, 230235. R nd RW, Kurze T (1965) F ci l nerve pr
eserv tion
y posterior foss tr nsme t l microdissection in tot l remov l of c
oustic neurom s. Journ l of Neurology, Neurosurgery nd Psychi try 28, 311316. Ru
ssell DS, Ru
enstein LJ (1977) P thology of Tumours of the Nervous System, 4th e
dn. Willi ms & Wilkins, B ltimore. Zulch KJ (1986) Br in Tumours, Their Biology
nd P thology, 3rd edn. Springer Verl g, Berlin.
Clinic l present tion
The cysts usu lly present following long history of symptoms rel ted to their
position. Cr ni l nerve
norm lities such s trigemin l neur lgi nd hemif ci
l sp sm m y occur with cere
ellopontine ngle epidermoid tumours nd the supr se
ll r cyst will produce visu l imp irment with optic trophy nd often
itempor
l hemi nopi . Le k ge of epidermoid cyst contents m y result in chemic l meni
ngitis, nd in p tients with posterior foss dermoid cysts,
cteri l meningitis
m y occur through the derm l sinus connecting the cyst with the skin.
R diologic l investig tions
The CT sc n of n epidermoid cyst is ch r cterized
y low-density lesion th t

does not enh nce. The dermoid cyst will lso h ve re s which re even less dens
e th n CSF, indic ting the presence of f t (Fig. 7.19). MRI h s superceded CT fo
r ccur te preoper tive ev lu tion nd pl nning. Epidermoid lesions re usu lly
m nifest s low sign l on T1 nd high sign l on T2 im ges, lthough depending on
lipid content, v ri
le sign l intensities m y
e seen within the s me lesion.
CHAPTER 8
8
Pituit ry tumours
Pituit ry denom s ccount for 810% of ll intr cr ni l tumours. In 1886 Pierre M
rie rst m de the connection
etween cromeg ly nd pituit ry denom s. P tients
m y present either with signs of endocrine distur
nce or with compression of th
e dj cent neur l structures, especi lly the optic p thw ys.
Cl ssi c tion
Historic lly, three m in types of pituit ry denom s were de ned
y their cytopl s
mic st ining ch r cteristics: chromopho
ic, cidophilic nd
sophilic the impli
c tion
eing th t these tumours were either hormon lly in ctive, secreted growth
hormone, or produced drenocorticotrophic hormone (ACTH), respectively. The dev
elopment of immunoperoxid se techniques nd electron microscopy h s provided m
ore re ned cl ssi c tion of pituit ry denom s
sed on the speci c hormone th t is pr
oduced. This cl ssi c tion is shown in T
le 8.1.
P thology
Pituit ry denom s rise from the nterior lo
e ( denohypophysis) of the pituit
ry gl nd which develops from R thkes pouch, n ectoderm l diverticulum rising fr
om the roof of the stomodeum, immedi tely in front of the
uccoph rynge l mem
r
ne. The posterior lo
e (neurohypophysis/p rs nervos ) rises from the infundi
ul
um developing from the oor of the dienceph lon (Fig. 8.1). The tumour rises with
in the pituit ry foss . If
it is less th n 10 mm in di meter it is known s micro denom . The tumour m y gr
ow loc lly within the sell nd c use erosion nd remodelling of the oor of the s
ell nd posterior clinoid processes (m cro denom ). The tumour usu lly spre ds
superiorly into the supr sell r cisterns, where it m y c use compression of the
optic p thw ys, p rticul rly the optic chi sm. Further growth superiorly c uses
compression of the hypoth l mus nd, if l rge enough, o
struction of the 3rd ven
tricle, resulting in hydroceph lus (Figs 8.2 nd 8.3). The tumour m y lso grow
l ter lly out of the sell into the c vernous sinus. Occ sion lly the l ter l ex
tension m y
e suf cient to c use distur
nce of the cr ni l nerves in the c verno
us sinus. Uncommonly the tumour penetr tes further l ter lly, into the tempor l
lo
e. The tumour m y infrequently extend inferiorly through the oor of the pituit
ry foss into the sphenoid sinus, resulting in CSF rhinorrhoe . The loc liz tio
n of micro denom s within the pituit ry foss corresponds somewh t with the regi
on l distri
ution of the norm l denohypophyse l cells. Prol ctin- nd growth ho
rmonesecreting micro denom s tend to occur l ter lly, where s most denom s secr
eting ACTH occur in the centr l zone. The pituit ry hormones re synthesized in
the rough endopl smic reticulum nd re p ck ged in the Golgi pp r tus. After p
ck ging they c n
e visu lized
y either electron microscopy or immunoperoxid s
e st ining s secretory gr nules within the cytopl sm. The hormone is rele sed f
rom the cell
y exocytosis, following fusion of the gr nule with the cell mem
r
ne 109
110
CHAPTER 8

T
le 8.1 Cl ssi c tion of pituit ry denom s. Percent ge of tumours 40 20 20 15 5

12 1 12
Hormone secreted Prol ctin Growth hormone Null cell (no hormone) ACTH Prol ctin
nd growth hormone FSH/LH TSH Acidophil stem cell (no hormone)
which usu lly occurs t the v scul r pole of the cell. Adenom s m y either
e de
nsely or sp rsely gr nul ted nd this will determine their st ining properties (
Fig. 8.4). Multiple endocrine neopl si type 1 (Werners syndrome) is n utosom l
domin nt disorder representing the inherited occurrence of
oth
enign nd m li
gn nt neopl sms involving the pituit ry gl nd, p r thyroid gl nd nd p ncre s.
Function l types of pituit ry denom
Prol ctin cell denom
Prol ctin is 23 500-D polypeptide hormone produced
y the prol ctin-secreting
cells (l ctotrophs) situ ted predomin ntly in the posteroACTH, drenocorticotrophic hormone; FSH, follicle-stimul ting hormone; LH, lutei
nizing hormone; TSH, thyroid-stimul ting hormone.
3rd ventricle Optic chi sm B s l cisterns (CSF) Pituit ry gl nd Oculomotor nerve
Trochle r nerve C vernous sinus Ophth l mic nerve (V1) M xill ry nerve (V2)
Infundi
ulum Ar chnoid Dur Sphenoid Anterior lo
e Pituit ry foss B s l cistern
s (CSF) Posterior lo
e Clivus
Sphenoid sinus
Intern l c rotid rtery
A
ducent nerve
Fig. 8.1 Pituit ry gl nd.
Fig. 8.2 An tomic l rel tions of the pituit ry gl nd.
Fig. 8.3 L rge pituit ry tumour compressing the hypoth l mus nd 3rd ventricle.
PITUITARY TUMOURS
111
( )
is prim rily under the in uence of n inhi
itory gent, prol ctin-inhi
iting f ctor,

elieved to
e dop mine. Prol ctinom s re the most frequently occurring pituit
ry denom s. The m jority present s micro denom s in young fem les with sympto
ms of hypersecretion c using menorrhoe nd g l ctorrhoe ; impotence m y
e the
only symptom in m les. This expl ins the much gre ter surgic l frequency of pro
l ctinom s in women, nd why these tumours m y grow to l rge size in m les nd
elderly fem les. Three-qu rters of prol ctin-secreting tumours re chromopho
e

y light microscopy, the rest
eing either we kly cidophilic or composed of m
ixture of cells. The serum levels of prol ctin will
e elev ted nd levels excee
ding 2000 ng/ml re suggestive of n inv sive prol ctinom . A minor elev tion of
serum prol ctin, up to level of twice norm l, is not necess rily di gnostic o
f prol ctin-secreting denom , s ny lesion of the hypoth l mus, pituit ry or
pituit ry st lk which interferes with the production or rele se of prol ctin-in
hi
iting f ctor m y c use some incre se in the serum prol ctin.
Growth hormone cell denom

Growth hormone is single ch in 21 000-D polypeptide produced
y cells situ te

d princip lly in the l ter l p rt of the gl nd. Growth hormone denom s represen
t pproxim tely 1520% of pituit ry denom s. Under light microscopy the fe tures
re either cidophil or chromopho
e, depending on the extent of the cytopl smic
gr nules. The denom s m y
e composed of either densely or sp rsely gr nul ted
cells, occurring with pproxim tely equ l frequency;
out 10% of tumours show
mixture of
oth cell types (Fig. 8.4). Hyperprol ctin emi occurs quite frequen
tly in growth hormone-producing denom s nd m y result either from n denom p
roducing
oth hormones or from growth hormoneproducing denom
eing of suf cien
t size to interfere with the rele se of prol ctin-inhi
iting f ctor, there
y ele
v ting the serum prol ctin second rily.
(
)
Fig. 8.4 Electron microscopy. ( ) Mem
r ne-
ound electron-dense gr nules of grow
th hormone synthesized in the endopl smic reticulum nd p ck ged
y the Golgi p
p r tus ( 20 000). (
) Immunogold l
elling of gr nules with ntiserum g inst gr
owth hormone ( 75 000).
l ter l hypophysis nd constituting 1520% of denohypophyse l cells. The
est kno
wn physiologic l functions of prol ctin re stimul tion of
re st growth nd pro
motion of l ct tion. Its role in the m le is poorly understood
ut it is import
nt in sperm togenesis. Unlike the secretion of other pituit ry hormones which is
controlled prim rily
y hypoth l mic rele sing hormone, prol ctin secretion
112
CHAPTER 8
ACTH (corticotrophic) denom s
ACTH is single ch in polypeptide which stimul tes the dren l cortex nd promo
tes secretion of cortisol nd rel ted steroids. Adenom s producing ACTH re the
c use of Cushings dise se. The denom s re densely gr nul ted with
sophilic cy
topl sm. ACTH-producing tumours constitute pproxim tely 15% of denom s, over 8
0% of which re micro denom s. A
out 1520% of denohypophyse l cells re corticot
rophs, loc ted in the centr l component of the nterior lo
e of the pituit ry gl
nd, nd it is not surprising th t most micro denom s re loc ted centr lly r th
er th n l ter lly, s occurs with the other hormoneproducing micro denom s.
with extensive penetr tion of the pituit ry c psule, dur l sinuses nd surroundi
ng
one. Most inv sive pituit ry denom s re sp rsely gr nul ted or chromopho
i
c nd re either hormon lly in ctive or prol ctin producing. Met st sizing pitui
t ry c rcinom , either extr cr ni lly or throughout the CSF p thw y, is extremel
y r re.
Clinic l present tion (T
le 8.2)
The presenting fe tures re due to: the size of the tumour endocrine distur
nce
. He d che occurs princip lly in p tients with cromeg ly nd is uncommon in oth
er types of pituit ry tumour.
Gon dotroph cell denom
Follicle-stimul ting hormone (FSH) nd luteinizing (LH) cells represent pproxim
tely 10% of the norm l pituit ry cells nd re sc ttered throughout the denohy
pophysis. Adenom s resulting from these cells re very uncommon nd most re fun
ction lly silent.
Visu l f ilure
C reful ssessment of the visu l elds, the visu l cuity nd the optic fundi is e
ssenti l. Supr sell r extension of the pituit ry tumour c uses compression of th
e optic chi sm resulting in
itempor l hemi nopi . The
itempor l hemi nopi i

niti lly involves the upper qu dr nts,
efore extending to the lower qu dr nts o
f the visu l eld. If the chi sm is pre xed, th t is, pl ced more nteriorly th n us
u l, homonymous hemi nopi m y occur due to compression of the optic tr ct. Bi
l ter l centr l scotom s result from the tumour pressing on the posterior p rt o
f the chi sm where the m cul r
res decuss te. Prim ry optic trophy will
e evid
ent in p tients with long-st nding compression of the chi sm. Ocul r p lsies occ
ur in
out 10% of p tients nd re due to inv sion of the c vernous sinus. The
3rd nerve is the most frequently ffected, followed
y the 6th nd 4th cr ni l n
erves. F ci l p in results from compression of the trigemin l nerve, usu lly the
ophth lmic division, s result of c vernous sinus inv sion.
Thyrotroph cell denom
Thyroid-stimul ting hormone (TSH)-producing tumours re r re.
Null cell denom s
Twenty to thirty per cent of pituit ry denom s h ve no clinic l or
iologic l e
vidence of hyperfunction. Mild hyperprol ctin emi m y occur second ry to distor
tion of the pituit ry st lk. Most (75% of) null cell denom s re chromopho
ic,
with few or no cytopl smic gr nules. Approxim tely 20% of null cell tumours show
m rked ccumul tion of mitochondri nd re c lled oncocytom s. Clinic lly, null
cell denom s re ggressive nd, s they re hormon lly silent, they m y grow t
o l rge size, so th t p tients present with visu l distur
nce. A
out 10% of p
ituit ry denom s re inv sive,
Endocrine
norm lities
Endocrine distur
nce is due to either hypopituit rism or excess secretion of
p rticul r pituit ry hormone.
PITUITARY TUMOURS
113
T
le 8.2 Clinic l m nifest tions of pituit ry tumours. M ss effects He d ches (es
peci lly cromeg ly) Superior extension Chi sm l syndrome (imp ired visu l cuit
y nd elds) Hypoth l mic syndrome (distur
nce in thirst, ppetite, s tiety, slee
p nd temper ture regul tion; di
etes insipidus uncommon; in ppropri te ADH syn
drome uncommon) O
structive hydroceph lus L ter l extension Cr ni l 3rd, 4th, 6t
h, diplopi Cr ni l 5th, f ci l p in Tempor l lo
e dysfunction Inferior extensio
n N soph rynge l m ss CSF rhinorrhoe Endocrine effects Hyperpituit rism GH gig nt
ism/ cromeg ly PRL hyperprol ctin emic syndrome ACTH Cushings dise se TSH thyroto
xicosis Hypopituit rism GH child: shortness of st ture, hypoglyc emi PRL dult
fem le: f ilure of postp rtum l ct tion ACTH hypocortisolism (Addisons) TSH hypot
hyroidism LH/FSH hypogon dism Acute deterior tion Pituit ry poplexy GH, growth
hormone; PRL, prol ctin; TSH, thyroid-stimul ting hormone; LH, luteinizing hormo
ne; FSH, follicle-stimul ting hormone; ACTH, drenocorticotrophic hormone.
m y
e clinic lly evident in the l rger tumours. The endocrine secretions re no
t equ lly depressed
ut there is selective f ilure nd the order of suscepti
i
lity is s follows: growth hormone, gon dotrophin, corticotrophin, thyroidstimul
ting hormone. Gon dotrophic de ciency prior to pu
erty ret rds the development of
second ry sex ch r cteristics; dult men h ve poor
e rd growth, women suffer f
rom menorrhoe nd
oth sexes h ve loss of li
ido nd de cient pu
ic nd xill ry
h ir. The
iochemic l
norm lity is m nifest
y low oestrogen nd ndrogen p
roduction with reduced urin ry 17-ketosteroids. Hypopituit rism initi lly result
s in v gue symptoms, including l ck of energy, undue f tigu
ility, muscle we kn
ess nd norexi nd, when prolonged nd severe, it will c use low
lood pressur
e. Clinic l hypothyroidism is m nifest
y physic l nd ment l sluggishness nd
preference for w rmth. When the hypopituit rism is severe, episodic confusion o
ccurs nd the p tient will
ecome drowsy. It is essenti l to recognize the fe tu
res of severe pituit ry insuf ciency s n endocrine crisis c n
e precipit ted
y

minor stressful events occurring during hospit l investig tion or s result o

f n intercurrent infection. Pituit ry poplexy results from spont neous h emorr
h ge into pituit ry tumour. It is ch r cterized
y sudden, severe he d che fol
lowed
y tr nsient or more prolonged loss of consciousness with fe tures of neck
stiffness, vomiting nd photopho
i . The condition is simil r to su
r chnoid h
emorrh ge resulting from ruptured neurysm,
ut is often ssoci ted with p r
lysis of one or more of the ocul r muscles (usu lly
il ter l) nd cute visu l
deterior tion. An cute endocrine crisis m y
e precipit ted
y the poplexy (Fi
g. 8.5).
Hypopituit rism Hypopituit rism results from f ilure of the hormones secreted
y
the denohypophysis nd it gives rise to the clinic l fe tures rst descri
ed
y
Simmonds in 1914. Pituit ry gl nd f ilure does not occur if the tumour is micr
o denom ,
ut
Prol ctinom The prol ctin-secreting tumour m y
e micro denom or m cro denom
within the pituit ry foss . The p tients re usu lly women who present with in
fertility ssoci ted with menorrhoe nd g l ctorrhoe , lthough the tumour m y
occ sion lly c use infertility in men. L rge
114
CHAPTER 8
prol ctinom s occur in the elderly nd in m les, nd these c n c use endocrine d
istur
nce ssoci ted with hypopituit rism nd visu l f ilure.
Fig. 8.5 MRI showing pituit ry tumour extending from exp nded pituit ry foss in
to the supr sell r cisterns with compression of optic chi sm.
Acromeg ly Acromeg ly results from growth hormonesecreting pituit ry denom w
hich, s descri
ed previously, consists of cells th t st in either s cidophils
, chromopho
es or
oth. The onset of cromeg ly is slow nd insidious, usu lly d
uring the 3rd nd 4th dec des of life, with
oth sexes
eing ffected equ lly. T
he clinic l fe tures (T
le 8.3) include
one nd soft tissue ch nges th t re e
vident s n enl rged supr cili ry ridge, enl rged front l sinuses nd incre sed
m ndi
ul r size, which will c use the chin to project (progn thism) (Fig. 8.6).
The h nds nd feet enl rge, nd the skin
ecomes co rse nd gre sy
T
le 8.3 Clinic l m nifest tions of growth hormone-producing pituit ry tumours.
Endocrine Effect of excess GH on tissue growth nd intermedi ry met
olism Skin
nd su
cut neous tissue overgrowth Skelet l overgrowth Visceromeg ly Incre sed
BMR, he t intoler nce, hyperhydrosis C r
ohydr te intoler nce Di
etes mellitus
Associ ted pituit ry dysfunction Owing to pituit ry compression or destruction H
ypopituit rism Associ ted thyroid dysfunction Goitre Thyrotoxicosis toxic nodul
r goitre, Gr ves dise se Associ ted multiple endocrine neopl si type 1 Prim ry h
yperp r thyroidism Tumours of the endocrine p ncre s syndromes Neurologic l Rel
ted to tumour m ss effects Rel ted to GH hypersecretion Acrop r esthesi s Entr p
ment neurop thies Peripher l neurop thy Myop thy Ophth lmologic l Rel ted to tum
our m ss effects Rel ted to GH hypersecretion Gl ucom Exophth lmos L rynge l Vo
ice ch nges Cord x tion Sleep disorders Skelet l Acr l ch nges Arthrop thy C rdio
v scul r Hypertension C rdiomeg ly Congestive he rt f ilure Conduction defects
nd rrhythmi s Derm tologic l Acr l ch nges Hyperhydrosis Acne Hypertrichosis Hy
perpigment tion Ac nthosis nigric ns
PITUITARY TUMOURS
115
p tients compl in of l ck of energy, physic l we kness nd l ssitude. Supr sell

r extension of the tumour occurs in
out 15% of c ses nd m y result in compres

sion of the optic p thw ys. A pituit ry denom with excessive growth hormone se
cretion occ sion lly presents in childhood nd results in gig ntism.
Cushings dise se Cushings dise se is due to ACTH-producing pituit ry denom s. Ove
r 80% of the tumours re micro denom s nd the rem inder re m cro denom s invol
ving the whole of the sell or with extr sell r extension. The onset is insidiou
s nd the dise se m y ffect children or dults. Severe o
esity occurs, the skin
is tense nd p inful, nd purple stri e ppe r round the trunk. F t is deposit
ed, p rticul rly on the f ce (moon f ce), neck, cervicodors l junction (
uff lo
hump) nd trunk. The skin
ecomes purple colour due to v sodil t tion nd st s
is. Spont neous
ruising is common. The skin is gre sy, cne is common nd f ci
l h ir excessive. P tients compl in of excessive f tigue nd we kness. There is
w sting nd ccidity of the muscles. Osteoporosis predisposes to spont neous fr c
tures. Glucose toler nce is imp ired, the serum pot ssium is low nd v scul r hy
pertension occurs. If untre ted, 50% of c ses re f t l in 5 ye rs.
Cushings syndrome There is excessive cortisol production, due to n ACTH-producin
g pituit ry denom (Cushings dise se) in 90% of c ses. Other c uses of Cushings s
yndrome re n dren l denom or c rcinom , or n ectopic source of ACTH produc
tion such s from n o t cell c rcinom of the lung or
err nt drenocortic l t
issue occurring outside the dren l gl nd. NelsonS l ss syndrome This consists o
f n ACTH-producing pituit ry denom in p tient who h s undergone
il ter l o
r su
tot l dren lectomy. Before the development of CT sc nning nd tr ns-spheno
id l microsurgery, p tients with Cushings dise se
Fig. 8.6 A 58-ye r-old fem le with cromeg ly. A
ove is photogr ph t ken when
she w s 25 ye rs old.

nd swe ts profusely. The voice
ecomes ho rse nd gruff nd thor cic kyphosis o
ccurs s result of osteoporosis. Other pro
lems ssoci ted with cromeg ly inc
lude hypertension, c rdi c hypertrophy nd di
etes. He d che is often severe in
p tients with pituit ry tumours c using cromeg ly nd
116
CHAPTER 8
often underwent tot l dren lectomy when pneumoenceph logr phy h d f iled to rev
e l pituit ry tumour. However, cceler ted growth of n existing denom is in
duced
y the loss of norm l corticosteroid feed
ck. Unlike the denom s of Cush
ings dise se,
out h lf of p tients with NelsonS l ss syndrome h ve m cro denom
s. P tients h ve m rked cut neous hyperpigment tion due to secretion of either

et mel nocyte-stimul ting hormone nd/or
et lipotrophin.
L
or tory investig tions
R dioimmuno ss y will help to identify the hormone
eing secreted. Serum prol ct
in level in p tients with prol ctinom s will v ry from just
ove the upper limi
t of norm l to v lues gre ter th n 20 000 mIU/l (norm l 70550 mIU/l). The levels
m y show consider
le v ri tion in p rticul r p tient nd prol ctin levels gre
ter th n 2000 mIU/l re lmost lw ys indic tive of pituit ry tumour. As ment
ioned previously, hyperprol ctin emi m y
e ssoci ted with other pituit ry tum
ours nd h s
een noted in some p tients with cromeg ly. Null cell tumours m y

e ssoci ted with mild hyperprol ctin emi due to distortion of the pituit ry s
t lk or impingement on the hypoth l mus. Serum growth hormone is me sured
y r d
ioimmuno ss y, the norm l v lues
eing less th n 5 mIU/l in m les nd less th n
10 mIU/l in fem les. Growth hormone exerts its effects on peripher l tissues ind
irectly vi som tomedins polypeptides produced prim rily
y the liver nd
ro
l s
ts. Serum som tomedin C (insulinlike growth f ctor I, IGF-I) is more ccur te
indic tor of growth hormone
io ctivity th n the serum growth hormone levels. Pr
ovoc tive tests of growth hormone secretion re useful in con rming cromeg ly. Mo

st p tients with cromeg ly do not show the norm l suppression of growth hormone
following glucose lo d. Other provoc tive tests utilize thyrotrophinrele sing h
ormone nd growth hormonerele sing hormone. In contr st to other pituit ry tumou
rs th t rely
on im ging studies s the foremost di gnostic investig tion, c reful endocrino
logic l ssessment is critic l in the di gnosis of Cushings dise se, especi lly
s in ne rly 50% tumour is not evident on MRI. There re three essenti l steps
in the di gnosis of Cushings dise se due to pituit ry p thology. 1 Con rm excess se
cretion of cortisol (norm l 120650 nmol/l). 2 Distinguish ACTH-dependent from ACT
H-independent c uses of hypercortisol emi . 3 Distinguish pituit ry-
sed Cushin
gs dise se from ectopic st tes of ACTH production. A 24-hour urine specimen is th
e simplest initi l me ns of est
lishing hypercortisol emi . The investig tion o
f Cushings dise se involves the me surement of ACTH
y r dioimmuno ss y of s mple
s of
oth peripher l
lood nd
lood from the petros l sinus. A differenti l lev
el of ACTH in petros l vein
lood nd peripher l
lood will help con rm the presen
ce of pituit ry
sis for the ACTH production, especi lly fter dministr tion
of corticotrophin-rele sing hormone (CRH). A dex meth sone suppression test wil
l help di gnose Cushings syndrome nd its c use. The urine- nd pl sm -free corti
sol is me sured nd is norm lly suppressed following dministr tion of low-dose
dex meth sone (0.5 mg 6-hourly). The levels will
e suppressed following high-do
se dex meth sone (2 mg 6hourly) in pituit ry-dependent Cushings dise se. There wi
ll
e f ilure of suppression of the levels with high-dose dex meth sone if the
Cushings syndrome is due to other th n pituit ry c uses.
R diologic l investig tions
High-resolution CT sc nning nd m gnetic reson nce im ging using thin slices nd
intr venous contr st re the ppropri te investig tion. Pituit ry micro denom s
re usu lly hypodense nd m y c use upw rd
ulging nd convexity of the upper

order of the gl nd in dults, devi tion of the pituit ry st lk nd thinning of t
he sell r oor on the side of the tumour (Fig. 8.7). High-qu lity CT sc nning is

le to demonstr te tumours s sm ll s 4 mm in di meter. M cro denom s enPITUITARY TUMOURS
117
(
) ( )
Fig. 8.7 ( ) CT showing hypodense micro denom in pituit ry gl nd. (
) MRI showi
ng micro denom .
Fig. 8.8 Axi l nd coron l CT sc ns showing l rge pituit ry tumours.
h nce fter intr venous contr st nd the ex ct n ture of the extr sell r extensi
on c n
e
est ppreci ted with direct coron l sc ns (Fig. 8.8). MRI h s improve
d the identi c tion of micro denom s, which ppe r s low-density foc l lesions on
T1-weighted sc ns nd high intensity on T2-weighted sc ns (Fig. 8.9). M cro den
om s usu lly ppe r s isointense on the T1-weighted im ges nd moder tely hyper
intense on the T2im ges (Fig. 8.10). H emorrh ge into tumour, such s occurs f
ollowing pituit ry poplexy, shows s high-intensity re s
ec use of meth emogl
o
in on the T1- nd T2-weighted sc ns intermingled with low-density regions due
to h emosiderin (see Figs 8.5 nd 8.9). Dyn mic sc ns t ken t 30-second interv
ls following intr venous g dolinium m y help demonstr te sm ll micro denom s. Pl
in skull X-r ys m y show enl rgement of the sell with thinning erosion or
ulg
ing of its contours (Fig. 8.11). In the p st ngiogr phy h s
een performed to e
xclude incident l neurysms nd to determine the position of the intern l c roti
d rteries in the c vernous sinuses,
ut this inform tion c n now
e o
t ined s
tisf ctorily from good-qu lity MRI nd, if necess ry, m gnetic reson nce ngiogr

 phy.
118
CHAPTER 8
( ) ( )
(
)
Fig. 8.9 MRI sc n (T1) showing l rge pituit ry tumour with re of recent h emor
rh ge.
(
)
Fig. 8.10 Enh nced MRI showing l rge pituit ry tumour extending up to the 3rd ve
ntricle.
Fig. 8.11 Pl in l ter l skull X-r y showing thinning of the dorsum sell e nd de
struction of the pituit ry foss in p tient with l rge pituit ry tumour.
PITUITARY TUMOURS
119
Fig. 8.12 Hypoth l mic gliom .
Differenti l di gnosis
The m jor differenti l di gnoses re: cr nioph ryngiom supr sell r meningiom (
rising from the tu
erculum sell e). Uncommon m sses round the supr sell r regi
on lso include optic nerve nd hypoth l mic gliom (Fig. 8.12), gi nt neurysm
rising from the c rotid rtery, R thkes cleft cysts, supr sell r germinom s nd
chordom s.
Tre tment
The o
jectives of tre tment of p tients with pituit ry tumours depend on whether
the p tient h s presented with fe tures of endocrine distur
nce or pro
lems re
l ted to compression of dj cent neur l structures. The methods of tre tment use
d re: 1 Oper tive procedures: ( ) tr ns-sphenoid l excision (
) tr nscr ni l ex
cision. 2 R diother py. 3 Medic l tre tment with ntisecretory drugs.
Surgic l excision
This will
e used s the prim ry method of tre tment for: l rge tumours c using
compression of dj cent neur l structures, p rticul rly the visu l p thw ys GH-secreting tumours c
using cromeg ly ACTH-secreting tumours c using Cushings dise se the occ sion l t
re tment of prol ctin-secreting denom , either micro denom or m cro denom c
on ned within the sell , when medic l tre tment using
romocriptine is not toler t
ed. Most tumours c n
e excised vi the tr nssphenoid l ppro ch to the pituit r
y foss (Fig. 8.13). The development of the surgic l microscope nd uoroscopic r
diogr phy h s m de this s fe procedure. The sphenoid sinus is usu lly entered
using unil ter l tr ns-sept l ppro ch, with the incision either in the n s l
mucos or su
l
i lly. The mucos is re ected from the n s l septum nd oor nd the
sphenoid is opened. The nterior w ll of the sell is removed nd the pituit ry
foss entered. Micro denom s (tumours less th n 10 mm in di meter) m y
e evide
nt on the surf ce of the gl nd or m y
ecome evident only once the gl nd is inci
sed. These tumours c n
e completely excised, preserving pituit ry function. The
supr sell r extension of the tumour c n
e gently co xed down into the pituit r
y foss
y slightly r ising the intr cr ni l pressure using V ls lv m noeuvre

or
y the n esthetist injecting sm ll increments of nitrous oxide nd oxygen m

ixture into the lum
r thec until the intr cr ni l pressure forces the supr sel
l r tumour into the oper tive eld. This will lso h ve the ddition l
ene t th t t
he intr cr ni l g s will provide pneumoenceph logr m, outlining the rem ining
supr sell r extension of tumour. A tr nscr ni l oper tion is occ sion lly necess
ry, p rticul rly where there is su
front l or retrocliv l extension of the tu
mour. Postoper tive m n gement requires c reful ttention to the uid
l nce nd
hormon l st tus. Endocrine de ciency in the immedi te postoper tive period will re
quire repl cement with p renter l hydrocortisone nd possi
ly the use of v sopre
ssin for the tre tment of di
etes insipidus, which often occurs t le st tr nsi
ently fter the excision of l rge pituit ry tumour. In the e rly postoper tive
period queous v so120
CHAPTER 8
the postoper tive endocrine studies demonstr te residu l excessive hormone secre
tion.
Tumour Sphenoid sinus
Medic l tre tment
Tre tment of pituit ry denom s is undert ken to restore the endocrine st tus of
the p tient
y repl cement of either the pituit ry hormone itself or the hormon
e of the pituit ry-dependent gl nds. This will
e necess ry preoper tive proce
dure in p tients with evidence of hypopituit rism nd will frequently
e necess
ry fter the surgic l excision of m cro denom . Prol ctin-secreting pituit ry
tumours re tre ted with
romocriptine, dop mine gonist. This is the preferre
d tre tment for symptom tic prol ctin-secreting micro denom nd m y
e used e
ither s the de nitive tre tment of l rger prol ctin-secreting tumours or in conju
nction with surgery. Some p tients show poor toler nce to
romocriptine, s it m
y c use intr ct
le n use , vomiting nd postur l hypotension, nd these p tien
ts will require surgic l tre tment of the tumour.
N s l speculum
Endotr che l tu
e ( )
Cr nioph ryngiom
This tumour m y occur t ny ge, lthough ne rly h lf occur in the rst 20 ye rs
of life. They re thought to rise from the epitheli l remn nts of R thkes pouch.
The tumours occur in the region of the pituit ry foss nd extend through the s
upr sell r cisterns to the hypoth l mus. The m jority re cystic, nd the uid is
often yellow nd sp rkling with cholesterol cryst ls. The cyst m y
e l rger th
n the solid component, which is often p le nd crum
ly, consisting of epitheli l
de
ris. There re two histologic l types of tumours. The d m ntinous type rese
m
les d m ntinom of the j w nd is encountered in virtu lly ll children. The
p pill ry type, so-c lled dult cr nioph ryngiom , occurs in
out one-third of
dults nd is r re in children.
(
)
Fig. 8.13 ( ) Di gr m of oper tive exposure in tr nssphenoid l resection of pitu
it ry tumour. A selfret ining retr ctor is inserted nd the nterior w ll of the
sphenoid sinus is removed. (
) Intr oper tive Xr y showing the retr ctor in pos
ition nd the forceps in the pituit ry foss .
pressin (Pitressin ) should
e given
y intr muscul r or su
cut neous injection
nd, if the di
etes insipidus persists,
y the intr n s l route. Other long-term
hormon l repl cements m y include cortisone cet te (12.525 mg twice d ily), thy

roxine nd testosterone.
R diother py
Postoper tive r diother py m y
e used if there h s
een su
tot l excision of
the tumour or if
Clinic l present tion
Clinic l fe tures include:
PITUITARY TUMOURS
121
r ised intr cr ni l pressure visu l imp irment endocrine dysfunction.
R ised intr cr ni l pressure This is common, p rticul rly in children, who prese
nt with he d che, vomiting nd p pilloedem . Visu l imp irment This is due to p
pilloedem , chi sm l compression or com
in tion of
oth. P pilloedem is due t
o hydroceph lus s result of 3rd ventricul r o
struction
y the tumour. The vi
su l eld defect is frequently simil r to th t produced
y pituit ry tumour,

itempor l hemi nopi ,
ut homonymous defects re more common th n in pituit ry
denom . Endocrine
norm lities These re frequent in children nd consist of: h
ypogon dism stunting of growth di
etes insipidus. Endocrine f ilure due to cr n
ioph ryngiom rising in dults is essenti lly simil r to th t c used
y pitui
t ry tumour, except th t di
etes insipidus occurs more commonly in p tients pre
senting with cr nioph ryngiom .
( )
Investig tions
The CT sc n usu lly shows cystic tumour in the supr sell r region with c lci c t
ion (Fig. 8.14). Tumours in dults m y
e solid nd re less c lci ed th n those s
een in younger p tients. MRI is useful in showing the full extent of the tumour
(Fig. 8.15). Ch nges in the sell turcic re seen in pproxim tely 50% of p tie
nts. Supr sell r tumours nd the ssoci ted hydroceph lus press downw rds on the
dorsum sell e nd nterior clinoids nd m y enl rge the sell . Ne rly 90% of tu
mours in children h ve r diogr phic lly identi
le c lci c tion in the tumour, wher
e s only 40% of dults h ve r diologic lly demonstr
le c lci c tion. The c lci c ti
on consists of ggreg tes of sm ll ecks of c lcium nd m y
e curviline r, outlin
ing portion of the cyst w ll.
(
)
Fig. 8.14 CT sc n. Cr nioph ryngiom .
On occ sions cr nioph ryngiom nd pituit ry tumours need to
e distinguished fr
om R thkes cleft cyst (Fig. 8.16). Both cr nioph ryngiom s nd R thkes cleft cys
ts re thought to rise from em
ryonic remn nts of R thkes pouch. By the 6th week
of em
ryonic life the residu l lumen of R thkes pouch is reduced to n rrow cle
ft th t gener lly regresses. Persistent enl rgement of this cleft is s id to
e
the c use of R thkes cleft cyst. These cysts re epitheliumlined cysts cont ining
mucoid m teri l. They re usu lly con ned to the sell ,
ut on occ sions c n
122
CHAPTER 8
form l rge cystic tumours extending into the supr sell r cisterns (Fig. 8.16).
Tre tment

Preoper tive visu l nd endocrine ssessment is essenti l. The st nd rd tre tmen

t for cr nioph ryngiom is oper tive, with n ttempt t m xim l resection of th
e tumour. However, complete resection m y not
e possi
le due to the extent of t
he tumour nd the intim te tt chment to hypoth l mic vit l structures. The usu
l surgic l ppro ch is through pterion l cr niotomy or
ifront l cr niotomy with sep r tion of the front l lo
es
nd division of the l min termin lis. Tumours extending into the 3rd ventricle m
y lso need to
e ppro ched through the corpus c llosum. An extended tr nssphe
noid l ppro ch is sometimes dvised for those tumours extending down to the oor
of the pituit ry foss . Postoper tive m n gement will include c reful ttention
to uid nd electrolyte
l nce s m ny p tients h ve t le st tr nsient di
ete
s insipidus following surgery. Other hormones m y need repl cement depending on
endocrinologic l ssessment. The role of postoper tive r diother py in p tients
with su
tot l resection is controversi l
ut r diother py m y
e
ene ci l in de
cre sing the production of cyst uid nd del ying recurrence of the tumour.
Empty sell syndrome
The empty sell refers to communic ting extension of the su
r chnoid sp ce in
to the pituit ry foss (Fig. 8.17). This m y occur s result of n incomplete
n tomic l form tion of the di phr gm sell e which llows the r chnoid to hern
i te directly into the pituit ry foss or s second ry phenomenon following ei
ther pituit ry surgery or r diother py.
Fig. 8.15 MRI. Cr nioph ryngiom .
Fig. 8.16 MRI showing l rge R thkes cleft cyst.
PITUITARY TUMOURS
123
Norm l
Empty sell
Ar chnoid
B s l cisterns (CSF) Di phr gm sell e
CSF
Fig. 8.17 Empty sell . The su
r chnoid sp ce is
le to enter the sell through
n incomplete di phr gm sell e.
Dur
Anterior lo
e pituit ry
Anterior lo
e pituit ry
There is good re son to reg rd the empty sell s n n tomic l v ri nt r ther t
h n syndrome. Bergl nd showed the presence of su
r chnoid sp ce within the se
ll in 20%, nd n tomic l defects of the di phr gm sell e of 5 mm or more in n
e rly 40%, of n utopsy series without pituit ry dise se. However, defects in t
he di phr gm re not the only requirement for form tion of n empty sell . Incr
e sed intr cr ni l pressure ssoci ted with
enign intr cr ni l hypertension or
long-st nding hydroceph lus will c use herni tion of the su
r chnoid sp ce into
the sell nd will result in the remodelling of the pituit ry foss to produce
the cl ssic glo
ul r ppe r nce on pl in X-r y nd CT sc n (Fig. 8.18). It is po

ssi
le th t the norm l v ri tions in CSF pressure m y
e tr nsmitted into the fo

ss through n incompetent di phr gm nd so result in the
ony ch nges.
( )
Clinic l present tion
Most p tients with the r diologic l fe tures of n empty sell re symptom tic.
The m jority of p tients presenting with symptoms re o
ese, middle- ged, hyper
tensive women. He d che is the most common symptom ssoci ted with the empty sel
l
ut the fe tures re so v ried th t their relev nce to the intr sell r su
r
chnoid sp ce is du
ious without n underlying c use for possi
le r ised intr cr
ni l pressure. Visu l eld defects nd endocrine
norm lities re su
tle nd unco
mmon in p tients with
(
)
Fig. 8.18 Empty sell . ( ) The cl ssic glo
ul r enl rged sell . (
) CSF within t
he sell .
124
CHAPTER 8
prim ry empty sell syndrome. In p tients with second ry empty sell , e.g. fol
lowing surgery or r diother py, eld defects m y
e more pronounced
ut re r rely
severe. The most serious consequence of n empty sell is spont neous CSF rhino
rrhoe . This usu lly occurs only if there h s
een n underlying c use of r ised
intr cr ni l pressure, such s
enign intr cr ni l hypertension. It is m n ged

y rep iring the le k in the oor of the sell with crushed muscle nd f sci l t
nd performing CSF shunt.
Further re ding
A

oud CF, L ws ER (1988) Di gnosis of pituit ry tumours. In: Young WF, Klee GG,
eds. Endocrinology nd Met
olism Clinics of North Americ . W B S unders, Phil
delphi , Vol. 17, 241277. Bergl nd RM, R y BS, Tor k RM (1968) An tomic l v ri ti
ons in the pituit ry gl nd nd dj cent structures in 225 hum n utopsy c ses. J
ourn l of Neurosurgery 28, 9399. Bl ck P McL, Zerv s NT, C ndi G (1987) Incidenc
e nd m n gement of complic tions of tr nssphenoid l oper tion for pituit ry de
nom s. Neurosurgery 20, 920924. Cushing H (1912) The Pituit ry Body nd its Disor
ders. Clinic l St tes Produced
y Disorders of the Hypophysis Cere
ri. JB Lippin
cott, Phil delphi . E
ersold MJ et l. (1983) Pituit ry poplexy tre ted
y tr n
ssphenoid l surgery. A clinicop thologic l nd immunocytochemic l study. Journ l
of Neurosurgery 58, 315320. H rdy J (1968) Tr nsphenoid l microsurgery of the no
rm l nd p thologic l pituit ry. Clinic l Neurosurgery 16, 185217. K ufm n B (196
8) The empty sell turcic : m nifest tion of the intr sell r su
r chnoid sp ce.
R diology 90, 931941.
K ye AH, Bl ck P McL (2000) Oper tive Neurosurgery. Churchill Livingstone, Londo
n, New York, Edin
urgh. K ye AH, G l
r ith JEK, King J (1981) Intr cr ni l press
ure in p tients with empty sell syndrome without
enign intr cr ni l hypertensi
on. Journ l of Neurosurgery 55, 453456. Kov cs K, Horv th E (1985) Morphology of
denohypophyse l cells nd pituit ry denom s. In: Imur H, ed. The Pituit ry Gl
nd. R ven Press, New York. L ws ER, R ndle RV, A

oud CF (1982) Surgic l tre tm
ent of cromeg ly: results in 140 p tients. In: Givens JR, ed. Hormone Secreting
Pituit ry Tumours. Ye r Book. Medic l Pu
lishers, Chic go. Molitch ME, F hl
usc
h R (1995) Medic l versus surgic l tre tment of gi nt pituit ry prol ctinom s. I
n: AlMefty O, Origit no TC, H rkey HL, eds. Controversies in Neurosurgery. Thiem
e, New York. Ross D, Wilson CB (1988) Results of tr nssphenoid l microsurgery fo
r growth hormone secreting pituit ry denom in series of 214 p tients. Journ l
of Neurosurgery 68, 854867. Scheith uer BW (1984) Surgic l p thology of the pitui

t ry. The denom s P rt I. In: Sommers SC, Rosen PP, eds. P thology Annu l P rt
I. Appleton-CenturyCrofts, Connecticut, 317374. Scheith uer BW (1984) Surgic l p
thology of the pituit ry. The denom s P rt II. In: Sommers SC, Rosen PP, eds. P
thology Annu l P rt I. Appleton-CenturyCrofts, Connecticut, 269329. Th p r K, L
ws ER (1995) Pituit ry tumours. In: K ye AH, L ws ER, eds. Br in Tumours. Church
ill Livingstone, Edin
urgh. Trum
le HC (1951) Pituit ry tumours: o
serv tions on
tumours which h ve spre d widely
eyond the con nes of the sell . British Journ l
of Surgery 39, 724. Wilson CB (1984) A dec de of pituit ry microsurgery. Journ l
of Neurosurgery 61, 814833. Editori l (1982) The intr sell su
r chnoid sp ce.
L ncet July 31, 249250.
CHAPTER 9
9
Su
r chnoid h emorrh ge
The sudden onset of severe he d che in p tient should
e reg rded s su
r c
hnoid h emorrh ge until proven otherwise. Su
r chnoid h emorrh ge occurs when

leeding is prim rily within the su
r chnoid sp ce r ther th n into the
r in it
self. It represents
out 510% of ll non-tr um tic intr cr ni l h emorrh ge with
n incidence of pproxim tely 15 per 100 000 popul tion. Apoplectic de th h s

een mentioned in the e rliest medic l writings
ut its rel tionship to intr cr n
i l h emorrh ge nd cere
r l neurysm w s not est
lished until the l tter p rt
of the seventeenth century. The introduction of cere
r l ngiogr phy
y Moniz n
d Lim in Lis
on in 1927 llowed the di gnosis of cere
r l neurysm to
e m de i
n living p tients who h d sust ined su
r chnoid h emorrh ge. Pioneering surgery
in the 1930s nd 1940s,
y Kr yen
uhl in Switzerl nd nd D ndy in North Americ
, showed th t neurysms could
e tre ted oper tively, lthough t th t time with
consider
le mor
idity nd mort lity. Consequent improvements in microsurgic l
techniques nd neuro n esthesi h ve consider
ly improved the s fety of surgery
.
rh ge
ecomes more frequent th n rteriovenous m lform tions over the ge of 20
ye rs. R re c uses of su
r chnoid h emorrh ge include
leeding from tumour,

leeding disorders,
lood dyscr si s nd rupture of spin l rteriovenous m lfor
m tion (T
le 9.1). The etiology of su
r chnoid h emorrh ge rem ins undiscover
ed in pproxim tely 15% of c ses fter thorough clinic l nd r diogr phic study.
These p tients often h ve ssoci ted intr cr ni l v scul r therosclerosis nd
hypertension.
Su
r chnoid h emorrh ge presenting fe tures (T
le 9.2)
He d che
The sudden onset of severe he d che of type not previously experienced
y th
e p tient is the h llm rk of su
r chnoid h emorrh ge. A rel tively sm ll le k f
rom n neurysm m y result in minor he d che, sometimes referred to s the sent
inel he d che, s this m y
e the w rning episode of su
sequent m jor h emorrh
ge from the neurysm. N tur lly, recognition of possi
le minor w rning h emorrh
ge is essenti l to vert possi
le l ter c t strophic
leed, lthough m ny re
only recognized in retrospect.
C uses of su
r chnoid h emorrh ge
The most common c use of su
r chnoid h emorrh ge in dults is rupture of
err
y neurysm. Su
r chnoid h emorrh ge in children is much less common th n in the
dult popul tion nd the most common p edi tric c use is rupture of n rteriov
enous m lform tion. Cere
r l neurysm s c use of su
r chnoid h emorDiminished conscious st te
Most p tients h ve some deterior tion of their conscious st te following su
r c
hnoid h emorrh ge. This v ries from only slight ch nge when the h emorrh ge h

s
een minor to poplectic de th resulting from m ssive h em125

126
CHAPTER 9
T
le 9.1 C uses of su
r chnoid h emorrh ge (%). Cere
r l neurysm Arteriovenou
s m lform tion Undiscovered Other r re c uses Spin l rteriovenous m lform tion
Tumour Blood dyscr si 70 10 15 5
T
le 9.2 Su
r chnoid h emorrh ge presenting fe tures. He d che Diminished cons
cious st te Meningism Neck stiffness, vomiting, photopho
i , fever Foc l neurolo
gic l signs Intr cere
r l h emorrh ge Foc l pressure
y neurysm V sosp sm Fund
l ch nges Su
hy loid h emorrh ge Retin l h emorrh ge P pilloedem Fig. 9.1 Intr
cere
r l h em tom in tempor l lo
e due to rupture of middle cere
r l rtery
neurysm.
orrh ge. It is common c use of sudden de th in young dults.
Meningism
Blood in the su
r chnoid cere
rospin l uid will c use the fe tures of meningism
he d che, neck stiffness, photopho
i , fever nd vomiting. Irrit tion of the ner
ve roots of the c ud equin , which occurs when the
lood extends down to the lu
m
r thec , m y result in sci tic -type p in nd low
ck discomfort.
Foc l neurologic l signs
Foc l neurologic l signs m y occur in su
r chnoid h emorrh ge due to concomit nt intr cere
r l h emorrh ge, the loc l pressur
e effects of the neurysm itself, or cere
r l v sosp sm. A cere
r l neurysm usu
lly lies within the su
r chnoid cisterns
ut the neurysm m y
ecome dherent
to the dj cent
r in p renchym due to dhesions, frequently resulting from pre
vious le k ge of
lood. A h emorrh ge from n neurysm in these circumst nces m
y lso extend into the
r in nd the position of the intr cere
r l h em tom wil
l determine the type of neurologic l de cit. A middle cere
r l rtery neurysm fre
quently ruptures into the tempor l lo
e, resulting in hemip resis nd ph si if
the domin nt hemisphere is involved (Fig. 9.1). Anterior communic ting rtery
neurysms m y h emorrh ge into the front l lo
es with su
sequent kinetic mutism
(Fig. 9.2). Defective conjug te ocul r movement m y result from h emorrh ge into
front l lo
e, persistent devi tion usu lly
eing tow rds the side of the lesi
on nd purposeful g ze defective w y from th t side. Occ sion lly, n neurysm
m y lso rupture into the su
dur l sp ce, resulting in su
dur l h em tom nd

r in compression c using l ter lizing neurologic l signs. An rteriovenous m lf
orm tion usu lly lies t le st p rti lly within
SUBARACHNOID HAEMORRHAGE
127
Fig. 9.3 Gi nt intern l c rotid rtery neurysm.
Fig. 9.2 Front l intr cere
r l h em tom with
lood in the Sylvi n ssure nd vent
ricles from ruptured nterior communic ting rtery neurysm.
the
r in p renchym , so th t when it ruptures intr cere
r l
leeding is frequen
tly ssoci ted with the su
r chnoid h emorrh ge. Foc l neurologic l signs m y r
esult from the position of the neurysm itself. An neurysm rising from the int
ern l c rotid rtery t the origin of the posterior communic ting rtery (known
s posterior communic ting rtery neurysm) m y c use pressure on the 3rd cr n
i l nerve. P tients with n enl rging neurysm in this position m y present with

fe tures of 3rd cr ni l nerve p lsy (ptosis, pupil dil t tion, extr ocul r mu
scle p lsy) prior to su
r chnoid h emorrh ge. It is vit l th t the correct di
gnosis of n enl rging cere
r l neurysm is m de in this situ tion, so s to v
oid the possi
le c t strophic effects of su
r chnoid h emorrh ge. The m jor dif
ferenti l di gnosis of the etiology of n pp rently isol ted 3rd cr ni l nerve
p lsy is n isch emic lesion such s those resulting from di
etes mellitus or
therosclerosis. Pupil size is useful guide in differenti ting
etween these c
uses. The pupil is usu lly dil ted, with n exp nding neurysm which compresses
the superior spect of the nerve th t cont ins the
p r symp thetic pupill ry
res rising from the nucleus of EdingerWestph l in the
mid
r in. An exp nding neurysm usu lly results in more p in th n the isch emi
ssoci ted with di
etes mellitus, lthough this is n unreli
le guide. If ther
e is ny dou
t
out the c use of the 3rd nerve p lsy then ngiogr phy must
e p
erformed expeditiously. In p tient with imp ired conscious st te, or in one wi
th other
norm l neurologic l signs suggesting m ssive h emorrh ge, 3rd cr ni
l nerve p lsy m y
e second ry to tempor l lo
e herni tion. A gi nt neurysm (d
e ned s l rger th n 2.5 cm in di meter) m y c use compression of dj cent neur l
structures resulting in foc l signs (Fig. 9.3). A l rge neurysm of the intern l
c rotid rtery or nterior communic ting rtery will c use compression of the o
ptic nerve or chi sm, respectively, resulting in visu l f ilure. L rge verte
ro

sil r neurysms m y c use
r instem compression. Cere
r l v sosp sm following s
u
r chnoid h emorrh ge does not usu lly result in clinic l m nifest tions for 2
or 3 d ys fter the initi l
leed so th t, lthough it m y
e the c use of su
s
equent foc l signs resulting from
r in isch emi , it is not the c use of foc l
signs immedi tely fter the h emorrh ge.
Optic fundi Mild p pilloedem is common within the rst few d ys of h emorrh ge
e
c use of the sudden
128
CHAPTER 9
elev tion of intr cr ni l pressure resulting from hydroceph lus or cere
r l oede
m . A tr nsient communic ting hydroceph lus often occurs fter su
r chnoid h em
orrh ge due to
lood
locking the r chnoid villi. In
out 10% of c ses the hyd
roceph lus persists nd is severe enough to require CSF shunt. Ophth lmoscopy
m y reve l fund l h emorrh ges, p rticul rly in severe su
r chnoid h emorrh ge.
Sm ll, sc ttered retin l h emorrh ges usu lly resolve s tisf ctorily, lthough
the l rge su
hy loid h emorrh ges m y
re k into the vitreous, resulting in perm
nent visu l defect.
su
r chnoid h emorrh ge m y
e misdi gnosed s either migr ine or tension he d
che. A full neurologic l ex min tion should
e performed with p rticul r ttenti
on given to the presence of neck stiffness, ltered conscious st te, pupill ry s
t tus nd fund l h emorrh ge. Clinic l gr ding systems h ve
een
sed on the se
verity of the he d che nd neck stiffness nd on the level of conscious st te. T
he two m jor systems re the Hunt nd Hess cl ssi c tion nd the World Feder tion
of Neurologic l Surgeons (WFNS) system (T
le 9.3).
Investig tions
The m jor differenti l di gnosis is meningitis, lthough minor h emorrh ge is
often misdi gnosed s migr ine. Con rm tion of the clinic l di gnosis of su
r chn
oid h emorrh ge should
e undert ken s soon s possi
le. Computerized tomogr ph
y (CT) sc nning (Fig. 9.4) is the
est initi l investig tion s it will con rm the
di gnosis in over 85% of c ses. It will lso provide ddition l inform tion on
ssoci ted p thology
Clinic l ssessment

The di gnosis is usu lly o
vious when the history is o
t ined from the p tient,
rel tive or friend. The cl ssic sudden onset of severe he d che with fe tures of
meningism nd decre sed conscious st te is ch r cteristic of su
r chnoid h e
morrh ge. However, dif culty m y occur when the h emorrh ge h s
een minor nd, tr
gic lly,
T
le 9.3 Su
r chnoid h emorrh ge gr ding systems. Hunt nd Hess gr ding system
* Gr de Description 1 2 3 4 5 Asymptom tic, or minim l he d che nd slight nuch
l rigidity Moder te to severe he d che, nuch l rigidity, no neurologic l de cit (e
xcept cr ni l nerve p lsy) Drowsiness, confusion or mild foc l de cit Stupor, mode
r te to severe hemip resis, possi
le e rly decere
r te rigidity nd veget tive d
istur
nces Deep com , decere
r te rigidity, mori
und
WFNS gr ding system Gr de Gl sgow Com Score (GCS) 1 2 3 4 5 15 1413 1413 127 63
Motor de cit No de cit except cr ni l nerve p lsy No de cit Any de cit With or without
foc l neurode cit Com with or without
norm l posturing
* Serious systemic dise se such s hypertension, di
etes, severe rterioscleros
is, chronic pulmon ry dise se, nd v sosp sm on ngiogr phy result in pl cement
in next less f vour
le c tegory.
SUBARACHNOID HAEMORRHAGE
129
the su
r chnoid h emorrh ge nd will determine the su
sequent tre tment. Intr rteri l digit l su
tr ction ngiogr phy h s consider
ly reduced the risks of c
onvention l ngiogr phy nd should
e undert ken s soon s the di gnosis h s
e
en con rmed nd it is cle r th t the p tient will survive the initi l h emorrh ge.
Cere
r l neurysm
Cere
r l neurysms re the most common c use of su
r chnoid h emorrh ge in the
dult popul tion, with m xim l incidence in the 4th nd 5th dec des of life,
lthough they c n occur t ny ge.
Fig. 9.4 Blood in the Sylvi n ssure nd
s l cisterns indic tive of su
r chnoid
h emorrh ge.
Surgic l n tomy
The gre t m jority of neurysms rise t the
r nch points of two vessels, usu l
ly t n cute ngle, nd re situ ted m inly on the circle of Willis nd the tr
unks of the l rge rteries which supply it. A few rise from its immedi te
r nc
hes
ut neurysms on peripher l vessels re r re (Fig. 9.5). The m jority of ne
urysms occur in const nt positions on the circle of Willis nd
out 85% occur o
n the nterior h lf of the circle (T
le 9.4). Aneurysms rise t pproxim tely
equ l frequency from the intern l c rotid rtery, nterior communic ting rtery
nd middle cere
r l rtery. Those ssoci ted with the intern l c rotid rtery mo
st frequently rise t the origin
such s intr cere
r l h emorrh ge nd hydroceph lus, nd on the position of the
h emorrh ge, which is helpful if there is more th n one neurysm. Arteriovenous
m lform tion c using su
r chnoid h emorrh ge c n frequently
e di gnosed on the
CT sc n. If there is ny dou
t th t su
r chnoid
lood is present on the CT sc
n, s m y occur following more minor h emorrh ges, lum
r puncture is essenti
l. The presence of x nthochromi (yellow st ining) in the CSF will con rm su
r ch
noid h emorrh ge. X nthochromi resulting from
re kdown of h emoglo
in in the r
ed
lood cells occurs within 68 hours fter the initi l h emorrh ge nd it will c
on rm th t the
lood in the CSF is not due to tr um from the lum
r puncture need
le. A further method frequently suggested to exclude tr um from the p ss ge of

the lum
r puncture needle s c use of
loody CSF is to llow the CSF to drip
into three consecutive tu
es; if the
lood f ils to cle r in the l st tu
e su
r c
hnoid h emorrh ge is con rmed. However, this method will result in m ny f lse posi
tive di gnoses. The CSF should lso
e immedi tely ex mined for the presence of
white
lood cells nd org nisms. Cere
r l ngiogr phy will con rm the c use of
Anterior cere
r l Anterior communic ting Intern l c rotid Middle cere
r l Poster
ior communic ting
Posterior cere
r l B sil r
Superior cere
ell r Anterior inferior cere
ell r Posterior inferior cere
ell r V
erte
r l
Fig. 9.5 Usu l sites of cere
r l neurysms.
130
CHAPTER 9
T
le 9.4 Position of cere
r l neurysm. Anterior circle of Willis Anterior comm
unic ting rtery Middle cere
r l rtery
ifurc tion or trifurc tion Intern l c r
otid rtery Posterior communic ting rtery Termin l
ifurc tion Anterior choroid
l rtery Ophth lmic rtery Intr c vernous Peric llos l rtery Posterior circul
tion (15%) Termin l
sil r rtery most common Verte
ro
sil r junction Posterio
r inferior cere
ell r rtery Anterior inferior cere
ell r rtery Superior inferi
or cere
ell r rtery Posterior cere
r l rtery
P thogenesis of cere
r l neurysms
The common type of cere
r l neurysm resulting in su
r chnoid h emorrh ge is
s ccul r neurysm, which is lso known s
erry or congenit l neurysm. Fusi
form neurysms occur in the intr cr ni l circul tion, p rticul rly the verte
ro

sil r rteries or intern l c rotid rteries, nd re due to diffuse therom tou
s degener tion of the rteri l w ll, frequently ssoci ted with hypertension. My
cotic neurysms result from septic em
oli. They m y
e situ ted on peripher l ve
ssels, re frequently multiple nd h ve high risk of h emorrh ge. The s ccul r
or
erry neurysm rises t the junction of vessels where there is congenit l
de ciency in the muscle co t. The el stic l yer in cere
r l rteries, in contr st
with rteries elsewhere, is limited to the intern l l min , m king these vessel
s more suscepti
le to we kening effects of degener tion. Fr gment tion nd disso
lution of the intern l el stic mem
r ne occurs t the site of neurysm developme
nt. The com
in tion of the muscle defect nd the discontinuity of the underlying
intern l el stic mem
r ne is pro

ly necess ry for the form tion of s ccul r
neurysm. Other f ctors th t incre se the risk of neurysm form tion include t
herom nd hypertension. There is n incre sed incidence of therom in the vess
els of the circle of Willis nd hypertension in p tients with ruptured neurysms
. It is pro

le th t these f ctors pl y role in the growth of the neurysm n
d its su
sequent rupture in some p tients.
of the posterior communic ting rtery (the so-c lled posterior communic ting rt
ery neurysm), less frequently t the termin l
ifurc tion, nd occ sion lly t
the origin of the ophth lmic rtery, the nterior choroid l rtery or in the c v
ernous sinus. Middle cere
r l rtery neurysms rise from the middle cere
r l r
tery t its
ifurc tion or trifurc tion in the Sylvi n ssure (Fig. 9.6). Less com
monly n neurysm m y rise from the peric llos l rtery t the genu of the corp
us c llosum. Approxim tely 15% of neurysms rise from the posterior h lf of the
circle of Willis, the most common position
eing the
sil r rtery, most frequ
ently t the termin l
ifurc tion into the posterior cere
r l rteries. However,
n neurysm m y rise from ny of the m in
r nches of the verte
r l or
sil r
rteries, in p rticul r the posterior inferior cere
ell r rtery, nterior infe

rior cere
ell r rtery or superior cere
ell r rtery (Fig. 9.7).

Multiple neurysms Aneurysms occur in more th n one position in pproxim tely 15
% of c ses.
Rel ted dise ses There is no de nite heredit ry
sis to the development of intr c
r ni l neurysms, lthough n epidemiology study h s shown n incre sed incidenc
e of pproxim tely seven-fold in rst-degree rel tives of p tients who h ve h d n
neurysm l su
r chnoid h emorrh ge, with lifetime risk of 25% of developing
n neurysm l su
r chnoid h emorrh ge. Aneurysms do occur in ssoci tion with he
redit ry syndromes such s EhlersD nlos syndrome, co rct tion of the ort nd po
lycystic kidney dise se.
SUBARACHNOID HAEMORRHAGE
131
( )
(
)
(c)
(d)
Fig. 9.6 ( ) Anterior cere
r l rtery neurysm. (
) Middle cere
r l rtery neur
ysm. (c) Posterior communic ting rtery neurysm. (d) Termin l intern l c rotid
rtery neurysm.
( )
(
)
(c)
Fig. 9.7 ( ) Termin l
sil r rtery neurysm. (
) Aneurysm rising from junctio
n of
sil r rtery nd superior cere
ell r rtery. (c) Posterior inferior cere

ell r rtery neurysm.
M n gement of ruptured cere
r l neurysm
The m n gement of p tients following rupture of cere
r l neurysm is determine
d
y three m jor f ctors. 1 Severity of the initi l h emorrh ge.
2 Re
leeding of the neurysm. 3 Cere
r l v sosp sm. Severity of the initi l h em
orrh ge A
out 30% of ll p tients suffering su
r chnoid h emorrh ge from ru
ptured neurysm
132
CHAPTER 9
either h ve n poplectic de th or re deeply com tose s result of the initi
l h emorrh ge. Re
leeding This occurs in
out 50% of p tients within 6 weeks n
d 25% of p tients within 2 weeks of the initi l h emorrh ge. A
out h lf the p ti
ents th t h ve su
sequent h emorrh ge will die s result of the re
leed. Aft
er the rst ye r the risk of further h emorrh ge from the neurysm is
out 23% p
er ye r. The only cert in w y to prevent n neurysm re
leeding is to exclude it
from the circul tion. Anti
rinolytic gents (such s epsilon mino c proic cid
or tr nex mic cid) decre se the risks of re
leeding
ut, s they re ssoci ted
with incre sed incidence of throm
osis (such s deep vein throm
osis nd pulmon

 ry em
olus) nd n incre sed risk of cere
r l throm
osis ssoci ted with v sosp
sm, these gents re now r rely used. Cere
r l v sosp sm Angiogr phic v sosp sm
(Fig. 9.8) occurs in
out 50% of p tients following su
r chnoid h emorrh ge
nd in 25% it results in serious neurologic l complic tions. There is direct co
rrel tion
etween the mount of
lood noted in the
s l cisterns on the CT sc n
, the risk of developing v sosp sm nd its severity. Although the sp sm m y prin
cip lly ffect the vessels most dFig. 9.8 Sp sm of the nterior cere
r l rteries following su
r chnoid h emorrh
ge from n nterior communic ting rtery neurysm.
j cent to the ruptured neurysm, gener lized v sosp sm occurs frequently. The cl
inic l m nifest tions resulting from v sosp sm will
e determined
y the vessels
which re most severely ffected. Sp sm of the intern l c rotid rtery nd midd
le cere
r l rteries produces hemip resis nd ph si in the domin nt hemisphere
. V sosp sm of the nterior cere
r l vessels c uses p r lysis of the lower lim
s
nd kinetic mutism. Severe v sosp sm m y c use widespre d cere
r l isch emi s
o th t the p tient m y
ecome o
tunded; if the v sosp sm is suf ciently severe it
will result in de th. V sosp sm does not usu lly occur until 2 or 3 d ys fter t
he initi l h emorrh ge nd its onset is r rely del yed
eyond 14 d ys. The c use
of del yed cere
r l v sosp sm rem ins o
scure
ut it is cert inly rel ted to v
soconstrictor su
st nces in the CSF s result of the h emorrh ge. V so ctive s
u
st nces isol ted from
oth the
lood clot surrounding the sp stic vessels nd
the dj cent CSF include oxyh emoglo
ulin rele sed from the erythrocytes, seroto
nin, throm
ox ne A2, prost gl ndins (F2 lph nd E2), ngiotensin nd hist mine.
In ddition, unidenti ed v soconstrictor su
st nces h ve
een isol ted from incu

tes of
rinogen, pl telets, erythrocytes nd
lood/CSF mixtures. The contr ctile
process ultim tely depends on the v il
ility of cytosolic ctiv tor c lcium i
ons. Until recently there h s
een no s tisf ctory tre tment for est
lished cer
e
r l v sosp sm. If the neurysm h s
een surgic lly occluded from the circul ti
on then hypertensive ther py com
ined with hypervol emi m y overcome the hypope
rfusion due to n rrowing of the cere
r l
lood vessels nd reverse the isch emic
effects. C lcium ch nnel
locking gents such s nimodopine nd nifedipine re
frequently used in su
r chnoid h emorrh ge to prevent nd tre t v sosp sm, lth
ough there is still some dou
t s to their effectiveness. Nimodopine, su
stitu
ted 1,4-dihydropyridine, is c lcium nt gonist th t
locks the in ux of extr cellu
l r c lcium, the prim ry source of c lcium for contr ction of l rge cere
r l rt
eries. Tri ls in North Americ , Brit in nd Sc ndin vi h ve shown promising res
ults when the c lcium ch nnel
locking gents re
SUBARACHNOID HAEMORRHAGE
133
used prophyl ctic lly, lthough there is de
te s to whether their effect is du
e to their ction on the cere
r l vessels or due to
r in protection effect. Fol
lowing the ngiogr m th t con rms cere
r l neurysm, decision is then m de s
to the de nitive tre tment of the neurysm. This will involve either: surgery with
clipping of the neurysm or endov scul r o
liter tion of the neurysm.
Surgery for ruptured neurysm The timing of the oper tion is critic l in o
t ini
ng optim l results following su
r chnoid h emorrh ge. Although
etter oper tive
results m y
e chieved when the surgery is del yed, the longer the oper tion i
s deferred the gre ter the risk th t the neurysm will re
leed. In gener l, the
oper tion is performed s soon s possi
le fter the cere
r l ngiogr m. In the
p st surgery w s voided when the p tient h d clinic l or ngiogr phic lly sever
e v sosp sm,
ut it is now recognized th t it is
est to clip the neurysm even
in the presence of clinic l or r diologic l v sosp sm s with the neurysm exclu
ded from the circul tion the sp sm c n
e tre ted using hypertensive hypervol em
ic ther py nd endov scul r techniques. Surgery is usu lly not performed on p ti

ents who re com tosed or h ve fe tures of decere
r te posturing response, unles

s the CT sc n shows l rge intr cere
r l h em tom resulting from the ruptured
neurysm which needs to
e ev cu ted, or hydroceph lus s c use of the poor ne
urologic l st te.
Preoper tive m n gement In those p tients in whom it h s
een elected for some r
e son to del y surgery, the m n gement should include c reful ttention to the f
ollowing. Posture. The p tient should lie t in quiet room with su
dued lightin
g. Every ttempt should
e m de to void environment l situ tions which could c
use sudden elev tion of the p tients
lood pressure nd thus incre se the risk of
rupture of the neurysm. Sed tion using
r
itur tes or di zep m m y
e necess
ry if the p tient is git ted.
Blood pressure control. The
lood pressure is frequently elev ted immedi tely f
ter the h emorrh ge nd should
e c refully controlled. Initi lly this should
e
done using intr venous medic tion nd utilizing v sodil ting gents (such s hy
dr l zine or glyceryl trinitr te) nd
et
lockers. Although it is essenti l to
control high
lood pressure, s this m y le d to rupture of the neurysm, hypote
nsion m y result in cere
r l isch emi , p rticul rly when v sosp sm is present.
The ppropri te desir
le
lood pressure will depend upon the premor
id level. F
luids nd electrolytes. Correct hydr tion is essenti l to void electrolyte dist
ur
nce; in ddition, overhydr tion m y precipit te cere
r l oedem nd insuf cien
t uids m y incre se the risk of cere
r l throm
osis ssoci ted with v sosp sm. El
ectrolyte distur
nces m y lso occur following su
r chnoid h emorrh ge due to
in ppropri te ntidiuretic hormone (ADH) secretion, which results in hypon tr em
i . P in relief. Simple n lgesic medic tion or codeine phosph te is
est used f
or controlling the he d ches resulting from su
r chnoid h emorrh ge. Surgic l p
rocedures The surgic l procedures v il
le re: occlusion of the neck of the n
eurysm reinforcement of the s c of the neurysm proxim l lig tion of feeding v
essel. H emorrh ge from n neurysm is due to rupture of the fundus of the neur
ysm l s c. Therefore, the
est surgic l procedure is to occlude the neck of the
neurysm, there
y isol ting the neurysm from the circul tion. In
rief, the ope
r tion involves cr niotomy which is usu lly
sed on the pterion (pterion l cr
niotomy) for neurysms of the nterior circul tion. This type of cr niotomy m y
lso
e used for neurysms rising from the termin l
sil r rtery, lthough s
ome surgeons prefer n ppro ch under the tempor l lo
e vi tempor l cr niotom
y. Microsurgic l techniques, utilizing the oper ting microscope nd microneurosu
rgic l instruments, re employed. Access to the
s l cisterns m y
e ided
y w
ithdr wing CSF either using ventricul r dr in or from the lum
r thec . The r
ch134
CHAPTER 9
noid round the
s l cisterns is opened, the neck of the neurysm identi ed nd d
issected nd clip pl ced cross the neck to exclude the neurysm from the circ
ul tion. During the dissection of the neurysm it is essenti l th t vit l dj ce
nt vessels, including the perfor ting rteries, re not injured, s d m ge to th
ese vessels m y result in severe neurologic l dis
ility. Occ sion lly it is not
possi
le to s fely pl ce clip cross the neck of the neurysm, usu lly s r
esult of
r nches of the p rent vessel either rising from the neurysm or
eing
insep r
le from the fundus. In this c se the w ll of the neurysm m y
e reinf
orced
y num
er of techniques, including wr pping the w ll with crushed muscle
, g uze or cotton wool or com
in tion of these. R pidly solidifying polymer (
neurysm cement) m y
e poured round the neurysm to provide it with solid cov
ering. Although lig tion of the common or intern l c rotid rtery in the neck w
s commonly used for tre tment of neurysms of the intern l c rotid rtery, impro
ved microneurosurgic l techniques h ve m de this oper tion lmost o
solete. The
procedure h s lso
een used for intr c vernous neurysms
ut these re now
est
tre ted
y endov scul r techniques usu lly performed
y r diologist. Lig tion

of the intern l c rotid rtery m y
e performed for gi nt intern l c rotid r

tery neurysm which is not men
le to direct surgery,
ut n extr cr ni lintr cr
ni l
yp ss procedure m y need to
e performed prior to the occlusion to preven
t cere
r l isch emi . Postoper tive m n gement The usu l postcr niotomy oper tiv
e m n gement pplies, with speci l ttention to
e given to the neurologic l st
te, hydr tion, posture, oxygen tion nd
lood pressure. Anticonvuls nt medic tio
n is recommended for 3 months to 1 ye r. Steroid medic tion is sometimes used in
the initi l postoper tive course to control cere
r l oedem , lthough its effec
tiveness is not proven. The m jor speci c postoper tive pro
lem results from del y
ed cere
r l v sosp sm. As indic ted previously, prophyl ctic c lcium ch nnel
lo
cking gents m y
e of use in preventing this complic tion. The tr nscr ni l Dop
pler, utilizing
non-inv sive ultr sound, m y give useful inform tion on the degree of intr cr ni
l v sosp sm. Symptom tic v sosp sm c n
e tre ted using hypervol emic hypertens
ive ther py. This tre tment ent ils c reful monitoring nd requires the tr nsfer
of the p tient to n intensive c re unit. Recently, endov scul r techniques to
dil te the vessels in sp sm or dminister intr - rteri l p p verine into the int
r cr ni l vessels h ve
een used to tre t cere
r l v sosp sm postoper tively wit
h some success.
Endov scul r procedures for ruptured neurysms
Over the p st 10 ye rs endov scul r techniques (using det ch
le coils) h ve
ee
n used to o
liter te cere
r l neurysms. These h ve
een investig ted in intern
tion l tri ls, nd h ve proven to
e effective in excluding the neurysm from th
e circul tion. The technique is usu lly performed
y speci list intervention l
r diologist, nd lmost lw ys under gener l n esthesi . As with surgery, it i
s recommended th t the neurysm is coiled s soon s possi
le fter the ngiogr m
h s
een performed to con rm the presence of n neurysm (Fig. 9.9). The recent IS
AT tri l showed the possi
le superiority of endov scul r coiling over surgery,
lthough there h s
een some de
te s to these ndings. The m in concern rem ins
s to whether the o
liter tion of the neurysm is perm nent, s the neurysm c n
recur, p rticul rly with imp ction of the coils into the fundus of the neurysm, s
ome months fter the initi l tre tment. In most centres check ngiogr m is per
formed t 6, 12 nd 24 months fter the tre tment. The decision s to whether n
neurysm should
e clipped
y surgeon or coiled
y n intervention l neuror diolo
gist is
est m de jointly
y the tre ting speci list, cere
rov scul r neurosurge
on nd neuror diologist. The intervention l neuror diologist will
se his decis
ion on the ccess to the neurysm nd the con gur tion of the neurysm. Access to
the neurysm m y
e imp ired
y
SUBARACHNOID HAEMORRHAGE
135
the neurysm whilst m int ining p tency of the vessels. At The Roy l Mel
ourne H
ospit l pproxim tely 50% of neurysms since the ye r 2000 h ve
een tre ted
y
coiling.
M n gement of n unruptured neurysm
Multiple neurysms occur in 15% of p tients who present following su
r chnoid h
emorrh ge. In gener l, n unruptured neurysm will
e clipped t the s me time
s the surgery for the ruptured neurysm, provided it c n
e performed through t
he s me cr niotomy. The indic tions for surgery re controversi l for n unruptu
red neurysm occurring in p tient who h s suffered su
r chnoid h emorrh ge
from nother neurysm, or for n unruptured neurysm found incident lly. The de

te reg rding the optim l m n gement of p tients with n unruptured neurysm rev
olves round the rel tive risk of rupture of the neurysm vs. the risk of tre tm
ent,
y either surgery or n endov scul r ppro ch. In the p st the risk of h em
orrh ge from n unruptured neurysm w s usu lly quoted t 23% per ye r. However,

in 1998 the New Engl nd Journ l of Medicine pu
lished l rge study of the n tur
l history of unruptured intr cr ni l neurysms which indic ted th t the risk of
h emorrh ge w s very much lower, p rticul rly for neurysms less th n 10 mm in
di meter nd those rising from the middle cere
r l rtery. There h s
een consi
der
le de
te in the neurosurgic l liter ture reg rding the ver city of the soc lled ISUIA (Intern tion l Study of Unruptured Intr cr ni l Aneurysms) study, w
ith some experts questioning the methodology. In gener l, the risk of rupture wi
ll depend on the size of the neurysm, on the con gur tion of the neurysm, in p r
ticul r if there is d ughter s c tt ched to the fundus, on positive f mily hi
story for neurysm l su
r chnoid h emorrh ge nd on the ge of the p tient. Sym
ptom tic neurysms of ll sizes should
e considered for tre tment.
( )
(
)
Fig. 9.9 ( ) Endov scul r tre tment of intern l c rotid rtery neurysm. Figure
(
) shows the neurysm excluded from the circul tion following the endov scul r
insertion of coils.
stenosis or tortuosity within the c rotid rtery (for nterior circul tion neur
ysms) nd verte
ro
sil r rtery (for posterior circul tion neurysms). The dome
to neck r tio is n import nt consider tion in deciding whether the con gur tion of
the neurysm is ppropri te for coiling. In gener l, most neuror diologists pre
fer the r tio to
e 2 : 1 or gre ter. New techniques in intervention l r diology
including the use of stents nd three-dimension l coils h ve incre sed the num

er of neurysms th t c n
e tre ted
y endov scul r techniques. At present, over
80% of termin l
sil r neurysms c n
e tre ted
y endov scul r techniques,
u
t only
out h lf of the nterior circul tion neurysms re men
le to coiling. M
ost intervention l r diologists do not coil middle cere
r l rtery neurysms, s
there is dif culty in o
liter ting
Arteriovenous m lform tion
Arteriovenous m lform tions re the most common c use of su
r chnoid h emorrh g
e in
136
CHAPTER 9
children. Other types of v scul r m lform tions of the
r in include the followi
ng. C pill ry tel ngiect si
leed infrequently
ut m y result in f t l h emorrh
ge, p rticul rly in the pons. C vernous h em ngiom (see Fig. 9.12) often c use
minor loc l extr v s tions of
lood
ut m jor h emorrh ge is uncommon. P tients
frequently present following n epileptic seizure if the h em ngiom is in the
cere
r l hemispheres. Posterior foss h em ngiom s m y present with
r instem
stroke. Venous m lform tions. The rteriovenous m lform tion is the most common
v scul r m lform tion. Although it ccounts for pproxim tely 60% of ll su
r c
hnoid h emorrh ge in children,
y the 3rd dec de it is responsi
le for 20% nd

y the 5th dec de for less th n 5%.
Surgic l n tomy
Most rteriovenous m lform tions re situ ted in the cere
r l hemispheres, ltho
ugh they m y occur in the posterior foss involving either the cere
ellum or
r
instem nd they show consider
le v ri tion in size. The m lform tions involving
the cere
r l hemispheres frequently form pyr mid l m ss, the
se of which m
y re ch the cortic l surf ce with the pex pointing tow rds the l ter l ventricl
e. There re frequently multiple, enl rged rteries feeding the m lform tion nd
rteri lized dr ining veins extend super ci lly to the superior s gitt l sinus or
tr nsverse sinus or deeply into the deep cere
r l venous system.

R diologic l investig tions for rteriovenous m lform tions (Figs 9.109.12)

An rteriovenous m lform tion is often pp rent on the CT sc n
ec use of the vi
vid enh ncement of the enl rged feeding vessel nd rteri lized dr ining veins
fter intr venous contr st. Cere
r l ngiogr phy is
est performed using digit l
su
tr ction ngiogr phic techniques nd is essenti l for dequ te ev lu tion of
the m lform tion. Precise determin tion of the position of the m jor feeding nd
dr ining vessels is vit l prior to surgery. M gnetic reson nce im ging is v l
u
le id in determining the ex ct position of the rteriovenous m lform tion n
d the vessels. Preoper tive occlusion of ccessi
le m jor feeding vessels close
to the m lform tion
y n intervention l r diologist m y
e useful if the proced
ure is technic lly fe si
le. A ow-directed c theter is positioned in the rtery,
which is occluded using cy no cryl te glue or polymerizing coll gen mixture.
Clinic l present tion
H emorrh ge. This is the most frequent rst symptom of n rteriovenous m lform ti
on nd, lthough the
leeding m y
e su
r chnoid, there is commonly n intr cer
e
r l component. The rteriovenous stulous communic tion results in the developme
nt of neurysms within the lesion, enl rgement of the rteries which feed the m
lform tion nd, consequently, the possi
le second ry development of s ccul r ne
urysms on the m jor feeding vessels. The h emorrh ge ssoci ted with n rteriov
enous m lform tion m y quite often
e due to rupture of s ccul r neurysm on t
he feeding vessel. Epilepsy. This is the second most common presenting m nifest
tion of n rteriovenous m lform tion. He d che. Migr ine ch r cteristics re p
rticul rly ssoci ted with he d che due to rteriovenous m lform tion. Progressi
ve neurologic l de cit. For ex mple, slowly progressive hemip resis m y occur in
l rge m lform tion due either to loc l isch emi induced
y the shunt or to i
ncre sing size of the lesion.
M n gement
As with cere
r l neurysms the im of tre tment is to void either n initi l h
emorrh ge or re
leed from the m lform tion. There h s
een controversy over the
risk of h emorrh ge nd the mor
idity nd mort lity ssoci ted with rupture of
n rteriovenous m lform tion. Recent studSUBARACHNOID HAEMORRHAGE
137
( )
(
)
(c)
Fig. 9.10 ( ) The rteriovenous m lform tion enh nces vividly on the CT sc n ft
er intr venous contr st nd the m jor dil ted feeding vessels c n
e seen. (
) T
he MRI shows the position of the m lform tion in coron l nd xi l pl nes nd fu
rther inform tion
out the feeding vessels nd dr ining veins (c).
( )
(
)
Fig. 9.11 Cere
r l ngiogr phy (digit l su
tr ction ngiogr m) demonstr tes the
v scul r n tomy of the rteriovenous m lform tion. ( ) The m jor feeding vessel
s re shown on the rteri l ph se. (
) The dr ining veins re demonstr ted on th
e venous ph se.
ies h ve shown th t the ch nce of h emorrh ge for
oth ruptured nd unruptured

rteriovenous m lform tions is
out 3% e ch ye r nd th t the com
ined mor
idity

nd mort lity of e ch h emorrh ge is t le st 40%. However, unlike cere
r l ne
urysms, h emorrh ge from n rteriovenous m lform tion r rely c uses symptom tic
v sosp sm. Surgic l excision, provided it is technic lly fe si
le nd would not
result in dis
ling neurologic l de cit, should
e performed if the m lform tio
n h s h emorrh ged. Unruptured rteriovenous m lform tions should
e considered
for excision if surgery is unlikely to produce signi c nt neurologic l de cit.
Surgery for rteriovenous m lform tions The principles of the oper tion involve
isol tion nd occlusion of the princip l feeding rteries followed
y meticulous
dissection of the m lform tion, with occlusion nd division of the numerous sm
ll feeding vessels. The dr ining veins should
e lig ted only fter ll the feed
ing vessels h ve
een occluded, since prem ture o
struction to the rteri lized
venous out ow will result in precipitous swelling nd rupture of the v scul r m
ss. The surgic l m n gement of gi nt rteriovenous m lform tions is fr ught with
consider
le risk. The lesions m y
e surrounded
y chronic lly isch emic
r in
ste l
y the
138
CHAPTER 9
Neon tes present shortly fter
irth with cy nosis nd he rt f ilure due to the
shunt through the m lform tion. Inf nts nd young children present with seizures
nd hydroceph lus due to o
struction of the cere
r l queduct. Adults m y prese
nt with multiple su
r chnoid h emorrh ge.
Su
r chnoid h emorrh ge of unknown etiology
In
out 15% of p tients the c use of su
r chnoid h emorrh ge rem ins uncle r,
despite clinic l nd r diologic l investig tion. M ny of these p tients re hype
rtensive nd h ve evidence of intr cr ni l rteri l therosclerosis, lthough th
is is not inevit
le. Most p tients m ke good recovery following the su
r chn
oid h emorrh ge nd re
leeding is uncommon.
Fig. 9.12 C vernous h em ngiom .
m lform tion nd
rupt occlusion of the shunt through the m lform tion h s led
in some c ses to oedem nd h emorrh ge in the dj cent
r in, phenomenon firs
t descri
ed
y Spetzler nd which w s c lled the norm l perfusion pressure
re kt
hrough theory. Methods th t h ve
een employed to void this complic tion include
preoper tive nd intr oper tive em
oliz tion nd st ged excision of the m lform
tion. The use of r diosurgic l techniques, involving either the g mm knife (
highly focused co
lt source of irr di tion) or stereot ctic r diosurgery using
line r cceler tor, h s
een dvoc ted for the tre tment of sm ll (less th n 3
cm di meter), unruptured nd surgic lly in ccessi
le rteriovenous m lform tion
s with complete ngiogr phic o
liter tion in gre ter th n 80% of lesions with
di meter of 3 cm or less t 3 ye rs fter r di tion. However, s the r diother p
y effect is slow, the p tient rem ins t risk from h emorrh ge for some ye rs.
M n gement
It is essenti l to exclude c use for the su
r chnoid h emorrh ge nd this m y
ent il repe ting the cere
r l ngiogr phy, p rticul rly if the initi l ngiogr
phy h s
een in some w y imperfect nd especi lly if not ll the m jor vessel
r
nches h ve
een shown dequ tely. Systemic c uses for the su
r chnoid h emorrh
ge must
e excluded, s well s r re c uses such s pituit ry poplexy. The p t
ient is m n ged symptom tic lly with
ed rest until the he d che h s settled.
Further re ding
Chy tte D, Fode N, Sundt T (1988) E rly versus l te intr cr ni l neurysm surger
y in su
r chnoid h emorrh ge. Journ l of Neurosurgery 69, 326331. Dorsch NWC, Ki

ng MT (1994) A review of cere
r l v sosp sm in neurysm l su
r chnoid h emorrh

ge. Journ l of Clinic l Neuroscience 1, 1926. Dorsch NWC, King MT (1994) A review
of cere
r l v sosp sm in neurysm l su
r chnoid h emorrh ge. Journ l of Clinic
l Neuroscience 1, 7892. Dorsch NWC, King MT (1994) A review of cere
r l v sosp s
m in neurysm l su
r chnoid h emorrh ge. Journ l of Clinic l Neuroscience 1, 15
1160.
Vein of G len m lform tion
This unusu l m lform tion results when rteries feed directly into the vein of G
len nd produces distinct clinic l syndromes depending on the ge t which the
dise se presents.
SUBARACHNOID HAEMORRHAGE
139
Dr ke CJ (1984) E rly times in neurysm surgery. Clinic l Neurosurgery 32, 4150.
Freedm n WA (1995) Line r cceler tor r diosurgery for rteriovenous m lform tio
ns, the rel tionship of size to outcome. Journ l of Neurosurgery 82, 180189. Hero
s RC, Tu YK (1987) Is surgic l ther py needed for unruptured rteriovenous m lfo
rm tions? Neurology 37, 279286. Heros RC, Zerv s NT, V rsos V (1983) Cere
r l v s
osp sm fter su
r chnoid h emorrh ge: n upd te. Ann ls of Neurology 14, 599603.
J ne JA et l. (1985) The n tur l history of neurysms nd rteriovenous m lfor
m tions. Journ l of Neurosurgery 62, 321323. K ye AH, Bl ck P McL. (2000) Oper ti
ve Neurosurgery. Churchill Livingstone, London, New York, Edin
urgh. Intern tion
l su
r chnoid neurysm tri l (ISAT) of
neurosurgic l clipping versus endov scul r coiling in 2143 p tients with rupture
d intr cr ni l neurysms: r ndomised tri l. L ncet 360, 12671273. Ljunggren B,
Br ndt L (1985) Timing of neurysm surgery. Clinic l Neurosurgery 33, 147176. M r
ire JN, Aw d IA (1995) Intr cr ni l c vernous m lform tions: lesion
eh viour
nd m n gement str tegies (Review). Neurosurgery 37, 591605. The Intern tion l Stu
dy of Unruptured Aneurysm Investig tors (1998) Unruptured intr cr ni l neurysms
risk of rupture nd risks of surgic l intervention. New Engl nd Journ l of Medi
cine 339 (24), 17251733. Weir B (2002) Unruptured intr cr ni l neurysms; revie
w. Journ l of Neurosurgery 96, 342. Weir B, M cDon ld MD (1992) Cere
r l v sosp s
m. Clinic l Neurosurgery 40, 4056.
CHAPTER 10
10
Stroke
Stephen M. D vis
Introduction nd terminology
Stroke is the third most common c use of de th in most Western countries nd the
commonest c use of chronic dult dis
ility. In m ny Western countries, there h
s
een n impressive f ll in popul tion-
sed stroke mort lity over the p st fe
w dec des, ver ging 45% e ch ye r. This h s
een chie y ttri
uted to the more eff
ective recognition nd tre tment of hypertension nd other risk f ctors. Despite
this progress, with the incre sing life expect ncy of the popul tion, m rked
incre se in stroke prev lence h s
een predicted. Stroke is gener lly dise se
of geing,
ut young dults re lso ffected, with somewh t different p thoge
netic spectrum. The over ll ppro ch to stroke h s undergone dr m tic ch nge i
n recent ye rs, with import nt recent dv nces in prevention str tegies, the wid
espre d introduction of cute stroke units, the pplic tion of new im ging techn
ologies nd the introduction of throm
olysis for selected p tients with cute is
ch emic stroke. The term stroke is used to descri
e sudden neurologic l deficit
of v scul r etiology l sting more th n 24 hours. A tr nsient isch emic tt ck (

TIA) indic tes tr nsient neurologic l deficit of v scul r origin l sting less
th n 24 hours, lthough m ny p tients with TIAs l sting more th n minutes h ve i
n f ct suffered some neuron l d m ge nd this time definition h s
een recently
ch llenged. Stroke is cl ssified s cere
r l inf rction or isch emic stroke, sig
nifying isch emic
r in d m ge, or cere
r l h emorrh ge, where the prim ry p tho
logy involves v scul r rupture nd extr v s tion of 140

lood into the surrounding tissues. The term h emorrh gic inf rction is used to de
scri
e n inf rct into which there h s
een second ry extr v s tion of
lood.
Although su
r chnoid h emorrh ge (SAH) m y not
e ssoci ted with foc l neuro
logic l deficit, it is usu lly c tegorized s stroke su
type. Old nd misle di
ng terms such s CVA (cere
rov scul r ccident) nd RIND (reversi
le isch emic neuro
logic l deficit) h ve gener lly
een
ndoned.
Stroke prevention
Stroke prevention involves prim ry nd second ry str tegies. Prim ry prevention
includes lifestyle modi c tion nd tre tment of risk f ctors in individu ls who h
ve not experienced cere
rov scul r symptoms. Risk f ctor m n gement c n
e c teg
orized into the high risk ppro ch (detecting nd tre ting p tients t high risk
of stroke, such s those with tri l
rill tion or hypertension) nd the m ss or
popul tion ppro ch (such s reducing s lt int ke nd hence ttempting to lower

lood pressure in the entire popul tion). These two ppro ches re complement r
y, lthough the popul tion ppro ch h s the potenti l of much gre ter imp ct on
stroke r tes in society s whole. Second ry prevention involves the use of str
tegies in symptom tic individu l fter stroke or TIA, gener lly t ilored to
the speci c type of cere
rov scul r p thology. Most stroke prevention studies h v
e focused on reduction in the incidence of stroke, neurologic l dis
ility nd
other v scul r endpoints. Recently,
STROKE
141
T
le 10.1 Modi
le risk f ctors for isch emic stroke. Risk f ctor Smoking Hypert
ension Di
etes He rt dise se (p rticul rly tri l
rill tion) Hypercholesterol e
mi Rel tive risk of stroke 1.04.0 2.04.0 2.08.0 6.08.0 1.02.0 Bene ts proven Yes Yes Y
es Yes Yes
prevention of v scul r dementi h s lso
een recognized s n import nt dditio
n l go l.
Prim ry prevention
Prim ry prevention str tegies t rget the modi
le risk f ctors for stroke (T
le
10.1). In the consider tion of risk f ctors nd stroke, the popul tion ttri
ut

le risk is useful concept in ev lu ting the over ll import nce of risk f ct
or, com
ining the rel tive risk of the individu l f ctor nd its prev lence.
Age nd gender In gener l, the frequency of stroke incre ses with dv ncing ge.
Over ll, m le gender is ssoci ted with incre sed stroke risk. Among older p ti
ents there is slightly higher r te of stroke in fem les,
ut this m y simply r
e ect the gre ter longevity of women. Hypertension Hypertension is the most import
nt risk f ctor for
oth cere
r l inf rction nd h emorrh ge. The popul tion tt
ri
ut
le risk from hypertension h s
een estim ted s 26%. M ny popul tion stud
ies h ve demonstr ted n incre sed frequency of stroke with
oth systolic nd di
stolic hypertension. Hypertension is correl ted with common p thogenetic stroke
mech nisms. These include c rdi c dise se (risk of cere
r l em
olism), intr cer
e
r l sm ll vessel dise se (l cun r inf rction, intr cere
r l h emorrh ge), extr
cr ni l therosclerosis (throm
oem
olism), development of cere
r l neurysms (s
u
r chnoid h emorrh ge) nd rupture of

deep perfor ting vessels (intr cere
r l h emorrh ge). A met - n lysis in 1990 sh
owed th t modest reduction of systolic
lood pressure (BP) reduces stroke risk

y
out 40%. This
enefit lso extends to those with mild hypertension. Most of
these e rlier tri ls of ntihypertensive gents involved
et -
lockers nd diure
tics, r ther th n the newer gents such s the ngiotensinconverting enzyme inhi

itors, ngiotensin II receptor
lockers nd c lcium ch nnel nt gonists. Block
de of the ngiotensin receptor h s theoretic l ppe l, with potenti l
enefits

eyond
lood pressure reduction. However, there is still uncert inty
out the re
l tive
enefits of different cl sses of ntihypertensive gents. Some still dvo
c te diuretics s first-line ntihypertensive gents. One study suggested cl ssspecific
enefits for ngiotensin receptor
lock de over
et -
lockers,
ut rece
nt met n lysis shows th t the degree of
enefit is not rel ted to drug cl ss,

ut r ther the extent of
lood pressure reduction.
Atri l
rill tion V lvul r he rt dise se h s long
een recognized s c use of c
ere
r l em
olism. The decline in the incidence of rheum tic he rt dise se, ssoc
i ted with 17-fold incre se in stroke risk in those with tri l
rill tion, m y
h ve contri
uted to the reduction in popul tion-
sed stroke mort lity. Non-v l
vul r tri l
rill tion (NVAF) is now the most common c use of c rdiogenic cere
r
l inf rction in Western countries, ssoci ted with 68 times incre se in stroke
risk. NVAF is ge-rel ted, ffecting pproxim tely 1.7% of the
142
CHAPTER 10
popul tion ged 60 ye rs nd rising to 11% in those older th n 75 ye rs. Clinic
l tri ls h ve demonstr ted rel tive risk reduction of
out 70% in the stroke
r te in p tients with NVAF without prior cere
rov scul r symptoms, tre ted with
w rf rin. In compli nt p tients, there re low risks of m jor h emorrh gic compl
ic tions with c reful monitoring, with pproxim tely 1% m jor h emorrh ge r te
per ye r, iming t n intern tion l norm lized r tio (INR) in the 23 r nge. The
re is modest
ene t with spirin,
ut it is only
out h lf s effective s w rf
rin. Aspirin is gener lly used when p tients re not c ndid tes for ntico gul
tion, nd in those under the ge of 60 ye rs with tri l
rill tion,
ut without
risk f ctors or echoc rdiogr phic fe tures of structur l he rt dise se (termed lo
ne tri l
rill tion). W rf rin is often com
ined with spirin to convey ddition
l
ene t in p tients with prosthetic he rt v lves nd tri l
rill tion or prior th
rom
o-em
olism.
tion. Chronic smoking lso lowers cere
r l
lood

ow.

Hypercholesterol emi Hyperlipid emi , nd in p rticul r hypercholesterol emi ,

is much we ker risk f ctor for stroke th n isch emic he rt dise se in epidemio
logic l studies. However, tri ls of the HMG-CoA reduct se inhi
itors (st tins),
which produce potent cholesterol reduction, h ve
een shown in high-risk p tient
s (gener lly with isch emic he rt dise se) nd cholesterol r nging from norm l t
o high levels, to m rkedly reduce the risk of stroke. The ef c cy of these drugs m
y lso re ect other ph rm cologic l ctions, including effects on the endothelium
nd therosclerotic pl que. He vy lcohol use He vy lcohol us ge is ssoci ted
with n incre sed risk of
oth isch emic nd h emorrh gic stroke, p rticul rly
su
r chnoid h emorrh ge. In contr st, some studies h ve suggested th t low to m
oder te lcohol int ke is stroke-protective. Miscell neous f ctors Polycyth emi
is n import nt nd tre t
le risk f ctor for cere
r l inf rction. Leisure-time
physic l ctivity h s
een linked with reduced risk of isch emic stroke. There
h s
een interest in the ther peutic role of vit mins such s folic cid nd B6,
given epidemiologic l links
etween homocystin emi nd stroke,
ut further tri
l results re w ited. Asymptom tic c rotid dise se C rotid
ruits occur in t
le st 4% of symptom tic dults over the ge of 40 ye rs, of which only propor
tion re due to severe intern l c rotid stenosis. There h ve
een num
er of r

ndomized controlled tri ls to determine whether c rotid end rterectomy (CEA) is

indic ted in p tients with severe
ut symptom tic c rotid stenosis (Fig. 10.1).
The most de nitive of these indic ted th t there w s n 11% stroke r te in the he
misphere ipsil ter l to 60% or gre ter c rotid stenosis, reduced signi c ntly to
5%
y CEA in good surgic l h nds. However, this study
Di
etes Di
etes mellitus is ssoci ted with two- to vefold incre se in the r
te of stroke. This is due to
oth cceler ted therogenesis in the extr cr ni l
nd intr cr ni l rteries (m crov scul r dise se) nd the development of sm ll v
essel, l cun r inf rcts (microv scul r dise se). The prognosis of cute stroke i
n di
etic nd other hyperglyc emic p tients is worse th n in those with norm l

lood sug r levels, pro

ly due to the production of excessive l ct te nd incr
e sed tissue d m ge. Optim l control of
lood glucose is likely to reduce the v
scul r complic tions of di
etes. Smoking Smoking is n import nt risk f ctor fo
r stroke, p rticul rly isch emic stroke nd su
r chnoid h emorrh ge. It t le s
t dou
les stroke risk in
oth men nd women. Like di
etes, smoking cceler tes
therogenesis nd h s intr v scul r effects on pl telet dhesion nd viscosity.
The intr v scul r effects ppe r to
e p rticul rly import nt, evidenced
y su

st nti l reduction of stroke risk within 24 ye rs of smoking cess STROKE
143
indic tions for c rotid end rterectomy nd the introduction of cere
r l ngiopl
sty/stenting.
Fig. 10.1 Arteri l digit l su
tr ction ngiogr m demonstr ting severe, proxim l
intern l c rotid rtery stenosis ( rrow).

lso con rmed th t the n tur l history of

vely
enign. A l rge num
er of oper tions
ut 83 to prevent one stroke per ye r. The
stenosis therefore rem ins controversi l,
y selected p tients.

symptom tic c rotid stenosis w s rel ti

re required in stroke prevention,
o
pl ce of CEA for symptom tic c rotid
nd should only
e considered in highl

Second ry prevention
Second ry prevention str tegies fter stroke or TIA, unlike the prim ry preventi
on techniques, re t ilored to the underlying stroke p thology. The r nge of sec
ond ry prevention str tegies continues to exp nd (T
le 10.2). These include the
introduction of num
er of new ntipl telet str tegies, proof of the ef c cy of
w rf rin in non-v lvul r tri l
rill tion, cl ri c tion of the
Antipl telet str tegies The
ene ts of spirin were est
lished
y pivot l studies
more th n 25 ye rs go, with rel tive risk reduction of
out 22% for the com
posite outcomes of stroke, de th or myoc rdi l inf rction. Met - n lysis of the
10 second ry prevention tri ls where spirin w s tested g inst pl ce
o in secon
d ry stroke prevention showed no discerni
le difference
etween high, medium nd
low doses of spirin. The consensus mongst stroke clinici ns tod y is th t dos
es in the low 50325 mg r nge re preferred, given th t
leeding risk is lower th n
the high-dose regimens. The effectiveness of spirin depends on the inhi
ition o
f pl telet cyclo-oxygen se,
ut other ntipl telet str tegies with differing ct
ions h ve lso
een proven in stroke prevention. Unlike spirin, clopidogrel inh
i
its pl telet ADP. It w s shown in the CAPRIE tri l to
e more effective th n
spirin in over ll v scul r protection in high-risk p tients, with rel tive ris
k reduction of 8.7% for clopidogrel over spirin. If one ssumes
out one-qu
rter reduction of ll v scul r events in high-risk p tients with spirin, this c
n
e improved to pproxim tely one-third with clopidogrel. However,
ec use of
the sm ll
solute risk reduction ( pproxim tely 0.5% per ye r) nd the cost of
the drug, clopidogrel tends to
e used s second-line ther py in p tients who r

e either intoler nt of spirin or re spirin f ilures. Clopidogrel nd spirin

h ve n dditive or synergistic effect
ec use of their different ctions. This
com
in tion ppro ch h s
een proven in cute coron ry syndromes nd is currentl
y
eing tested in p tients with prior TIA or stroke. Dipyrid mole reduces pl tel
et ggreg tion
y r ising the nti ggreg ting effects of cyclic AMP nd cyclic G
MP. A synergistic effect
etween spirin nd dipyrid mole w s demonstr ted in th
e ESPS 2 tri l. The rel tive stroke risk w s reduced
y 18% with spirin, 16% wi
th dipyrid mole, nd n pp rently dditive 37% with the com
in tion of the two
ther pies. This com
in tion ntipl telet ppro ch is lso widely used.
144
CHAPTER 10
T
le 10.2 Ther peutic opportunities in second ry prevention. Str tegy Antipl te
let nd ntico gul nt str tegies Aspirin Clopidogrel Dipyrim dole Com
in tion n
tipl telet str tegies Glycoprotein II
/III inhi
itors W rf rin Surgic l nd int
ervention l str tegies C rotid end rterectomy Angiopl sty/stenting Indic tion Le
vel of evidence
Non-v lvul r tri l
rill tion, v lvul r he rt dise se Symptom tic c rotid stenos
is (< 70%) Asymptom tic c rotid stenosis Not yet proven; possi
le role in p tien
ts with extr cr ni l, intr cr ni l symptom tic therosclerotic stenosis No r ndo
mized, controlled tri ls to d te con rming
ene ts
I II II II (com
ined spirin/ dipyrim dole) Disc rded I I
I II III
Qu lity of evidence r tings Level I: Evidence is o
t ined from system tic revi
ew of ll relev nt r ndomized controlled tri ls. Level II: Evidence is o
t ined
from t le st one properly designed r ndomized controlled tri l. Level III: Evid
ence is o
t ined from well-designed controlled tri ls without r ndomiz tion; fro
m welldesigned cohort or c secontrol n lytic l studies. Level IV: Opinions of re
spected uthorities,
sed on clinic l experience, descriptive studies or report
s of expert committees.
The pl telet glycoprotein II
/III receptor is the n l common p thw y of pl telet
ggreg tion. Or l pl telet GPII
/III nt gonists prevent the
inding of
rinoge
n to pl telets
ut h ve proved h z rdous in p tients with coron ry or cere
rov s
cul r dise se nd h ve now
een
ndoned.
W rf rin The Europe n Atri l Fi
rill tion tri l comp red w rf rin, spirin nd p
l ce
o in second ry stroke prevention. W rf rin w s su
st nti lly more effective
th n spirin nd is the recommended str tegy in ntico gul tion c ndid tes. How
ever, w rf rin is not superior to spirin in p tients with symptom tic cere
r l
therosclerosis.
C rotid end rterectomy Two l rge tri ls (the North Americ n Symptom tic C rotid
End rterectomy Tri l NASCET nd the Europe n C rotid Surgery Tri l ECST) showed
m jor
enefits for c rotid end rterectomy over optim l medic l ther py in p tien
ts with gre ter th n 70% c rotid stenosis nd either TIA or non-dis
ling stroke
. In the NASCET study, n
solute risk reduction of 17% over 18 months w s chi
eved, indic ting th t one stroke could
e prevented for every six p tients. Cere

rov scul r ngiopl sty/stenting Percut neous tr nslumin l ngiopl sty is incre
singly used for symptom tic nd symptom tic c rotid stenosis, usu lly com
ined
with
STROKE

145
endov scul r stenting. However, there is limited proof of ef c cy nd s fety comp
red with end rterectomy. One tri l showed th t the
ene ts nd risks of surgery n
d ngiopl sty/stenting were pproxim tely equiv lent. L rge tri ls re now
eing
conducted in p tients with symptom tic c rotid stenosis, comp ring stenting wit
h end rterectomy. Dist l protection devices, which tr p em
olic de
ris t the ti
me of the procedure, represent n import nt dv nce. Stenting h s lso
een used
in sm ll series of p tients with symptom tic intr cr ni l stenoses, in whom opt
im l medic l ther py h s f iled.
to detect inf rction or non-v scul r p thology, c rotid duplex Doppler to di gno
se m jor c rotid dise se, nd ECG, sometimes followed
y echoc rdiogr phy, to di
gnose tri l
rill tion or nother c rdioem
olic source.
C rotid-territory TIAs
These re due to tr nsient isch emi in the retin or cere
r l hemisphere. Tr ns
ient monocul r
lindness ( m urosis fug x) is due to tr nsient reduction in reti
n l perfusion produced
y em
olism or h emodyn mic f ilure. The p tient often de
scri
es sh de pulled down over one eye. In clinic l pr ctice it is vit l to de
termine whether visu l distur
nce is truly monocul r, indic ting retin l isch
emi , or
inocul r, often implic ting the verte
ro
sil r circul tion. Hemisphe
ric symptoms most commonly consist of tr nsient dysph si nd v rying degrees of
hemip resis or hemisensory distur
nce, either singly or in com
in tion.
Acute stroke
It is estim ted th t pproxim tely 20 million strokes occur round the world e c
h ye r, with 5 million de ths. M ny stroke p tients re left with signi c nt neuro
logic l dis
ility th t c n thre ten their independence,
ility to work nd qu
lity of life. Better cute stroke tre tments re improving this dism l picture.
Verte
ro
sil r TIAs The spectrum of tr nsient isch emic tt cks nd stroke
Tr nsient isch emic tt cks (TIAs) nd completed cere
r l inf rcts re c used
y
simil r p thologic l mech nisms, most commonly l rge vessel therosclerotic dis
e se, c rdioem
olism nd sm ll vessel l cun r dise se. P tients with TIAs nd co
mpleted inf rcts, whether l rge or sm ll, h ve simil r prognosis, with 510-fo
ld nnu l incre se in stroke risk. Both conditions should
e reg rded s medic l
emergencies. While TIAs
y convention l st less th n 24 hours, most l st only m
inutes. The m jority of TIAs l sting more th n 12 hours produce tissue d m ge on
sensitive
r in im ging techniques, such s m gnetic reson nce im ging. The old
24hour de nition is therefore incre singly criticized nd is not clinic lly useful
. The recognition of p tients with minor isch emic de cits presents vit l opport
unity for prevention of m jor stroke. P tients with TIAs should
e urgently ev l
u ted within 24 hours of the episode. Investig tions would typic lly include C
T or MR sc n These re often more complex th n c rotid territory events nd usu
lly include two or more of the following symptoms:
inocul r visu l distur
nce
vertigo diplopi t xi
il ter l we kness or p r esthesi e de fness tinnitus m
nesi . These symptoms re produced
y tr nsient isch emi of the
r instem, occi
pit l nd medic l tempor l lo
es nd upper spin l cord.
Cl ssi c tion nd p thogenesis of stroke
Cere
r l inf rction (isch emic stroke)
Cere
r l inf rction ccounts for pproxim tely 80% of stroke p tients nd m y
e
cl ssi ed ccording to n tomic l loc tion or p thogenesis. It
146
CHAPTER 10
is useful to incorpor te
oth cl ssi c tions when considering stroke in p rticul

 r p tient.
An tomic l cl ssi c tion (T
le 10.3) The n tomic l loc tion refers to the speci c
rteri l territory (e.g. intern l c rotid vs. verte
ro
sil r, nterior cere
r l
vs. middle cere
r l) or speci c loc tion within the
r in (e.g. l ter l medull ry
syndrome, ventr l pontine inf rction or intern l c psul r inf rction). Inf rcti
on most commonly occurs in the middle cere
r l rteri l territory nd c n
e cl
ssi ed s cortic l or deep (su
cortic l). The cortic l middle cere
r l syndromes d
epend on whether sm ll
r nch h s
een occluded, or whether one or
oth of the
m in two divisions of the middle cere
r l rtery is involved, the superior or i
nferior division. Su
cortic l inf rcts occur in the territory of the deep perfor
ting vessels supplying the intern l c psule, th l mus,
s l g ngli nd
r ins
tem. The occlusion of single perfor ting vessel produces sm ll deep inf rct,
less th n 1.5 cm in di meter, c lled l cun r inf rct, p rticul rly if ssoci
ted with one of the ve cl ssic l clinic l
syndromes (see
elow). The o
struction of the origins of sever l of the deep per
for ting
r nches c n produce l rger su
cortic l inf rct termed stri toc psu
l r inf rct.
P thogenetic cl ssi c tion Gre ter emph sis is now pl ced on the p thogenesis of c
ere
r l inf rction, s this is useful for the selection of second ry prevention
ther pies. This cl ssi c tion is often referred to s the TOAST system, fter the
TOAST tri l. 1 L rge rtery therosclerosis. 2 C rdiogenic em
olism. 3 L cun r i
nf rction. 4 R re c uses (e.g. dissection, v sculitis, prothrom
otic st tes). 5
Uncl ssi ed: despite dequ te investig tion due to in dequ te investig tion.
L rge rtery therosclerosis (Figs 10.110.3) The development of extr cr ni l the
rosclerotic pl que produces progressive rteri l stenosis. Su
sequent pl que c
omplic tions include ulcer T
le 10.3 An tomic l stroke syndromes. Arteri l territory Intern l c rotid rte
ry Middle cere
r l rtery occlusion Stroke syndrome M y
e symptom tic. Mixture
of middle nd nterior cere
r l rtery syndromes Contr l ter l hemiplegi ( rm
often more ffected th n leg), hemi n esthesi , homonymous hemi nopi , ph si ,
in ttention, cortic l sensory loss Hemip resis, chie y in the leg Homonymous hemi
nopi , disconnection syndromes, hemi n esthesi , mnesi , mid
r in nd th l mic
syndromes Qu drip resis,
ul
r p r lysis, imp ired g ze, cortic l
lindness, co
m Qu drip resis,
ul
r p r lysis,
sent horizont l
ut ret ined vertic l g ze
. Norm l conscious st te;
le to
link to comm nd (locked in syndrome) Ipsil ter
l t xi , Horners syndrome, nyst gmus, f ci l num
ness, 9th nd 10th nerve p lsie
s, contr l ter l spinoth l mic loss
Anterior cere
r l rtery occlusion Posterior cere
r l occlusion Verte
ro
sil r
throm
osis (
sil r occlusion) Ventr l pontine inf rction
L ter l medull ry syndrome
STROKE
147
tion, intr pl que h emorrh ge nd superimposed pl telet
rin throm
us form tion. St
roke is most often due to the development of throm
us (Fig. 10.4) followed
y pr
op g tion nd dist l throm
oem
olism into the intr cr ni l vessels, sometimes em

olism composed of therom tous de
ris, or h emodyn mic f ilure due to the reduc
tion of cere
r l perfusion in the rteri l
order zones (
orderzone or w tershed
inf rction). Prim ry intr cr ni l therosclerosis nd therothrom
osis is r re
in C uc si n popul tions,
ut more common in Afric n, Afric nAmeric n nd Asi n
stroke p tients (Fig. 10.5). Clinic l fe tures include demonstr tion of relev nt
rteri l p thology with 50% or gre ter

stenosis nd the
sence of c rdi c source. Anterior circul tion inf rcts typi
c lly involve the cere
r l cortex. Prodrom l TIAs in the s me rteri l territory
re nother pointer. C rdiogenic em
olism (Fig. 10.6) A v riety of c rdi c dise
ses ffecting the c rdi c w lls, v lves or ch m
ers c n le d to cere
r l em
oli
sm. These include: non-v lvul r tri l
rill tion (the most common) v lvul r he r
t dise se myoc rdi l inf rction with ventricul r throm
us form tion post-c rdi c
surgery (v lvul r surgery or coron ry rtery
yp ss gr fts) prosthetic c rdi c
v lves infective endoc rditis tri l myxom c rdiomyop thy sept l defect with p
r doxic l em
olism. Clinic l fe tures include deline tion of c rdi c source n
d l ck of evidence of l rge rtery dise se. Simil rly, the cere
r l cortex is us
u lly involved. L cun r inf rction (Fig. 10.7) The occlusion of single deep perf
or ting rteries supplying the intern l c psule,
s l g ngli or
r instem c n
le d to the development of sm ll l cun r inf rcts. These re most commonly the r
esult of hypertensive dise se, which produces loc lized rteri l w ll p thology,
termed lipohy linosis, in these sm ll penetr ting rteries, or loc lized micro
therom . L cun r inf rcts re
Fig. 10.2 C rotid ultr sound of the c rotid
ifurc tion, demonstr ting n ulcer
ted stenotic pl que ( rrows) t the common c rotid
ifurc tion.
Fig. 10.3 Diffusion-weighted im ging sc n (left im ge) showing l rge cute middl
e cere
r l rtery (MCA) cortic l inf rct. M gnetic reson nce ngiogr phy (right
im ge) shows l ck of ow in the left MCA.
148
CHAPTER 10
Fig. 10.6 Echoc rdiogr m demonstr ting clot (CL) in left ventricle (LV).
Fig. 10.4 Intern l c rotid therothrom
osis from utopsy specimen. Fig. 10.7 Dif
fusion-weighted im ging sc n showing cute left th l mic l cun r inf rct (hyperi
ntense lesion).
less often due to em
olism from proxim l source such s extr cr ni l theroscl
erosis or intr c rdi c throm
us. Cl ssic l l cun r syndromes include pure motor
hemip resis, pure sensory stroke, sensorimotor stroke, t xic hemip resis nd th
e dys rthri /clumsy h nd syndrome. Clinic l pointers include clinic l di gnosis
of one of the cl ssic l syndromes, usu lly exclusion of l rge rtery or c rdi
c source nd ide lly neuroim ging con rm tion of sm ll, deep inf rct.
Fig. 10.5 M gnetic reson nce ngiogr phy demonstr ting severe dist l verte
r l/p
roxim l
sil r rtery stenosis ( rrow).
Oxfordshire cl ssi c tion Another commonly used stroke cl ssi c tion, termed the Oxf
ordshire cl ssi c tion, divides isch emic stroke into TACI (tot l nterior circul
tion inf rction) (Fig. 10.8), PACI (p rti l nterior circul tion inf rction), PO
CI (posterior circul tion inf rction) nd LACI (l cun r inf rction).
STROKE
149
Fig. 10.8 CT sc n showing severe left middle cere
r l cortic l inf rction. Note
sp ring of nterior nd posterior cere
r l rteri l territories. Fig. 10.9 CT sc
n showing hypertensive put min l h emorrh ge.
This cl ssi c tion is lso useful nd the su
types correl te with prognosis.
Cere
r l h emorrh ge (h emorrh gic stroke)

Cere
r l h emorrh ge is gener lly cl ssi ed into intr cere
r l nd su
r chnoid h
emorrh ge.
Intr cere
r l h emorrh ge (Figs 10.910.11) Non-tr um tic (prim ry) intr cere
r l
h emorrh ge is most commonly due to hypertension, which le ds to rupture of deep
perfor ting rteries in the put men, th l mus, centr l white m tter,
r instem
nd cere
ellum. The precise mech nism of this v scul r rupture is uncert in,
ut
m y
e rel ted to the development of sm ll Ch rcotBouch rd micro neurysms on the
vessel w lls of these end- rteries, or direct rupture of vessels ffected
y li
pohy linosis. Lo
r h emorrh ge refers to super ci l v scul r rupture within the cer
e
r l lo
es, outside these deep rteri l territories. It is sometimes due to n
underlying structur l lesion, such s n rteriovenous m lform tion, cere
r l n
eurysm, tumour, v sculop thies or co gul tion disorders. Amyloid
Fig. 10.10 CT sc n showing front l nd occipit l lo
r h emorrh ges in p tient
with myloid ngiop thy.
150
CHAPTER 10
identi ed t ngiogr phy. Some of these idiop thic
leeds re due to perimesenceph
lic h emorrh ge.
Modern principles of cute stroke m n gement
In Austr li , the USA nd Europe, guidelines for cute stroke m n gement h ve
e
en developed,
sed on the evidence from l rge controlled clinic l tri ls. Key p
rinciples of cute stroke m n gement include: 1 Urgent recognition of stroke sym
ptoms
y the p tient or their c rer. 2 Urgent m
ul nce tr nsport to hospit l
with dequ te di gnostic f cilities nd org nized stroke c re. 3 Urgent tri ge
nd investig tion in the emergency dep rtment including CT
r in sc nning. 4 Asse
ssment for cute stroke ther py, p rticul rly throm
olysis. 5 Admission to spe
ci lized stroke unit.
Fig. 10.11 CT sc n showing right cere
ell r h emorrh ge. The
r instem nd 4th v
entricle re compressed.
Hospit l rriv l times: Time is Br in
ngiop thy is n import nt c use of lo
r cere
r l h emorrh ge in elderly p tien
ts nd is due to myloid deposition in the w lls of the cere
r l rteries. These
h emorrh ges m y
e multiple nd typic lly occur in p tients who re normotensi
ve nd m y show fe tures of Alzheimers dise se. Clinic l clues to ICH include the
fe tures of sudden rise in intr cr ni l pressure with depressed conscious st
te, he d che nd vomiting. The mort lity is much higher th n in isch emic stroke
. However, p tients with ICH m y
e surprisingly lert nd well looking. Convers
ely, p tients with isch emic stroke m y h ve e rly depression of conscious st te
. Hence, neuroim ging, usu lly with CT, is m nd tory in ll c ses to r pidly di
gnose ICH. Del yed hospit l rriv l times re worldwide pro
lem. New ther pies
such s tPA (see
elow) re time-dependent. In m ny centres, only qu rter of
stroke p tients rrive within 3 hours. Much of this del y is due to pu
lic ignor
nce nd confusion
out the n ture of stroke nd the need for urgent ev lu tion
nd tre tment. However, profession l nihilism is nother
rrier.
Clinic l ssessment of stroke
The following questions should
e considered in the m n gement of ny p tient wi
th presumed stroke. Is it stroke? Is it n inf rct or h emorrh ge? Is the p
tient eligi
le for throm
olytic ther py or other urgent intervention? Wh t is th
e rteri l or n tomic l loc liz tion nd p thogenesis?
Su
r chnoid h emorrh ge (see Ch pter 9) Su
r chnoid h emorrh ge is cl ssi ed cc

ording to p thologic l c use nd site. The two most import nt identi
le c uses i
nclude rupture of
erry neurysm nd rteriovenous m lform tion,
ut in up to
15% of c ses no
leeding c n
e
Stroke nd pseudostroke Non-v scul r p thologies (pseudostroke) such
STROKE
151

s cere
r l tumour, su
dur l h em tom ,
scess, migr ine, met
olic distur
nce
s nd epilepsy c n mimic the stroke process. All p tients with suspected stroke
require n urgent CT or MR sc n to exclude non-cere
rov scul r disorders, s wel
l s to differenti te
etween inf rct nd h emorrh ge (Figs 10.8 nd 10.9). Lum

r puncture is reserved for those c ses where meningitis is considered (usu lly
fter CT sc n) or where the di gnosis of su
r chnoid h emorrh ge is still con
templ ted fter norm l CT sc n.
The distinction
etween inf rct nd h emorrh ge The distinction
etween cere
r l
h emorrh ge nd inf rction is vit l, s some h emorrh ges re considered for su
rgic l ev cu tion, while p tients with isch emic stroke m y
e considered for th
rom
olysis or ntico gul tion. While there re clinic l pointers (see
ove) thi
s differenti tion is usu lly
sed on CT sc n ndings. Although CT sc nning rem in
s the workhorse for cute stroke ssessment, recent studies indic te th t MRI is
t le st s sensitive for intr cere
r l h emorrh ge s CT nd f r more sensitiv
e for cute isch emi . P tients with isch emic stroke often h ve e rly inf rct c
h nges on CT, lthough these m y
e su
tle. MRI with diffusionweighted im ging (
DWI) is incre singly used s this technique llows sensitive di gnosis of cere

r l isch emi (Figs 10.3 nd 10.7). Is the p tient eligi
le for throm
olysis? T
he throm
olytic gent tissue pl sminogen ctiv tor (tPA) h s now
een licensed i
n most p rts of the world s the rst proven stroke drug ther py, given intr venou
sly within 3 hours of stroke onset in selected p tients with isch emic stroke. T
he pprov l of this cute ther py followed the positive results of two-p rt pi
vot l tri l, conducted in the USA. Other Europe n tri ls testing tPA up to 6 hou
rs h ve shown m rked trend to
ene t over risk, nd met - n lysis con rms the 3-ho
ur window for tPA. Use of tPA incre ses the risk of symptom tic h emorrh gic com
plic tions
y three- to fourfold. M jor e rly inf rct ch nges on CT, for ex mple
gre ter th n one-third of the

re of the middle cere
r l rtery, re ssoci ted with higher risk of h emorr
h gic tr nsform tion. Up to 10% of isch emic stroke p tients c n
e tre ted in w
ell-org nized centres. In contr st to the tPA tri ls, three tri ls ev lu ting th
e role of intr venous streptokin se produced neg tive results, chie y linked to th
e su
st nti l risk of intr cere
r l h emorrh ge ssoci ted with the drug. Direct
infusion of throm
olytics vi intr - rteri l c theters h s
een shown to
e eff
ective in one tri l up to 6 hours fter middle cere
r l rtery occlusion. There
is interest in experiment l mech nic l devices which c n
re k up throm
i, with
much lower risk of cere
r l h emorrh ge.
Loc tion nd p thogenesis of inf rction Cortic l inf rcts (Fig. 10.3). B sed on
the clinic l ex min tion, distinction should
e m de
etween n inf rction in
the c rotid or in the verte
ro
sil r territory. With reg rd to c rotid territor
y inf rction, the presence or
sence of cortic l signs dysph si , pr xi , nos
ognosi (un w reness or deni l of the stroke), sensory, motor or visu l gnosi
(in ttention), c lculi , right/left confusion, dysgr phi or cortic l sensory l
oss (loss of two-point discrimin tion, stereognosis, dysgr ph esthesi ) suggest
s n em
olic source from either the extr cr ni l vessels or the he rt, r ther th
n l cun r inf rction. Su
cortic l inf rcts (Fig. 10.7). As indic ted e rlier, l
cun r inf rcts (less th n 1.5 cm in di meter) rely on di gnosis of one of the c
l ssic l l cun r syndromes. Cortic l signs re not present. L cun r syndromes re e

ct sm ll vessel occlusions in the intern l c psule, th l mus nd
r instem. H em

orrh ge Intr cere
r l h emorrh ge (Fig. 10.9). As previously discussed, the r pi
d onset of stroke with e rly depression of conscious st te f vours the di gnos
is of prim ry intr cere
r l h emorrh ge. Prim ry intr cere
r l h emorrh ge nd
neurysm l su
r chnoid h emorrh ge c n overl p in their clinic l present tions
. For ex mple,
erry neurysm c n rupture prim rily into the
r in p renchym ,
presenting s n intr cere
r l h emorrh ge, where s prim ry
r in
152
CHAPTER 10
h emorrh ge c n rupture directly into the ventricul r system nd present with m
rked meninge l fe tures due to su
r chnoid
lood. For su
r chnoid h emorrh ge
see Ch pter 9.
Urgent ssessment in the emergency dep rtment
R pid tri ge of suspected stroke p tients should
e performed in n emergency de
p rtment, with skilled medic l ev lu tion nd urgent investig tions. These invol
ve
lood tests, including
lood glucose me surement to exclude hypoglyc emi (
which c n mimic cute stroke), nd n ECG to di gnose tri l
rill tion or cute
myoc rdi l inf rction, which c n reve l the c use of cere
r l em
olism. Most imp
ort ntly, CT
r in sc n must
e performed urgently in ll p tients to differen
ti te
etween cere
r l inf rction nd cere
r l h emorrh ge. In ddition, CT sc n
ning is essenti l to exclude the other
r in p thologies th t c n simul te strok
e. As noted
ove, m ny centres re now using cute MRI with DWI nd m gnetic re
son nce ngiogr phy (MRA). Computed tomogr phic ngiogr phy (CTA) is nother use
ful tool. Duplex Doppler sc nning is of v lue in the di gnosis of l rge vessel
therosclerosis, while echoc rdiogr phy (usu lly less urgent) c n id in the di g
nosis of intr c rdi c throm
i nd v lvul r or c rdi c w ll kinetic
norm lities
. Tr nsoesoph ge l echoc rdiogr phy is superior to tr nsthor cic echoc rdiogr ph
y.
system tic overview of the
ene ts of stroke units, it w s shown th t mort lity co
uld
e reduced
y
out 25%, lso with evidence of reduced dis
ility. In dditi
on, stroke units h ve
een found to reduce
ed st y nd hence hospit l costs. Th
e components of te m m n gement in the stroke unit include: 1 Acute medic l, sur
gic l nd nursing c re. 2 Di gnosis of the p thologic l mech nism of the stroke
nd the institution of t ilored second ry prevention str tegies. 3 Prevention, e
rly detection nd tre tment of complic tions such s spir tion pneumoni , pres
sure sores, hyperglyc emi , seizures nd sepsis. 4 Use of me surement instrument
s nd stroke registers. 5 E rly integr ted neuroreh
ilit tion with ev lu tion o
f premor
id st tus nd ther py go ls. 6 Involvement, educ tion, nd support for
p tient nd f mily. 7 E rly, coordin ted disch rge pl nning.
Acute medic l c re
Three key, evidence-
sed dv nces include the use of tPA, spirin (see
elow)
nd stroke unit c re. In the cute ph se, close monitoring of vit l nd neurologi
c l signs is p r mount, given th t
out third of stroke p tients deterior te
fter dmission to hospit l. C rdi c monitoring is useful for m ny cute stroke
p tients. Initi l clinic l ev lu tion should involve ssessment of the p tients f
unction nd clinic l st te
efore the stroke, the stroke type nd p thogenesis,
document tion of the n ture nd severity of neurologic l de cits, nd comor
id dis
e ses. E rly mo
iliz tion, r nge of motion exercises for hemiplegic lim
s, frequ
ent turning, uid nd nutrition l m inten nce, dysph si m n gement, void nce of
spir tion, prevention of deep venous throm
osis (DVT) nd pneumoni , m n gement
of incontinence, tre tment of urin ry tr ct infections nd other c uses of feve
r, nd m inten nce of skin integrity re ll key pl nks in the te m tre tment of
the cute stroke p tient.

Acute stroke units

One of the most import nt developments in cute stroke m n gement in recent ye r
s h s
een the proven v lue of stroke units,
sed on the results of clinic l tr
i ls which h ve comp red org nized expert c re in speci l unit to the m n geme
nt of p tients in gener l medic l w rds. The usu l stroke unit model involves
geogr phic l re in the hospit l, incorpor ting skilled multidisciplin ry str
oke te m, led
y neurologist or other physici n with expertise in stroke m n g
ement. V scul r surgeons nd neurosurgeons should
e re dily v il
le for consu
lt tion nd intervention in selected c ses. In
STROKE
153
P tients should h ve oximetry,
ut there is no evidence of the v lue of routine
supplement l oxygen
y m sk or n s l prongs. P tients should
e well hydr ted us
ing norm l s line r ther th n glucose-cont ining solutions,
ec use of the h z r
ds of even mild degrees of hyperglyc emi . In gener l,
lood pressure should not

e cutely lowered,
ec use this c n compromise cere
r l perfusion in cute str
oke. Hypertension is usu l in cute stroke nd this usu lly settles spont neousl
y over 23 d ys. We routinely use compression stockings nd usu lly low-dose hep r
in, or low molecul r weight hep rin or hep rinoid, in DVT prophyl xis. Airw y s
sessment should
e n urgent priority to void spir tion pneumoni . We gener ll
y use n sog stric feeding fter 2448 hours if the p tients
ul
r function is comp
romised. Adverse prognostic f ctors include dv ncing ge, depression of conscio
us st te, severity of neurologic l de cit, conjug te g ze p lsy nd e rly urin ry
incontinence. C uses of mort lity in stroke p tients re predomin ntly neurologi
c l (tr nstentori l herni tion) in the 1st week, nd due to second ry systemic f
ctors in the 2nd nd 3rd weeks such s pneumoni , pulmon ry em
olism nd c rdi
c c uses.
tion l Stroke Tri l (IST) ev lu ted unmonitored, su
cut neous hep rin up to 48 h
ours fter stroke onset nd showed th t slight reduction in recurrent stroke w
s offset
y n incre sed risk of h emorrh gic tr nsform tion. The r te of e rly
, recurrent em
olism in p tients with tri l fi
rill tion w s much lower in rece
nt tri ls th n in num
er of e rlier clinic l studies. System tic overview of t
he v il
le evidence le ds to the conclusion th t hep rin is not gener lly indi
c ted for most p tients with cute isch emic stroke,
ut hep rin should
e consi
dered for selected p tients with nondis
ling isch emic stroke nd very high r
isk of recurrent em
olism.
Acute spirin
Two l rge tri ls (the Intern tion l Stroke Tri l nd the Chinese Acute Stroke Tr
i l)
oth showed th t, s for cute myoc rdi l inf rction, spirin dministered
within 48 hours of stroke onset produced modest improvement in outcomes t 6 m
onths. Hence, spirin should
e used routinely in cute isch emic stroke, unless
throm
olysis is
eing used.
Progressing stroke
A deterior ting neurologic l de cit is seen in
out one-third of stroke p tients.
Cere
r l oedem , with progressive elev tion of intr cr ni l pressure, is n imp
ort nt c use of de th. However, this oedem is gener lly cytotoxic nd clinic l
tri ls h ve demonstr ted th t corticosteroids re of no v lue in either cere
r l
inf rction or h emorrh ge. Intr venous m nnitol nd glycerol re sometimes empi
ric lly used,
ut their v lue h s not
een proven. In highly selected young p ti
ents with severe, e rly
r in oedem , surgic l decompression with hemicr niectom
y should
e considered.
Neuroprotective ther pies
A complex c sc de of
iochemic l ch nges occurs in stroke, second ry to the init

i l isch emi . Isch emic
r in injury is ssoci ted with elev ted levels of the
excit tory neurotr nsmitters glut m te nd sp rt te. These le d to excessive st
imul tion of the N-methyl D- sp rt te (NMDA) receptor on the cell surf ce. This
ctiv tion is followed initi lly
y n in ux of sodium nd w ter into the cells n
d secondly
y sudden rise in intr cellul r c lcium. A r nge of neuroprotective
compounds h s
een designed to inhi
it v rious points in the excitotoxic c sc d
e. These include c lcium ch nnel nd NMDA nt gonists, glut m te rele se inhi
it
ors, glycine nt gonists, free r dic l sc vengers, inhi
itors of the neutrophil
in ux into the isch emic region nd v rious growth f ctors. To d te, none of these
compounds h ve proven effective in dequ tely
Hep rin
Hep rin h s
een the most widely used unproven ther py in most countries. The In
tern 154
CHAPTER 10
powered, Ph se III clinic l tri ls,
ut there re sever l ongoing studies. Other
ppro ches include the com
in tion of throm
olysis with neuroprotective drugs.
Young dult nd r rer c uses of stroke
Stroke in young dults is due to wide spectrum of c uses. Those presenting wit
h cere
r l h emorrh ge usu lly h ve n underlying lesion such s n neurysm or
v scul r m lform tion. P tients presenting with cere
r l inf rction h ve r nge
of p thologies somewh t different to those in older ge groups, including: migr
ine or l contr ceptive pill mitr l v lve prol pse cere
r l v sculitis extr cr n
i l rteri l dissection
romuscul r dyspl si moy -moy dise se hyperco gul
ilit
y st tes. Consequently, these p tients require more intensive investig tion th n
m ny older stroke p tients. Some form of cere
r l ngiogr phy nd tr nsoesoph g
e l echoc rdiogr phy re virtu lly m nd tory in ll c ses. M ny p tients will l
so require lum
r puncture, to look for evidence of n underlying in mm tory co
ndition, nd det iled h em tologic l investig tions directed t di gnosis of h
yperco gul
ility st te.
Cere
r l hemorrh ge
While routine surgic l ev cu tion of h em tom is unproven, we consider ev cu ti
on in selected p tients with cere
r l h emorrh ge, p rticul rly in the cere
ellu
m, s well s younger p tients with lo
r h emorrh ge. The gener l principles of
cute stroke m n gement pply equ lly to intr cere
r l h emorrh ge s to inf rc
tion.
Prevention of recurrent stroke second ry stroke prevention
P tients with symptom tic high-gr de c rotid stenosis should
e considered for s
u
cute c rotid end rterectomy. While surgery is w ited, or if end rterectomy i
s in ppropri te, ntipl telet ther py should
e instituted. In c rdioem
olic str
oke, there is uncert inty s to the optim l timing of ntico gul tion, p rticul
rly in p tients with tri l
rill tion. Our ppro ch is to employ hep rin cutely
if the p tient h s mild de cit nd there is high risk of recurrent em
olism.
M ny p tients with tri l
rill tion re commenced on w rf rin 710 d ys fter onse
t, without prior hep rin,
ec use of the risk of h emorrh gic tr nsform tion. L
cun r inf rcts re less commonly em
olic nd gener lly h ve good prognosis.
Migr ine
Migr ine is well-recognized c use of stroke in young dults,
ut the precise m
ech nism of inf rction is uncle r. Although v sosp sm is usu lly postul ted, it
h s only r rely
een demonstr ted on cere
r l ngiogr phy in p tients with migr
ine nd stroke. The di gnosis of migr ine s the c use of inf rction should only

e m de in migr ineur who h s persisting neurologic l de cit, in the w ke of

 cl ssic l tt ck, nd where other c uses h ve
een excluded
y det iled invest
ig tions, including ngiogr phy nd echoc rdiogr phy.
Post-stroke reh
ilit tion
While
eyond the scope of this review, reh
ilit tion fter stroke is of immense
import nce. M ny models of c re h ve
een developed, including home-
sed c re
s well s the more common inp tient nd outp tient models. Incre singly, comp r
tive tri ls of different ther peutic protocols re
eing conducted to provide
more r tion l evidence-
sed ppro ch to post-stroke reh
ilit tion. In optim l
stroke c re, the reh
ilit tion te m should
e integr ted into the cute stroke
unit.
Or l contr ceptive pill
Or l contr ceptive gents, p rticul rly the higher-dos ge oestrogen-cont ining f
orms which c n incre se
lood co gul
ility, h ve
een linked with stroke. Howev
er, the rel tive risk of
STROKE
155
the or l contr ceptive pill is pro

ly very sm ll, nd should not
e ssumed to

e the c use of young dult stroke without exclusion of other c uses.
C rdi c c uses
Mitr l v lve prol pse is common echoc rdiogr phic nding in young women,
ut ls
o gures prominently in clinic l series of young dult stroke p tients. Another c
rdi c em
olic source in young dults is p tent for men ov le (PFO) with p r doxi
c l em
olism from the venous circul tion. The investig tion of young dult strok
e p tients with tr nsoesoph ge l echoc rdiogr phy (TOE) not infrequently shows P
FO t higher r te th n those seen in control popul tion,
ut their precise s
igni c nce nd ther peutic implic tions re often uncert in. There is n incre sed
risk when PFO is ssoci ted with tri l sept l neurysm. There re current tri
ls ev lu ting endov scul r devices to close these lesions.
tients with extr cr ni l rtery in mm tion, he d che, systemic symptoms nd n el
ev ted erythrocyte sediment tion r te. The chief complic tion is isch emic optic
neurop thy due to inf rction of the optic nerve,
ut cere
r l inf rction occ si
on lly occurs due to involvement of the verte
r l rteries. Ophth lmic herpes zo
ster c n
e followed
y middle cere
r l rteritis nd inf rction.
Dissection of the extr cr ni l nd intr cr ni l rteries
Dissections re often due to tr um , lthough the preceding injury m y
e extrem
ely mild. Recognized c uses include motor c r ccidents with torsion l neck or s
e t
elt injuries, nd cervic l m nipul tion. Spont neous dissections lso occur
, some of these c ses h ving n underlying rteriop thy such s
romuscul r dyspl
si . C rotid rtery dissection m y
e ssoci ted with ocul r p in nd Horners sy
ndrome. Neurologic l de cits m y follow due to intim l throm
us superimposed on th
e ruptured lining of the rtery nd dist l throm
oem
olism. The l ter l medull r
y syndrome is common clinic l present tion of verte
r l rtery dissection. Di
gnosis is m de
y ngiogr phy or MRA, which shows n rrowed or t pered rtery,
the string sign, sometimes with the form tion of n rteri l neurysm. MRA m y sho
w intr mur l throm
us. Antico gul tion is often used to prevent su
sequent throm

oem
olic events. Intr cr ni l rteri l dissections re much r rer, nd c n pres
ent with
r in isch emi or su
r chnoid h emorrh ge.
Cere
r l v sculitis
Cere
r l v sculitis used to
e commonly seen in p tients with n underlying
s
l meningitis due to tu
erculosis or syphilis
ut it is now seen m inly with sep
tic, in mm tory conditions. These include multisystem disorders such s poly rter

itis nodos . Isol ted centr l ngiitis (gr nulom tous ngiitis) is n septic v
sculitis ssoci ted with multifoc l cere
r l inf rcts nd high mort lity. The
c use is unknown. As with the other types of intr cr ni l rteritis, some p tien
ts h ve
e ding of rteries on ngiogr phy, n excess of cere
rospin l uid lymphocy
tes nd n elev ted erythrocyte sediment tion r te. De nitive di gnosis often depe
nds on
r in
iopsy,
ut neg tive
iopsy does not exclude the condition. Highdose steroids re used, sometimes in com
in tion with other immunosuppressive g
ents, p rticul rly cyclophosph mide. Cere
r l rteritis c n lso
e c used
y il
licit drug use with heroin, or l or intr venous mphet mines, coc ine nd other
gents. Gi nt cell rteritis (tempor l rteritis) is seen in older p Fi
romuscul r dyspl si
This rteriop thy chie y ffects fem les nd most commonly involves the dist l por
tions of the extr cr ni l c rotid rtery. It m y
e ssoci ted with ren l
romusc
ul r dyspl si . It is usu lly symptom tic,
ut is ssoci ted with n incre sed
risk of
oth TIAs nd cere
r l inf rction. There is lso n incre sed risk of su

r chnoid h emorrh ge due to ssoci ted
erry neurysms. The isch emic events
re pro

ly
156
CHAPTER 10
Fig. 10.12 Cere
r l ngiogr phy demonstr ting moy -moy dise se.
Fig. 10.13 S gitt l MRI sc n showing fe tures of cere
r l venous throm
osis, wit
h high sign l ( rrows) due to
lood clot in the superior s gitt l sinus.
due to throm
oem
olism nd occ sion lly rteri l dissection. Angiogr phy demonst
r tes cl ssic s w tooth ppe r nce. Tre tment is usu lly conserv tive with sp
irin.
Cere
r l venous throm
osis
Cere
r l venous throm
osis is r re type of stroke with v ri
le clinic l m nif
est tions,
ut incre singly recognized with the widespre d use of MRI. Septic n
d septic syndromes re recognized. Septic throm
osis is now very r re. It most
commonly involves the c vernous sinus
ut c n lso ffect the superior s gitt l
nd l ter l sinuses. Sources of infection include the f ce, p r n s l sinuses, m
iddle e r infection nd
cteri l meningitis. Aseptic cere
r l venous throm
osis
is most commonly seen in conditions ssoci ted with hyperco gul
ility st tes s
uch s the postp rtum period, presence of the lupus ntico gul nt or the or l co
ntr ceptive pill. Clinic l fe tures r nge from the insidious development of he d
che nd p pilloedem , to more fulmin nt foc l syndromes in the c vernous sinus
region, hemiplegi , depressed conscious st te, fever, seizures, sinus t chyc rdi
nd meningismus. (Fig. 10.13). Ther py in septic c ses is directed g inst the
c us tive infection. Tre tment in septic c ses involves the use of e rly ntic
o gul tion, which h s
een shown to improve the outcome. Tr nsdur l throm
olysis
is lso used in selected c ses, p rticul rly when the p tients condition worsens
, despite hep rin.
Moy -moy dise se (Fig. 10.12)
Moy -moy dise se is r re o
liter tive rteri l condition where the termin l i
ntern l c rotid rteries re occluded nd there is ne, tel ngiect tic we
of n
stomotic, intr cr ni l vessels which produces the cl ssic ngiogr phic puff of s
moke ppe r nce (Fig. 10.12). The posterior circul tion is usu lly sp red. It is
ssoci ted with n incre sed risk of either cere
r l inf rction due to
r in isc
h emi or h emorrh ge due to rupture of the
norm l tel ngiect tic vessels. The
c use of the condition is unknown nd medic l ther py is ineffective. Surgic l
rev scul riz tion procedures constitute the m inst y of ther py.

Hyperco gul
ility st tes

Disorders of
lood h emost sis nd co gul tion re sometimes recognized s the c
use of stroke in young dults. V rious r re
norm lities of the co gul tion pr
ocess h ve
een identi ed in some stroke p tients. These include the presence of t
he lupus ntico gul nt, which is n ntiphospholipid nti
ody, ctiv ted protein
C resist nce, nd de ciencies of protein S nd C.
STROKE
157
Further re ding
Al
ers GW, C pl n LR, E ston JD, F y d PB, Mohr JP, S ver JL, Sherm n DG; TIA Wo
rking Group (2002) Tr nsient ischemic tt ck propos l for new de nition. New Eng
l nd Journ l of Medicine 347, 17131716. Antipl telet Tri lists Coll
or tion (199
4) Coll
or tive overview of r ndomised tri ls of ntipl telet tre tment, P rt 1
. Prevention of de th, myoc rdi l inf rction, nd stroke
y prolonged ntipl tel
et ther py in v rious c tegories of p tients. British Medic l Journ l 343, 139142
. Atri l Fi
rill tion Investig tors (1994) Risk f ctors for stroke nd ef c cy of
ntithrom
otic ther py in tri l
rill tion. An lysis of pooled d t from ve r ndo
mized controlled tri ls. Archives of Intern l Medicine 154, 14491457. Blood Press
ure Lowering Tre tment Tri lists Coll
or tion (2003) Effects of different
loodpressure-lowering regimens on m jor c rdiov scul r events: results of prospectiv
ely designed overviews of r ndomised tri ls. L ncet 362, 15271535. Bucher HC, Gri
f th LE, Guy tt GH (1998) Effect of HMG-CoA reduct se inhi
itors on stroke. A met
n lysis of r ndomized controlled tri ls. Ann ls of Intern l Medicine 128, 8995.
CAPRIE Steering Committee (1996) A r ndomised,
linded, tri l of clopidogrel vs.
spirin in p tients t risk of ischemic events (CAPRIE). L ncet 348, 13291339. C
AVATAS Investig tors (2001) Endov scul r vs. surgic l tre tment in p tients with
c rotid stenosis in the C rotid nd Verte
r l Artery Tr nslumin l Angiopl sty S
tudy (CAVATAS): r ndomised tri l. L ncet 357, 17291737. Diener H, Cunh L, For

es C, Sivenius J et l. (1996) Europe n Stroke Prevention Study 2. Dipyrid mole
nd cetyls licylic cid in the second ry prevention of stroke. Journ l of Neuro
logic l Science 143, 113. Europe n C rotid Surgery Tri lists Coll
or tive (ECST)
Group (1991) MRC Europe n C rotid Surgery Tri l: interim results for symptom tic
p tients with severe (7099%) or with mild (020%) c rotid stenosis. L ncet 337, 12
351243. Executive Committee for the Asymptom tic C rotid Atherosclerosis Study (1
995) End rterectomy for symptom tic c rotid rtery stenosis. Journ l of the Ame
ric n Medic l Associ tion 273, 14211428.
Furl n A, Hig shid R, Wechsler L, Gent M, Rowley H, K se C, Pessin M, Ahuj A,
C ll h n F, Cl rk WM, Silver F, River F (1999) Intr - rteri l prourokin se for
cute ischemic stroke. The PROACT II study: r ndomized controlled tri l. Proly
se in Acute Cere
r l Throm
oem
olism. Journ l of the Americ n Medic l Associ tio
n 282, 20032011. He rt Protection Study Coll
or tive Group (2002) MRC/BHF he rt
protection study of cholesterol lowering with simv st tin in 20536 high risk ind
ividu ls: r ndomized pl ce
o-controlled tri l. L ncet 360, 722. Intern tion l S
troke Tri l Coll
or tive Group (1997) The Intern tion l Stroke Tri l (IST): r
ndomised tri l of spirin, su
cut neous hep rin,
oth, or neither mong 19 435
p tients with cute ischemic stroke. L ncet 349, 15691581. M dden KP, K r nji PN
, Ad ms HP Jr, Cl rke WR (1995) Accur cy of initi l stroke su
type di gnosis in
the TOAST study. Tri l of ORG 10172 in cute stroke tre tment. Neurology 45, 197
51979. Mohr JP, Thompson JL, L z r RM, Levin B, S cco RL, Furie KL, Kistler JP, A
l
ers GW, Pettigrew LC, Ad ms HP Jr, J ckson CM, Pullicino P (2001) W rf rin-Asp
irin Recurrent Stroke Study Group. A comp rison of w rf rin nd spirin for the
prevention of recurrent ischemic stroke. New Engl nd Journ l of Medicine 345, 14
441451. The N tion l Institute of Neurologic l Disorders nd Stroke rt-PA Stroke
Study Group (1995) Tissue pl sminogen ctiv tor for cute ischemic stroke. New E
ngl nd Journ l of Medicine 333, 15811587. North Americ n Symptom tic C rotid End
rterectomy Tri l Coll
or tors (1991) Bene ci l effect of c rotid end rterectomy i

n symptom tic p tients with high gr de c rotid stenosis. New Engl nd Journ l of
Medicine 325, 445453. Stroke Unit Tri lists Coll
or tion (1997) Coll
or tive sys
tem tic review of the r ndomised tri ls of org nized inp tient (stroke unit) c r
e fter stroke. British Medic l Journ l 314, 11511159. Whisn nt JP (1997) Modelli
ng of risk f ctors for ischemic stroke: The Wills Lecture. Stroke 28, 18401844. Z
immet PZ, Al
erti KGMM (1997) The ch nging f ce of m crov scul r dise se in noninsulin-dependent di
etes mellitus: n epidemic in progress. L ncet 350, 14.
CHAPTER 11
11
Ar chnoid cyst
Development l
norm lities
There re num
er of neurosurgic l conditions th t re development l in origin
nd th t involve the cr nium, intr cr ni l contents nd spin l column. The more
import nt of these will
e descri
ed in this ch pter.
Ar chnoid cysts re
enign development l cysts th t occur long the cr niospin l
xis. Bright ( fter whom Brights dise se w s n med) w s the rst to ccur tely des
cri
e the condition in 1831. In 1964 Ro
inson descri
ed l rge series of middle
cr ni l foss r chnoid cysts nd erroneously postul ted th t the prim ry defec
t w s genesis of the tempor l lo
e; he l ter revised his opinion nd recognized
th t the cysts were r chnoid m lform tions. In 1958 St rkm n recognized th t t
he cysts were development l, were intr - r chnoid in loc tion, nd th t they resul
ted from splitting nd duplic tion of the r chnoid mem
r ne. The cysts cont in
cle r, colourless uid which resem
les norm l cere
rospin l uid; they occur in ch r
cteristic loc tions. Sylvi n ssure 50% Cere
ellopontine ngle 10% Qu drigemin l
10% Supr sell r 10% Vermi n 8% Cere
r l convexity 5% Other 7%
Sylvi n ssure The Sylvi n ssure is the most common site for r chnoid cysts nd sy
mptoms m y
ecome m nifest t ny ge. There is m rked m le predomin nce. The
most common presenting fe tures re: 1 R ised intr cr ni l pressure: ( ) he d ch
es (
) n use (c) vomiting. 2 Seizures. H emorrh ge into the cyst following mino
r he d injury, lthough uncommon, will c use sudden onset of neurologic l symp
toms nd signs due to r ised intr cr ni l pressure nd compression of the underl
ying
r in. However, s with r chnoid cysts in ny other loc tion, the cyst m y
rem in symptom tic throughout life. Cere
ellopontine ngle The clinic l fe tur
es re simil r to those of n coustic neurom , with sensorineur l he ring loss
s the most common initi l symptom. A l rge cyst m y c use minor imp irment of 5
th nerve function, with depression of the corne l re ex nd, r rely, t xi due to
cere
ell r compression. Supr sell r r chnoid cysts The m jority of cysts in th
is position present in children nd dolescents nd the clinic l m nifest tions
re due to: hydroceph lus visu l imp irment endocrine dysfunction. The hydroceph
lus results from protrusion of
Clinic l fe tures
The presenting fe tures depend on the position of the r chnoid cyst. 158
DEVELOPMENTAL ABNORMALITIES
159
the cyst into the 3rd ventricle nd occlusion of the for men of Monro. Visu l f
ilure results from compression of the optic p thw ys, s well s long-st nding r
ised intr cr ni l pressure c using optic trophy. Endocrine dysfunction m y
e
due to intr sell r extension of the cyst nd compression of the pituit ry gl nd,
or long-st nding pressure on the hypoth l mus, nd is m nifest s hypopituit ri
sm, growth ret rd tion or isosexu l precocious pu
erty.

Cere
r l convexity In dults r chnoid cysts over the convexity present with sei
zures, he d ches or progressive hemip resis. The presenting fe ture in inf nts
m y
e symmetric l enl rgement of the he d. Convexity nd Sylvi n ssure cysts s
lightly predispose the p tient to su
dur l h em tom form tion. Qu drigemin l ci
stern The cysts rising in the supr collicul r region mimic pine l m sses nd th
e most common presenting symptom is o
structive hydroceph lus with r ised intr c
r ni l pressure.
( )
R diologic l investig tions
The computerized tomogr phy sc n or m gnetic reson nce im ging will show the cys
t in the ch r cteristic position nd the uid will h ve the s me density s CSF (F
igs 11.1 nd 11.2). The
one windows on CT sc n or pl in skull X-r ys m y show r
emodelling nd erosion of dj cent
one. The Sylvi n ssure r chnoid cyst is ch r
cteristic lly ssoci ted with exp nsion of the middle cr ni l foss , elev tion
of the lesser wing of the sphenoid, nd outw rd exp nsion nd thinning of the sq
u mous portion of the tempor l
one. The supr sell r r chnoid cyst extending in
to the 3rd ventricle nd c using hydroceph lus m y
e dif cult to differenti te fr
om dil ted 3rd ventricle due to queduct stenosis. MRI, p rticul rly s gitt l
views, will help to differenti te the conditions.
(
)
Fig. 11.1 ( ) Ar chnoid cyst rising in the Sylvi n
oid cyst c using o
structive hydroceph lus.

ssure. (
) Supr sell r r chn

Tre tment
Ar chnoid cysts re frequently di gnosed s n incident l nding on CT sc n. Surge
ry is not necess ry if they re completely symptom tic, with no distortion or e
nl rgement of the ventricul r system; the p tient should
e c refully reviewed
t regul r interv ls. There re two m jor surgic l procedures for r chnoid cysts
. 1 Cr niotomy, excision of the cyst w ll nd opening of the mem
r nes to llow
dr in ge into the
s l cisterns.
160
CHAPTER 11
2 Shunting of the cyst into the peritone l c vity. The type of surgic l procedur
e will depend on the position of the cyst, the presenting fe tures nd the surge
ons preference.
Chi ri m lform tions nd syringomyeli
Chi ri m lform tions nd syringomyeli re complex development l m lform tions w
ith wide spectrum of severity; they m y present t ny st ge of life. The cond
itions re linked
Fig. 11.2 MRI. Posterior foss r chnoid cyst.
closely through
oth their underlying p thophysiology nd their clinic l present
tion. The Chi ri m lform tion results from
norm lities t the cr niocervic l
junction involving the c ud l cere
ellum, medull nd upper cervic l region. In
1881 nd 1885 Chi ri reported the nom ly of the cere
ellum nd medull o
long t
nd descri
ed three types of m lform tion. The type I m lform tion consists of
c ud l displ cement of the cere
ell r tonsils
elow the for men m gnum into the
upper cervic l c n l. The type II m lform tion comprises c ud l displ cement of
the cere
ell r vermis, 4th ventricle nd medull o
long t
elow the for men m
gnum. This is simil r to the c se reported
y Arnold in 1894 nd consequently is

 lso known s the ArnoldChi ri m lform tion (Fig. 11.3). The type III m lform tion
involves c ud l displ cement of the cere
ellum nd
r instem into high cervic
l meningocele. Chi ri lso descri
ed type IV
norm lity, comprising two c se
s of hypopl stic cere
ellum. It is usu l for Chi ri type I m lform tions to pres
ent clinic lly in dults nd m ny of the presenting fe tures re rel ted to the
common ssoci tion of syringomyeli . The Chi ri type II m lform tion h s n even
higher incidence of ssoci tion with syringomyeli nd it is lmost inv ri
ly
present in p tients with myelomeningocele. Other frequent ssoci tions re hydro
ceph lus due to queduct stenosis, tresi or forking of the queduct, fusion of
the superior nd inferior colliculi on
oth sides into
Type I Chi ri Fourth ventricle (norm l position)
Type II Chi ri Fourth ventricle (displ ced c ud lly)
Medull (norm l position)
Cere
ell r tonsils
Medull displ ced c ud lly Herni ted cere
ell r vermis
Fig. 11.3 M jor fe tures of Chi ri types I nd II m lform tion.
DEVELOPMENTAL ABNORMALITIES
161
single
e ked structure, nd sm ll nd crowded posterior foss . Supr tentori l no
m lies include enl rgement of the m ss intermedi , microgyri nd heterotopi s,
which m y involve
oth the cere
r l hemispheres nd cere
ellum. Cr ni l l cun e
or mesoderm l defects of the skull (luckensch del) re common nd the r diogr p
hic ppe r nce is of multiple punched-out re s which usu lly resolve in the rst 6
months of life. The Chi ri type II m lform tion m y lso
e ssoci ted with nom
lies of the c rdiov scul r system, g strointestin l system (imperfor te nus)
nd genitourin ry system. Syringomyeli is c vit tion within the spin l cord. How
ever, the c vit tion occurring in ssoci tion with the Chi ri m lform tion is us
u lly c lled hydromyeli , s it is dil t tion of the centr l c n l which is li
ned
y ependym . The term syringomyeli is reserved for c vit tion lying outside
the centr l c n l re lined
y gli l tissue.
returns to norm l the reverse should occur, with ow from the intr cr ni l c vity
into the spin l c vity. If this equ liz tion of pressures is imp ired nd del ye
d
y dhesions nd tissue in the for men m gnum, pressure differenti l is cre
ted
etween the intr cr ni l nd intr spin l uid comp rtments nd ltern tive p t
hw ys develop, such s through p tent o
ex into the spin l centr l c n l. In
ddition, the pressure differenti l promotes progressive c ud l displ cement of c
ere
ell r tissue through the for men m gnum.
Clinic l present tion
The clinic l fe tures of the Chi ri type II m lform tion present in inf ncy, chi
ldhood or dolescence. A Chi ri type I m lform tion c uses symptoms presenting i
n dolescence nd dulthood nd the neurologic l fe tures re lmost lw ys due
to the development of syrinx within the spin l cord or lower
r instem. In inf
ncy the
norm lity will
e pp rent with its ssoci tion with myelomeningocele
. Progressive hydroceph lus m y develop. Severe
r instem dysfunction m y result
in episodic pnoe , depressed g g re ex, nyst gmus nd sp stic p resis of the upp
er lim
s. In childhood the type II m lform tion m y
e m nifest
y nyst gmus, sp
stic p r lysis nd
ul
r dysfunction. In dolescence the symptoms m y
e due t
o either type I or type II Chi ri m lform tion. The fe tures will involve pr
ogressive sp stic p r lysis of the upper nd/or lower lim
s nd m y lso include

the fe tures of syringomyeli , including suspended thermo n esthesi sensory lo

ss, trophic ch nges in the h nds nd upper extremities nd
ul
r pro
lems due
either to extension of the syrinx into the lower
r instem or to the direct pres
sure from the Chi ri type II m lform tion itself. Adult symptoms re prim rily d
ue to com
in tion of the Chi ri type I m lform tion nd syringomyeli . The ch
r cteristic symptoms include occipit l he d che, ex cer
ted
y coughing, nd ne
ck nd rm p in. Nyst gmus is common nd m y
e either horizont l or vertic l.
Aetiology
G rdner popul rized the hydrodyn mic theory of the origin of syringomyeli (hydr
omyeli ) ssoci ted with Chi ri m lform tion. In
rief, the theory proposes th t
the CSF is un
le to p ss freely out of the 4th ventricle s the norm l p thw y
s h ve either f iled to open or re o
liter ted due to crowding in the posterior f
oss . The norm l puls tions of CSF re consequently tr nsmitted down the centr l
c n l of the spin l cord. The close ssoci tion of the Chi ri type II m lform t
ion nd meningomyelocele led to theory th t spin l cord tethering resulted in
the c ud l displ cement of the cr ni l structures s result of growth. Altern
tively, the m lform tion m y result from hind
r in m ldevelopment during e rly f
et l life. Willi ms h s proposed differenti l pressure
etween the intr cr ni
l nd intr spin l uid comp rtments s nother mech nism for the development of th
e m lform tion. With V ls lv m noeuvre, the engorgement of the spin l epidur
l veins c uses rise in the intr spin l pressure. The spin l su
r chnoid sp ce
is compressed nd pressure w ve moves into the intr cr ni l c vity. When the
spin l pressure
162
CHAPTER 11
( )
Fig. 11.4 C pe-like distri
ution of p in nd temper ture loss typic lly resulting
from syrinx or other intr medull ry lesion in the cervic l nd upper thor cic re
gions.
(
)
Down
e t nyst gmus, ex cer
ted
y the p tient looking down nd out, is ch r cte
ristic of cr niocervic l junction
norm lity. The ch r cteristic clinic l fe
tures resulting from syringomyeli include: dissoci ted sensory loss (loss of p
in nd temper ture sens tion with preserv tion of joint position sense) occurrin
g in c pe-like distri
ution (Fig. 11.4). The sensory loss will often result in p
inless injury of the ngers or h nds nd Ch rcot joints m y develop we kness nd
w sting of the sm ll muscles of the h nd progressive long tr ct signs resulting
in sp stic p resis of the lower lim
s nd p r lysis of the upper lim
s
ul
r fe
tures, if the syrinx extends into the lower
r instem.
Fig. 11.5 ( ) Chi ri type II m lform tion with dyspl stic cere
ellum extending t
hrough for men m gnum to C2 level nd c ud l displ cement of 4th ventricle nd m
edull . There is syrinx (hydromyeli ) in the cervic l nd upper thor cic spin
l cord. (
) Chi ri type I m lform tion with cervic l syrinx (hydromyeli ).
g tions of cr niocervic l junction
norm lities. S gitt l nd coron l sc ns wil
l show displ cement of the cere
ellum into the upper cervic l c n l, with c ud l
displ cement of the 4th ventricle nd
r instem in the type II m lform tion. MR
I will lso show the other ssoci ted intr cr ni l
norm lities (Fig. 11.5). Ho
wever, it m y not dequ tely demonstr te the underlying n tomy of the
ony stru
ctures. MRI h s virtu lly repl ced CT sc nning with intr thec l contr st, nd my
elogr phy is no longer necess ry.

R diologic l investig tions

MRI h s revolutionized the r diologic l investiSyringomyeli (hydromyeli ) As discussed e rlier, intr spin l c vit tion c n
e
either hydromyeli , where the syrinx is dil t tion of the centr l c n l nd th
ere is usu lly comDEVELOPMENTAL ABNORMALITIES
163
munic tion with the CSF p thw ys, or the noncommunic ting syringomyeli , where t
he syrinx is sep r te from the centr l c n l. Hydromyeli (communic ting syringo
myeli ) usu lly results from Chi ri type I nd type II m lform tions nd occ sio
n lly from
sil r r chnoiditis. Non-communic ting syringomyeli m y
e due to
previous tr um or neopl sms, or ssoci ted with spin l r chnoiditis. Occ sion
lly, no underlying
norm lity c n
e found s c use of the syrinx. The most v
lu
le r diologic l investig tion is MRI, which will show the extent of the syr
inx nd ssoci ted
norm lities. This investig tion h s lmost completely repl
ced the use of CT sc n with w ter-
sed contr st injected into the su
r chnoid
sp ce. Del yed post-contr st CT sc nning shows intr medull ry op ci c tion s well
s the enl rged cord.
the posterior foss decompression
ut others prefer to perform the oper tion onl
y if there is continuing progression of neurologic l dis
ility.
Cr nioverte
r l junction
norm lities
The cr nioverte
r l junction region involves the for men m gnum, the dj cent oc
cipit l
one nd the tl s nd xis verte
r e. Numerous congenit l or cquired

norm lities occur in this region which m y result in compression of the underly
ing neur l structures. Some
norm lities, including spin
i d of the nterior o
r posterior rch of the tl s, rem in symptom tic.
B sil r inv gin tion (impression)
B sil r inv gin tion is deform tion of the
siocciput in which there is n up
w rd indent tion or inv gin tion of the
se of the skull, including the rim of
the for men m gnum, occipit l condyles nd neigh
ouring
one, into the posterior
foss . The clivus is often shortened nd the inv gin tion reduces the di meter
of the for men m gnum. The odontoid process frequently projects into the nterio
r p rt of the for men m gnum so th t its di meter is reduced still further. Ther
e is frequent ssoci tion with congenit l fusion of the cervic l verte
r e to
e ch other or to the occiput. Acquired
sil r impression m y
e due to softenin
g of the
one
y dise se. The most common c use is P gets dise se
ut it m y lso
occur in osteom l ci , hyperp r thyroidism nd osteogenesis imperfect . The cli
nic l fe tures resulting from
sil r impression re due to compression of the n
eur l structures t the cervicomedull ry junction the medull o
long t , the cr
ni l nerves, the cervic l roots nd the spin l cord. The clinic l fe tures m y i
nclude progressive qu drip resis, dysph gi , respir tory dif culties, nyst gmus
(sometimes down
e t) nd su
occipit l he d che due to irrit tion of the 2nd cerv
ic l nerve. Useful r diologic l investig tions include CT sc n (with s gitt l re
construction nd intr thec l contr st) (Fig. 11.6), MRI nd pl in X-r ys.
Tre tment
There re two m jor surgic l procedures. 1 Posterior foss nd upper cervic l de
compression. 2 Shunting of the syrinx. The posterior foss oper tion involves de
compression
y removing the posterior rim of the for men m gnum, posterior rch
of the tl s nd l min e of the upper cervic l spine, extending down to
elow th
e level of the descent of the cere
ell r tonsil. The dur is opened widely, s i
t often cts s constricting
nd, p rticul rly t the level of the for men m
gnum. A v riety of other procedures m y
e performed t th t time, including dis

section of the su
r chnoid dhesions, opening of the for men of M gendie into t
he 4th ventricle, plugging the enl rged opening of the centr l c n l t the o
ex
with tissue, nd pl cing stent through the for men of M gendie. These procedu
res must
e done with meticulous microsurgery techniques to void further d m ge
to this extremely sensitive re . The dur is closed using gr ft of cervic l
f sci . The syringomyeli (hydromyeli ) m y
e shunted into either the su
r chn
oid sp ce or the peritone l or pleur l c vity. Some surgeons prefer to c rry out
this procedure t the s me time s
164
CHAPTER 11
Fig. 11.6 S gitt l reconstruction of CT sc n (with intr thec l contr st) demonst
r ting the odontoid process extending through the for men m gnum.
Ch m
erl ins line
Fig. 11.8 Atl nto xi l su
lux tion in rheum toid rthritis.
Atl nto xi l disloc tion
H rd p l te McGregors line
Fig. 11.7 Pl in X-r y me surement for
sil r inv gin tion.
V rious me surements t the
se of the skull c n
e used to di gnose the nom l
y. Ch m
erl ins line joins the tip of the dors l lip of the for men m gnum to the
dors l m rgin of the h rd p l te nd, on l ter l skull X-r y or s gitt l MRI,
should norm lly lie
ove the tip of the odontoid process of the xis nd p ss
through the ventr l lip of the for men m gnum. McGregors line joins the h rd p l
te to the most c ud l portion of the occipit l curve. In
sil r inv gin tion th
e tip of the odontoid lies more th n 4.5 mm
ove this line (Fig. 11.7).
Disloc tions t the tl nto xi l joint c n result from congenit l m lform tions,
involving in p rticul r the congenit l fusion of the occiput to the tl s nd f
usions of C2 nd C3. Multiple congenit l cervic l fusions occur in the KlippelFei
l syndrome. These types of fusions will incre se the str in on the lig ments of
dj cent verte
r e, resulting in inst
ility. Atl nto xi l disloc tion m y lso
result from in mm tory conditions such s rheum toid rthritis or following tr um
(Fig. 11.8).
D ndyW lker cyst
The D ndyW lker cyst is cystic enl rgement of the 4th ventricle, usu lly ssoci
ted with hypopl si or p rti l genesis of the cere
ellum nd hydroceph lus of
the 3rd nd l ter l ventricles. It is pro

le th t the prim ry c use is n em
r
yologic l f ilure of the for min l outlets of the 4th ventricle to open, resulti
ng in cystic enl rgement of the 4th ventricle nd hydroceph lus. However, the D
ndyW lker cyst is sometimes ssoci ted with other congenit l
norm lities, such
s genesis of the corpus c llosum nd queduct stenosis, nd it h s
een sugges
ted th t it m y represent cere
ell r dysr phism. The clinic l fe tures re usu
lly pp rent in inf ncy nd result prim rily from hydroceph lus.
Pl ty
si
Pl ty
si is sometimes erroneously used synonymously with
sil r impression. P
l ty
si refers to n o
tuse
s l ngle joining the pl ne of the clivus with t
he pl ne of the nterior foss of the skull; it is s id to
e present if the ng
le exceeds 145. Although pl ty
si is often present with
sil r impression it c
uses no symptoms
y itself.
DEVELOPMENTAL ABNORMALITIES

165
Myelomeningocele
Meningocele
Dyspl stic nerve roots
S c

Fig. 11.10 Myelomeningocele nd meningocele. Fig. 11.9 D ndyW lker cyst in poster
ior foss with hydroceph lus of l ter l nd 3rd ventricles.
rologic l involvement nd the lesion is symptom tic throughout life. The cyst m
y
e di gnosed in childhood, p rticul rly if it does not produce signi c nt hydro
ceph lus, nd the m jor presenting fe tures re t xi nd del yed motor develop
ment. The di gnosis is m de
y CT sc n (Fig. 11.9) or MRI. The st nd rd tre tmen
t is now shunting of the cyst
ut, if queduct stenosis is present, ventricul
r shunt m y lso need to
e inserted.
Myelomeningocele
Myelomeningocele is the most common nd import nt form of spin l dysr phism pres
enting during the neon t l period (Fig. 11.10). It is ch r cterized
y protrusio
n of the neur l elements through verte
r l defect into meninge l-lined s c.
Typic lly, the cord t this level is not fused
ut is in its ttened em
ryologic
l st te, with the nerve roots rising from the ventr l surf ce nd the open cent
r l c n l lying dors lly. The m jor dis
ility from this condition results from
the irreversi
le neurologic l de cit c used
y this spin l cord
norm lity. Depen
ding on the level of the defect the spin l cord or conus nd c ud equin m y
e
involved. Although myelomeningocele m y occur t ny level, it is most common i
n the lum
r nd lum
os cr l segments. This disorder occurs in pproxim tely 1 i
n 1000 live
irths. There is gre ter frequency mong whites th n
l cks, with
slight fem le predomin nce. There is f mili l incidence: if one mem
er of
f mily is ffected the risk of the disorder occurring in su
sequent offspring is

out 5%. Se son l out
re ks of myelomeningocele h ve
een reported nd there
re ethnic
Spin l dysr phism
Spin l dysr phism results from incomplete or f ulty closure of the dors l midlin
e em
ryologic l structures. The m jor forms of spin l dysr phism re: 1 Myelomen
ingocele. 2 Meningocele. 3 Lipomyelomeningocele. 4 Occult spin l dysr phism: ( )
dermoid tumours (
) di stem tomyeli (c) intr spin l lipom (d) hypertrophic lum
termin le. Spin
i d occult is
ony de cit usu lly found in the l min e of the
lum
os cr l spine nd due to midline fusion defect. It is n incident l r dio
logic l nding nd is present in up to 20% of dults. In the v st m jority there i
s no neu166
CHAPTER 11
differences, with higher incidence of the condition in the western United King
dom, northern Indi nd Egypt. Pren t l di gnosis of myelomeningocele nd other
open neur l tu
e disorders is possi
le
y me suring lph -fetoprotein in the mn
iotic uid t 1416 weeks nd
y pren t l ultr sound ex min tion. The widespre d use

of incre singly ccur te ultr sound ex min tion in the rst trimester of pregn nc
y h s led to detection of myelomeningocele, nd in m ny centres detection is fol
lowed
y p rent l decision to termin te the pregn ncy. Consequently there h s
e
en dr m tic decre se in the incidence of children
orn with this type of nom
ly. The ultr sound ndings c n
e con rmed
y MRI. Myelomeningocele is frequently s
soci ted with other congenit l
norm lities, most commonly the Chi ri type II m
lform tion, queduct stenosis (forking) nd hydromyeli (syringomyeli ). The m
n gement of myelomeningocele involves: 1 Assessment of the s c nd its coverings
. 2 Neurologic l ev lu tion. 3 Ex min tion for other ssoci ted conditions: ( )
within the CNS, e.g. hydroceph lus (
) extr cr ni l nom lies, e.g. g strointest
in l, urin ry. 4 Counselling nd c reful discussions with the p rents. 5 Surgic
l procedures. Decisions concerning whether p tient should undergo surgic l p
rocedure with closure of the s c re mong the most dif cult in neurosurgic l pr c
tice. On the one h nd, the child h s right to life
ut there is lso right t
o life of suf cient qu lity to
e worth living. The immedi te decision is whethe
r to close the myelomeningocele s c. In 1959 physici ns in Shef eld undertook pr
ogr mme of immedi te closure in ll neon tes, with ggressive tre tment of the h
ydroceph lus nd other m lform tions. However, when the series w s ex mined, onl
y 7% h d less th n grossly crippling dis
ility nd m y
e considered to h ve q
u lity of life not inconsistent with self respect, e rning c p city, h ppiness
nd even m rri ge. Consequently, selective criteri
were developed nd the children excluded from tre tment were those with: p r lys
is t L2 to L3 or
ove m rked hydroceph lus kyphosis other m jor congenit l
n
orm lities or
irth injuries. However, lthough when these criteri re used,
l rge num
er of untre ted inf nts do not live long, signi c nt minority do, nd
there is gre t concern
out their qu lity of life. The initi l oper tion ims t
o: preserve ll the neur l tissue nd reduce it into the interverte
r l c n l; u
ntether the spin l cord o
t in w tertight closure of dur lining the s c cover
the defect with muscle, f sci nd skin. It will su
sequently
e necess ry to m
ke decisions concerning the tre tment of hydroceph lus nd other ssoci ted m l
form tions. The continued c re of these children requires multidisciplin ry p
pro ch including urologists, orthop edic surgeons, physici ns, physiother pists
nd soci l workers. The children frequently h ve severe urologic l pro
lems, whi
ch m y result in ren l f ilure s dolescents. The musculoskelet l disorders, in
cluding t lipes nd hip disloc tion, require c reful orthop edic m n gement to m
ximize ny residu l lower lim
function.
Lipomyelomeningocele
This is much less common disorder th n myelomeningocele. The m ss is covered

y skin nd the lipom tous tissue extends intr dur lly nd is intim tely interwov
en with the rootlets of the c ud equin nd the conus medull ris, which is not
usu lly fused. Neurologic l ex min tion is usu lly norm l t
irth nd progressi
ve neurologic l de cits occur, resulting from growth nd tethering of the spin l c
ord. The most common symptoms include
l dder nd
owel distur
nce,
ck p in
nd progressive p r lysis in the legs with foot deformities nd loss of sens tion
. Surgery is del yed until
out 4 months. The lipom is removed s completely
s possi
le
DEVELOPMENTAL ABNORMALITIES
167
without end ngering the neur l tissue; the prim ry surgic l im is to untether t
he spin l cord.
Meningocele
This is much less common th n myelomeningocele nd is ch r cterized
y cystic
lesion cont ining only meninges nd CSF; it does not cont in ny neur l tissue.

Occult spin l dysr phism

Occult spin l dysr phism includes num
er of spin l disorders, the v st m jorit
y of which re situ ted in the lum
r region nd produce progressive neurologic
l dysfunction, often s result of tethering of the spin l cord. The more commo
n lesions include: intr spin l lipom s ( nd lipomyelomeningocele) dermoid tumour
s di stem tomyeli . The underlying intr spin l lesion c n often
e suspected fro
m n overlying skin lesion such s dimple, sinus tr ct, f tty m ss, h em ngiom
or
norm l tuft of h ir. The neurologic l symptoms re usu lly rst noted in ch
ildhood or dolescence. There is usu lly slowly progressive neurologic l dysfu
nction involving:
owel nd
l dder distur
nce progressive we kness of the legs
nd foot deformities
ck p in sensory dis
ility in the lower lim
s progressiv
e scoliosis. Depending on the level of the
norm lity, the neurologic l ex min
tion will show evidence of either lower nd/or upper motor neurone d m ge. Lipom
s re the most frequent form of occult spin l dysr phism. The lum
os cr l lipom
presents t
irth s soft su
cut neous m ss, usu lly in the midline nd cove
red
y skin. In its most severe form lipomyelomeningocele the lipom will involv
e neur l tissue nd will extend through spin
i d . Less severe
norm lities w
ill
e ssoci ted with low-lying conus nd
with tethering of the cord
y n enl rged lum termin le or f tty tissue. Di stem
tomyeli is condition in which the spin l cord is
i d nd the two hemicords re
sep r ted
y
ony spur or dur l
nd. Progressive neurologic l dysfunction wi
ll occur due to tr ction on the cord th t is tr ns xed during growth periods (Fig.
11.11). The cord is occ sion lly tethered
y shortened, thickened lum termin l
e hypertrophic lum termin le nd the only ssoci ted
norm lities re spin
i d
occult nd n occ sion l h iry p tch over the lower spine. R diologic l ssessm
ent will utilize pl in Xr y, CT sc n (with intr thec l contr st) nd MRI. Pl in
X-r ys will show r nge of verte
r l
norm lities including spin
i d , hemiver
te
r e,
norm l spin l curves, di stem tomyelic spurs nd widened interpedicul
r dist nces. The surgic l tre tment is imed t: removing the underlying p tholo
gic l c use while preserving neur l tissue untethering the cord.
Cr niosynostosis
Cr niosynostosis is prem ture closure of cr ni l sutures nd occurs in 1 in 3000

irths. It m y occur s condition involving single cr ni l suture or s p r
t of complex syndrome involving multiple fusion
norm lities. The posterior f
ont nelle h s usu lly closed
y 23 months of ge nd the nterior font nelle
y

out 1618 months. The
r in ce ses to grow t 1012 ye rs of ge, t which time the
cr ni l sutures re o
liter ted
y rm
rous tissue. Complete ossi c tion of the sut
ures does not occur until fter the 3rd dec de of life. The clinic l fe tures of
cr niosynostosis re rel ted to: the cr ni l deformity r ised intr cr ni l pres
sure the presence of ssoci ted congenit l
norm lities.
S gitt l synostosis This is
y f r the most common type of prim ry cr niosynosto
sis; the incidence is gre ter th n
168
CHAPTER 11
th t of ll other types of cr niosynostosis com
ined. M les re most commonly f
fected nd the condition is usu lly not ssoci ted with other congenit l
norm
lities. Prem ture fusion of the s gitt l suture results in the he d exp nding in
the occipitofront l di meter nd produces long n rrow he d (sc phoceph ly). C
ompens tory growth long the metopic nd coron l sutures c uses the forehe d to
exp nd l ter lly (front l
ossing).
( )
(
)

Coron l synostosis Coron l synostosis occurs more commonly in fem les. The he d
exp nds superiorly nd l ter lly (
r chyceph ly). This produces short nterior
cr ni l foss , sh llow or
its, hypertelorism nd elev tion of the forehe d. Cho
n l tresi is common. Bil ter l coron l synostosis commonly occurs s one of s
ever l congenit l defects in Crouzons nd Aperts syndromes. If only one coron l su
ture fuses prem turely there will
e n symmetric l cr ni l deformity. Metopic
synostosis Metopic synostosis produces n rrow, tri ngul r forehe d (trigonocep
h ly) ssoci ted with hypotelorism. L m
doid synostosis L m
oid synostosis is un
common. There is symmetric l ttening of the posterior cr nium.
Oper tive tre tment
Surgery is performed to: correct the cr ni l deformity relieve the effects of r
ised intr cr ni l pressure. If only one suture is fused, compens tory growth lo
ng the open suture lines will reduce the risk of r ised intr cr ni l pressure,
lthough there h ve
een studies showing r ised intr cr ni l pressure, usu lly on
ly moder te, in children with single suture fusion. However, if two or more cr n
i l sutures fuse prem turely there is risk of incre sed intr cr ni l pressure
s growth proceeds. This m y le d to ment l nd motor ret rd tion nd to optic
trophy.
(c)
Fig. 11.11 Lum
r di stem tomyeli with
one spur nd dur l
nd ( rrow) p ssing
through the c ud equin .
DEVELOPMENTAL ABNORMALITIES
169
The oper tive procedure h s usu lly involved strip cr niectomy of the fused su
ture. However, s the long-term results h ve
een somewh t dis ppointing, the tr
end is now to undert ke more m jor v ult reconstruction to o
t in perm nent
effect. Surgery is usu lly
est del yed until the child is 36 months old,
ut if
two or more cr ni l sutures re fused then it m y
e undert ken e rlier to minim
ize the effects of
r in compression. The oper tion involves resection of the f
fected sutures. It should
e c refully pl nned nd meticulously performed to min
imize
lood loss, which is the gre test risk of the procedure.
Further re ding
Del sh w JB et l. (1989) Cr ni l v ult growth in cr niosynostosis. Journ l of N
eurosurgery 70, 159166. Dyste GN, Menezes AH, V nglider JC (1989) Symptom tic Chi
ri m lform tions. An n lysis of present tion, m n gement nd long term outcome
. Journ l of Neurosurgery 71, 159168. G rdner WJ (1965) Hydrodyn mic mech nism of
syringomyeli : its rel tionship to myelocoele. Journ l of Neurology, Neurosurge
ry nd Psychi try 28, 247259. Hockley AD, W ke MJ, Goldin H (1988) Surgic l m n g
ement of cr niosynostosis. British Journ l of Neurosurgery 2, 307314. Hoffm n HJ
et l. (1982) Investig tion nd m n gement of supr sell r r chnoid cysts. Journ
l of Neurosurgery 57, 597602. Hoffm n HJ, Hendrick EB, Humphreys RP (1975) M nif
est tions nd m n gement of Arnold Chi ri m lform tion in p tients with myelomen
ingocoele. Childs Br in 1, 255259. Hoffm n HJ, Hendrick EB, Humphreys RP (1976) Th
e tethered spin l cord: its prote n m nifest tions,
di gnosis nd surgic l correction. Childs Br in 2, 145151. Humphreys RP (1985) Spi
n l dysr phism. In: Wilkins RH, Rengoch ry SS, eds. Neurosurgery. McGr w-Hill, N
ew York, 20412052. K ye AH, Bl ck P McL (2000) Oper tive Neurosurgery. Churchill
Livingstone, London, New York, Edin
urgh. Levy WJ, M son L, H hn JF (1983) Chi r
i m lform tion presenting in dults: surgic l experience in 127 c ses. Neurosu
rgery 12, 377390. Little JR, Gomez MR, McC rty CS (1973) Infr tentori l r chnoid
cysts. Journ l of Neurosurgery 39, 380386. Lor
er J (1971) Results of tre tment
of myelomeningocoele: n n lysis of 524 unselected c ses, with speci l referenc

e to possi
le selection for tre tment. Development l Medicine nd Child Neurolog

y 18, 279303. M pstone T (1994) M n gement of tethered cord syndrome. Neurosurger
y Qu rterly 4, 8291. M tson DD (1969) Neurosurgery in Inf ncy nd Childhood. Ch r
les C Thom s, Spring eld. Milhor t TH (1978) P edi tric Neurosurgery. Contempor ry
Neurology Series. F A D vis, Phil delphi . Milhor t TH, Miller JI, Johnson WD (
1993) An tomic l
sis of syringomyeli . Neurosurgery 32, 748754. Ro
inson RG (19
64) The tempor l lo
e genesis syndrome. Br in 87, 87106. Ro
inson RG (1971) Cong
enit l cysts of the
r in: r chnoid m lform tions. Progress in Neurologic l Sur
gery 4, 133174. Shillito J, M tson DD (1968) Cr niosynostosis: review of 519 su
rgic l p tients. P edi trics 41, 829853. St rkm n SP, Brown TC, Linell EA (1958)
Cere
r l r chnoid cysts. Journ l of Neurop thology nd Experiment l Neurology 1
7, 480500. Willi ms B (1980) On the p thogenesis of syringomyeli : review. Jour
n l of the Roy l Society of Medicine 73, 298806.
CHAPTER 12
12
Infections of the centr l nervous system
Infections involving the nervous system present in v riety of w ys, m ny of wh
ich result in de th or severe mor
idity if not di gnosed nd tre ted promptly. T
he infections usu lly occur s the result of h em togenous spre d or direct exte
nsion from dj cent
one, soft tissue or sinuses. A l rge v riety of p thogens
re involved, including viruses, fung l gents nd
cteri . The most common infe
ctions involving the neurosurgeon re: cute
cteri l meningitis cere
r l
sce
ss. Neurosurgeons re involved in the m n gement of nervous system infections
e
c use p tients frequently present with m nifest tions of r pidly progressive n
eurologic l illness, s well s the systemic m nifest tions of sepsis. Infection
m y involve ny p rt of the nervous system or its coverings nd c n
e cl ssi ed
in the following w y. 1 Cr ni l v ult infections. 2 Extr dur l
scess/empyem .
3 Su
dur l
scess/empyem . 4 Meningitis. 5 Br in: ( )
r in
scess (
) enceph
litis.
Meningitis
B cteri l meningitis is serious, life-thre tening infection of the meninges. V
ir l meningitis is the more common infection
ut is usu lly self-limiting, nd t
he neurosurgeon is r rely involved. Most of the common org nisms th t c use
c1
70
teri l meningitis re rel ted to the p tients ge nd to the presence nd n ture
of ny underlying predisposing dise se. Although few types of
cteri l org ni
sms ccount for most c ses of cute pyogenic meningitis there is wide r nge of
org nisms th t m y
e responsi
le. T
le 12.1 shows the most common c uses of

cteri l meningitis rel ted to ge. The
cteri re ch the meninges nd cere
ros
pin l uid
y three m in routes. 1 H em togenous spre d from extr cr ni l foci of
infection. 2 Retrogr de spre d vi infected throm
i within emiss ry veins from i
nfections dj cent to the centr l nervous system, such s sinusitis, otitis or m
stoiditis. 3 Direct spre d into the su
r chnoid sp ce, such s from osteomyeli
tis of the skull nd infections of the p r n s l sinuses. The CSF is good cult
ure medium which will support the growth of m ny microorg nisms. Norm l CSF cont
ins very low concentr tions of immunoglo
ulins nd complement components nd is
devoid of polymorphonucle r ph gocytes. Furthermore, ph gocytosis is imp ired

y the low opsonic ctivity of CSF nd org nisms such s Streptococcus pneumoni e
, H emophilus in uenz e type B, Group B streptococci, Escherichi coli nd Kle
sie
ll pneumoni e h ve polys cch ride c psule th t hinders ph gocytosis.
Clinic l present tion
B cteri l meningitis is usu lly n cute illness with r pid progression of clini
c l signs. M ny c ses re preceded
y symptoms of n upper

INFECTIONS OF THE CENTRAL NERVOUS SYSTEM

171
T
le 12.1 Common org nisms c using prim ry
cteri l meningitis rel ted to ge.
Age Neon te (04 weeks) 412 weeks 3 months5 ye rs Over 5 ye rs nd dults Org nism
Group B streptococcus, Escherichi coli Group B streptococcus, Streptococcus pne
umoni e, S lmonell , *H emophilus in uenz e, Listeri monocytogenes H emophilus in u
enz e*, Streptococcus pneumoni e, Neisseri meningitidis Streptococcus pneumoni
e, Neisseri meningitides
*In countries with routine H emophilus in uenz e immuniz tion this org nism is r
re c use of meningitis.
respir tory tr ct infection. The m jor presenting fe tures re: high fever menin
gismus, including he d ches, neck stiffness, photopho
i , vomiting nd n ltere
d ment l st te in terms of clinic l present tion. Although p tients re usu lly
lert t the commencement of the illness they will frequently
ecome drowsy nd
confused. If tre tment is not commenced promptly there m y
e further deterior t
ion of the conscious st te s result of direct septic effect on the underlyi
ng
r in, septic throm
osis of the cere
r l rteries nd veins, or the developme
nt of hydroceph lus. Foc l neurologic l signs m y develop s result of cortic
l inf rction second ry to throm
osis. In inf nts, neon tes, the elderly nd the
immunocompromised, the present tion of
cteri l meningitis m y
e different. Ne
ck stiffness nd fever re often
sent nd the present tion includes listlessne
ss nd irrit
ility in the young nd confusion or o
tund tion in the elderly/ im
munocompromised. A c reful se rch should
e m de for skin r sh. Meningococc l
infection frequently h s coexisting petechi l r sh, which occurs less frequent
ly in other
cteri l (e.g. st phylococc l
cter emi , H emophilus in uenz e infe
ction) or vir l infections. The origin l source of infection, e.g. sinusitis,

cteri l endoc rditis, otitis medi or m stoiditis, m y
e evident nd m ny p tie
nts h ve evidence of ph ryngitis
cteri l meningitis sometimes follows n upper
respir tory tr ct infection.
Di gnosis
The di gnosis is m de
y CSF ex min tion o
t ined
y lum
r puncture which shoul
d
e performed immedi tely the di gnosis is suspected. If the p tient is drowsy,
h s other signs of r ised intr cr ni l pressure or there re foc l neurologic l
signs, n urgent computerized tomogr phy sc n must
e performed prior to the lu
m
r puncture to exclude n intr cr ni l sp ceoccupying lesion. The CSF fe tures
on lum
r puncture re: M croscopic lly cloudy CSF. R ised white cell count. Th
is is usu lly in excess of 500 cells/mm3 nd is predomin ntly polymorphonucle
r leukocytosis. The protein level is gre ter th n 0.8 g/l; it is often su
st nti
lly higher. The glucose level is less th n 2 mmol/l, frequently much lower. A u
seful index is the CSF : serum r tio which is less th n 0.4 in
cteri l infec
tion. The Gr m st in will
e positive in over 70% of p tients if common p thogen
s re involved, nd in pproxim tely 50% of p tients for Gr mneg tive
cilli. O
ther tests which should
e performed on the CSF include: ex min tion for Cryptoc
occus neoform ns using n Indi ink prep r tion nd n gglutin tion test for cr
yptococc l ntigen investig tion for Myco
cterium tu
erculosis (ZiehlNeelsen st
in for cid-f st
cilli) nd moe
e.
172
CHAPTER 12
Dif culties rise in di gnosis of p rti lly tre ted
cteri l meningitis
ec use t
he CSF culture is often neg tive. Other investig tions include:
lood cultures r
diologic l investig tions to detect the source of infection chest X-r y, CT sc

n or skull X-r y for sinusitis. The differenti l di gnosis includes: 1 Other typ
es of meningitis: ( ) vir l (
) fung l (Cryptococcus neoform ns) (c) moe
ic (d)
tu
erculous (e) c rcinom tous (Ch pter 6). 2 Su
dur l empyem . 3 Su
r chnoid h
emorrh ge (Ch pter 9). 4 Vir l enceph litis (especi lly herpes simplex enceph l
itis).
Tre tment
High-dose intr venous nti
iotic ther py should
e selection of the nti
iotic depends on: the
kely org nism involved, t king into ccount the
e of infection CSF micro
iology studies the
tion into the CSF.

e commenced immedi tely, nd th

initi l expect tion of the most li
ge of the p tient nd the sourc
nti
iotic th t h s the
est penetr

There re m ny nti
iotic regimes
ut if there is no o
vious site of infection i

niti l ther py should commence immedi tely s follows. Neon tes (under 3 months)
cefot xime or ceftri xone plus
enzyl penicillin or moxy-/ mpicillin. Three m
onths to 15 ye rs cefot xime or ceftri xone. 15 ye rs to dult
enzyl penicillin
plus cefot xime/ceftri xone. Add v ncomycin if Gr m-positive streptococci re s
een nd there is ny suspicion of intermedi te nd/or resist nt Streptococcus pn
eumoni e. When the org nism h s
een identi ed the most ppropri te nti
iotic sho
uld
e used, depending on sensitivities nd the
ility of the nti
iotic to pen
etr te into the CSF. T
le 12.2 shows the CSF penetr tion of nti
iotics. The us
u l speci c ntimicro
i l ther py following identi c tion of the org nism is: Pneumo
coccus or meningococcus
enzyl penicillin (child: 60 mg/kg up to 1.82.4 g intr ve
nously 4-hourly). If p tient is sensitive to penicillin use cefot xime (child: 1
5 mg/kg 6-hourly or ceftri xone 100 mg/kg d ily). A
out h lf the p tients with m
eningococc l meningitis h ve petechi e or purpur . Su
clinic l or clinic l disse
min ted intr v scul r co gul tion often ccomp nies meningococc emi nd m y pro
gress
T
le 12.2 Anti
iotic penetr tion into cere
rospin l uid. Good penetr tion with o
r without meninge l in mm tion Chlor mphenicol* Metronid zole Third-gener tion ceph
losporins Ceftri xone/Cefot xime Good penetr tion only with meninge l in mm tion
Penicillins Penicillin Amoxycillin Fluclox cillin Rif mpicin Trimethoprim/sulph
methox zole V ncomycin C r
penems Meropenem
F ir to poor penetr tion First-gener tion ceph losporins Aminoglycosides Gent mi
cin To
r mycin
*Limited v il
ility in most countries. R rely used.
INFECTIONS OF THE CENTRAL NERVOUS SYSTEM
173
to h emorrh gic inf rction of the dren l gl nds, ren l cortic l necrosis, pulmo
n ry v scul r throm
osis, shock nd de th. The nti
iotic ther py must
e ccomp
nied
y intensive medic l supportive ther py. H. in uenz e moxy-/ mpicillin if o
rg nism is suscepti
le. If the p tient is llergic or org nism-resist nt use cef
ot xime or ceftri xone. Listeri
enzyl penicillin or moxy-/ mpicillin plus tri
methoprim nd sulf methox zole. Hospit l- cquired meningitis v ncomycin plus cef
ot xime, ceftri xone or meropenem.
Complic tions of
cteri l meningitis
Complic tions re more likely to occur if tre tment is not commenced immedi tely
. The m jor complic tions re s follows. Cere
r l oedem . Seizures. Hydroceph l
us communic ting hydroceph lus. This m y occur e rly in the dise se or s l te
m nifest tion. Su
dur l effusion, p rticul rly in children. Most resolve spont
neously
ut some m y require dr in ge. Su
dur l empyem . A r re complic tion th
t usu lly requires dr in ge. Br in
scess, which occurs s r re complic tion

of meningitis.
overt signs of meningitis. There is frequently coexisting ventriculitis. The d
i gnosis is con rmed
y ex min tion of the CSF vi lum
r puncture or occ sion l
ly
y withdr w l of CSF from the reservoir mech nism of the shunt. The tre tment
consists of dministr tion of nti
iotics nd remov l of the shunt. The most fr
equently isol ted org nisms in meningitis following neurosurgic l oper tions re
St ph. ureus, St ph. epidermidis nd Gr mneg tive ero
ic
cilli. The clinic
l fe tures of
cteri l meningitis m y
e m sked, or confused with the underlyin
g neurosurgic l illness nd oper tion. Although the meningitis m y present s
r pidly fulmin ting infection, it is lso possi
le for the clinic l fe tures to
evolve slowly. The di gnosis must
e suspected if there is unexpl ined fever, im
p ired conscious st te, seizures or neck stiffness following surgery. CSF must

e o
t ined nd tre tment with the ppropri te nti
iotics commenced. However, it
m y
e dif cult to identify the c us tive org nism, s perioper tive prophyl ctic
nti
iotics m y m ke isol tion of the org nism dif cult.
Br in
scess
Cere
r l
scess m y occur t ny ge, m y
e single or multiple nd, lthough m
ost re supr tentori l, c n lso occur in the cere
ellum or
r instem.
B cteri l meningitis following neurosurgic l procedures
B cteri l meningitis m y complic te ny intr dur l neurosurgic l procedure, ofte
n with dev st ting consequences. It is much fe red complic tion following inse
rtion of CSF shunt (Ch pter 3). The m jority of shunt infections re c used
y
St phylococcus epidermidis nd diphtheroids, species th t re present in the no
rm l skin or . However, other p thogens such s St ph. ureus, pneumococcus nd H
emophilus species m y lso
e involved. Unlike prim ry
cteri l meningitis, th
e clinic l present tion is frequently su
cute or chronic nd p tients present w
ith low-gr de fever
efore developing the more
P thogenesis
Pyogenic in mm tion of the
r in le ding to cere
r l
scess m y result from: h e
m togenous spre d from known septic site or occult focus direct spre d from n
dj cent infected p r n s l or m stoid sinus tr um c using penetr ting wound
. The met st tic
r in
scesses rising
y h em togenous dissemin tion of infec
tion re frequently multiple nd develop t the junction of white nd grey m tte
r. The incidence in e ch p rt of the
r in is proportion l to its region l
lood
ow, so th t most
scesses occur in the
174
CHAPTER 12
distri
ution of the middle cere
r l rtery, princip lly the p riet l lo
e, ltho
ugh they c n lso
e found in the cere
ellum nd
r instem. The most common sour
ces of infection include skin pustules, chronic pulmon ry infection (e.g.
ronch
iect sis), diverticulitis, osteomyelitis nd
cteri l endoc rditis. P tients wi
th congenit l he rt dise se nd who h ve right-to-left shunt re p rticul rly
prone to
r in
scesses
ec use their
lood does not lter through the c pill ry

eds within the lungs. The site of origin of the h em togenous spre d is unknown
in pproxim tely 25% of p tients. Direct spre d from p r n s l sinuses, m stoid
ir cells or the middle e r re the most common p thogenic mech nisms in most s
eries. Infection from the p r n s l sinuses spre ds,
y retrogr de throm
ophle
i
tis, through the diploic veins into either the front l or tempor l lo
e. The
s
cesses re usu lly single nd re loc ted super ci lly. Front l sinusitis m y c us
e
r in
scess in the front l lo
e, sphenoid sinusitis n
scess in either t
he front l or tempor l lo
e, m xill ry sinusitis n
scess in the tempor l lo
e
nd ethmoid sinusitis n
scess in the front l lo
e. Middle e r infection m y
spre d into the tempor l lo
e nd, uncommonly, cere
ell r
scess m y result f

rom infection spre ding from the m stoid ir cells. The mech nism of
scess for
m tion is either
y erosion of the dj cent
one nd spre d through the dur or
due to retrogr de septic throm
ophle
itis in n emiss ry vein. A cere
r l
sces
s m y result from cr niocere
r l tr um which h s c used penetr ting
r in inj
ury, p rticul rly if foreign
odies such s
one or h ir h ve
een impl nted in
the
r in. A less common
ut well-documented c use of
r in
scess results from
infection spre ding from skull tongs used in skelet l tr ction for cervic l dis
loc tion.
circul tion nd which surround the re of developing infective necrosis. The ne
crotic centre enl rges nd pus is formed
y the rele se of enzymes from the in mm
tory cells. At the periphery of this necrotic centre
ro
l sts l y down reticu
lin network nd, s the
scess enl rges, coll gen c psule develops. The w ll
of the
scess develops more slowly on the ventricul r side
ec use of the poore
r v scul rity of the deep white m tter comp red with the cortic l grey m tter. C
onsequently, the
scess tends to enl rge into the deep white m tter, nd it m y
rupture into the l ter l ventricle.
B cteriology
In the pre nti
iotic er
r in
scess w s c used predomin ntly
y St ph. ureus
nd streptococci. After the introduction of nti
iotics the incidence of st phy
lococc l
scesses declined nd most
scesses were thought to
e due to strepto
cocci, lthough up to 50% of culture results in some series were sterile. When imp
roved n ero
ic culture techniques
ec me v il
le the incidence of positive cu
ltures incre sed, nd m ny of the
scesses previously thought to
e sterile wer
e found to h ve
een c used
y n ero
ic org nisms, p rticul rly streptococci n
d B cteroides species. Meticulous culture techniques h ve resulted in consider

le improvement in the de nition of the
cteri l spectrum in
r in
scesses nd h
ve con rmed th t streptococci re the most common org nisms isol ted in
r in
s
cesses of ll origins,
ut the ex ct
cteri l or depends on the c use of the

scess (T
le 12.3). Streptococci re isol ted from pproxim tely 80% of
r in

scesses. The most common single species is the
et -h emolytic c r
oxyphilic Str
ep. milleri, micro erophilic streptococcus which grows in n ero
ic culture n
d lso 10% c r
on dioxide. The m jor h
it t of Strep. milleri is the liment ry
tr ct, including the mouth nd dent l pl que. The ssoci tion
etween front l l
o
e
scesses, Strep. milleri nd sinusitis indic tes th t the source of infecti
on in m ny
r in
scesses is the upper respir tory tr ct, the org nism p ssing
into the
r in from the p r n s l sinuses.
Histop thology
The
scess
egins s sm ll re of foc l in mm tion cere
ritis consisting of p
olymorphonucle r leukocytes, lymphocytes nd pl sm cells, which migr te from th
e peripher l
lood
INFECTIONS OF THE CENTRAL NERVOUS SYSTEM
175
T
le 12.3 Cere
r l
scess p thogenesis nd princip l org nisms. History Sinusi
tis front l Site of
scess Front l lo
e Predomin nt org nisms Aero
ic nd n er
o
ic streptococci Streptococcus milleri H emophilus species Mixed or Aero
ic nd
n ero
ic streptococci Enteric
cteri or Entero
cteri B cteroides fr gilis
H emophilus species Aero
ic streptococci An ero
ic streptococci Entero
cteri S
t phylococcus ureus St phylococcus ureus
M stoiditis, otitis
Tempor l lo
e
H em togenous, cryptogenic

Br in
Tr um
Br in
Otogenic
scesses usu lly yield mixed or , including
cteroides (B cteroides
fr giles), v rious streptococci nd mem
ers of the Entero
cteri ce e (Escherich
i coli, Proteus nd Pseudomon s species). Met st tic h em togenous infection m
y
e due to v rious ero
ic nd n ero
ic streptococci, entero
cteri nd other
Gr m-neg tive
cilli. St ph. ureus is often the p thogen in
scesses resulti
ng from tr um nd c n lso
e seen in postoper tive
scesses.
the
scess involves n eloquent intr cere
r l loc tion it m y present when quit
e sm ll. Altern tively, n
scess in the front l lo
e m y re ch l rge size
e
fore producing ny m jor neurologic l de cit. A
out h lf the p tients with
r in

scess h ve systemic symptoms, including fever, t the time of the di gnosis. M
rked toxic symptoms m y
e ttri
ut
le to the
scess rupturing into the ventri
cle or ssoci ted meningitis.
Di gnosis
CT sc nning h s
een responsi
le for dr m tic reduction in the mort lity from
cere
r l
scess
ec use of its
ility to di gnose single nd multiple
scesse
s nd to loc lize the lesion ccur tely. The CT sc n or m gnetic reson nce im gi
ng ppe r nce is typic lly ring-enh ncing m ss often surrounded
y consider
l
e oedem (Figs 12.112.4). The enh ncing c psule is usu lly thinner dj cent to th
e ventricle comp red with the more super ci l c psule. The lesions m y
e multiple
(Fig. 12.2
) or multilocul ted (Fig. 12.3). In the e rly st ges of development
of the
scess, when the infection is loc lized s cere
ritis, the CT sc n or MRI
ppe r nce will
e n re of low
Presenting fe tures
P tients present with fe tures of: 1 An intr cr ni l m ss: ( ) r ised intr cr ni
l pressure (
) foc l neurologic l signs, e.g. hemip resis, dysph si (c) epilep
tic seizures. 2 Systemic toxicity fever nd m l ise. 3 Clinic l fe tures of the
underlying source of the infection sinusitis,
cteri l endoc rditis, diverticul
itis. The clinic l fe tures develop over 24 weeks, lthough slower progression
is not unusu l. If
176
CHAPTER 12
density which enh nces fter intr venous contr st
ut without the typic l ring pp
e r nce nd usu lly with m rked dj cent oedem . MRI is more sensitive investi
g tive tool nd c n help differenti te
etween n
scess nd tumour. If the
s
cess is due to h em togenous spre d it is usu lly loc ted t the grey/white m tt
er junction (Fig. 12.1).
Peripher l
lood ex min tion m y show leukocytosis. R ised erythrocyte sedimen
t tion r te nd positive
lood cultures m y
e o
t ined if there is coexisting
septic emi .
M n gement
The principles of tre tment re to: identify the
cteri l org nisms institute
nti
iotic ther py dr in or excise the
scess. A specimen of the pus is essenti
l for ccur te identi c tion of the org nism so th t the ppropri te nti
iotic th
er py c n
e commenced. Occ sion lly, the org nism c n
e identi ed from positiv
e
lood culture or other o
vious source of infection. A lum
r puncture is
sol

utely contr indic ted ( nd of no di gnostic
ene t) in the presence of cere
r l

scess.
Fig. 12.1 Cere
r l
scess e rly in its development. A sm ll contr st-enh ncing
lesion surrounded
y lowdensity cere
r l oedem .
Surgic l tre tment Surgic l tre tment of the
scess involves either: spir tion
of the
scess through
urr hole, with repe ted spir tions s required or ex
cision of the
scess. Dr in ge of the
scess through
urr hole, if necess ry
using CT-guided stereot xis, is s fe, effective w y of o
t ining the pus nd
emptying the
scess. It is frequently necess ry to repe t
( )
(
)
Fig. 12.2 ( ) Typic l ring-enh ncing cere
r l
scess in the front l lo
e with s
urrounding cere
r l oedem . (
) Multiple cere
r l
scesses.
INFECTIONS OF THE CENTRAL NERVOUS SYSTEM
177
Fig. 12.3 Multilocul ted cere
r l
scess.
Fig. 12.4 CT sc n. L rge cere
r l
scess.
Anti
iotic ther py As soon s pus h s
een o
t ined, nti
iotic ther py should c
ommence. The initi l choice of nti
iotic,
efore culture results re v il
le,
will depend on the pro

le c use of the
r in
scess nd the Gr m st in. The
usu l initi l tre tment is high-dose penicillin, ceftri xone nd metronid zole.
Strep. milleri is the org nism most frequently found in
scesses of sinusitic o
rigin involving the front l lo
e; it is highly sensitive to penicillin. A
scesse
s of otitic origin, usu lly occurring in the tempor l lo
e, re c used
y wide
r nge of ero
ic nd n ero
ic
cteri . A com
in tion of penicillin with cefot
xime, ceftri xone or metronid zole should
e used. A
scesses of met st tic or c
ryptogenic origin m y
e c used
y streptococci or
y mixture of
cteri , nd

ro d-spectrum ther py, including penicillin, should
e used until
cteriologi
c l results re v il
le. As m ny of the org nisms re resist nt to penicillin,
it h s
een suggested th t penicillin se-st
le penicillin (e.g. uclox cillin)
should
e su
stituted. For postsurgic l
r in
scess it is recommended th t v
ncomycin
e used with cefot xime or ceftri xone. As soon s the culture results
re v il
le the ppropri te nti
iotic should
e used intr venously in high do
ses nd selection should
e m de
e ring in mind the nti
iotics penetr tion into
the
r in nd CSF. Corticosteroid ther py (dex meth sone) m y
e necess ry to r
educe cere
r l oedem nd should
e considered in p tients who re drowsy or h v
e deterior ting neurologic l st te despite surgery nd nti
iotic tre tment. A
nticonvuls nt medic tion should
e commenced s there is n incidence of seizure
s in
etween 30 nd 50% of c ses.
the spir tions nd this is
est ev lu ted
y follow-up CT sc ns. Surgic l excis
ion of the
scess should
e considered if: there is persistent re ccumul tion o
f pus despite repe ted spir tions the
scess is in n ccessi
le site there is
well-formed
rous c psule which f ils to coll pse despite repe ted spir tions
there is cere
ell r
scess.
Prognosis
The improvements in nti
iotic ther py nd CT sc nning h ve dr m tic lly reduced
the mort lity of
cteri l
scesses from 50% to less th n 10%.

Epidur l
scess
Cr ni l epidur l
scess results following:
178
CHAPTER 12
tr um surgery cr niotomy or insertion of skull tr ction tongs p r n s l sinusit
is or m stoiditis. The condition is frequently ssoci ted with osteomyelitis of
the cr ni l v ult.
penetr ting wounds, or m y follow surgery. In inf nts su
dur l empyem m y occur
s n infection of the su
dur l sp ce following meningitis.
Micro
iology
The most common
sitis is Strep.
r n ero
es nd
th t indic ted

org nism responsi
le for su
dur l empyem following front l sinu

milleri. However, other ero
ic nd n ero
ic streptococci, othe
St ph. ureus m y lso
e responsi
le, with p ttern simil r to
for intr cere
r l
scesses.

Clinic l fe tures
The clinic l fe tures of n epidur l
scess re prim rily those of osteomyeliti
s, with cute loc lized p in nd tenderness nd loc lized pitting oedem of the
sc lp over the ffected re , descri
ed
y Perciv l Pott nd known s Potts puffy
tumour. There re usu lly systemic symptoms of infection. When the epidur l colle
ction enl rges the p tient will st rt to h ve symptoms of r ised intr cr ni l pr
essure nd, if the m ss is l rge enough, ssoci ted symptoms of neurologic l d
e cit. The most common org nisms re ero
ic nd n ero
ic streptococci nd St ph.
ureus.
Clinic l present tion
In contr st to p tients with extr dur l
scesses, p tients presenting with su
d
ur l empyem s re usu lly seriously ill,
eing toxic nd fe
rile, with fe tures
of meninge l irrit tion. They frequently h ve r pidly progressive neurologic l s
igns, including depressed conscious st te, hemip resis nd dysph si . Epileptic
seizures occur in most p tients. The cl ssic present tion is p tient with hi
story of cute front l sinusitis who develops severe he d ches nd high fever n
d h s r pid neurologic l deterior tion with seizures.
Tre tment
Tre tment involves ev cu tion of the pus, excision of ny infected
one nd surg
ic l er dic tion of the underlying c use (e.g. sinus infection). High-dose nti

iotic ther py should
e commenced with di-/ uclox cillin plus gent mycin. This mus
t
e utilized until the org nism is identi ed nd sensitivity determined. The di g
nosis is m de on CT sc n or MRI, which will show the extr dur l collection of pu
s s well s osteomyelitis nd the infected sinuses.
Di gnosis
The differenti l di gnosis includes: vir l enceph litis
cteri l meningitis
r
in
scess septic c vernous sinus throm
osis.
Su
dur l
scess
A su
dur l
scess is n uncommon
ut potenti lly life-thre tening infection wit
h possi
le serious neurologic l sequel e in the p tients who survive. The
sces
s follows infection in the p r n s l sinuses, p rticul rly front l sinusitis nd
, less commonly, infection in the m stoid ir cells. The infection in the su
dur
l sp ce is n extension of the sinusitis through emiss ry veins nd retrogr de
throm
ophle
itis. Su
dur l empyem m y lso result from
Investig tions

Evidence of underlying sinusitis will
e present on pl in skull X-r y nd CT sc

n. The CT sc n or MRI ndings m y
e su
tle, s the
scess is usu lly iso- or hyp
odense. The interhemispheric collection c n e sily
e overlooked nd thus must

e considered in p tients with this sort of clinic l present tion. There m y
e c
ontr st enh ncement of the underlying in med mem
r nes nd
INFECTIONS OF THE CENTRAL NERVOUS SYSTEM
179
evidence of underlying cere
r l oedem , lthough these fe tures re not univers
l. Lum
r puncture should not
e performed if the di gnosis is suspected clinic
lly, s it is h z rdous nd frequently not di gnostic. There is usu lly pleocy
tosis in the CSF with elev ted protein,
ut the sug r m y
e norm l nd org nism
s c nnot usu lly
e cultured.
Tu
erculosis
Tu
erculosis involving the
r in is unusu l in Western countries
ut is not unco
mmon in Indi , Afric , Asi , the Middle E st, South Americ nd e stern Europe.
In developed countries tu
erculosis is more likely to present in p tients who r
e immunocompromised HIV-positive nd immunosuppressed p tients. The infection m
y occur s: tu
erculous meningitis intr cr ni l tu
erculom s.
Tre tment
Su
dur l empyem is surgic l emergency. The principles of tre tment, s for n
y
scess, re: tre t the source of sepsis sinus surgery (if indic ted) dr in th
e
scess identify the org nism tre t with ppropri te high-dose nti
iotics. Th
e surgic l techniques used to dr in the
scess re either multiple
urr holes,
cr niotomy or cr niectomy. The dv nt ge of the multiple
urr hole technique is
th t the pus m y
e widespre d nd
il ter l nd the p tient is frequently serio
usly ill. However, it m y
e dif cult to dr in the pus dequ tely through
urr hol
es, s the underlying oedem tous
r in m y swell up to occlude the hole. In ddi
tion, it is dif cult to provide dequ te dr in ge for the p r f lcine pus, which f
requently lies
etween the f lx nd the medi l spect of the hemispheres, throug
h
urr holes. The su
dur l sp ce should
e irrig ted with nti
iotic solution t
the time of surgery nd su
dur l c theters should
e left in pl ce for further
dr in ge of pus nd postoper tive nti
iotic irrig tion. It is rgu
le whether
the nti
iotic irrig tion is useful
ut it is pro

ly worthwhile, in view of th
e seriousness of the situ tion. High-dose systemic nti
iotic ther py should
e
commenced s soon s pus is o
t ined for culture. The initi l ther py will depen
d on the underlying c use, s for cere
r l
scess,
e ring in mind th t the mos
t common infecting org nism is penicillin-sensitive Strep. milleri from front l
sinusitis. Further nti
iotic ther py will depend on the results of the culture.
Anticonvuls nt ther py is import nt s the p tients h ve high incidence of se
izures.
Tu
erculous meningitis
Tu
erculous meningitis is usu lly su
cute illness; p tients present with he d
ches, confusion nd fe tures of meningitis. It occurs more frequently in childr
en nd will result in second ry p thologic l ch nges nd neurologic l de cit, incl
uding:
s l r chnoiditis c using hydroceph lus visu l f ilure due to r chnoid
itis round the optic p thw ys multiple cr ni l nerve p lsies due to the
s l
r chnoiditis rteritis c using cere
r l inf rction. CSF ex min tion will show: l
ymphocytic pleocytosis (100500 cells/mm3) elev ted protein (gre ter th n 0.8 g/l)
low glucose (less th n 2 mmol/l) low chloride (less th n 110 mmol/l) cid-f st

cilli in 20% of p tients using ZiehlNeelsen st in. The de nitive di gnosis is o
ften only m de on culture of M. tu
erculosis, which m y t ke up to 6 weeks. Anti
tu
erculous ther py should
e commenced if tu
erculous meningitis is suspected,
s it is inv ri
ly f t l within 6 weeks nd is often more r pidly f t l. The n
titu
erculous medic tion includes isoni zid, rif mpicin, eth m
utol nd pyr zin

mide. Hydroceph lus should
e tre ted with ventriculoperitone l shunt. Steroid
ther py h s
een used to diminish the risk of r chnoid dhesions nd rteritis

ut is pro

ly of little
ene t. A
rief course m y
e indic ted in p tients wit
h
180
CHAPTER 12
r ised intr cr ni l pressure. The serum sodium in older p tients m y drop, pro


ly due to in ppropri te ntidiuretic hormone secretion, nd should
e tre ted

y uid restriction.
Cere
r l cryptococcosis presents s: meningitis meningoenceph litis cere
r l gr
nulom .
Intr cr ni l tu
erculom
An intr cr ni l tu
erculom or
scess origin tes
y h em togenous spre d from t
u
erculous lesions in other p rts of the
ody, especi lly the lung. They re fre
quently multiple nd re predomin ntly loc ted in the posterior foss in childre
n nd young dults,
ut m y occur throughout the cere
r l hemispheres. The clini
c l present tion is simil r to n intr cr ni l tumour, with fe tures of r ised i
ntr cr ni l pressure, foc l neurologic l signs nd epileptic seizures. Systemic
symptoms of tu
erculosis, such s fever, excessive perspir tion nd leth rgy, oc
cur in less th n 50% of c ses. The CT sc n or MRI ppe r nce of tu
erculous gr
nulom will show n re of low ttenu tion with surrounding v sogenic oedem .
The enh ncement on this occ sion tends to
e peripher l. Once g in there m y
e
multiple lesions. There is usu lly surrounding oedem nd the lesions m y
e mu
ltiple. The tu
erculom is occ sion lly c lci ed. The preoper tive di gnosis is us
u lly ppreci ted only fter recognition of tu
erculous foci elsewhere in the
o
dy. The optim l tre tment is surgic l excision of the tu
erculom , if it is in
surgic lly ccessi
le region, nd ntitu
erculous chemother py.
Meningitis The most common present tion is meningitis, which is usu lly su
cute
, nd the p tients present with incre sing he d ches followed
y vomiting, seizu
res nd imp ired conscious st te. P pilloedem occurs in up to h lf of p tients
nd cr ni l nerve p lsies m y develop. Meningoenceph litis This will develop if
the meninge l infection extends long the VirchowRo
in sp ces into the
r in. Int
r cere
r l gr nulom s These re uncommon in cryptococc l infection
ut m y devel
op in conjunction with meningitis or in isol tion (Fig. 12.5). P tients present
with symptoms of r ised intr cr ni l pressure, convulsions nd neurologic l de cit
including lower cr ni l nerve p lsies. The CSF studies will show:
Cere
r l cryptococcosis
Cryptococcosis (torul ) is fung l infection th t m y involve the CNS. Cryptoco
ccus neoform ns is commonly found in vi n h
it ts, nd p rticul rly mong pige
ons. The usu l port l of entry is
y inh l tion of the ir
orne cryptococcus. Up
to h lf of p tients with CNS involvement h ve n underlying predisposing condit
ion such s AIDS, intr venous drug use, s rcoidosis, lymphom or prolonged stero
id ther py, nd some p tients lso h ve cryptococc l lesions in the lung.
Fig. 12.5 Cryptococc l gr nulom . A contr stenh ncing m ss with surrounding oede
m .
INFECTIONS OF THE CENTRAL NERVOUS SYSTEM
181
elev ted pressure pleocytosis usu lly lymphocytes elev ted protein decre sed glu
cose (in 50%) Cryptococcus neoform ns on wet prep r tion st ined with Indi ink

positive l tex cryptococc l gglutin tion test which detects cryptococc l c psul
r ntigen in CSF.
Tre tment
Tre tment consists of nticryptococc l ther py using mphotericin B, 5- ucytosine
or ucon zole. Intr cere
r l gr nulom s m y need to
e excised nd thor cotomy m
y
e necess ry for lung lesion. Hydroceph lus is common complic tion of cry
ptococc l infection of the CNS system nd should
e tre ted with shunt.
Fig. 12.6 Hyd tid d ughter cyst or
rood c psule.
Hyd tid
Hyd tid dise se is endemic in rur l re s, p rticul rly those involved with shee
p nd c ttle r ising such s the western region of Victori in Austr li , South
Americ nd South Afric . Echinococcus gr nulosus is sm ll t peworm,
out 6 m
m long nd with pproxim tely four segments, which lives in the sm ll
owel of c
nines. The ov re shed in the f eces of dogs nd the intermedi te hosts re c
ttle nd sheep, lthough hum ns m y lso serve in this c p city. Following inges
tion the egg c psule is digested nd the hexoc nth oncosphere penetr tes the int
estin l mucos nd p sses into the port l circul tion. Most re tr pped in the l
iver (65%) or lungs (20%) nd less th n 5% p ss to the
one or CNS. The em
ryos
lodge within the c pill ries nd will develop into cysts, which progressively in
cre se in size. The cyst is filled with the cle r hyd tid fluid, round which is
n inner nucle ted germin tive l yer nd n outer op que non-nucle ted l yer wi
th delic te l min tions. D ughter cysts develop from the germin tive l yer (Fig.
12.6). The infl mm tory re ction occurring in the tissue which surrounds the l
min ted l yer does not usu lly occur in the
r in.
The neurosurgeon is involved when the hyd tid cyst lodges in the
r in, verte
r
e or or
it.
Intr cere
r l hyd tid cyst
Intr cere
r l hyd tid cyst presents s m ss lesion with slowly developing neur
ologic l involvement. Hyd tid cysts in the
r in re usu lly single nd l rge n
d re often prim ry. In r re inst nces they re em
olic nd multiple nd c n ri
se from cysts in the left ventricle of the he rt. Cysts c n lso rise from rupt
ure of cere
r l cyst nd second ry seedings. The MRI or CT sc n (Fig. 12.7) sh
ows hypodense cyst with minim l or low enh ncement round the m rgins. The sur
gic l tre tment is excision nd gre t c re should
e t ken to remove the cyst in
t ct. If the contents re spilled the hyd tid dise se m y
e dissemin ted throug
h the CNS nd n phyl ctic shock h s occ sion lly
een reported. Or
it l involve
ment is usu lly m nifest
y unil ter l proptosis. If the verte
r l column is inv
olved there will
e destruction of c ncellous
one with verte
r l coll pse nd p
ossi
le cord compression. If cere
r l hyd tid dise se is suspected, clinic l ex
min tion nd r diologic l investig tion m y show involvement of the liver nd lu
ng. Serologic l investig tions help in the di gnosis.
182
CHAPTER 12
Fig. 12.8 MRI following g dolinium showing intr cere
r l toxopl smosis.
Fig. 12.7 Hyd tid cyst in front l lo
e extending into the l ter l ventricle.
Medic l ther py is with prolonged use of l
end zole.
AIDS
Acquired immune de ciency syndrome (AIDS) is due to the T-cell lymphotrophic virus
type III, lso known s the hum n immunode ciency virus (HIV) type I. The virus

tt cks the p tients immune system, rendering the p tient prone to opportunistic i
nfection or m lign ncy. The virus is lso neurotrophic nd c n involve the CNS d
irectly. Approxim tely 10% of p tients re di gnosed due to CNS symptoms t pres
ent tion nd up to 70% of p tients h ve centr l nervous symptoms t de th. CNS m
nifest tions h ve sever l c uses. Second ry infection. The most common infectio
n is Toxopl sm gondii, which is m ss lesion nd m y present with fe tures ind
istinguish
le from tumour. The lesions m y
e solit ry or multiple nd re us
u lly ring-enh ncing on CT
Fig. 12.9 MRI of herpes simplex enceph litis with m jor involvement of the right
tempor l lo
e nd less severe ch nges in the left tempor l lo
e.
sc n nd MRI (Fig. 12.8). Cere
r l toxopl sm infections re tre ted with sulf d
i zine nd pyrimeth mine. Cryptococcus neoform ns is the second most common c us
e of CNS infection nd other infections include tu
erculosis, C ndid l
ic ns,
herpes simplex enceph litis (Fig. 12.9) nd progressive multifoc l leukoenceph l
op thy. M lign nt dise se of the
r in m y
e either prim ry CNS lymphom , secon
d ry lymphom from systemic dise se or second ry K posis s rcom . The AIDS virus
m y infect the nervous system directly, c using the AIDS dementi complex in
INFECTIONS OF THE CENTRAL NERVOUS SYSTEM
183
over 90% of p tients. This m y present s
eh viour l ch nge simil r to presen
ile dementi . Tr nsverse myelitis nd peripher l neurop thy m y lso occur. The
dvis
ility of
iopsy of intr cere
r l lesions in AIDS p tients nd the possi
l
e
ene ts re not cle r t this st ge, s there is no s tisf ctory tre tment of th
e underlying dise se process. A re son
le ppro ch is to tre t HIV-positive p t
ients with cere
r l lesions th t h ve the r diologic l ppe r nce typic l of tox
opl smosis with ther py for Toxopl sm infection, nd to reserve
iopsy for p ti
ents who do not respond to ther py, or if the r diologic l ppe r nce is typic
l for toxopl smosis.
lesions or hydroceph lus. Medic l tre tment is with l
end zole or pr ziqu ntel.
Steroids re used in the e rly st ge of tre tment to ttenu te the cute in mm t
ory response.
Herpes simplex enceph litis
This vir l infection c n present with r pid onset of fever, he d ches, vomiting
nd progressive neurologic l deterior tion. The type 1 virus is the c us tive or
g nism in most of the dult c ses. It presents s n cute necrotizing enceph li
tis nd h s high mor
idity nd mort lity in the
sence of e rly ntivir l the
r py. P tients present with symptoms of meningitis, progressive deterior ting
neurologic l st te nd seizures. Di gnosis c n
e re ched with electroenceph log
r phy th t reve ls foc l slowing, periodic spikes or sh rp nd w ve p tterns. CT
sc nning c n
e norm l initi lly or reve l oedem -like ch nges in the tempor l
or front l lo
es. This c n
e ssoci ted with re s of h emorrh ge. MRI is the i
nvestig tion of choice, reve ling the sign l ch nges within the tempor l lo
e wi
th oedem nd h emorrh ge (Fig. 12.9). The di gnosis is con rmed
y the presence o
f mononucle r cells in the CSF nd the detection of vir l DNA. E rly tre tment w
ith ciclovir is the tre tment of choice nd c n
e life-s ving. Very r rely doe
s the p tient develop r ised pressure to the extent of requiring intr cr ni l pr
essure monitoring, ventricul r dr in or decompressive cr niotomy.
Neurocysticercosis
This is the most common p r sitic dise se of the centr l nervous system. It is c
used
y the l rv l st ge of t peworm T eni solium. Hum n
eings re the only
de nitive host for this p r site, whilst
oth hum ns nd pigs re the intermedi t
e hosts for the em
ryonic form. Infest tion occurs
y e ting poorly cooked infes
ted food or through cont min tion of food
y fertilizer cont ining the eggs. The

l rv penetr tes the intestin l w ll nd g ins ccess to
r in, muscle or eye v

i the
loodstre m. There re multiple forms of neurocysticercosis, the commones
t form
eing p renchym l dise se. However, the cyst c n lso
e concentr ted in
the su
r chnoid sp ce, intr ventricul r sp ce, intr sell r region or spin l c n
l. Neurocysticercosis commonly h s multiple cysts t v rious st ges of the dise
se process from c lci ed lesions to cysts of v rying sizes in v rying loc tions
nd often v ri tions in terms of enh ncement (when present). The lesions c n pres
ent with hydroceph lus, com , convulsions nd neurologic l de cits. A lum
r punct
ure ex min tion of the CSF c n
e di gnostic provided it is not contr indic ted.
This cyst c n occ sion lly
e found in the CSF; however, the presence of lymp
hocytosis, eosinophili nd positive complement x tion test will contri
ute to
the di gnosis. Surgic l intervention is indic ted for m ss
Further re ding
Becker GL et l. (1980) Amoe
ic
scesses of the
r in. Neurosurgery 6, 192194. B
ell WE (1981) Tre tment of
cteri l infections of the centr l nervous system. A
nn ls of Neurology 9, 313327. Brown E (1987) Antimicro
i l prophyl xis in neurosu
rgery. British Journ l of Neurosurgery 1, 159162. Ch n KH, M nn KS, Yue DP (1989)
Neurosurgic l spects of cere
r l cryptococcosis. Neurosurgery 25, 4448. Everett
ED, St us
ugh LJ (1980) Antimicro
i l gents nd the centr l nervous system. N
eurosurgery 6, 691714.
184
CHAPTER 12
G rvey G (1983) Current concepts of
cteri l infections of the centr l nervous
system. Journ l of Neurosurgery 59, 735744. Gotv i P, De Louvis J, Hurley R (1987
) The
cteriology nd chemother py of cute pyogenic
r in
scess. British Jou
rn l of Neurosurgery 1, 189204. H ines SJ (1989) Ef c cy of nti
iotic prophyl xis
in cle n neurosurgic l oper tions. Neurosurgery 24, 401405. Hl vin ML, K minski H
J, Fensterm ker RA, White RJ (1994) Intr cr ni l suppur tion: modern dec de of
postoper tive su
dur l empyem nd epidur l
scess. Neurosurgery 34, 974981. M
mp l m TJ, Rosen
lum ML (1988) Trends in the
m n gement of
cteri l
r in
scesses: review of 102 c ses over 17 ye rs. Ne
urosurgery 23, 451458. M ndel GL, Dougl s RG, Bennett JE (1985) Principles nd Pr
ctice of Infectious Dise ses, 2nd edn. John Wiley, New York. Rich rds P (1987)
AIDS nd the neurosurgeon. British Journ l of Neurosurgery 1, 163172. Rosen
lum M
L, Levy RM, Bredesen DE (1986) Neurologic l implic tions of the cquired immunod
e ciency syndrome (AIDS). Clinic l Neurosurgery 34, 419445. Steph nov S (1988) Surg
ic l tre tment of
r in
scesses. Neurosurgery 22, 724730.
CHAPTER 13
13
Low
ck p in nd leg p in
A
out 90% of the popul tion suffer from low
ck p in t some time nd 30% of th
ese will develop leg p in due to lum
r spine p thology. The critic l f ctor in
ssessing p tients with low
ck p in is whether there re lso fe tures of lum

os cr l nerve root compression, such s leg p in or foc l signs of neur l compre
ssion in the lower lim
s. In gener l, neurosurgeons re princip lly concerned wi
th lum
r spine p thology th t c uses nerve root compression. Sci tic is the cl
inic l description of p in in the leg due to lum
os cr l nerve root compression
which is usu lly in the distri
ution of the sci tic nerve. Sci tic w s rst menti
oned in n Egypti n m nuscript d ted 25003000 BC. In 1934 Mixter nd B rr est
li
shed th t prol psed lum
r interverte
r l disc w s commonly the c use of sci t
ic . Lum
r c n l stenosis, n rrowing of the lum
r spin l c n l, is the other

m jor spin l c use of leg p in. In 1949 Ver
iest speci c lly de ned the clinic l si
gni c nce of the n rrow spin l c n l nd the syndrome of intermittent neurogenic c
l udic tion of the legs. A lum
r disc prol pse c n occur t ny ge in dults

ut is uncommon in teen gers. The symptoms of lum
r c n l stenosis usu lly comme
nce fter the 5th dec de. Although lum
r c n l stenosis nd lum
r disc prol ps
e m y
e present in the s me p tient, they e ch produce distinct clinic l enti
ty which will
e descri
ed sep r tely.
Sci tic
Aetiology (T
le 13.1)
The most common c use of sci tic is lum
r disc prol pse c using nerve root c
ompression. Sci tic -type p in m y lso occur s result of
ony compression of
the nerve root, usu lly
y n osteophyte, nd is often ssoci ted with lum
r c
n l stenosis or spondylolisthesis. N rrowing of the l ter l recess of the spin l
c n l m y lso occur in conjunction with lum
r c n l stenosis, nd m y c use co
mpression of nerve root. Sci tic m y occ sion lly
e c used
y tumours of the
c ud equin or
y pelvic tumours, such s spre d from c rcinom of the rectum.
An tomy nd p thology
Ne rly 75% of the lum
r exionextension nd of tot l spin l movement occurs t the
lum
os cr l junction, 20% of lum
r exionextension occurs t the L4/5 level nd t
he rem ining 5% is t the upper lum
r levels. Consequently, it is not surprisin
g th t 90% of lum
r disc prol pses occur t the lower two lum
r levels; the mo
st frequently ffected disc is t the L5/S1 level. The lum
r disc consists of
n intern l nucleus pulposus surrounded
y n extern l l min r
rous cont iner, th
e nnulus
rosus. A disc prol pse m y consist of the nucleus pulposus
ulging, wi
th the nnulus
eing stretched
ut int ct. Altern tively, the nucleus m y ruptur
e through the nnulus nd sequestr te s free fr gment under the posterior lon
gitudin l lig ment or lie in the extr dur l sp ce. Prol pse of 185
186
CHAPTER 13
T
le 13.1 C uses of sci tic . Prol psed lum
r disc Lum
r spondylosis (osteoph
yte) Lum
r c n l stenosis (l ter l recess) Lum
r spondylolisthesis C ud equin
tumours (e.g. ependymom ) Pelvic tumours (e.g. rect l c rcinom ) Spin l rteri
ovenous m lform tion (r re)
the disc is usu lly in posterol ter l direction, s the posterior longitudin l
lig ment prevents direct posterior herni tion. Less frequently the disc m y her
ni te l ter lly to tr p the nerve in the neur l for men. A prol psed interverte

r l disc c uses compression of the nerve which runs long the posterior spect o
f the disc nd down under the pedicle of the verte
r
elow (Fig. 13.1). Consequ
ently, n L4/5 posterol ter l interverte
r l disc prol pse will usu lly compress
the L5 nerve root, which runs c ud lly cross the disc to enter the neur l for
men
elow the L5 pedicle. Simil rly, lum
os cr l (L5/S1) disc prol pse will us
u lly ffect the S1 nerve root. The less common l ter l disc prol pse will c use
compression of the nerve root t one level higher th n expected (e.g. L4 nerve
root compression due to L4/5 l ter l disc prol pse). In the c se of l rge disc
prol pse, there m y
e evidence of more th n one nerve root compression. C ud
equin compression m y result if the disc herni tion is suf cient to rupture the p
osterior longitudin l lig ment nd produce posterior centr l disc prol pse.
( )
(
)
Fig. 13.1 The di gr m shows ( ) posterol ter l lum
r disc propl pse c using c
ompression of lum
r nerve root p ssing cross the disc to enter the neur l c n

l
elow the pedicle nd (
) l ter l disc prol pse c using compression of the n
erve root p ssing
ene th the pedicle
ove the disc prol pse.
P tient ssessment
The p tient suffering from sci tic will
e in o
vious discomfort, which will
e
re ected
y movements nd posture when lying supine. The p tient lies tilted, usu
lly to the side opposite to the sci tic , with the ffected hip nd knee slight
ly exed t king pressure off the stretched nerve. The p in is worse on movement, c
oughing, sneezing or str ining. Although
ck p in
m y
e present, the import nt fe ture is the p in which r di tes down the leg in
the distri
ution of the ffected nerve. The p in usu lly r di tes into the
utt
ock, long the posterol ter l spect of the thigh nd c lf into the foot (S1 ner
ve root); it m y extend into the dorsum of the foot nd gre t toe (L5 nerve root
). An L3/4 disc herni tion m y produce p in in the posterior thigh
ut, s with
n L2/3 disc prol pse, the p in is frequently long the nterior spect of the t
high. L4 root p in frequently r di tes into the nterior spect of the lower leg
. Depending on the degree of nerve root compression, the p tient m y compl in of
sensory distur
nce such s num
ness or tingling in the leg or foot, nd we kne
ss m y
e present. The history must include n ssessment of sphincter function,
s l rge disc prol pse m y c use c ud equin compression.
Ex min tion fe tures
Lum
r
ck movements m y
e restricted nd scoliosis m y
e seen, usu lly con
c ve to the side of the ffected leg. Str ight leg r ising (L segues test) will

e restricted on the ffected side nd, in severe c ses, p in in the ffected leg
will
e reproduced when the opposite leg is r ised.
LOW BACK PAIN AND LEG PAIN
187
T
le 13.2 Segment l innerv tion of lower lim
muscul ture. L1 L2 L3 L4 L5 S1 S2
S3 Pso s m jor; pso s minor Pso s m jor; ili cus; s rtorius; gr cilis; pectineu
s; dductor longus; dductor
revis Qu driceps; dductors (m gnus, longus,
revi
s) Qu driceps; tensor f sci e l t e; dductor m gnus; o
tur tor externus; ti
i l
is nterior; ti
i lis posterior Gluteus medius; gluteus minimus; o
tur tor inter
nus; semimem
r nosus; semitendinosus; extensor h llucis longus; extensor digitor
um longus nd peroneus tertius; popliteus Gluteus m ximus; o
tur tor internus; p
iriformis;
iceps femoris; semitendinosus; popliteus; g strocnemius; soleus; per
onei (longus nd
revis); extensor digitorum
revis Piriformis;
iceps femoris;
g strocnemius; soleus; exor digitorum longus; exor h llucis longus; some intrinsic
foot muscles Some intrinsic foot muscles (except
ductor h llucis; exor h lluci
s;
revis; exor digitorum
revis; extensor digitorum
revis)
Ex min tion of the neurologic l dis
ility should proceed in n ordered f shion.
Initi lly se rch is m de for w sting in speci c muscle groups, p rticul rly the q
u driceps, c lf muscles, extensor digitorum
revis muscle nd the sm ll muscles
of the foot. The p tient is then ex mined for we kness in e ch of the muscle gro
ups (T
les 13.2 nd 13.3). We kness of dorsi exion of the foot nd extension of t
he gre t toe (extensor h llucis longus) is most commonly c used
y prol psed L
4/5 interverte
r l disc with involvement of the L5 nerve root; severe c ses m y
result in complete foot drop. Pl nt r exion we kness is c used
y compression o
f the S1 nerve root, usu lly due to prol psed lum
os cr l disc. However, pl nt
r exion is very strong movement nd ny we kness m y
e dif cult to elicit unles
s tested
y sking the p tient to st nd on the toes of the ffected side. A l rg
e prol psed disc t the L4/5 level m y result in some pl nt r exion we kness
ec
use of compression of the S1 nerve root, nd simil rly l rge lum
os cr l disc
prol pse m y
e ssoci ted with dorsi exion we kness due to L5 nerve root compress
ion. The deep tendon re exes should
e c refully tested s they provide o
jective

evidence of nerve root compression. The nkle jerk is depressed or
sent when t
he S1 nerve root is compressed, usu lly
y lum
os cr l disc prol pse.
T
le 13.3 Segment l innerv tion of lower lim
joint movements. Hip Flexors, dd
uctors, medi l rot tors Extensors,
ductors, l ter l rot tors Extensors Flexors
Dorsi exors Pl nt r exors Invertors Evertors Intrinsic muscles L1, 2, 3 L5, S1 L3,
4 L5, S1 L4, 5 S1, 2 L4, 5 L5, S1 S2, 3
Knee Ankle Foot
Sens tion should
e tested in the foot nd leg (Fig. 13.2). At the end of the ex
min tion the p tient should
e turned prone so th t the
uttocks c n
e inspect
ed for trophy of the glute l muscles, sens tion c n
e tested long the
ck of
the legs nd in the peri n l region, nd n l tone c n
e ssessed. A rect l ex
min tion should
e performed if there re clinic l fe tures suggestive of pel
vic tumour.
188
CHAPTER 13
S4 L1 Ventr l xi l line L2 S2 L3 Extension forw rds from dors l xi l line L4 L
5 L3 S3
L2
Dors l xi l line
L3/4 prol psed interverte
r l disc P in in the nterior thigh W sting of the qu
driceps muscle We kness of the qu driceps function nd dorsi exion of foot Diminis
hed sens tion over nterior thigh, knee nd medi l spect of lower leg Reduced k
nee jerk.
M n gement
Most p tients with sci tic chieve good p in relief with simple conserv tive tr
e tment nd less th n 20% will require surgery. The likelihood of symptom tic re
lief without surgery is rel ted to the p thology of the disc prol pse. A
ulging d
isc is likely to settle with simple conserv tive me sures,
ut sci tic due to
nucleus pulposus th t h s herni ted out of the disc sp ce nd sequestr ted outsid
e the nnulus will pro

ly need surgery for s tisf ctory relief of symptoms.
L4
L5
S1 L5 L5 S1
Fig. 13.2 Segment l distri
ution of nerves of the lum
r nd s cr l plexuses to
the skin of the nterior nd posterior spect of the lower lim
.
Summ ry of clinic l fe tures
Clinic l loc liz tion of the disc prol pse should
e possi
le in the m jority of
p tients with sci tic . The following fe tures re typic l (
ut not inv ri
le)
of disc herni tion. L5/S1 prol psed interverte
r l disc P in long the posterio
r thigh with r di tion to the heel We kness of pl nt r exion (on occ sion) Sensor
y loss in the l ter l foot A
sent nkle jerk. L4/5 prol psed interverte
r l disc
P in long the posterior or posterol ter l thigh with r di tion to the dorsum o
f the foot nd gre t toe We kness of dorsi exion of the toe or foot P r esthesi
nd num
ness of the dorsum of the foot nd gre t toe Re ex ch nges unlikely.

Conserv tive tre tment Most p tients chieve good p in relief following
ed rest
, usu lly for period of
out 710 d ys, nd the use of simple n lgesic gents
nd non-steroid l nti-in mm tory medic tion. Although tr ction is sometimes reco
mmended it pro

ly h s only limited
ene t nd m y result in lower leg complic ti
ons. Resolution of the p in is pro

ly due to com
in tion of some resorption
of the prol psed disc m teri l, the oedem of the nerve decre sing nd possi
le
d pt tion of the p in
res to pressure. Spin l m nipul tion is not recommended
nd the concept th t disc prol pse c n
e reduced
y m nipul tion is myth. Init
i lly, the only necess ry investig tions re pl in lum
r spine X-r y nd n e
rythrocyte sediment tion r te (ESR). The lum
r spine X-r y will di gnose n ss
oci ted spondylolisthesis which m y contri
ute to the sci tic , nd it lso help
s to exclude sinister p thology, such s met st tic tumour involving the spin l
verte
r e. The ESR will lso exclude systemic dise se. Some clinici ns dvoc te
the use of high-dose corticosteroids in the conserv tive m n gement of sci tic
due to lum
r disc prol pse. Whilst
LOW BACK PAIN AND LEG PAIN
189
steroid ther py m y help to give tr nsient p in relief, the limited
ene t is pro


ly outweighed
y the possi
le complic tions of corticosteroid tre tment. Chemo
nucleolysis h s, in the p st,
een dvoc ted s tre tment for lum
r disc prol
pse. It involves the intr disc l injection of proteolytic enzyme, such s chy
mop p in, which dissolves disc m teri l. Chymop p in w s rst isol ted in 1941 nd
h s
een used intermittently since 1963 in clinic l studies. There is sm ll r
isk of serious n phyl ctic re ction following intr disc l injection. Although c
hymop p in dissolves the norm l nucleus pulposus it h s high f ilure r te in t
he tre tment of prol psed disc, s it f ils to ffect the extruded disc m teri l
, nd further nerve compression m y occur following chemonucleolysis from the di
sc dissolving nd coll psing, resulting in n rrowing of the interverte
r l neur
l for men. The procedure is not recommended for use t this time.
mo
ilizing despite dequ te relief with
ed rest. In this group of p tients phys
iother py nd limited tri l of spin l
r ce might
e tried,
ut they usu lly
h ve only limited success. Neurologic l de cit. A signi c nt we kness or incre sing
mount of we kness is n indic tion for e rly investig tion nd surgery. Centr
l disc prol pse. P tients with
il ter l sci tic or other fe tures indic ting
centr l disc prol pse, such s sphincter distur
nce nd diminished perine l se
ns tion, should
e investig ted promptly. An cute centr l disc prol pse m y le
d to cute, severe, irreversi
le c ud equin compression nd should
e investig
ted nd tre ted s n emergency. Tumour. Surgery is indic ted if the clinic l f
e tures suggest th t tumour could
e the c use of sci tic .
Indic tions for surgery P in. The most common indic tion for surgery in p tients
with disc prol pse is p in in the following situ tions. Inc p cit ting p in des
pite 710 d ys of
ed rest. Continuing episodes of recurrent p in when
Investig tions
Lum
r myelogr phy (Fig. 13.3) w s the timehonoured investig tion for lum
r dis
c prol pse. The use of w ter-solu
le non-ionic contr st m teri l voids the risk
of the postmyelogr m r chnoiditis previously seen with the oil-
sed
( )
(
)
Fig. 13.3 Lum
r myelogr m using w ter-solu
le contr st medium showing ( ) poste
rol ter l L4/5 disc prol pse nd (
) complete
lock due to l rge centr l L5/S1
disc protrusion.

190
CHAPTER 13
mediums. Although myelogr phy is now much s fer, there is very sm ll risk of r
e ction to the contr st medium, p rticul rly epileptic seizures. Some p tients s
uffer he d ches following the myelogr m. These re due to the lum
r puncture (C
h pter 2) nd/or the effects of the contr st m teri l. High-qu lity computerized
tomogr phy sc nning (Figs 13.4 nd 13.5) nd m gnetic reson nce im ging (Fig. 1
3.6) h ve l rgely superseded myelogr phy for the di gnosis of lum
r disc prol p
se. The MRI is especi lly helpful in showing the size, con gur tion nd position o
f the disc prol pse, s well s ny ssoci ted nerve root or thec l compression.
In ddition the MRI will lso demonstr te p thology t other discs, such s deg
ener tive ch nges s evidenced
y decre sed sign l in the disc on the T2-weighte
d sc ns.
Oper tive procedure for lum
r disc prol pse
The oper tion involves excision of the disc prol pse with decompression of the
ffected nerve root. In the p st the oper tion usu lly ent iled complete or p r
ti l l minectomy, identi c tion of the compressed nerve root, its mo
iliz tion off
the disc prol pse nd excision of the herni ted disc. However, with improvement
s in instrument tion nd m gni c tion, e.g. the oper ting microscope, most disc pr
ol pses c n
e excised with minim l distur
nce to the norm l
ony n tomy nd w
ith the remov l of only sm ll mount of
one, usu lly from the dj cent l min
e on the side of the prol pse. A full l minectomy m y occ sion lly
e necess ry
prior to the disc excision of l rge centr l disc prol pse c using c ud equin
compression. A percut neous lum
r discectomy to remove the nucleus pulposus is
sometimes dvoc ted for the
ulging lum
r disc. However, if the disc is
ulging th
e sci tic will ne rly lw ys settle with conserv tive tre tment nd surgery is
not necess ry. A percut neous discectomy of the intr disc l contents will f il t
o relieve the sci tic if the disc h s ruptured through the nnulus,
ec use it
will not remove the herni ted disc m teri l c using the nerve root compression.
( )
(
)
Fig. 13.4 ( ) CT sc ns of lum
r spine showing posterol ter l disc prol pses. (

) CT sc n of left posterol ter l disc prol pse fter intr thec l contr st.
Postoper tive mo
iliz tion
Most p tients commence w lking the d y fter surgery nd re disch rged from hos
pit l on d y 2 or 3 following the oper tion. A gently gr du ted mo
ilizing progr
mme should
e c refully
LOW BACK PAIN AND LEG PAIN
191

nd str ining for the rst 4 weeks. A gr du ted ctive exercise progr mme c n comm
ence fter the rst month.
Prognosis following surgery
The results following lum
r disc surgery re directly rel ted to the ccur cy o
f the preoper tive clinic l ev lu tion. Excellent results c n
e chieved if: th
ere is good history of sci tic there re good signs of nerve root irrit tion
the investig tions show evidence of herni ted disc t surgery the nerve root i
s stretched
y disc prol pse the p tient is well motiv ted. If ny of the
ov
e criteri re
sent the results following surgery re dis ppointing. Recurrent

sci tic following surgery occurs in
out 10% of c ses nd is usu lly due to f
urther disc prol pse, either t the s me level or t nother level. The principl
es of m n gement re simil r to those descri
ed for the initi l tre tment of sci
tic . Recurrent sci tic is sometimes due to dhesions developing round the ne
rve root c using perineur l
rosis. The tre tment is conserv tive, with judicious
use of
ed rest followed
y gentle mo
ilizing exercises, simple n lgesic medic
tion nd non-steroid l nti-in mm tory gents. Surgery to excise the dhesions i
s successful in relieving the p in in less th n 60% of p tients. R rely, recurre
nt sci tic is due to intr dur l r chnoiditis. Tre tment is conserv tive nd su
rgery to divide the intr dur l dhesion is r rely successful.
Fig. 13.5 CT sc n of left l ter l disc prol pse.
Lum
r c n l stenosis
The p tient with lum
r c n l stenosis usu lly compl ins of p in r di ting diffu
sely into the legs, p rticul rly when st nding or w lking. The p in m y
e dif
fuse che, or is sometimes descri
ed s h ving
urning qu lity; it is usu lly re
lieved with sitting nd p tients often dopt posture of
ending the
ody forw
rd when w lking to help relieve the discomfort. The p in m y
Fig. 13.6 MRI of lum
r disc prol pse.
expl ined to the p tient, often with the help of physiother pist. Gentle
ck
strengthening exercises commence fter 10 d ys, nd the p tient should void pro
longed periods of sitting, lifting
192
CHAPTER 13

e simil r to th t descri
ed
y p tients with v scul r occlusive dise se, lthou

gh key fe ture is the presence of p in when st nding only. On occ sions there
m y
e fe tures of sci tic like p in in ssoci tion with the diffuse p in of lum

r c n l stenosis due to entr pment of nerve root
y n osteophyte or within
the l ter l recess of the c n l. The p tient often compl ins of su
jective feeli
ng of we kness nd of diffuse num
ness nd tingling r di ting down the legs. Sphin
cter dif culties m y occur if the stenosis is p rticul rly severe.
Ex min tion ndings
The ex min tion of the lower lim
s nd
ck often reve ls little or no
norm li
ty. Foc l w sting m y occur in the lower lim
s if the compression is severe, nd
the nkle jerks m y
e depressed or
sent. De nite sensory distur
nce or we kne
ss occur only in the most severe c ses. The peripher l pulses should
e checked
s the symptoms m y mimic those due to peripher l v scul r dise se.
Fig. 13.7 CT sc n of lum
r spine showing severe lum
r c n l stenosis.
P thology nd n tomy
The stenosis of the lum
r c n l m y involve reduction of the s gitt l di meter
of the c n l, n rrowing of the l ter l recess nd stenosis of the neur l for men.
The p thology is frequently due to com
in tion of congenit l c n l stenosis n
d degener tive p thology, such s lum
r spondylosis with hypertrophy of the f c
et joints nd lig mentum vum, osteophyte form tion nd thickening of the l min e
c using further n rrowing of the c n l nd
ulging of the discs such th t the s
p ce for the neur l elements
ecomes compromised. The most frequently ffected l
evels re L4/5 nd L3/4. The lum
os cr l level m y
e involved,
ut this is less
common. The stenosis m y lso
e rel ted to degener tive spondylolisthesis, p
rticul rly t the L4/5 level.
usu lly str ightforw rd,
ut it should
e con rmed
y r diologic l investig tions.

Highqu lity CT sc nning (Fig. 13.7) nd MRI (Fig. 13.8) h ve repl ced the need
for myelogr phy (Fig. 13.9). All these r diologic l studies demonstr te the c n
l stenosis. The myelogr m will show m rked indent tion of the contr st column n
d, if the stenosis is severe, there m y
e complete
lock to the ow of contr st
. The MRI will show the extent nd severity of the stenosis s well s other p t
hology rel ted to the lum
r discs such s degener tive dise se nd prol pse. Th
e clinic l fe tures of lum
r c n l stenosis do not respond f vour
ly to conser
v tive tre tment, nd surgery is lmost inv ri
ly successful in relieving the s
ymptoms. The oper tion consists of decompressive lum
r l minectomy extending
over the whole region of the stenosis with decompression of the lum
r thec nd
nerve roots. The p tient c n
e mo
ilized promptly fter the oper tion nd co
urse of gently gr du ted ctive exercises prescri
ed, usu lly with the help of
physiother pist.
B ck p in
Low
ck p in without leg p in or signs of nerve root compression is common pr
o
lem. The usu l present tions re:
M n gement
The clinic l di gnosis of lum
r c n l stenosis is
LOW BACK PAIN AND LEG PAIN
193
Fig. 13.8 MRI showing severe c n l stenosis.
cute low
ck p in, often following minor tr um chronic or recurrent low
ck
p in. Acute sudden-onset
ck p in, following recognized episode of tr um , is
usu lly due to soft tissue str in. If the injury w s severe it m y h ve c used
fr cture or disc herni tion. The m n gement of p tients with n cute onset of

ck p in following tr um involves: history nd ex min tion to exclude symptom
s nd signs of nerve root compression
Fig. 13.9 Lum
r myelogr m showing lum
r c n l stenosis.
194
CHAPTER 13
r diologic l ev lu tion to exclude fr cture or disc herni tion (if severe tr um
) conserv tive m n gement with initi l
ed rest followed
y gentle mo
iliz tion
nd simple n lgesic medic tion. Most of the p in nd stiffness should settle f
ter few d ys, lthough mild discomfort m y linger for some weeks. The more dif c
ult pro
lem is chronic or recurrent
ck p in, where the p tient gives history
of less severe or even trivi l tr um . In some c ses no p thologic l c use will

e found. The most common etiology is degener tive dise se which includes: lum

r spondylosis spondylolisthesis degener tive disc dise se. Other uncommon
ut
import nt c uses of low
ck p in which, in the e rly st ges, m y present withou
t p in r di ting into the legs or r dicul r signs, include: spin l tumours (Ch p
ter 15) thor cic disc prol pse (Ch pter 15) spin l
scess (Ch pter 15) rteriov
enous m lform tion (Ch pter 15). These serious
ut unusu l c uses m y present wi
th cute or chronic
ck p in
ut ne rly lw ys h ve other fe tures, e.g. sympto
ms or signs of nerve root involvement, which would lert the
clinici n to the possi
ility of more sinister
sis for the
ck p in. Intr -

domin l p thology should lso
e considered in p tients presenting with
ck p
in, especi lly: p ncre tic dise se p ncre titis or tumours ortic neurysm ren l
dise se c lculus, infection or tumour. Lum
r spondylosis, degener tive dise
se involving the verte
r l column, is the most common demonstr
le c use of low


ck p in. The rthritic process m y involve ny of the spin l joints nd
e ss

oci ted with degener tive disc dise se. Low
ck p in, without fe tures of sci t
ic , is only r rely c used
y disc prol pse, nd then only if the prol pse is l
rge nd centr l.
Spondylolisthesis
Spondylolisthesis is su
lux tion of one verte
r l
ody on nother, usu lly inv
olving the L4 or L5 levels, nd m y
e due to congenit l defects involving the n
eur l rch or to degener tive ch nges. Spondylolysis descri
es defect in the p
rs inter rticul ris, often the precondition for spondylolisthesis (Fig. 13.10).
V rious cl ssi c tions h ve
een used to c tegorize spondylolisthesis; most su
di
vide the
( )
Fig. 13.10 Lum
r spondylolysis with
il ter l defects in the p rs inter rticul
ris.
(
)
LOW BACK PAIN AND LEG PAIN
195
T
le 13.4 Cl ssi c tion of spondylolisthesis. Congenit l Dyspl stic Isthmic
Congenit l de ciency of superior f cet of s crum or inferior f cet of 5th lum
r v
erte
r Lesion in p rs inter rticul ris Lytic f tigue fr cture Elong ted
ut int
ct p rs Acute fr cture In dults, usu lly t L4/5; c use of lum
r c n l sten
osis
Degener tive Tr um tic P thologic l
P gets dise se, neopl stic, osteogenesis imperfect nd chondropl si
forms into those of congenit l nd those of degener tive origin (T
le 13.4). Th
e congenit l dyspl stic v riety results from congenit l de ciencies of the superio
r f cets of the s crum or the inferior f cets of the 5th lum
r verte
r . The lu
m
os cr l junction is inc p
le of withst nding the trunc l forces imposed
y th
e erect st nce nd there is gr du l forw rd slipp ge of the 5th verte
r l
ody.
This is frequently ssoci ted with spin
i d occult of L5 or S1. The congenit l
isthmic c tegory involves defect of the p rs inter rticul ris, either lytic
f tigue fr cture or, r rely, when the inter rticul ris fr cture occurs followin
g severe tr um . A further su
type is when the p rs is elong ted
ut int ct. Deg
ener tive spondylolisthesis, lso known s pseudospondylolisthesis, results from
severe loc lized rthritis of the f cets ( pophyse l joints) of the slipped ver
te
r e. R diologic l investig tions, including pl in Xr ys, CT sc n nd MRI, wil
l show the type of spondylolisthesis, the mount of slipp ge nd the ssoci ted
n rrowing of the neur l c n ls (Fig. 13.11). The degree of su
lux tion is common
ly descri
ed
y the percent ge of slip (T ill rd method) or ssigned gr de (IIV
)
sed on the num
er of qu rters of the dj cent
ody sp nned
y the slip.
Fig. 13.11 CT sc n showing lum
r spondylolisthesis
p in, which m y r di te into the
uttocks,
ut p tients often compl in of tight
feeling in the upper thighs. Symptom tic children nd dolescents often h ve g
it distur
nce, the so-c lled tight h mstring syndrome. The verte
r l slipp ge m
y produce compression of the lum
r nerve roots in the neur l for men. This c us
es sci tic , the symptoms of which m y
e indistinguish
le from those due to di
sc prol pse. N rrowing of the
ony c n l m y result in clinic l symptoms of lum

r c n l stenosis.
Clinic l present tion The presenting fe tures involve
ck p in nd leg p in. Th
e initi l symptom is usu lly
ck
Tre tment Children nd dolescents In the m jority of children nd dolescents
196
CHAPTER 13
symptom tic spondylolisthesis responds to conserv tive tre tment. The following
indic tions re guidelines for lum
r fusion. P in unrelieved
y conserv tive me
sures. Progression of su
lux tion on seri l r diologic l studies. Su
lux tion o
f gre ter th n 30%. Tight h mstring g it. The usu l surgic l procedure is spin
l fusion. Only r rely is l minectomy necess ry, nd it should never
e perfor
med unless the spine is fused s there will
e progressive slip. Adults In mos
t p tients conserv tive ther py involving short periods of
ed rest during ex ce
r
tions of discomfort, gentle mo
ilizing exercises, simple n lgesic medic tion
nd non-steroid l nti-in mm tory medic tion will
e suf cient. If some p in persi
sts following
ed rest period with properly tted lum
r
r ce m y
e of v lue
. Surgery involves either l minectomy to decompress the neur l structures nd/
or spin l fusion to prevent inst
ility. The indic tions for l minectomy inclu
de: symptom tic spin l c n l stenosis (th t is, symptoms of lum
r c n l stenosi
s) clinic l fe tures of nerve root compression (e.g. sci tic ) unrelieved
y con
serv tive ther py. A l minectomy decompresses the lum
r thec nd nerve roots,
usu lly with s tisf ctory relief of lower lim
symptoms. However, l minectomy
m y incre se the inst
ility nd some surgeons prefer to com
ine decompressive
l minectomy with spin l fusion. An intertr nsverse fusion
etween the tr nsve
rse processes h s
een the tr dition l method of fusion,
ut more recently inter
n l pedicle screw x tion nd/or inter
ody c ges pl ced in the emptied disc sp ce
e
tween the verte
r l
odies h ve
ecome the preferred method. Spin l fusion, with
out l minectomy, is occ sion lly indic ted nd should
e considered in p tients
with the following conditions. Inc p cit ting low
ck p in unrelieved
y
conserv tive tre tment, where the r diologic l ndings show rel tive
sence of
degener tive dise se s c use for the p in. This is n uncommon situ tion sinc
e, in most c ses, it is not possi
le to identify th t the spondylolisthesis is t
he sole c use of the
ck p in. Documented progressive su
lux tion. This is unco
mmon in dults
ut is de nite indic tion for spin l fusion. In gener l, the tre
tment of symptom tic gr de I spondylolisthesis
y fusion nd x tion rem ins contr
oversi l nd should
e considered on n individu l
sis. Symptom tic p tients w
ith gr de II slip usu lly
ene t from surgery, nd symptom tic p tients with gr
de III or IV
ene t gre tly.
Further re ding
Bogduk N (2004) M n gement of chronic low
ck p in. Medic l Journ l of Austr li
180 (2), 7984. Br nch CL, H ndley EN, Ducker T (1995) Tre tment of lum
os cr l
spondylolisthesis. In: Al-Mefty O, Origit no TC, H rkey HL, eds. Controversies i
n Neurosurgery. Thieme, New York. H rdy RW (1982) Lum
r disc dise se. Semin rs
in Neurologic l Surgery. R ven Press, New York. K ye AH, Bl ck P McL (2000) Oper
tive Neurosurgery. Churchill Livingstone, London, New York, Edin
urgh. M roon J
C, Young P, T rlov E, H ines SJ (1995) Tre tment of lum
r disc protusion, percu
t neous discectomy, microdiscectomy, conserv tive tre tment. In: Al-Mefty O, Ori
git no TC, H rkey HL, eds. Controversies in Neurosurgery. Thieme, New York. Mixt
er W, B rr J (1934) Rupture of the interverte
r l disc with involvement of the s
pin l c n l. New Engl nd Journ l of Medicine 211, 210214. Sherm n FC, Rosenth l R
K, H ll JE (1979) Spine fusion for spondylolysis nd spondylolisthesis in childr
en. Spine 4, 5967. Vier
iest H (1954) A r dicul r syndrome from development l n r
rowing of the lum
r verte
r l c n l. Journ l of Bone nd Joint Surgery 36B, 2302

37. Wiltse LL, Newm n PH, M cN
I (1976) Cl ssi c tion of spondylolysis nd spond
ylolisthesis. Clinic l Orthop edics 177, 2329.
CHAPTER 14
14
Cervic l disc dise se nd cervic l spondylosis
Cervic l spine disorders predomin ntly c use neck p in nd/or rm symptoms. Cerv
ic l disc prol pse nd cervic l spondylosis re the two common cervic l spine di
sorders. Degener tive ch nges in the verte
r l column re the
sic underlying p
thologic l processes in
oth these conditions. Although the two conditions m y

e distinct clinic l entities, the sh red common p thogenetic mech nism results
in spectrum of clinic l present tion depending upon whether the degener tive d
ise se h s resulted prim rily in disc rupture or cervic l spondylosis. As in the
lum
r region the critic l clinic l fe ture depends on whether there is nerve r
oot entr pment c using rm p in nd/or foc l signs of neur l compression in the
upper lim
. Cervic l cord compression due to disc prol pse or cervic l spondylos
is is discussed in Ch pter 15.
An tomy nd p thology
The structure of the cervic l disc is essenti lly the s me s in the lum
r regi
on nd consists of n intern l nucleus pulposus surrounded
y the extern l
rous
l min , the nnulus
rosus. The role of tr um in the degener tive process nd di
sc herni tion is not cle r. It is pro

le th t repetitive excessive stresses do
ex cer
te the norm l geing process nd c use disc degener tion. Although it i
s frequently possi
le to identify some minor episode of tr um prior to the onse
t of n cute disc prol pse, re dily identi
le episode of more m jor tr um s
the precipit ting event is much less frequent. The cervic l disc prol pse is us
u lly in the posterol ter l direction,
ec use the strong posterior longitudin l
lig ment prevents direct posterior herni tion. The posterol ter l disc herni ti
on will c use compression of the dj cent nerve root s it enters nd p sses thr
ough the interverte
r l neur l for men. Unlike the lum
r region, the nerves p s
s directly l ter lly from the cervic l cord to their neur l for men, so th t the
herni tion compresses the nerve t th t level (Fig. 14.1). The rr ngement of t
he cervic l nerve roots nd the rel tionship to the verte
r l
odies differ from
the lum
r region the C1 nerve root le ves the spin l c n l
etween the skull (
the for men m gnum) nd the tl s, nd the C8 root, for which there is no corres
ponding num
ered verte
r , p sses through the C7/T1 for men. Consequently, C5/
6 disc prol pse will c use compression of the C6 nerve root, C6/7 prol pse c u
ses compression of the C7 nerve root nd the C7/T1 disc 197
Cervic l disc prol pse
In the 1934 report of their experiences with ruptured interverte
r l discs, Mixt
er nd B rr descri
ed four c ses with cervic l disc dise se. Prol pse of n inte
rverte
r l disc is less common in the cervic l region th n in the lum
r re . T
he disc herni tion occurs most frequently t the C6/7 level nd slightly less co
mmonly t the C5/6 level. Disc herni tion
ove these levels nd t the C7/T1 le
vel is much less common. The predomin nt frequency of disc prol pse t C6/7 nd
C5/6 is due to the force exerted t these levels which ct s fulcrum for the
mo
ile spine nd he d.
198
CHAPTER 14
lesions, middle nger ( nd sometimes index nger) in C7 lesions, nd little nd ring
ngers in C8 lesions. The p tient m y notice we kness of the rm, p rticul rly if
the C7 root is ffected, s this c uses we kness of el
ow extension nd the mov

ement h s only very little supply
y other nerve roots (C8). A severe C5 root le
sion m y c use we kness of shoulder
duction nd the p tient m y compl in of di
f culty in elev ting the rm.
Ex min tion fe tures
Fig. 14.1 Posterol ter l cervic l disc prol pse c using compression of the dj c
ent nerve root.
prol pse c uses compression of the C8 nerve root. Occ sion lly cervic l disc m
y herni te directly posteriorly, c using compression of the dj cent cervic l s
pin l cord (Ch pter 15) which is neurosurgic l emergency.
Clinic l present tion
The ch r cteristic presenting fe tures of p tient with n cute cervic l disc
herni tion consist of neck nd rm p in nd the neurologic l m nifest tions of c
ervic l nerve root compression. Although the p in usu lly
egins in the cervic l
region it ch r cteristic lly r di tes into the perisc pul r re nd shoulder
nd down the rm (
r chi l neur lgi ). The neck p in commonly regresses while the
r di ting rm p in
ecomes more severe. It is usu lly descri
ed s deep,
oring o
r ching p in nd the p tient is usu lly severely distressed nd de
ilit ted
y th
e discomfort. The distri
ution of the p in is widespre d nd conforms to sclerot
omes (segment l distri
ution to muscle nd
one) r ther th n to derm tomes. The
p tient frequently compl ins of sensory distur
nce, p rticul rly num
ness or ti
ngling in the distri
ution of the derm tome ffected. The loc tion of the sensor
y distur
nce is more useful th n the p in s n indic tion of root level: thum

( nd sometimes index nger) in C6
Cervic l spine movements will
e restricted nd the he d is often held rigidly t
o one side, usu lly moder tely exed, nd tilted tow rds the side of the p in in s
ome p tients
ut occ sion lly w y from it in others. L ter l tilt rel xes the r
oots on the side of the conc vity
ut diminishes the interverte
r l for min e,
nd exion slightly sep r tes the posterior p rt of the interverte
r l sp ce nd le
ssens the tension in the prol pse. If the disc herni tion is long st nding there
m y
e w sting in the ppropri te muscle group, p rticul rly the triceps in C
7 root lesion. The p tient is then ex mined for we kness in e ch of the muscle g
roups (T
les 14.1 nd 14.2). We kness of el
ow extension nd nger extension is m
ost commonly c used
y C6/7 prol pse with compression of the C7 nerve root. Le
ss commonly, disc herni tion with compression of the C5 root will c use we kness
of shoulder
duction, compression of the C6 root will c use mild we kness of e
l
ow exion, nd compression of C8 m y c use we kness of the long exor muscles, tri
ceps, nger extensors nd intrinsic muscles. The deep tendon re exes provide o
jecti
ve evidence of nerve root compression in the following distri
ution. Biceps re ex
C5 Br chior di lis (supin tor) re ex C6 Triceps re ex C7 Sens tion should
e tested
in the rm nd h nd nd the sensory loss will
e ch r cteristic for the nerve ro
ot involved (Fig. 14.2) lthough there m y
e some overl p. A full neurologic l
ex min tion must
e performed nd p rticul r c re t ken to ssess the
CERVICAL DISC DISEASE AND CERVICAL SPONDYLOSIS
199
T
le 14.1 Segment l innerv tion of upper lim
muscul ture. C3, 4 C5 C6 C7 Tr pe
zius; lev tor sc pul e Rhom
oids; deltoids; supr spin tus; infr spin tus; teres
minor;
iceps Serr tus nterior; l tissimus dorsi; su
sc pul ris; teres m jor; p
ector lis m jor (cl vicul r he d);
iceps; cor co
r chi lis;
r chi lis;
r chio
r di lis; supin tor; extensor c rpi r di lis longus Serr tus nterior; l tissimu
s dorsi; pector lis m jor (stern l he d); pector lis minor; triceps; pron tor te
res; exor c rpi r di lis; exor digitorum super ci lis; extensor c rpi r di lis longu
s; extensor c rpi r di lis
revis; extensor digitorum; extensor digiti minimi Pe
ctor lis m jor (stern l he d); pector lis minor; triceps; exor digitorum super ci l

is; exor digitorum profundus; exor pollicis longus; pron tor qu dr tus; exor c rpi
uln ris; extensor c rpi uln ris;
ductor pollicis longus; extensor pollicis lon
gus; extensor pollicis
revis; extensor indicis;
ductor pollicis
revis; exor p
ollicis
revis; opponens pollicis Flexor digitorum profundus; intrinsic muscles
of the h nd (except
ductor pollicis
revis; exor pollicis
revis; opponens poll
icis); hypothen r muscles
C8
T1
T
le 14.2 Segment l innerv tion of upper lim
joint movements. Shoulder A
ducto
rs nd l ter l rot tors Adductors nd medi l rot tors Flexors Extensors Supin to
rs Pron tors Flexors nd extensors Long exors nd extensors Intrinsic muscles C5
C6, 7, 8 C5, 6 C7, 8 C6 C7, 8 C6, 7 C7, 8 C8, T1
C6 C6
C3 C4 T2 C5 T2 C5 C4
El
ow Fore rm Wrist Digits H nd
T1
T1
C8
C8 C7
C6
presence in the lower lim
s of long tr ct signs, such s incre sed tone, pyr m
id l p ttern of we kness, hyperre exi or n upgoing pl nt r response. If there is
cervic l disc herni tion these fe tures will indic te th t it is compressing
the spin l cord.
C7
Fig. 14.2 Upper lim
derm tome distri
ution.
Summ ry of clinic l fe tures
Clinic l loc liz tion of disc prol pse is possi
le in
200
CHAPTER 14
most p tients with
r chi l neur lgi due to cervic l disc prol pse. The followi
ng fe tures re typic l (
ut not inv ri
le) for disc herni tion: C6/C7 prol pse
d interverte
r l disc (C7 nerve root) We kness of el
ow extension A
sent triceps
jerk Num
ness or tingling in the middle or index nger. C5/6 prol psed interverte

r l disc (C6 nerve root) Depressed supin tor re ex Num
ness or tingling in the th
um
or index nger Occ sion lly mild we kness of el
ow exion. C7/T1 prol psed inter
verte
r l disc (C8 nerve root) We kness m y involve long exor muscles, triceps, ng
er extensors nd intrinsic muscles Diminished sens tion in ring nd little nger
nd on the medi l
order of the h nd nd fore rm Triceps jerk m y
e depressed.
M n gement
Most p tients with rm p in due to n cute soft cervic l disc herni tion chiev
e good p in relief with conserv tive tre tment. This should include
ed rest,

cervic l coll r, simple n lgesic medic tion, non-steroid l nti-in mm tory medic
tion nd muscle rel x nts. M nipul tion of the neck is potenti lly h z rdous n
d is contr indic ted. The following re indic tions for further investig tion n
d surgery. 1 P in: ( ) continuing severe rm p in for more th n 10 d ys without

ene t from conserv tive ther py (
) chronic or rel psing rm p in. 2 Signi c nt we
kness in the upper lim
th t does not resolve with conserv tive ther py. 3 Evide
nce of centr l disc prol pse c using cord compression this should
e investig
ted urgently.
Differenti l di gnosis
The clinic l fe tures of n cute cervic l disc prol pse, with severe neck nd
rm p in nd commonly diminished sens tion in the derm tome of the ffected cervi
c l root, re so ch r cteristic th t in the v st m jority of c ses the di gnosis
is self-evident. The most common c use of r di ting rm p in, other th n cute
prol pse, is spondylosis
ut, s h s
een indic ted, disc prol pse nd spondylos
is re spects of one continuing degener tive process nd, in the cervic l regio
n, the distinction
etween them
ecomes
lurred. Other unlikely
ut possi
le dif
ferenti l di gnoses include: cervic l nerve root compression
y spin l tumour
(e.g. meningiom , neuro
rom ) (Ch pter 15) thor cic outlet syndrome (Ch pter 17)
P nco sts tumour in ltr ting the roots of the
r chi l plexus peripher l nerve entr
pments, such s c rp l tunnel syndrome, medi n nerve entr pment in the cu
it l
foss nd t rdy uln r p lsy (Ch pter 17).
R diologic l investig tions
High-qu lity MRI is now the investig tion of choice nd h s lmost completely re
pl ced
oth myelogr phy nd CT (Fig. 14.3). The cervic l myelogr m using w ter-

sed non-ionic iodine contr st m teri l w s most useful investig tion for dete
rmining the presence nd site of the disc herni tion (Fig. 14.4). CT sc nning
y
itself is frequently not helpful,
ut if performed following intr thec l iodine
contr st it will demonstr te disc herni tion, nd sm ller volumes of intr the
c l contr st re necess ry th n with myelogr phy (Fig. 14.5).
Oper tive procedure
The two most commonly performed oper tions for cervic l disc prol pse re: 1 Cer
vic l for minotomy with excision of the disc prol pse. 2 Anterior cervic l disce
ctomy, with su
sequent fusion. Cervic l for minotomy. This involves fenestr tion
CERVICAL DISC DISEASE AND CERVICAL SPONDYLOSIS
201
( )
(
)
(c)
Fig. 14.3 MRI of cervic l disc prol pse. ( ) Cervic l xi l T1-weighted im ge (
rrow shows disc prol pse). (
,c) S gitt l MRI showing disc prol pse compressing
the spin l thec nd distorting the cervic l cord.
of the
one posteriorly, to provide direct ccess to the cervic l nerve root nd
disc prol pse. A sm ll mount of
one from the l ter l m rgins of the dj cent
l min nd rticul r f cets is removed to identify the nerve root in the for men
. Further
one c n then
e removed from round the nerve root to enl rge the neu
r l c n l. The nerve root is gently retr cted nd the disc herni tion excised. T
he m jor dv nt ges of the technique re th t the nerve is directly decompressed

oth
y remov l of the disc herni tion nd
y enl rgement of the for men, nd c
ervic l fusion is not necess ry. The m jor dis dv nt ge is the possi
ility of

recurrent disc herni tion,
ut this is very uncommon. In gener l, the results of
the procedure re very s tisf ctory, with excellent relief of rm p in nd, pro
vided the nerve h s not
een irrep r
ly d m ged
y long-st nding disc herni tio
n, return of full strength to the rm. Anterior cervic l discectomy. This involv
es n nterior ppro ch to remove the cervic l disc nd the prol pse. Some surge
ons perform form l fusion t the level using
one t ken from the ili c crest,
o
vine
one, rti ci l
one, or n interverte
r l c ge, usu lly lled with
one chips.
The fusion
202
CHAPTER 14
Fig. 14.5 CT myelogr m showing posterol ter l cervic l disc protrusion.
Cervic l spondylosis
Fig. 14.4 Cervic l myelogr m showing posterol ter l cervic l disc protrusion w
ith compression of the cervic l nerve root.
m y
e supplemented
y met l (usu lly tit nium) pl te screwed onto the nterio
r verte
r l surf ce,
ridging the disc sp ce. Some surgeons do not perform for
m l fusion, s spont neous
rous or
ony fusion will occur cross the disc sp ce
provided ll the disc h s
een excised. The m jor dis dv nt ge is th t the fusio
n will result in ddition l stress t the dj cent cervic l levels, there
y rend
ering them more prone to degener tive dise se. An nterior ppro ch with disc ex
cision is m nd tory for centr l disc protrusion.
Cervic l spondylosis is degener tive rthritic process involving the cervic l
spine nd ffecting the interverte
r l disc nd zyg pophyse l joints. R diologic
l ndings of cervic l spondylosis re present in 75% of people over 50 ye rs of
ge who h ve no signi c nt symptoms refer
le to the cervic l spine.
P thologic l ch nges
The degener tive process resulting in cervic l spondylosis nd its progression o
ccur in most c ses l rgely s result of the inevit
le stresses nd tr um s th
t occur to the cervic l spine s result of the norm l ctivities of d ily liv
ing. It is pro

le th t the process is ggr v ted
y repetitive or chronic tr u
m , s m y occur in some occup tions, nd s result of n episode of severe tr
um . The process princip lly involves the interverte
r l discs nd zyg pophyse
l joints. Reduced w ter content nd fr gment tion of the nucle r portion of the
cervic l discs re n tur l geing processes. As the disc degener tes there is gr
e ter stress on the rticul r c rtil ges of the verte
r l end-pl tes nd osteoph
ytic spurs develop round the m rgins of the disintegr ting end-pl tes, projecti
ng posteriorly into the spin l c n l nd nteriorly into the preverte
r l sp ce.
Postoper tive c re Wh tever ppro ch is used, the p tient is encour ged to mo
il
ize the d y fter surgery. A soft cervic l coll r m y
e useful in the rst week
fter for minotomy to minimize the neck p in. A rm coll r is usu lly worn for th
e rst 46 weeks fter nterior discectomy, or until there is evidence of fusion. Th
e prognosis for p in relief following the oper tion is excellent provided the di
gnosis h s
een ccur te nd the nerve decompressed.
CERVICAL DISC DISEASE AND CERVICAL SPONDYLOSIS
203
The degener tive process involving the zyg pophyse l joints will lso le d to os
teophyte form tion. The interverte
r l for men m y
e n rrowed
y these osteophy
tes, so c using compression of the nerve root. The osteophyte form tion th t c u
ses compression of the nerve in the neur l for men, nd which is seen round

ulging nnulus, is sometimes c lled h rd disc protrusion, s distinct from the

cute soft cervic l disc herni tion. The spondylitic process m y c use n rrowing of
the spin l c n l s result of osteophyte form tion, p rticul rly the form tio
n of hypertrophic
ony ridges t the nterior interverte
r l sp ces of the spin
l c n l nd hypertrophy of the lig ment fl vum. This m y result in compression
of the underlying cord. Such compression is m xim l during hyperextension of the
neck nd m y c use cervic l myelop thy (Ch pter 15).
disc prol pse, in th t the p in r di tes diffusely into the perisc pul r re n
d shoulder, nd into the upper lim
in scler tom l distri
ution. There m y
e
other fe tures of nerve root compression, including num
ness nd tingling in the
ppropri te derm tome distri
ution, nd we kness of the rm. Although the clini
c l fe tures m y
e lmost indistinguish
le from those due to n cute soft dis
c prol pse, the process is usu lly not s cute nd the p tient often h s hist
ory of intermittent or chronic p in. W sting of muscle group in the ppropri t
e nerve root distri
ution is more common
ec use of the longer history,
ut the
ex min tion ndings will otherwise
e simil r to those seen with n cute soft dis
c protrusion. Cervic l myelop thy. This m y result from cervic l spondylosis c u
sing n rrowing of the spin l c n l with compression of the underlying spin l cor
d. The fe tures of progressive we kness nd sensory dis
ility re descri
ed in
Ch pter 15.
Presenting fe tures
There re three m jor m nifest tions of cervic l spondylosis, depending on wheth
er there is compression of cervic l nerve root or the spin l cord. 1 Neck p in
. 2 R di ting rm p in. 3 Cervic l myelop thy (Ch pter 15). Neck p in. This is t
he most common clinic l m nifest tion of cervic l spondylosis nd its onset m y

e precipit ted
y minor tr um . The p in usu lly settles over period of few
d ys or weeks
ut frequently recurs nd is ssoci ted with incre sing stiffness
of the neck. R di ting rm p in. Br chi l neur lgi (r di ting rm p in) result
s from nerve root
eing compressed in the neur l for men
y osteophyte form ti
on, with su
sequent n rrowing of the
ony c n l. The p tient frequently h s hi
story of intermittent neck p in s result of cervic l spondylosis for some mon
ths or ye rs, nd the onset of the rm p in m y
e precipit ted
y n episode of
minor tr um . The clinic l fe tures re simil r to the neur lgi c used
y n
cute soft
R diologic l ndings
Pl in cervic l spine X-r ys (Fig. 14.6) show: n rrowing of the disc sp ce (the C
5/C6 nd C6/C7 levels re the most commonly ffected) osteophyte form tion with
encro chment into either the spin l c n l or neur l for men reduced mo
ility t
positions of fusion nd incre sed mo
ility t dj cent levels. The indic tions f
or further r diologic l investig tions depend on the clinic l present tion. Alth
ough CT sc n will cle rly show the
ony ch nges seen on the pl in cervic l spine
X-r ys, it is not indic ted for the investig tion of cervic l spondylosis which
is c using only neck p in. Nerve root entr pment, c using rm p in, is
est vis
u lized
y high-qu lity MRI. A CT sc n following intr thec l contr st or cervi
c l myelogr m with w ter-
sed non-ionic iodine contr st will lso show the nerv
e root compression,
ut re now only r rely necess ry s MRI provides such excel
lent visu liz tion of the p thology nd nerve root compression. The r diologic l
ssessment of cervic l myelop thy is discussed in Ch pter 15.
204
CHAPTER 14
gesics. During n cute episode the p tient m y
e more comfort
le in soft ce
rvic l coll r. As the p in su
sides the p tient should
e encour ged to perform
simple mo
ilizing exercises which m y
e
est undert ken with the supervision of
physiother pist. If the episodes
ecome frequent nd severe the p tient m y n

eed to consider ch nge of lifestyle, p rticul rly work pr ctices nd recre tio
n l
eh viour, which might
e ggr v ting the cervic l spondylosis.
Fig. 14.6 Cervic l spondylosis. There is n rrowing of the C5/6 nd C6/7 disc sp
ces, osteophyte form tion nd su
lux tion t the C4/5 level.
Differenti l di gnosis
Neck p in There re numerous possi
le c uses of neck p in, depending on the mode
of clinic l present tion nd the presence of neurologic l signs in the lim
s. T
he most common c use of neck p in is minor muscul r or lig mentous str in whic
h usu lly follows minor tr um . If there h s
een m jor injury then fr cture
disloc tion or cute disc herni tion should
e considered nd excluded. Other r
re c uses of neck p in re spin l tumours or spin l
scess. The other possi
le
di gnoses in p tient presenting with rm p in h ve
een descri
ed e rlier in
the ch pter.
Arm p in The symptoms frequently settle with the m n gement descri
ed
ove. The
following re indic tions for surgery. Severe p in th t does not settle with co
nserv tive tre tment over 23 weeks. Chronic or recurrent p in. Progressive we kne
ss in the rm which c uses function l dis
ility. The most frequently involved n
erve root producing signi c nt function l we kness is the C7 root,
ut the C8 or C
5 roots m y lso result in function l dis
ility s result of long-st nding ro
ot compression. The choice of surgic l procedure is simil r to th t for n cute
soft disc prol pse. Cervic l for menotomy, with decompression of the nerve root
, excision of the osteophytes nd enl rgement of the neur l for men, is n effec
tive surgic l technique. As the spondylitic process is often t multiple levels,
two roots often need to
e decompressed. Some surgeons f vour n nterior ppro
ch nd cervic l discectomy with excision of the osteophyte extending into the n
eur l for men. The decompression is followed
y fusion s descri
ed in the pre
vious section on cervic l disc prol pse. Cervic l myelop thy (See Ch pter 15.)
M n gement
Neck p in due to cervic l spondylosis The p in usu lly resolves with simple cons
erv tive me sures, including the use of non-steroid l nti-in mm tory medic tion
nd simple n lFurther re ding
Ad ms CBT, Logue V (1971) Studies in cervic l spondylitic myelop thy: 1. Movemen
t of the cervic l roots, dur nd cord nd their rel tionship to the course of t
he extr thec l roots. Br in 94, 557568. Hoff J (1980) Cervic l spondylosis. In: W
ilson CB, Hoff
CERVICAL DISC DISEASE AND CERVICAL SPONDYLOSIS
205
JT, eds. Current Surgic l M n gement of Neurologic l Dise se. Churchill Livingst
one, New York. K ye AH, Bl ck P McL (2000) Oper tive Neurosurgery. Churchill Liv
ingstone, London, New York, Edin
urgh. Lees F, Aldren-Turner JW (1963) N tur l h
istory nd prognosis of cervic l spondylosis. British Medic l Journ l 2, 16071610
. Lunsford LD et l. (1980) Anterior surgery for cervic l disc dise se. P rt 1.
Tre tment of l ter l cervic l disc herni tion: 253 c ses. Journ l of Neurosurger
y 53, 111.
M rtins AN (1976) Anterior cervic l discectomy with nd without inter
ody
one g
r ft. Journ l of Neurosurgery 44, 290295. Simeone FA, Vise WM, Gro
D, Henderson
F (1995) Tre tment of soft cervic l disc herni tion. In: AlMefty O, Origit no TC
, H rkey HL, eds. Controversies in Neurosurgery. Thieme, New York. Zeidm n SM, D
ucker TB (1992) Cervic l disc dise ses (Review). Neurosurgery Qu rterly 2, 116159
.

CHAPTER 15
15
Spin l cord compression
Compression of the spin l cord is common neurosurgic l pro
lem. Although the i
niti l clinic l m nifest tions v ry consider
ly, if the condition is unrecogniz
ed nd untre ted the eventu l outcome will inevit
ly
e dis
ling p r lysis nd
sphincter distur
nce. Spin l cord compression requires e rly di gnosis nd urg
ent tre tment if these dis strous consequences re to
e voided. The compressio
n m y occur t ny position from the cervicomedull ry junction to the conus medu
ll ris. Although compression of the c ud equin is not strictly spin l cord com
pression, the p thophysiology nd tre tment is so simil r th t it is considered
with cord compression. Spin l tr um m y lso c use cord compression
ut will
e
discussed sep r tely in the next ch pter (Ch pter 16).
P thology
The spin l cord m y
e compressed
y lesions th t re: extr dur l (80%) intr dur
l, extr medull ry (15%) intr medull ry (5%). The m jor groups of p thologic l c
uses re: 1 Tumour: ( ) met st tic (
) prim ry. 2 Degener tive: ( ) disc prol p
se (
) osteoporosis/spondylosis. 3 Infection: ( ) verte
r l
ody (
) disc sp ce
206
(c) extr dur l (d) intr dur l. 4 H em tom : ( ) spont neous (tr um ) (
) rterio
venous m lform tion. 5 Development l: ( ) syrinx (
) rteriovenous m lform tion
(c) r chnoid cyst. Although there is l rge r nge of possi
le c uses of cord c
ompression, in clinic l pr ctice the l rge m jority re due to the following. 1
Extr dur l: ( ) met st tic tumour (
) extr dur l
scess. 2 Intr dur l, extr med
ull ry: ( ) meningiom (
) schw nnom . 3 Intr medull ry: ( ) gliom ( strocytom
nd ependymom ) (
) syrinx. T
le 15.1 shows list of the possi
le c uses of s
pin l cord compression nd their prim ry positions.
Presenting fe tures
There re two m jor presenting fe tures th t re the h llm rks of spin l cord co
mpression. 1 P in. 2 Neurologic l de cit. There is consider
le v ri tion in the m
nner in which these two m jor fe tures present, depending on: the site of the c
ompression nd the involvement of dj cent nerve roots
SPINAL CORD COMPRESSION
207
T
le 15.1 Spin l cord compression. Extr dur l Met st tic tumour Lymphom Myelom
Leuk emi Prim ry verte
r l
ody tumour Chordom Disc prol pse Osteoporosis/sp
ondylosis Extr dur l
scess Extr dur l h em tom Intr dur l, extr medull ry Men
ingiom Schw nnom Arteriovenous m lform tion Spin l seeding from intr cr ni l t
umour (medullo
l stom , ependymom ) Intr medull ry Gliom ependymom , strocytom
Arteriovenous m lform tion, h em tom A
scess Met st tic tumour Syrinx H em ng
iom
compression, with involvement of the thor cic nerve roots, will often
e ssoci
ted with p in r di ting round the chest w ll. This girdle p in is n import nt fe
ture ssoci ted with lesion which m y c use cord compression. Where s
ck p
in in gener l is non-speci c common symptom, usu lly ssoci ted with degener tiv
e dise se, girdle p in should rouse the suspicion of n underlying sinister c use
. The p in is often ggr v ted
y coughing nd str ining. Centr l p in due to spin
l cord compression itself is n unple s nt diffuse dull che, often with
urni
ng qu lity, nd is frequently descri
ed with dif culty. It m y involve lim
or si

de of the trunk, depending on the segment involved. Flexion or extension of the

neck m y c use electric shock or tingling r di ting down through the
ody to the e
xtremities of the lim
s. This is c lled Lhermittes sign, nd is typic lly ssoci
ted with cervic l cord involvement, either
y compressive lesion or due to n
in mm tory process.
the speed of the compression the p thologic l c use nd the n ture of the compre
ssive lesion the involvement of the
lood supply of the spin l cord.
Neurologic l de cit The neurologic l fe tures of spin l cord compression consist o
f: progressive we kness sensory distur
nce sphincter distur
nce.
Motor imp irment The level of p r lysis will depend on the position of the cord
compression. Thor cic cord compression will result in progressive p r p resis
of the lower lim
s nd if the cervic l cord is involved the upper lim
s will ls
o
e ffected. The compression of the corticospin l p thw ys will result in n u
pper motor neurone we kness with little or no w sting, incre sed tone, incre sed
deep tendon re exes nd positive B
inski response. As the cord
ecomes more seve
rely compressed complete p r plegi will result. The we kness h s n initi l py
r mid l p ttern, with the exor movements
eing most severely ffected, whilst the
extensor movements, e.g. hip extension, knee extension nd pl nt r exion, re rel
tively preserved.
P in P in is common e rly fe ture of cord compression nd often precedes the o
nset of ny neurologic l distur
nce, sometimes
y m ny months. P in is due to i
nvolvement of loc l, p insensitive structures, such s the
one of the verte
r l
column. P in in the
ck m y lso
e c used
y sp sm of the erector spin e musc
les. P in of spin l root origin is due to involvement of the nerve root t the l
evel of the compression. In cervic l compression nerve root involvement will c u
se p in r di ting into the upper lim
in the distri
ution of the nerve root. Tho
r cic cord
208
1
Purific tion of Supercoiled Pl smid
Anthony P. Green 1. Introduction Current technologies for the purific tion of su
percoiled pl smids re limited. The use of cesium chloride gr dients in the pres
ence of ethidium
romide is time consuming, l
or intensive, requires the use of
known mut gens nd is not conducive to l rge sc le. As result, first-gener ti
on high-perform nce liquid chrom togr phy (HPLC) methods
sed on nion-exch nge
nd size exclusion h ve
een developed
ut re difficult to ccommod te product
ion t l rge sc le nd still result in compromised purity (1,2). The success of
DNA v ccines in nim l models nd the initi tion of hum n tri ls (3,4) h s led t
o need to incre se the level of supercoiled pl smid purity s well s the meth
odology utilized to produce these pl smids t l rge sc le. Sever l p r meters of
the purific tion process need to
e ddressed:
The
ility to prep re supercoiled pl smid t purity levels ccept
le for clini
c l m teri l. The
ility to prep re clinic l gr de supercoiled pl smid th t wil
l
e sc l
le in order to produce gr m qu ntities of product. The
ility to pre
p re clinic l gr de supercoiled pl smid in ccord nce with cGMP principles. The

ility to develop v lid ted ss ys to ssess purity, yield, nd cont min tion l
evels.
Ch llenges to the successful development of purific tion process c n
e divide
d into
iologic l nd pr ctic l. The
iologic l ch llenge rises from the spectr
um of
iomolecules th t must
e purified w y from the supercoiled pl smid produ
ct ( T
le 1). Addition lly, the spectrum of nucleic cid cont min nts nd pl sm
id isoforms within th t spectrum, s shown in T
le 2, must
e removed. The remo
v l of the rel xed DNA, DNA c ten nes s well s endotoxins (5,6) re p rticul
r pro
lem requiring ddition l steps in the process.
From: Methods in Molecul r Medicine, vol. 29, DNA V ccines: Methods nd Protocol

s Edited
y: D. B. Lowrie nd R. G. Wh len Hum n Press Inc., Totow , NJ

1
2
T
le 1 Constituents in Crude Lys te Pl smid DNA Chromosom l DNA RNA Lipids Endo
toxin Proteins C r
ohydr tes T
le 2 Pl smid DNA Forms Monomer supercoiled Nicke
d Line r Dimers C ten nes
Green
The pr ctic l ch llenge rises
ec use the purific tion process th t is develope
d must produce highly pure product t high yield nd must
e reproduci
le, sc l

le nd economic l ( see Note 1). We descri
e new purific tion process th t h
s
een used to gener te clinic l m teri l using propriet ry non-porous polymer
resin, PolyFlo , which uses principles of ion-p ir reverse-ph se chrom togr phy
to chieve sep r tion
sed on size nd ch rge density. The process c n
e perfo
rmed using either cetonitrile (ACN) or eth nol (EtOH). Simult neous remov l of
cont min ting endotoxins, chromosom l DNA, RNA, proteins, nd pl smid isoforms d
uring purific tion is unique dv nt ge of this resin. This process meets the c
h llenges for purity, yield, reproduci
ility, nd sc l
ility (1). 2. M teri ls
2.1. Crude St rting M teri l The prep r tion of crude st rting m teri l from
io
m ss is typic lly performed using cid/
se extr ction (7). This cl ssic lk lin
e-lysis process provides m teri l signific ntly reduced in protein, lipid nd ch
romosom l DNA. Newer protocols h ve
een dopted to improve the initi l purity e
ven further, including the use of temper ture shift during ferment tion (8) or t
he ddition of second cet te precipit tion (NH 4Ac) to reduce the RNA
urden
(9). We descri
e two purific tion methods: n ACN process using st rting m teri
l in which minim l efforts h ve
een m de to reduce the RNA
urden, nd (2) n E
tOH process in which the st rting m teri l h s
een reduced for RNA
y nion-exc
h nge chrom togr phy nd/or di filtr tion. The protocols descri
ed do not incorp
or te the use of RN se ( see Note 2).
2.2. ACN Purific tion M teri ls
1. Gl ss
orosilic te chrom togr phy column p cked with PolyFlo resin (see Note
3). 2. St rting m teri l ( mmonium cet te supern t nt) (see Note 4).
Purific tion of Supercoiled Pl smid
3
3. 1.0 M TEAA (triethyl mine cet te) pH 7.0. 4. 0.5 M KPO4 pH 7.0. 5. 1.0 M TBA
P (tetr
utyl mmonium phosph te); Aldrich Chemic ls (Milw ukee, WI) no.# 26, 810
-0; 1 M in H2O). 6. 100% Acetonitrile (ACN, Americ n Chemic l Society (ACS) gr d
e or equiv lent). 7. TES (20 mM Tris-HCl pH 8.0, 1.0 mM EDTA, 5.0 mM N Cl). 8. E
quili
r tion
uffer: 0.1 M TEAA pH 7.2, 6% ACN. 9. W sh
uffer I: TES, 5% ACN. 1
0. W sh
uffer II: 0.1 M KPO4, 2.0 mM TBAP, 5% ACN. 11. W sh
uffer III: 0.1M KP
O4, 2.0 mM TBAP, 15% ACN. 12. Elution
uffer: 0.1 M KPO4, 2.0 mM TBAP, 25% ACN.
13. S nitiz tion
uffer: 0.5 N N OH.
2.3. Eth nol Purific tion M teri ls
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Gl ss
orosilic te chrom togr phy column p ck
ed with PolyFlo resin. RNA-reduced pl smid s mple. 0.5 M KPO4 pH 7.0. 1.0 M TBAP
(tetr
utyl mmonium phosph te; Aldrich Chemic ls # 26, 810-0; 1 M in H2O). Eth
nol (EtOH, ACS gr de or equiv lent). TES (0.02 M Tris-HCl pH 8.0, 1.0 mM EDTA, 5
.0 mM N Cl). Equili
r tion
uffer: 0.1 M KPO4 pH 7.0, 2.0 mM TBAP, 1% eth nol. W
sh
uffer I: TES, 7% eth nol. W sh
uffer II: 0.1 M KPO4, 2.0 mM TBAP, 5% eth n
ol. Elution
uffer: 0.1 M KPO4, 2.0 mM TBAP, 25% eth nol. S nitiz tion
uffer: 0
.5 N N OH.

2.4. Post-Purific tion M teri ls

Millipore Pellicon II (Bedford, MA) or A/G Technology (Needh m, MA) di filtr tio
n/ultr filtr tion technologies re pplic
le for
uffer exch nge nd concentr t
ion.
3. Purific tion Protocols
A schem tic of the two protocols is shown in Fig. 1.
3.1. ACN Protocol
1. Adjust to o
t in line r flow r te of 150 cm/h nd equili
r te column in 3 co
lumn volumes of equili
r tion
uffer. 2. Prep re s mple
y diluting 1/5 with TES
nd djusting to 0.1 M TEAA using 1 M TEAA stock solution. S mple lo d should

e no more th n 0.5 mg/mL of resin (lo d concentr tion is
sed on tot l A260nm).
3. Lo d s mple nd w sh with equili
r tion
uffer until the monitor returns to

seline (~2 column volumes). Collect w sh.
4
Green
Fig.1. Schem tic flow-ch rt of PolyFlo purific tion process. 4. W sh with ~3 col
umn volumes of w sh
uffer I (TES, 5% ACN). M ke sure monitor returns to
selin
e. Collect w sh. 5. W sh with ~3 column volumes of w sh
uffer II (0.1 M KPO4, 2
.0 mM TBAP, 5% ACN). 6. W sh with ~3 column volumes of w sh
uffer III (0.1 M KP
O4, 2.0 mM TBAP, 15% ACN) until the monitor returns to
seline. Collect w sh. 7
. Elute product with 10-column volumes line r gr dient from 0.1 M KPO4, 2.0 mM
TBAP, 15% ACN to 0.1 M KPO4, 2.0 mM TBAP, 25% ACN. Collect elution fr ctions. T
his is the purified product. 8. Cle n column
y running 2 column volumes of s ni
tiz tion
uffer (0.5 N N OH). Turn pump off nd let sit in 0.5 N N OH for 1 h. R
e-equili
r te column with 3 column volumes of equili
r tion
uffer. Monitor pH to
ssure th t ll the N OH h s
een removed. 9. Purified s mple from step 8 m y

e processed through concentr tion nd/or
uffer exch nge steps. It is recommende
d to di filter g inst 0.5 M N - cet te pH 7.8 to remove residu l TBAP.
3.2. ACN Protocol Results The results of the ACN protocol re shown in Fig. 2. N
o difference in the purity of the product is seen using st rting m teri l repres
enting 1 g or 100 g of
iom ss. The RNA is elimin ted. Despite the signific nt q
u ntities of rel xed DNA, >50% of tot l pl smid, this cont min nt is removed in
the w sh step. The fin l purity is >90%. 3.3. Eth nol Protocol
1. Adjust pump speed to o
t in line r flow r te of 150 cm/hr nd equili
r te c
olumn in 3 column volumes of equili
r tion
uffer.
Purific tion of Supercoiled Pl smid
5
Fig. 2. PolyFlo chrom togr phy of pl smid using ACN process. (A) Chrom togr phic
tr cing for pplic tion of nucleic cid s mple extr cted from 1.0 g E. coli cel
ls. (B,C) 1% Ag rose gel n lysis of resolved pe ks from 1.0 g
iom ss (B) nd 1
00 g
iom ss (C) . L ne 1 = 1 g nucleic cid s mple; l nes 4 nd 5 = TES nd ACN
w sh; l nes 6 nd 7 = 15% (v/v) ACN/TBAP w sh; l nes 8 nd 9 = 15-25% (v/v) ACN
gr dient; nd l ne 10 = 50% ACN strip. Reprinted from (1). 2. Prep re s mple for
lo ding
y djusting to 2 mM TBAP nd 1% eth nol. S mple lo d should
e no more
th n 0.5 mg/mL of resin (lo d concentr tion is
sed on tot l A260nm). S mple s
hould
e <0.5 M N Cl. 3. Lo d s mple nd w sh with equili
r tion
uffer until th
e monitor returns to
seline. Collect flow through. 4. W sh with ~3 column volu
mes of w sh
uffer I (TES, 6% eth nol). M ke sure monitor returns to
seline. C
ollect w sh. 5. W sh with ~3 column volumes of w sh
uffer II (0.1 M KPO4, 2.0 m
M TBAP, 5% eth nol). 6. Elute product with 10-column volumes line r gr dient f
rom 0.1 M KPO4, 2.0 mM TBAP, 5% Eth nol to 0.1 M KPO4, 2.0 mM TBAP, 25% eth nol.

Collect elution fr ctions. This is the purified product. 7. Cle n column
y run
ning 2 column volumes of s nitiz tion
uffer (0.5 N N OH). Turn pump off nd let
sit in 0.5 N N OH for 1 h. Re-equili
r te column with 3 column volumes of equili

r tion
uffer. Monitor pH to ssure th t ll the N OH h s
een removed.
6
Green
Fig. 3. PolyFlo Chrom togr phy of pl smid using EtOH process. Crude lys te w s r
educed for RNA
y di filtr tion g inst 1020 vol TES using Millipore XL mem
r n
e (100,000 MWCO) prior to pplic tion onto 1 4 cm PolyFlo column. (A) Chrom to
gr phic tr cing t 254 nm; (B) 1% Ag rose gel of resolved pe ks. L ne 1 = st rti
ng m teri l; l ne 2 = 1% EtOH flow-through pe k; l ne 3 = 6% EtOH w sh pe k; nd
l ne 4 = 525% EtOH gr dient. 8. Purified s mple from step 7 m y
e processed thr
ough concentr tion nd/or
uffer exch nge steps. It is recommended to di filter
g inst 0.5 M N - cet te pH 7.8 to remove residu l TBAP.
3.4. Eth nol Protocol Results
The results of the Eth nol protocol re shown in Fig. 3. The residu l RNA is eli
min ted. Despite the signific nt qu ntities of rel xed DNA, >60% of pl smid, thi
s cont min nt is removed in the w sh step. The fin l purity is >95%. 4. Notes 4.
1. Org nic Solvent The choice of org nic solvent for chrom togr phy is predic te
d on the mount of cont min ting RNA. In gener l, if the RNA
urden is less th n
50%, the eth nol process m y
e employed. This c n
e ccomplished through nio
nexch nge chrom togr phy, di filtr tion or RN se tre tment. A rigorous test of t
he mount of cont min ting RNA
elow which the eth nol process c n
e used h s n
ot
een performed. If RNA reduction is chieved
y RN se digestion, the s mple m
ust
e di filtered or di lyzed to remove excess ri
onucleotides prior to PolyFlo
chrom togr phy.
Purific tion of Supercoiled Pl smid
7
Fig. 4. Endotoxin
inding to PolyFlo column (1 4 cm). (A) Tot l endotoxin unit
s (EU) were determined in the flow-through (solid squ res) nd gr dient elution
(open circles) fter lo ding s mple
uffer w s spiked with incre sing levels of
endotoxin. (B) An lysis of endotoxin levels in purified pl smid prep r tions t
defined interv ls during 100 consecutive pplic tions of single PolyFlo chrom
togr phy column. Reprinted from (1).
4.2. Endotoxin
PolyFlo h s n
endotoxin re
m nner ( Fig.

Remov l
extremely hydropho
ic surf ce. As such, signific nt qu ntities of
removed s p rt of the purific tion process nd in reproduci
le
4).

4.3. Specific tions

No specific tions for the purity of supercoiled pl smid, the levels of residu l
cont min nts or even the methods for ev lu ting purity h ve
een codified (10).
While m ny methods c n
e used to n lyze pl smid DNA, it is only recently th t
these methods h ve
een pplied to pl smid DNA s potenti l ph rm ceutic l pro
duct (11). T
le 3 descri
es some of the t rget specific tions nd methods used
within the industry.
4.4. Multiple Chrom togr phy Runs
PolyFlo is chemic lly inert polymer th t withst nds rigorous s nitiz tion proc
edures which llows for multiple runs. One hundred consecutive pplic tions of c
rude pl smid with no ch nge in purity or cont min tion levels h ve

8
T
le 3 T rget Specific tions P r meter Purity % Monomer supercoiled Purity Cont
min nts RNA Genomic DNA Endotoxin Protein T rget specific tion >95 1.82.0 <1% <1
% <100EU/mg Neg tive Testing method 1% Ag rose gel A260:A280
Green
1% Ag rose gel Slot-
lot hy
ridiz tion LAL gel clot SDS-PAGE

een documented (1). The resin c n
e s nitized to remove ny residu l nucleic

cid, protein, lipid nd endotoxin
y exposure to 0.5 N N OH lone or in com
in t
ion with 0.1 M HCl.
4.5. Potenti l Interferences
PolyFlo resin is sensitive to detergents
ut not ch otropes or s lts. The use of
PEG in the lysis process does not ffect PolyFlo perform nce (1). However, dete
rgents such s SDS, in concentr tions >0.005% prevent
inding to the resin nd s
hould
e voided.
4.6. Process Optimiz tion
As with ll chrom togr phic procedures, there re sever l key steps th t will f
fect results. Using PolyFlo in the chrom togr phy of pl smids, the key elements
re the org nic solvent concentr tion in the lo ding
uffer nd the column w sh
steps. For ex mple, if the lo d concentr tion of org nic is too high, product wi
ll
e lost in the flow-through fr ction. If the lo d concentr tion is too low, t
hen the RNA will
ind nd will not
e elimin ted in the flow-through. As conse
quence, tr ce RNA levels m y
e seen throughout the chrom togr phy. The s me con
sider tions c n
e pplied to the w sh concentr tion. References
1. Green, A. P., Prior, G. P., Helveston, N. M., T ittinger, B. E., Liu, X.-F.,
nd Thompson, J. A. (1997). Prep r tive purific tion of supercoiled pl smid DNA
for ther peutic pplic tions. BioPh rm 10, 5262. 2. Horn, N. A., Meek, J. A., Bud
h zi, G., nd M rquet, M. (1995). C ncer gene ther py using pl smid DNA: purifi
c tion nd DNA for hum n clinic l tri ls. Hum. Gene Ther. 6, 565573.
Purific tion of Supercoiled Pl smid
9
3. Ulmer, J. B., Donnelly, J. J., nd Liu, M. (1996). Tow rd the development of
DNA v ccines. Curr. Opin. Biotechnol. 7, 653658. 4. Donnelly, J. J., Ulmer , J. B
., Shiver, J. W., nd Liu, M. (1997). DNA v ccines. Ann. Rev. Immunol. 15, 617648
. 5. We
er, M., Mller, K., Welzeck, M., nd Schoor, J. (1995). Effects of lipopol
ys cch ride on tr nsfection efficiency in euk ryotic cell. BioTechniques 19, 9309
40. 6. Wicks, I. P., Howell, M. L., H ncock, T., Kohs k , H., Olee, T. -W., nd
C rson, D. (1995). B cteri l lipopolys cch ride copurifies with pl smid DNA: imp
lic tions for nim l models nd hum n gene ther py. Hum. Gene Ther. 6, 317323. 7.
Birn
um, H. C. nd Doly, J. (1979). A r pid lk line extr ction procedure for
screening recom
in nt pl smid DNA. Nucleic Acids Res. 7, 15131523. 8. L hij ni, R
., Hulley, G., Sori no, G., Horn, N., nd M rquet, M. (1996). Highyield producti
on of pBR322-derived pl smids intended for hum n gene ther py
y employing tem
per ture-controll
le point mut tion. Hum. Gene Ther. 7, 19711980. 9. Thompson,
nd Bl kesley, (1983). Purific tion of nucleic cids
y RPC-5 n log chrom togr p
hy: perist ltic nd gr vity-flow pplic tions. Meth. Enzymol. 110, 123127. 10. Of
fice of V ccine Rese rch nd Review (1996). Points to consider on pl smid DNA v
ccines for preventive infectious dise se indic tions, Food nd Drug Administr ti
on, Bethesd , MD. 11. Midd ugh, C. R., Ev ns, R. K., Montgomery, D. L., nd C si
miro, D. R. (1998). An lysis of pl smid DNA from ph rm ceutic l perspective. J
. Ph rm. Sci. 8, 130146.
2

Production of Pl smid DNA in Industri l Qu ntities According to cGMP Guidelines

Jo chim Schorr, Peter Moritz, nd M rtin Schleef
1. Introduction Within the l st five ye rs, the exponenti l growth of rese rch
ctivities on the development of genetic v ccin tion nd gene ther py h s m de it
necess ry to develop n e sy, cost-effective, industri l sc le process for prod
uction of pl smid DNA (see Note 1). One m in issue is th t the process should co
nform to cGMP guidelines nd
e ccept
le to the FDA or other n tion l regul to
ry gencies. The cGMP environment should
e implemented independently of the int
ended use of the DNA product. A typic l pplic tion would
e the supply of genet
ic inform tion th t is missing within the cell, e.g.,
ec use of genetic dise
se like cystic fi
rosis (CF). In such c ses the ther py h s
een performed
y tr n
sferring liposome-pl smid DNA complexes to the lung epithelium to express the

sent chloride ch nnel gene (CFTR) for the restor tion of the dr ining system of
this tissue (1). The more preventive ppro ch of gene medicine could involve v cci
n tion using pl smid DNA either
y su
cut neous or intr muscul r injection (26) o
r other techniques (for n overview see ref. 7, nd this volume). The expression
of immunogenic epitopes c n c use
oth humor l nd CTL response (8,9), nd Ch p
ter 6. In ll c ses, it is essenti l to
e
le to use ther peutic gent (the

iologic, usu lly pl smid DNA) free of ny other m teri ls. Such cont min tion i
ncludes components used in the isol tion process nd coming from the org nism fr
om which the pl smid is isol ted, m inly residu l proteins, RNA, nd genomic DNA
of the host cell. In this ch pter, we descri
e the development of ph rm ceuti
c l m nuf cturing process to isol te pl smid DNA st rtFrom: Methods in Molecul r
Medicine, vol. 29, DNA V ccines: Methods nd Protocols Edited
y: D. B. Lowrie
nd R. G. Wh len Hum n Press Inc., Totow , NJ
11
12
Schorr, Moritz, nd Schleef
ing from technology (Qi gen, Hilden, Germ ny) th t h s
een shown over the p s
t ye rs to
e tool in rese rch nd development th t fulfills the requirements
descri
ed in Su
he ding 1. (see Notes 24).
1.1. The Host Cell Selection
A single ppropri te host str in for ll rese rch work or industri l sc le ph rm
ceutic l m nuf cturing does not exist (see Note 5). An ppropri te str in shoul
d
e clone derived from host str in stock th t is completely ch r cterized
nd free of ny cont min tion. It should
e s fe for the environment, for the iso
l ted product, the exposed p tients, nd the employees doing the m nuf cturing w
ork s well s he lth c re personnel. Consider
le experience in the field of mo
lecul r cloning nd DNA techniques h s
een o
t ined with Escherichi coli (E. c
oli), nd E. coli K12 fulfills the needs for s fe, wellch r cterized host str
in for DNA production. System tic n lysis of over 20 different E. coli su
str i
ns demonstr ted th t very l rge qu lit tive nd qu ntit tive differences exist
mong ll the su
str ins tested. These differences m inly concern p rticul rly th
e mount of pl smid DNA per gr m of
iom ss nd the pl smids isoform distri
utio
n. Pl smid isoforms consist of supercoiled molecules, dimers, or conc temers or
c ten nes (ch ins of two or more pl smids), s well s line r or nicked pl smids
. The o
served differences in isoforms depend on the pl smid s well s on the h
ost str in. This me ns th t not only the genetic
ckground of the host is respo
nsi
le for the differences of the pl smid isoform distri
ution,
ut the pl smid
itself lso contri
utes to cert in extent.
1.2. Growth Conditions
B cteri l cultures for the purpose of pl smid isol tion were performed in
tc
h mode, using culture
ottle volumes of up to m ximum of 2 L. Studies were don

e to determine the growth medium nd conditions for optim l
cteri l growth nd

pl smid yields, resulting in optic l density (O.D. 600) v lues of round 36 O.D.
units in complex
cteri l growth medi . For purposes such s rese rch gr de pl
smid prep r tions for cloning, sequencing, nd tr nsfection experiments, this p
rocedure is dequ te nd the fin l n lysis of the prep red DNA is usu lly n g
rose gel electrophoresis, DNA qu ntific tion nd identity test (restriction dig
estion). These test criteri re not stringent enough for ph rm ceutic l purpose
s, nd the procedure of the m nuf cturing h d to
e dr stic lly modified. The fi
rst point to consider in the development of ph rm ceutic l gr de process is th
t the
tch culture method h s no type of online monitoring or regul tion. The
growth conditions re djusted
efore inocul ting the medium nd left unch nged,
usu lly for
etween 1620 h. No pH monitoring or djustment is
Production of Pl smid DNA
13
performed, oxygen nd c r
on source re lso neither monitored nor regul ted. Es
senti l su
str tes re depleted nd toxic products ccumul te. Degr ded cellul r
components, including pl smids, ccumul te in overgrown cultures nd cell de th
follows. To overcome these pro
lems for the isol tion of recom
in nt proteins,
high perform nce ferment tion technology h s
een developed over the l st few ye
rs. Ferment tion processes require different growth medi th n
tch cultures.
The possi
ility of monitoring the growth conditions llows for the introduction
of essenti l medi components
efore they re exh usted (feeding), nd for the m
inten nce of const nt pH control nd oxygen supplies. Besides the effects of
such regul tion, the culture process
ecomes more defined nd the ph rm ceutic l
requirements on document tion c n
e fulfilled. A further fe ture of the fermen
t tion technology for the l rge-sc le pl smid production, is the potenti l of hi
gh-density ferment tion to yield l rge mounts of
iom ss. Experiment l work on
the composition of
cteri l growth medi for
ottle cultures nd ferment tion d
emonstr tes th t the choice of ferment tion conditions nd growth medi strongly
influence the yields of pl smid th t re o
t ined from E. coli cells. One m in
focus w s put on the mount of pl smid per cell (copy num
er), th t c n
e monit
ored on-line
y c pill ry gel electrophoresis (10).
1.3. Downstre m Processing
The isol tion of
iomolecule from the
cteri l culture (usu lly referred to
s downstre m processing or DSP) is performed to sep r te pl smid DNA from other
undesired components present within the source of m teri l. These undesir
le co
mponents re genomic DNA, RNA, proteins, lipids, lipopolys cch rides (LPS) or en
dotoxins, components of the cell w ll nd int ct
cteri (see Note 6). The lk
line lysis (11) w s modified nd is reproduci
ly performed in sc les up to five
liter
cteri l culture (ultr pure 100 chrom togr phy system, Qi gen). The most
import nt fe ture of this technique is the form tion of complex of most of the
undesired components mentioned
ove, which c n e sily
e removed
y centrifug
tion (rese rch sc le) or flo ting nd filtr tion (rese rch nd industri l sc le)
. The resulting cle red lys te is pplied to n industri l sc le process chrom tog
r phy column with nion exch nge resin, to specific lly
ind the neg tively ch r
ged pl smid DNA nd (under ppropri te
uffer conditions) not to
ind residu l u
ndesired components (e.g., protein, RNA, nucleotides, LPS). Such nion exch nge
chrom togr phy is not limited in sc le (comp red to ppro ches such s gel filtr
tion). Moreover, in the c se of specific types of resin m teri l, possessing de
nse, high surf ce ch rge, one-step process c n
e used.
14
Schorr, Moritz, nd Schleef
As n ddition l ph rm ceutic l requirement, process for the complete, r pid r

emov l of LPS molecules w s developed (see Su
he ding 2.5.). Endotoxins such s
E. coli LPS c n h ve cytotoxic effects on m mm li n cells in vitro nd in vivo (
1215) nd if present in l rge enough mounts in vivo c n c use symptoms of toxic
shock syndrome nd ctiv tion of the complement c sc de (16). In our process dev
elopment we focused on the use of only non-toxic su
st nces, nd in p rticul r
voided ny potenti lly c rcinogenic or immunogenic re gents. Addition lly, the e
nvironment w s controlled nd the resulting liquid w ste w s
iodegr d
le.
1.4. Qu lity Assur nce nd Qu lity Control When we
eg n our work on DNA m nuf c
turing, the only criteri for the qu lity of pl smid DNA were those of typic l r
ese rch work. Usu lly the qu lity for this rese rch gr de m teri l w s estim ted
using n lytic l gel electrophoresis, restriction enzyme digestion, nd DNA seq
uence re dings. We therefore est
lished set of qu lity criteri (17) th t is
now well ccepted
y the scientific community. Relev nt issues from this work we
re discussed t the Food nd Drug Administr tion (FDA)/World He lth Org niz tion
(WHO) conference on Nucleic Acid V ccines, Fe
ru ry 57, 1996 t the N tion l Ins
titute of Allergy nd Infectious Dise ses/N tion l Institutes of He lth (NIAID/N
IH) (Bethesd , MD) (18). An overview of the regul tory spects for design, m nuf
cturing, qu lity ssur nce, nd qu lity control of v ccin tion vectors re summ
rized in the WHO Guidelines for Assuring the Qu lity of DNA V ccines (WHO Technic
l Report, J n. 17, 1997). The design of the production process focused on its
ccept nce
y n tion l nd intern tion l uthorities such s the FDA (W shington,
DC), Medicines Control Agency (UK) nd others, nd h d to fulfill the ppropri
te cGMP regul tions (see Note 7).
2. M teri ls 2.1. Buffers
P1: 50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 100 g/mL RN seA. P2: 200 mM N OH, 1% (w/v
) SDS. P3: 3.0 M KAc, pH 5.5. QBT: 750 mM N Cl, 50 mM MOPS, pH 7.0, 15% (v/v) is
oprop nol, 0.15% (v/v) Triton-X-100. 5. QC: 1.0 M N Cl, 50 mM MOPS, pH 7.0, 15%
(v/v) isoprop nol. 6. QN: 1.6 M N Cl, 50 mM MOPS, pH 7.0, 15% (v/v) isoprop nol.
1. 2. 3. 4.
2.2. Tr nsform tion nd Host Cells Prep re competent cells such s E. coli K12 D
H5 (Life Technologies, Eggenstein, Germ ny), DH10B (Life Technologies) or TG1 (#
6056; Deutsche
Production of Pl smid DNA
15
S mmlung von Mikroorg nismen und Zell Culturen, Br unschweig, Germ ny), tr nsfor
m the pl smid DNA, nd select recipients on g r pl tes cont ining the ppropri
te selection f ctor.
2.3. Ferment tion Cultiv te cells using suit
le fermenter, such s Biost te
B
iore ctor (B. Br un Biotech, Melsungen, Germ ny) with working volume of 5
L. Use, e.g., the complex
cteri l growth medi LB with ddition l s lt (19). 2
.4. Cell H rvest Cells c n
e h rvested
y
tch centrifug tion t 4600g for 15
min t 4C.
1. Beckm n J2-21 centrifuge with JA-10E rotor. 2. 500 mL Polypropylene
ottles
(N lgene, Rochester, NY).
2.5. The Anion Exch nge Chrom togr phy System Perform nion exch nge chrom togr
phy (Qi gen) to specific lly
ind dou
le str nded DNA. Single str nded DNA, RNA,
nucleotides, proteins, LPS. nd other cont min nts do not
ind to the chrom tog
r phic resin under ppropri te conditions.
1. For sm ll-sc le prep r tions (e.g., test runs using the produced
iom ss), 50
0 g
tches of DNA use the Qi gen EndoFree Pl smid Kit (Ref. #12362). 2. For l rg
er sc le prep r tions, use n nion-exch nge chrom togr phy column for the isol
tion of up to 100 mg pl smid DNA (e.g., ultr pure 100 column #11100, Qi gen) nd
LPS-free processing
uffers (#11910, Qi gen).

3. Methods The complete process of pl smid DNA production is performed under wel
ldocumented conditions nd in the c se of GMP m nuf cturing under controlled env
ironment l conditions. The following ex mples of the process we use will give so
me insight into the steps performed (Fig. 1).
3.1. The Host Cell Selection
To o
t in pure nd well-ch r cterized production str in c p
le of high yields
of DNA, the selection of n ppropri te E. coli K12 pl smid host cell clone is
essenti l. Besides good micro
iologic l pr ctices nd the use of st nd rd oper t
ing procedures (SOPs), well est
lished qu lity ssur nce nd qu lity control
system is of gre t relev nce, since ll further process steps depend on this.
1. Check the DNA received
y the Qi gen DNA Production F cility for l rge-sc le
m nuf cturing for its identity first (size, restriction p ttern, sequence), nd
if it is s tisf ctory, rele se it for further processing. 2. Tr nsform the DNA t
o E. coli K12 host cells, nd select individu l colonies for further cultiv tion
.
16
Schorr, Moritz, nd Schleef
Fig. 1. Flow ch rt of cGMP pl smid m nuf cturing procedure. QA indic tes the typ
es of qu lity ssur nce tests th t must
e performed t cert in steps to ensure
consistent qu lity nd reproduci
ility, nd to fulfill the needs of process docu
ment tion. 3. Use 3 mL of n overnight culture of cells for sm ll-sc le pl smi
d isol tion (QIAprep, Qi gen). In c se of l rge num
ers of clones, use n utom
ted device for the isol tion of DNA in 96-well form t (BioRo
ot 9600, Qi gen).
4. Identify ppropri te cell clones
y comp ring them nd selecting those with
high pl smid yield nd proper pl smid isoform distri
ution for further product
ion steps (Fig. 2).
Production of Pl smid DNA
17
Fig. 2: Comp rison of pl smid pUC21 DNA produced in different E. coli K12 host c
ells. The upper p nel shows the undigested DNA with its different isoforms. The
lower p nel shows s control the s me mount of DNA fter n EcoRI digestion.
The molecul r size m rker (M) is HindIII digested -DNA. Ge: 1% (w/v) agarose in
TAE, pH 8.0, run at 5V/cm and stained with ethidium bromide.
5. Further purify the seected cone by two singe-coony passages and check it
for identity and absence of microbioogica contaminants. Use it subsequenty fo
r the inocuation of a cuture to prepare a gycero stock of between 100500 via
s. This stock is caed Master Ce Bank (MCB); it is necessary to be abe to re
produciby inocuate cuture media from the MCB in the foowing process step an
d any future manufacturing run. 6. Perform an extensive quaity assurance progra
m to check the quaity of this MCB. Test the identity, pasmid content, as we
as absence of microbioogica contaminants before proceeding with the foowing
step. An important additiona requirement is the compete sequencing of the DNA
construct at this stage to excude any difference to the origina pasmid and to
have a data backup for postproduction sequencing. 7. Use vias of the MCB to in
ocuate a fresh cuture to produce an equay arge set of stocks (100500 vias),
which are required for the reproducibe inocuation of the fermentation-precut
ures. This second gycero stock is caed Manufacturing Working Ce Bank (MWCB
). Perform the same tests for Quaity Assurance (QA) as with the MCB.
3.2. Fermentation A fermentation process for E. coi ces carrying pasmids in
a certain copy number must be we characterized, reproducibe, easy to monitor

and reguate; If possibe it shoud run automaticay. The MCB and MWCB describe
d above are the backbones for any reproducibiity. Further important issues are
18
Schorr, Moritz, and Scheef
the types of fermenter, reguation and growth medium used. Batches of 5200 L are
routiney run, and if required, further scaing-up is possibe.
1. Use an appropriate amount of the MWCB to inocuate a pre-cuture in E. coi g
rowth medium. 2. Use the pre-cuture to inocuate the fermenter for an overnight
run at 37C with controed pH (7.5) at maximum aeration. 3. Harvest the ces by
use of a fow-through centrifuge and determine the biomass content (wet and dry
weights).
3.3. Lysis of Bacteria To isoate the pasmid DNA from the E. coi ces, a modi
fied akaine ysis procedure (11) is used. This step is of critica importance
to reduce contaminants such as protein, RNA, genomic DNA, and ce wa residues
. Here we describe, as a piot scae exampe, the approach of isoating up to 10
0 mg pasmid DNA starting from 60 g wet weight biomass (see aso the protoco su
ppied with the Qiagen utrapure100 kit).
1. Thoroughy resuspend 60 g biomass in 1000 mL buffer P1 in a 5-L gass botte.
2. Add 1000 mL of buffer P2, mix the compete voume and incubate it at room te
mperature for 5 min. 3. Add 1000 mL of buffer P3 and mix it carefuy. 4. Incuba
te the ysate for 30 min at room temperature to aow the faky white precipitat
e of SDS, protein, genomic DNA, and ce residue to rise to the surface. 5. Care
fuy pump the ysate out of the botte. 6. Fiter the ysate through a QIAfite
r unit (Qiagen), mixed with 1/10 voume of buffer ER (Qiagen), and coect the fi
trate for subsequent chromatography.
3.4. Anion Exchange Chromatography
The anion exchange chromatography coumns are oaded by pumping ysate with a pe
ristatic pump or preferaby with a process chromatography system for better mon
itoring of the process.
1. Equiibrate the Qiagen utrapure 100 coumn with 350 mL buffer QBT at a fow
rate of 10 mL/min. 2. Load the coumn at a fow rate of approximatey 4 mL/min o
vernight. 3. Wash the charged coumn with 3 L of LPS-free buffer QC at a fow ra
te of 20 mL/min. 4. Eute pasmid DNA with 400 mL LPS-free buffer QN at a fow r
ate of 3 mL/min. 5. Precipitate the DNA with 0.7 voumes of isopropano at 4C and
centrifuge at 20,000g for 30 min in LPS-free centrifuge bottes. 6. Wash the DN
A peet with LPS-free 70% EtOH and rinse. 7. Dry the DNA peet and resuspend i
t in the appropriate buffer system for further appications.
Production of Pasmid DNA 3.5. Quaity Assurance
19
The foowing QA is performed within the manufacturing process as an in-process
contro (IPC).
1. Restriction Anaysis: Digest the pasmid DNA to competion by use of differen
t restriction enzymes, foowing the instructions of the suppiers. Use agarose
ge eectrophoresis to confirm that the tota DNA size and moecuar weight of f
ragments are consistent with those expected from the knowedge of the sequence.
2. Sequencing: Determine the compete nuceotide sequence of both DNA strands by
DNA sequencing. Perform a steps foowing SOPs and document the data in a seq
uencing report. 3. Pasmid Stabiity: Monitor the presence or absence of a pasm
id containing an antibiotic resistance marker by inocuating a defined amount of
ces on both antibiotic and non-seective agar pates. If ces are not abe t
o grow on seective pates, the percentage of cones growing on both media repre
sents the pasmid stabiity. 4. DNA Quaity: In addition to the anayses of fragme

nt identity and sequence, use spectrophotometric scans between 220320 nm for the
detection of sat and organic contamination (20) within the DNA. Inspect the app
earance of a sampe in an agarose ge eectrophoresis. Important features are th
e isoform distribution (by agarose ge eectrophoresis) and the DNA concentratio
n. Aso determine the content of RNA, genomic DNA and LPS by HPLC, Southern bot
and the kinetic QCL test kit (BioWhittaker, Wakersvie, MD) respectivey. 5.
DNA Quantity: Determine the DNA concentration by spectrophotometric anaysis and
cacuation from its absorbance at 260 nm.
4. Notes
1. For arge scae DNA production, we focused on the deveopment of a technoogy
for industria-scae manufacturing of nuceic acids that combines cost effectiv
eness with the fexibiity to insta the system in every research aboratory (p
iot scae) or GMP faciity (industria scae). 2. A major consideration in the
deveopment of this technoogy was to avoid timeconsuming centrifugation and mu
tipe chromatographic coumn runs. Centrifugation of arge voumes to cear bact
eria ysates can now be repaced by just one passage through a fitration unit
that makes it possibe to fiter arge voumes of bacteria ysate. 3. The proce
ss incudes the estabishment of Master Ce Banks and Master Working Ce Banks
; fermentation and downstream processing are monitored at a stages by extensiv
e in-process contros. 4. The three most important factors which need to be cons
idered in the process deveopment for pasmid DNA production are: seection of t
he optima host strain, optimization of growth conditions, and the nuceic acid
preparation method.
20
Schorr, Moritz, and Scheef
5. A arge set of different E. coi host strains has been studied to identify st
rains producing arge amounts of pasmid DNA per ce with the highest quaity.
Quaity criteria for the seection of a host strain are the homogeneity of the p
asmid DNA isoated from the host strain (>90% covaenty cosed circe), and th
e endotoxin content of the DNA purified from a specific host strain. 6. Endotoxi
ns (LPS) are major contaminants of nuceic acids, especiay pasmid DNA prepara
tions. Due to their negativey charged phosphate groups, endotoxins tend to co-p
urify with nuceic acids. It has been demonstrated that LPS contamination of DNA
has a direct infuence on transfection efficiency into many types of cutured c
es, and that different ces show variabe sensitivity to this contamination (
13). 7. The Qiagen procedure has been approved to produce DNA for human cinica
Phase I studies in the UK (1) and other European countries, as we as in the U
nited States by the FDA (21). A drug master fie (DMF) for the cinica grade ma
nufacturing process is fied with the FDA.
References
1. Capen, N. J., Gao, X., Hayes, P., Easwarapu, R., Fisher, G., Kinrade, E., e
t a. (1994). Gene therapy for cystic fibrosis in humans by iposome-mediated DN
A transfer: UK reguatory process and production of resources. Gene Therapy 1, 1
39147. 2. Davis, H. L., Whaen, R. G., and Demeneix, B. A. (1993). Direct gene tr
ansfer into skeeta musce in vivo: factors affecting efficiency of transfer an
d stabiity of expression. Hum. Gene Ther. 4, 151159. 3. Manthorpe, M., CorneferJensen, F., Hartikka, J., Fegner, J., Runde, A., Margaith, M., and Dwarki, V
. (1993) Gene therapy by intramuscuar injection of pasmid DNA: studies on fire
fy uciferase gene expression in mice. Hum. Gene Ther. 4, 411418. 4. Miche, M.L., Davis, H. L., Scheef, M., Mancini, M., Tioais, P., and Whaen, R. G. (199
5) DNA-mediated immunization to the hepatitis B surface antigen in mice: aspects
of the humora response mimic hepatitis B vira infection in humans. Proc. Nat
. Acad. Sci. USA 92, 53075311. 5. Davis, H. L., Miche, M.-L., Mancini, M., Sche
ef, M., and Whaen, R. G. (1994) Direct gene transfer in skeeta musce: pasmi
d DNA-based immunization against the hepatitis B surface antigen. Vaccine 12, 15

031509. 6. Woff, J. A., Wiiams, P., Acsadi, G., Jiao, S., Jani, A., and Chong,
W. (1991) Conditions affecting direct gene transfer into rodent musce in vivo.
BioTechniques 11, 474485. 7. Woff, A. J. (1994) Gene TherapeuticsMethods and App
ications of Direct Gene Transfer, Birkhuser, Boston. 8. Schirmbeck, R., Bhm, W.,
Ando, K., Chisari, F. V., and Reimann, J. (1995) Nuceic acid vaccination primes
hepatitis B surface antigen-specific cytotoxic T ymphocytes in nonresponder mi
ce. J. Viro. 69, 59295934.
Production of Pasmid DNA
21
9. Davis, H. L., Schirmbeck, R., Reimann, J., and Whaen, R.G. (1995) DNA-mediat
ed immunization in mice induces a potent MHC cass I-restricted cytotoxic T ymp
hocyte response to hepatitis B virus surface antigen. Hum. Gene Ther. 6, 14471456
. 10. Schmidt, T., Friehs, K., and Fasche, E. (1996) Rapid determination of p
asmid copy number. J. Biotech. 49, 219229. 11. Ish-Horowics, D., and Burke, J. F.
(1981). Rapid and efficient cosmid coning. Nuceic Acid Res. 9, 29892998. 12. C
otten, M., Baker, A., Satik, M., Wagner, E., and Busche, M. (1994). Lipopoysa
ccharide is a frequent contamination of pasmid DNA preparations and can be toxi
c to primary ces in the presence of adenovirus. Gene Ther. 1, 239246. 13. Weber
, M., Mer, K., Wezeck, M., and Schorr, J. (1995) Effects of ipopoysaccharide
on transfection efficiency in eukaryotic ces. BioTechniques 19, 930940. 14. Wi
cks, I. P., Howe, M. L., Hancock, T., Kohsaka, H., Oee, T., and Carson, D. A.
(1995) Bacteria ipopoysaccharide copurifies with pasmid DNA: impications f
or anima modes and human gene therapy. Hum. Gene Ther. 6, 317323. 15. Morrison,
D. C. and Ryan, J. L. (1987) Endotoxins and disease mechanisms. Annu. Rev. Med.
38, 417432. 16. Vukajovich, S. W., Hoffman, J., and Morrison, D. (1987) Activat
ion of human serum compement by bacteria ipopoysaccharides: Structura requi
rements for antibody independent activation of the cassica and aternative pat
hways. Mo. Immuno. 24, 319331. 17. Schorr, J., Moritz, P., Seddon, T., and Sch
eef, M. (1995) Pasmid DNA for human gene therapy and DNA vaccines. NY Acad. Sci
. 772, 271273. 18. Smith, H. A., Godentha, K. L., Voge, F. R., Rabinovich, R.,
and Aguado, T. (1997) Workshop on the contro and standardization of nuceic ac
id vaccines. Vaccine 15, 931933. 19. Mier, J. H. (1972) Experiments in Moecua
r Genetics, Cod Spring Harbor Laboratory Press, Cod Spring Harbor, NY, p. 443.
20. Wifinger, W.W., Mackey, K., and Chomczynski, P. (1997) Effect of pH and io
nic strength on the spectrophotometric assessment of nuceic acid purification.
BioTechniques 22, 474481. 21. Isner, J. M., Wash, J., Symes, A., Pieczek, A., Ta
keshita, S., Lowry, J., et a. (1995) Arteria gene therapy for therapeutic angi
ogenesis in patients with periphera artery disease. Circuation 91, 26872692.
3
Deveopment and Characterization of Lyophiized DNA Vaccine Formuations
Nancy L. Shen, Jukka Hartikka, Nancy A. Horn, Marston Manthorpe, and Magda Marqu
et
1. Introduction The potentia appications of using pasmid DNA for immunization
and other gene therapy approaches have been discussed in an increasing number o
f pubications in the past few years. Injection of mouse musce with naked DNA (
pasmid DNA in saine) resuted in significant episoma expression from a number
of encoded reporter genes such as firefy uciferase, choramphenico acetytra
nsferase, and -g l ctosid se (1). DNA v ccin tion h s
een shown to induce neutr
lizing nti
odies g inst the gene product, helper T-cell responses of the Th1 p
henotype, nd cytotoxic T lymphocyte responses (2). V ccin tion with pl smid DNA
stimul tes immunogenicity nd provides protection g inst v rious infectious di
se ses in pre-clinic l nim l models. Ex mples include hep titis B in chimp nzee
s (3),
ovine herpes virus in mice (4), influenz A virus in ferrets (5), hum n
immunodeficiency virus in rhesus monkeys (6), Myco
cterium tu
erculosis in mice
(7,8), m l ri in mice (9,10), nd genit l herpes simplex virus in guine pigs

(11). Recently, DNA v ccines for the protection g inst influenz (Merck Rese rc
h L
or tories, R hw y, NJ), m l ri (Vic l Inc., S n Diego, CA), nd HIV (Apoll
on Inc., Phil delphi , PA), h ve entered ph se I hum n clinic l tri ls. R pid pr
ogress h s
een m de in the re s of djuv nts for DNA v ccines (12), route of i
mmuniz tion (13), industri l sc le ferment tion nd ph rm ceutic l gr de purific
tion (14). One m jor interest in the commerci l development of DNA v ccines, es
peci lly for developing countries, is to incre se DNA v ccine st
ility t room
From: Methods in Molecul r Medicine, vol. 29, DNA V ccines: Methods nd Protocol
s Edited
y: D. B. Lowrie nd R. G. Wh len Hum n Press Inc., Totow , NJ
23
24
Shen et l.
temper ture, to reduce the requirement for costly cold stor ge, nd to extend pr
oduct shelf-life. Freeze-drying, or lyophiliz tion, h s
een used in ph rm ceuti
c l processes to prolong product st
ility, p rticul rly for protein products (1
5,16). Freezedrying is used for n ttenu ted virus v ccine g inst yellow fever
(17) nd live rinderpest virus v ccine for c ttle (18). The freeze-drying pro
cess c n
e divided into three successive st ges: freezing, prim ry drying, nd
second ry drying. After freezing the product, the prim ry drying process involve
s lowering pressure nd supplying he t for w ter v por su
lim tion. During the s
econd ry drying st ge, the residu l
sor
ed moisture ev por tes from the dried
m teri l. In this ch pter, we descri
e lyophilized DNA v ccine formul tion th
t provides cute protection during lyophiliz tion nd permits full recovery of
product ctivity.
1.1. Screening Buffer nd pH
To screen excipients used in lyophilized v ccine DNA formul tions, we ev lu ted

uffers nd pH using n in vivo reporter gene ss y. Pl smid DNA VR1223 encoding
gene for lucifer se (19,20) w s formul ted nd injected intr muscul rly into
dult mouse rectus femoris muscle t 50 g DNA in 50 L volume. Injections were perf
ormed on 5 mice (10 muscles) for e ch formul tion. Lucifer se enzyme ctivity w
s me sured 7 d post-injection. The results re shown in T
le 1. There w s no st
tistic l difference in expression from ny of the tested formul tions
y non-p
r metric M nn-Whitney r nk sum test (p < 0.05). Either pH 6.0 or pH 7.0 w s ppr
opri te. Simil rly, either phosph te-
uffered s line (PBS) or citr te-
uffered s
line permitted gene expression. There were no dverse effects detected in injec
ted mice.
1.2. Screening Lyoprotect nts
Lyoprotect nt, required component in lyophilized formul tion, provides prote
ction of
iologic l molecules from freezing nd drying processes nd gives mech
nic l support to the finished product. To screen nd select lyoprotect nts, v ri
ous sug rs nd polymers were dded to the liquid formul tion nd tested in mouse
muscles. The results re summ rized in T
le 2. Consider
le v ri tion in lucif
er se enzyme expression w s noted mong v rious concentr tions of sug rs or poly
mers, nd mong simil r experiments performed
y using different
tches of mice
. There w s no st tistic l difference in expression from ny of the tested lyopr
otect nts comp red to PBS, pH 7.0,
y non-p r metric M nn-Whitney r nk sum test
(p < 0.05). However, 2- to 3-fold enh ncement w s o
served from formul tions c
ont ining sug r lyoprotect nt or sug r/polymer lyoprotect nt. The sug rs include
d treh lose, m nnitol, l ctose, sucrose, nd sor
itol; nd the polymers included
polyvinyl pyrrolidone (PVP)
Lyophilized DNA V ccine
T
le 1 Screening Buffers nd pH Used in DNA V ccine Formul tions Aver ge  SEM: n

g of lucifer se per muscle 96.4  17.5 228.2  76.9 216.9  37.6 178.6  67.9 137.9  35.7
82.5  17.4
25
Buffers nd pH 0.9% N Cl 0.9% N Cl, 10mM sodium phosph te, pH 7.0 0.9% N Cl, 10
mM sodium citr te, pH 7.0 0.45% N Cl, 10mM sodium citr te, pH 7.0 0.45% N Cl, 10
mM sodium citr te, pH 6.0 L ct ted Ringers
uffer
Tot l num
er of muscles 10 10 10 10 10 10
All formul tions
se w s extr cted
hese v lues were
nn-Whitney r nk

cont ined VR1223 pl smid DNA t 1 mg/mL concentr tion. Lucifer

from mouse leg muscle nd ss yed 7 d post-injection. None of t
signific ntly different from s line control
y non-p r metric M
sum test (p < 0.05).

K-30 nd polyethylene glycol (PEG)-3350. No dverse effect on these mice w s o
s

erved during the 7 d following injection of the test formul tions. Treh lose, PV
P K-30, nd PEG-3350 were chosen for further v ccine formul tion development. Tr
eh lose is non-reducing dis cch ride of glucose found in sever l org nisms th
t re
le to survive desicc tion (21). Treh lose h s
een demonstr ted to prote
ct cells from freezing injury nd is n effective drying protect nt (22). Commer
ci l formul tions cont ining treh lose nd PVP h ve shown good recovery of enzym
e ctivity fter freeze-drying nd more th n one ye r stor ge st
ility t 28C (23
).
1.3. Freeze-Drying Cycle Development
Next, we ch r cterized two proposed formul tions
y freeze-drying microscopy (24
) nd ex mined the effect of lyophiliz tion p r meters on the finished freeze-dr
ied product to determine freeze-drying cycle. Sodium phosph te cryst llizes du
ring freezing nd c uses pH shifts (25). Sodium chloride h s r ther low eutect
ic temper ture nd requires r ther low prim ry drying temper ture (25). Theref
ore, sodium phosph te nd sodium chloride were removed from the proposed lyophil
ized formul tion,
ut
oth components were included l ter in the reconstituting
re gent. Freeze-drying microscopic n lysis w s performed on two formul tions,
nd the results indic ted th t the retention of c kelike structure in the dried r
egion w s o
served
elow the coll pse temper ture. The coll pse temper ture for
formul tion A (VR1223 DNA [1 mg/mL]/12% treh lose [w/v]/2% PVP [w/v]) w s 26C. The
coll pse temper ture for formul tion B (VR 1223 DNA [1 mg/mL]/15% treh lose [w/
v]/2% PVP [w/v]/ 2% PEG [w/v]) w s 27C. A complete loss of structure w s o
served

ove the coll pse temper ture (Dr. Mich el Pik l, University of Connecticut, St
orrs,

26
T
le 2 Screening Lyoprotect nts
y Using In Vivo Mouse Muscle Lucifer se Ass y
Aver ge  SEM: ng of lucifer se per muscle 143.7  23.4 197.5  47.1 105.1  53.7 100.9 
52.7 50.1  19.8 357.9  111.5 81.4  31.6 166.1  58.6 49.9  22.8 298.8  105.6 162.2  50.
211.6  52.2 170.4  43.0 84.7  25.9 207.5  38.9 120.8  54.1 159.3  80.8 167.8  52.9 12
.1  40.5
Shen et l.
Lyoprotect nts PBS/pH 7.0 PBS/4% treh lose PBS/12% treh lose PBS/15% treh lose P
BS/18% treh lose PBS/12% m nnitol PBS/15% m nnitol PBS/20% m nnitol PBS/12% l ct
ose PBS/15% l ctose PBS/20% l ctose PBS/4% sucrose PBS/2% sor
itol PBS/2% PVP PB
S/2% PEG PBS/4% treh lose/1% PEG/1% PVP PBS/4% m nnitol/1% PEG/1% PVP PBS/4% l c
tose/2% PEG PBS/12% l ctose/0.9%
enzoyl lcohol
Tot l Percent of num
er of PBS muscles control 50 20 10 10 10 10 10 10 10 10 10
20 20 20 20 10 10 10 10 100 137 73 70 35 249 57 116 35 208 113 147 119 59 144 8

4 111 117 89
All formul tions cont ined VR1223 pl smid DNA t concentr tion of 1 mg/mL. The
pH of ll formul tions w s 7.0 Tot l num
er of muscles w s from 15 experiments.
None of the results were signific ntly different from PBS control
y non-p r me
tric M nn-Whitney r nk sum test (p < 0.05).
CT, person l communic tion). Therefore, the prim ry drying temper ture w s m int
ined
elow the coll pse temper ture
y djusting the ch m
er pressure to 53 m Hg
. As the su
lim tion of ice w s completed, the product temper ture w s incre sed
to +35C for 12 h during the second ry drying. This process resulted in drying of
1 mL of liquid DNA formul tion within 2 d.
1.4. Physic l Chemic l Ch r cteriz tion
A summ ry of results from tests on the finished product is shown on T
le 3. The
lyophilized product is white c ke nd p rti lly det ched from the gl ss vi l. T
he c ke cont ins pproxim tely 2% w ter
y weight. The lyophilized c ke c n
e r
econstituted with 1 mL of PBS, pH 7.0  0.2. The lyophilized
Lyophilized DNA V ccine
T
le 3 Physic l-Chemic l Test on Lyophilized nd Reconstituted Vi ls Physic l-c
hemic l tests Gener l ppe r nce Moisture content Solu
ility Reconstituted ppe
r nce pH Ag rose gel /ethidium
romide st ining Qu ntit tion of DNA Results
27
White c ke, p rti lly det ched from gl ss vi ls 2% <1 min Cle r colorless odorle
ss solution 7.0  0.1 Identic l to the liquid pl smid DNA st nd rd 98  2% recovery
VCL1102 pl smid DNA w s lyophilized in formul tion A (12% treh lose, 2% PVP) or
formul tion B (15% treh lose, 2% PVP, 2% PEG). The freeze-dried vi l w s reconst
ituted with 1 mL PBS, pH 7.0.
c ke dissolves within 1 min nd forms cle r, colorless, nd odorless solution.
There w s no detect
le loss of DNA determined
y spectrophotometric
sorption
t 260 nm w velength. Supercoiled pl smid DNA structure w s ret ined without p
p rent degr d tion, s reve led
y g rose gel electrophoresis with ethidium
ro
mide st ining.
1.5. In Vitro Bio ss ys of the Reconstituted Lyophilized DNA Formul tion
Two pl smids, VCL1005 (26) nd VCL1102 (27), were lyophilized nd tested for in
vitro potency. Both pl smids re currently
eing used in hum n nti-c ncer clini
c l tri ls (26,27). The VCL1005 encoding hum n HLA-B7 gene w s lyophilized in fo
rmul tion A (12% treh lose/2% PVP), nd reconstituted with 1 mL PBS, pH 7.0  0.2.
The reconstituted VCL1005 formul tion w s complexed with DMRIE/DOPE (1,2-dimyri
styl-oxypropyl-3-dimethylhydroxyethyl mmonium
romide/dioleoylphosph tidyleth n
ol mine) nd used to tr nsfect UM449 hum n mel nom tumor cells (28). T
le 4 in
dic tes th t the HLA-B7 is synthesized nd expressed on the cell surf ce of UM44
9 t 48 h fter tr nsfection. The level of HLA-B7 expression w s not signific nt
ly different from the expression of the non-lyophilized DNA control. Pl smid VCL
1102 encoding the gene for hum n IL-2 w s lso lyophilized in formul tion A in
ddition to formul tion B (15% treh lose/2% PVP/2% PEG). Freeze-dried vi ls were
reconstituted with PBS, pH 7.0  0.2 nd complexed with DMRIE/DOPE, nd
oth formu
l tions were used to tr nsfect UM449 cells. The culture supern t nt w s ss yed
for hum n IL-2
y ELISA (27). T
le 5 indic tes th t IL-2 is synthesized nd sec
reted in cell culture
28
T
le 4 Pl smid VCL1005 Lyophilized in Formul tion A S mples Control, non-lyophi
lized VCL1005 Lyophilized VCL1005, reconstituted Ass y reference Corrected me n

fluorescence 32.71 34.65 35.52 S.D. 0.16 4.17 0.81

Shen et l.
Num
er of repe ts 2 2 2
Percent of control 100 106 N/A
VCL1005 w s lyophilized in formul tion A (12% treh lose, 2% PVP) nd reconstitut
ed in 1 mL PBS, pH 7.0. HLA-B7 expression w s me sured
y FACS n lysis
y using
n in vitro cell tr nsfection ss y.
T
le 5 Pl smid VCL1102 Lyophilized in Fomul tion A or B S mples Aver ge correct
ed me n I.U./mL 1957 2508 1720 2485 S.D. 635 710 297 524 Num
er of repe ts 4 4 4
4 Percent of control 100 128 88 N/A
Control, non-lyophilized VCL1102 Lyophilized VCL1102 in formul tion A Lyophilize
d VCL1102 in formul tion B Ass y reference
Lyophilized VCL 1102 w s reconstituted in 1 mL PBS, pH 7.0. Hum n IL-2 expressio
n in the supern t nt of UM449 cells fter tr nsfection w s me sured
y ELISA. Fo
rmul tion A is: 12% treh lose, 2% PVP. Formul tion B is: 15% treh lose, 2% PVP,
2% PEG.
supern t nts 48 h post-tr nsfection. There is no signific nt difference in IL-2
expression
etween lyophilized VCL1102 nd non-lyophilized control DNA.
1.6. In Vivo Bio ss ys of the Reconstituted Lyophilized DNA Formul tion
A p nel of lyophilized VR1223 pl smid DNA formul tions were ev lu ted for lucife
r se expression in mice. Lyophilized formul tions cont ined treh lose, PVP, nd
PEG lyoprotect nts. Freeze-dried vi ls were reconstituted with PBS, pH 7.0  0.2
nd injected into mouse rectus femoris muscles. Lucifer se ctivities were ss ye
d t 7 d fter injection. Results, s shown in T
le 6, indic te th t the level
of muscle expression from reconstituted formul tion A (12% treh lose/2% PVP) is
simil r to non-lyophilized DNA control. In ddition, there is st tistic l diff
erence in expression
etween formul tion A nd formul tion F (8% treh lose/2% PV
P/2% PEG)
y non-p r metric M nnWhitney r nk sum test (p < 0.05). The reconstitu
ted formul tion A yields 3fold higher expression th n reconstituted formul tion
F. There w s no detect
le dverse side effect on mice for 7 d fter injection.

Lyophilized DNA V ccine

T
le 6 Pl smid VR1223 Lyophilized Formul tions Aver ge  SEM: ng of lucifer se pe
r muscle 202.9  54.1 265.9  60.4 184.5  63.5 172.7  56.2 136.0  50.6 131.4  61.4 98.4 
41.9 90.7  21.1
29
Lyophilized formul tions Control, non-lyophilized VR1223 in PBS, pH 7.0 Lyophili
zed VR1223 in formul tion A Lyophilized VR1223 in formul tion B Lyophilized VR1
223 in formul tion C Lyophilized VR1223 in formul tion D Lyophilized VR1223 in f
ormul tion E Lyophilized VR1223 in formul tion F Lyophilized VR1223 in formul t
ion G
Tot l num
er of muscles 10 10 10 10 10 10 10 10
Lyophilized formul tions were reconstituted with 1 mL PBS, pH 7.0 nd injected i
n mouse leg muscle. Lucifer se ctivity w s ss yed 7 d fter injection. Formul
tion A is: 12% treh lose, 2% PVP. Formul tion B is: 15% treh lose, 2% PVP, 2% PE
G. Formul tion C is: 10% treh lose, 2% PVP, 2% PEG. Formul tion D is: 12% treh l
ose, 2% PVP, 2% PEG. Formul tion E is: 15% treh lose, 2% PVP. Formul tion F is:

8% treh lose, 2% PVP, 2% PEG. Formul tion G is: 10% treh lose, 2% PVP. The resu
lt with formul tion A is signific ntly different from formul tion F
y non-p r m
etric M nn-Whitney r nk sum test (p < 0.05).
1.7. Conclusion nd Perspectives of Lyophilized DNA V ccines
The experiment l work presented in this ch pter descri
es development of lyoph
ilized n ked DNA v ccine for potenti l use in hum n gene ther py. Freeze-dried l
ive or ttenu ted vir l v ccines for yellow fever nd rinderpest dise ses in c t
tle were previously reported (17,18). In this report, we ev lu ted v rious sug r
s s lyoprotect nts, p rticul rly the non-reducing dis cch rides, such s treh l
ose, nd w ter solu
le polymers, such s PVP nd PEG. A freezedrying cycle for p
l smid DNA formul tion w s developed
sed on freezedrying microscopic n lysis
nd n ex min tion of the effect of lyophiliz tion p r meters. Typic l finished
c ke w s white nd re dily dissolved in PBS, pH 7.0. Recovery of full product c
tivity w s demonstr ted
y pl smid DNA gene expression from m mm li n cell cultu
re in vitro nd from mouse muscle in vivo. Thus, the freeze-drying process did n
ot introduce ny dverse effect on the integrity nd potency of pl smid DNA. The
results suggest th t lyophilized DNA v ccine formul tion is commerci lly fe s
i
le. St
ility, sterility, nd toxicity studies re currently
eing pursued nd
re required prior to clinic l development. The procedures descri
ed herein m y

e used to prep re s fe nd effective lyophilized DNA v ccine with shelf-li
fe th t is ppropri te for clinic l use.
30
Shen et l.
2. M teri ls 2.1. Chemic ls All ingredients in these formul tions re pproved d
rug su
st nces for injection.
1. Treh lose, m nnitol, l ctose, PVP K-30, PEG-3350, sor
itol, nd sucrose re U
SP gr de nd from Spectrum Qu lity Products (New Brunswick, NJ). 2. USP gr de st
erile w ter for injection (SWFI) nd sodium chloride (5%) in SWFI re from B xte
r He lthc re (Round L ke, IL). 3. Sug r nd
uffer stock t concentr tions of 204
0% re prep red in SWFI nd filtered through 0.22 m filters (N lgene, Rochester,
NY). 4. Pl smid DNA is replic ted nd isol ted from Escherichi coli DH10B str i
n (29). Supercoiled pl smid is purified
y column chrom togr phy (30). Pl smid c
onstructs re precipit ted
y eth nol (29), re-solu
ilized in SWFI, nd stored
t 20C. Pl smid endotoxin levels re <30 endotoxin units/mL
sed on LAL gel clot
ss y (Associ tes of C pe Cod, Woods Hole, MA) nd the spectrophotometric A260/A2
80 r tios re
etween 1.8 nd 2.0.
2.2. Cont iners nd Equipment
1. The 5-mL gl ss vi ls (Type 1 gl ss), 13-mm gr y
utyl stoppers, luminum crim
p se ls, nd crimper re o
t ined from West Co. (Lionville, PA). 2. A pilot fr
eeze-dryer (Duro-stop MP) is v il
le from FTS Kinetics (Stone Ridge, NY). 3. K
rl Fisher w ter titr tion Aqu st r pp r tus is v il
le from EM Science (Gi


stown, NJ). 4. A micropl te luminometer, Dyn tech Model ML250 is v il
le from
An lytic l Luminescence L
s (S n Diego, CA). 5. St tistic l softw re such s Si
gm St t version 2.0 is v il
le from J ndel Scientific Softw re (S n R f el, CA
). 6. FACS c n/LYSIS II system is v il
le from Becton Dickinson (Mount in View
, CA).
2.3. Formul tions nd Filling Vi ls Prep re formul tions in
iologic l s fety
hood
y mixing concentr ted sterile stock solutions including desired pl smid DN
A constructs, sug r, polymer,
uffer, nd 5% sodium chloride.
1. Prep re 0.2 M sodium phosph te
y dissolving 3.86 g of sodium phosph te di
s
ic nd 0.77 g of sodium phosph te mono
sic in 100 g of SWFI. Prep re PBS, pH 7.
0  0.2
y mixing SWFI, 0.2 M sodium phosph te t pH 7.0  0.2, nd 5% sodium chlori
de (in SWFI) to chieve fin l concentr tions of sodium phosph te t 10 mM nd so
dium chloride t 0.9%. Filter stock solutions through 0.22 m filter. 2. W sh n

d rinse gl ss vi ls nd stoppers with SWFI. B ke gl ss vi ls t 225C in depyrog

en tion oven for t le st 4 h. Autocl ve gl ss vi ls nd stoppers
efore formul
tion filling. 3. Dispense formul tions in liquots of 1 mL into 5-mL gl ss vi ls
(Type 1 gl ss, West Co.).
Lyophilized DNA V ccine 2.4. Pl smid Constructs
31
1. Pl smid DNA VCL1005 (26) cont ins the Rous S rcom virus long termin l repe t
(RSV-LTR) promoter/enh ncer th t drives the tr nscription nd tr nsl tion of th
e MHC cl ss I hum n leukocyte ntigen B7 (HLA-B7) gene nd the chimp nzee 2 micro
glo
ulin gene in pUC19
ck
one. Pl smid VCL1005 lso possesses poly denyl tio
n sequences from
ovine growth hormone nd k n mycin resist nce gene s
cter
i l selection m rker. 2. Pl smid VCL1102 (27) cont ins the coding sequence for t
he hum n interleukin2 (IL-2) gene th t is downstre m from the CMV immedi te-e rl
y gene promoter/ enh ncer nd 5 untr nsl ted sequences in pUC18
ck
one. 3.
Pl smid VR1223 (19,20) encodes the Photinus pyr lis lucifer se gene under the co
ntrol of hum n CMV immedi te-e rly promoter with intron A in p rti lly deleted
pBR322. VR1223 cont ins k n mycin-resist nce sequence s
cteri l selectio
n m rker.
3. Methods 3.1. Freeze-Drying Methods
1. Determine the coll pse temper ture of the formul tion
y using freeze-drying
microscopic n lysis (24) (see Note 1). 2. Pl ce the vi ls on the freeze-drier s
helves t room temper ture nd su
sequently equili
r te t 1C for
out 30 min. 3.
Cool the shelves to 55C nd hold th t temper ture for 2 h. 4. C rry out prim ry d
rying t product temper ture of
out 32C, or 5C
elow the coll pse temper ture.
5. C rry out second ry drying t 35C. Complete the drying fter djusting the ch
m
er pressure to
etween 55120 m Hg. 6. Insert the stoppers into the vi ls under v
cuum in the freeze-dryer. Crimp-se l the freeze-dried vi ls nd store them t 28C
.
3.3. Physic l-Chemic l Ch r cteriz tion
1. Visu lly inspect the lyophilized c ke for the color, texture, nd volume under
tr nslucent light g inst
l ck
ckground. 2. An lyze the moisture content
using K rl Fisher w ter titr tion. 3. Reconstitute the freeze-dried c ke with 1
mL of PBS, pH 7.0  0.2 nd visu lly inspect the solu
ility. 4. Estim te pl smid D
NA recovery using g rose gel (0.8%) electrophoresis (29) longside control of
non-lyophilized pl smid DNA st nd rd for comp rison. Determine pl smid DNA conc
entr tion nd recovery
y spectrophotometric
sorption t 260 nm (29).
3.4. In Vivo Bio ss ys
1. Inject formul ted pl smid VR1223 (50 g) intr muscul rly in the rectus femoris
muscle of fem le 4- to 12-wk-old BALB/c mice (H rl n Spr gue-D wley) (see Note 2
).
32
Shen et l.
2. At 7 d post-injection, extr ct the lucifer se from the entire qu driceps musc
le nd ss y using n utom ted micropl te luminometer (20). 3. Perform st tisti
c l comp risons using the non-p r metric M nn-Whitney r nk sum test.
3.5. In Vitro Bio ss ys
1. Reconstitute freeze-dried vi ls cont ining pl smid DNA VCL1005 with 1 mL PBS,
pH 7.0  0.2. 2. Complex the pl smid DNA (5 g) with DMRIE/DOPE in 1:1 lipid mixtur
e to yield DNA/lipid m ss r tio of 1:1 to enh nce introduction of the DNA into
cells (28). 3. Use the DNA/lipid mixture to tr nsfect UM-449 hum n mel nom cel

ls (see Note 3). 4. H rvest tr nsfected cells fter 48 h of incu
tion nd su
je
ct them to immunofluorescent st ining with HLA-B7 monoclon l nti
ody (hy
ridom
BB7.1, ATCC # HB56) nd flow cytometric n lysis (26). 5. Reconstitute freeze-d
ried VCL1102 with 1 mL PBS, pH 7.0  0.2 to fin l DNA concentr tion of 1 mg/mL.
6. Complex the pl smid DNA (30 g) with 6 g of DMRIE ( s DMRIE/DOPE 1:1 lipid mixtu
re) to yield DNA/lipid m ss r tio of 5:1. Dilute the DNA/lipid mixture to 2 g D
NA/mL nd tr nsfect IL-2 deficient UM449 cells (28). 7. H rvest the culture supe
rn t nt fter 48 h nd test in triplic te for the presence of IL-2 using n Enzy
me Amplified Sensitivity Immuno ss y kit (Medgenix Di gnostics, Fleurus, Belgium
) (27).
4. Notes
1. The microscopic n lysis of our freeze-dried Formul tions A nd B w s perform
ed t Dr. Mich el Pik ls l
or tories t the School of Ph rm cy, University of Co
nnecticut, Storrs, CT. 2. Anim l c re throughout the study is in compli nce with
the Guide for the Use nd C re of L
or tory Anim ls (U.S. Dep rtment of He lth
nd Hum n Services, N tion l Institutes of He lth, NIH Pu
lic tion No. 86-23, rev
ised 1985) s well s with loc l Institution l Anim l C re nd Use Committee req
uirements. 3. UM-449 hum n mel nom cells do not express HLA-B7 molecules nd r
e deficient in expressing 2-microglo
ulin. They were o
t ined s gift from M rk
C meron, Alfred Ch ng, nd G ry N
el, University of Michig n, Ann Ar
or, MI.
Acknowledgments The uthors would like to th nk Rese rch Production for providin
g DNA, Qu lity Control for n lytic l support, Fr ncine Cornefert-Jensen nd P m
el Str uch for technic l support, nd George Gr y for continued guid nce nd su
pport.
Lyophilized DNA V ccine
References
33
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, J., Rundell, A., M rg lith, M., nd Dw rki, V. (1993) Gene ther py
y intr mus
cul r injection of pl smid DNA: studies on firefly lucifer se gene expression in
mice. Hum. Gen. Ther. 4, 419431. 20. H rtikk , J., S wdey, M., Cornefert-Jensen,
F., M rg lith, M., B rnh rt, K., Nol sco, M., V hlsing, H. L., Meek, J., M rque
t, M., Ho
rt, P., Norm n, J., nd M nthorpe, M. (1996) An improved pl smid DNA
Expression vector for direct injection into skelet l muscle. Hum. Gene Ther. 7,
12051217. 21. El
ein, A. D. (1974) The met
olism of ,-treh lose. Chem. Biochem. 30
, 227256. 22. Liu, Q., Schmidt, R. K., Teo, B., K rplus, P. A., nd Br dy, J. W.
(1997) Molecul r dyn mics studies of the hydr tion of ,-treh lose. J. Am. Chem. 11
9, 78517862. 23. Shen, N. L. L., K ci n, D. L., Putn m, J. G., nd D vid, W. M. (
1997) Method of m king st
ilized enzyme compositions for nucleic cid mplific
tion. United St tes P tent Num
er: 5,614,387. 24. Pik l, M. J. nd Sh h, S. (199
0) The coll pse temper ture in freeze drying: dependence on me surement methodol
ogy nd r te of w ter remov l from the gl ssy ph se. Int. J. Ph rm. 62, 165186. 2
5. M cKenzie, A. P. (1977) The physico-chemic l
sis for the freeze-drying proc
ess. Dev. Biol. St nd. 36, 5167. 26. Lew, D., P rker, S. E., L timer, T., A
i, A
. M., Kuw h r -Rundell, A., Doh, S. G., Y ng, Z., L f ce, D., Gromkowski, S. H.,
N
el, G. J., M nthorpe, M., nd Norm n, A. J. (1995) C ncer gene ther py using
pl smid DNA: ph rm cokinetic study of DNA following injection in mice. Hum. Gen
e Ther. 6, 553564. 27. P rker, S. E., Kh ti
i, S., M rg lith, M., Anderson, D., Y
nk uck s, M., Gromkowski, S. H., L timer, T., Lew, D., M rquet, M., M nthorpe,
M., Ho
rt, P., Hersh, E., Stopeck, A. T., nd Norm n, J. (1996) Pl smid DNA gen
e ther py: studies with the hum n interleukin-2 gene in tumor cells in vitro nd
in the murine B16 mel nom model in vivo. C ncer Gene Ther. 3, 175185. 28. Felgn
er, J. H., Kum r, R., Sridh r, C. N., Wheeler, C. J., Ts i, Y.J., Border, R., R
msey, P., M rtin, M., nd Felgner, P. L. (1994). Enh nced gene delivery nd mech
nism studies with novel series of c tionic lipid formul tions. J. Biol. Chem.
269, 25502561. 29. S m
rook, J., Fritsch, E. F., nd M ni tis, T. (eds.) (1989)
Molecul r Cloning: A L
or tory M nu l, 2nd ed., Cold Spring H r
or L
or tory P
ress, Cold Spring H r
or, NY. 30. Horn, N. A., Meek, J. A., Bud h zi, G., nd M
rquet, M. (1995) C ncer gene ther py using pl smid DNA: purific tion of DNA for
hum n clinic l tri ls. Hum. Gene Ther. 6, 565573.
4
Repe ted Use of Qi gen Columns in L rge-Sc le Prep r tion of Pl smid DNA
Derek Gregory, Ric rdo E. T scon, nd Dougl s B. Lowrie 1. Introduction The prep
r tion of l rge mounts of high-purity int ct pl smid DNA is signific nt expe
nse in DNA v ccine rese rch. In our l
or tory, mice re typic lly e ch immunize
d
y injection of 100 g on four occ sions, so th t n experiment with 50 mice req
uires 20 mg DNA s minimum. The Qi gen tip10,000 (Gig ) column is intended to
deliver up to 10 mg of high copy-num
er pl smid DNA from Escherichi coli. We h

ve found th t
y repe tedly regener ting the column nd re pplying the flowthrou
gh volume of DNA extr ct, we c n re dily o
t in 24-fold incre sed yields, up to 4
0 mg, from the st nd rd 2.5 L
roth culture. 2. M teri ls
1. Qi gen tip-10,000 Gig column kit (C t. No. 12191; five columns with
uffers
per kit) is v il
le from Qi gen Ltd., Cr wley, West Sussex, UK. 2. Luri Berli
ni (LB)
roth: B cto-tryptone, 25 g; B cto-ye st extr ct (Difco L
or tories, Su
rrey, UK) 12.5 g; N Cl 25 g; dissolve in 2.5 L distilled w ter, djust the pH to
7.5 with N OH, nd sterilize
y utocl ving. Dispense the medium
y h lf-fillin
g sterile conic l fl sks nd dd the selective nti
iotic ppropri te to the pl
smid of interest. 3. LB g r pl tes: these re prep red s
ove
ut cont ining
15 g/L B cto- g r nd dispensed into sterile Petri dishes fter ddition of the
nti
iotic.
3. Methods
1. Stre k out the tr nsfected
cteri on two LB g r pl tes, one cont ining the
recommended concentr tion of nti
iotic nd one cont ining dou
le the concentr
tion. Incu
te the pl tes for 24 h t 37C.
From: Methods in Molecul r Medicine, vol. 29, DNA V ccines: Methods nd Protocol
s Edited
y: D. B. Lowrie nd R. G. Wh len Hum n Press Inc., Totow , NJ
35
36
T
le 1 Sequenti l Yields of Pl smid DNA P ss l Yield (mg) LPS content
Immunog
enicityc

Gregory, T scon, nd Lowrie
P ss 2 7.1 <0.05 1.6  0.3
P ss 3 6.1 <0.005 2.4  0.2
P ss 4 4.0 <0.005 3.5  0.5
P ss 5 2.3 <0.0005 NT
Tot l yield 28.4
8.9 <0.05 NT
pCMV4.65 (1) w s recovered from E. coli lys te
y p ssing the lys te through Q
i gen tip-10,000 column five times (Qi gen).
Endotoxin units/microgr m ss yed

y limulus endotoxin ss y kit. c B l
/c mice were injected intr muscul rly wit
h 2 50 g DNA four times t 4-wk interv ls, then killed fter 2 wk. Splenocytes fr
om two to three mice were pooled, cultured for 48 h with or without recom
in nt
ntigen (10 g/mL), then supern t nts were ss yed for interferon-
y enzyme-linked
immunosor
ent assay (1). Results are shown as n/mL  SD increment in the presenc
e of antien.
2. Select five or six typical colonies rowin at the hihest concentration of a
nti
iotic and inoculate them separately into 10 ml LB
roth and incu
ate the cul
tures for 810 h. 3. Make minipreps of plasmid DNA from these samples and select t
he culture showin the hihest yield. 4. Inoculate 1.0 or 0.5 L volumes of LB
r
oth (total 2.5 L) with 1.0 or 0.5 mL of the selected culture and incu
ate the cu
ltures on an or
ital flask shaker overniht at 37C. 5. Prepare the
acterial lysa
te and pass it throuh the Qiaen column as descri
ed in the kit
ut retainin t
he flowthrouh lysate. 6. After elutin the plasmid from the column accordin to
the protocol, wash the column with 100 mL sterile distilled water and then with
a 50 mL equili
ration
uffer. The column is now ready for reuse (see Note 1). 7

. Rerun the flow-throuh lysate throuh the column, elute the retained plasmid,
wash and reequili
rate the column as
efore. 8. Repeat this step a further three
times (see Note 2). Typical yields are shown in Ta
le 1.
4. Notes
1. The columns can
e sealed with parafilm and stored at 4C. 2. The column can
e
used for a further 2.5 L of culture (of the same plasmid) with somewhat reduced
yield
efore the flow
ecomes too restricted.
Reference
1. Tascon, R. E., Colston, M. J., Rano, S., Stavropoulos, E., Greory, D., and
Lowrie, D. B. (1996) Vaccination aainst tu
erculosis
y DNA injection. Nat. Med
. 2, 888892.
5
The Immunoloy of DNA Vaccines
Thomas Ttin, Jonathan Austyn, Walter J. Storkus, and Louis D. Falo Jr.
1. Introduction The surprisin o
servation that direct inoculation of an express
ion plasmid encodin a forein protein into the skin of mice resulted in the ind
uction of anti
ody responses, demonstrated that injection of naked DNA could resul
t in antien expression in an immunoenic form (1). This o
servation and the su

sequent demonstration that intramuscular injections of plasmid DNA encodin infl
uenza nucleoprotein could protect mice aainst a challene with live influenza v
irus have opened up new avenues for vaccine development (23). Immunization with p
lasmid DNA has
een shown to activate
oth humoral and cellular immune responses
, includin the eneration of antien-specific CD8+ cytotoxic T cells as well as
CD4+ T helper cells (4). An increasin num
er of studies usin experimental ani
mal models have demonstrated that plasmid DNA immunization can promote effective
immune responses aainst numerous viruses, includin influenza, ra
ies, HIV, HB
V, HCV, and HSV; several
acteria, includin: Myco
acterium tu
erculosis, Mycopl
asma pulmonis, and Borrelia
urdorferi; as well as parasites, such as malaria a
nd leishmania (4). Phase I clinical vaccine trials are currently
ein performed
for HIV, HBV, and influenza virus. With the molecular identification of tumor a
ntiens (5), there has also
een increasin interest in the development of DNA-

ased immunization for cancer. Preclinical studies demonstrate that DNA-
ased imm
unizations taretin model tumor antiens such as chicken oval
umin (6), -alacto
sidase (7), or CEA (8) induce protective immune responses leadin to rejection o
f a su
sequent, normally lethal challene with antien-expressin tumor cells.
From: Methods in Molecular Medicine, vol. 29, DNA Vaccines: Methods and Protocol
s Edited
y: D. B. Lowrie and R. G. Whalen Humana Press Inc., Totowa, NJ
37
38
Ttin et al.
Vaccines consistin of naked plasmid DNA have several potential advantaes over
alternative immunization approaches relyin on the delivery of purified or recom

inant proteins, or live attenuated or recom
inant viruses. They offer the promi
se of a readily delivera
le, molecularly defined reaent that results in antien
synthesis in situ,
ut that is neither infectious nor capa
le of replication. I
mportantly,
oth humoral and cell-mediated immune responses may
e elicited aai
nst multiple defined antiens simultaneously. Furthermore, it may
ecome possi
l
e to manipulate the nature of the resultin immune response throuh the co-deliv
ery of enes encodin immunomodulatin cytokines or costimulatory molecules. Gen
etic constructs can
e modified, allowin for the removal or insertion of transm
em
rane domains, sinal sequences, or other residues that affect the intracellul
ar traffickin and su
sequent processin of antien. The sequence may also
e mo

dified
y sitedirected mutaenesis, permittin sinle amino-acid exchanes desi

ned to enhance the antienic potency of individual epitopes or to a
olish unwant
ed physioloic effects of the wild-type protein. Importantly, plasmid DNA, encod
in suita
le antiens or immune modulators, can readily and economically
e cons
tructed and produced in lare quantities with a hih deree of purity and sta
il
ity. 2. DNA Vaccines: Antien Expression, Processin, and Presentation It is now
well esta
lished that injection of plasmid DNA throuh various methods and rout
es can induce
oth humoral and cell-mediated immune responses. Sinificant titer
s of neutralizin anti-viral anti
odies and potent cytotoxic T lymphocyte (CTL)
responses have
een documented in a num
er of experimental animal models (4,9).
Importantly, antien-specific anti
odies and CTL reactivity could
e detected in
rodents loner than 1 yr after immunization (9). These studies suest that pla
smid DNA immunization may promote lon-lastin humoral and cellular immune respo
nses qualitatively similar to live attenuated or recom
inant viral vaccines with
out the safety hazards of inoculation of live virus.
2.1. Effects of Variations in Gene Delivery and Expression
Plasmid DNA immunization has
een accomplished usin epidermal, mucosal, intramu
scular, and intravenous routes of administration (3). Striated muscle initially
was considered to
e the only tissue capa
le of efficiently takin up and expres
sin free plasmid DNA in aqueous solution (1012). This in vivo ene transfer meth
od was extensively studied
y ene therapists as a simple way to deliver recom
i
nant proteins such as Factor IX, rowth hormone, or -1- ntitrypsin. Tr nsfection
of muscle fi
ers could re dily
e demImmunology of DNA V ccines
39
onstr ted using reporter genes such s -g l ctosid se or firefly lucifer se. Inde
ed, -g l ctosid se ctivity could
e detected in up to 5% of myofi
rils nd lucif
er se ctivity w s demonstr ted up to 19 mo following intr muscul r injection of
pl smid (11). An lyses
y PCR nd Southern
lotting reve led th t the injected
pl smid persisted episom lly, presum
ly due to the low turnover of myocytes in
vivo. The gene gun h s
een of p rticul r interest s n ltern tive to intr mus
cul r injection s the mode of delivery of pl smid DNA. The gene gun propels DNA
-co ted gold p rticles directly into the cytopl sm of cells in t rget tissue
y
me ns of electric l disch rge or helium pulse (1,3,13,14). Expression of
iolist
ic lly delivered DNA encoding -g l ctosid se or lucifer se w s found to
e tr nsi
ent in
om
rded epidermis or liver, nd low levels of long-term expression coul
d
e o
served in the dermis (13). Both direct injection of pl smid DNA in s line
nd p rticle-medi ted delivery of pl smid DNA h ve now
een extensively investi
g ted s immuniz tion methods. P rticle-medi ted delivery of DNA to the epidermi
s of mice using the gene gun required up to 5000 times less DNA th n intr muscul
r or intr derm l inocul tion of DNA in queous solutions for the induction of i
mmune responses (14). Following injection of pl smid DNA in s line, cells presum

ly t ke up DNA from extr cellul r sp ces. In contr st, using the gene gun, DNA
is delivered directly into the cell cytopl sm. These differences in gene delive
ry re
elieved to ccount for the f ct th t efficient tr nsfection nd inductio
n of immune responses c n reproduci
ly
e chieved with n nogr m qu ntities of p
l smid DNA using the gene gun, where s injection of pl smid DNA in s line usu ll
y requires 25100 g (15,16). It is import nt to note, however, th t the tr nsfectio
n efficiency of the t rget tissue does not necess rily correl te with the effici
ency of immuniz tion. Indeed, one of the striking results of e rly studies ex mi
ning different routes of DNA delivery w s th t intr derm l injection of free pl
smid DNA c n elicit potent immune responses to the encoded ntigen despite the r
el tively low efficiency of DNA upt ke nd expression in skin when comp red to m
uscle (17). Recent evidence suggests th t the site nd method of DNA delivery m
y ffect the n ture of the immune response induced g inst ntigens encoded
y p
l smid DNA (16,18). The two most common t rgets for gene delivery, skin nd musc

le, re gener lly considered to h ve signific nt differences in immunocompetency

. The skin nd mucous mem
r nes re the site where most foreign ntigens re nor
m lly encountered, nd these tissues h ve highly developed immune surveill nce f
unctions. The epidermis cont ins numerous
one-m rrow-derived L ngerh ns cells (
LC), which re profession l ntigen presenting cells speci lized for the initi t
ion of immune responses (19). Resident LC c n efficiently t ke up nd process fo
reign ntigens. In ddition,
oth ker tinocytes nd LC c n secrete proinfl mm to
ry cytokines such s IL-1,
40
Tting et l.
TNF-, nd GM-CSF in response to llergens, infection, or injury. This cytokine mi
lieu c n induce the m tur tion nd migr tion of LC to region l lymph nodes, wher
e they present ntigen to T cells. In contr st, muscle is not gener lly consider
ed to h ve speci lized immune system nd it cont ins rel tively few, if ny, r
esident dendritic cells, m croph ges, or lymphocytes. Interestingly, intr derm l
or intr muscul r injection of DNA in s line h s
een found to preferenti lly in
duce Th1
i s of the immune response in mice, with the exp nsion of IFN--produc
in CD4+ T cells and the production of anti
odies of the IG2a isotype (16,20,21
). Surprisinly, plasmid DNA immunization usin the ene un taretin skin or m
uscle (16,21) induces immune responses with a Th2
ias. There also appear to
e
differences in the dependency of the immune response on ene expression in the t
aret tissue for intramuscular needle vs. epidermal ene un DNA delivery (18).
Excision of an injected muscle
undle within 10 min of DNA inoculation did not a
ffect the manitude or duration of anti
ody responses. In contrast,
iopsy of th
e skin taret site up to 24 h after ene un
om
ardment completely a
roated th
e anti
ody response in the majority of mice. In these studies, ene un delivery
to the skin was performed with only 1  of plasmid DNA, whereas intramuscular in
jection utilized 100  of DNA in 50 L of saline. The authors interpreted these res
ults to suest that ene un-
om
ardment of skin resulted primarily in antien
expression
y epidermal cells, which appeared to
e required for the induction o
f immune responses. On the other hand, intra-muscular injection was thouht to l
ead to the rapid movement of DNA or DNA-transfected cells out of the injected mu
scle, so that the immunostimulatory events leadin to anti
ody production and CT
L reactivity took place primarily in distal tissue. Taken toether, these studie
s demonstrate that variations in ene delivery can have important qualitative ef
fects on the nature of the resultin immune response. At this point, the relativ
e contri
ution of DNA delivery varia
les includin the taret site, the method of
delivery, and/or the quantity and adjuvant properties of DNA deliveredto the o
s
erved differences in the nature of the immune response remains unclear.
2.2. Processin and Presentation of Genetically Introduced Antiens
The precise mechanisms involved in the activation of immune responses to antien
s expressed in vivo followin injection of plasmid DNA have not yet
een determi
ned. In the past decade, important insihts into the molecular nature of antien
processin, presentation, and reconition have shed new liht on the induction
of immune responses. The mechanism
y which lymphocytes reconize antien has
e
en outlined in sinificant detail in a num
er of recent review articles (2225) (i
llustrated in Fi. 1). CD8+ cytolytic T lymphocytes
Immunoloy of DNA Vaccines
41
Fi. 1. MHC class I and class II restricted antien-processin pathway.
reconize a non-covalent trimeric complex consistin of a major histocompati
ili
ty complex (MHC) class I heavy chain allele, 2-microlo
ulin, and an 812 amino aci

d lon peptide antien. CD4+ helper T lymphocytes, in contrast, reconize 1225 am

ino acid lon peptide antiens presented in the context of dimeric MHC class II
molecules. Most of the peptides presented
y MHC class I molecules are derived f
rom endoenously synthesized protein antiens processed in the cytosol
y multic
atalytic proteasomes. The resultin short peptides are delivered into the endopl
asmic reticulum
y the transporter associated with antien presentation (TAP). I
n the endoplasmic reticulum,
42
Ttin et al.
compati
le peptides interact with nascent class I heavy chains and 2microlo
uli
n-eneratin transport-competent trimeric complexes that are su
sequently delive
red to the cell surface where they
ecome accessi
le to CD8+ T cell scrutiny. In
contrast, exoenous proteins taken up into endocytic compartments from the extr
acellular milieu are the major source of antiens presented in the context of MH
C class II molecules. Internalized antien is denatured
y low pH and deraded

y endosomal and lysosomal proteases, exposin peptide sements that
ind to MHC
class II molecules. The resultin class IIpeptide complexes are transported to th
e mem
rane for detection
y CD4+ T cells. One result of this eneral sereation
of processin pathways is that protein antiens introduced into the extracellul
ar fluids (as
y injection) typically do not ain access to the cytosol and are
therefore enerally excluded from the MHC class I processin pathway. This repre
sents a theoretical hurdle for protein-
ased vaccine desin and may explain why
the elicitation of stron CTL responses with non-livin protein antiens has en
erally
een pro
lematic. Several studies, however, have found that cell-associat
ed antiens can elicit CD8+ T-cell responses in vivo, and accumulatin evidence
suests that some phaocytic cell types, such as macrophaes or immature dendri
tic cells (DC), can take up exoenous proteins for class I-restricted presentati
on (24). Once a protein antien is denatured and/or diested, the a
ility of a 
iven peptide to
ind to, and
e presented
y, a iven MHC class I or class II al
lele is determined
y structural motifs within the peptide sequence. These motif
s allow for sufficient compati
ility
etween peptide amino acid side-chains and
micropockets formed within the peptide-
indin roove of the MHC molecule (25).
The deree of intermolecular compati
ility determines the affinity of peptide fo
r an individual MHC molecule, the correspondin half-life of peptide MHC complexe
s and, to a lare deree, the likelihood that the peptideMHC complex is immunoen
ic. In addition to the reconition of peptideMHC liand
y the T-cell receptor, T
-cell primin requires the presentation of antien
y professional antienpresen
tin cells (APCs) capa
le of providin co-stimulation. Presentation of antiens to
T cells in the a
sence of appropriate costimulatory sinals can result in activ
ation induced cell death
y apoptosis (26). Dendritic cells are the most potent
APCs identified to date (27,28). They are capa
le of capturin antiens in the p
eriphery, and then miratin to T-cell-rich areas of secondary lymphoid orans,
where they can efficiently stimulate naive or quiescent T cells. Upon encounter
with antien, DCs undero a maturational process, which is associated with their
capacity to activate T cells. This includes the upreulation of MHC, costimulat
ory, and adhesion molecule expression on their cell surface. Factors associated
with the extraordinary capacity of dendritic cells to function as antien-presen
tin cells are summarized in Fi. 2.
Immunoloy of DNA Vaccines
43
Fi. 2. Dendritic cells as professional antien-presentin cells.
Because enetic immunization can elicit
oth humoral and cellular immune respons
es, the DNA-encoded antien presuma
ly ains access to
oth the MHC class I and

class II antien processin pathways, and is also capa
le of interactin with im

munolo
ulin molecules on the surface of B-cells. Initially, the efficient induc
tion of CD8+ CTL was considered to
e a result of endoenous antien synthesis i
n vivo, which presuma
ly permitted antien to access the class I restricted anti
en processin pathway of transfected cells. However, the predominant transfecte
d cell type followin intramuscular DNA injection is the myocyte. Myocytes expre
ss only low levels of MHC class I molecules and lack expression of costimulatory
molecules, such as B7.1 and B7.2. Presentation of antiens
y muscle cells alon
e would
e expected to cause either immunoloical inorance or tolerance.
2.3. A Critical Role for Professional Antien Presentin Cells To determine the
role of professional APCs in the induction of CD8+ CTL responses followin DNA i
mmunization, several roups have performed studies usin
one marrow-chimeric mi
ce (2932). They utilized the influenza nucleoprotein (NP) as a model antien, sin
ce CTL epitopes have
een defined for multiple strains (H-2
, H-2d, and H-2k). P
arent F1
one marrow chimeras were enerated in which H-2
d (C57BL/6 BALB/c) rec
ipient mice were reconstituted with
one marrow from either H-2
(C57BL/6) or H2d (BALB/c) animals. CTL responses followin immunization with plasmid DNA encod
in influenza NP via intramuscular or intradermal inoculations were restricted t
o the MHC haplotype of the donor
one marrow alone, and not to the other recipie
nt MHC haplotype, which was also expressed
y the recipients myocytes
44
Ttin et al.
Fi. 3. Cross-primin in enetic immunization: immune induction requires antien
presentin
y professional APCs.
(2932) (illustrated in Fi. 3A). Thus, CTL could only
e stimulated if antien wa
s presented in the context of MHC molecules on the
one marrow-derived professio
nal antien-presentin cells, rulin out the possi
ility that myocytes or kerati
nocytes were directly activatin cytotoxic T cells. This view was further suppor
ted
y transplantation experiments, in which NP-transfected myo
lasts (H-2k) wer
e transplanted into F1
one marrow chimeras consistin of H-2k d (C3H DBA/2) rec
ipient mice which had received
one marrow from H-2d (DBA/2) donors (33). Here,
the resultin influenza nucleoprotein-speImmunoloy of DNA Vaccines
45
Fi. 4. Genetic immunization: mechanisms of antien presentation.
cific CTL were restricted to the MHC haplotype of the
one marrow donors (H-2d,
see Fi. 3B). Thus, specific CTL responses could
e induced as a result of the t
ransfer of antien from the transplanted nucleoprotein-expressin muscle cells (
H-2k), to a enetically disparate
one-marrow-derived APC (H2d) capa
le of prese
ntin the NP-derived antien in the context of its own MHC class I molecules (cr
oss-primin). Toether, these data stronly suest that the initiation of cellmediated immune responses followin plasmid DNA immunization requires the presen
tation of antien
y professional APCs. How professional APCs acquire antien is
much less clear. Professional APCs could acquire the antien either throuh dir
ect transfection and expression within the APC or via the uptake of exoenous an
tien synthesized within other transfected cell types (illustrated in Fi. 4). M
uscle transplantation studies descri
ed previously (18) suest that antien-exp
ression
y transplanted myoctes can
e sufficient for T-cell activation (33). Ho
wever, previously descri
ed studies demonstrate that the eneration of an immune
response after intramuscular injection in vivo does not require persistence of
the transfected taret muscle tissue, suestin that the mechanism of immune in

duction after ene delivery in vivo is less clear (18). The same studies suest
a strikin difference in dependency on ene expression in the taret tissue, de
pendin on the taret site, since excision of ene un tareted skin for as lon
as 24 h after delivery prevented immunization, whereas excision of transfected
muscle as soon as 10 min after injection did not appear to diminish the immune r
esponse.
46
Ttin et al.
We have directly demonstrated that cutaneous DNA immunization usin the ene un
results in the direct transfection of skin-derived dendritic cells in vivo (34)
. These transfected APCs traffic to and localize in the drainin lymph nodes, wh
ere they continue to express endoenously synthesized antien in the T-cell-rich
areas of drainin lymph nodes. Su
sequently, and consistent with these o
servat
ions, Son et al. (35) have reported that splenic dendritic cells contained prov
iral DNA and expressed antienic proteins after direct administration of antien
-encodin enes usin retroviral vectors. Casares et al. (36) demonstrated that
DC miratin out of DNA injected tissue appear to carry plasmid DNA and trier
immune responses to the transene. However, whether transfection of APCs is crit
ical for the efficient induction of immune responses followin in vivo plasmid D
NA immunization remains unclear. The relative contri
utions of direct transfecti
on of APCs vs re-uptake and presentation of antien synthesized in
ystander cel
ls remains to
e determined. Clearly, very low num
ers of antien-presentin den
dritic cells are sufficient for immune induction. Early studies usin epidermal
LC have demonstrated that injection of as few as 10 LC are sufficient for induct
ion of allospecific immune responses (37). Timares-Le
ow et al. (38) have recent
ly reported a direct comparison of the efficiency of transfected, adoptively tra
nsferred skin-derived fi
ro
last vs dendritic cell lines in eneratin immune re
sponses aainst transene encoded antiens. In this model, in which transfected
cells are injected su
cutqaneously into A/J mice, 100-fold less dendritic cells
are required for immunization. The presentation
y APCs of endoenously synthesi
zed antien and exoenously acquired antien is not mutually exclusive, and
oth
mechanisms may
e operatin followin in vivo ene delivery. Thouh the relativ
e contri
ution of each mechanism to primin is unclear, it is not unlikely that
antien release from transfected non-APCs could potentiate or prolon the result
in immune response, especially when these transfected cells are tareted
y the
first wave of antien-specific cytotoxic T cells. Evidence for immune-mediated
destruction of muscle fi
ers followin direct ene transfer with antien-express
in plasmid DNA has indeed
een found and support this hypothesis (39).
2.4. Genetic Enineerin of the Immune Response
There are several examples of attempts to modulate the immune response elicited

y plasmid DNA immunization throuh the co-delivery and expression of antien wi
th other immunoloically relevant molecules. For example, simultaneous delivery
of plasmid DNA encodin GM-CSF enhances immune responses resultin from plasmid
DNA immunization (40,41). This is consistent with the involvement of DCs in the
activation of the immune system since GM-CSF is important for the rowth, differ
entiation, and maturation of denImmunoloy of DNA Vaccines
47
dritic cells, at least in vitro. In addition, several roups have recently shown
the adjuvant effects of co-delivery of plasmids encodin immunostimulatory cyto
kines includin IL-2, IL-7 or IL-12 (4245). Adjuvant administration of plasmids e
ncodin T-cell rowth factors such as IL-2 and IL-7, and Th1-
iasin cytokines s
uch as IFN- nd IL-12, m y
e expected to selectively enh nce the induction of ce

ll-medi ted immunity. Another potenti l str tegy for the enh ncement of T-cell r
esponses is the djuv nt dministr tion of pl smids encoding costimul tory molec
ules such s B7.1 or B7.2, presum
ly to provide the second sign l required for
optim l T-cell ctiv tion (46,47). Co-delivery of costimul tory molecule genes f
or B7.1, nd p rticul rly for B7.2, h ve
een shown to enh nce the induction of
cell-medi ted immunity following intr muscul r injection of pl smid-encoded nti
gen (41,45,48). These o
serv tions h ve led investig tors to question whether ex
pression of such costimul tory molecules
y muscle cells endows them with the c
p
ility to directly stimul te T cells (49). Whether or not expression of sing
le co-stimul tory molecule lone c n convert muscle cell or other som tic cell
type to profession l APC is uncle r,
ut test
le. The possi
ility of indirec
t effects of mem
ers of the B7 f mily should lso
e considered. Sign ling vi t
he B7-CD28 p thw y prevents T-cell poptosis (26) nd m y
e expected to promote
second ry exp nsion of primed CTLs. In ddition, B7 f mily mem
ers lso ppe r
to induce cytokine production, p rticul rly IFN-,
y NK cells leadin to enhanced
immune responses (our own unpu
lished o
servations). The o
servation that immun
ostimulatory DNA sequences (ISS) containin unmethylated CpG dinucleotide motifs
in a particular
ase context can contri
ute to effective induction of an immune
response is particularly relevant for DNA vaccines. Studies usin -alactosidase
as a model antien demonstrated that the Th1
ias of the immune response induce
d
y plasmid DNA immunization, with the expansion of IFN--producin CD4+ T cells
and cytotoxic T cells, was maximal when the plasmid DNA
ack
one contained such
ISS (50). Concomitant injection of ISS-deficient plasmid DNA encodin the antie
n with non-codin ISS-enriched plasmid DNA also induced a Th1-
iased response, i
ndicatin that the ISS-enriched plasmid DNA exerts an adjuvant effect. Several 
roups have reported that
acterial DNA containin ISS is capa
le of activatin N
K cells and macrophaes in vitro leadin to the production of IFN-/, IFN-, TNF-, IL1, IL-6, IL-12, nd IL-18 (5056). Both the type I interferons nd IL-12 re known
to enh nce the gener tion nd incre se the cytotoxicity of NK cells nd CD8+ CT
L nd to promote T helper 1-like phenotype (ch r cterized
y high IFN- producti
on) in the antien-specific differentiation of naive CD4+ T cells. Local IFN- pro
duction
y NK cells is capa
le of activatin macro48
Ttin et al.
phaes, enhancin antien uptake and presentation. Thus, injection of hih doses
of ISS-containin plasmid DNA activates innate immunity, and leads to the produ
ction of Th1-
iasin cytokines within the skin or muscle, promotin cell-mediate
d immunity. Interestinly, the potent immunostimulatory effects of Freunds adjuva
nt may depend on the ISS-enriched myco
acterial DNA. Indeed, the use of plasmid
DNA-containin ISS as an adjuvant can provide, in part, the immunostimulatory ef
fects of Freunds adjuvant without the severe inflammatory and toxic side effects
(56). It has
een reported that plasmid DNA immunization
y
iolistic cutaneous
delivery induces immune responses with a Th2
ias (16,21). This miht reflect th
e fact that these ene un immunizations use only 1/100th as much plasmid DNA co
mpared to saline DNA immunization. This amount of plasmid DNA may not contain su
fficient quantities of ISS to induce the specific cytokine milieu capa
le of pro
motin a Th1 immune response. We have o
served that plasmids encodin the Th1-
i
asin cytokines IFN- or IL-12 c n shift the type of immune response elicited
y c
ut neous gene gun delivery tow rds Th1 phenotype (86). In conclusion,
y choos
ing n ppropri te injection vehicle nd desired route of delivery, nd co-deliv
ering pl smids encoding immunomodul ting cytokines or costimul tory molecules, i
t m y
e possi
le to t ilor the induction of the immune response in vivo tow rds
effector mech nisms th t re
est suited for given p thogen or tumor. 3. Dend
ritic Cells s Biologic l Adjuv nts for DNA Immuniz tion The est
lishment of cu
lture conditions th t llow for the in vitro gener tion of l rge num
ers of immu
nostimul tory DC from precursor popul tions in
one m rrow nd
lood h s stimul
ted signific nt interest in the use of DC s
iologic l djuv nt, p rticul rly

for c ncer v ccines (5763). Mouse DC h ve
een isol ted from spleen, or grown fr
om
one m rrow cells cultured in GMCSF nd IL-4. Hum n DC h ve
een grown from C
D34+ progenitors isol ted from cord
lood,
one m rrow, or peripher l
lood. In
gener l, culture of these CD34+ progenitors in GM-CSF nd TNF- induces growth nd
differenti tion of DC, nd yields c n
e incre sed
y inclusion of c-kit lig nd
(stem cell f ctor [SCF]) nd Flt-3 lig nd, which exp nd the progenitor pool. DC
c n lso
e gener ted
y culture of hum n
lood monocytes in GM-CSF nd IL-4 (m
onocyte-derived DC). Cultured murine
one m rrow-derived DC, when doptively tr
nsferred, c n effectively present tumor ntigen peptides to the immune system le
ding to the induction of prophyl ctic nd ther peutic cell-medi ted immunity g
inst experiment l murine tumors th t express the s me ntigen (6468). Prim ry tu
mor ntigen-specific T-cell responses could lso efficiently
e induced in vitro
in hum n model using cultured, peptide-pulsed, utoloImmunology of DNA V ccines
49
gous DC s stimul tors nd peripher l
lood leukocytes from he lthy donors or c
ncer p tients s responders (6971). Clinic l tri ls using cultured, peptide-pulse
d DC th t re doptively tr nsferred for the tre tment of met st tic mel nom h
ve lre dy
een initi ted t the University of Pitts
urgh nd t other instituti
ons. The insertion of cDNA encoding n entire tumor ntigen into cultured DC is
currently
eing explored
y num
er of l
or tories s n ltern tive to the us
e of peptides for DC-
sed tumor immunother py(7288). A m jor strength of such ge
ne-
sed v ccines comp red to peptide-
sed ppro ches is th t their pplic tion
does not require prior knowledge of the p tient HLA-h plotype or of specific Tcell epitopes. In ddition, they m y promote
oth cl ss I- nd cl ss II-restrict
ed T-cell responses. The re liz tion th t the induction of immune responses foll
owing pl smid DNA immuniz tion is likely to require ntigen present tion
y prof
ession l APCs provides n ddition l r tion le for DC doptive tr nsfer str tegi
es. Endogenous ntigen synthesis within DC ensures direct ccess to the MHC cl s
s I ntigen processing p thw y. After homing to T-cell-rich re s of second ry l
ymphoid org ns, DCs could present ppropri te MHC cl ss I-
inding epitopes to n
ive T cells in the context of costimul tion. V ccines consisting of murine
one
m rrow-derived DC ex vivo genetic lly modified to express tumor ntigens using v
ir l vectors (72,8083), pl smid DNA (87), or RNA (92), re
le to promote tumor
ntigen-specific T cell responses nd prophyl ctic cell-medi ted immunity g ins
t tumors in experiment l mouse models. Of import nce, v ccin tion with cultured
DC th t h d
een infected ex vivo with recom
in nt denovir l vectors w s shown
to circumvent the pro
lem of neutr lizing nti
odies, severely limiting the repe
ted direct dministr tion of denoviruses in vivo (78). Thus, cultured DC m y

e n ide l vehicle for the implement tion of denovir l vector-
sed str tegies
for immuniz tion. Hum n cultured DC h ve lso
een tr nsduced using retrovir l v
ectors (7477), denovir l vectors (78,79), poxviruses (84), or pl smid DNA (85,86
).
3.1. Retrovir l Vectors
Retrovir l vectors h ve
een used to deliver genes to DC. The potenti l dv nt g
e of this ppro ch is th t the genes m y
e expressed throughout the life of the
cell, nd c n
e tr nsmitted to the cell progeny. However, integr tion of retro
vir l vectors into the chromosom l DNA of t rget cells requires th t the cells u
ndergo cell division. Hence, there h s
een p rticul r interest in the use of re
trovir l-medi ted gene tr nsfer into hum n DC grown from prolifer ting CD34+ pro
genitors. Murine
one m rrow-derived DC c n
e retrovir lly tr nsduced
y co-cul
ture with ecotropic producer cell lines (72) or
y centrifug l tr ns50
Tting et l.

duction with retrovir l supern t nt on d 2,3, nd 4 of the DC culture (73). The

use of ecotropic retrovir l p ck ging cells or supern t nt with high titer is
critic l, since murine stem cells do not express the receptor for mphotropic re
trovir l envelope proteins. Adoptive tr nsfer of DCs expressing model tumor n
tigen c n induce ntigen specific immunity le ding to the prophyl ctic nd ther
peutic ntitumor immunity (72) (T
le 1). Intr tumor l injection of DC expressin
g IL-12 genes c n medi te effective ther py of est
lished tumors (73). Hum n DC
developing in culture from
one m rrow nd cord
lood CD34+ progenitors h ve
e
en tr nsduced with mouse CD2 s reporter gene (74). The m ture DC were shown t
o exhi
it norm l, m ture phenotype (e.g., CD40, CD80, CD83,CD86, p55, S100 exp
ression) nd function ( llogeneic MLR, SEA super ntigen, nd tet nus toxoid rec
ll responses). Aver ge tr nsduction efficiencies of 11.5% for cells grown from

one m rrow progenitors, nd 21.2% for cord
lood progenitors, were reported for
the CD1 + DC progeny. Import ntly, the proportion of DC nd other myeloid popul
tions th t developed in the retrovir lly tr nsduced cultures w s simil r to th t
in mock tr nsduced cultures, indic ting th t retrovir l tr nsduction does not i
mpose
i s on development of one cell type over nother. Tumor- ssoci ted gene
s h ve
een expressed in hum n DC following retrovir l-medi ted tr nsduction (75
,76). The MUC-1 gene, encoding t rget ntigen for
re st c rcinom , w s introd
uced into DC
y centrifug l tr nsduction grown from cord
lood CD34+ progenitors
. A tr nsduction efficiency of >13.5% w s reported for the CD1 + progeny (75). I
mport ntly, the MUC-1 product w s found to exhi
it the s me non-pol rized mem
r
ne expression in DC s in tumor cells, nd the DC were competent stimul tors of
llogeneic MLR responses. The mel nom - ssoci ted MART-1 gene h s lso
een expr
essed in DC grown from CD34+ progenitors derived from peripher l
lood of p tien
ts with m lign nt mel nom (76). Tr nsduction efficiencies were reported to
e h
igher following coculture of the developing cells with one p ck ging cell line (
PG13; 2228%) comp red to nother (PA317), nd higher th n when the cells were inc
u
ted with retrovir l supern t nts. The tr nsduced DC stimul ted the production
of IFN-g mm from tumor-infiltr ting lymphocyte cell lines, nd induced ntigen
-specific CTL from utologous T cells, indic ting th t they were c p
le of nti
gen present tion. However, s pointed out (74), retrovir l infection nd st
le
gene expression w s not directly demonstr ted in this study, nd it is possi
le
th t ntigen rele sed
y tr nsduced cells could h ve
een cquired nd presented

y nontr nsduced cells. Hum n monocyte-derived DC h ve lso
een retrovir lly t
r nsduced with the reporter genes L cZ nd CAT (77). In view of the usu l requir
ement for cell division for retrovir l integr tion, it is interesting th t hig
h tr nsImmunology of DNA V ccines
51
duction efficiency of 4260% w s reported for CD1 + cells nd th t the gene produc
ts were still detect
le fter 20 d culture. Interestingly, s previously descri

ed, splenic DC h ve
een implic ted in the induction of immunity following intr
muscul r injection of retrovir l vectors encoding n HIV protein or ov l
umin i
n mice (35). DC isol ted from the spleens of these mice were shown to cont in pr
ovirus, to express retrovir lly encoded proteins, to present ov l
umin to spec
ific T cell line, nd to induce CTL specific for the HIV protein (i.e., s pepti
de-MHC complexes) fter doptive tr nsfer to n ive mice. However, it is uncle r
whether DC within the muscle were tr nsduced prior to their migr tion into the s
pleen, or whether the retrovirus entered the
lood fter intr muscul r injection
nd tr nsduced DC within the spleen.
3.2. Adenovir l Vectors
Adenovir l vectors c n
e used for gene tr nsfer in nondividing cells,
ut their
utility m y
e limited
y tr nsient gene expression nd immune responses g ins
t the virus itself. In one study (78) hum n monocyte-derived DC were infected wi

th denovirus (AdV) type 5 str ins t multiplicity of infection (MOI) of 100:1

. Although only 15% of the cells were found to express e rly or l te virus protei
ns, these cells were shown to induce virus-specific CD4+ nd CD8+ T prolifer tiv
e responses nd CTL when they were cultured with n ive T cells in restimul tion
ss ys. The pp rently low infectivity of DC in this is
ro dly consistent with
findings from nother report in which n E1deleted, replic tion-incompetent AdV
type 5 vector w s used to tr nsfer genes to hum n monocyte-derived DC (79) (T
l
e 2). To chieve gene expression in 95% or more of the cells, MOIs gre ter th n
1000 were required. While mel nom cell line w s more re dily tr nsduced, thes
e vir l lo ds resulted in cytop thic effects th t were not seen for DC; for ex m
ple, >90% of the DC rem ined vi
le fter exposure to virus t n MOI of 10,000
where s 100% of the mel nom cells were killed. This report demonstr ted th t hi
gh level expression of reporter genes (L cZ nd lucifer se) IL-2, nd IL-7 could

e chieved
y AdV-medi ted tr nsduction t these high MOIs. In contr st, v r
iety of physic l methods for gene tr nsfer, including DNA/liposome complexes, el
ectropor tion nd c lcium phosph te precipit tion, were rel tively inefficient o
r ineffective in the s me study. Adenovir l vectors h ve lso
een used to tr ns
duce immort lized murine DC lines or cultured
one m rrow-derived DC with model
tumor ntigens such s -g l ctosid se or ov l
umin (8083). Adoptive tr nsfer of tr
nsduced DC lso promotes tumor- ntigen-specific CTL responses nd prophyl ctic
nd ther peutic nti-tumor immunity. However, none of the studies reported so f
r h ve employed tumor ntigens simil r to those found for hum n c ncers, which
52
Tting et l.
T
le 1 Ex Vivo Gene Tr nsduction of Dendritic Cells: Retrovir l Tr nsduction Re
f. (72) Vector: Gene: Retrovir l vector MFG, MLV LTR, in ecotropic CRE p ck ging
cell line i) Bet -g l (L cZ) s model tumor ntigen ii) Hum n HER-2/neu s cont
rol Cell: Murine
one m rrow progenitors cultured in GM-CSF nd IL-4 Mode: Co-cu
lture with p ck ging cell lines for the first 2 d of DC gener tion n lysis nd
use on d 7 Findings: Gene expression, ntigen present tion in vitro induction of
CTL nd prophyl ctic/ther peutic ntitumor immunity in vivo Ref. (73) Vector: G
ene: Cell: Mode: Findings: Ref. (74) Vector: Gene: Cell: Mode: Findings: Ref. (7
5) Vector: Gene: Cell: Mode: Retrovir l vector MFG, MLV, LTR, in mphotropic BIN
G p ck ging cell line i) Bet -g l (L cZ) reporter gene ii) Hum n MUC-1 Hum n cor
d
lood CD34+ progenitors cultured in GM-CSF, TNF-, IL-4 nd SCF Infection t d 1
nd d 2 of culture (2 cycles) cells incu
ted with supern t nt from cell line,
2 h, n lysis e.g., t d 14 Gene expression, norm l DC, phenotype nd (incre sed
) function Retrovir l vector MFG, MLV LTR, in mphotropic CRIP p ck ging cell li
ne Mouse CD2 reporter gene DC from hum n
one m rrow nd cord
lood CD34+ progen
itors cultured in GM-CSF, TNF-, SCF, nd Flt-3 lig nd Infection t d 3 of culture
cells incu
ted with cell line plus poly
rene, 24 g n lysis t d 10d 13 Gene ex
pression, norm l DC phenotype nd function Retrovir l vector MFG, MLV, LTR, tr n
sient tr nsfection of ecotropic BOSC23 or ecotropic CRE p ck ging cell lines i)
Hum n CD80 ii) Murine IL-12 Murine
one m rrow progenitors cultured in GM-CSF n
d IL-4 Infection t d 2, d 3, nd d 4 of culture (3 cycles)
y centrifug l tr ns
duction (2 h t 32C) with supern t nt from cell lines, n lysis on d 7 Gene expre
ssion, ntitumor effect in vivo
Findings:
Immunology of DNA V ccines
T
le 1 (continued) Ref. (76) Vector: Gene: Cell: Mode: Findings: Ref. (77) Vect
or: Gene: Cell: Mode: Findings:
53
Retrovir l vector MFG, MLV, LTR, in mphotrophic PA317 or PG13 (gi

on pe leuke

mi virus envelope) p ck ging cell lines i) Murine CD80 ii) Hum n MART-1 Hum n c
ord
lood CD34+ progenitors cultured in GM-CSF, TNF-, nd SCF Infection t d 1, d
2, nd d 3 of culture (3 cycles) cells incu
ted with cell lines or supern t nt
from cell lines plus poly
rene, 6 h, n lysis on d 12 Gene expression, Antigen
present tion in vitro
Retrovir l pl smid MFG, MLV, LTR, in mphotropic CRIP p ck ging cell line Bet -g
l (L cZ) reporter gene DC from hum n
lood monocytes cultured in GM-CSF nd IL4 Infection t pprox d 7d 8 of culture cells incu
ted with supern t nt from cel
l line, 4 h; 3 cycles of infection on successive d ys Gene expression

ll ppe r to derive from norm l germ line-encoded genes. It will
e critic l to

ev lu te whether the inherent immunogenicity ssoci ted with denovir l vectors
will help or suppress the induction of immune responses to these self molecules,
which m y
e consider
ly less immunogenic.
3.3. N ked DNA
Non-vir l gene delivery methods h ve sever l import nt dv nt ges of potenti l c
linic l interest:
1. More th n one gene c n re dily
e tr nsfected simult neously, llowing for co
tr nsfection of genes encoding distinct tumor ntigens nd/or immunostimul tory
cytokines. 2. Only the gene of interest is tr nscri
ed without immunologic l int
erference from vir l proteins
oth in vitro nd in vivo. 3. There is no risk of
recom
in tion ssoci ted with replic tion-deficient vir l vectors. 4. Insertion
of foreign DNA into the genome is less likely due to the tr nsient n ture of gen
e tr nsfer. 5. The ppro ch uses highly purified DNA, which c n re dily
e produ
ced in l rge qu ntities, nd is very st
le.
54
Tting et l.
T
le 2 Ex Vivo Gene Tr nsduction of Dendritic Cells: Adenovir l Tr nsduction Re
f. (79) Vector: Gene: Cell: Mode: Findings: Ref. (80) Vector: Gene: Cell: Mode:
Findings: Ref. (81) Vector: Gene: Cell: Mode: Findings: Ref. (82) Vector: Gene:
Cell: Mode: Findings: Adenovir l vector, CMV promoter/enh ncer, SV40 polyA termi
n tion sign l (E1-deleted, replic tion-deficient, Ad-5 vector) i) Lucifer se nd

et -g l (L cZ) s reporter genes ii) Polyom middle T ntigen Murine
one m rr
ow progenitors cultured in GM-CSF nd IL-4 Incu
tion with denovir l vector for
2 h on d 6, n lysis fter 24 h or injection Gene expression, induction of CTL
nd nti-tumor immunity Adenovir l vector, CMV promoter/enh ncer, SV40 polyA ter
min tion sign l (E1-deleted, replic tion-deficient, Ad-5 vector) MART-1 Murine

one m rrow progenitors, cultured in GM-CSF nd IL-4 Incu
tion with denovir l v
ector in RPMI plus 2% FCS for 2 h on d 6, i.v. injections d 0, 7, nd 14 Gene ex
pression, induction of CTL nd nti-tumor immunity Adenovir l vector, RSV promot
er/enh ncer, SV40 polyA termin tion sign l (E1-deleted, replic tion-deficient, A
d-5 vector) i) Bet -g l (L cZ) s reporter genes ii) Ov l
umin Immort lized DC l
ine JAWS II nd Freshly isol ted splenic DC Incu
tion with denovir l vector;
n lysis fter 48 h or injection d 0 nd 7 Gene expression, induction of CTL nd
nti-tumor immunity Adenovir l vector, CMV promoter/enh ncer, SV40 polyA termin
tion sign l (E1-deleted, replic tion-deficient, Ad-5 vector) i) Lucifer se nd

et -g l (L cZ) s reporter genes ii) IL-2, IL-7 DC from hum n
lood monocytes cu
ltured in GM-CSF nd IL-4 Incu
tion with denovir l vector in RPMI plus 2% hum
n AB serum for 2 h on d 6; n lysis fter 24 h Gene expression
Immunology of DNA V ccines
T
le 2 (continued) Ref. (83) Vector: Gene: Cell: Mode: Findings:
55

Adenovir l vector, CMV promoter / enh ncer, SV40 polyA termin tion sign l (E1-/E
3-deleted, replic tion-deficient, Ad-5 vector) Bet -g l (L cZ) s model tumor n
tigen Immort lized DC lineXS52 nd murine
one m rrow progenitors cultured in GM
-CSF nd IL-4 Incu
tion with denovir l vector for 2 h on d 6, n ylsis fter 2
4 h or injection Gene expression, induction of CTL nd nti-tumor immunity
Hum n monocyte-derived DC h ve
een tr nsduced with pl smid DNA encoding reporte
r genes nd mel nom - ssoci ted hum n tyrosin se
y liposome-
sed tr nsfectio
n (85) (T
le 3). Low level expression of the reporter genes (CAT, nd L cZ enco
ding -g l ctosid se) nd tyrosin se w s detected t the protein level. Furthermor
e, DC tr nsfected with the tyrosin se gene were shown to cluster with CTL line
specific for tyrosin se peptide-MHC cl ss I peptide, nd to induce secretion
of TNF
y these ntigen-specific T-cells, suggesting th t the ntigen expressin
g DC were c p
le of ntigen present tion. We h ve o
t ined simil r results with
peripher l
lood-derived DC tr nsfected with pl smid DNA encoding mel nom nti
gens
y p rticle
om
rdment using the gene gun (86). The use of p rticle-medi t
ed gene tr nsfer consistently llows the insertion of genes encoding ntigens in
to DC without interference from vir l proteins, l
eit with consider
ly lower t
r nsduction efficiency when comp red to vir l vectors. Five different mel nom
ntigens were ev lu ted: MART-1/Mel n-A, pmel 17/gp100, tyrosin se, MAGE-1, nd M
AGE-3. Our results suggest th t non-vir l gene delivery system c n lso
e use
d to present given mel nom - ssoci ted ntigen in n immunogenic form t
y gen
e-modified DC nd promote the induction of prim ry mel nom -re ctive CTL in vitr
o. The doptive tr nsfer of murine
one m rrow-derived DC genetic lly engineered
to express tumor ntigens
y p rticle-
om
rdment (87) w s c p
le of inducing
ntigen-specific CTL nd protective nti-tumor immunity. In comp r tive study,
mouse splenic DC or m croph ges were tr nsduced with pl smid DNA encoding HSV-1
proteins (88). Following intr muscul r injections of tr nsduced cells into n iv
e mice, the induction of nti-HSV immunity w s ssessed. It w s found th t mice
injected twice t weekly interv ls with tr nsduced DC were protected g inst
su
sequent ch llenge with
56
T
le 3 Ex Vivo Tr nsduction of Dendritic Cells: N ked DNA Ref. (85)
Tting et l.
i) N ked DNA, CMV promoter (Str t gene, Heidel
erg, Germ ny) ii) cDNA euk ryotic
expression vector (pCEP4; Invitrogen, Amstel, The Netherl nds) Gene: i) Bet -g
l (L cZ) nd CAT reporter genes ii) Hum n tyrosin se Cell: DC from hum n
lood m
onocytes cultured in GM-CSF nd IL-4 Mode: Tr nsfection t d 7 of culture, cells
incu
ted with DNA plus c tionic liposomes (Lipofectin; Life Technology); 24 h
n lysis 25 d fter tr nsfection Findings: Gene expression, ntigen present tion
in vitro Ref. (86) Vector: Gene: N ked DNA, CMV promoter (pCI; Promeg , M dison,
WI) i) Lucifer se nd GFP reporter genes ii) MART-1, gp100/pmel 17, tyrosin se,
MAGE-1, MAGE-3 Cell: DC from hum n
lood monocytes cultured in GM-CSF nd IL-4
Mode: Cells
iolistic lly tr nsfected using the gene gun Findings: Gene expressi
on, ntigen present tion nd induction of prim ry CTL in vitro Ref. (87) Vector:
Gene: N ked DNA, CMV promoter (pCI; Promeg ) i) Lucifer se nd GFP reporter gen
es ii) HPV16-E7, murine p53 Cell: DC from murine
one m rrow precursors cultured
in GM-CSF nd IL-4 Mode: Cells
iolistic lly tr nsfected using the gene gun int
r venously injections on d 0 nd 7 nd n lysis t d 14 Findings: Gene expressio
n, induction of CTL nd protective ntitumor immunity in vivo Ref. (88) Vector:
Gene: Cell: Mode: N ked DNA, CMV promoter (pcDNAI; Invitrogen, S n Diego, CA) HS
V-1 gB or ICP-27 Mouse prim ry spleen DC or m croph ges Cells incu
ted with vec
tor plus DOTAP, 3 h; i.m. injections on d 0 nd 7 nd n lysis t d 14 Findings:
Gene expression, immunity in vivo
Vector:

HSV-1. This enh nced immunity ppe red to
e ssoci ted m inly with n incre sed
Th1 CD4+ T cell response s demonstr ted
y the production of high titers of Ig
G2
ut not IgG1 nti
odies in vivo even fter just one intr muscuImmunology of DNA V ccines
T
le 4 Ex Vivo Gene Tr nsduction of Dendritic Cells: RNA Ref. (92) Vector:N/A G
ene: Cell: Mode: Findings: Ov l
umin, tumor ntigens Mouse prim ry spleen DC Cel
ls incu
ted with RNA plus DOTAP, 24 h Induction of CTL responses nd immunity in
vivo
57
l r injection. Furthermore, splenocytes from the v ccin ted mice produced IFN-g
nd IL-2,
ut not IL-4, when they were restimul ted in vitro. Enh ncement of nt
igen-specific T-cell prolifer tive responses in vitro nd DTH responses in vivo
w s lso o
served. The level of immunity induced
y v ccin tion with tr nsduced
DC w s not s high s th t elicited
y the virus itself,
ut w s t le st s pot
ent, nd in sever l ss ys consider
ly stronger th n th t induced following int
r muscul r injection of the vectors lone. Moreover, tr nsduced m croph ges were
found to
e ineffective t inducing immunity, despite the f ct th t these cells
were shown to express vector-specific mRNA nd protein.
3.4. RNA
Sever l reports indic te th t mRNA c n
e used for in vivo genetic v ccin tion i
n mice. For ex mple, virus-specific CTL were induced following immuniz tion with
liposome-enc psul ted, in vitro synthesized, RNA encoding influenz nucleoprote
in (89); nti
odies were elicited following intr muscul r injection of in vitro
synthesized RNA encoding hum n c rcinoem
ryonic ntigen (90); nd nti
odies wer
e produced fter delivery of hum n -1- ntitrypsin mRNA
y gene gun into the skin
epidermis (91). Mouse splenic DC h ve
een pulsed with RNA in vitro nd h ve
ee
n found to induce CTL in vitro nd immunity in vivo (92) (T
le 4). It w s shown
th t DC pulsed with in vitro synthesized RNA for ov l
umin, or with tot l or po
lyA+ RNA from ov l
umin-expressing tumor cells, could stimul te ntigen-specific
CTL responses in vitro. Furthermore, mice v ccin ted with DC pulsed with RNA fr
om the l tter source were found to
e protected g inst su
sequent ch llenge w
ith the tumor cells. In ddition, v ccin tion of mice with DC th t were pulsed w
ith tot l or polyA+ RNA from poorly immunogenic nd highly met st tic mel nom
cell line resulted in dr m tic reduction of lung met st ses. The potenti l d
v nt ge of this ppro ch is, for ex mple, th t it should
e possi
le to o
t in
nd mplify RNA from even very sm ll mount of tumor tissue for DC-
sed clinic
l immunother py.
58
Tting et l.
4. Summ ry Despite some uncert inties, it is cle r from the studies outlined
o
ve th t genes c n
e delivered to DC
y v riety of me ns nd expressed in func
tion l (e.g., enzymes nd cytokines) or immunogenic (e.g., vir l nd tumor ntig
ens) forms. Wh t is not yet cle r re the rel tive efficiencies of these individ
u l techniques for delivery nd expression of defined genes. System tic, comp r
tive studies will
e required using st nd rdized re gents nd cle rly defined pr
otocols. The immune responses resulting from the doptive tr nsfer of cultured D
C gene-modified ex vivo using different vir l nd non-vir l vector systems shoul
d
e c refully ch r cterized in nim l models nd comp red to those induced
y d
irect inocul tion of pl smid DNA in vivo. Such studies re now underw y in sever
l l
or tories nd the first results should
e forthcoming over the next ye r o
r two. The further ev lu tion of immunogenic vir l vectors such s denoviruses
will lso
e import nt. These vectors c n tr nsduce DC very efficiently nd h ve

een effective in murine models in vivo. The expression of immunogenic vir l pr

oteins might severely limit their pplic
ility or, conversely, might provide no
n-specific help for the induction of immune responses to the encoded ntigen. Wi
th further improvements in gene tr nsfer technology, str tegies m y lso
e deve
loped to t rget the delivery of genes directly to DC in vivo, thus o
vi ting the
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se, B. T. (1997) Enh ncement of immune response to n ked DNA v ccine
y immuniz
tion with tr nsfected dendritic cells. J. Leukocyte Biol. 61, 125132. M rtinon, F
., Krishn n, S., Lenzen, G., M gne, R., Gom rd, E., Guillet, J.-G., et l. (1993
) Induction of virus-specific cytotoxic T lymphocytes in vivo
y liposome-entr p
ped mRNA. Eur. J. Immunol. 23, 17191722. Conry, R. M., LoBuglio, A. F., Wright, M
., Sumerel, L., Pike, M. J., Joh nning, F., et l. (1995) Ch r cteriz tion of
messenger RNA polynucleotide v ccine vector. C ncer Res. 55, 13971400. Qiu, P., Z
iegelhoffer, P., Sun, J., nd Y ng, N. S. (1996) Gene gun delivery of mRNA in si
tu results in efficient tr nsgene expression nd genetic immuniz tion. Gene Ther
. 3, 262268. Boczkowski, D., N ir, S. K., Snyder, D., nd Gil
o , E. (1996) Dendr
itic Cells pulsed with RNA re potent ntigen-presenting cells in vitro nd in v
ivo. J. Exp. Med. 184, 465472.
84.
85.
86.

87.
88.
89.
90.
91.
92.
6
Methodology Used in DNA-B sed Prophyl ctic nd Ther peutic Immuniz tion Ag inst
Hep titis B Virus in Chimp nzees
Alfred M. Prince nd Betsy Brotm n 1. Introduction 1.2. Chimp nzee Welf re Chimp
nzees,
ec use of their ne r-hum n n ture, h ve speci l needs th t must
e met

y those who c rry out medic l rese rch with them. Perh ps the most import nt of
these is the need for comp nionship. Chimp nzees kept lone
ecome o
viously de
pressed, nd m nifest stereotypic
eh vior such s compulsive rocking. We h ve f
ollowed policy of keeping chimp nzees in groups of two or more, whenever possi

le. This h s virtu lly prevented overt depressive symptom tology. This policy h
s not signific ntly ffected studies on hep titis B nd C (1). Addition l me su
res to prevent depression nd enrich the lives of these nim ls include providin
g v riety of food sources, including those th t require consider
le ingenuity
to ccess; e.g., coconuts or p lm nuts th t c n
e opened only
y the use of h
mmer nd nvil, or two stones with j m or must rd held in 50-mL dispos
le cen
trifuge tu
e in pipe
out 2 m outside of the c ge which c n
e re ch
le with
the use of long thin twig. This fishing ctivity resem
les closely the hunting
for termites in the wild nd is f vorite ctivity. Chimp nzees, especi lly du
lts, re very physic l, eng ging in sometimes frightening displ ys. These re im
port nt for the nim ls well-
eing nd require th t c ges
e l rge, ide lly t le
st 20 ft long, nd t le st 12 ft high. Ropes nd tires on ch ins re import nt
pl y o
jects.
From: Methods in Molecul r Medicine, vol. 29, DNA V ccines: Methods nd Protocol
s Edited
y: D. B. Lowrie nd R. G. Wh len Hum n Press Inc., Totow , NJ
65
66
Prince nd Brotm n
The foregoing sources of enrichment re essenti l to the hum n conduct of experi
ments with chimp nzees. 2. Prelimin ry Studies of Immunogenicity An import nt pr
elimin ry to c rrying out studies on prophyl xis or immunother py in the chimp n
zee model is to investig te the immunogenicity of hep titis B surf ce ntigen-en
coding pl smids (HBsAg) in mice. This w s done in series of import nt studies

etween 19931996 (27). These studies used v riety of pl smid constructs, most co
mmonly under the control of the cytomeg lovirus (CMV) immedi te e rly promoter.
Doses of 100 g/mouse given in two intr muscul r sites ppe red to
e optim l. Pre
tre tment of muscle with c rdiotoxin cceler ted the nti
ody response
ut pro


ly h d no effect on its m gnitude (2). Although single injection of DNA produ
ced strong ntiHBs response, which did not diminish with time,
ooster DNA i
njection resulted in 10- to 200-fold further incre se of titer (7). In study
th t w s n import nt precursor of ttempted immunother py in the chimp nzee mo
del, M ncini et l. immunized mice tr nsgenic for HBsAg with single injection
of 100 g of pl smid DNA encoding HBsAg nd Pre-S2 determin nts (3). This resulted

in dis ppe r nce of circul ting HBsAg nd hep titis B (HBV) messenger RNA in
the liver, in the
sence of ny liver histop thology. P ssive tr nsfer experime
nts showed th t the effect w s modul ted
y T cells. Unfortun tely, these dr m t
ic results were not confirmed in tri ls with five other tr nsgenic lines (F. Chi
s ri, person l communic tion). The re sons for this discrep ncy need to
e furth
er investig ted. Only single study h s investig ted the immunogenicity of HBsA
gencoding pl smids in chimp nzees (8). E ch nim l w s immunized t 0, 8, 16, n
d 27 wk, with one nim l receiving 2 mg/dose nd the second 400 g. The nim ls we
re immunized with Biojector needleless injection system for the priming dose
nd for the 8- nd 27-wk
oosters (Bioject, Se ttle, WA). The 16 wk-
ooster w s
dministered
y needle nd syringe fter pretre tment of the injection site
y in
jection of 25% sucrose. At 52 wk
oth nim ls received n ddition l
oost with
convention l ye st derived HBV v ccine. Anti-HBs responses were qu ntit ted
y t
hree sep r te ss ys nd expressed s mIU/mL
y comp ring it to the World He lth
Org niz tion Intern tion l St nd rd. This is n essenti l form of st nd rdiz ti
on if comp rison of immunogenicity is to
e m de
etween different l
or tories.
The nim l receiving 2 mg doses re ched ne rly 10,000 mIU/mL fter the first
o
oster nd 190,000 mIU/mL one mo fter the v ccine
ooster. The response in the
nim l receiving 400 g doses w s much lower, with no nti
ody detect
le
efore th
e first
oost, nd tr nsient low-level responses fer e ch
ooster. Unfortun tel
y the design of the study did not permit comp r tive ev lu Hep titus B Virus in Chimp nzees
67
tion of the Biojector in comp rison to needle injection into sucrose-pretre ted
muscle. 3. Prophyl ctic Immuniz tion of New
orn Chimp nzee Ag inst HBV Only si
ngle study h s
een done to ev lu te the protective effic cy of DNA
sed immuniz
tion (9). We chose to ttempt to protect new
orn chimp nzees g inst ch lleng
e with HBV. The r tion le is th t, in Asi ,
out 40% of tr nsmissions of HBV le
ding to c rrier-st te infections origin te in infected mothers, with tr nsmissi
on occuring mostly during the first d y of life. Thus, in Asi it is essenti l t
h t HBV immuniz tion
e given on the d y of
irth. The design of this study r is
ed the methodologic l question of whether the

ies should
e t ken from their
mothers nd r ised
y hum n surrog tes. Bec use m ny chimp nzees who re not r i
sed
y chimp nzee mothers h ve difficulty resoci lizing with chimp nzees nd l t
er
ecoming good mothers themselves, we chose to return the chimps to their moth
ers immedi tely fter e ch immuniz tion nd fter ch llenge. There w s very sm
ll risk th t the

ies could infect their mothers fter ch llenge. This did no
t occur. Both mothers nd

ies re undou
tedly h ppy with our decision. The pl
smid used in this study encoded HBsAg nd the Pre-S2 determin nt under the cont
rol of the CMV promoter (6). On the d y of
irth nd t 6 nd 24 wk e ch of the
new
orn chimp nzees received 1.0 mg divided into four sites intr muscul rly. Blo
od s mples (12 mL) were o
t ined t 2 wk interv ls. The nim ls were ch llenged
t 33 wk with 100 chimp nzee infectious doses (CID50) of our st nd rd ch llenge s
tock of HBV su
type dw. This stock h s
een titr ted in chimp nzees more th t 1
0 yr go nd h s since
een stored in liquots t 70C. Anti-HBs responses were we
k, pe king t 2030 mIU/mL, nd tr nsient. At the time of ch llenge, little or no
nti-HBs w s detect
le. Nevertheless,
y st nd rd criteri , the nim ls rem ine
d uninfected fter ch llenge developing no HBsAg or nti
ody to the core protein
( nti-HBc). An unimmunized control developed
oth of these m rkers. After ch ll
enge,
oth immunized nim ls m de n mnestic nti-HBs responses. These were show
n to
e result from minim l post-ch llenge replic tion of HBV, demonstr
le only

y polymer se ch in re ction (PCR). We concluded from this study th t non-steri
lizing,
ut protective, immunity h d
een chieved. We now need to determine whe
ther this c n
e chieved when the ch llenge is lso done on the d y of
irth. T
his would mimic the requirements of the hum n situ tion. 4. DNA C n ry Pox-B sed
Immunother py of Chronic HBV Infection The extr ordin ry results reported
y M
ncini et l. (3) stimul ted us to ttempt immunother py of the one chronic c rri

er v il
le in our colony. This nim l h s

68
Prince nd Brotm n

een chronic lly infected since 1985. We used the following constructs in this
ttempt.
4.1. Pl smid Constructs
Pl smid pJW-So, which encodes HBsAg,
ut not Pres-1 nd PreS2, w s used for the
first DNA-
sed immuniz tion. Both pl smids were purified
y dou
le
nding in C
sCl-ethidium
romide gr dients . This pl smid utilized the vector pJW4303 (10),
which w s kindly provided to us
y Dr. J mes Arthos. This expression system is

sed on the CMV immedi te e rly promoter element for tr nscription l control nd
cont ins TPA-mimic le der sequence of 23 mino cids th t is cloned in fr me
with the mino terminus of expressed proteins
y me ns of unique NheI site. Th
e vector provides
ovine growth hormone poly denyl tion sign l for messenger R
NA expression. High-level secretion of HBsAg
y this vector w s o
served fter t
r nsfection of COS-1 cells: 33 ng/mL of supern t nt
y qu ntit tive Ausri , 3 d
fter tr nsfection. The HBV sequence w s o
t ined
y PCR from hum n pl sm , su

type yw, with n upstre m primer th t provided NheI site, nd hy
ridized to
the second mino cid codon of the HBsAg sequence open re ding fr me t the min
o end. The first mino cid of the HBsAg sequence w s omitted. At the c r
oxyl e
nd of the HBsAg gene, 3 primer gener ting BglII site nd including the TAA
stop codon w s used. PJW4303-So w s constructed
y direction lly cloning the pro
duct of this PCR t the NheI nd B mH1 sites of pJW4303. The vector pCDNA III, o

t ined from Invitrogen (C rls
d, CA) employs the CMV promoter of pJW4303 s we
ll s the 3 BGH poly denyl tion site
ut l cks the TPA le der element. The pCDN
AIII-So clone w s constructed
y PCR from the HBsAg gene s HindIII/BglII fr g
ment. The fr gment w s cloned into pCDNA t unique HindIII/B mHI. This clone w s
used for the second DNA immuniz tion, s it w s not cle r which of the foregoin
g pl smids w s more immunogenic. Tr nsfection of pCDNA II into Cos-1 cells did n
ot reve l secretion of HBsAg into the supern t nt.
4.2. C n ry Pox Construct
The c n ry pox construct ALVAC HBV L;M (vCP 157, Virogenetics Inc., Renssel er,
NY) encodes HBsAg, PreS-1, nd PreS-2. This construct uses s vector the ALVAC
v ccine str in of c n ry pox (11). ALVAC HBV L;M cont ins two expression c sset
tes, which encode two different forms of the surf ce ntigen of the yw str in o
f HBV: the L form (HBsAg, Pre-S1, nd Pre-S2), nd the M form (HBsAg nd Pre-S2)
. The c n ry pox vector w s used for the fin l
oosts.
Hep titus B Virus in Chimp nzees 4.3. Immuniz tion
69
Two milligr ms of pJW-So pl smid DNA w s given in four intr muscul r sites t wk
0. Four weeks l ter, the nim l w s simil rly injected with 2.0 mg pCDNA III-So
. On weeks 15 nd 28, the nim l w s
oosted with the recom
in nt c n rypox vect
or ALVAC HBV L;M 4 108 PFU in four sites intr muscul rly nd the s me qu ntity i
ntr venously. The c n ry pox vector w s used for the fin l
oosts, s it h s
ee
n shown th t recom
in nt pox virus vectors give superior n mnestic responses, c
omp red with DNA lone (12).
4.4. Results
Qu ntit tive PCR reve led 1000-fold drop in vir l lo d in pl sm
eginning 1 w
k fter the ALVAC
ooster. The levels rem ined undetect
le
y qu ntit tive PCR
for 18 wk; however,
orderline qu ntities of HBV DNA were detected during this t
ime
y nested PCR, t le st 70 wk of follow-up to d te. HBsAg declined
out fou

rfold fter the ALVAC
ooster
ut r pidly returned to norm l levels. No
norm l
ities in tr ns min se levels were seen. 5. Discussion Our results indic te th t
DNA-
sed immuniz tion c n protect even new
orn chimp nzees g inst overt infect
ion with HBV. Future experiments will
e needed to determine whether this protec
tion lso will extend to virus ch llenge t the time of
irth. This would
e req
uired for protection of new
orn inf nts from m tern lly derived infections. Prot
ection g inst HBV infections is usu lly rel ted to levels of nti-HBs, 10 mIU/m
L
eing considered to
e protective level of nti
ody (8). In our study, the n
ew
orn nim ls m de tr nsient nti-HBs responses. Little or no nti
ody w s pres
ent t the time of ch llenge. It is likely th t the protection o
served depended
on cytotoxic T cells, lthough this could not
e ev lu ted due to the sm ll siz
e of the nim ls. The ttempt to control viremi in chronic lly infected nim
l
y DNA nd ALVAC-
sed immunother py w s encour ging, t le st 70 wk of follow
up. Acknowledgments The generosity of M rion Perkus ( t Virogenetics, Troy, NY)
nd P steur Merieux Conn ught, UK, in providing the ALVAC vector is gr tefully
cknowledged. The pJW-So nd pCDNA III-So pl mids were prep red
y Dr. Chuck T ck
ney.
70
References
Prince nd Brotm n
1. Prince, A. M., Good ll, J., Brotm n, B., Dienske, H., Schelekens, H., nd Eic
h
erg, J. W. (1989) Appropri te conditions for m inten nce of chimp nzees in stu
dies with
lood-
orne viruses: n epidemiologic l nd psychosoci l perspective.
J. Med. Prim tol. 18, 2742. 2. D vis, H. L., Michel, M.-L., nd Wh len, R. G. (19
93) DNA-
sed immuniz tion induces continuous secretion of hep titis B surf ce
ntigen nd high levels of circul ting nti
ody. Hum. Molec. Genet. 2, 18471851. 3
. M ncini, M., H dchouel, M., D vis, H. L., Wh len, R. G., Tioll is, P., nd Mic
hel, M.-L. (1996) DNA-medi ted immuniz tion in tr nsgenic mouse model of the h
ep titis B surf ce ntigen chronic c rrier st te. Proc. N tl. Ac d. Sci. USA 93,
12,49612,501. 4. D vis, H. L., Michel, M.-L., M ncini, M., Schleef, M., nd Wh l
en, R. G. (1994) Direct gene tr nsfer in skelet l muscle: pl smid DNA-
sed immu
niz tion g inst the hep titis B virus surf ce ntigen. V ccine 12, 15031509. 5.
Michel, M.-L., D vis, H. L., Schleef, M., M ncini, M., Tioll is, P., nd Wh len,
R. G. (1995) DNA-medi ted immuniz tion to the hep titis B surf ce ntigen in mi
ce: spects of the humor l response mimic hep titis B vir l infection in hum ns.
Proc. N tl. Ac d. Sci. USA 92, 53075311. 6. M ncini, M., D vis, H., Tioll is, P.
, nd Michel, M.-L. (1996) DNA-
sed immuniz tion g inst the envelope proteins
of the hep titis B virus. J. Biotech. 44, 4757. 7. D vis, H. L., M ncini, M., Mic
hel, M.-L., nd Wh len, R. G. (1996) DNA-medi ted immuniz tion to hep titis B su
rf ce ntigen: longevity of prim ry response nd effect of
oost. V ccine 14, 91
0915. 8. D vis, H. L., McCluskie, M. J., Gerin, J. L., nd Purcell, R. H. (1996)
DNA v ccine for hep titis B: evidence for immunogenicity in chimp nzees nd comp
rison with other v ccines. Proc. N tl. Ac d. Sci. USA 93, 72137218. 9. Prince, A
. M., Wh len, R., T ylor, P. E., nd Brotm n, B. (1997) Protective effic cy of D
NA-
sed immuniz tion g inst HBV in new
orn chimp nzees, in V ccines 97 (Brown
, F., Burton, D., Doherty, P., Mek l nos, J., nd Norr
y, E., eds.), Cold Spring
H r
or L
or tory Press, Cold Spring H r
or, NY, pp. 141144. 10. Lu, S., Arthos,
J., Montefiori, D. C., Y sutomi, Y., Must f , F., Johnson, E., et l. (1996) Si
mi n immunodeficiency virus DNA v ccine tri l in m c ques. J. Virol. 70, 39783991
. 11. T rt gli , J., Perkus, M. E., T ylor, J., Norton, E. K., Audonnet, J. C.,
Cox, W. I., et l. (1992) NYVAC: highly ttenu ted str in of v ccini virus. V
irology 188, 217232. 12. Leong, K. H., R msey, A. J., Morin, M. J., Ro
inson, H.
L., Boyle, D. B., nd R msh w, I. A. (1995) Gener tion of enh nced immune respon
ses
y consecutive immuniz tion with DNA nd recom
in nt fowlpox virus, in V cci
nes 95 (Brown, F., Ch nnock, R., Norr
y, E., eds.), Cold Spring H r
or L
or tor
y Press, Cold Spring H r
or, NY, pp. 327331.

7
Intr muscul r nd Intr derm l Injection of DNA V ccines in Mice nd Prim tes
He ther L. D vis 1. Introduction There re sever l different w ys to dminister
pl smid DNA v ccines (1). Those most commonly used include intr muscul r (i.m.)
injection or intr derm l (i.d.) injection of pure pl smid DNA (n ked DNA), or
iol
istic introduction of DNA-co ted gold p rticles into the epidermis with gene gu
n. It is lso possi
le to deliver n ked or liposome-formul ted DNA to mucos l sur
f ces. The chief dv nt ges of the injection methods re th t they do not requir
e ny speci lized equipment nd they re e sy to perform nd gener lly effic cio
us. However, they do c use p in in non- nesthetized nim ls. In contr st, the ge
ne gun ppro ch requires speci l prep r tion to co t the DNA onto the gold p rti
cles, which must then
e stored under dry conditions to prevent the DNA from det
ching, nd speci lized equipment (the Accell gene gun itself is v il
le from
Bio-R d, Hercules, CA). However, the gene gun is p inless nd it llows immune r
esponses to
e induced with very sm ll qu ntities of DNA, pp rently
ec use of
the high efficiency of tr nsfection with direct delivery of the DNA into cells.
It is not completely cle r which is the
est method for DNA-
sed immuniz tion,

ec use of very few comp r tive studies using the s me DNA v ccine in the s me
nim l model. In ddition, numerous other f ctors, including the skill of the inv
estig tor c n gre tly influence the outcome of the tr nsfection procedure (2). I
mmuniz tion of mice is gener lly effective with i.m. injection of pl smid DNA (n
ot t king into ccount differences in doses). In this c se single dministr ti
on of DNA encoding most ntigens c n induce strong
From: Methods in Molecul r Medicine, vol. 29, DNA V ccines: Methods nd Protocol
s Edited
y: D. B. Lowrie nd R. G. Wh len Hum n Press Inc., Totow , NJ
71
72
D vis
immune responses th t l st for the life of the nim l (3). In contr st, the gene
gun usu lly requires repe ted dministr tions for sust ined immune responses in
mice. Intr derm l injection of pl smid DNA c n work s well s i.m. injection,

ut this depends he vily on the skill of the investig tor. For l rger nim ls, i
.m. injection of pl smid DNA v ccine c n lso
e highly effective with singl
e dministr tion, for ex mple, in dogs (Henry B ker, person l communic tion). In
other c ses, repe ted i.m. immuniz tions re required to tt in nd m int in hi
gh nti
ody titers, s we h ve found with the chimp nzee (4), m king this ppro
ch more comp r
le to the i.d. nd gene gun ppro ches. Somewh t
etter results
c n
e o
t ined for i.m. injection of DNA v ccines in l rger nim ls using nee
dleless injection system such s the Biojector (Bioject Inc., Portl nd, OR), whic
h gives
etter distri
ution of the injected su
st nce (5). For some l rge specie
s, intr derm l injection m y work
etter th n i.m., s seems to
e the c se for
the Aotus monkey (6). In summ ry, it ppe rs th t the optim l DNA delivery metho
d depends on the nim l species, the ntigen
eing expressed, nd the protective
correl tes for th t dise se. Thus, for given ntigen nd nim l species, the

est route of dministr tion will pro

ly h ve to
e determined empiric lly. Ne
vertheless, the experience g ined
y others c n
e used s guide to choose the

est method of immuniz tion for given nim l model. The following descri
es
re descri
ed methods to dminister n ked DNA
y i.m. or i.d. injection to rodent
s nd non-hum n prim tes. 2. M teri ls 2.1. Injection with Needle nd Syringe
1. Appropri te nesthesi or tr nquilizer for nim l (e.g., h loth ne [H loc r
o
n L
or tories, River Edge, NJ] or Met f ne [J nssen Ph rm ceutic ls, North York,
ON, C n d ] for mice, ket mine for prim tes) (see Note 1). 2. R zor, prefer
ly
electric, to sh ve skin over injection site (see Note 2). 3. U-100 insulin syri
nge (100, 50 or 30 units = 1, 0.5, or 0.3 mL, respectively) with tt ched 29G1/2
needle (Becton Dickinson, Fr nklin L kes, NJ) or tu
erculin syringe fitted with
28G needle (Becton Dickinson) (see Note 3). 4. Polyethylene tu
ing (PE 20, i.d.

= 0.38 mm). 5. Pl smid DNA v ccine dissolved in endotoxin-free s line (Sigm , S

t. Louis, MO) t ppropri te concentr tion (e.g., 0.1 mg/mL to deliver 10 g in 10
0 L, or 1 mg/ mL for delivery of 100 g in s me volume) (see Notes 4 nd 5).
2.2. Injection with Biojector
1. Appropri te nesthesi for nim l (e.g., h loth ne or Met f ne for mice). 2.
R zor, prefer
ly electric, to sh ve skin over injection site (see Note 2). 3. B
iojector needleless injection system with syringes nd CO2 c rtridges (Bioject I
nc.) (see Note 6).
DNA V ccine Delivery
3. Methods 3.1. I.M. Injection of Mice with Needle nd Syringe
73
1. Prep re needle
y pl cing piece of polyethylene tu
ing over the needle nd
cutting it c refully to length such th t only 23 mm of needle protrudes (
sic
lly just the
eveled portion should protrude). This serves s guide for how de
ep the needle should
e inserted into the muscle. 2. Dr w DNA solution into the
syringe nd ensure there re no ir
u

les. For
il ter l injection, you will
need 100 L for e ch mouse (see Note 5). 3. Anesthetize mice (see Notes 1 nd 7).
4. Sh ve the fur over the nterior, l ter l nd medi l surf ces of the leg from

ove the knee to the nkle. 5. Position the mouse on its
ck, nchor one foot
to the t
le with your thum
(of the opposite h nd th n you hold the syringe)
nd use your index finger of the s me h nd to stretch the
ody nd nchor the
d
omen. M ke sure the nterior ti
i l ridge (
ony ridge running vertic lly
etween
the knee nd nkle) is f cing directly upif the leg is rot ted you will h ve p
oor injection nd poor results. 6. We h ve found the
est muscle to inject is th
e ti
i lis nterior (TA) muscle (see Notes 8 nd 9). Insert the needle t 45 n
gle into the TA
out 3 mm l ter l (to the outside) of the nterior ti
i l tu
er
osity (
ump on the ti
i l ridge
out h lfw y
etween the knee nd the nkle).
M ke sure the syringe is ligned with the
ody xis nd not ngled to either si
de (in which c se the DNA m y end up in the interstiti l sp ce). The needle shou
ld
e inserted until the PE tu
ing rests g inst nd slightly depresses the skin
,
ut dont press so h rd th t the skin is deeply indented
y the PE tu
ing. Slowl
y inject (i.e., over 510 s) 50 L of the DNA solution (see Note 10) without ch ngin
g the pressure of the PE tu
ing on the leg (this t kes some pr ctice), hold the
needle in pl ce for few seconds, then c refully withdr w the needle. A sm ll d
rop of liquid m y form over the injection site. For
il ter l injection, repe t
in the s me m nner for the opposite leg (see Note 9).
3.2. I.M. Injection of Prim tes with
1. Sh ve the re over the injection
ve
een used successfully) nd wipe
olume of DNA (usu lly
etween 200 L
dle completely into the muscle
elly
the needle slowly s you do so (
ut
ll DNA h s
een injected).

Needle nd Syringe
site (the deltoid,
iceps, nd qu driceps h
with n lcohol sw
. 2. Dr w the desired v
nd 1 mL) into the syringe. 3. Insert the nee
t 45 ngle nd inject slowly, withdr wing
it should still
e in the muscle
elly when

3.3. I.M. Injection with Biojector

1. Anesthetize or tr nquilize the nim l if necess ry nd sh ve the re over th
e injection site (the deltoid,
iceps, nd qu driceps h ve
een used successfull
y). Wipe the site with n lcohol sw
. 2. Dr w the desired volume of DNA (usu l
ly
etween 200 L nd 1 mL) into the ppropri te size of syringe (see Note 6) usin
g the det ch
le needle provided for this purpose. Det ch the needle nd disc rd
.
74
D vis

3. M ke sure the CO2 c rtridge in the Biojector injection device h s enough pres
sure (there is n indic tor for this), then insert the syringe into the device.
4. Press the end of the syringe firmly g inst the skin, m king sure th t it is
perpendicul r to the surf ce of the skin, nd pull the trigger.
3.4. I.D. Injection
1. Anesthetize or tr nquilize the nim l if necess ry nd sh ve the re over th
e injection site. In mice, the skin over the nterior
domin l w ll or the lowe
r
ck re frequently used; in prim tes, the skin over the
ck or the lim
s m y

e used. 2. Dr w DNA solution into the syringe nd ensure there re no ir
u


les. For e ch site, the m ximum volume th t you c n comfort
ly inject is 2025 L.
Most i.d. injections re given t sever l (i.e., 28) sites. 3. Stretch the skin t
ut
etween thum
nd index finger nd with the syringe lmost p r llel to the s
kin, insert the needle with
evel up into the skin just until the
evel is compl
etely within the skin (
out 34 mm). Slowly rot te the needle 90 so th t the
evel
is now f cing sidew ys. Slowly inject the DNA solution. Once the desired volume
h s
een injected (it will form
lip on the skin), rot te the needle 90 to the

evel-up position nd slowly withdr w (see Note 11).
4. Notes
1. Mice should
e nesthetized since, when w ke, they m y contr ct their muscle
s nd squeeze the DNA solution out. L rger nim ls m y
est
e tr nquilized to
void their struggling or
iting during injection. 2. Sh ving is essenti l for go
od visu liz tion of l ndm rks when choosing the injection site), nd when the in
jection is in process. Regul r veterin ry or doggrooming clippers re suit
le f
or l rge nim ls; however, they re
it l rge for mice, especi lly if the teet
h re widely sp ced. The electric r zors th t
r
ers use to trim side
urns re
ide l for mice, s they h ve sm ll he d with closely sp ced teeth, nd re oft
en rech rge
le, so there is no interference with the cord. M nu l r zors re cu
m
ersome nd slow to use nd e sily cut the delic te skin of mouse. 3. The ins
ulin syringes,
y virtue of their fused needle, h ve virtu lly no de d sp ce. Th
ey re prefer
le to the tu
erculin syringes, which h ve
out 100 L of de d sp c
e (w sting DNA), nd often develop ir
u

les, which c n m ke ccur te volume d
elivery difficult. The most ccur te volumes re possi
le with the 0.3 mL insuli
n syringe,
ut this h s to
e relo ded more frequently th n the 1 mL syringe. M
ke sure you h ve U-100 insulin syringes so th t the units will correspond to vol
ume (1 unit = 10 L). Some countries do not use U-100 insulin nd you m y not
e

le to get these syringes t your ph rm cy. In this c se you m y use U-60 syring
es (1 unit = 16.7 L)
ut you will h ve to c lcul te the correct num
er of units f
or the desired volume nd m rk it on the side with fine-tip m rker. Since it i
s
est to h ve the
evel pointing down nd the num
er sc le up to see it, you m
y h ve to disc rd m ny insulin syringes where the needle h s
een fused on in th
e wrong orient tion.
DNA V ccine Delivery
75
4. Most investig tors f mili r with in vitro tr nsfection methods routinely diss
olve their DNA in TE. This could
e used for i.d. injection,
ut should not
e u
sed for i.m. injection, since EDTA is chel ting gent nd c n kill muscle fi
e
rs whose function is c lcium-dependent. Some investig tors simple dissolve the p
l smid DNA in w ter; however, this c n le d to rel xing of the DNA from the pref
erred super-coiled st te to rel xed st te. Physiologic l s line (0.9% N Cl) is
fine s long s the solution will not
e kept for long s the pH m y ch nge. Ph
osph te
uffered s line m y lso
e used. The m ximum concentr tion possi
le is

out 10 mg/mL. 5. The effective dose of DNA v ccine delivered
y i.m. injection
to mouse is usu lly 10100 g. This m y
e injected unil ter lly or divided into t
wo equ l p rts nd injected
il ter lly; the l tter is preferred
ec use it c n

compens te for one
d injection. In either event, e ch TA muscle should
e inje

cted with 50 L. Thus, dministr tion of 100 g dose unil ter lly would
e 50 L t
2 mg/mL, or
il ter lly, tot l of 100 L t 1 mg/mL. 6. The ddress for Bioject
Inc. is 7620 SW Bridgeport Rd., Portl nd, OR 97224 USA, Tel: (503) 639-7221, FAX
: (503) 620-6431. The pp r tus costs
out $1000 (US). Syringes re single use
only nd must
e purch sed sep r tely. CO2 c rtridges provide the propulsion to
depress the syringe plunger, nd e ch will do for
out 10 injections. Syringes
come in v rious sizes (17) which refer to the perture size nd thus the depth of
penetr tion of the liquid. A #1 h s the sm llest perture nd most superfici l
delivery, #7 is l rgest nd deepest. The Bioject device is too powerful for us
e on mice (even with the #1),
ut the #1 syringe c n
e used for i.m. injection
of r ts. In l rger nim ls, higher num
ers m y
e required for i.m. injection (e
.g., #2 for Aotus monkey, #3 for Rhesus m c que, #47 for chimp nzee, depending on
the site). A too-low num
er for the species will give su
cut neous injection,
which is not desired. Bioject provides guide for which syringe size to use fo
r i.m. injection of hum ns of different size nd
ody weights. This, t
est, c
n
e only guide for other nim ls which m y h ve very different thickness of
skin or connective tissue surrounding the muscle. The only w y to know for sure
is to perform dissection fter tri l, or, to void h ving to kill the nim
l, to t ke n X-r y fter injection of r dioop que su
st nce. It is lso possi

le to give n i.d. injection with the Biojector using speci l sp cer nd syri
nge #1. 7. It is optim l to use 68-wk-old mice (weight 1921 g) s they re l rge e
nough to inject the 50-L volume into the TA muscle. Much older mice m y give poor
er immune responses. For given ntigen, different str ins of mice will respond
differently, thus the
est str in to use m y h ve to
e determined empiric lly,
lthough BALB/c mice seem to respond well to most ntigens. Fem les m y give
e
tter immune responses th n m les, nd re nicer to work with s they re less g
gressive nd c n e sily
e group housed. 8. M ny people inject DNA v ccines into
the qu driceps muscle of the mouse
ec use this is the one muscle th t every no
n- n tomist knows, nd there is the misconception th t it comprises l rge musc
le nd therefore impossi
le to miss.
76
D vis
In f ct, the qu driceps is four sep r te muscles with common tendon of inserti
on, so there is risk of injecting
etween two of these muscle
ellies into the
interstiti l sp ce, which would give very poor immune response. In ddition,
the skin of the mouse is loose nd f tty over the thigh (it looks like their leg
s st rt t the knee), nd this incre ses the risk of injecting the DNA su
cut ne
ously, which will lso give poor results. We h ve o
t ined
est results with the
TA muscle, which is the fleshy muscle
elly
etween the knee nd the nkle nd
to the outside of the nterior ti
i l ridge (if you pull your toes nd nkle up,
you c n feel this muscle contr ct on yourself). It is l rger th n ny of the in
dividu l
ellies of the qu driceps, nd is immedi tely
elow the skin of the leg
, which is t ut nd thin, llowing one to ctu lly see the muscle
elly enl rgin
g s you inject the DNA. 9. It is strongly recommended th t you first pr ctice y
our injection using colored liquid (e.g., Indi ink or tryp n
lue). Do this o
n freshly killed mice nd fter e ch injection remove the skin nd dissect the m
uscle out to see how well you did. All of the colored su
st nce should
e in the
TA, the
elly of which will
e uniformly colored. If it is under the skin nd i
n the sp ce under the TA muscle, it is not good injection. M ny people inject
one side of the
ody
etter th n the other. To determine if this is the c se for
you, inject sever l left nd right TA muscles with 10 g of lucifer se reporter
gene construct in 50 L volume nd ss y the muscles 35 d l ter for lucifer se
ctivity. With th t dose, we routinely me sure lucifer se ctivity t 3 d in the
TA muscle of
out 500,000 rel tive light units per second per milligr m protein
(2). One hint is to use your thum
to push the plunger on the syringe s th t w
ill give you much
etter control th n using your index finger. Another hint: if

you ccident lly pull the needle out fter it w s inserted
ut
efore you inject
ed, try to reinsert the needle into the s me hole, otherwise the DNA you inject
will come out of this hole. This is nother re son for sh ving the fur off, s y
ou will never find the hole otherwise. 10. We h ve found th t volume of 50 L is
ide l for injection of the TA s this llows distri
ution of the DNA throughout
the entire muscle without c using excessive d m ge. L rger volumes will c use d
m ge nd h ve excessive pressure
uildup, which then c use DNA le k ge. Sm ller
volumes c n give more v ri
le results. 11. With n i.d. injection, the
lip sh
ould persist in the skin for t le st sever l minutes. If it dis ppe rs quickly
or never forms, you h ve given su
cut neous injection, which is not likely to
work.
References
1. D vis, H. L. nd Br zolot Mill n, C. L. (1997). DNA-
sed immuniz tion g ins
t hep titis B virus. Springer Semin. Immunop thol. 19, 195209. 2. D vis, H., Wh l
en, R., nd Demeneix, B. (1993) Direct gene tr nsfer into skelet l muscle in viv
o: f ctors ffecting efficiency of tr nsfer nd st
ility of expression. Hum n G
ene Ther. 4, 151159.
DNA V ccine Delivery
77
3. D vis, H. L., M ncini, M., Michel, M.-L., nd Wh len, R. G.(1996) DNA-medi te
d immuniz tion to hep titis B surf ce ntigen: longevity of prim ry response nd
effect of
oost. V ccine 14, 910915. 4. D vis, H. L., McCluskie, M. J., Gerin, J
. L., nd Purcell, R. H. (1996). DNA v ccine for hep titis B: evidence for immun
ogenicity in chimp nzees nd comp rison with other v ccines. Proc. N tl. Ac d. S
ci. USA 93, 72137218. 5. D vis, H. L., Michel, M. L., M ncini, M., Schleef, M.,
nd Wh len, R. G.(1994) Direct gene tr nsfer in muscle with pl smid DNA for the p
urpose of nucleic cid immuniz tion. V ccine 12, 15031509. 6. Gr mzinski, R. A.,
Br zolot Mill n, C. L., O
ldi , N., Hoffm n, S. L., nd D vis, H. L. (1998) Imm
une response to hep titis B DNA v ccine in Aotus monkeys: comp rison of v cc
ine formul tion, route nd method of dministr tion. Mol. Med. 4, 109118.
8
Veterin ry DNA V ccines
Sylvi v n Drunen Littel-v n den Hurk, R lph P. Br un, nd Lorne A. B
iuk 1. In
troduction 1.1. Desired Ch r cteristics of Veterin ry DNA V ccines V ccin tion h
s relied, in gener l, on two technologies for the production of ntigenic m ter
i l suit
le for the gener tion of protective immune response; live infectious
gents nd in ctiv ted or su
unit v ccine prep r tions. Live infectious gents
gener lly re most effic cious,
ut there is lw ys risk f ctor when using thi
s type of v ccine. The m jor dv nt ge of in ctiv ted nd su
unit prep r tions i
s their s fety; however, limited effic cy nd dur tion of immunity nd/or high c
ost m y limit the usefulness of these types of v ccines. DNA v ccines represent
new nd potenti lly powerful ppro ch to the development of su
unit v ccines.
Veterin ry v ccines h ve their own set of desired ch r cteristics. In ddition t
o the o
vious requirement for effic cy, v ccines for veterin ry use h ve to
e r
el tively inexpensive, st
le under field conditions nd e sy to dminister. Whe
re me t qu lity is n issue, s for food-producing nim ls, the method of delive
ry is import nt in th t it is undesir
le to h ve needle tr cts or v ccine resid
ues in the relev nt tissues. This is one of the re sons why the currently prefer
red route of injection is su
cut neous or intr derm l, r ther th n intr muscul r
. Furthermore, from m n gement viewpoint, it is desir
le to immunize once nd
t n e rly ge. Fin lly, multiv lent v ccines re preferred, in order to reduc
e expenses incurred
y v ccin tion nd h ndling. Sever l ch r cteristics of pl s
mid DNA m ke it n excellent vector for delivery of genes encoding protective n
tigens to nim ls. These include gre t

From: Methods in Molecul r Medicine, vol. 29, DNA V ccines: Methods nd Protocol
s Edited
y: D. B. Lowrie nd R. G. Wh len Hum n Press Inc., Totow , NJ
79
80
v n Drunen Littel-v n den Hurk, Br un, nd B
iuk
e se nd speed of production, e sy qu lity control, non- integr tion of the DNA
nd l ck of immunogenicity of the vector itself. In ddition, there is evidence
th t,
y using DNA immuniz tion, it m y
e possi
le to immunize t n e rly ge
with one dose of multi-component v ccine, nd induce long-lived immunity. Thus
, over the lifetime of the nim l, the expenses incurred
y v ccin tion nd tre
tment could
e signific ntly lower.
1.2. Consider tions for Development of Veterin ry DNA V ccines
The prim ry t rgets for DNA immuniz tion in livestock nim ls re infectious ge
nts such s viruses,
cteri , nd p r sites. The use of DNA immuniz tion to com

t utoimmune dise se or tumors is not of gre t relev nce for livestock nim ls

ec use of their short life sp ns,
ut m y
e import nt for comp nion nim ls.
To develop n ppropri te v ccine it is cruci l to underst nd the p thogenesis o
f the p rticul r gents. In gener l, immune responses gener ted
y v ccines re
designed to replic te responses found in nim ls during n tur l infection. One m
jor dv nt ge of DNA immuniz tion is th t this technology is extremely vers til
e in the types of responses th t c n
e gener ted, nd thus c n
e more re dily
d pted to specific situ tions. An import nt initi l consider tion for DNA immun
iz tion is pl smid delivery. Bec use the site nd method of delivery will ffect
the type of immune response, n ppropri te procedure must
e chosen th t
l n
ces the pr ctic l spects of v ccine delivery to l rge nim ls, with the desire
to gener te the most protective response possi
le. Intr muscul r injection, lth
ough popul r in m ny nim l models, m y
e undesir
le for livestock
ec use of
the potenti l effects on me t qu lity. Delivery of pl smid into the epithelium (
skin or mucos l surf ces) is considered to h ve the most promise
ec use of the
immunocompetence of these tissues. Addition lly,
ec use these tissues re the s
ites of entry
y most p thogens, immuniz tion t these sites is expected to
e m
ore effective. Sever l methods m y
e used to modul te the type of response gene
r ted
y DNA immuniz tion. Experiments in which different forms of the ntigen
re expressed
y the pl smid demonstr te th t cell- ssoci ted ntigens (either cy
topl smic or mem
r ne
ound) m y
i s response more tow rds cellmedi ted res
ponse in comp rison to secreted ntigens. Secreted ntigens, however, c n still
gener te cell-medi ted responses, nd under cert in conditions, show enh nced

ilities to gener te humor l responses.The method of delivery, nd the co-express
ion of cytokine or immunomodul tory products, m y lso ffect the type of respon
se. We h ve found th t the types of responses produced v ry with the ntigen, n
im l species, nd route of immuniz tion. Thus, e ch pl smid must
e ev lu ted se
p r tely for its
ility to induce the desired responses in the t rget species
nd most import ntly to protect g inst ch llenge from the infectious gent.
Veterin ry DNA V ccines
81
Although m ny ntigens h ve
een found to work well when dministered
y polynuc
leotide immuniz tion, there re genes th t do not function effectively in produc
ing n immune response. When t rgeting v ccines for specific dise ses the choice
of ntigens m y
e limited; thus cert in genes m y need to
e re-engineered. Ge
nes from
cteri or RNA viruses re norm lly not tr nscri
ed in the nucleus of
nim l cells nd thus m y either cont in in ppropri te sequences, or l ck sequen
ces import nt for proper functioning in the complex sequence of events involved

in RNA tr nscription, m tur tion nd tr nsport. The presence of 5 intron such

s th t commonly found with the hum n cytomeg lovirus immedi te e rly promoter,
in our experience, is cruci l for the expression of genes from
cteri nd RNA
viruses. Further d pt tions of genes such s ltering codon
i s, or removing
in ppropri te post-tr nscription l modific tion sites m y lso
e employed to ge
t stronger expression of function l proteins. Proteins m y lso
e modified to e
nh nce their ntigenicity. T rgeting proteins to specific cell popul tions, form
tion of l rge p rticles or chimer s, or co-expression of other genes m y enh nc
e immunogenicity of proteins,
ut these re likely to
e specific for e ch prote
in nd this emph sizes the import nce of testing e ch pl smid sep r tely. The ex
periment l designs further descri
ed in this ch pter re prim rily
sed on our
experience using rumin nt (sheep, c ttle) model. In order to reduce expenses,
prelimin ry screening of DNA v ccine c ndid te is conducted in the mouse model
to insure th t the constructs function in vivo. Once the immunogenicity of the
pl smid-expressed proteins h s
een confirmed, it is gener lly possi
le to test
the effic cy of veterin ry DNA v ccine using the n tur l or very simil r hos
t s the nim l model. We h ve used
oth sheep nd c ttle s models to test indu
ction of immunity nd protection g inst respir tory dise se in c ttle. The immu
ne responses o
served in out
red nim ls re gener lly not s homogeneous s tho
se o
served in mice, so l rger num
ers re required to o
t in st tistic lly sign
ific nt results. In m ny respects, the methods c n
e re dily d pted to other s
pecies such s pigs, go ts, horses, nd comp nion nim ls. Gener lly, the ss ys
used to n lyze the immune responses induced
y DNA immuniz tion of l rge nim
ls re simil r to those used in the mouse model, lthough there re some limit t
ions with respect to v il
ility of re gents. However, one of the most import n
t consider tions for using pl smid DNA s veterin ry v ccine, is th t the meth
ods nd routes of delivery of pl smid DNA developed for the mouse model gener ll
y need to
e d pted for use in t rget species. Following gener l introduction
on the methods nd protocols used, specific det ils re descri
ed for immunizin
g nim ls with pl smids encoding protective ntigen of
ovine herpesvirus-1 (B
HV-1), glycoprotein D (gD).
82
v n Drunen Littel-v n den Hurk, Br un, nd B
iuk
2. M teri ls 2.1. Construction, Production, nd Purific tion of Pl smids Use st
nd rd molecul r
iology techniques s descri
ed
y S m
rook et l. (1).
2.2. In Vitro An lysis of Protein Production
1. Dul
eccos Minim l Essenti l Medium (DMEM) nd Opti-MEM re v il
le from Gi
c
o-BRL (Gr nd Isl nd, NY). 2. Fet l
ovine serum (FBS) is v il
le from Sigm Ch
emic l Co. (St Louis, MO). 3. Lipofectin is v il
le from Gi
co-BRL.
2.3. Delivery of Pl smids
1. Speci l equipment: the Helios Gene-gun system is v il
le from Bio-R d L
or
tories (Hercules, CA); Minitome cryosectioner is v il
le from D mon/IEC Divis
ion (Needh m Heights, MA); fluorescent microscope. 2. 18- nd 27-G ge needles n
d tu
erculin syringes re v il
le from Becton Dickinson (Fr nklin L kes, NJ).
3. A pl smid expressing green fluorescent protein (GFP) is v il
le from Qu n
tum Biotechnologies (L v l, Qu
ec, C n d ). 4. Phosph te-
uffered s line (PBS): 0
.01 M N 2HPO4/KH2PO4, 0.15 M N Cl/KCl, pH 7.3 (8 g N Cl, 0.2 g KCl, 1.15 g N 2HP
O4, 0.2 g KH2PO4 per liter). Store t room temper ture, disc rd if solution
eco
mes cloudy or precipit tes.
2.4. Humor l Immunity
1. Speci l equipment: n enzyme-linked immunosor
ent ss y (ELISA) re der is v
il
le from Bio-R d L
or tories (Mississ ug , Ont rio, C n d ). 2. Polystyrene
microtiter pl tes (Immulon 2) re v il
le from Dyn tech L
or tories (Ch ntill
y, VA). 3. Co ting
uffer: 0.01 M N 2CO3/N HCO3, pH 9.6 for ELISA; 0.05 M N 2CO3

/ N HCO3, pH 9.8 for B cell ELISPOT. Store t room temper ture for up to 2 mo. 4
. PBST: PBS with 0.05% Tween-20. M ke this fresh for e ch experiment. 5. Anti
od
ies: ppropri te lk line phosph t se-conjug ted second ry nti
odies or
iotiny
l ted second ry nti
ody nd strept vidin- lk line phosph t se; v il
le from v
rious comp nies such s Kirkeg rd nd Perry L
or tories (G ithers
urg, MD), Z
ymed (South S n Fr ncisco, CA), nd Bioc n Scientific (Mississ ug , Ont rio, C n
d ). Store t 4C or 20C. 6. Su
str te p-nitrophenyl phosph te (PNPP) is v il
le
from Sigm Chemic l Co.: dilute 100 PNPP stock in PNPP
uffer; 100 stock: 1 g PN
PP in 10 mL 1% dieth nol mine
uffer. Store t 20C. 7. Dieth nol mine
uffer, 1% (
10 mL dieth nol mine, 990 mL ddH2O, 1 mL 500 mM MgCl2, pH to 9.8 with conc. HCl)
. Store t 4C. 8. Stop solution: 0.3 M ethylenedi minetetr cetic cid (EDTA) (11
1.6 g N 2 EDTA, 13 g N OH, 800 mL ddH2O, pH 8.0; m ke up to 1 L). Store t room
temper ture.
Veterin ry DNA V ccines
83
9. Nitrocellulose pl tes re v il
le from Millipore (Bedford, MA), or Polyfilt
ronics Inc. (Rockl nd, MA). 10. Complete medium for sheep; AIM V (Gi
co-BRL), su
pplemented with 2% FBS (Sigm ), 50 g/mL gent mycin nd 5 105 mM 2-merc ptoeth nol:
for c ttle; MEM (Gi
co-BRL), supplemented with 10% FBS (Sigm ), 2 mM L-glut min
e, 50 g/mL gent mycin nd 5 10 5 mM 2-merc ptoeth nol. Store t 4C for up to 6 mo.
11. ELISPOT su
str te: SIGMAFAST (Sigm ) 5-
romo-4-chloro-3-indolyl phosph te (BC
IP)/nitro
lue tetr zolium (NBT) t
lets. Dissolve one t
let in 10 mL ddH2O, m
ke fresh.
2.5. Cellul r Immunity
1. Speci l equipment: A Coulter counter is v il
le from Coulter Electronics In
c. (Hi le h, FL) or use hemocytometer, Cell h rvester ( v il
le from Sk tron
Inc., Sterling, VA) nd Stereoscope (Olympus S2 series or other);
enchtop centr
ifuge (CS6R; Beckm n). 2. V cut iner tu
es with EDTA(K3) dditive, or no dditive
s re v il
le from Beckton Dickinson. 3. Citr te
uffer (0.05 M) (28.8 g dextr
ose, 44 g N -citr te, 16 g citric cid, nhydr., per L). Store t 4C. 4. PBS/EDTA
: PBS with 0.1% N 2EDTA. Store t room temper ture. 5. Isotonic 60% Percoll: Per
coll is v il
le from Ph rm ci (Mississ ug , Ont rio, C n d ); m ke Percoll is
otonic
y dding one p rt 10X PBS to 9 p rts of Percoll; then m ke 60%
y diluti
ng 6 p rts Percoll with 4 p rts PBS. Store t 4C. 6. AIM V (Gi
co-BRL), supplemen
ted with 2% FBS (Sigm ), 50 g/mL gent mycin nd 5 105 mM 2- merc ptoeth nol. Store
t 4C for up to 6 mo. 7. Ficoll-P que PLUS is v il
le from Ph rm ci . Store t
4C. 8. H nks B l nced S lt Solution (HBSS): 0.001 M N 2HPO4/KH2PO4, 0.15 M N Cl/K
Cl, 0.001 M C Cl2, 0.001 M MgSO4, 0.1% dextrose (8 g N Cl, 0.4 g KCl, 0.14 g C C
l2, 0.2 g MgSO4 in 45 mL ddH2O + 0.06 g N 2HPO4 nhydr., 0.06 g KH2PO4, 1 g dext
rose in 45 mL ddH2O, mix slowly nd check pH 7.3, m ke up to 1 L). Store t 4C. 9
. MEM (Gi
co-BRL), supplemented with 10% FBS (Sigm ), 2 mM L-glut mine, 50 g/mL g
ent mycin nd 5 10 5 mM 2-merc ptoeth nol. Store t 4C for up to 6 mo. 10. Dex met
h sone is v il
le from Sigm . 11. 96-well round-
ottom nd 24-well fl t
ottom
pl tes (Cost r) re v il
le from Fisher Scientific (Nepe n, Ont rio, C n d ).
12. [methyl-3H]Thymidine is v il
le from Amersh m (O kville, Ont rio, C n d )
; dilute in AIM V (sheep) or MEM (c ttle) to 20 Ci per milliliter nd store t 4C.
13. Filterm ts re v il
le from Sk tron Inc. (Sterling, VA). 14. Anti
odies:
r

it nti-
ovine IFN (non-commercial), which also cross-reacts with sheep IFN. 1
5. Nitrocellulose plates, anti
odies, ELISPOT su
strate.
84
van Drunen Littel-van den Hurk, Braun, and Ba
iuk
2.6. Challene and Protection
Special equipment: ULTRA-NEB 99 ne
ulizer is availa
le from DeVil
iss (Barrie, On

tario, Canada). 3. Methods

3.1. Construction, Production and Purification of Plasmids
Plasmids that will direct the expression of the desired ene in animal systems a
re constructed usin standard molecular
ioloy techniques (1). A popular promot
er, and one we have most commonly used, is the human cytomealovirus (HCMV) imme
diate early promoter that also has a 5 intron, desinated intron A. We have als
o used the promoter from the Rous sarcoma virus (RSV) lon terminal repeat, whic
h is a reasona
ly stron promoter,
ut which induces lower levels of protein exp
ression in vitro than the HCMV promoter. Genes are inserted into restriction sit
es that follow the promoter reion, and the inserted sequences must contain the
start and stop codons of the protein. After the inserted ene the plasmid contai
ns 3 sequences, such as those derived from the
ovine rowth hormone ene, whic
h include the polyadenylation sinal.
1. O
tain enes
y restriction diests of other plasmids, or from framents ene
rated
y polymerase chain reaction (PCR) usin proofreadin polymerases to reduc
e potential sequence modifications. 2. Cut vector at an appropriate site, and li
ate the vector and frament toether
y DNA liase. 3. Transform competent Esch
erichia coli with the liation mixture. 4. Grow overniht on anti
iotic-containi
n aar plates to select for transformed
acteria. 5. Grow overniht cultures fr
om sinle colonies, purify the plasmids and characterize
y restriction diestio
n. 6. Verify the construct
y in vitro expression of the protein. 7. Other modif
ications to the enes may
e made
efore or after insertion of the enes into th
e expression plasmid, with confirmation
y in vitro expression and/ or sequencin
 of the relevant reions of the plasmid. 8. To purify lare quantities of plasm
id, row overniht
acterial cultures and collect
acteria
y centrifuation (se
e Note 1). Plasmid may
e purified from
acterial pellets
y usin ion exchane
resins such as those o
tained from Qiaen (Chatsworth, CA), or
y cesium chlorid
e centrifuation (1). 9. Assess plasmid purity
y aarose el electrophoresis (1
) and
y determinin the ratio of the a
sor
ance at 260 nm over 280 nm, which sh
ould
e at least 1.8. Restriction diests are done to verify the identity of the
plasmid.
Veterinary DNA Vaccines
85
3.2. In Vitro Analysis of Protein Production Before usin plasmid constructs in
vivo, protein production is confirmed in vitro
y transient transfection assays.
A num
er of different cell lines and transfection aents may
e used with satis
factory results. Routinely, we use COS-7 cells. The transfection efficiency in o
ther cells, such as Madin Dar
y
ovine kidney (MDBK) cells, is enerally found t
o
e lower than in COS-7 cells.
1. Grow COS-7 cells to 50% confluency in DMEM supplemented with 10% fetal
ovine
serum (FBS). 2. For a 35-mm tissue culture plate, dilute 20  of lipofectin to 1
00 L in OptiMEM and leave at room temperature for 30 min. 3. Dilute the DNA to 2 
/100 L of Opti-MEM, mix with 20  of lipofectin in 100 L of Opti-MEM and incu
ate a
t room temperature for 15 min (see Note 2). 4. Wash cells with Opti-MEM, dilute
the 200 L of lipofectin-DNA complex to 1 mL with Opti-MEM and add dropwise to the
cells. 5. After 5 h, remove Opti-MEM solution and add fresh DMEM containin 10%
FBS to the cells. 6. One to three days later, harvest the transfected cells and
supernatant medium and determine the levels of forein protein production in th
e cells and/or supernatant medium, dependin on whether you are dealin with cel
l-associated or secreted proteins. This may
e done
y immunofluorescence (2),

y immunoprecipitation after radio-la
elin the cells (3),
y Western
lottin (4
,5), or
y ELISA (6). Secreted proteins may have to
e concentrated
y immunopre
cipitation
efore analysis
y Western
lottin. The percentae of transfected ce
lls may
e determined
y FACS analysis (2).
3.3. Delivery of Plasmids For all types of delivery, animals are awake and may

e restrained in a chute for cattle, with a halter if required, or
y an attendan

t for sheep. Hair may
e removed
y clippers to assist vaccine delivery. Vaccina
tin into sites that are infected
y pathoens, or share drainin lymph nodes wi
th the site of infection, is expected to
e the
est approach to induce protecti
on. 3.3.1. Intramuscular (IM) Injection
1. Make up plasmid (typically 500 ) in 2 mL of saline. 2. Usin a 3-mL syrine a
nd a 1 1/2 in. 18-aue needle, inject plasmid into one site in either the lute
al or lonisimus dorsal muscle in cattle. We have not used intramuscular injecti
ons in sheep (see Note 3).
3.3.2. Intradermal (ID) Injection
1. Adjust plasmid concentration to 1 m/mL in saline. 2. Usin a 1-mL syrine an
d 1/2 in. 27-ae needle, inject plasmid into 5 sites (100 L per site) in the ski
n on the outside of the ear or the le of sheep or cattle (see Note 4).
86
van Drunen Littel-van den Hurk, Braun, and Ba
iuk
3.3.3. Gene Gun Delivery
Delivery of plasmid
y a ene un may
e desira
le
ecause of the lower amounts
of DNA that are used. Gold particles are coated with the desired plasmid, and pa
rticles are shot into the cells of the skin. Use of marker enes (i.e., chloroamph
enicol acetyltransferase, -alactosidase, GFP) is recommended to determine the
e
st conditions for delivery of plasmid. For lare animals, sites with little hair
, such as around the tail, axillary reions of the les, or in the mouth, may
e
chosen. Other sites can
e used
ut may require clippin and/or shavin. Furthe
r hair removal can
e achieved
y usin an electric shaver or a
lade razor with
soap. Some reddenin of the skin has at times
een found after delivery of old
particles,
ut the animals have not shown any discomfort (see Note 5). We have
recently
eun to use the Helios ene-un system (Bio-Rad) that uses a helium di
schare to deliver old particles. To determine the appropriate firin condition
s we use a plasmid expressin a GFP to coat the old particles, and then use a v
ariety of firin conditions, skin treatments, and sites to deliver the DNA-coate
d old (see Note 5).
1. Coat old particles (1.6 m) with plasmid as descri
ed
y the manufacturer. 2.
Prepare site(s)
y clippin and/or shavin, if required, and fire old particles
coated with GFP plasmid. Firin pressures for the mouse a
domen are
etween 1502
00 psi, and for sheep and cattle, firin pressures of 300400 psi have
een used.
3. Twenty-four hours after deliverin plasmid, take skin samples and fix in form
alin for 2 h, then wash with PBS and freeze in 30% sucrose at 70C. 4. Section the
frozen samples and view them with a fluorescent microscope, to determine the re
ion and level of ene expression.
3.4. Humoral Immunity
Humoral or anti
ody responses can
e measured in a num
er of ways. One of the mo
st common methods for detection of anti
ody responses is ELISA. Althouh ELISAs
are typically used to quantitate anti
ody levels in the serum, they can also
e
used with other samples such as nasal secretions. Isotype and su
class of anti
o
dies may also
e determined
y ELISA, and this can
e an important indicator of
the type of response enerated
y the vaccine. Sinle anti
ody-secretin cells c
an
e detected usin B-cell ELISPOT assays. These assays can help localize the p
lasma cells secretin anti
odies and characterize these anti
odies. Neutralizati
on assays, which measure the a
ility of anti
odies to
lock or neutralize the in
fectious aent are an important assessment of the functionality of the anti
ody
response. Since neutralization assays are very specific for different pathoens,
we do not descri
e protocols here.
Veterinary DNA Vaccines 3.4.1. ELISA Protocol for Sheep and Cattle Immunized wit
h Plasmid-Encodin BHV-1 D

87
Many different types of ELISAs have
een descri
ed (6). We use the followin en
eral procedure. Polystyrene plates are coated with the appropriate antien and i
ncu
ated with serially diluted ovine or
ovine sera. Affinitypurified alkaline p
hosphatase (AP)-conjuated or horseradish peroxidase (HRPO)-conjuated secondary
anti
odies aainst total IG are used as the detectin anti
ody. The reaction i
s visualized with PNPP or 2,2-azino-di-(3ethyl-
enzthiazoline sulfonate [6]) (A
BTS). Anti
ody isotypes and su
classes are determined in an indirect ELISA usin
lycoprotein D (D)-coated plates and isotype- or su
class-specific monoclonal
anti
odies (see Note 6).
1. Coat polystyrene microtiter plates with 200 L of D (0.25 /mL) per well in coa
tin
uffer and incu
ate plates at room temperature overniht. Glycoprotein D is
an affinity-purified preparation. 2. Wash the plates 4 times with PBST. 3. Incu

ate plates for 2 h at room temperature with 200 L per well of ovine/
ovine sera
serially diluted in PBST. 4. Wash the plates 4 times with PBST. 5. Incu
ate plat
es with 200 L per well of affinity-purified alkaline phosphatase (AP)-conjuated
oat anti-ovine/
ovine IG at a dilution of 1:7,500. 6. Wash the plates 4 times
with PBST. 7. Add 150 L per well of su
strate PNPP. 8. After 1 h, add 50 L per wel
l of 0.3 M EDTA to stop the reaction. 9. Read reaction with an ELISA reader at 4
05/490 nm.
3.4.2. B cell ELISPOT Assay Specific for BHV-1 D
The presence of anti
ody secretin B cells may
e determined in B-cell ELISPOT (
7) assays usin isolated cells from tissues such as
one marrow, Peyers patches
or lymph nodes where plasma cells are typically found. Cells are incu
ated on ni
trocellulose plates that have
een coated with the desired antien. Anti
odies s
ecreted from these cells
ind to the antien on the plate. A
iotinylated second
ary anti
ody, followed
y a streptavidin AP conjuate (or an AP-conjuated secon
dary anti
ody) are then added and the anti
odies are visualized usin a su
strat
e for AP that forms insolu
le complexes. These complexes appear as spots on the pl
ate, which represent sinle anti
ody secretin B cells. Steps 13 are done under s
terile conditions.
1. Coat nitrocellulose plate with 150 L of antien (45 /mL D, 10 /mL BHV-1) per w
ell in coatin
uffer overniht at 4C. Wells coated with a nonspecific antien sh
ould also
e included.
88
van Drunen Littel-van den Hurk, Braun, and Ba
iuk
2. Wash plate 3 times with sterile PBST, 3 times with sterile PBS, and then
loc
k with 150 L/well of complete medium at room temperature for 12 h (or 30 min at 37C
) prior to platin cells. 3. O
tain a sinle-cell suspension from your tissue of
choice and dilute to 1 and 0.5 107 cells/mL in complete medium. Discard
lockin
 solution from the plate and add duplicate or triplicate samples of cells in 10
0 L. Incu
ate the plate on a level surface in a humidified CO2 incu
ator at 37C, f
or 612 h, without distur
in it. 4. Wash the plate 6 times with PBST to remove al
l cells. Add secondary anti
odies specific for different isotypes or IG su
clas
ses. Make anti
ody solutions up in PBST containin 1% FBS (or BSA) usually at 1:
6000 to 1:8000. Incu
ate for 12 h at room temperature or loner at 4C. For
iotiny
lated anti
odies o to step 5, for alkaline phosphatase conjuated anti
odies pr
oceed to step 6. 5. Wash plate 3 times with PBST. Add streptavidin alkaline phos
phatase conjuate in PBST at 1:1000. Incu
ate at room temperature for 12 h. 6. Wa
sh plate 4 times with PBST, and once with PBS. Add the NBT/BCIP su
strate, wait
for spots to appear (1/41h) and wash with ddH2O
efore
ackround is too stron.
Let the surface dry and then count the spots with a stereoscope.
3.5. Cellular Immunity

A second component of the immune response is the cellular immune response, which
is crucial for control of most infectious diseases. One method to measure the c
ellular response to vaccination is lymphoproliferation. Co-culture of isolated l
ymphocytes with antien causes lymphocytes specific for the antien to prolifera
te, sinifyin a response to the vaccine. The second method descri
ed here is th
e measurement of cytokines produced
y stimulated T lymphocytes. Specific types
of cytokines are secreted in response to infectious aents and vaccines, and the
cytokine ELISPOT is a
le to identify and quantitate the cytokine secretion from
T lymphocytes (see Note 7).
3.5.1. Proliferation Assays for Sheep and Cattle Immunized with Plasmid Encodin
BHV-1 D
In order to detect activated T cells, antien-specific proliferation assays are
performed. In mice, lymphoproliferation is usually measured in splenocytes. Howe
ver, in sheep and cattle, this assay is enerally performed with mononuclear cel
ls isolated from peripheral
lood (PBMCs), which is collected in vacutainer tu
e
s containin an anti-coaulant. The PBMCs are isolated and purified from
lood

y radient centrifuation, washed, counted and cultured in the presence or a
sen
ce of antien. This may
e crude or purified antien;
Veterinary DNA Vaccines
89
however, it is important to do a dose titration. After 3 d in culture, the cells
are pulsed with [methyl- 3H]thymidine. The cells are harvested 18 h later and t
hymidine uptake is measured
y scintillation countin. Proliferative responses a
re calculated as the means of triplicate wells and expressed as stimulation inde
x or increased cpm over medium control. Dependin on the species, different assa
y conditions are favored. The entire assay is carried out under sterile conditio
ns.
3.5.1.1. ISOLATION OF PBMCS FROM WHOLE OVINE BLOOD
1. Transfer
lood (2040 mL from vacutainer tu
es with EDTA[K3] additive) into 50mL polypropylene centrifue tu
es. 2. Centrifue at 1500 for 20 min at 10C in a

enchtop centrifue with
rake turned off. 3. Remove the
uffy-coat layer (white
layer of cells, approximately two-thirds from the top) and mix with PBS/EDTA to
a total volume of 10 mL usin a pre-wet pasteur pipet, then layer over 5 mL iso
tonic 60% Percoll in a 15-mL polypropylene centrifue tu
e. 4. Centrifue at 250
0 for 20 min at 20C with
rake off. 5. Remove the mononuclear cell
and (white l
ayer in the middle) with a pre-wet pasteur pipet and transfer into another 15 mL
centrifue tu
e. There should
e four distinct layers: erythrocytes and ranulo
cytes (
ottom), next Percoll, then lymphocytes, monocytes and platelets formin
a distinct white
and at the interface
etween Percoll and PBS. 6. Wash 2 times
with 10 mL of PBS/EDTA and 2 times with PBS, and centrifue cells for 7 min at 2
50. 7. Resuspend last cell pellet in 10 mL of AIM V containin 2% FBS and count
cells with a hemocytometer or Coulter counter. 8. Dilute cells in AIM V contain
in 2% FBS to o
tain 3.5 x 106 cells/mL for proliferation and 6 106 cells/mL for
ELISPOT assay (see Note 8).
3.5.1.2. ISOLATION OF PBMCS FROM WHOLE BOVINE BLOOD
1. Transfer
lood (4050 mL from vacutainer tu
es prefilled with 1/10 volume citra
te
uffer) into 50 mL polypropylene centrifue tu
es. 2. Centrifue at 1000 for
30 min at 20C with the
rake turned off. 3. Remove the
uffy coat layer in appro
x 5 mL, mix it with 5 mL HBSS and layer over 5 mL Ficoll-Paque in a 15-mL polypr
opylene centrifue tu
e. 4. Centrifue at 1500 for 45 min at 20C in a
enchtop c
entrifue with the
rake off. 5. Remove the mononuclear cell
and (white layer i
n the middle) and transfer it into another 15 mL centrifue tu
e. 6. Wash 3 time
s with 10 mL of HBSS and centrifue cells for 8 min at 250. 7. Resuspend the la
st cell pellet in 10 mL of MEM containin 10% FBS and count the cells with a hem
ocytometer or Coulter counter. 8. Dilute the cells in MEM containin 10% FBS to

o
tain 3.5 106 cells/mL for proliferation and 6 106 cells/mL for ELISPOT assay (
see Note 8).
90
van Drunen Littel-van den Hurk, Braun, and Ba
iuk
3.5.1.3. PROLIFERATION ASSAY 1. Prepare the appropriate antiens for stimulation
: BHV-1 D at 0.1 and 1 /mL or UV-irradiated BHV-1 at 5 105 and 5 104 plaque-for
min units/mL. To round-
ottom 96-well plates add 100 L of antien per well. Cont
rol wells contain medium only. Dilute the antiens in appropriate medium (AIM V/
2% FBS for sheep and MEM/10% FBS for cattle; see Note 9). Plates may
e frozen a
t 20C. 2. Prepare a suspension of 3.5 106 cells/mL PBMCs, as descri
ed in Su
headi
n 3.5.1.1., step 8, and in Su
headin 3.5.1.2., step 8. 3. To the antien-conta
inin plates add 100 L of cell suspension per well (3.5 105 cells). Each sample i
s tested in triplicate. 4. Incu
ate in a humidified CO2 incu
ator at 37C for 3 d.
5. Add 20 L (0.4 Ci) of [methyl-3H] thymidine per well and incu
ate ON (18 h) at
37C in a humidified CO2 incu
ator. 6. Cells may
e frozen at this time until they
are ready for processin. This freezethaw lyses the cells very effectively. 7.
Harvest the cells on filtermats usin a semi-automatic cell harvester and count
the incorporation of [methyl- 3H] thymidine in a scintillation counter. 8. Calcu
late the stimulation index (SI) as counts per minute in the presence of antien/
counts per min in the a
sence of antien, where an SI of >2 is considered positi
ve. Alternatively, the level of proliferation may
e expressed as increased coun
ts a
ove those o
tained with the medium control.
3.5.2. Cytokine ELISPOT for Sheep and Cattle Immunized with Plasmid Encodin BHV
-1 D ELISPOT assays are used to measure the production of cytokines from activa
ted T cells. Presently, we routinely measure ovine and
ovine IFN,
ut this assay
may
e used to measure any cytokine provided that the appropriate anti
odies ar
e availa
le. PBMCs are isolated as descri
ed for the proliferation assay, cultur
ed for 2448 h in the presence and a
sence of the appropriate antien, washed and
resuspended to the appropriate concentration in culture medium (see Note 9). Nit
rocellulose plates are coated with an IFN-specific monoclonal anti
ody. Different
dilutions of PBMCs are added to triplicate wells, such that cytokine secreted f
rom the PBMCs may
ind to the anti
ody coated on the wells. After an overniht i
ncu
ation, the plates are washed and incu
ated with IFN-specific ra

it serum, fo
llowed
y a
iotinylated secondary anti
ody and streptavidin- alkaline phosphata
se. Su
strate consistin of BCIP and NBT is used to visualize the spots, each of
which represent sinle IFN- secretin cells. The num
er of ELISPOTS from antien
-stimulated cells are compared to those produced
y unstimulated cells.
DAY 1: 1. Isolate PBMCs from ovine or
ovine
lood. 2. Pre-incu
ate 6 106 cells
in 1 mL in the a
sence or presence of D (0.5 /mL) in 24-well plates for 2448 h a
t 37C in a humidified CO2 incu
ator. Set up triplicate wells for each sample.
Veterinary DNA Vaccines
91
3. Coat 96-well nitrocellulose plates over niht at 4C with
ovine IFN-specific an
ti
ody at a 1/400 dilution in coatin
uffer.
DAY 2:
1. Wash nitrocellulose plate 3 times with sterile PBST. 2. Block with AIM V/2% F
BS (sheep) or MEM/10% FBS (cattle) for 2 h at 37C in a humidified CO2 incu
ator.
3. Harvest cells, centrifue for 78 min at 250, then count and dilute them to 1
107, 5 106, and 1 106/mL. 4. Wash nitrocellulose plate 3 times with sterile PBST
and add 100 L of cells to triplicate wells. 5. Incu
ate over niht at 37C on a le
vel shelf in a humidified CO2 incu
ator.

DAY 3:
1. Wash the plates 2 times in cold PBST, 1 time in ddH2O, and 1 time in PBST (al
low extra time for the first wash to effectively lyse the cells). 2. Add ra

it
anti-
ovine IFN at a 1:100 dilution in PBST-0.1% BSA. 3. Incu
ate for 24 h at room
temperature and wash 3 times in PBST. 4. Add
iotinylated rat anti-ra

it IG a
t a 1:1000 dilution in PBST-0.1% BSA (see Note 10). 5. Incu
ate for 2 h at room
temperature and wash 3 times in PBST. 6. Add streptavidin-alkaline phosphatase a
t a dilution of 1:1000 in PBST-0.1% BSA. 7. Incu
ate for 2 h at room temperature
and wash plates 4 times with PBST, and once with PBS. 8. Add the NBT/BCIP su
st
rate, wait for spots to appear (1/41h) and wash with ddH2O
efore the
ackround
is too stron. Let plates dry and count the spots (cytokine-secretin cells) wit
h a stereoscope.
3.6. Challene and Protection
Even thouh immune responses induced in mice and/or the natural host provide a 
ood indication of the efficacy of a particular DNA vaccine, the ultimate oal is
to protect the animals from infection, which needs to
e tested
y challenin
them with the appropriate pathoen. This is particularly important in situations
where the immune response is not entirely
alanced, in which case a protective
immune response may not have
een enerated. In contrast, it is also possi
le to
o
serve protection in the a
sence of any measura
le immune response. Most of ou
r research on DNA immunization has
een carried out with a protective antien of
BHV-1. Althouh initial efficacy studies may
e done in mice and sheep, protect
ion can only
e properly assessed in cattle, the natural host of BHV-1 (8).
1. Challene: Expose each calf to 4 min of an aerosol of 107 pfu/mL of BHV-1 str
ain 108. We enerate the aerosol with a DeVil
iss ne
ulizer.
92
van Drunen Littel-van den Hurk, Braun, and Ba
iuk
2. Followin challene on d 0, attendin veterinarians clinically evaluate calve
s each mornin for 10 d. Body weihts and rectal temperatures are measured daily
. In addition, animals are iven a nasal score and a clinical score
etween 0 (n
ormal) and 4 (severe). The clinical score is
ased on the levels of depression,
conjunctivitis, and rhinitis. Finally, a sick score is determined
ased on their
rectal temperature, depression, conjunctivitis, and rhinitis. 3. Collect nasal
secretions daily
y cotton swa
to determine the amount of virus shed. 4. O
tain

lood samples for ELISA and/or cellular assays 1014 d after challene.
4. Notes
1. Construction, production, and purification of plasmids: Yields of plasmid may

e improved
y limitin
acterial rowth, such as
y rowin overniht cultures
with reduced aeration. Fillin the flask one-half, rather than one-third, full
of medium works well in reducin aeration. Other plasmids can
e very difficult
to row and may require rowth at 30C rather than 37C for
oth the initial screeni
n for positives and lare scale rowth. 2. In vitro analysis of protein product
ion: The optimal DNA-Lipofectin ratio varies from 12  of DNA to 220  of lipofectin
and should
e experimentally determined for different systems. Other transfecti
on aents like lipofectamine (Gi
co-BRL) may
e more efficient. 3. Intramuscular
(IM) injection: Injections into the muscle do not seem as efficient in lare an
imals as compared to mice, and we find intradermal injections may induce 10-fold
stroner immune responses. 4. Intradermal (ID) injection: The injected solution
should form
le
s in the skin, which differentiates ID from su
cutaneous inject
ion. Bleedin should not occur as the DNA solution may leak. There are alternati
ves to needle injection, such as the Biojector jet injection system (Bioject Inc
., Portland, OR), which delivers solution into skin or muscle without a needle.
We have no experience with these devices and cannot descri
e their use other tha
n to acknowlede their availa
ility. 5. Gene un delivery: Depilatory treatments
such as Nair are extreme irritants on sheep and cattle skin and should never
e

used. To enhance penetration efficiency of the old particles, pressin scotch

tape over the site repeatedly after shavin removes some of the keratin layer an
d improves penetration of the old particles. 6. Humoral immunity: Positive and
neative sera should
e run on each ELISA plate to allow comparison of samples o
n different plates. ELISA results may
e expressed either as a
sor
ance (optical
density) values or as titers, which may
e expressed as the hihest dilution, r
esultin in a readin of two to three standard deviations a
ove the value of the
neative control. Non-specific
ackround stainin may show up, which may resul
t from factors like impurities in the antien preparation, cross-reactivity of t
he anti
odies, or inappropriate dilution of the conjuate. In addition to correc
tin these pro
lems, it is possi
le to reduce the
ackround
y addin a
lockin
 step after coatin of the plates with antien. FBS, BSA, horse serum, or elat
in at concentrations of 110% may
e
Veterinary DNA Vaccines
93
7.
8.
9.
10.
used as
lockin aents. The dilutions for use of commercial conjuates are usua
lly provided,
ut may have to
e experimentally determined, specifically if the
assay does not appear to
e sensitive enouh or if there is non-specific
ackro
und. Cellular immunity: It is very important to include positive and neative an
imals in all cellular immunity assays
ecause of the varia
ility inherent in the
se experiments. This varia
ility is
oth due to the out
red nature of the animal
s and to the nature of the sample. In contrast to splenocytes, the PBMCs isolate
d from peripheral
lood are su
ject to cell traffickin, and therefore, contain
a small, possi
ly varia
le, fraction of the activated lymphocytes. If the animal
s are to
e euthanized, spleens and/or local lymph nodes may
e collected at the
end of the experiment and processed to assess antien-specific proliferation an
d/or cytokine production. Cells, once isolated, should
e processed and plated a
s quickly as possi
le, as they
ein to clump if left standin too lon. Leavin
them at room temperature, and keepin them fairly dilute can help reduce clumpi
n. Severe clumpin may
e reversed somewhat
y incu
ation at 37C. For cattle and
sheep over the ae of 3 mo dexamethasone (110 nM) added to the culture medium fo
r proliferation, and ELISPOT assays can help to control nonspecific stimulation.
The dilutions for use of commercial conjuates are usually provided,
ut may ha
ve to
e experimentally determined.
References
1. Sam
rook, J. E., Fritsch, F., and Maniatis, T. (eds.) (1989). Molecular Cloni
n: A La
oratory Manual, 2nd ed. Cold Sprin Har
or La
oratory Press, Cold Sprin
 Har
or, NY. 2. Colian, J. E., Kruis
eek, A. M., Marulies, D. H., Shevach, E.
M., Stro
er, W., and Coico R. (1992). Current Protocols of Immunoloy: Immunofl
uorescence and Cell Sortin. Greene Pu
lish. Assoc. and Wiley, New York, pp. 5.0
.1.5.4.19. 3. Ausu
el, F. M., Brent, R., Kinston, R. E., Moore, D. D., Seidman,
J. G., Smith, J. A., and Struhl, K. (1993). Current Protocols of Molecular Biolo
y: Purification of Proteins
y Precipitation. Greene Pu
lish. Assoc. and Wiley,
New York, pp. 10.16.1.10.16.9. 4. Ausu
el, F. M., Brent, R., Kinston, R. E., Mo
ore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1993). Current Protoco
ls of Molecular Bioloy: Detection of Proteins. Greene Pu
lish. Assoc. and Wiley
, New York, pp. 10.6.1.10.8.14. 5. Burnette, W. M. (1981). Western
lottin: electr
ophoretic transfer of proteins from sodium dodecyl sulfate polyacrylamide els t

o unmodified nitrocellulose and radioraphic detection with anti
ody and radioio

dinated protein A. Anal. Biochem. 112, 195203. 6. Tijssen, P. (1985). Practice an
d theory of enzyme immunoassays, in La
oratory Techniques (Burdon, R. H. and van
Knippen
er, P. H., eds.), Elsevier, New York, pp. 449500.
94
van Drunen Littel-van den Hurk, Braun, and Ba
iuk
7. Colian J. E., Kruis
eck A. M., Marulies D. H., Shevach E. M., Sto
er W., an
d Coico, R. (1992). Current Protocols in Immunoloy: Immunoloical Studies in Hu
mans. Greene Pu
lish. Assoc. and Wiley, New York, pp. 7.0.1.7.31.7. 8. Van Drunen
Littel-van den Hurk, S., Tikoo, S. K., van den Hurk, J. V., Ba
iuk, L. A., and
Van Donkersoed, J. (1997). Protective immunity in cattle followin vaccination
with conventional and marker
ovine herpesvirus-1 vaccines. Vaccine 15, 3644.
9
DNA-Based Immunization of Neonatal Mice
Cynthia L. Brazolot Millan and Heather L. Davis 1. Introduction Immunization of
neonates carries three inherent questions: (i) Is the neonatal immune system mat
ure enouh to elicit an immune response, and if not, how early can immunization

e carried out? (ii) If an immune response is not elicited, will tolerance
e in
duced? (iii) Can offsprin of immune mothers
e immunized aainst an antien des
pite hih levels of passively transferred maternal anti
odies to that antien? D
espite the earlier fear that the immature neonatal immune system would
e toleri
zed
y too early delivery of antiens, and at least one report that DNA
ased imm
unization of new
orn mice did in fact lead to tolerance (1), a num
er of recent
pu
lications have demonstrated that the neonatal murine immune system is indeed
immunocompetent and can respond appropriately to vaccination. Various antiens,
delivered as purified protein (2), DNA vaccines (35), live retrovirus (6), or his
tocompati
ility antiens on transplanted cells (7), have
een shown to induce
o
th humoral and cellular immune responses in mice immunized within the first few
days of life. Similarly, in our own la
oratory, we have successfully immunized n
ew
orn mice with plasmid DNA vaccines encodin the hepatitis B virus (HBV) surfa
ce antien (HBsA). With this antien, we find that the DNA vaccine induces anti

odies aainst HBsA (anti-HBs) in mice injected as early as 12 h after
irth, w
hereas immunization with recom
inant HBsA protein does not (Brazolot Millan, et
al., in press). The strenth of the humoral response enerated
y DNA-
ased imm
unization on the first day of life is the same as that with injection of DNA at
3 or 7 d. By comparison, injection of recom
inant protein, with or without alum,
at d 1, 3, or 7 does not elicit a detecta
le humoral immune response, nor does
it prime a response, even at a dose sufficient
From: Methods in Molecular Medicine, vol. 29, DNA Vaccines: Methods and Protocol
s Edited
y: D. B. Lowrie and R. G. Whalen Humana Press Inc., Totowa, NJ
95
96
Brazolot Millan and Davis
to immunize adult mice. Furthermore, we have evidence that DNA- (
ut not protein
-)
ased immunization of neonates
orn to anti-HBs positive mothers is capa
le o
f primin an immune response, despite the presence of hih levels of passively t
ransferred maternal anti
odies. Althouh a primary humoral response was not dete
cted in these neonates (i.e., no measura
le anti-HBs after the maternally derive
d anti
odies had disappeared), a pronounced anamnestic response was o
served in
the mice as youn adults upon challene with recom
inant HBsA protein (Brazolot M
illan, et al., in press). Thus, in our HBV model, DNA-
ased immunization does no
t induce tolerance in neonatal mice and, in fact, it can
e used to immunize ver

y youn neonates, whether or not they har
or passively transferred anti-HBs anti

odies. 2. Materials
1. Vehicle to dissolve DNA such as 0.15 M NaCl (Sima, St. Louis, MO). 2. Alhydr
oel 85 (Superfos Biosector, Ved
aek, Denmark; o
tained throuh Cedarlane, Horn
y,
Ontario, Canada). 3. Heparin, 50 units/mL (Sima). 4. Insulin syrines (3/10 cc
) with an attached 29G1/2 needle (Becton Dickinson, Franklin Lakes, NJ). 5. Plas
mid DNA prepared usin Qiaen anion-exchane chromatoraphy columns (Qiaen Gm
H
, Hilden, Germany). In our HBV vaccine model, we use a plasmid construct encodin
 the major protein (S) of the HBV envelope under the control of the cytomealov
irus (CMV) immediate early promoter (pCMV-S) (8) (see Note 1). 6. Protein antie
n in suita
le medium for injection (i.e., in saline, alone or adsor
ed to alum).
For our HBV vaccine model we use recom
inant HBsA, purified from yeast (Genzym
e Dianostics, San Carlos, CA) (see Note 1).
3. Method 3.1. Formulations
1. Dilute plasmid DNA (stored at 20C until time of immunization) in physioloical
saline (0.15 M NaCl) to desired concentration and keep on ice until injected int
o the mice. We use pCMV-S DNA (8) at 0.5 m/mL. 2. Dilute protein in physioloic
al saline (0.15 M NaCl) to desired concentration. If an adjuvant is desired, add
alum (as Alhydroel; 2.5 L/ protein), mix, then maintain on ice for 30 min prior
to immunization. We use recom
inant HBsA at 50 /mL.
3.2. Immunizations
1. Pups may
e immunized very soon after
irth. We typically carry this out with
in the first 12 h of life (see Note 2). Wear loves at all times while handlin
pups and their mothers. 2. First remove the mother from the cae and keep her an
esthetized with Halothane as (Halocar
on La
oratories, River Ede, NJ) while ma
nipulatin her offsprin.
DNA Vaccines in Neonates
97
3. Remove all pups from the nest
ut do not anesthetize them for injection. Take
one pup at a time and drape it over your index finer (of the opposite hand tha
n you will inject with). Use your thum
to restrain the hind les aainst your i
ndex finer and your middle finer to anchor the head and upper
ody. Insert the
needle throuh the lateral thih (in a posterior-to-anterior direction) so that
the point ends in the quadriceps muscle mass (anterior thih). Inject the DNA v
accine in 10 L per site. We typically carry out
ilateral injections and divide t
he DNA vaccine dose equally
etween the two injection sites,
ut still at a volu
me of 10 L/site (see Notes 3 and 4). 4. Place the immunized pup
ack into the nes
t in the cae. Once all manipulations are complete, the still-anesthetized mothe
r is placed on top of the litter to recover there.
3.3. Blood Collection
1. Blood can
e collected from pups
einnin at 1 week followin immunization.
One-week-old mice are sacrificed
y decapitation, and the
lood is collected int
o a microfue tu
e containin 10 L of heparin (50 units heparin/mL) (see Note 5).
2. Beinnin at 2 wk of ae, the mice do not have to
e killed as it is possi
l
e to o
tain
lood
y retro-or
ital puncture usin a heparinized Pasteur pipet (s
ee Note 5).
4. Notes
1. We find that a DNA vaccine encodin HBsA successfully elicits antien-specif
ic immune responses even in the immature immune system of neonatal mice. Other i
nvestiators report similar successes for DNA vaccines encodin several other an
tiens (35), however, tolerization was reported for a DNA vaccine encodin a mala
rial antien (1). Thus, it is not yet clear how many and which DNA vaccines will
work in neonatal mice. Usin the reaents and techniques descri
ed a
ove, we ro

utinely achieve 8090% success in immunizin neonates with HBsA-expressin DNA (p

CMV-S). Thus, investiators could use this model to control for their injection
technique if they find that their DNA vaccine expressin a different antien doe
s not appear to
e workin in their hands. 2. In our experience of handlin new

orn pups and their mothers, we rarely o
serve maternal rejection of offsprin. O
ur practice is that we do not handle the pups until the mother has finished ivi
n
irth to her entire litter and has reassem
led a nest in the cae and retriev
ed the pups into it. Also, the mother never sees us handle her pups as she is re
moved from the cae and kept anesthetized while the pups are injected, and we th
en place the still-anesthetized mother directly onto her litter followin all in
jections. These precautions seem to adequately prevent any awareness of distur
a
nce. To avoid transferrin human odors to the pups, they are handled only with 
loved hands. However, if pro
lems consistently arise with a mother rejectin her
manipulated offsprin, a small da
of Vicks VapoRu
placed on her nose will pre
vent her from detectin forein odors on her pups. 3. Youn mouse pups are fairl
y translucent and the introduction of the needle should
e o
served carefully. I
t is easy to insert the needle too far and thus exit the le from the other side
. Nevertheless, the needle must also
e inserted far enouh.
98
Brazolot Millan and Davis
The entire
evel of the needle must
e within the lim
, and an extra 12 mm depth
is advisa
le. 4. Care must
e taken not to stretch the
ody of the pup too tiht
ly over your finer or to apply too much pressure durin immo
ilization as this
will force expulsion of the DNA as soon as you inject it. This can also happen i
f the DNA is injected too fast or the needle is withdrawn too quickly. Thus, the
pluner of the syrine should
e depressed relatively slowly, and the needle sh
ould
e left in situ for a few seconds followin immunization, prior to
ein wi
thdrawn. 5. Approximately 50 L of
lood may
e routinely o
tained from a 1-wk-old
mouse; however, it must
e killed (
y decapitation) in order to recover the
lo
od. Beinnin at 2 wk of ae, mice do not have to
e killed
ut rather can
e
l
ed
y retroor
ital puncture. For 2- and 3-wk-old mice, only 100200 L of whole
loo
d should
e removed from the pup; however, from 4 wk of ae onward, 300500 L of
l
ood may
e routinely removed. Althouh 2- and 3-wk-old mice are small, their eye
s may
e
led usin Pasteur pipets, without risk of
lindin. Capillary tu
es or
heparinized lass micropipets may
e used,
ut in our experience, it is unneces
sary to attempt
lood collection with the smaller and more fraile tu
es, as Pas
teur pipets work quite adequately. It is worth cautionin that youn mice often
seem to take a lon time to succum
to the effects of halothane,
ut once anaest
hetized, they seem to
ecome deeply anesthetized (danerously so), very quickly.
As well, neonatal mice that appear to
e over-anesthetized can recover amazinl
y fast when removed from the as.
References
1. Mor, G., Yamshchikov, G., Sedeah, M., Takeno, M., Wan, R., Houhten, R. A.,
Hoffman, S., and Klinman, D. M. (1996) Induction of neonatal tolerance
y plasm
id DNA vaccination of mice. J. Clin. Invest. 98, 27002705. 2. Forsthu
er, T., Yip
, H. C., and Lehmann, P. V. (1996) Induction of TH1 and TH2 immunity in neonatal
mice. Science 271, 17281730. 3. Bot, A., Bot, S., Garcia-Sastre, A., and Bona, C
. (1996) DNA immunization of new
orn mice with a plasmid-expressin nucleoprotei
n of influenza virus. Viral Immunol. 9, 207210. 4. Bot, A., Antohi, S., and Bona,
C. A. (1997) Immune response of neonates elicited
y somatic transene vaccinat
ion with naked DNA. Frontiers Biosci. 2, 173188. 5. Wan, Y., Xian, Z., Pasquini
, S., and Ertl, H. C. J. (1997) Immune response to neonatal enetic immunization
. Viroloy 228, 278284. 6. Sarzotti, M., Ro

ins, D. S., and Hoffman, P. M. (1996
) Induction of protective CTL responses in new
orn mice
y a murine retrovirus.
Science 271, 17261728. 7. Ride, J. P., Fuchs, E. J., and Matziner, P. (1996) Ne
onatal tolerance revisited: Turnin on new
orn T cells with dendritic cells. Sci

ence 271, 17231726. 8. Davis, H. L., Michel, M.-L., and Whalen, R. G. (1993) DNA
ased immunization for hepatitis B induces continuous secretion of antien and h
ih levels of circulatin anti
ody. Human Mol. Genet. 2, 18471851.
10
Intramuscular Injection of DNA Vaccines in Fish
Jol Heppell and Heather L. Davis 1. Introduction The DNA-
ased immunization techn
oloy has only
een applied to fish very recently. Thouh a preliminary study sh
owin reporter ene expression in fish muscles was pu
lished in 1991 (1), the fi
rst demonstration of an immune response to plasmid-encoded antien was not repor
ted until five years later (2). Thus, relatively little is known a
out the admin
istration methods, immunoloical responses, and protective efficacy of DNA vacci
nes in aquatic animals. In some instances, results o
tained with other classes o
f verte
rates (mammals and
irds) can
e applied directly to fish,
ut
ecause o
f physioloical, immunoloical and structural differences
etween these animals,
this is not necessarily true. For example, it was shown recently that short spe
cific DNA sequences (CpG motifs), in a particular nucleotide context, act as imm
unostimulants (3,4). No study has
een reported yet assessin these immunostimul
atory sequences in fish,
ut it would
e unlikely that the same sequences are ef
fective in all animal species (Weeratna et al., this volume). Different techniqu
es have
een used to introduce DNA into es and em
ryos of fish to create trans
enic animals. For vaccination purposes, direct injection with a needle is the s
implest and most effective method. Particle
om
ardment (i.e.,
iolistic introdu
ction of DNA-coated old particles usin a ene un which shoots old particles
at hih speed into the animals superficial tissue) has also
een tested,
ut it w
as shown to
e less effective (5). Thouh the ene un technique could
e improv
ed for la
oratory use, it is unlikely that it will
e applied for mass vaccinati
on, due to economical and practical constraints. Other routes of administration
have
een used in fish with antien-
ased vaccines (6). Successful transfection
of fry or adult fish cells usin such methods as immersion (
y dip,
ath, or spr
ay), or oral administration, with pure
From: Methods in Molecular Medicine, vol. 29, DNA Vaccines: Methods and Protocol
s Edited
y: D. B. Lowrie and R. G. Whalen Humana Press Inc., Totowa, NJ
99
100
Heppell and Davis
DNA solution, or mixed with carrier molecules or microcapsules, has never
een r
eported. Experiments conducted in our la
oratory showed that immersion of fish i
n naked DNA solution (with or without prior hyperosmotic shock), direct applicat
ion on the ills, and delivery into the diestive tract
y intu
ation do not pro
vide satisfactory results. However, it will
e important to develop these altern
ative methods of vaccination to immunize small fish on a commercial scale. This
chapter summarizes methods for direct transfer of plasmid DNA into fish for the
purpose of vaccination. In particular, plasmid constructs, administration method
s, and expression of injected enes are discussed in liht of results o
tained w
ith mammals. 2. Materials
1. Appropriate anesthesia for fish (tricaine or
enzocaine). 2. U-100 insulin sy
rines (0.3 cc) with attached 29G1/2 needle (Becton Dickinson, Franklin Lakes, N
J) (see Note 1). 3. Polyethylene tu
in (PE 20, ID = 0.38). 4. Purified plasmid
DNA dissolved in sterile saline (0.15 M NaCl) or phosphate
uffered saline (PBS)
(see Notes 2 and 3).
3. Method
1. Dilute DNA solution, if needed, in the same solution used to dissolve it. For
each fish, 50  of plasmid DNA diluted in a volume of 10 to 25 L is appropriate f
or initial testin of the vaccine (see Notes 4 and 5). 2. Prepare appropriate nu

m
er of syrines
y insertin needles in polyethylene tu
in and cuttin it to a

lenth such that only 23 mm of the needle protrudes. The tu
in will prevent the
needle from oin too deep into the fish tissue (see Note 6). 3. Aspirate DNA s
olution into syrines and eliminate air
u

les. Syrines can
e refilled a few
times if several fish have to
e injected with the same vaccine. 4. Anesthetize
fish
y immersion in 0.0084% tricaine (also called MS-222 or 3-amino
enzoic acid
ethyl ester) or 0.01%
enzocaine (see Note 7). 5. The DNA vaccine is to
e inje
cted intramuscularly. Remove fish from the anesthetic
ath and insert needle in
the flanks, approximately midway
etween the dorsal fin and the lateral line, wi
th the needle pointin toward the tail. Inject slowly, and wait for 12 s
efore w
ithdrawin the needle, to prevent loss of DNA solution (see Notes 810). 6. Immedi
ately transfer fish into well oxyenated water for recovery and test for immune
response when appropriate (see Note 11).
4. Notes
1. Insulin syrines can
e used to deliver small volumes accurately. Alternative
ly, when lare num
ers of fish have to
e injected, it miht
e more convenient
to use a device that can automatically deliver pre-selected volumes repeatedly,
without havin to refill the device. A repeater pipet fitted with a 28G needle i
s a ood alternative.
DNA Vaccines in Fish
Ta
le 1 List of Promoters Tested
y Injection into Fish Muscle Promoter Glucocor
ticoid-responsive mouse mammary tumor virus (MMTV) Cytomealovirus (CMV) immedia
te early (alone or with translational promoter) Carp -actin Fish species Rain
ow
trout (Oncorhynchus mykiss) Rain
ow trout (Oncorhynchus mykiss) Ze
ra fish (Brac
hydanio rerio) Rain
ow trout (Oncorhynchus mykiss) Tilapia (Oreochromis niloticu
s) Common carp (Cyprinus carpio) Common carp (Cyprinus carpio) Common carp (Cypr
inus carpio) Common carp (Cyprinus carpio) Rain
ow trout (Oncorhynchus mykiss)
101
Reference 7
7,8 8 7 9 1 1 1 1 5
SV40 early Ra

it -cardiac myosin heavy chain (MHC) Human MxA Artificial (
ased o
n human MxA) Herpes simplex virus thymidine kinase (tk) with the CMV enhancer
2. DNA should
e dissolved and diluted to the desired concentration with sterile
isotonic and non-toxic
uffer to avoid any extended or permanent tissue damae.
3. Plasmid constructs currently used to express forein enes in fry and adult
fish tissues are similar to those employed for mammals. Several promoters have

een tested in fish (Ta
le 1), and althouh expression of reporter enes under co
ntrol of these promoters was detected in all cases, efficiency varied reatly. C
omparison of the different studies to rank promoters accordin to the expression
level o
tained is not possi
le,
ecause conditions under which they were tested
are too different,
ut, overall, the cytomealovirus promoter seemed to ive ve
ry ood results (5,7,8). No comparisons of the other controllin elements (e..,
polyadenylation sinal and introns) have
een reported,
ut there is no indicat
ion that ene expression from plasmid DNA in piscine muscle cells will
e sinif
icantly different from that in mammalian myofi
ers. 4. The total volume of DNA t
o inject per fish should
e kept small. We have shown that dilution of DNA in la
rer volumes increases consistency
etween individual fish,
ut decreases the ex
pression level of the reporter ene (Ta
le 2). Anderson et al. (7) tested larer
volumes (100 and 200 L) and came to the same conclusion. The reater variations
o
served
etween individual fish with smaller volumes could come from the inaccu
racy of the deliverin device and/or the loss of DNA. When very small volumes of
DNA are injected into muscles, reat care should
e taken to prevent DNA from l
eakin out of the animal when the needle is retracted. Hence, it is important th

at fish are well anesthetized
efore injections.

102
Heppell and Davis
Ta
le 2 Total Luciferase Activity in Rain
ow Trout Muscle Injected with 1  of Lu
ciferase-Encodin Plasmid, Accordin to the Volume of Saline (0.15 M NaCl) Used
to Suspend DNA Volume of DNA injected per fish (L) 5 10 25 50 100
aMean
Luciferase activity (RLU/sec)a 416 554 327 621 174 525 76 867 47 451      173 583 89
936 31 206 14 368 13 476
luciferase activity calculated in relative liht unit per second in muscle  stand
ard error of the mean. For each volume tested, n 5.
Ta
le 3 Optimal Dose of DNA to Inject Intramuscularly for Maximum Expression of
Reporter Genes Reporter ene used CATa luciferase luciferase luciferase
aChloramphenicol
Optimal dose of DNA () 50 25 50 110
acetyltransferase.
Reference 1 7 5 8
5. The dose-response of forein ene expression followin intramuscular injectio
n has
een studied in different fish species (Ta
le 3). Maximum activity for a 
iven ene is reached with the same dose of plasmid DNA, independently of the pro
moter used (7). Injection of larer amounts of DNA did not result in increased e
xpression levels. DNA is pro
a
ly taken up only
y a limited num
er of cells, ei
ther due to cell mem
rane damae or other causes. To date, there is no indicatio
n that results will vary reatly
etween fish of different species or size,
ut
ae could
e an important factor (1). Nevertheless, the optimal dose of DNA is n
ot directly proportional to the size of animals to
e immunized. To test the imm
une response to a plasmidencoded antien, a startin dose of 2550  DNA per fish a
ppears to
e sufficient. 6. To minimize tissue damae and injuries to animals, w
e prefer to limit penetration of the needle to 23 mm. This is particularly import
ant when very small fish (<3 ) have to
e injected. The reported depth of injec
tion varies from 2 mm (5) to 10 mm (1). Larer fish can
e injected without dept
h-limitin devices. 7. Fish have to
e completely anesthetized to make handlin
easier, and to avoid muscle contractions which could expel DNA out of the animal
. This is very important when small volumes are injected. Avoid leavin fish for
too lon in the anesthetic
ath as they may die.
DNA Vaccines in Fish
103
8. Various routes of injection were tested,
ut intramuscular administration see
ms to work
est. Very low or no expression of reporter enes was detected when D
NA was injected in the peritoneum or the ills (5; J. Heppell and H. L. Davis, u
npu
lished results). 9. The most common sites for intramuscular injection are th
e flanks, usually midpoint
etween the dorsal fin and the lateral line, or immed
iately rostroventral to the dorsal fin. Injections close to the caudal fin ive
similar expression level,
ut are less convenient and less consistent, possi
ly
due to loss of DNA solution caused
y manipulation of fish or muscular contracti
on when animals come out of anesthesia. 10. Before insertion of the needle, the
site of injection can
e wiped with cotton swa
s or other clean a
sor
ent materi
al, to remove excess mucus. On very small fish, mucus can make the needle slip o

n the fish scales, causin injuries to the animal. 11. Experiments with the luci
ferase reporter ene showed that expression can last for several weeks (5,7,8).
This was shown to
e sufficient for induction of protective immune responses (2)
. Specific anti
odies can
e detected in sera from DNAvaccinated fish,
ut this
is not an a
solute prediction of protection. Delay
efore anti
odies can
e dete
cted varies accordin to the species and temperature. In rain
ow trout kept at 1
2C, specific anti
odies were detected
y ELISA startin at 2 wk post-injection (J
. Heppell and H. L. Davis, unpu
lished results).
References
1. Hansen, E., Fernandes, K., Goldspink, G., Butterworth, P., Umeda, P. K., and
Chan, K. C. (1991) Stron expression of forein enes followin direct injectio
n into fish muscle. Fed. Eur. Biochem. Soc. 290, 7376. 2. Anderson, E. D., Mouric
h, D. V., Fahrenkru, S. C., LaPatra, S., Shepherd, J., and Leon, J. C. (1996)
Genetic immunization of rain
ow trout (Oncorhynchus mykiss) aainst infectious h
ematopoietic necrosis virus. Mol. Marine Biol. Biotech. 5, 114122. 3. Krie, A. M
. (1996) Lymphocyte activation
y CpG dinucleotide motifs in prokaryotic DNA. Tr
ends Micro
iol. 4, 7376. 4. Pisetsky, D. S. (1996) Immune activation
y
acterial
DNA: a new enetic code. Immunity 6, 303310. 5. Gmez-Chiarri, M., Livinston, S.
K., Muro-Cacho, C., Sanders, S., and Levine, R. P. (1996) Introduction of forei
n enes into the tissue of live fish
y direct injection and particle
om
ardmen
t. Dis. Aquat. Or. 27, 512. 6. Newman, S. G. (1993) Bacterial vaccines for fish.
Annual Rev. Fish Dis. 3, 145185. 7. Anderson, E. D., Mourich, D. V., and Leon,
J. C. (1996) Gene expression in rain
ow trout (Oncorhynchus mykiss) followin in
tramuscular injection of DNA. Mol. Marine Biol. Biotech. 5, 105113. 8. Heppell, J
. and Davis, H. L. (1997) Expression of forein enes in fish followin direct D
NA injection, in Fish Vaccinoloy (Guddin, R., Lillehau, A., Midtlyn, P. J.,
and Brown, F., eds.), Develop. Biol. Stand. 90, Karer, Basel, Switzerland, p. 4
64. 9. Rahman, A. and Maclean, N. (1992) Fish transene expression
y direct inj
ection into fish muscle. Mol. Marine Biol. Biotech. 1, 286289.
11
Development of DNA Vaccines for Salmonid Fish
Eric D. Anderson and Jo-Ann C. Leon 1. Introduction Vaccination of fish aainst
many different pathoenic oranisms has made it possi
le to rear Atlantic salmo
n in net pen caes and produce fish commercially around the world. In fact, vacc
ine use is critical for the continued rowth of the aquaculture industry and res
earchers are continually lookin to develop new and improved vaccines for a wide
variety of fish pathoens. Fish vaccines have
een formulated from killed or at
tenuated pathoens, recom
inant viral proteins or peptides, and most recently, p
lasmid DNA encodin viral proteins (17). The use of DNA vaccines for the control
of viral diseases of fish is particularly appealin since this type of vaccine e
liminates the need to purify the viral pathoen or immunoprotective antien. Oth
er advantaes are the elimination of any possi
ility of reversion to virulence s
ince the DNA vaccine encodes only a portion of the viral enome, the relative st
a
ility of the DNA preparation; the ease of DNA preparation; and, importantly, t
he elicitation of a ro
ust immune response in fish. We have found that injection
of fish with a DNA vaccine (pCMV4-G) encodin the lycoprotein ene of infectio
us hematopoietic necrosis virus (IHNV), a pathoenic fish rha
dovirus, induces a
stron protective immune response to virus challene (5,6). The process of deve
lopin this vaccine is outlined in Fi. 1. First, the experimental parameters th
at overned the expression of forein enes in fish after the injection of naked
DNA were determined. These parameters included DNA dose, volume of injection fl
uid, first appearance of expressed protein, duration of expression, and tissues
of expression (5). Then, the efficacy of the DNA vaccine for IHNV was tested in
rain
ow trout. In this case, anti
ody production as well as protection aainst t
he lethal
From: Methods in Molecular Medicine, vol. 29, DNA Vaccines: Methods and Protocol
s Edited
y: D. B. Lowrie and R. G. Whalen Humana Press Inc., Totowa, NJ

105
106
Anderson and Leon
Fi. 1. Flow diaram showin the steps in developin a DNA vaccine.
effects of virus infection were used as measurements of vaccine efficacy (6). In
this chapter, we summarize the practical considerations, experimental procedure
s, and o
stacles that may
e encountered in developin a DNA vaccine for fish.
1.1. Transene Expression in Rain
ow Trout (Oncorhynchus mykiss)
The selection of the plasmid that will encode the antien (A) or reporter prote
in is the initial step in DNA vaccine desin. While the criteria used in selecti
n a plasmid may vary, the selection will in most cases
e
ased on the properti
es of the promoter/enhancer that will drive A expression. Any requirements for
expression in specific tissues, the induci
ility and strenth of the promoter, a
nd possi
le licensin prohi
itions on the use of forein DNA in food animals all
have
earin on the selection of the plasmid vector used in the vaccine constru
ction. Our studies indicate that the level of ene expression followin the dire
ct injection of DNA into rain
ow trout depends on the promoter used to drive the
forein ene expression. The cytomealovirus immediate early promoter (CMV-IEP)
consistently produced very hih levels of
DNA Vaccines for Salmonid Fish
107
luciferase activity in fish muscle tissue. This result with CMV-IEP is consisten
t with the results o
tained in a num
er of different animal systems (8) and is t
he pro
a
le reason that plasmids containin the CMV-IEP are widely used for DNA
vaccines. Identifyin the minimum effective vaccine dose is important for econom
ic as well as safety reasons. In practical terms, small fish are vaccinated in 
roups of hundreds to thousands of animals. The use of a sinle small dose of DNA
can make the operation less cum
ersome and ensure that the cost of vaccine prep
aration is not prohi
itive. When the reporter plasmid, pCMV-Luc, was used to det
ermine what quantity of DNA was required for maximum luciferase activity in rain

ow trout, we found that
etween 2550  of DNA injected into the dorsal musculatur
e of 50  rain
ow trout produced maximum luciferase activity (5). There was cons
idera
le varia
ility in luciferase activity when fish were injected with 100 L of
solution containin 25  of DNA. When the same amount of DNA was injected in a l
arer volume, i.e., 200 L, reproduci
le measurements of luciferase activity were
o
tained. Thus, larer injection volume provided reater reproduci
ility. In stu
dies that measured vaccine efficacy throuh the induction of protective immune r
esponse, the smallest dose tested was 10  vaccine injected in a 200 L volume in 1
 fish (6). The minimum effective dose was not determined in these studies. Hep
pell and colleaues (9) have shown recently that DNA vaccine doses at 0.1  in 5 L
will also induce protective immunity in rain
ow trout at 1  averae weiht. Th
e duration of plasmid driven-antien production required to induce a stron prot
ective immune response in fish is unknown. Because continuous antien production
may lead to tissue necrosis or immune tolerance, and
ecause the continued pres
ence of plasmid DNA may
e perceived as pro
lematic in animals destined for huma
n consumption, the lenth of time that the plasmid DNA and its encoded antien p
ersisted in transfected cells in fish was determined. When 1 and 50  fish were
injected with 10 or 25  of pCMVLuc, maximum luciferase activity was o
served 7 d
later. For the small fish, luciferase activity declined to approximately 10% of
the maximum over a 115-d test period. For the larer fish, the activity remaine
d near 50% of the maximum for 28 d and diminished to just 10-fold a
ove
ackrou

nd
y 63 d postinjection when the experiment was terminated. Thus, in each case,

the presence of antien in transfected muscle tissue was lon-lived. No ross h
istoloical a
normalities were o
served durin the experiments and there was no
evidence of immune tolerance. Despite these initial findins that DNA vaccines d
o not have any ill effects on the injected fish, safety concerns may lead to the
desin of future vaccines that incorporate reulatory components limitin the d
uration of antien and DNA persistence in tissues.
108
Anderson and Leon
The sites of expression and the fate of the injected DNA should
e tested when d
evelopin the vaccine. We accomplished this
y usin two plasmids, pCMVGal with t
he reporter ene -alactosidase fused to the CMV-IEP, and pCMV4-Luc. In 50- fish
injected with 25  of pCMVGal in 200 L, reporter activity was confined to muscle t
issues in the area surroundin the path of injection. When 1- fish were injecte
d with 10  of pCMVGal in 200 L, evidence of the enzyme was found alon the injecti
on path and in scattered muscle cells up to 5 mm distant from the site of inject
ion. When the small fish were injected with 10  of pCMV4-Luc, luciferase activit
y was o
served in numerous tissues at 35 d postinjection. The amount of reporter
protein activity recovered from the tissues was varia
le and maximum activity wa
s recovered from muscle cells alon the injection path. The role that these tran
sfected tissues play in the overall efficacy of the DNA vaccine is unknown
ut i
t may
e sinificant that reporter activity was also recovered from two primary
fish lymphoid orans, the kidney and spleen. Thus, when injectin small fish wit
h DNA, special consideration should
e placed on determinin which cell types ar
e transfected with the DNA vaccine. The physical nature of the DNA in the transf
ected cells of the internal orans is unknown. However, in transfected muscle ce
lls, we found that the vaccine was maintained as non-replicatin, supercoiled pl
asmid DNA. We found no evidence of plasmid interation into the host enome.
1.2. DNA Vaccination Methods for testin vaccine efficacy and the immune respons
e elicited
y the vaccine are necessarily dependent upon the system under invest
iation. Several excellent sources of information on eneral vaccine testin in
fish are availa
le (10; see Note 1). We will discuss here only protocols and res
ults relevant to the development of a DNA vaccine for fish. Several
asic points
should
e considered when choosin a model system for the development of a fish
DNA vaccine. There should
e availa
le a cloned ene(s) encodin the immunoprot
ective antien(s) for the pathoen, i.e., the antien should
e identified and i
mmunoloical reaents for detectin the antien should
e availa
le. It also is
essential that the proression of the disease caused
y the pathoen under inves
tiation is well characterized and, importantly, reproduci
le in the la
oratory.
In eneral, the efficacy of fish vaccines is relatively easy to determine
ecau
se salmonid fish can
e reared and vaccinated in lare num
ers. After vaccinatio
n and a suita
le period of time for immune response development (34 wk in salmoni
d fish species), the vaccinated fish are challened with a lethal dose of the pa
thoen and efficacy is measured
y the difference in the percentae of survivin
fish in the vacciDNA Vaccines for Salmonid Fish
109
nated vs control fish. The major limitation to our understandin of how fish vac
cines work is that there are very few tools availa
le to examine the fish immune
response at the cellular level. This limitation is particularly relevant
ecaus
e efficacy and the cellular immune response has
een shown to
e directly relate
d, for DNA vaccines in mammals (8). The first step taken in the development of a
DNA vaccine for IHNV was the demonstration that the G and nucleocapsid protein

(N) DNA vaccines, pCMVG and pCMV-N, were functional. Expression of the IHNV G an
d N proteins
y pCMV-G and pCMV-N was confirmed
y immunohistochemical stainin
of epithelioma papulosum cyprini (EPC) cells transiently transfected with the re
spective plasmids. The transfected cells reacted with the appropriate monoclonal
anti
odies to N or G. Protein synthesis was similarly confirmed in muscle tissu
e in vivo in thin sections prepared from vaccinated fish. Both the pCMV-G and pC
MV-N transfected cells contained viral protein throuhout the cytoplasm. When th
e pCMV-G transfected cells were
riefly reacted with the color su
strate, the G
protein was seen primarily at the cell surface. These results demonstrated that
the plasmids was correctly constructed. The fish humoral immune response induced

y each DNA vaccine was examined in 1  rain
ow trout inoculated with a solutio
n containin 5  each of pCMV-G and pCMV-N, or 10  of pCMV-G, or 10  of pCMV-N. T
he appropriate control plasmid, pCMV-Luc, was also included in the study. There
were hih levels of IHNV
indin anti
ody (ELISA) titers in serum collected 4-wk
-postinjection in the fish receivin the com
ination of pCMV-G and pCMV-N. These
titers remained hih throuhout the 14-wk test period. AntiIHNV anti
ody in fis
h receivin pCMV-G was first o
served at 6-wkpostinjection with a peak titer at
8 wk. A comparison of anti-IHNV anti
ody titers
etween the roup receivin only
pCMV-G versus the roup receivin the com
ination vaccine at 8-wk-postinjection
showed that the serum from the co-injected roup contained an averae of 26-fol
d reater anti-IHNV anti
ody titer than the fish receivin pCMV-G alone. However
, the increase in anti
ody titer in the co-injected roup did not correlate with
an increase in vaccine efficacy as measured
y survival after lethal virus chal
lene. The relative percentae survival of fish followin challene with IHNV wa
s 75% for pCMV-G and 78% for pCMV-G with pCMV-N. Only the sera from fish that re
ceived pCMV-G contained IHNV-neutralizin activity and the activity was very low
and near the limits of assay detection. This would suest that the cellular im
mune response in vaccinated fish is the important component in the immune respon
se that confers protection. Alternatively, in vitro measurements of virus neutra
lizin anti
odies may not
e an accurate predictor of in vivo neutralizin activ
ity.
110
2. Materials 2.1. Plasmids and DNA Purification
Anderson and Leon
1. Special equipment and materials: Plasmid DNA, spectrophotometer, standard
ac
terioloical supplies, electrophoresis apparatus, and materials/reaents necessa
ry for analyzin electrophoresed DNA. 2. For initial testin, we recommend usin
either pCDNA3 (Invitroen) or a similar plasmid. These plasmids should
e enin
eered to encode either the antien of choice, or one of the reporter proteins, -
alactosidase or luciferase. These reporter proteins provide a sensitive assay fo
r expression, have low endoenous
ackround in fish, and are relatively simple
to measure. 3. LB
roth/aar: For 1 L of media, add 10 
acto-tryptone, 5 
ac
to-yeast extract, and 5  NaCl to dou
le-distilled water (ddH2O). Adjust the pH
to 7.5 with sodium hydroxide. Sterilize
y autoclavin. For solid media add 15 
aar to 1 L of LB
roth and autoclave. When the autoclaved solution cools to 505
5C add 50 /mL ampicillin when appropriate. 4. 1000 ampicillin stock: Dissolve 0.5
 ampicillin in 10 mL 70% ethanol. Filter sterilize the solution and dispense i
nto 1 mL volumes and store at 20C. 5. TE: 10 mM Tris-HCl, 1 mM EDTA, pH 7.4. This
reaent should
e made with the hihest rade water availa
le and this should
e
at least ddH2O althouh triple distilled, reverse osmosis-treated water is
ett
er. Autoclave, filter sterilize, and dispense in small volumes. Store at room te
mperature or 20C. 6. 3 M sodium acetate, pH 5.2. Sterilize the solution
y autocla
vin and dispense into 10 mL portions and store at room temperature. 7. 95% and
70% ethanol. 8. DNA purification column and reaents: For DNA purification, use
the Qiaentip 500 (Qiaen). The necessary reaents are supplied with the columns
or can
e prepared as outlined in the Qiaen-tip 500 manual.

2.2. Cell Transfection

1. Special equipment and materials: A standard cell culture facility and materia
ls must
e availa
le. 2. Fish cells: Two different cell lines are recommended, c
hinook salmon em
ryo cells CHSE-214 [11; ATCC (American Type Culture Collection)
cat no. CRL 1681], and epithelioma papulosum cyprini (EPC) cells derived from t
he common carp (12). The EPC cells are not availa
le throuh ATCC
ut can
e o
t
ained from us upon request. 3. Fish cell line maintenance medium: Eales minimum
essential medium with Earles salts supplemented with 10% fetal calf serum, 100 IU
/mL penicillin, 100 /mL streptomycin, and 2 mM L-lutamine. 4. Transfection medi
um: Opti-MEM 1 reduced serum medium (Gi
co-BRL, Gaithers
ur, MD). 5. PBS: 0.14
M NaCl, 10 mM sodium phosphate, pH 7.4. Sterilize the PBS
y passae throuh a 0
.2 m filter and store the solution at room temperature.
DNA Vaccines for Salmonid Fish
111
6. Lipid: For transfection we commonly use lipofectamine (Gi
co-BRL). In our han
ds, lipid-facilitated transfection of fish cells is much more efficient than cal
cium chloride methods.
2.3. Analysis of Transfected Tissue Culture Cells 2.3.1. Common Reaents
1. Special equipment : Standard cell culture facility and materials, fume hood.
2. 1% lutaraldehyde cell fixin solution: For 10 mL, add 0.1 lutaraldehyde to
a solution containin 0.1 M sodium phosphate and 1 mM MCl2, pH 7.0 (1% fixin
reaent). The solution is toxic and should
e prepared and used under a fume hoo
d. Generally we prepare a fresh solution as needed. 3. Phosphate-
uffered saline
(PBS).
2.3.2. Histochemical Detection of -alactosidase
1. Stock X-Gal solution: For a 20% solution dissolve 2.0 X-Gal (5-
romo-4chloro
-3-indolyl--D-alactoside) in N, N dimethylformamide. Dispense the X-Gal solutio
n into 1.5 mL tu
es, wrap in aluminum foil to protect the solution from liht, a
nd store at 20C. 2. -Galactosidase detection solution: This is made fresh as needed

y dilutin the stock X-Gal solution to 0.2% in the followin solution: 10 mM s
odium phosphate
uffer (pH 7.0), 150 mM NaCl, 1 mM MCl2, 3.3 mM potassium ferro
cyanide, and 3.3 mM potassium ferricyanide. The solution is toxic, liht sensiti
ve, and should
e stored at 4C.
2.3.3. Luciferase Assays
1. Special equipment: Sinle photon counter, tissue homoenizer. 2. Lysis
uffer
: 0.1 M potassium phosphate (pH 7.8), 1% Triton X-100, 1 mM dithiothreitol (DTT)
, 2 mM EDTA. 3. Assay
uffer: 30 mM Tris-HCl (pH 7.8), 3 mM ATP, 15 mM manesium
sulfate, 10 mM DTT, and 1 mM coenzyme A or 1 mM inoranic pyrophosphate. 4. 1 m
M D-luciferin: Dissolve the D-luciferin in ddH20, adjust the pH to 6.0-6.3. Disp
ense the solution into 1.5 mL tu
es and store in the dark at 20C.
2.3.4. Antien Detection
1. 2. 3. 4. Special reaent: Antien-specific monoclonal anti
ody. Biotinylated
anti-mouse anti
ody. Avidin-alkaline phosphatase conjuate reaent. Equili
ratio
n
uffer: 100 mM Tris-HCl, pH 8.2. This solution should
e sterilized
y autocla
vin. 5. Alkaline phosphatase color development solution: Numerous reaents are
availa
le commercially. We recommend the Vectastain red su
strate (Vector La
ora
tories, Burliname, CA).
112
Anderson and Leon
6. Blockin solution: 5% non-fat powdered milk in PBS. 7. Rinsin solution: 0.5%

non-fat powdered milk in PBS.

2.4. Injection of Fish
1. Special la
oratory needs and materials: Fish-rearin facility and supplies, p
athoen-free fish. 2. Injection: For delivery of the DNA use a 27G 1/2 needle an
d a 1-mL tu
erculin syrine or a repeat pipette. 3. Injection solution: The DNA
should
e suspended in PBS. 4. Anesthetic: Water containin 100 /mL of tricaine
methane sulfonate (MS222; Arent Chemical La
oratories, Redmond, WA). The anesth
etic solution is used once and then discarded. 5. Anesthetic overdose: Water con
tainin 300 /mL MS222.
2.5. Analysis of Fish Injected with DNA
1. Special equipment and supplies: fish-rearin facility, histoloy la
oratory,
sinle photon monitor, tissue homoenizer, fish dissection tools. 2. Fish fixati
ve: 10%
uffered formalin, which is commercially availa
le or prepared
y diluti
n 37% formaldehyde in PBS and adjustin the solution to pH 7.4.
2.5.1. Preparation of DNA from Fish Tissues
1. Special equipment and reaents: Mortar and pestle, liquid nitroen. 2. Diest
ion
uffer: 100 mM sodium chloride, 10 mM Tris-HCl (pH 8.0), 25 mM EDTA (pH 8.0)
, 0.5% sodium dodecyl sulfate, 0.1 /mL proteinase K. 3. PBS. 4. 25:24:1 phenol/c
hloroform/isoamyl alcohol equili
rated in TE. 5. 7.5 M ammonium acetate. For ste
rilization, autoclave the solution. 6. 95% and 70% ethanol (ethanol diluted with
sterile distilled water to 70%). 7. 1 /mL DNase-free RNase.
2.5.2. Analysis of DNA
y Hy
ridization
The southern
lottin method is used to determine the physical nature of the DNA
vaccine in transfected fish cells. This method involves standard protocols outl
ined in Sam
rook et al. (13)
3.1. Plasmid DNA Preparation
A num
er of protocols are availa
le for preparin DNA vaccines. We have tested r
elatively crude preparations of DNA encodin reporter proteins in fish and the r
esultin protein activity is similar to that achieved with hihly purified DNA.
All of the protocols have merit. We use Qiaen-tip 500 columns
ecause they are
simple, quick, and consistently provide very pure DNA. The followin outline is
for preparation of approximately 500  of pure plasmid DNA.
DNA Vaccines for Salmonid Fish
113
1. Streak E. coli DH5 tr nsformed with the pl smid on LB- g r pl tes cont ining 5
0 g/mL mpicillin. Incu
te t 37C overnight. 2. Inocul te 10 mL LB- mpicillin (50
g/mL) medium in 50-mL fl sk with 23 colonies from the
ove pl te. Incu
te 812
h t 37C with vigorous sh king, 225250 rpm. 3. Inocul te the 10 mL from
ove into
90115 mL LB- mpicillin medium in 500-mL fl sk. Incu
te 812 h t 37C with vigoro
us sh king, 225250 rpm. 4. Tr nsfer the
cteri l culture to sterile
ottle nd
pellet the
cteri
y lowspeed centrifug tion for 10 min. 5. Dec nt the fluid,
invert the
ottles, nd pl ce on p per towels
riefly to collect rem ining flui
d. 6. Purify the DNA from the
cteri l pellet with Qi gen-tip 500 column. Fol
low the m nuf cturers protocol supplied with the column. Resuspend the columnpuri
fied DNA in 300 L TE nd tr nsfer to 1.5-mL tu
e. 7. To the purified DNA s mple
, dd 30 L sodium cet te nd 750 L 95% eth nol. Vortex the solution
riefly nd p
ellet the DNA in microcentrifuge (m ximum speed) for 15 min. 8. Aspir te the f
luid off from the DNA pellet, dd 500 L 70% eth nol, vortex for 15 s, nd spin th
e DNA in microcentrifuge for 15 min. 9. Repe t step 8. Air dry the DNA in st
erile environment, e.g., under l min r flow hood or suit
le sterile cont inme
nt environment. Resuspend the DNA in 200300 L of TE. 10. Using spectrophotometer
, determine the DNA concentr tion nd purity of the s mple
y me suring the opti

c l density of the solution t 260 nm nd 280 nm. 11. Adjust the DNA s mple to 25
g/mL
y dilution in TE. Store the DNA s mple t 4C. 12. The fin l critic l step i
s to demonstr te th t the pl smid is int ct nd predomin tely circul r. This sho
uld
e determined
y gel electrophoresis of 200300 ng of the pl smid DNA (see Not
e 2).
3.2. Cell Tr nsfection Protocol
The recommended cell lines, CHSE-214 nd EPC, re commonly used
y fish he lth s
peci lists. The temper ture toler nce of the cell lines re
ro d (11,12). In ge
ner l, the CHSE-214 cells should
e m int ined t 15C nd the EPC cells t 28C.
1. Split the cells from newly confluent monol yers into 9.4 cm2 (6-well pl tes)
wells such th t the following d y the monol yers h ve re ched 90% confluency. Th
e m inten nce temper ture t this point should
e the s me s th t for the p tho
gen for which the v ccine is
eing developed. 2. Thirty minutes prior to tr nsfe
ction, rinse the cells twice in Opti-MEM 1 followed
y ddition of 23 mL of OptiMEM 1. 3. For lipofect mine tr nsfection prep re the DNA nd lipid complex s ou
tlined in the protocol supplied
y the m nuf cturer. The optimum tr nsfection ef
ficiency
114
Anderson nd Leong
of the fish cells in 9.4-cm2 well using lipofect mine is chieved using 1 g DNA
nd 6 L or 9 L lipid for CHSE-214 nd EPC cells, respectively. 4. Incu
te the ce
lls with the lipid DNA complex for 24 h. 5. Rinse the cells twice with Opti-MEM
1 nd then dd 3 mL cell line m inten nce medi .
3.3. Procedures for An lysis of Tr nsfected Tissue Culture Cells 3.3.1. Cell Pre
p r tion
1. At the design ted times (typic lly 4872 h post-tr nsfection), rinse the tr nsf
ected cells twice with PBS. 2. Under fumehood spir te the PBS nd dd 2 mL of
1% glut r ldehyde to e ch well. Let the cells sit for 30 min t room temper tur
e. 3. Disc rd the fix tive (this solution is toxic), dd 5 mL PBS nd pl ce the
cells on rocking pl tform for 1 min. Aspir te the PBS nd repe t this step fou
r more times.
3.3.2. Histochemic l Detection of -g l ctosid se
1. To e ch well dd 1 mL of -g l ctosid se detection solution. 2. Incu
te the ce
lls t 37C for 24 h. 3. Aspir te the solution, dd 5 mL PBS nd pl ce the cells on
rocking pl tform for 1 min. Aspir te the PBS nd repe t the rinse two times.
4. Aspir te the PBS nd dd 1.5 mL glycerol to the wells. 5. Count the num
er of
tr nsfected cells. The tr nsfected cells re st ined
lue (Fig. 2).
3.3.3. Lucifer se Ass y
1. Add 200 L lysis
uffer to e ch well. Tritur te the solution
riefly nd pl ce
in 1.5-mL tu
e. Add n ddition l 200 L lysis
uffer to the well nd tr nsfer t
he supern t nt to the s me tu
e. 2. Using 10 L of the lysed cells m ke 10-fold se
ri l dilutions in 90 L lysis
uffer. 3. Me sure the lucifer se ctivity in 100 L o
f diluted s mple
y dding 100 L ss y
uffer, gently mix, then dd 100 L D-lucife
rin, mix g in, nd pl ce in photon counter. 4. The lucifer se ctivity should

e djusted
y c li
r tion with lucifer se st nd rds (see Note 3).
3.3.4. Antigen Detection
This protocol is slight modific tion of Drolet et l. (14).
1. Add 3 mL
locking re gent to e ch well. Pl ce the pl tes on rocker pl tform
nd git te gently for 1 h. 2. Aspir te the
locking solution nd dd 1 mL of t
he ppropri te concentr tion of ntigen-specific monoclon l nti
ody. Incu
te w
ith gentle git tion on rocker pl tform for 1 h. 3. Aspir te the prim ry monoc
lon l nti
ody, dd 5 mL rinsing solution, nd git te on rocker pl tform for

1 min. Repe t the rinse step n ddition l four times.

DNA V ccines for S lmonid Fish
115
Fig. 2. Tissue section st ined for -g l ctosid se ctivity. The d rkly st ining m
uscle tissue expressing -g l ctosid se is shown cross the middle of the figure.
4. Aspir te the rinsing solution, nd dd 1 mL of ppropri tely diluted
iotinyl
ted nti-mouse nti
ody. Incu
te with gentle git tion on rocker pl tform fo
r 1 h. 5. Rinse the cells, s in step 3. 6. Add 1 mL of vidin- lk line phosph t
se conjug te nd incu
te on the rocker pl tform for 1 h. 7. Rinse the cells,
s in step 3. 8. Flood the wells with equili
r tion
uffer nd git te on the roc
ker for 2 min. 9. Aspir te the wells, nd 1 mL of color su
str te. 10. Aspir te
the wells, dd 2 drops of glycerol, nd pl ce coverslip over the cells. 11. Co
unt the num
er of tr nsfected cells. Using Vector red su
str te, the cells cont
ining ntigen will st in red.
3.4. Injection of Fish with Pl smid DNA
Speci l c re should
e t ken to ensure th t minimum mount of time is used whe
n v ccin ting fish. Prior to v ccin tion, pilot experiment should
e performed
to determine how r pidly the v ccin tion process c n
e done
y n individu l.
A work st tion should
e designed so th t the fish c n e sily nd swiftly
e mov
ed
etween the stock t nk, nesthetizing t nk, nd fin l holding t nk.
116
Anderson nd Leong
Fig. 3. Injection of DNA into fish. (A) Schem tic di gr m showing injection site
t the posterior end of the dors l fin into the dors l muscul ture. (B) Cross s
ection of fish showing loc tion of the m jor org ns.
1. Equili
r te the DNA/PBS solution to the w ter temper ture in which the fish
re
eing held. 2. Anesthetize 1020 fish t time with MS222. The fish should sto
p moving in 23 min. Remove the fish quickly from the nesthetic. 3. A single fish
c n
e injected while holding it in gloved h nd. L rge fish should
e injecte
d on t
le covered with wet pl stic. 4. Inject the fish midw y
etween the nt
erior end of the dors l fin nd the l ter l line (see Figs. 3 nd 4). Gently inj
ect the solution into the muscle tissue of the fish. After the solution h s
een
injected w it 23 s. The injection process should t ke no longer th n 1020 s. 5. A
s quickly s possi
le, pl ce the fish
ck in holding t nk.
DNA V ccines for S lmonid Fish
117
Fig. 4. Photogr ph of r in
ow trout fry nd tu
erculin syringe used to inject DN
A into the fish.
3.5. An lysis of Fish Injected with DNA The fish to
e n lyzed should
e euth n
ized
y n overdose of MS222. Fish to
e fixed prior to histochemistry should
e
pl ced in 10%
uffered form lin fter surgic lly opening their
domens from th
eir liver to their rectum. Fish to
e used for histochemic l ss ys on fresh tis
sues should
e processed immedi tely s outlined. 3.5.1. Histochemic l Detection
of -G l ctosid se in Fixed nd Em
edded Tissue (see Note 4)
1. Keep the fish in the form lin fix tive for 24 h t 4C. 2. Remove the fish from
the fix tive nd rinse them in continuously flowing ddH2O for 20 min. 3. Incu

te the rinsed fish in -g l ctosid se detection solution for 48 h t room temper tu

re. 4. Rinse the fish s in step 2. 5. Pl ce the fish in fix tive for 24 h. 6. R
inse the fish in flowing ddH2O for sever l hours. 7. P r ffin em
ed the fish. 8.
For microscopic ex min tion t ke 6 mm sections from the p r ffin
lock.
118 3.5.2. Lucifer se Ass ys
Anderson nd Leong
1. Surgic lly remove sm ll uniform portion of tissue from the fish. The ex ct
size of tissue t ken will depend on the outcome of Su
he ding 3.5.1. 2. Pl ce th
e tissue in 14-mL polypropylene tu
e cont ining lysis
uffer (1:5, wt/vol). 3.
Numerous methods re v il
le to mince the tissue. We use polytron homogeniz
er (Brinkm n Instruments, West
ury, NJ) set t 13,000 rpm for 20 s. 4. Cl rify t
he homogen tes
y centrifug tion t 10,000g for 10 min. 5. Prep re the s mples
nd me sure lucifer se ctivity s descri
ed
ove.
3.5.3. Immunohistochemic l Detection of Antigen (see Note 5)
1. Le ve the fish in fix tive for t le st 24 h. 2. Rinse, p r ffin em
ed, nd c
ut sections s descri
ed
ove. 3. For ntigen detection the slides should
e pl
ced on slide tr y, in humid pl stic
ox. E ch of the solutions should
e d
ded slowly to the slide forming droplet th t completely covers the thin sectio
n.
3.5.4. Prep r tion of DNA from Fish Tissue
1. Surgic lly remove sm ll uniform portion of tissue from the fish. 2. As quic
kly s possi
le, pl ce the tissue in liquid nitrogen. Once the tissue is frozen,
it c n
e processed s outlined
elow or stored t 70C. 3. Grind the frozen tissu
e to co rse powder using mort r nd pestle. 4. Pl ce the powdered tissue in
tu
e cont ining digestion
uffer (100 mg tissue/1 mL digestion
uffer). 5. Inc
u
te the s mples on sh king pl tform t 50C for 1216 h. The s mple should
e ge
ntly mixed to void she ring of the rele sed DNA. 6. Add n equ l volume of phen
ol/chloroform/iso myl lcohol to the s mple nd gently mix. 7. Centrifuge the s
mple to sep r te the org nic nd queous ph ses. 8. Tr nsfer the queous (upper)
ph se to new tu
e nd repe t steps 6 nd 7 nd com
ine the s mples. 9. To the
queous ph se dd 1/2 vol mmonium cet te nd 2 vol 95% eth nol. Gently mix. 1
0. Immedi tely centrifuge the s mple to pellet the DNA. 11. Aspir te the supern
t nt nd rinse the DNA pellet with 70% eth nol. Repe t this step two times with
centrifug tion
etween rinses to re-pellet the DNA. 12. Suspend the DNA in TE to
fin l concentr tion of 15 g/mL. Store the DNA t 4C.
4. Notes
1. The procedures descri
ed for the development of DNA v ccine for fish is
s
ed prim rily on our experience with s lmonid fish species. It is import nt to re
mem
er th t there re m ny different fish species. Some fish prefer h
it ts of
cle r,
DNA V ccines for S lmonid Fish
119
highly oxygen ted w ter nd others c n occupy tur
id, low oxygen-level w ter; so
me c n live in m rine qu tic environments t temper tures of 24C nd others prefe
r freshw ter temper tures t 2832C. Genetic studies on fish immunoglo
ulins nd MH
C genes indic te th t there is consider
le diversity in gene rr ngements nd i
n the immune response mong the different fish species. Even the presence of dif
ferent immunoglo
ulin isotypes is unresolved t present for fish. Thus, some of
the experiment l procedures, such s the period of time required for n immune r
esponse to develop
efore virus ch llenge, will v ry with the fish species. Alth
ough there re some re gents v il
le commerci lly for detecting r in
ow trout
immunoglo
ulin, m ny investig tors purify fish immunoglo
ulin nd m ke their own

 ntiser for nti
ody detection. Also, while higher verte
r tes immune systems
sh re some common fe tures with fish, one should not ssume th t the immune resp
onse is completely simil r nd ll ss ys should
e tested empiric lly. Fin lly,
it should
e noted th t in
red s lmonid fish str ins re not v il
le commerci
lly lthough G ry Thorg rd t W shington St te University (Pullm n, WA) h s de
veloped some in
red r in
ow trout lines. Without in
red lines, it difficult to d
evelop ppropri te ss ys for the r in
ow trout cellul r immune response. Th t i
s not the c se for c tfish cells where continuous cell lines re v il
le throu
gh Norm n Miller nd Willi m Clem t the University of Mississippi t J cksonvil
le, Mississippi. B sic immunologic l reference nd l
or tory m nu ls for the st
udy of the fish immune system re v il
le (1517) nd m ny of the protocols c n

e modified for p rticul r fish species. 2. The Qi gen-tip 500 column c n
e u
sed t le st twice if it is not llowed to dry out
etween uses. For multiple us
e, prep re sever l crude prep r tions of DNA cont ining m ximum of 500 g DNA. P
ss the crude prep r tion cross the column. Immedi tely equili
r te the column
with 2 vol of equili
r tion
uffer nd repe t the isol tion of DNA. 3. For c li

r tion of lucifer se ctivity, st nd rd curve should
e gener ted using purifi
ed firefly lucifer se (An lytic l Luminescence L
or tory, Ann Ar
or, MI). This
step is necess ry
ec use lucifer se ctivity c n v ry depending on uncontroll

le environment l conditions. Once st nd rd curve h s
een gener ted, selected
control st nd rds should
e used e ch time n ss y is performed so th t the ct
ivity of the lucifer se in the test s mple c n
e estim ted. Prep re the lucifer
se in 0.1 M phosph te
uffer, pH 7.8. To gener te the st nd rd curve use 10fold
dilutions, from 0.011 pg, of purified lucifer se in 100 L of lysis
uffer. 4. In

oth control nd pCMVG l injected fish, intense
-g l ctosid se st ining is o
ser
ved throughout the stom ch nd the intestine. This st ining is, more th n likely
, due to the
cteri inh
iting the intestin l tr ct. Therefore, if desired, pr
ior to immersion in the X-G l, the stom ch nd intestine c n
e surgic lly remov
ed. This step helps elimin te
leeding of the st ining solution into surrounding
tissues. Also, there is no
ckground lucifer se ctivity in the stom ch or int
estine, so if me surements of protein ctivity in these org ns re critic l the
fish should
e injected with pCMV-Luc.
120
Anderson nd Leong
5. Extensive non-specific st ining c n occur when either tissue culture cells or
fish sections re st ined for ntigen. This pro
lem c n usu lly
e elimin ted

y keeping the s mples moist t ll times. For ex mple, when spir ting the rinse
solution from tissue culture cells, hold the pipette for spir tion in one h nd
nd squirt
ottle with rinse solution in the other h nd, spir te the solutio
n quickly nd immedi tely squirt the w sh solution into the well.
References
1. Leong, J. C. (1993) Molecul r nd
iotechnologic l ppro ches to fish v ccine
s. Curr. Opin. Biotechnol. 4, 286293. 2. Leong, J. C. nd Fryer, J. L. (1993) Vir
l v ccines for qu culture. Ann. Rev. Fish Dis. 93, 225240. 3. Emmenegger, E. C.
, Hu ng, C., L ndolt, M., L P tr , S., nd Winton, J. R. (1995) Immune responses
to synthetic peptides representing ntigenic sites on the glycoprotein of infec
tious hem topoietic necrosis virus. Vet. Res. 26, 374378. 4. Mourich, D. V. nd L
eong, J. C. (1991) M pping of the immunogenic regions of the IHNV glycoprotein i
n r in
ow trout nd mice, in Proceedings of the Second Intern tion l Symposium o
n Viruses of Lower Verte
r tes, Corv llis, OR, July 2931, pp. 93100. 5. Anderson,
E. D., Mourich, D. V., F hrenkrug, S. C., L P tr , S. C., Shep rd, J., nd Leong
, J. C. (1996) Genetic immuniz tion of r in
ow trout (Oncorhynchus mykiss) g in
st infectious hem topoietic necrosis virus. Mol. M r. Biol. Biotechnol. 5, 114122
. 6. Anderson, E. D., Mourich, D. V., nd Leong, J. C. (1996) Gene expression in
r in
ow trout (Oncorhynchus mykiss) following intr muscul r injection of DNA. M
ol. M r. Biol. Biotechnol. 5, 105113. 7. Gomez-Chi rri, M., Livinston, S. K., Mur

o-C cho, C., S nders, S., nd Levine, R. P. (1996) Introduction of foreign genes
into the tissue of live fish
y direct injection nd p rticle
om
rdment. Dis.
Aqu t. Org. 27, 212. 8. Ertl, H. nd Xi ng, Z. (1996) Novel v ccine ppro ches.
J. Immunol. 156, 35793582. 9. Heppell, J., Lorenzen, N., Lorenzen, E., Jensen, K.
E., Wu, T., nd D vis, H. T. (1997) DNA v ccines for fish: protection g inst v
ir l hemorrh gic septicemi virus in trout. A
str ct in First Intern tion l Vete
rin ry V ccines nd Di gnostics Conference. M dison, WI, July 2731, 1997. 10. Ell
is, A. E., ed. (1988) Fish V ccin tion. Ac demic Press, S n Diego, CA. 11. Fryer
, J. L. , Yush , A., nd Pilcher, K. W. (1965) The in vitro cultiv tion of tissu
e nd cells of P cific S lmon nd Steelhe d Trout. Ann. N.Y. Ac d. Sci. 126 ( rt
.1), 566586. 12. Tom sec, J. nd Fij n, N. (1971) Virusne
olesti ri
(vir l dis
e se of fish). Fin l Report on Rese rch under P rt of Project 6n/1966. Z gre
,
Yugosl vi . 13. S m
rook, J., Fritsch, E. F., nd M ni tis, T. (1989) Molecul r
Cloning: A L
or tory M nu l. Cold Spring H r
or L
. Press, Cold Spring H r
or
, NY. 14. Iw m , G. nd N k nishi, T., eds. (1996). The Fish Immune System: Org
nism, P thogen, nd Environment. Ac demic Press, S n Diego, CA.
DNA V ccines for S lmonid Fish
121
15. Drolet, B. S., Rohovec, J. R., nd Leong, J. C. (1993) Serologic l identific
tion of infectious hem topoietic necrosis virus in fixed tissue culture cells

y lk line phosph t se immunocytochemistry. J. Aqu t. Anim. He lth 5, 256269. 16.
Stolen, J. S., Fletcher, T. C., Anderson, D. P., K tt ri, S. L., nd Rowley, A
. F., eds. (1992) Techniques in Fish Immunology-2. SOS Pu
lic tions, F ir H ven,
NJ. 17. Stolen, J. S., Fletcher, T. C., Rowley, A. F., Anderson, D. P., K tt r
i, S. L., Zelikoff, J. T., nd Smith, S. A., eds. (1994) Techniques in Fish Immu
nology-3. SOS Pu
lic tions, F ir H ven, NJ.
12
CTL An lysis for Tumor V ccines
Antonio Ros to, G
riell Mil n, Ann lis Z m
on, P ol Z novello, nd Dino Coll
vo 1. Introduction M ny studies h ve
een conducted with the im to stimul te
ther peutic immune response g inst tumors. In most c ses, efforts h ve
een di
rected tow rd the induction of tumor-specific cytotoxic T lymphocytes (CTL),
ec
use this T lymphocyte su
popul tion is considered to pl y m jor role in the d
estruction of tumor cells (1). In p rticul r, v ccin tion protocols h ve
een de
signed to incre se the immunogenicity of int ct c ncer cells
y using djuv nts
or engineering tumor cells with cytokine or costimul tory molecule genes. A seco
nd line of rese rch h s employed immuniz tion with tumor ssoci ted ntigens (TAA
). These ntigens re expressed from derepressed or mut ted genes in tumor cells
, nd re recognized
y CTL in the form of peptides ssoci ted with MHC cl ss I
molecules. Genes encoding TAA h ve
een inserted into recom
in nt vir l vectors,
which re then used to infect the hosts cells nd induce expression of the tr ns
gene. Moreover, immuniz tion with purified TAA peptides or with ntigen-presenti
ng cells, such s dendritic cells, pulsed with TAA peptides h ve
een proposed.
An innov tive ppro ch for c ncer immunother py is offered
y DNA v ccin tion,
procedure th t h s lre dy
een used successfully to induce humor l nd cell-me
di ted immune responses to consider
le num
er of vir l,
cteri l, nd p r si
tic gents (2). Sever l fe tures m ke DNA v ccin tion p rticul rly relev nt to t
he field of c ncer ther py: i) the numerous genes encoding TAA th t h ve recentl
y
een cloned, nd therefore might
e used to construct DNA tumor v ccines; ii)
the synthesis of TAA within the hosts cells, which m y f vor the present tion of
TAA epitopes to CTL in the context of the hosts MHC cl ss I molecules; iii) the g
ener tion of strong ntigen-specific CTL response.
From: Methods in Molecul r Medicine, vol. 29, DNA V ccines: Methods nd Protocol
s Edited
y: D. B. Lowrie nd R. G. Wh len Hum n Press Inc., Totow , NJ
123

124
Ros to et l.
In this ch pter, we provide some guidelines nd suggestions to follow in order t
o induce high levels of TAA-specific CTL,
y injecting DNA-expression vectors co
nt ining genes encoding tumor ntigens, nd we will descri
e the technic l steps
to ev lu te lytic ctivity of this T cell su
popul tion.
1.1. Choosing the TAA nd Experiment l Model
The prototypes of hum n TAA re mem
ers of the mel nom - ssoci ted ntigen (MAGE
) f mily, origin lly detected on mel nom cells
ut lso sh red
y other neopl s
i s. These TAA ( nd the more recently descri
ed BAGE nd GAGE ntigens) re gene
r ted
y tr nscription l ctiv tion of norm l genes not expressed in norm l tiss
ues, with the exception of testis. A second group of ntigens is represented
y
differenti tion ntigens, which re expressed not only
y c ncer cells
ut lso

y norm l cells of the s me histotype. Fin lly, the third group of ntigens ris
es from mut tion of norm l genes which m y encode new ntigens specific for indi
vidu l tumors (3). Genes encoding e ch of these different TAA might
e cloned in
to pl smid vector nd used s potenti l DNA v ccine to induce n immune resp
onse c p
le of protecting g inst or elimin ting tumors
e ring the relev nt n
tigen. Most of the inform tion o
t ined thus f r in c ncer immunother py is deri
ved from n lysis of the immune re ctivity elicited in mice g inst model tumor
ntigens, very often represented
y xenogenic proteins th t re not even rel ted
to mouse proteins (4,5). In theory, lmost ny protein might constitute poten
ti l TAA if it is
le to induce n immune response when rtifici lly introduced
into tumor cell. Technic l procedures descri
ed here re
sed on experience
cquired with n experiment l tumor expressing TAA encoded
y norm l mouse g
ene, P1A, origin lly isol ted from the m stocytom cell line P815 (6), nd more
recently found to
e widely expressed
y different tumor cell line ges (7). The
P1A ntigen is recognized
y specific CTL clones in the context of the MHC cl ss
I molecule Ld. Therefore, this TAA, which sh res m ny ch r cteristics with hum
n ntigens of the MAGE, GAGE nd BAGE f milies, represents useful experiment l
model to develop nd study the effic cy of new v ccin tion str tegies th t coul
d find potenti l pplic tions for ther py of hum n tumors.
1.2. Pl smid Vectors nd V ccin tion Procedures
Most of the work done on DNA v ccin tion h s employed pl smid vectors driven
y
the strong vir l CMV or RSV promoters. We h ve found th t the CMV promoter usu l
ly drives higher levels of TAA expression comp red to other euk ryotic promoters
. Thus, cloning the TAA gene of interest into CMV-driven pl smid vector might

e the first choice. Once the gene is cloned,
CTL An lysis for Tumor V ccines
125
it is necess ry to c refully ev lu te its expression from the pl smid in vitro.
It is dvis
le to confirm the sequence of the pl smid insert, especi lly if the
TAA gene w s mplified
y PCR. Expression of the TAA from the pl smid must lw
ys
e n lyzed in tr nsient tr nsfections in terms of mRNA production nd protei
n expression, using specific nti
odies in order to visu lize the ntigen. If sp
ecific nti
odies re not v il
le, the TAA protein might
e t gged
y modifying
the gene
y the ddition of short sequence coding for t g epitope recognized

y commerci lly v il
le nti
ody. Our studies c rried out with TAA, modifie
d with 6- mino cid t g referred s AU-1, showed th t this procedure does not
modify the physic l properties of the TAA or lter the correct processing nd pr
esent tion of ntigenic epitopes. In f ct, we o
served th t cells st
ly tr nsfe
cted with the AU-1-modified P1A gene expressed the TAA nd were lysed
y CTL clo

nes specific for the relev nt epitope (8). The site of pl smid injection into th
e mouse is point th t lso h s to
e t ken into ccount. Sever l routes of dm
inistr tion h ve
een tried
y others (2) nd ourselves,
ut we h ve concluded t
h t intr muscul r inocul tion (i.m.), e.g., into the qu driceps or ti
i lis nte
rior (TA) muscles, rem ins the
est choice to induce CTL sensitiz tion. In this
reg rd, the muscle to
e inocul ted must
e cle rly visu lized in order to m xim
ize the efficiency of the inocul tion; therefore, to g in ccess to the qu drice
ps, sm ll incision of the overlying skin must
e performed. Inocul tion of the
TA does not require surgic l procedures nd c n
e c rried out tr nscut neously
(provided th t surrounding fur is c refully removed with trimmer or depil t
ory cre m) nd h s now
ecome our st nd rd procedure. The num
er of pl smid inoc
ul tions necess ry to produce n efficient nd homogeneous CTL response m y v ry
depending on the mouse str in nd the strength of the ntigen under study. For
ex mple, in DBA/2 mice, 3 i.m. inocul tions of 100 g of pl smid expressing the we
kly immunogenic ntigen P1A were needed to induce CTL gener tion, l
eit t v
ery v ri
le extent, in the m jority of v ccin ted mice (8). Interestingly, the
s me protocol of immuniz tion gener ted very efficient nd homogeneous CTL res
ponse in ll tre ted B l
/c mice. Applic tion of the s me protocol to immunize m
ice with pl smid vectors encoding the strong env nd g g vir l ntigens of Molon
ey-murine leukemi virus (M-MuLV) gener ted high CTL levels in ll v ccin ted n
im ls (Ros to et l., unpu
lished results). V rious pretre tment methods h ve
e
en proposed to improve the efficiency of the DNA immuniz tion, such s the use o
f
upiv c ine or hypertonic sucrose solutions (9). In our h nds, however, these
procedures did not enh nce the effic cy of DNA v ccin tion with the TAA-coding p
l smids descri
ed
ove. Inste d, dr m tic improvement, in terms of CTL gener
tion, w s chieved
y
126
Ros to et l.
pretre ting the TA muscle with c rdiotoxin: in this c se, single pl smid injec
tion, c rried out 5 d fter c rdiotoxin tre tment, w s sufficient to induce impr
essively high levels of CTL in B l
/c mice nd lso
yp ssed the limited respons
iveness o
served in sever l DBA/2 nim ls (Ros to et l., unpu
lished results).
A det iled description of how to perform c rdiotoxin tre tment nd pl smid inocu
l tion currently c n
e found t The DNA v ccine We
site (9).
1.3. An lysis of the CTL Response in Vitro nd in Vivo
Det iled protocols for performing CTL n lysis in llogeneic or vir l experiment
l systems h ve
een reported elsewhere (10,11). Here we will descri
e method
for gener ting nd n lyzing CTL in
ulk cultures following i.m. immuniz tion wi
th pl smid DNA coding for TAA. This n lysis m y
e c rried out
y setting up
mixed leukocyte tumor cell culture (MLTC), o
t ined
y restimul ting splenocytes
from DNA-immunized mice with irr di ted tumor cells
e ring the relev nt TAA, o
r mixed leukocyte peptide cell culture (MLPC), which employs the ntigenic pep
tide s the stimul tor. CTL lytic ctivity is me sured using 51Cr-rele se ss
y. CTL n lysis in vitro must
e followed
y c reful ev lu tion of the c p cit
y of the immunized mice to reject syngeneic tumor expressing the relev nt TAA,
in order to ev lu te the over ll in vivo efficiency of the v ccin tion procedur
e nd to study whether correl tion exists
etween tumor regression nd CTL gen
er tion. 2. M teri ls 2.1. Prep r tion of Spleen Cells nd MLTC nd MLPC Restimu
l tion
1. Speci l equipment: 137Cs cell irr di tor. 2. Complete culture medium: Dul
ecc
os Modified E gle Medium (DMEM) supplemented with 10% (v/v) fet l c lf serum (FCS
), 2 mM L-glut mine, 100 U/mL e ch penicillin nd streptomycin, 10 mM HEPES [4-(
2-hydroxy-ethyl)-1-piper zine eth nesulfonic cid], 5 10-5 M -merc ptoeth nol. M
ke fresh s required nd sterilize
y filtr tion through 0.22 m filter. 3. Inco
mplete medium: DMEM supplemented with 3% (v/v) FCS, 100 U/mL e ch penicillin nd
streptomycin, 10 mM HEPES. M ke fresh s required nd sterilize
y filtr tion t

hrough 0.22 m filter. 4. Petri dishes (3 cm, sterile). 5. Cell str iner with n
outside di meter th t llows it to rest on the top of 50 mL conic l tu
e (100
m Nylon, sterile). 6. Polystyrene round-
ottom (10 mL) nd polypropylene gr du t
ed conic l (50 mL) tu
es with c ps. 7. 25 cm2 fl sks (tissue culture qu lity). 8
. Forceps nd scissors (sterile). 9. Syringe plunger (sterile).
CTL An lysis for Tumor V ccines
127
10. Neu
uer cell counting ch m
er. 11. Vit l dye: prep re 0.1% (w/v) Eosin Y
solution in s line solution (0.9% N Cl). 12. Stimul tor tumor cells: grow t 0.81
106/mL if in suspension or to slightly confluent st tus if dherent. 13. Pept
ide (95% purity) stock: prep re 1 mM solution in molecul r
iology gr de DMSO;
filter-sterilize, liquot t 100 L/vi l nd store t 80C.
2.2. 51Cr-Rele se Ass y
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Speci l equipment: -counter. Incomplete medium as
in Su
headin 2.1. 51Cr as sodium chromate (Na CrO ) in normal saline solution a
t 1 mCi/mL. 2 4 Polystyrene round-
ottom (10 mL) tu
es with caps. Round-
ottom 9
6-well plates with covers. Small plastic tu
es (3 cm tall) that fit into 96-well
plates. Multichannel pipet. Neu
auer cell countin cham
er. Eosin Y solution as
in Su
headin 2.1. Taret cells: row at 0.60.8 106/mL if in suspension or to a
su
confluent status if adherent. 11. Peptide as in Su
headin 2.1. 12. Triton X100: prepare a 5% (v/v) solution in saline solution. Store at room temperature.
3. Methods
3.1. Preparation of Spleen Cells and MLTC and MLPC Restimulation 3.1.1. Collecti
on of Spleens and Preparation of Lymphoid Cells
1. Euthanize mice and disinfect with 70% alcohol. Aseptically remove spleens usi
n sterile forceps and scissors and transfer into a 3-cm Petri dish containin 2
mL of incomplete medium. 2. Position a cell strainer onto a raduated conical t
u
e and rinse the mem
rane with 12 mL of incomplete medium. Transfer the spleen o
nto the cell strainer and ently press with a pluner to allow dissociation of s
plenocytes from spleen stroma (see Notes 1 and 2). Rinse the mem
rane of the cel
l strainer with 20 mL of incomplete medium, allowin cells to collect into the c
onical tu
e (see Note 3). Discard the cell strainer with the spleen stroma. 3. C
arefully pipet the cell suspension to
reak clumps. Centrifue the sinle-cell s
uspension for 57 min at 250. Resuspend the cell pellet with 20 mL of incomplete
medium; place an aliquot of the cells in a Neu
auer countin cham
er after stain
in with Eosin Y and count via
le cells (see Note 4). Centrifue cell suspension
as a
ove and resuspend the pellet in complete culture medium, adjustin the cel
l concentration to 107/mL.
128 3.1.2. Preparation of Stimulator Tumor Cells
Rosato et al.
1. Collect cells from culture
y centrifuin for 57 min at 250 in a 10-mL polys
tyrene round-
ottom tu
e. Wash once with 10 mL of incomplete medium, as descri
e
d in Su
headin 3.1., step 3. 2. Resuspend pellet in 10 mL of incomplete medium
and count via
le cells in a Neu
auer countin cham
er as descri
ed a
ove. 3. Cen
trifue cell suspension and remove 9 mL of medium, leavin 1 mL over the pellet,
then irradiate the pellet at 60100 Gy (see Note 5). 4. Wash the cells twice as d
escri
ed a
ove and recount. Resuspend the pellet in complete culture medium, adj
ustin the cell concentration to 2 105/mL.
3.1.3. MLTC Setup
1. In a 25-cm2 culture flask, add 10 mL of complete culture medium (see Note 6),

2.5 mL of splenocyte cell suspension (2.5 107 total) and 2.5 mL of irradiated s
timulator tumor cells (0.5 106 total) (see Note 7). 2. Position the flask vertic
ally and cultivate for 5 d in an incu
ator at 37C, with 5% CO2 and 95% humidity (
see Note 8).
3.1.4. MLPC Setup
1. In a 25-cm2 culture flask, add 12.5 mL of complete culture medium, 2.5 mL of
splenocyte cell suspension (2.5 107 total) and 15 L of sterile peptide stock solu
tion (1 M final concentration). 2. Position the flask vertically and cultivate f
or 5 d in an incu
ator at 37C, with 5% CO2 and 95% humidity.
3.2. 51Cr-Release Assay 3.2.1. Preparation of Effector Cells
1. Without distur
in the cells, remove 5 mL of culture medium from the top of t
he MLTC or MLPC flasks. 2. Resuspend cells from the
ottom of the flask and tran
sfer the entire contents into a 10-mL polystyrene round-
ottom tu
e (see Note 9)
, then centrifue once and resuspend cells in 10 mL of incomplete medium. 3. Cou
nt via
le cells with Eosin Y and adjust cell concentration to 1.8 106/mL in inco
mplete medium (see Note 10).
3.2.2. Preparation of Taret Cells
3.2.2.1. 51Cr LABELING
1. After countin, collect 1 106 taret cells from the culture into a 10 mL poly
styrene round-
ottom tu
e (see Note 11), centrifue and carefully remove all med
ium away from the pellet.
CTL Analysis for Tumor Vaccines
129
2. Gently resuspend the pellet with 10 L of FCS. Add 100 L of 51Cr and incu
ate at
37C for 1 h, with periodic entle shakin (see Note 12). 3. After la
elin, wash
cells three times with incomplete medium and count via
le cells. Adjust cell co
ncentration to 2 104/mL.
3.2.2.2. PEPTIDE PULSING OF 51Cr-LABELED TARGET CELLS
1. After la
elin, wash cells two times with incomplete medium, resuspend the pe
llet in 1 mL of incomplete medium and add 1 L of peptide from stock solution (1 M
final concentration). 2. Incu
ate at 37C for 0.5 h, then wash three times and adj
ust cell concentration to 2 104/mL (see Note 13).
3.2.3. Coincu
ation of Effector and Taret Cells
1. Distri
ute 150 L of effector cell suspension in the first row of a round-
otto
m 96-well plate (see Note 14); run each sample in triplicate. 2. Add 100 L of inc
omplete medium to the next 3 or 5 rows, dependin on the num
er of dilutions you
may want to carry out. 3. With a multichannel pipet, transfer 50 L of cell suspe
nsion from the first row to the next one and mix well to allow complete dilution
. Repeat the same operation for the followin rows, and discard 50 L of cell susp
ension from the last dilution. At this point each well should contain 100 L of a
3-fold diluted effector cell suspension. 4. Add 100 L of taret cell suspension t
o wells containin effector cells (see Note 15). 5. To determine spontaneous rel
ease (amount of isotope released
y taret cells in the a
sence of effectors) an
d maximum release (total radioactivity incorporated
y taret cells), add 100 L o
f taret cell suspension to 6 additional empty wells. Fill 3 of these wells with
100 L of incomplete medium (spontaneous release) and the remainin 3 with 100 L o
f Triton X-100 solution (maximum release). 6. Centrifue plates for 35 min at 150
 and incu
ate for 4 h at 37C, with 5% CO2 and 95% humidity.
3.2.4. Collection of Supernatant and Calculation of Lytic Activity
1. After incu
ation, collect 100 L of supernatant with a multichannel pipet and t
ransfer into plastic tu
es (see Note 16). Determine radioactivity in a -counter.

2. Calculate the specific 51Cr release as follows: experimental release spontane

ous release % specific release = 100 maximum release spontaneous
release, indicatin the effector-to-taret ratio on the x-axis (from 90:1 to 3:
1 or 0.3:1, dependin on whether 4 or 6 dilutions have
een carried out) and the
correspondin percentae of specific lysis on y-axis.
130
4. Notes
Rosato et al.
1. Care should
e taken to remove most of the fi
rous material from the spleen

efore pressin with the pluner, as the presence of liament tissue may reduce c
ell recovery. 2. If a cell strainer is not availa
le, a 10-cm Petri dish can
e
equally useful. In this case, the spleen can
e pressed on the plastic surface,
the stroma removed and cell clumps resuspended
efore transferrin to a tu
e. 3.
Careful rinsin of the cell strainers nylon mem
rane is an important step to avo
id loss of splenocytes. 4. Via
ility of splenocytes and correct countin must
e
carefully checked to allow reproduci
le results and comparative analysis amon
different immunized mice. 5. Irradiation is usually the
est method to
lock pro
liferation of tumor cells and results in ood via
ility for 2472 h. This aspect i
s important to induce optimal stimulation of responder spleen cells. Alternative
ly, mitomycin C can
e used to inhi
it division of tumor cells,
ut in this case
, cell recovery and via
ility are inferior and stimulatin capacity is reduced.
6. A critical inredient for successful restimulation of splenocytes is the FCS.
Different
atches should
e tested for optimal results
ecause they may vary r
eatly in their capacity to support cell proliferation or may even
e excessively
rich and induce acquisition of non-specific cytotoxic activity. 7. A responder
(splenocytes) to stimulator (irradiated cancer cells) ratio of 50, as descri
ed,
is usually optimal for most experimental tumors. However, it must
e kept in mi
nd that a lare num
er of tumors are endowed with immunosuppressive capacity and
that excessive quantities of such cells in culture can completely
lock differe
ntiation of splenocyte into CTL. Therefore, careful titration of tumor cell num

er to add to the culture will allow the determination of optimal stimulation rat
io. 8. If a lare num
er of effector cells are needed, MLTC and MLPC can
e set
up in 75 cm2 flasks, triplin the num
er of splenocytes and irradiated tumor cel
ls to
e added. Vertical positionin of flasks allows collection of cells in a n
arrower surface and
etter cell-cell interactions. 9. If the entire culture is n
ot needed for the 51Cr release assay, only an aliquot may
e used, leavin the r
emainder for the next day. In fact, one can measure lytic activity as many as 6
d after set-up unless complete exhaustion of culture medium is o
served, and cul
tures may
e retested if the first attempts encounter technical difficulties. 10
. The presence of a hih num
er of
last cells in cultures is usually a ood si
n of active proliferation and stimulation and is enerally followed
y relevant
cytotoxic activity. Nonetheless, some cultures may appear devoid of any stimulat
ion and present hih levels of cytotoxicity. 11. Cells to
e la
eled must not
e
overrown
ecause this reduces isotope incorporation and increases spontaneous
release (which should not exceed 20%). The optimal cell concentration for la
eli
n is that reported in Su
headin 2.2., and counts measured in 1 min from 2 103
taret cells should rane
etween 20004000.
CTL Analysis for Tumor Vaccines
131
12. La
elin of cells rowin in suspension does not present particular pro
lems
,
ecause their num
er can
e easily evaluated at any moment. Adherent cells, wh
ich must
e trypsinized in order to
e counted
efore la
elin, sometimes form l
are clumps durin the la
elin step, which cannot
e re-counted later. In this
case, the clumps can simply
e resuspended and can
e assumed not to have under

one any loss in cell num
ers. Adherent cells usually incorporate hih levels of
isotope. 13. Peptide pulsin of taret cells can also
e carried out
efore or d
urin the la
elin period. 14. When many cultures have to
e tested, the distri

ution of effector cell suspensions into the 96-well plate may require some time,
thus allowin time for the cells to settle down at the
ottom of the well. Ther
efore,
efore startin dilutions, care must
e taken to mix well, in order to tr
ansfer a volume containin a homoeneous quantity of effector cells. 15. After d
istri
utin the taret cells, a
rief centrifuation allows rapid contact
etwee
n cells and can
e desinated as the startin time of the test. 16. Collection o
f supernatant from wells is a critical step, as removal of pelleted material wil
l completly ruin the test. To correctly collect the supernatant, the plate shoul
d
e slihtly inclined (for example,
y placin the plate cover under one side)
and the multichannel pipet (whose tips must
e alined properly) should
e kept
at a 45 anle, with the tips touchin the well walls. If dou
ts exist as to wheth
er the pellet has
een distur
ed, it is
etter to dischare the volume into the
wells, centrifue the plate and try aain.
References
1. Roth, C., Rochlitz, C., and Kourilsky, P. (1994) Immune response aainst tumo
rs. Adv. Immunol. 57, 281351. 2. Donnelly, J. J., Ulmer, J. B., Shiver, J. W., an
d Liu, M. A. (1997) DNA vaccines. Annu. Rev. Immunol. 15, 617648. 3. Boon, T. and
Van der Bruen, P. (1996) Human tumor antiens reconized
y T lymphocytes. J.
Exp. Med. 183, 725729. 4. Jaffee, E. M. and Pardoll, D. M. (1996) Murine tumor a
ntiens: is it worth the search? Curr. Opin. Immunol. 8, 622627. 5. Restifo, N. P
. (1996) The new vaccines:
uildin viruses that elicit antitumor immunity. Curr
. Opin. Immunol. 8, 658663. 6. Van den Eynde, B., Leth, B., Van Pel, A., De Plaen,
E., and Boon, T. (1991) The ene codin for a major tumor rejection antien of
tumor P815 is identical to the normal ene of syneneic DBA/2 mice. J. Exp. Med.
173, 13731384. 7. Ramarathinam, L., Sarma, S., Maric, M., Zhao, M., Yan, G., Ch
en, L., and Liu, Y. (1995) Multiple lineaes of tumors express a common tumor an
tien, P1A,
ut they are not cross-protected. J. Immunol. 155, 53235329. 8. Rosat
o, A., Zam
on, A., Milan, G., Ciminale, V., DAostino, D. M., Macino, B., Zanovel
lo, P., and Collavo, D. (1997) CTL response and protection aainst P815 tumor ch
allene in mice immunized with DNA expressin the tumor-specific antien P815A.
Hum. Gene Ther. 8, 14511458.
132
Rosato et al.
9. Whalen, R. G. (1997) The DNA vaccine we
. enwe
/dnavax/ dnavax.html. 10. Hud
son, L. and Hay, F. C. (1989) Mixed-lymphocyte reaction (MLR) and cellmediated c
ytolysis (CMC), in Practical Immunoloy (3rd ed.) Blackwell Science, Oxford, UK,
pp. 160162. 11. Bachmann, M. F. (1997) Evaluation of lymphocytic choriomeniniti
s virusspecific cytotoxic T cell responses, in Immunoloy Methods Manual (Lefkov
its, I., ed.), Academic Press, pp. 19191933.
13
The Use of Bone Marrow-Chimeric Mice in Determinin the MHC Restriction of Epito
pe-Specific Cytotoxic T Lymphocytes
Akiko Iwasaki and Brian H. Bar
er 1. Introduction Plasmid DNA immunization has e
mered as a promisin vaccine stratey aainst infectious aents, as well as a p
otential intervention for the treatment of cancer, autoimmunity, and allery (1)
. Until recently, however, the cellular events
y which injected plasmid DNA eli
cits potent anti
ody and cytotoxic T-lymphocyte (CTL) responses were larely unk
nown. Upon intramuscular (i.m.) injection of naked DNA, predominant expression o
f transfected DNA occurs in the myofi
ers (2),
ut no direct transfection of ant
ien presentin cells (APC) has
een reported. There are essentially three diffe
rent mechanisms
y which CTLs can
e primed
y the injected DNA (3). The first p
ossi
ility is that the transfected muscle cells directly activate CTLs
y presen

tin the antienic peptide on their MHC class I molecules. Alternatively, the pr
imin of CTLs may
e mediated
y professional APC takin up antien released fro
m muscle cells. Finally, CTL primin may involve direct transfection of APC occu
rrin, al
eit at low level, and that the CTLs are activated
y the transfected A
PC. In an effort to identify the key cellular su
set(s) responsi
le for the indu
ction of CTL responses
y plasmid DNA immunization, we created and immunized a s
et of
one marrow-chimeric mice (Fi. 1). Bone marrow-chimeric mice have proven
to
e a valua
le tool for determinin the relevant cell type(s) involved in the
activation of CTLs (4). In a
one marrow chimera, only the
one marrow derived,
hemopoietic cells express the MHC haplotype of the donor oriin, whereas non-
on
e marrow derived cells such as muscle cells and skin keratinocytes
ear the host
MHC molecules. By immunizin defined chiFrom: Methods in Molecular Medicine, vo
l. 29, DNA Vaccines: Methods and Protocols Edited
y: D. B. Lowrie and R. G. Wha
len Humana Press Inc., Totowa, NJ
133
134
Iwasaki and Bar
er
Fi. 1. Schematic diaram for the eneration and immunization of
one marrowchim
eric mice.
meric mice and assessin the specificity of the CTLs enerated, one is a
le to d
etermine which of these cell type(s) were involved in stimulatin naive CTLs upo
n DNA immunization. We and others have demonstrated
y the use of
one marrow-ch
imeric mice that the key cells in the presentation of DNA-encoded antien
y
ot
h ene un-mediated epidermal injection (5) and
y needle i.m. injection (5,6) o
f plasmid DNA are
one marrow-derived. Thus, this represents another example of
how this technique can
e used to o
tain vital information a
out the nature of c
ells responsi
le for the presentation of antiens to naive T cells. The main o
s
tacle to the more extensive use of
one marrow-chimeric mice has
een the diffic
ulties associated with the successful production of these mice. Pro
lems can ari
se from death of the infection-prone lethally irradiated mice, and from complica
tions from raft vs host disease (GvHD)
y the donor
Epitope-Specific Cytotoxic T Lymphocytes
135

one marrow-derived cells. Here we descri
e a comprehensive procedure for enera

tin
one marrow-chimeric mice, and point out certain measures that may
e taken
to prevent commonly encountered pro
lems. Further, the procedures for DNA immun
ization and CTL assays that can
e performed with the fully reconstituted mice a
re also descri
ed here. 2. Materials
2.1. Construction of Bone Marrow-Chimeric Mice 2.1.1. Production of Anti
odies f
or the Depletion of T Lymphocytes in Donor Mice
1. In vitro culture equipment: CELLMAX system (Cellco, Germantown, MD). 2. Cultur
e media: RPMI-1640 supplemented with 10% FCS, penicillin (100 U/mL), streptomyci
n (100 /mL), and L-lutamine (2 mM). 3. Lactate assay kit (Sima, St. Louis, MO)
. 4. Hy
ridomas that secrete T-cell depletion anti
odies: anti-CD4 (YTS-191) and
anti-CD8 (YTS-169) (7). These anti
odies are
oth derived from hy
ridomas which
secrete rat IG2
anti
ody isotype.
2.1.2. Depletion of T Lymphocytes with Anti
odies Aainst CD4 and CD8 in Donor M
ice
1. Special equipment: flow cytometer. 2. 1 mL Tu
erculin syrine fitted with 27G
1/2 needle. 3. Concentrated anti
odies aainst CD4 (YTS-191) and CD8 (YTS-169) 

enerated usin the CELLMAX system. 4. Donor in
red mice. 5. Detection anti
odies
for CD4 (FITC-conjuated anti-CD4) and CD8 (Phycoerythrin-conjuated anti-CD8)
(Becton Dickinson, San Jose, CA). 6. Protein concentration filter, Centriprep co
ncentrators (Amicon, Beverly, MA).
2.1.3. Isolation and Injection of Bone Marrow Cells from the Donor Mice
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Special equipment: -irradiation source suita
le fo
r use with experimental animals. Sterile Petri dishes (60 15 mm style). Cold pho
sphate-
uffered saline (PBS) in 50 mL tu
es on ice. Pair of surical scissors an
d forceps. 70% ethanol in a squirt
ottle. 3 mL Tu
erculin syrine fitted with 2
3G11/2 needle. Sterile cell strainer. Culture media as in Su
headin 2.1.1. Infr
ared heatin lamp. Mouse holder.
136
Iwasaki and Bar
er
2.1.4. Treatment of Mice Before and After Lethal Irradiation
1. Anti
iotics: Clavulin-125F (SmithKline Beecham, Oakville, ON, Canada) stock s
olution. Dissolve 1 of Clavulin in 40 mL of 0.9% sterile NaCl under laminar flo
w hood. This stock solution can
e kept at 4C for up to 10 d. 2. Autoclaved water

ottles. 3. Clidox (chlorine dioxide), freshly made. Once the
ase and the acti
vator are mixed, it must
e used within 14 d.
2.1.5. Plasmid DNA Immunization of Reconstituted Mice
1. Endotoxin-free plasmid DNA purified from
acterial sources. Qiaen Meaprep c
olumns are recommended. Store lyophilized DNA at 20C. Each injection requires 100 
of DNA. 2. Sterile saline. 3. Insulin syrine. 4. Bone marrow-chimeric mice. 5.
70% Ethanol in a squirt
ottle. 6. Gauze.
2.2. Peptide Epitope Specific Cytotoxic T-Lymphocyte Assay 2.2.1. In Vitro Re-st
imulation of Splenocytes
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Special equipment: -Irradiation source suita
l
e for use with cells in suspension. Stimulators: syneneic naive mice. Responder
s:
one marrow-chimeric mice immunized with DNA vaccine. 70% ethanol in a squirt

ottle. Pair of surical scissors and forceps. Cold PBS in 10 mL tu
es on ice.
Sterile cell strainer. Culture media as in Su
headin 2.1.1. Sterile 3 mL tu
erc
ulin syrines. 50 mL tissue culture flasks. Chemically synthesized and purified
MHC class I-restricted peptide from the antien encoded
y the DNA immunoen, re
suspended at 100 /mL in sterile PBS. 12. -Mercaptoethanol.
2.2.2. 51Cr-Release Assay
1. 2. 3. 4. Special equipment: amma counter, Skatron cell harvester (Sterlin,
VA). 96-well v-
ottomed plates with lids. Multichannel pipettor. Culture media a
s in Su
headin 2.1.1., and a separate media made with 25% FCS content. 5. [51Cr
] Na2CrO4 in sodium chloride solution. 6. MHC class I-restricted peptide of the
DNA encoded antien, resuspended at 0.1 /mL in sterile PBS.
Epitope-Specific Cytotoxic T Lymphocytes
3. Methods
137
3.1. Construction of Bone Marrow-Chimeric Mice 3.1.1. Production of Anti
odies f
or the Depletion of T Lymphocytes in Donor Mice
We descri
e here a practical method of o
tainin a sterile, hihly concentrated
anti
ody usin the CELLMAX system.
1. Grow hy
ridomas in RPMI-1640 supplemented media to o
tain 5 107 cells. 2. Pel
let down 5 107 cells and resuspend in 15 mL of media. 3. In the mean time, equil
i
rate CELLMAX capillary module with fresh media for at least 48 h. 4. Inoculate

CELLMAX capillary module with 5 10 7 cells, makin sure that everythin that co
mes in contact with the module is sterile. 5. Monitor the rowth of the cells
y
lactate production, which corresponds to lucose consumption
y the cells. Medi
a should
e chaned when the lucose consumption reaches 50% of the startin lu
cose concentration. For RPMI-1640 with 2.0/L of lucose, reservoir media must

e chaned when the lucose level falls to 1.0/L. To monitor lactate production,
a simple spectrophotometric assay kit is availa
le from Sima (Lactate Reaents
and Lactate Standard Solution). 6. When lactate consumption level reaches 750200
0 m/day, harvest the extracapillary space (ECS)
y flushin out the ECS with st
erile syrines and collect the content into a 50 mL tu
e. 7. Centrifue the cell
s down at 210 for 5 min and collect the supernatant into a sterile tu
e. 8. Tes
t various concentrations of the ECS eluate for its a
ility to deplete T cell su

sets in vivo (Su
headin 3.1.2.).
3.1.2. Depletion of T Lymphocytes with Anti
odies Aainst CD4 and CD8
The most strinent assessment of T cell depletion can
e o
tained from the detec
tion of T cells in the lymph node, since the proportion and the concentration of
mature T cell population is the hihest in the lymph nodes, as opposed to
lood
, spleen, or
one marrow. In order to o
tain a sufficient num
er of cells to
e
used for flow cytometric analysis, mesenteric lymph nodes are harvested.
1. Draw up different concentrations of anti
odies into 1 mL syrines adapted wit
h 27G1/2 needles. We recommend usin 0.5 mL of the most concentrated form, and d
ilutions of 1/10 and 1/50. Typically, the supernatant from the hy
ridomas must

e concentrated to 150200 fold the volume of the total culture media used to row
the hy
ridomas in order for a 0.5 mL volume injected on d 0 and 3 to completely
deplete
oth su
sets of T cells. 2. Inject the anti
odies intraperitoneally into
naive mice at d 0 and d 3.
138
Iwasaki and Bar
er
3. Collect mesenteric lymph nodes and make sinle cell suspensions
y disruptin
the lymph node throuh a cell strainer. Wash the cells three times in cold PBS,
and count the num
er of cells. 4. Perform two-color stainin of cells usin ant
i-CD4 and anti-CD8 detection anti
odies conjuated with FITC or PE, respectively
. Analyze on flow cytometer. 5. Calculate the percent depletion
y comparin the
percentae of cells seen in either the CD4+ or the CD8+ sinle positive quadran
t from the undepleted mice. The depletion o
tained in vivo should
e close to 10
0%, in order to prevent GvHD
y the residual donor T cells. If required, concent
rate the anti
ody usin a concentrator system such as Centriprep (see Note 1).
3.1.3. Isolation and Injection of Bone Marrow Cells from the Donor Mice
Isolation of
one marrow cells from the femurs and ti
ia of donor mice is a la
o
rious procedure that requires meticulous care to remove flesh and tendon from th
e
ones without
reakin them under sterile condition. As much as possi
le, all
solutions are to
e kept on ice, and all procedures to
e done under laminar flo
w hood.
1. Sacrifice donor mice depleted of mature T cells in vivo. 2. Soak the mice in
70% ethanol, and make small incision in the a
dominal skin. 3. Remove the fur al
l the way to the ends of the hind les
y tearin apart the skin from the incisi
on point. 4. Cut off the feet and remove all remainin fur. Harvest the hind le
s and place them into sterile PBS on ice. 5. Trim off the muscle, fat, and tendo
n carefully, usin a pair of surical scissors and forceps. The femur and ti
ia
should have no remainin flesh
y this point. 6. Cut off
one endins (a
out 4 m
m off the ends of the
ones) with sharp scissors and place the middle part of th
e
one in a Petri dish containin fresh medium. Repeat this procedure until all
the
ones are processed. 7. Flush out the content of the
one marrow usin 23G11
/2 needles attached to a 3-mL tu
erculin syrine into a new Petri dish containin
 fresh media. When the content of the
one marrow from all the
ones are collec

ted, make sinle cell suspension usin the cell strainer and the end of a steril
e syrine pluner. 8. Wash the cells once with media. Count the num
er of cells
o
tained. Typically,
etween 107108
one marrow cells can
e o
tained from one mo
use. 9. Each recipient mouse requires
etween 5 1062 107
one marrow cells. Resus
pend the
one marrow cells in cold sterile PBS at an appropriate concentration t
hat contains the desired num
er of cells in 0.2 mL per mouse. 10. While the proc
edure for the donor
one marrow is takin place, irradiate the recipient mice at

etween 8001000 rad (lethal irradiation). The lethal dose depends on the mouse s
train. For example, BALB/c mice take less irradiation (825 rad) and C57B/6 mice
take more (925 rad). Take extra care not to expose the recipient mice to any pat
hoens.
Epitope-Specific Cytotoxic T Lymphocytes
139
11. Heat the irradiated recipient mice with a infrared heatin lamp for a few mi
nutes. Place a recipient mouse in a mouse holder and clean its tail with an alco
hol swa
. 12. Inject via the tail vein 0.2 mL of the
one marrow cell suspension
(2.5 1071 108 cells/mL). Pressure the tail with a auze to stop
leedin. 13. Pl
ace a fresh
ottle of water with anti
iotics in the caes. Do not feed the recip
ient mice for 24 h after irradiation.
3.1.4. Treatment of Mice Before and After Lethal Irradiation
Mice irradiated at a lethal dose are very prone to infection. One of the common
causes of death after irradiation of mice is Pseudomonas infection. In order to
avoid death of the mice
y
acterial infection, recipient mice should
e treated
with anti
iotic
efore and after the irradiation until complete reconstitution

y the donor
one marrow-derived cells takes place. Aain, all procedures are to

e done under a laminar flow hood.
1. Autoclave the drinkin water and allow it to cool down. 2. Make the anti
ioti
c Clavulin stock
y dissolvin 1  of Clavulin-125F into 40 mL of sterile 0.9% N
aCl solution. Mix well. 3. Add 5 mL of the stock solution to 100 mL of drinkin
water in a sterile
ottle. 4. Chane water with Clavulin three times a week.
3.1.5. Plasmid DNA Immunization of Reconstituted Mice Once the full reconstituti
on of the recipient mice is confirmed (Su
headin 4.3.), plasmid DNA immunoen c
an
e administered to these mice. The detailed methods and protocols for differe
nt ways of immunizin with DNA have
een descri
ed elsewhere in this volume. We
descri
e here a method that was used to o
tain CTLs
y i.m. injection of DNA.
1. Prepare DNA from
acterial culture usin Qiaen Meaprep columns. Detailed in
structions are iven in the Meaprep Kit. This kit allows endotoxin-free isolati
on of plasmid DNA. Store lyophilized DNA at 20C. 2. Resuspend the lyophilized DNA
in an appropriate volume of sterile saline to make concentration of 2 /mL (50 mL
per mouse). 3. Fill insulin syrines with DNA solution (num
er of mice injected
50 mL per mouse). 4. Wipe the le area with alcohol swa
. Inject 50 L of DNA per
quadriceps muscle.
3.2. Peptide/Epitope-Specific Cytotoxic T Lymphocyte Assay 3.2.1. In Vitro Re-st
imulation of Splenocytes In order to enerate CTLs specific for a particular cla
ss I MHC-restricted peptide, we descri
e here a method for re-stimulatin select
ively those memory CTLs that reconize a particular peptide derived from the ant
ien encoded
y the DNA immunoen.
140
3.2.1.1. STIMULATOR CELLS
Iwasaki and Bar
er
1. Sacrifice naive mice that express the MHC haplotype of the chimeric host. The

num
er of mice required for stimulator cells is half of the total num
er of res
ponder mice of that particular strain. 2. Collect spleens into PBS in 15 mL tu
e
s on ice. Make sinle cell suspension
y mashin it throuh cell strainers in a
Petri dish filled with 10 mL of media usin the head of a sterile syrine plune
r. 3. Transfer the cells to 15 mL tu
es and wash the splenocytes three times wit
h media. 4. Resuspend the cells in 10 mL media, and seal the lid tiht. Irradiat
e the cells at 2000 rads. 5. Wash the cells once with media. Resuspend cells in
2 mL of media. 6. Prepare a sterile peptide solution
y resuspendin 100  of the
antienic peptide in 1 mL of cold PBS, and filterin throuh a 0.2 micron filte
r. 7. Add the peptide solution to the stimulator cells. 8. Incu
ate at 37C for 1
h. 9. Resuspend the culture in appropriate volume which can
e transferred to th
e flasks containin the responders (1 mL per flask). 10. Transfer 1 mL aliquot o
f stimulator cells to the flasks containin responders.
3.2.1.2. RESPONDER CELLS
1. Sacrifice mice previously immunized with plasmid DNA. Optionally, collect
lo
od
efore sacrifice for detection of anti
ody responses. 2. Harvest spleens into
cold PBS in 15 mL tu
es. Make sinle cell suspension
y mashin it throuh cell
strainers in a Petri dish filled with 10 mL of media, usin the head of a steri
le syrine pluner. 3. Transfer the cells to 15 mL tu
es and wash the splenocyte
s three times with media. 4. Resuspend the cells in 10 mL of media. Transfer the
content into 50 mL tissue culture flasks. 5. Add stimulators. 6. Add enouh -mer
captoethanol to make the final concentration to 50 M in 15 mL total volume. 7. Ad
just the total volume to 15 mL with media. 8. Culture the cells with flasks stan
din upriht, undistur
ed for 67 d at 37C/5% CO2.
3.2.2. 51Cr-Release Assay
3.2.2.1. TARGET CELLS
1. Count the num
er of taret cells. Use tumor cell lines which express the MHC
class I allele to which the antienic peptide
inds. 2. Transfer the num
er of c
ells required to cover the num
er of wells in a 96-well plate. 104 taret cells
per well are incu
ated in triplicate (three columns) with twofold serial dilutio
ns of effector cells (from 100:1 to 0.78:1, eiht rows). 3. Centrifue the cells
at 210 for 5 min. Discard supernatant.
Epitope-Specific Cytotoxic T Lymphocytes
141
4. Resuspend the cells in 70 L of 25% FCS media. To one of the tu
es, add 10 L of
0.1 /L antienic peptide in PBS to
e used as the peptide pulsed taret. Save the
other tu
e as an unpulsed taret control. 5. Add 100 Ci of [51Cr] Na2CrO4 per tu

e. Incu
ate at 37C for 1 h. 6. Add 10 mL of media and incu
ate for 30 min more.
7. Wash the cells with media thorouhly (at least three times). Monitor the supe
rnatant for radioactivity
y a Geier counter to make sure that no more radioact
ivity is detected after the last wash. 8. Resuspend the cells at 105 cell/mL. Al
iquot 0.1 mL per well on top of effector cells.
3.2.2.2. EFFECTOR CELLS
1. Resuspend the culture that has
een incu
ated for 7 d. 2. Count the num
er of
cells. 3. Transfer the required num
er of cells (2 106 cells/well num
er of wel
ls) to 15 mL tu
es. 4. Centrifue the cells at 210 for 5 min. Discard supernata
nt. 5. Resuspend cells at 107 cell/mL in media. Aliquot 0.2 mL per well into the
top row only. 6. Fill the rest of the wells with 0.1 mL media. Usin a multicha
nnel pipettor, make serial dilutions
y transferrin 0.1 mL suspension from the
top row to the second row to the third row and so on until the last row. Since t
he top row contains 106 cells/well, serial dilution of that would ive effector
to taret ratios of : 100:1, 50:1, 25:1, 12.5:1, 6.25:1, 3.13:1, 1.56:1, and 0.7
8:1 in the 8 rows of 96 well plates. 7. To o
tain maximum possi
le 51Cr release
from each taret cell sampl, add to a num
er of wells 100 L of 2% Triton-X100. Al

so, to o
tain spontaneous 51Cr release values from the various taret cells, add
to a num
er of wells 100 L of media alone. 8. Add taret cells to the appropriat
e wells, includin those for maximum and spontaneous release. 9. Centrifue the
plates at 400 rpm for 3 min to settle the cells down to the
ottom of the wells.
10. Incu
ate the plates with lids on at 37C for 4 h. 11. Usin Skatron cell harv
ester, collect supernatant from each wells and count the supernatant on a amma
counter. 12. Maximum and spontaneous release can
e determined from wells that c
ontained either 2% Triton-X100 or medium alone, respectively. Specific lysis is
calculated as (experimental 51Cr releasespontaneous 51Cr release)/(maximum 51Cr r
eleasespontaneous 51Cr release) 100 %.
4. Notes
1. It is a
solutely crucial that the depletion of T cells o
tained in vivo to
e
close to 100% in order to prevent GvHD
y the residual donor T cells. The anti

ody
142
Iwasaki and Bar
er
concentration required to deplete T cells to completion can
e o
tained
y conce
ntratin the supernatant from CELLMAX
y Centriprep filter system. Typically, th
e supernatant from the hy
ridomas must
e concentrated to 150200-fold the volume
of the total culture media used to row the hy
ridomas in order for a 0.5 mL vol
ume injected on d 0 and 3 to completely deplete
oth su
sets of T cells. 2. The
survival of the irradiated mice depends on the cleanliness of the environment in
which they are maintained. Lethally irradiated mice are very prone to infection

y common
acteria such as Pseudomonas. Due to the irradiation-induced damae t
o the intestinal epithelia, it is recommended that mice not
e fed for the first
24 h after irradiation. Aside from the anti
iotic treatment indicated in Su
hea
din 3.1.4., scrupulous care must
e taken to handle mice with antiseptic techni
ques. For example, anythin that comes in contact with the mice should either
e
autoclaved or wiped with Clidox (chlorine dioxide) or other antiseptic aents.
Surical masks should
e worn
y all those who work with the mice, as well as cl
ean set of loves for each cae. All procedures should
e carried out strictly u
nder laminar flow hood. The first two weeks are the most critical period in dete
rminin whether mice will survive without infection. Later on, mice may die of G
vHD, in cases where the T cells from the donor mice were not properly depleted.
3. Normally, complete reconstitution of
one marrow injected mice requires
etwe
en 68 wk. Since the nature of the experiment requires that no recipientderived he
mopoietic cells
e present in these mice, and that reconstitution
y the donor

one marrow
e complete, it is recommended that the chimerism of these mice
e as
sessed after 6 wk. To determine the extent of reconstitution, peripheral
lood c
ells can
e stained for the MHC class I or class II molecules. For example, if t
he
one marrowchimeric mice are parent into F1, the peripheral
lood cells shoul
d only stain positively for the parental donor haplotype
ut not for the other a
llele expressed
y the F1 cells. Fi. 2 depicts the flow cytometric profile of p
eripheral
lood cells from reconstituted mice stained with anti-MHC class II ant
i
odies. The results clearly demonstrate complete reconstitution in H-2
H-2
xd an
d H-2dH-2
xd chimeric mice. 4. Althouh a num
er of CTL in vitro re-stimulation p
rotocols are availa
le, some of which are illustrated in this volume, we descri

e a method for selectively restimulatin memory CTLs specific for a sinle class
I MHC-restricted peptide. The presence of 50 M -mercaptoethanol in the stimulatio
n culture has
een found to
e very important for a proper rowth environment fo
r CTLs in our hands. Also, the flasks are stood upriht durin the 7 d incu
atio
n to maximize the interaction
etween the stimulator cells and responder cells.
Althouh other re-stimulation methods involve addition of rowth factors such as
IL-2 or supernatant from Con A stimulated splenocytes, we have not found this t
o
e necessary. Fi. 3 depicts representative lysis curves for CTLs o
tained, us
in the protocol descri
ed here, from H-2
H-2
xd and H-2dH-2
xd
one marrow-chimer

ic mice immunized with the plasmid DNA encodin the nucleoprotein of influenza.
Epitope-Specific Cytotoxic T Lymphocytes
143
Fi. 2. Anti-class II MHC stainin of peripheral
lood cells from the
one marro
w reconstituted mice. In order to confirm complete reconstitution
y donor
one
marrow, 7 wk after the
one marrow injection, peripheral
lood was collected fro
m F1(H-2
H-2d) (top), H-2
(H-2
H-2d) (middle), H-2d(H-2
H-2d) (
ottom), and sta
ined for MHC class II molecules, I-A
and I-Ad. The FACS profiles depicted here
are representative of all the chimeric mice used in this study.
References
1. Donnelly, J. J., Ulmer, J. B., Shiver, J. W., and Liu, M. A. (1997) DNA vacci
nes. Annu. Rev. Immunol. 15, 617648. 2. Wolff, J. A., Malone, R. W., Williams, P.
, Chon, W., Acsadi, G., Jani, A., and Felner, P. A.. (1990) Direct ene transf
er into mouse muscle in vivo. Science 247, 14651468. 3. Pardoll, D. M. and Becker
le, A. M. (1995) Exposin the immunoloy of naked DNA vaccines. Immunity 3, 1651
69. 4. Huan, A. Y. C., Golum
ek, P., Ahmadzadeh, M., Jaffee, E., Pardoll, D., a
nd Levitsky, H. (1994) Role of
one marrow-derived cells in presentin MHC class
I-restricted tumor antiens. Science 264, 961964.
144
Iwasaki and Bar
er
Fi. 3. CTL lysis curves of P815 (H-2d) cells pulsed with Kd-restricted NP147-15
5 peptide. Parent into F1
one marrow-chimeric mice, H-2
H-2
xd, and H-2dH-2
xd, w
ere immunized with influenza NP ene, and assayed for CTL lysis activity aainst
P815 (H-2d) cells pulsed with NP147-155. In this example, it is clear that only
reconstitution with H-2d
one marrow has ena
led the development of the H-2Kd r
estricted, peptide specific CTL response. 5. Iwasaki, A., Torres, C. A. T., Ohas
hi, P. S., Ro
inson, H. L., and Bar
er, B. H. (1997) Both ene un and intramusc
ular injection of plasmid DNA induce cytotoxic T-lymphocytes via
one marrow der
ived antien presentin cells. J. Immunol. 159, 1114. 6. Corr, M., Lee, D. J., Ca
rson, D. A., and Tihe, H. (1996) Gene vaccination with naked plasmid DNA: Mecha
nism of CTL primin. J. Exp. Med. 184, 15551560. 7. Co

old, S. P., Jayasuriya, A
., Nash, A., Prospero, T. D., and Waldmann, H. (1984) Therapy with monoclonal an
ti
odies
y elimination of T cell su
sets in vivo. Nature 312, 548551.
14
Immunostimulatory DNA Sequences
An Overview
John H. Van Uden and Eyal Raz 1. Introduction The
iochemical and enetic proper
ties of DNA have
een thorouhly investiated, yet only recently has it
een app
reciated that DNA carries more information than simply a
lueprint for the reul
ation and construction of proteins. Indeed, the immune systems of verte
rates ap
pear to have evolved the a
ility to distinuish the forein DNA of
acteria and
certain viruses from the self-DNA of the host, a new twist on the self vs non-se
lf detection system already well-known for forein proteins. Specifically, the f
requency of unmethylated CpG motifs (CpG denotes covalently linked CG dinucleoti
des, not C:G
ase pairs) is extensively suppressed in verte
rates, includin mam
mals (
y at least 20-fold [1]), whereas it is found at the usual frequency (1/16
) in most
acterial and viral DNA. There have now
een several reports (detailed
in Su
headins 2. and 4.) that
acterial DNA or synthetic oliodeoxyri
onucleot
ides (ODNs) containin
acterially derived sequences, stimulate the immune syste
ms of mice and humans to first mount innate, and then antienspecific (when fore
in antien is present), Th1-type responses. This adjuvant effect of
acterial i
mmunostimulatory DNA sequences (ISS) appears to
e important for the ro
ust Th1-

type immune response usually seen in enetic vaccination (2). Althouh the terms
CpG motif and ISS are enerally used synonymously in this field, CpG motifs are
defined structurally, whereas ISS are defined functionally (and therefore inclu
de non-CpG sequences that have
een found to
e stimulatory). A more complete un
derstandin of this phenomena should allow for more efficacious medical applicat
ions as well as a
etter view of the
asic mechanisms responsi
le for immune sti
mulation.
From: Methods in Molecular Medicine, vol. 29, DNA Vaccines: Methods and Protocol
s Edited
y: D. B. Lowrie and R. G. Whalen Humana Press Inc., Totowa, NJ
145
146
Van Uden and Raz
2. Early Reports of the Immunostimulatory Properties of Nucleic Acids As early a
s 60 years ao, Freund found that dead myco
acteria had antienindependent immun
ostimulatory a
ilities (3). Althouh several important immunostimulatory su
stan
ces have
een found since then in crude
acterial preparations, includin endoto
xin (pyroenic lipopolysaccharide [LPS]), muramyl dipeptide (MDP), exotoxins (su
ch as diphtheria, tetanus, and cholera toxins), and superantiens (such as staph
yloccocal enterotoxins and toxic shock syndrome toxin 1), it is likely that part
of the activity of Freunds adjuvant is due to the myco
acterial DNA constituent
(4). The first report of the direct immunostimulatory properties of nucleic acid
s came with the demonstration that dsRNA or its analoue, poly-inosinic, polycyt
idilic ri
onucleic acid (pIpC), are a
le to induce the type I interferons (IFN-/)
nd thus provoke n nti-vir l response (5,6). More recently, the IFN-induced, d
sRNA- ctiv ted protein kin se (PKR) h s
een shown to medi te this immune recogn
ition (7,8). This system is presumed to h ve evolved to recognize intr cellul r
dsRNA
ec use it is ssoci ted with the life cycle of v riety of p thogenic vi
ruses. The first reports of immunostimul tion
y rel tively pure fr ction of m
icro
i l DNA c me from Tokun g nd colle gues (914). They found in 1984 th t n
ucleic cid fr ction of Myco
cterium
ovis BCG (MY-1) h s potent nti-tumor ct
ivity in sever l mouse models, th t the DNA is solely responsi
le for the effect
, nd th t the DNA is not directly cytotoxic, so th t the effect is hostmedi ted
(9,10). It w s lso shown th t the ntitumor response is medi ted prim rily
y
n tur l killer (NK) cells ctiv ted in vivo
y the
cteri l DNA (11). In vitro
experiments h ve demonstr ted th t
cteri l DNA nd poly (dG,dC) ODNs ctiv te
m croph ges nd NK cells, nd stimul te the ntivir l response (inhi
ited
y nt
i-IFN-/ nti
odies) (13,15). Histologic lly, it w s shown th t injection of
cter
i l DNA le ds to e rly (d y 4) infiltr tion of m croph ges nd NK cells, nd l t
e (d 14) infiltr tion of del yed-type hypersensitivity (DTH) T cells (14,16). Pi
setsky nd coworkers discovered in 1991 th t DNAs from v riety of
cteri l sp
ecies re lso potent T-cell independent B-cell mitogens for multiple different
mouse str ins, where s DNAs from v rious m mm li n species re not (17). 3. Atte
mpts to Define the Sequences of DNA th t re Immunostimul tory Tokun g et l. f
irst demonstr ted th t p rticul r synthetic ODNs t ken from sequences in the myc
o
cteri l genome re
le to ccount for the immune ctiv tion seen with
cter
i l DNA. It w s found th t 6 of 13 synthetic 45-mer ODNs representing r ndomly s
elected sequences of the myco
cteri l genome
Immunostimul tory DNA Sequences
147
c used nti-tumor ctivity, NK
is of shorter overlappin ODNs
erwise inactive ODNs suested
contain CpG dinucleotides, are

ctiv tion, nd production of IFN-/ nd IFN-. Analys

as well as insertion of active sequences into oth
that certain palindromic hexamers, each of which
responsi
le for the immune activation
y the
act

erial DNA (1822). Since this oriinal preliminary characterization of the DNA seq
uences required for immunostimulation, more methodical studies to determine the
optimal and required sequences for activation have
een carried out. Krie and c
o-workers, usin mouse B cell stimulation as their primary model to screen over
150 sequences, have found that sinle-stranded ODNs containin the consensus seq
uence of a unmethylated hexamer of 5purine-purine-CG-pyrimidine-pyrimidine3 in
duce optimal B cell stimulation. Maximal effect was seen when the 5 purines wer
e GA and the 3 pyrimidines were TC or TT, and TGACGTT was found to
e the optim
al sequence for B cell activation (23). This roup found that the hexamer palind
romic structure is not an a
solute requirement, althouh the reported active pal
indromes were still found to have hih activity (23). In other studies, this con
sensus model has
een shown to hold also for stimulation of production of IL-6,
IL-12, and IFN- in mouse spleen cells (24) and for induction of
oth mouse and hu
man NK cell activity (25). The most universal requirement is for the inclusion o
f at least one CpG dinucleotide,
ut near-terminal CpGs are much less effective
(21,23). However, even this rule has exceptions, as some sequences without the C
pG dinucleotide retain su
-optimal ISS properties (26). Additionally, several r
oups have reported that methylation at the C-5 position of the cytosine in the C
pG motif (meCpG) almost completely a
olishes the immunoloic activity of ISS
ot
h in vitro and in vivo (23,25,2729). This phenomenon is particularly interestin

ecause verte
rate enomic DNA is hihly methylated at the cytosines in CpG moti
fs (approx 70%), whereas non-animal oranisms (includin pathoens) do not methy
late their CpG sequences to any lare deree (1,30), allowin the immune system
another way to reconize forein DNA. In fact, the dominant theory for the mecha
nism of CpG suppression in verte
rates is that, over evolutionary periods, meCpG
herita
ly mutates, primarily to TpG and its complement CpA (30). Methylation of
CpG motifs in eukaryotic promoter elements has
een shown to nearly completely
shut down transcriptional activity of the ene, and is a well-characterized meth
od of lonterm transcriptional reulation in development, X-chromosome inactivat
ion, and suppression of
oth invadin and endoenous viral and transposon enome
s (31). Most runs of multiple unmethylated CpG motifs (CpG islands) in the verte

rate enome are strictly associated with the promoters of actively transcri
ed
enes (1), so it appears that althouh there are potentially immunoloically act
ive CpG sequences in host enomes, evolution has only allowed
148
Van Uden and Raz
the
are minimum to remain. Active ISS may
e even further under-represented tha
n non-stimulatory CpG motifs; it has
een reported that four of the most stimula
tory CpG motifs are less than one-third as common as four of the least stimulato
ry CpG motifs amon human sequences in GenBank, while these sequences occur with
similar frequencies in E. coli (23,32). It is convenient to think of ISS as mod
ular 6-mer units that are necessary and sufficient for immunostimulation,
ut th
ere is evidence that distal flankin sequences are also important. It has
een f
ound,
y successive deletions of the 3 end of ISS-containin ODNs (ISS-ODNs) wi
th fixed 6-mer 5 flankin reions, that a minimum lenth of 18
ases is require
d, while maximal activity is seen with ODNs of 22
ases or loner (33). However,
it has also
een shown that a 15-mer ODN can efficiently stimulate NK activity
(25). Interestinly, cationic liposomal transfection of ODNs consistin solely o
f the 6-mer ISS motif is stimulatory, implyin that this is the minimal active u
nit inside the taret cell (22). As a further complexity, nuclease-resistant pho
sphorothioatemodified ODNs (ps-ODNs; see
elow) have
een demonstrated to stimul
ate without transfection when they are as short as eiht
ases in lenth (23). O
ne of several consistent hypotheses is that, while the 6-mer ISS is the minimal
required element, the flankin sequences may allow enouh time for the ODNs to e
nter the cell and trier the immunostimulatory sinalin cascade
efore exonucl
eases destroy the active ISS. The nucleotide composition of flankin sequences h
as also
een shown to affect the relative activity of ISS. Phosphodiester poly-G

repeats surroundin the 6-mer ISS have
een shown to
e a
le to sinificantly e
nhance the activity of ODNs on mouse splenocytes (21), an effect which appears t
o
e dependent on scavener receptor
indin and internalization of the ODNs (34
). Contrastinly, it has also
een reported that ps-ODNs are actually made less
active if they are flanked
y poly-G (25), and that ps-poly-G without any ISS (a
phosphorothioate homopolymer of G) actually inhi
its interferon- (IFN-) productio
n in response to
acterial DNA, Con A, or PMA/ionophore (35). Phosphorothioate-m
odified ODNs (ps-ODNs) have
een shown to
e approximately 200-fold more potent
than the natural phosphodiester ODNs (po-ODNs) without transfection, while maint
ainin nearly identical immunomodulatory properties (23). Ps-ODNs differ from th
eir natural counterparts in that they have a sulfur su
stituted for one of the t
wo non-
ridin oxyens of the phosphate that forms the link
etween nucleotides
. Thus, ps-ODNs are nuclease resistant and have much loner half-lives
oth in v
itro and in vivo (3639). These ps-ODNs have
een characterized extensively for us
e in the field of antisense, and are availa
le commercially. For the a
ove reaso
ns, ps-ODNs are ainin in popularity and are currently the ODN of choice in mos
t of the la
s workin in this field.
Immunostimulatory DNA Sequences
149
Considera
ly less has
een pu
lished a
out the optimization of ISS for humans. A
lthouh it is clear that many sequences that stimulate mouse cells also ive oo
d responses in human cells, it is equally clear that there is some deree of spe
cies-specificity (2,12,25,26,40). Furthermore, all of the sequence comparisons h
ave used in vitro data, usually usin peripheral
lood mononuclear cells (PBMCs)
instead of splenocytes (where the mouse work is enerally done), so it is diffi
cult to extrapolate to in vivo activities. We have found considera
ly more varia

ility in the responses of human donor cells to ISS than in cells from various m
ouse strains (E. Raz, unpu
lished data). One report has compared the efficacy of
a variety of different ODNs in stimulatin human peripheral
lood B cells, and
has found that, while the previously descri
ed ISS are potent, so are some CpG i
mmunostimulatory motifs that do not fit the 5pur-pur-CG-pyr-pyr3 model. Also,
they show that some sequences that do not contain any CpGs at all yield low leve
ls of stimulation (26). Interestinly,
y far the most potent ODN in this study
was derived from an antisense ODN to the rev ene of HIV-1. The unexpected and s
tron ISS effects of this ODN have
een independently characterized
y the orii
nal antisense roup (28,41,42). This and other examples of the unforeseen effect
s of ISS on experiments desined to address unrelated questions hihliht the ne
ed, not only for a
etter understandin of ISS,
ut also for a
roader dissemina
tion of this knowlede. Recently another paradim for the immune activity of DNA
has
een discovered. It has
een reported that some sequences containin repeat
in or alternatin Cs and Gs are a
le to specifically inhi
it the effects of ISS

oth in vitro and in vivo (named CpG-N
y the authors) (42a). Additionally, we
have found a different class of motifs that also potently inhi
it ISS at all lev
els,
ut these sequences have Th2-promotin effects in vivo and in vitro as well
(Nuyen, Malek, Van Uden, Tihe, and Raz, su
mitted for pu
lication). Therefore
, there seems to
e an important
alance
etween the ratios of different types o
f immunoloically active sequences in lare DNA (enomic or plasmid), and a
ett
er understandin of this will likely allow for the more rational desin of DNA v
accines and ene therapy vectors. 4. Effects of Immunostimulatory DNA Sequences
4.1. Th1-Biased Responses to Antiens Bacterial DNA (as well as plasmids and ODN
s derived from it) stimulates the innate arm of the immune system to activate se
veral non-specific antimicro
ial defense systems and sensitizes the specific arm
to mount
oth anti
ody and cell mediated antien-restricted responses aainst a
ny associated forein antien, as has
een demonstrated in enetic vaccination.
Althouh not complete, there is a clear
ias of the immune system toward antien
-specific Th1-type responses (characterized
y CD8+ CTL; CD4+ Th1 cells; IG2a>I
G1;

150
Van Uden and Raz
IFN->IL-4, IL-5, IL-10) and away from the opposin Th2-type responses (2,4,4351).
One prominent exception to this rule is the eneralized Th0/Th2type
ias seen wi
th intradermal inoculation usin particle-mediated transfer technoloies (the e
ne un) (52,53). This exception is likely due to the reatly reduced quantity (a
pproximately 1%) of adjuvant-like DNA delivered
y the ene un relative to inje
ction techniques (4,54). In fact, the Th1-inducin immunomodulatory properties o
f ISS may account for some of the effects seen in many enetic vaccination, anti
sense, and ene therapy studies (unpu
lished meta-analysis) (2,29,41). We have s
hown that ISS in the plasmid
ack
one of vectors used for enetic vaccination fu
nction as potent Th1-promotin adjuvants, and are required to allow the minute q
uantities of antien that are expressed to
e hihly immunoenic (2). Additional
ly, we and others have shown that ISS-containin plasmids or ODNs also function
as Th1-inducin adjuvants when coadministered with protein antiens, openin up
new possi
ilities of
oostin or modifyin current protein or su
unit vaccines (
54a54c).
4.2. Splenomealy The most strikin anatomical chane induced
y injection of
a
cterial DNA or immunostimulatory ODNs into mice is the massive transient splenom
ealy uniformly o
served within 2 wk. Althouh it has
een shown that
acterial
DNA is a powerful B cell mitoen (see Su
headin 4.6.), this pro
a
ly does not a
ccount for the several fold increases in spleen size o
served (41). Indeed, it h
as
een reported that the majority of the increased mass of the spleen can
e at
tri
uted to extramedullary hematopoesis in the spleen and not to alterations of
the quantity or quality of mature immune cells (54d) (and our unpu
lished o
serv
ations). This splenomealy has not
een documented in humans or nonhuman primate
s treated with hih doses of antisense ODNs (55). 4.3. NK Cell Activation As det
ailed in Su
headin 2., early studies found that
acterial DNA possesses potent
anti-tumor effects, which are larely attri
uta
le to activation of macrophaes
and NK cells (911,15,16). Purified NK cells were shown to
e refractory to stimul
ation with ISS, implyin that they are not directly activated. Indeed, anti
ody

lockin studies have revealed that NK cell activation is decreased
y
lockin
IL-12, TNF-, or IFN-/
, nd th t
locking ll three cytokines completely
rog tes
NK cytolytic ctivity (20,25,56). Simil rly, it w s shown th t cytokines from
dherent splenocytes stimul ted with
cteri l DNA re required for secretion of
IFN- from non-adherent splenocytes (presumed to
e NK cells) (56). Furthermore, B
and T cells are not required for activation of NK cells,
ecause the NK induci

ility of splenocytes from SCID mice is normal (25). In addition to
ecomin cyto
toxic, NK cells activated
y
acterially-derived DNA secrete lare amounts of IF
N- (24,57). In the early
Immunostimulatory DNA Sequences
151
response (within days), NK cells have
een shown to comprise over 90% of the IFN
- secretin cells (57), whereas later on, CD4+ T cells contri
ute sinificantly (
24). In addition to mouse cells, NK cells from human peripheral
lood mononuclea
r cells (PBMCs) are also activated
y ISS-ODNs (25).
4.4. Macrophae and Dendritic Cell Activation
Activated cytotoxic macrophaes are also seen early in the response to ISS. Alth
ouh macrophae activation in the response has
een shown to
e larely a
roate
d
y
lockin anti
odies aainst IFN- (13,15,20), other studies have documented d
irect activation of macrophaes and macrophae-like cell lines (2,27,56,58). It
seems likely that macrophaes are activated
y ISS directly,
ut that IFN- potent

ly auments their response. In addition to
ecomin cytotoxic, ISS-activated mac

rophaes also produce TNF-, IL-12, IL-18 ( lso known s IFN- inducin factor, IGIF
), IFN-, nd IFN- (2,4,27,56,58). In vitro, IL-12 nd IL-18 re potent stimul tors
of IFN-. Followin ISS stimulation of splenocytes, IL-12 reaches its maximum exp
ression level at 24 h, whereas IFN- peaks at a
out 48 h, suestin that primary
IL-12 production stimulates later IFN- production (56). The response is not limit
ed to mouse cells; primary human monocytes have also
een shown to
e activated

y ISS (2). Interestinly, this pattern of cytokine secretion sets up a local in
flammatory positive-feed
ack loop of macrophae and NK cell activation in respon
se to ISS. Macrophaes (and possi
ly other APCs or stromal cells) are activated

y
acterial DNA to secrete IFN-/, TNF-, nd IL-12, which in turn ctiv te NK cells
to secrete IFN-, which stimulates the macrophaes further, etc. A host of surfac
e molecules are also induced on macrophaes upon their activation
y ISS. For ex
ample,
one marrow derived macrophaes treated with ISS-ODN upreulate their exp
ression of class I MHC, CD40, ICAM-1, and CD16/32, all of which are important in
promotin antien processin and presentation (58a). Primary dendritic cells ar
e also activated
y ISS to produce IL-12, IL-6, TNF- nd incre se their expressio
n of cl ss II MHC nd B7-2 (58
,58c). ISS ctiv ted dendritic cells lso
ecome m
ore potent APCs, s seen in their
ility to ctiv te T cells in vitro in lloge
neic mixed lymphocyte re ctions nd with super ntigen stimul tion (58
). Thus, t
he profession l APCs of the immune system, m croph ges nd dendritic cells, re
directly nd potently ctiv ted through multiple mech nisms
y ISS.
4.5. Indirect T Cell Activ tion nd the Antigen-Specific Response
T cells lso contri
ute to this cytokine positive feed
ck loop. IFN-/, IL-12, IL18, TNF-, nd IFN- from macrophaes and NK cells have
een shown to
ias naive T h
elper cells toward the Th1 phenotype that is associated with cell152
Van Uden and Raz
mediated immunity (5964). When activated
y these cytokines and
y appropriate an
tien presented on MHC class II, Th1 cells secrete relatively lare amounts of I
FN-, which activates macrophaes in the locality of the Th1-reconized forein an
tien (delayed type hypersensitivity) (65). Bacterial DNA and ISS-ODNs do not ap
pear to directly activate T cells to undero proliferation or cytokine productio
n (17,66), as they do for B cells (see Su
headin 4.6.). Also, it has
een shown
that T cells are not required for the primary antien-independent activation of
NK cells, macrophaes, and B cells in response to ISS (17,2527,56,58,66). Nevert
heless,
oth CD4+ and CD8+ T cells have
een shown to
e instrumental, usin dep
letion and adoptive transfer studies, in the development and maintenance of the
characteristic Th1-type specific immunity induced
y enetic vaccination (51,67)
. Histoloical examination of reressin tumors injected with
acterial DNA reve
als that activated Th1 cells are seen in the lesion at late times (d 14), and th
at they are important for tumor rejection (68). Due to the fact that the T cell
stimulation is indirect and is therefore easier to detect in vivo than in vitro,
there are no pu
lished reports of T cell activation
y ISS in humans yet,
ut t
here are reports of Th1 responses to enetic vaccination in humans (68a,68
), wh
ich may
e mediated
y ISS in the vector
ack
one. Additionally, studies have sh
own Th1-like responses in non-human primates with enetic vaccination (6971).
4.6. B Cell Activation
In contrast to their indirect effects on T cells, immunostimulatory sequences ha
ve direct mitoenic and activatin effects on B cells. This was first descri
ed
in 1991 when lymphocytes depleted of T cells were shown to undero proliferation
in response to
acterial DNA (17). Su
sequently, B cell activation has
ecome t
he most thorouhly investiated effect of immunostimulatory sequences. B cells (

oth purified and in crude splenocyte cultures) have
een demonstrated to under
o polyclonal proliferation in response to
acterial DNA and ISS-ODNs, and this e

ffect is dependent upon unmethylated CpG motifs (ISS) (23,26,41,42,66,72). In fa

ct, over 95% of B cells in vitro enter the cell cycle under optimal conditions (
23). ISS also cause B cells to secrete hih titers of polyclonal IM anti
odies
independent of T cell help (23,26,41,73,74). Additionally, ISS-DNAs are a
le to
rescue the B cell line WEHI-231 from surface IM crosslinkin-induced rowth arr
est and apoptosis (75). These results imply that ISS can send an antien-indepen
dent costimulatory sinal, allowin rapid low-affinity immune responses,
ut als
o creatin the possi
ility of autoimmunity (see Su
headin 5.3.). Some of the ef
fects of activated B cells are mediated throuh the cytokines they release, and
ISS-stimulated B cells have
een shown to produce
oth IL-6
Immunostimulatory DNA Sequences
153
and IL-12 (24,73,74). The production of these cytokines coupled with the efficie
nt antien-presentation a
ilities of activated B cells suests that they could

e key players in initiatin the hallmark responses of the immunostimulatory eff
ects of DNA, includin anti
ody production and Th1 activation. Blockin IL-6 act
ion
y usin IL-6 knockout mice (Martin-Orozco and E. Raz, unpu
lished data), or
usin anti-IL-6 anti
ody, does not decrease the proliferative response. However
, anti-IL-6 does reduce IM secretion as much as 90%, indicatin that this may

e an autocrine stimulation (24,74). It is not clear whether the macrophae-produ
ced or B cell-produced IL-12 is quantitatively more important in induction of th
e Th1-type response (2,24,56), and it may depend in vivo upon the site of contac
t with
acterial DNA. Interestinly, low-level B cell receptor crosslinkin (sim
ulatin specific antien reconition
y surface IM) com
ined with ISS administr
ation syneristically potentiates proliferation, IL-6 production, and IM produc
tion
y B cells (23,74). This implies that while
acterial DNA is an antien-ind
ependent B cell activator, it also enhances antien-specific B cell responses. E
xoenous IFN- down-reulates IM in response to LPS,
ut with ISSODNs, IFN- actual
ly increases the amount of IL-6 and IM produced (73). The surface molecule prof
ile of B cells is also potently stimulated
y ISS. Class I MHC, class II MHC, B7
-1, B7-2, CD40, CD16/32, ICAM-1, IFN-R, and IL-2R are upreulated to varyin der
ees
oth in vivo and in vitro, and CD23 (FcR) is downr
gulat
d. This r
markabl
a
lt
ration in surfac
ph
notyp
is larg
ly cytokin
ind
p
nd
nt and would b

xp

ct
d to
nhanc
th
ability of B c
lls to act as activating APCs (58a). Human B
c
lls also r
spond to ISS-ODNs, and 95% of optimally stimulat
d B c
lls incr
as

th
surfac

xpr
ssion of th
activation mark
rs B7-2 and th
IL-2 r
c
ptor (26
). 5. Pot
ntial Sid
Eff
cts of Immunostimulatory DNA S
qu
nc
s As d
scrib
d abo
v
, immunostimulatory DNA s
qu
nc
s ar

xtr
m
ly pot
nt antig
n-ind
p
nd
nt act
ivators of a vari
ty of c
ll typ
s, launching both antibody-m
diat
d and c
ll-m

diat
d r
spons
s. Th
immun
syst
m hangs in a d
licat
balanc
, how
v
r, and
x
tr
m
activation or polarization toward
ith
r Th1 or Th2 r
spons
s could caus

unwant
d
ff
cts. Most of th
work in g
n
vaccination and g
n
th
rapy in g
n
r
al is aim
d at
v
ntually producing th
rap
utics for human us
, th
r
for
th
po
ssibl
adv
rs

ff
cts of ISS must b

xamin
d rigorously.
5.1. Provocation of a S
ptic
Anoth
r T-ind
p
nd
nt B c
ll
rly innat
r
spons
s against

ful in th
small quantiti
s

Shock-Lik
Syndrom

mitog
n, LPS, induc
s th
immun
syst
m to mount
a
gram-n
gativ
bact
ria, and is thus lik
ly to b
us
g
n
rat
d by controll
d local inf
ctions.

154
Van Ud
n and Raz
How
v
r, larg
amounts of LPS induc
a syst
mic inflammatory r
spons
that can l

ad to l
thal s
ptic shock. Th
inflammatory cytokin
s IL-1 and
sp
cially TNF- h
ve
een shown to
e the prim ry medi tors of LPS-induced septic shock, nd lth

ough not c us tive, n elev ted level of IL-6 is the most reli
le prognostic f
ctor for shock (76). As outlined e rlier, IL-6 nd TNF- h ve
een shown to
e pro
duced in l rge mounts
y
cteri l DNA. Two pu
lished reports h ve ddressed th
e issue of whether ISS c n promote systemic infl mm tory responses. In the first
study,
cteri l DNA or ISS-ODNs were injected i.v., followed
y su
leth l dose
s of LPS. They found th t TNF- nd IL-6 were m ssively incre sed rel tive to c lf
thymus (m mm li n DNA) pretre ted cells, nd th t the r te of leth l septic sho
ck w s incre sed from 0% to 75%. However, IFN- receptor knockout mice were almost
completely protected from this potentiation of the LPS response, suestin tha
t IFN- production from NK cells is important in this effect (57). The second stud
y to address this issue found that mice that were sensitized to TNF-
y pretre tm
ent with D-g l ctos mine underwent leth l shock when ch llenged with high doses
of
cteri l DNA or ISS-ODNs (without ddition l LPS). This effect w s
locked

y nti-TNF- nti
ody nd
y using TNF- receptor knockout mice,
ut not
y SCID
ei
ge mice (T, B, nd NK cell deficient). This implies th t in this model, the TNF-
produced
y m croph ges is more import nt th n the IFN- produced
y NK and T cell
s (58). Interestinly, these researchers also showed that certain modified ISS-O
DNs can retain the a
ility to stimulate IL-12 release
y macrophaes while losin
 their TNF- stimul ting
ility. These motifs re
le to serve s djuv nts for
Th1 response
ut h ve lost their
ility to induce toxic shock (76 ). Althoug
h these studies show th t ISS c n contri
ute to systemic infl mm tory re ction
when the system is otherwise primed, there h ve not
een ny reports of ny re
ction with
cteri l DNA or ISS-ODNs lone, reg rdless of dose in either mouse o
r hum n.
5.2. Loc l Infl mm tion
In ddition to systemic infl mm tion, excessive loc l infl mm tion c n lso c us
e serious pro
lems, s seen with
cteri lly derived djuv nts, such s complete
Freunds djuv nt (CFA), nd this is conceiv
le with purified
cteri l DNA. The
re h ve
een liter lly hundreds of in vivo genetic v ccin tion nd ntisense stu
dies with ctive ISS-cont ining pl smids nd ODNs, without ny grossly o
serv
l
e loc l infl mm tory responses reported. Addition lly, we h ve found th t intr d
erm l genetic v ccin tion does not induce loc l infl mm tory infiltr tes, despit
e the f ct th t it does provoke strong immunity (44), nd th t non-coding immu
nostimul tory pl smid, pUC-19, does not c use histologic l infl mm tion if deliv
ered lone intr derm lly (77). In conImmunostimul tory DNA Sequences
155
tr st, it h s
een shown th t while intr muscul r genetic v ccin tion with we
k immunogen (lucifer se) llows expression for gre ter th n 60 d without histolo
gic l infl mm tion, vector encoding strong immunogen (hep titis B surf ce n
tigen or -g l ctosid se) results in ne rly complete immune-dependent destruction
of the tr nsfected myofi
ers
etween 520 d with infl mm tory infiltr tes (78). It
is import nt to note th t this study shows th t ISS in the vector pl smids do n
ot c use destructive infl mm tion
y themselves; the response ppe rs to
e n
ntigen-specific cell-medi ted tt ck on the expressed ntigens. However, it h s

een demonstr ted th t delivering
cteri l DNA or ISS-ODNs without exogenous n
tigen to the lungs of mice c uses moder te incre ses in num
ers of neutrophils
nd other cells, s well s the infl mm tory cytokines TNF-, IL-6, nd MIP-2 in th
e l v ge fluid. Addition lly, it w s shown th t DNA from the lung fluid of cysti
c fi
rosis (CF) p tients, who suffer from recurrent
cteri l lung infections n
d chronic infl mm tion, is
le to reproduce the effects of ISS-cont ining DNA i
n mouse lungs (79). This indirectly implies th t DNA from lysed
cteri in the
lungs of chronic lly infected CF p tients m y contri
ute to the p thogenicity of
this dise se. It m y
e th t one current ther py used in CF, DN se instill tion
into the lungs of p tients, destroys the immunostimul tory effects of the
cte
ri l DNA present, in ddition to decre sing the viscosity of lung fluid to llow


etter cle r nce. However, this nd previous studies h ve not directly found

cteri lly-derived DNA to c use ny clinic lly signific nt d m ge to the lung or
ny symptoms of respir tory distress (79,80).
5.3. Autoimmune Dise se Induction Another potenti l dverse effect of the djuv
nticity of ISS is the possi
ility of precipit ting n utoimmune dise se in gene
tic lly or environment lly prone people. There is some evidence th t the immunos
timul tory properties of
cteri l DNA m y
e
le to pl y role in the develop
ment of utoimmune dise ses such s systemic lupus erthythem tosus (SLE). One pr
ominent fe ture of SLE is the presence of nti-DNA nti
odies th t re widely cr
oss-re ctive (sequence non-specific). Gilkeson nd coworkers h ve shown th t whe
n
cteri l DNA (
ut not m mm li n c lf thymus DNA) is given to mice complexed w
ith methyl ted BSA in CFA, IgM nd IgG nti-DNA nti
odies re produced t sust
ined levels,
ut re not cross-re ctive with m mm li n DNA (81,82). Addition lly
, this methyl- l
umin-
cteri l DNA complex in CFA provokes proteinuri nd prol
ifer tive glomerulonephritis in norm l BALB/c mice (83). However, when this mixt
ure is given to pre utoimmune NZB/NZW F1 mice ( model for genetic suscepti
ilit
y to SLE), nti-DNA nti
odies th t c n crossre ct with m mm li n DNA re produc
ed (84),
ut they do not incre se the severity of spont neous nephritis in these
nim ls. Unexpectedly, tre tment
156
V n Uden nd R z
with the complexed
cteri l DNA ctu lly gives signific nt protection from uto
immune nephritis nd prolongs surviv l rel tive to untre ted mice nd mice tre t
ed with complexed c lf thymus DNA (85). It is import nt to note, however, th t t
he CFA used s n djuv nt in e ch group lso includes myco
cteri l DNA, compli
c ting the interpret tion of these results. Another correl tion
etween SLE nd
ISS ctiv tion is th t hum n SLE is ssoci ted with high levels of IL-6, nd dis
e se initi tion nd progression in mouse models is dependent upon IL-6 (32), one
of the cytokines produced
y ISS ctiv tion. Also,
ec use
cteri lly derived
DNA stimul tes B cell polyclon l prolifer tion nd ctiv tion, s well s resist
nce to poptosis of cells th t recognize ntigen in the
sence of proper costi
mul tion, it could theoretic lly le d to exp nsion nd ctiv tion of utore ctiv
e B cells. ISS re p rticul rly efficient t stimul ting cell-medi ted immune re
sponses, nd precipit tion of one of the Th1-medi ted utoimmune dise ses is the
refore potenti l pro
lem. This w s ddressed recently using mouse model of m
ultiple sclerosis, experiment l llergic enceph lomyelitis (EAE). It w s found t
h t T cells th t h ve
een primed in vivo with myelin
sic protein (MBP) in CFA
c n
e ctiv ted in vitro to le ve quiescence nd
ecome c p
le of tr nsferrin
g EAE to recipient mice
y incu
tion with MBP nd
cteri l DNA, ISS-ODN, or LP
S (86). This Th1 cell ctiv tion w s found to
e entirely dependent upon IL-12 (
86). Although rel tively rtifici l system, this study supports the hypothesis
th t cert in types of infection m y contri
ute to the development of utoimmuni
ty nd shows th t the potent immunostimul tory properties of ISS-l den DNA re p
otenti lly d ngerous under conditions of extreme utoimmune sensitivity.
5.4. Reduction in Antigen Expression An ddition l concern is th t the interfero
n-medi ted ntivir l response gener ted
y ISS would
e expected to decre se nt
igen expression levels. Thus, it m y
e th t too much ISS djuv nt s either ODN
or in pl smid DNA could inhi
it the immune response. Indeed, neutr liz tion of
type I IFNs incre se expression levels from DNA v ccines in the interferon-sensi
tive cell line MG63 (R z, unpu
lished d t ) nd it h s
een reported th t coinje
ction of ISSODN with DNA v ccines in vivo c n reduce tr nsgene expression nd th
e ensuing immune response in dose-dependent m nner (86 ). Another expl n tion
proposed
y these uthors for the results is th t the ODNs re competing with th
e pl smid for upt ke nd therefore tr nsfection efficiency is reduced. This effe
ct could
e p rticul r for the phosphorothio te-modified ODNs or for the model

ntigen (hep titis B surf ce ntigen)
ec use it h s
een previously shown th t c
oinjection of the ISS-cont ining pl smid pUC19 with pl smid encoding the ntig
en is effective for incre sing the immune response to the
Immunostimul tory DNA Sequences
157

ntigen (2). One potenti l w y to circumvent the very serious issue of ntigen e
xpression level while still coupling the ntigen to the DNA-
sed djuv nt is to
cov lently conjug te the protein ntigen to ISS-ODN. We h ve found th t this p
pro ch is much more potent nd effective th n pl smid DNA v ccin tion or mixture
s of protein nd ISS-ODN in mice nd non-hum n prim tes (Tighe nd R z, m nuscri
pt in prep r tion). 6. Molecul r Mech nisms of Immunostimul tion
y DNA The mech
nism of ISS recognition
y cells nd the w y this recognition is tr nsl ted int
o cellul r ctiv tion is still l rgely unknown. Although it is likely th t there
re one or more ISS
inding proteins th t c n distinguish ISS from non-ISS, the
re is still no pu
lished experiment l evidence of this, or of ny other mech nis
m for cells to sense ISS. Nevertheless, sever l groups h ve reported evidence th
t the DNA must
e intern lized for ctivity, s opposed to ctiv ting surf ce
receptor. Phosphodiester (non-modified) ISS-ODNs re much more potent when lipo
som lly tr nsfected into cells (87), nd the minimum ctive size of these ODNs d
ecre ses from pproxim tely 16-mers to 6-mers (cont ining only the ISS without f
l nking sequences) when tr nsfected (22). Also, lthough no d t or methods were
shown, it h s
een reported th t ISS-ODNs cov lently linked to solid support
lose their stimul tory effects in mouse B cells, nd th t ODNs with nd without
ISS
ind the surf ce of B cells equ lly well (23). However, nother group found
th t ISS-ODNs
ound to seph rose
e ds did stimul te hum n B cells (26). Therefo
re, it is conceiv
le th t there re different mech nisms of ctiv tion in hum n
nd mouse B cells. However, further work is required to ddress this possi
ilit
y. The downstre m sign ling events of ISS ctiv tion h ve lso
een ex mined. It
h s
een reported th t ISS ctiv tion of B cells does not incre se tyrosine pho
sphoryl tion, inositol triphosph te (IP3) levels, or c lcium ion (C 2+) levels,
therefore these three common p thw ys of sign ling re unlikely to
e involved (
23). Inhi
itors of ctiv tion vi the PKC, PKA, nd NO p thw ys do not decre se
production of IL-6 in response to ISS,
ut ntioxid nts inhi
it to
seline leve
ls. Addition lly, this study showed th t the levels of re ctive oxygen species (
ROS) were incre sed within 20 min of tre tment (
efore IL-6 mRNA, which w s firs
t detected t 30 min) with ISS-ODN (74), implying th t the gener tion of ROS is
very e rly event nd th t it is required for ctiv tion. When ctiv ted
y ISS
-cont ining pl smid, the mouse m croph ge-like cell line RAW-264 expresses mRNA
of pl sminogen ctiv tor inhi
itor-2 nd, when costimul ted with IFN-, it express
es induci
le nitric oxide synthase. Additionally, these ISS-stimulated cells act
ivate transcription of reporter enes driven
y the HIV-1 lon terminal repeat (
HIV-1 LTR) pro158
Van Uden and Raz
moter. NF-B is one of the transcription factors that binds to and activates the H
IV-1 LTR, and has also been shown to be activated by ISS via gel-shift assays (2
7). Both NF-B and ROS are involved in the signaling pathways of a large number of
immune and inflammatory systems, including many of those observed as effects of
ISS (88,89). Additionally, ROS activate NF-B, so these studies may be providing
insight into the same signaling pathway (90). More recently the mitogen activate
d protein inase (MAPK) and stress inase pathways have been implicated in ISS s
ignaling. ISS-ODN have been shown to activate c-Jun NH2-terminal inase (JNK), J
NKK1, p38, activating transcription factor-2 (ATF-2), c-Jun, MAPK- activated pro
tein inase-2, and activator protein-1 (AP-1) (90a,90b). Blocade of the p38 pat

hway has been shown to bloc the subsequent signaling cascade and also the gener
ation of cytoines, and is therefore thought to be a crucial component (90a,90b)
. These signaling pathways are well-characterized regulators of immunological re
sponses, but more difficult to interpret is the finding that blocade of the aci
dification of endosomes by chloroquine, monensin, or bafilomycin A potently inhi
bits the entire signaling pathway and the subsequent production of cytoine (90a
,90b). It remains to be elucidated exactly how these various relatively nonspeci
fic immune signaling mechanisms combine to yield the characteristic rapid and co
ncerted Th1-promoting immune response that has been observed. The previously men
tioned study (Subheading 4.6.) that showed that ISS can rescue a B cell line fro
m anti-IgM induced apoptosis also found that the expression levels of several ge
nes associated with apoptosis are altered by ISS. Although the mRNA levels and p
rotein levels were not consistently regulated in the same fashion, myn, bcl2, an
d bcl-xL appear to be upregulated by ISS, whereas the IgM-induced downregulation
of c-myc is bloced by ISS (75), indicating that these growth and activation re
gulatory genes may be important in the response to bacterial DNA. There is also
some evidence that the cAMP system may be involved. Forsolin, a cAMP antagonist
, blocs IFN induction in response to ISS-ODN (22). It has been hypothesized tha
t, because the cAMP response element (CRE) consensus sequence, TGACGTCA, is so s
imilar to one of the maximally stimulatory ISS, TGACGTT, the ISS may exert its e
ffects by binding to the CRE binding protein complex (CREB/ATF) or a cellular an
alog. An additional similarity is that cytosine methylation of the CpG motif in
the CRE sequence prevents the binding of CREB/ATF to it, just as methylation of
the CpG motif in ISS abrogates its activity (32,91). 7. Clinical Applications of
the Th1 Bias There are several situations in which inducing a strong Th1 respon
se and reducing a Th2 response in an antigen-specific manner would be therapeuti
c.
Immunostimulatory DNA Sequences
159
For example, we and others have shown that IgE and other correlates of Th2mediat
ed allergy and asthma are downregulated in an antigen-specific manner by ISS-ODN
and by genetic vaccination, potentially leading to a new era in immunotherapy (
2,91a,91b,43,51,67). Additionally, Th1 responses (including CTL) are required to
clear many pathogens, and some invaders even bias the immune response toward in
effective Th2 responses against their dominant antigens (92). These would clearl
y be good targets for genetic vaccines with Th1-inducing ISS as adjuvants becaus
e protein vaccines and current adjuvants (which generally bias toward a Th2 resp
onse) are relatively ineffective against them. Many pathogens infect via the muc
osal route, and ISS has also been demonstrated to be a potent mucosal adjuvant (
9395). In addition to prophylactic genetic vaccination, some chronic infectious d
iseases such as those caused by hepatitis B virus, human immunodeficiency virus,
and herpes simplex virus may be treatable with therapeutic genetic vaccination
despite the state of functional tolerance that they facilitate in their hosts. I
SS-ODN can even be used without antigen as either a prophylactic or a therapeuti
c treatment for intracellular pathogens such as Leishmania major and Listeria mo
nocytogens (96,97) due to the activation of the antigen non-specific immune resp
onse. Similarly, cancer immunotherapy against tumor-associated antigens may be p
ossible, taing advantage of the strong CTL induced by genetic vaccination with
ISS as an adjuvant (98 and Cho and Raz, in preparation). 8. Summary and Perspect
ives Immunostimulatory DNA sequences provoe a rapid concerted activation of the
innate immune system by directly triggering non-specific responses from NK cell
s, macrophage/monocytes, and B cells (polyclonal). This adjuvant effect produces
a local environment that facilitates the anti-viral response (IFN-/, IFN-), cytoto
xic activity (NK cells and macrophaes), polyclonal neutralizin anti
odies (B c
ells), and induction of forein antien-specific Th1like responses (IFN-/, IFN-, IL
-12, and IL-18). The ensuin immunity is characterized
y lon-lastin antien r
estricted Th1 CD4+ cells, cytotoxic CD8+ T cells, and anti
ody (with relatively

more IG2a than IG1 in the mouse) in response to enetic vaccination or coadmin
istration of protein antien. This profile of responses is particularly valua
le

ecause it is difficult to achieve with typical vaccines and seems to
e exactl
y the one required to successfully clear intracellular pathoens (64). Therefore
, it appears that ISS can
e used as discrete DNA-
ased adjuvants. In eneral, w
hen more immunostimulation or Th1 deviation is advantaeous, such as in most en
etic vaccination applications, more numerous or potent ISS should
e used. Howev
er, when an immune inflammatory response or antiviral state would
e counter-pro
ductive, such as in ene replacement therapy,
160
Van Uden and Raz
or when Th2 responses are called for, ISS should
e enineered out of the vector
DNA (2,4). It is not yet clear which ISS are the most effective in humans, alth
ouh it is clear that human cells are at least similar to mouse cells in their r
eactivity to ISS. In addition to
ein a useful tool and important confound, the
reconition of ISS
y the immune system appears to
e a fundamental mechanism o
f
asic immune surveillance that is likely to
e key in the early host response
aainst invadin oranisms. Further characterization of the phenomenoloy and me
chanism of ISS activation of the immune response is therefore not only required
for clinical applications,
ut also likely to yield sinificant new insihts int
o as yet unforeseen areas of
ioloy. Acknowledments The authors are supported
in part
y rant and
y Dynavax Tecnoloies Corp. AI 40682 from the National Ins
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eoxynucleotides. Antisense Nucleic Acid Dru Devel. 8, 351. 87. Yamamoto, T., Ya
mamoto, S., Kataoka, T., and Tokunaa, T. (1994) Lipofection of synthetic oliod
eoxyri
onucleotide havin a palindromic sequence of AACGTT to murine splenocytes
enhances interferon production and natural killer activity. Micro
iol. Immunol.
38, 831836. 88. Baeuerle, P. A. and Henkel, T. (1994) Function and activation of
NF-kappa B in the immune system. Annu. Rev. Immunol. 12, 141179. 89. Lander, H.
M. (1997) An essential role for free radicals and derived species in sinal tran
sduction. Fase
. J. 11, 118124. 90. Sen, C. K. and Packer, L. (1996) Antioxidant
and redox reulation of ene transcription. Fase
. J. 10, 709720. 90a.Yi, A. K. a
nd Krie, A. M. (1998) Rapid induction of mitoen-activated protein kinases
y i
mmune stimulatory CpG DNA. J. Immunol. 161, 4493. 90
.Yi, A. K., Tuetken, R., Re
dford, T., Waldschmidt, M., Kirsch, J., and Krie, A. M. (1998) CpG motifs in
a
cterial DNA activate leukocytes throuh the pHdependent eneration of reactive o
xyen species. J. Immunol. 160, 4755. 91. Krie, A. M. (1996) An innate immune d
efense mechanism
ased on the reconition of CpG motifs in micro
ial DNA. J. La

. Clin. Med. 128, 128133. 91a.Goodman, J. S., Van Uden, J. H., Ko
ayashi, H., Bro
ide, D., and Raz, E. (1998) DNA immunotherapeutics: new potential treatment moda
lities for alleric disease. Int. Arch. Allery Immunol. 116, 177. 91
.Broide, D
., Schwarze, J., Tihe, H., Gifford, T., Nuyen, M. D., Malek, S., et al.
168
Van Uden and Raz
92. 93.
94.

95.
96.
97.
98.
(1998) Immunostimulatory DNA sequences inhi
it IL-5, eosinophilic inflammation,
and airway hyperresponsiveness in mice. J. Immunol. 161, 7054. Marrack, P. and K
appler, J. (1994) Su
version of the immune system
y pathoens. Cell 76, 323332.
Horner, A. A., Ronahy, A., Chen, P. M., Nuyen, M. D., Cho, H. J., Broide, D.,
and Raz, E. (1998) Immunostimulatory DNA is a potent mucosal adjuvant. Cell. Im
munol. 190, 77. Moldoveanu, Z., Love-Homan, L., Huan, W. Q., and Krie, A. M. (
1998) CpG DNA, a novel immune enhancer for systemic and mucosal immunization wit
h influenza virus. Vaccine 16, 1216. McCluskie, M. J. and Davis, H. L. (1998) Cp
G DNA is a potent enhancer of systemic and mucosal immune responses aainst hepa
titis B surface antien with intranasal administration to mice. J. Immunol. 161,
4463. Zimmermann, S., Eeter, O., Hausmann, S., Lipford, G. B., Rocken, M., Wa
ner, H., and Hee, K. (1998) CpG oliodeoxynucleotides trier protective and cu
rative Th1 responses in lethal murine leishmaniasis. J. Immunol. 160, 3627. Krie
, A. M., Love-Homan, L., Yi, A. K., and Harty, J. T. (1998) CpG DNA induces sus
tained IL-12 expression in vivo and resistance to Listeria monocytoenes challen
e. J. Immunol. 161, 2428. Weiner, G. J., Liu, H. M., Wooldride, J. E., Dahle,
C. E., and Krie, A. M. (1997) Immunostimulatory oliodeoxynucleotides containin
 the CpG motif are effective as immune adjuvants in tumor antien immunization.
Proc. Natl. Acad. Sci. USA 94, 10,833.
15
Immunostimulatory CpG Motifs and DNA Vaccines
Risini Weeratna, Arthur M. Krie, and Heather L. Davis 1. Introduction 1.1. Role
of Immunostimulatory Motifs Bacterial DNA,
ut not verte
rate DNA, causes direc
t stimulation of several components of the verte
rate immune system. This activa
tion is due to the presence of unmethylated CpG dinucleotides (1), which are pre
sent at the expected frequency in
acterial DNA,
ut are underrepresented (CpG su
ppression) and methylated in verte
rate DNA (2). The immunostimulatory effects in
clude direct induction of B cell proliferation and immunolo
ulin (I) secretion
(1), as well as activation of monocytes, macrophaes, and dendritic cells to up
reulate their expression of costimulatory molecules, which drive immune respons
es, and secretin a variety of cytokines, includin hih levels of IL-12 (3,4).
These cytokines then, in turn, stimulate natural killer (NK) cells to secrete IF
N- and to have increased lytic activity (5). Overall, CpG DNA induces a Th1 like
pattern of cytokine production dominated
y IL-12 and IFN- , with little secretio
n of Th2 cytokines (4,5). These effects can also
e o
tained with synthetic oli
onucleotides (ODN) (6,7) or plasmid DNA vectors (8) containin CpG immunostimula
tory motifs. From a teleoloical view, it appears likely that the rapid immune a
ctivation in response to CpG DNA may have evolved as one component of the innate
immune defense mechanisms that reconize structural patterns specific to micro

ial molecules. Not all CpG motifs are immunostimulatory, however. Not only is th
e particular sequence context of the unmethylated CpG dinucleotide important,
u
t there are species-specific motifs (9). Many motifs which work well on mouse ce
lls do not stimulate human cells, whereas those which do stimulate human cells w
ill also stimulate, at least to some deree, mouse cells. Also, the
est motif d
epends on the
ack
one used; the
est CpG motif with a phosphorothioate
From: Methods in Molecular Medicine, vol. 29, DNA Vaccines: Methods and Protocol
s Edited
y: D. B. Lowrie and R. G. Whalen Humana Press Inc., Totowa, NJ
169
170

Weeratna, Krie, and Davis

ack
one ODN will not necessarily
e the
est with a phosphodiester
ack
one. Al
thouh the effects of CpG motifs are not antien-specific, B cell activation
y
low concentrations of CpG DNA synerizes stronly with sinals delivered throuh
the B cell antien receptor for
oth B cell proliferation and I secretion (1).
This stron synery
etween the B cell sinalin pathways triered throuh the
B cell antien receptor and
y CpG DNA promotes antien-specific immune respons
es and suests utility of CpG DNA as a vaccine adjuvant.
1.2. CpG Motifs and DNA Vaccines
DNA vaccines composed of plasmid DNA have numerous CpG motifs (approx 1 per 20

ases), which
y virtue of their production in
acterial cells are unmethylated.
At least a proportion of these are immunostimulatory and it appears that such Cp
G sequences act as an autoadjuvant with DNA vaccines, and indeed that they may e
ven
e required for the successful induction of immune responses (5). It should

e possi
le to aument the immune responses of DNA vaccines
y the selective use
of CpG motifs. There are two different approaches that could
e used to do this
: (i) administration of CpG-containin ODN with the DNA vaccine, or (ii) direct
clonin of the CpG sequences into the DNA vaccine vector. 2. Methods and Pro
lem
s 2.1. Mixin CpG ODN with DNA Vaccines The easiest method to use CpG ODN as adj
uvants to DNA vaccines is to use synthetic CpG ODN alon with the plasmid DNA va
ccine. This approach has worked well with protein vaccines usin CpG ODN made wi
th a phosphorothioate
ack
one (see
elow), which is preferred over the naturall
y occurrin phosphodiester
ack
one
ecause it is resistant to nucleases (3). Ho
wever, pro
lems have arisen when this approach has
een tried with DNA vaccines.
We have found that coinjection of ODN with a phosphorothioate
ack
one and plas
mid DNA results in decreased ene expression in a dosedependent manner (as deter
mined with a luciferase reporter ene construct), and that this in turn results
in a dose-dependent decrease in immune responses to a DNA vaccine (as determined
with a hepatitis B surface antien-expressin plasmid). This effect is due to t
he synthetic
ack
one and not the CpG motif since it also occurs with a non-CpG
control phosphorothioate ODN. There are several possi
le reasons for the interfe
rence effect of the ODN on the plasmid. For example, it could
lock the entry of
the DNA into muscle cells, as has
een found in vitro (10). It miht also
lock
transcription of the transfected plasmid DNA or translation of the mRNA. When t
he ODN contains a CpG motif, the induced secretion of cytokines may also affect
expression. For example IFN-,
CpG and DNA Vaccines
171
a cytokine that is known to
e induced
y CpG ODN has
een reported to down-reu
late the expression
y viral promoters (1113). However, this cannot explain the r
esults with the non-CpG ODN. ODN with a phosphodiester
ack
one does not interfe
re with expression of forein enes from plasmid DNA injected into muscle. Unfor
tunately, they are deraded so quickly (10) that they do not have a sinificant
adjuvant effect in vivo (Davis et al., unpu
lished results). We are currently te
stin alternative modified
ack
ones to see if we can identify one that will not
interfere with ene expression yet will cause immune stimulation.
2.2. Clonin CpG Motifs into DNA Vaccine Vectors
There has
een confusion and disareement as to whether it is possi
le to aumen
t immune responses
y clonin CpG motifs into the plasmid DNA vectors. Althouh
the plasmids have a phosphodiester
ack
one, they are mildly nuclease-resistant

y virtue of
ein in a closed-circular form, and thus in theory, this should
e
possi
le. Indeed, a report
y Sato et al. (8) shows a sinificant improvement i
n immune responses
y the clonin of only two immunostimulatory CpG sequences in

to a DNA vector. However, this has not
een repeata
le in other systems (persona
l communications). It is possi
le that, for most antiens and doses used, an add
itional two motifs is simply too few to make a sinificant difference. When one
takes into consideration the fact that most DNA vaccine vectors already contain
200300 CpG motifs, several of which will
e immunostimulatory, it is unlikely tha
t an additional two motifs would have a sinificant impact on the immune respons
e. The optimal num
er of CpG motifs to
e cloned into a DNA vaccine is yet to
e
determined.
2.3. Use of CpG ODN as Adjuvant with Protein Vaccines
It is possi
le to aument
oth humoral and cell-mediated immune responses to ant
ien-
ased vaccines
y the addition of CpG ODN. For reasons discussed a
ove, thi
s is
est mixed directly with the antien for coadministration
y intramuscular,
su
cutaneous or intraperitoneal injection. It is
est to use synthetic ODN with
a phosphorothioate
ack
one, and for studies in mice, a ood pan-activatin seq
uence is TCCATGACGTTCCTGACGTT. Doses as small as 1  can
e effective, althouh w
e routinely use doses of 10100 . The ODN should
e ordered as a sodium salt rathe
r than an ammonium salt. If it arrives as a lyophilized powder, it may
e rediss
olved directly into saline, TE, or any other
uffer of choice. 3. Notes CpG ODN
appears to work extremely well with protein vaccines, indeed much
etter than al
um (6), the only adjuvant currently approved for human
172
Weeratna, Krie, and Davis
use, or even Freunds complete adjuvant (unpu
lished results). It should
e possi

le to improve the efficacy of DNA vaccines throuh the rational use of CpG motif
s; however, this area clearly needs further study. While few methods are actuall
y iven in this chapter, it is hoped that the description of the pro
lems we hav
e encountered will save time and frustration for someone enterin into this fiel
d. References
1. Krie, A. M., Yi, A., Matson, S., Waldschmidt, T. J., Bishop, G. A., Teasdale
, R., et al. (1995) CpG motifs in
acterial DNA trier direct B cell activation
. Nature 374, 543549. 2. Pisetsky, D. S. (1996) Immune activation
y
acterial DN
A: A new enetic code. Immunity 5, 303310. 3. Klinman, D., Yi, A., Beaucae, S. L
., Conover, J., and Krie, A. M. (1996) CpG motifs expressed
y
acterial DNA ra
pidly induce lymphocytes to secrete IL-6, IL-12 and IFN. Proc. Natl. Acad. Sci.
USA 93, 28792883. 4. Halpern, M. D., Kurlander, R. J., and Pisetsky, D. S. (1996)
Bacteria DNA induces murine interferon- production
y stimulation of interleuk
in-12 and tumor necrosis factor-cell. Immunol. 167, 72. 5. Cowdery, J. S., Chase
, J. H., and Krie, A. M. (1996) Bacterial DNA induces in vivo interferon- produc
tion
y NK cells and increases sensitivity to endotoxin. J. Immunol. 156, 4570.
6. Davis, H. L., Weeratna, R., Waldschmid, T. J., Tyrett, L., Schorr, J., and K
rie, A.M. (1998) CpG DNA is a potent enhancer in mice immunized with recom
inan
t hepatitis B surface antien. J. Immunol. 160, 870876. 7. Roman, M., Martinorozc
o, E., Goodman, S., Nuyen, M. D., Sato, Y., Ronahy, A., Korn
luth, R. S., Rich
man, D. D., Carson, D. A., and Raz, E. (1997) Immunostimulatory DNA sequences fu
nction as T helper-1-promotin adjuvants, Nature Med. 3, 849854. 8. Sato, Y., Rom
an, M., Tihe, H., Lee, D., Corr, M., Nuyen, M., Silverman, G. J., Lotz, M., Ca
rson, D. A., and Raz, E. (1996) Immunostimulatory DNA sequences necessary for ef
fective intradermal ene immunization. Science 273, 352354. 9. Krie, A. M. (1999
) CpG DNA: a novel immunomodulator. Trends Micro
iol. 7, 64,65. 10. Zhao, Q., Ma
tson S., Herrera C. J., Fisher E., Yu H., and Krie A. M. (1993) Comparison of c
ellular
indin and uptake of antisense phosphodiester, phosphorothioate and mix
ed phosphorothioate and methylphosphonate olionucleotides. Antisense Res. Devel
op. 3, 5366. 11. Guidotti, L. G., Guilhot, S., and Chisari, F. V. (1994) Interleu
kin-2 and alpha/
eta interferon down-reulate hepatitis B virus ene expression
in vivo
y tumor necrosis factor-dependent and -independent pathways. J. Virol.
68, 12651270. 12. Romero, R. and Lavine, J. E. (1996) Cytokine inhi
ition of the

Hepatitis B virus core promoter. J. Hepatol. 23, 1723. 13. Harms, J. S. and Split
ter, G. A. (1995) Interferon-amma inhi
its transene expression driven
y SV40
or CMV promoters
ut auments expression driven
y the mammalian MHC1 promoter.
Hum. Gene. Ther. 6, 12911297.
16
In Vitro Assay of Immunostimulatory Activities of Plasmid Vectors
Charles F. Reich and David S. Pisetsky 1. Introduction 1.1. Principles of DNA Va
ccination DNA vaccination represents a powerful new approach for the elicitation
of lon-lived protective immunity aainst a
road rane of protein antiens (1,
2). In this approach, the vaccine is a plasmid DNA vector that encodes a forein
protein to
e tareted for the induction of humoral or cellular responses. Foll
owin administration
y various routes, the plasmid is taken up
y cells to allo
w intracellular production of the protein for presentation to the immune system.
Althouh the traffickin of the plasmid and its protein product is not well und
erstood, the eneration of responses ultimately involves a
one marrow-derived a
ntien presentin cell (3). The effectiveness of DNA vaccines has
een impressiv
e and has
een su
jected to considera
le investiation to optimize this stratey
and improve vectors. While the intracellular production of antien may facilita
te immune responses, the vectors themselves may
e crucial for effectiveness. As
shown in recent studies, these vectors have immunostimulatory properties, which
can serve as adjuvants,
oth increasin the manitude of responses as well as t
heir cellular profile. The immunostimulatory properties of vectors, as with natu
rally-occurrin
acterial DNA, result from DNA sequences whose content differs

etween mammalian and
acterial DNA.
1.2. Immunoloical Properties of Bacterial DNA
Contrary to lon-held notions on the immunoloical inertness of DNA,
acterial D
NA displays potent immunostimulatory properties that are manifest in
oth in viv
o and in vitro systems. These properties are similar to those of endoFrom: Metho
ds in Molecular Medicine, vol. 29, DNA Vaccines: Methods and Protocols Edited
y
: D. B. Lowrie and R. G. Whalen Humana Press Inc., Totowa, NJ
173
174
Reich and Pisetsky
toxin and suest that, durin ordinary encounters with infectin oranisms,
ac
terial DNA can serve as a daner sinal and activate the innate immune system (4
,5). As defined oriinally in the murine system, these properties encompass indu
ction of cytokines, includin IFN-/, IL-12, IFN-, TNF-, nd IL-6; stimul tion of B c
ell mitogenesis nd immunoglo
ulin production; nd downstre m effects of cytokin
es, such s ctiv tion of NK cells or promotion of Th1 responses (611). The cytok
ine effects lso occur in hum n peripher l
lood cells, lthough direct mitoge
nic effect on hum n B cells h s not s yet
een descri
ed. Stimul tion
y
cter
i l DNA involves ctiv tion of NF-B by an anti-oxidant sensitive pathway that app
ears common to other immune activators (12,13).
1.3. DNA Sequences with Immunological Activity
The immunostimulatory properties of bacterial DNA reflect structural microhetero
geneity and the presence of short sequence motifs that are characteristic of pro
aryotic DNA. These motifs, also termed immunostimulatory sequences (ISS), or Cp
G motifs, have the general structure of two 5' purines, an unmethylated CpG dinu
cleotide, and two 3' pyrimidines. These motifs occur much more commonly in bacte
rial DNA than mammalian DNA for two reasons. In mammalian DNA, cytosine and guan
osine occur in tandem much less frequently than predicted by DNA base compositio
n, a phenomenon called CpG suppression. Furthermore, in mammalian DNA, unlie ba
cterial DNA, cytosine is commonly methylated (9,14,15). While the biological adv

antages of CpG suppression and cytosine methylation are not nown, they provide
the basis for a recognition system that allows distinction of euaryotic and pro
aryotic DNA for the selfnonself discrimination. In addition to CpG motifs, other
DNA sequences may influence the immunostimulatory properties of bacterial DNA a
s well as plasmids. Thus, runs of deoxyguanosine can directly stimulate murine B
cells as well as promote production of cytoines, as reflected in IFN- expressio
n. IFN- expression results from the activity of IL-12 and TNF-,
oth products of m
croph ges/monocytes. As shown using synthetic oligonucleotides, runs of dG, whi
le un
le to induce cytokine production themselves, c n enh nce the stimul tory
ctivity of ISS when juxt posed in the s me oligonucleotide (1618). This enh ncem
ent m y result from the incre sed upt ke of dG-rich oligonucleotides
y the m cr
oph ge sc venger receptor. This receptor h s
ro d lig nd specificity nd
inds
v riety of poly nions, including cetyl ted low density lipoprotein, fucoid n,
dextr n sulf te nd dG-rich oligonucleotides nd polynucleotides. By unconventi
on l hydrogen
onding
etween dG residues, dGrich compounds c n form four str nd
ed rr ys c lled qu druplex DNA (19). The incre se in m croph ge production of I
L-12 nd TNF-
y ISS presumIn Vitro Ass y of Pl smid Vectors
175

ly results from n incre sed upt ke into cells vi
inding to the m croph ge s
c venger.
1.4. Vectors s Source of ISS
As currently formul ted, pl smid vectors for v ccin tion represent potenti l s
ource of ISS
ec use: (i) pl smids h ve unmethyl ted cytosines due to prop g tio
n in
cteri ; nd (ii) pl smids displ y
cteri l DNA sequences
ec use of the
encoded foreign protein s well s genes for replic tion or nti
iotic resist nc
e. The import nce of vector ISS to the induction of v ccine responses h s
een c
le rly est
lished in in vivo experiments in mice. Thus, methyl tion of pl smid
vectors le ds to loss of v ccine ctivity. Furthermore, the potency of v ccine v
ectors v ries dr m tic lly depending on the content of ISS in nti
iotic resist
nce elements (20,21). In ddition to potenti ting over ll v ccine response, ISS
in the pl smid m y influence the gener tion of Th1 responses th t re ch r cteri
stic of DNA v ccine responses in mouse models. While IL-12 most likely c uses th
e Th1 predomin nce, IFN-/ m y lso contri
ute to this p ttern of T-cell responsive
ness. In this reg rd, the presence of ISS could complic te DNA v ccin tion
ec u
se of loc l infl mm tion, shift in the
l nce of Th1/Th2 cells, s well s stim
ul tion of nti-DNA nti
ody production. In these ctivities, pl smid DNA does n
ot differ from
cteri l DNA, which is norm lly-encountered foreign ntigen n
d ppe rs well-toler ted ordin rily. While
cteri l DNA c n induce nti
ody res
ponses in norm l individu ls, the nti
odies ppe r non-p thogenic
y virtue of
their isotype nd selectivity for
cteri l DNA (4).
1.5. In Vitro Assessment of Vector Activities
Ch r cteriz tion of in vitro immunostimul tory properties of pl smid vectors is
import nt, especi lly s v ccin tion techniques re refined nd vectors engineer
ed to promote effectiveness. Although the rr y of immunostimul tory ctivities
of
cteri l DNA is l rge, they reflect the presence of common motifs. This fe t
ure llows selective ss y of ctivities considered most cruci l for n djuv nt
effect. These ctivities include expression of IL-12 nd IFN- as well as B cell
mitoenesis. In contrast, assessment of DNA uptake is more pro
lematic
ecause t
he mechanisms of DNA uptake
y cells appear to depend on concentration as well a
s am
ient conditions. Since increased uptake should translate into increased imm
unostimulatory activity, however, either cytokine responses or B cell mitoenesi
s can
e used as surroate markers for uptake. In usin in vitro assays to asses
s ISS, certain caveats should
e considered. First, human responses appear more
sporadic than murine responses. This varia
ility could result from differences a

mon humans in their intrinsic capacity to respond to DNA. Alternatively, the
a

cterial DNA sequences causin opti176
Reich and Pisetsky
mal stimulation may differ in human and murine systems. Most of the work on immu
nostimulation has utilized olionucleotides that have
een characterized in the
murine system, suestin the need for a search for other active motifs for huma
n study. Furthermore, natural DNA sequences differ sinificantly in their activi
ties, raisin the possi
ility of active sequences in addition to CpG motifs; of
equal importance,
acterial DNA as well as plasmids may contain inhi
itory seque
nces that can affect the response to ISS. Althouh cytokine stimulation can
e a
ssayed in terms of either mRNA or protein, availa
le assays
y ELISA are conveni
ent and allow samplin of multiple specimens. The methods descri
ed utilize this
approach and have
een verified in the murine system with natural DNA, olionuc
leotides as well as plasmids. 2. Materials 2.1. Preparation of Mouse Splenocytes
1. Suita
le mice are availa
le from The Jackson La
oratory (Bar Har
or, ME) (see
Note 1). 2. 70% (v/v) ethanol. 3. Dissectin tools, scissors and forceps. 4. 60
mm diameter sterile petri dishes (Falcon, Los Aneles, CA). 5. Frosted microsco
pe slides. 6. RPMI 1640 medium with sodium
icar
onate and lutamine (Sima, St.
Louis, MO). 7. Heat inactivated fetal
ovine serum (HyClone, Loan, VT). 8. Red

lood cell lysis solution consistin of 1 part 0.17 M Tris pH 7.6 and 9 parts o
f 0.16 M ammonium chloride filtered throuh a .22 micron Nalene filter unit (Na
lene, Rochester, NY). 9. Sterile 15 mL conical polypropylene centrifue tu
es (
Costar, Cam
ride, MA). 10. Hemacytometer, microscope. 11. Sterile 96 well flat

ottom tissue-culture-treated cell culture clusters (Costar). 12. Control mitoe
ns, LPS, ConA (Sima). 13. Purified sterile DNA, olionucleotides (see Note 2).
14. Multi-channel pipettor with capacity of at least 100 L (Finnpipette) and ster
ile tips for the pipettor (USA/Scientific Plastics, Ocala, FL). 15. Sinle-chann
el pipettors with ranes of 540 L and 40200 L (Finnpipette) and sterile tips (USA/Sc
ientific). 16. Complete medium consistin of RPMI 1640, 5% (v/v) heat inactivate
d fetal
ovine serum, and 5 105 M 2-mercaptoethanol. 17. 5 and 10 mL sterile sero
loical pipets (Costar). 18. Laminar flow hood (Baker, Phillips
ur, NJ). 19. In
cu
ator, 37oC, 5% (v/v) CO2 (Forma Scientific, Marietta, OH).
2.2. Measurement of Proliferation
1. Thymidine [methyl-3H] 6.7 Ci/mmol aqueous solution (New Enland Nuclear, Bost
on, MA).
In Vitro Assay of Plasmid Vectors
177
2. Cells (mouse splenocytes or human peripheral
lood cells) plated in 96 well p
lates stimulated with mitoen. 3. Microharvester (Bellco Glass Inc., Vineland, N
J). 4. Glass fi
er strips for microharvester. 5. Distilled water. 6. Scintillati
on vials, 5.5 mL (USA/Scientific). 7. Scintillation fluid: Safety Solve (Researc
h Products International, Mt. Prospect, IL). 8. Scintillation counter: Packard T
ri-Car
(Downers Grove, IL).
2.3. General ELISA Reaents
1. 96 well polystyrene plates. Use Immulon II for cytokines, Microtiter for tota
l immunolo
ulin (Dynatech, Chantilly, VA). 2. 96 well polypropylene plates (Si
ma). 3. PBS pH 7.2, for washin plates. 4. PBS pH 7.2, 1% (w/v)
ovine serum al

umin, for
lockin plates. 5. Multichannel pipettor, 50300 L (Finnpipette), and ti
ps which hold at least 300 L (USA/Scientific). 6. PBS pH 7.2, containin 0.5% (w/
v)
ovine serum al
umin, 0.4% (v/v) Tween 20 (PBSBSAT). Make fresh daily or filter
sterilize and store it at 4C. Use this for dilutin samples, standards, and anti


odies. 7. 1.2 mL polypropylene tu
es in a 8 12 array rack with the same spacin
as 96 well polystyrene plates for dilutin standards and culture supernatants (
USA/ Scientific). 8. Citrate
uffer, 21 citric acid monohydrate/L water. Adjust
the pH to 4.2 with 50% (w/v) sodium hydroxide. 9. TMB: dissolve 3,3,5,5-tetra
methyl
enzidine dihydrochloride (Sima) to 0.75% (w/v) in water and filter to 0.
22 to remove any undissolved material. Store in 1 mL aliquots at 20C. 10. Hydroen
peroxide, 30% (v/v) (see Note 3). 11. Plate washer: Skatron Skan Washer (Skatro
n Instruments, Sterlin, VA). 12. Microplate reader with filters suita
le for re
adin at a wavelenth of 380 nm (Molecular Devices, Euene, OR).
2.4. Measurement of Cytokines
y ELISA
1. Coatin
uffer PBS pH 8.5. 2. Capture/
iotinylated detection anti
ody pairs a
nd standards purchased from Pharminen. Store anti
odies at 4C. Store standards d
iluted in sterile PBS, 1% (w/v)
ovine serum al
umin as aliquots frozen at 70C (Ta

le 1).
2.5. Measurement Total Murine Immunolo
ulins
y ELISA
1. PBS pH 7.2, for coatin
uffer. 2. Goat anti-mouse polyvalent immunolo
ulins
for use as capture anti
ody are availa
le from Sima. 3. Goat anti-mouse IG pe
roxidase conjuate, -chain-specific for measurin IG (Sima).
178
Ta
le 1 Reaents for Cytokine Assaysa Mouse Cytokines mIL2 mIL4 mIL6 mIL10 mIL12 p40
mIL12 p70 mIFN Human Cytokines hIL6 hIL12 p40 hIL12 p70 hTNF
Num
ers
Reich nd Pisetsky
C pture A
18161D 18191D 18071D 18141D 18491D 20011D 18181D
Detection A
(
iotin) 18172D 18042D 18082D 18152D 18482D 18482D 18112D
St nd rd 19211T 19231V 19251V 19281V 19401W, 19371W 19361V 19301T
18871D 20711D 20501D 18631D
18882D 20512D 20512D 18642D
19661V 19931V, 19721V 19721V 19761T
refer to Ph rmingen Rese rch Products C t log 19961997.
4. Go t nti-mouse IgM peroxid se conjug te, -ch in-specific for me suring IgM (S
igm ). 5. Purified mouse IgG (Sigm ). 6. TEPC 183, mouse IgM myelom protein (Si
gm ).
2.6. Prep r tion of Hum n Peripher l Blood Cells
1. Hep rinized whole
lood or commerci lly v il
le fresh
uffy co t (Interst t
e Blood B nk Inc.). 2. RPMI 1640 with l-glut mine nd sodium
ic r
on te (Sigm )
. 3. Ficoll-Hyp que lymphocyte isol tion medium (Ficoll-P que, Ph rm ci , Pisc t
w y, NJ). 4. Sterile 50 mL polypropylene conic l centrifuge tu
es (USA/Scientif
ic). 5. Complete medium consisting of RPMI 1640 + 10% (v/v) he t-in ctiv ted fet
l
ovine serum. 6. Hem cytometer nd microscope. 7. Sterile 96-well fl t
ottom
tissue culture tre ted cell culture clusters (Cost r). 8. Multi-ch nnel pipetto
r with c p city of t le st 100 L (Finnpipette) nd sterile tips for the pipettor
(USA/Scientific). 9. Single-ch nnel pipettors with r nges of 540 L nd 40200 L (Fin
npipette) nd sterile tips (USA/Scientific). 10. L min r flow hood (B ker). 11.
Incu
tor, 37C, 5% (v/v) CO2 (Form ).
2.7. Assessment of Endotoxin in DNA/Oligonucleotide Prep r tions

1. Qu ntit tive Chromogenic Limulus Ame
ocyte Lys te Kit (BioWhitt ker, W lkersv
ille, MD).
In Vitro Ass y of Pl smid Vectors
179
2. 96-well polystyrene microtiter pl tes. 3. Micropl te re der with filters suit

le for re ding t w velength of 405410 nm (Molecul r Devices). 4. Lipopolys c
ch ride (Sigm ). 5. Deoxyri
onucle se I (Sigm ).
3. Methods 3.1. Prep r tion of Mouse Splenocytes
1. S crifice mice
y cervic l disloc tion. 2. Disinfect the fur
y thoroughly s
tur ting it with 70% (v/v) eth nol. 3. With scissors m ke n incision in the
d
omin l skin nd, gr sping either side of this incision with gloved forefingers
nd thum
s, pull
ck the skin until the spleen c n
e o
served through the
dom
in l muscul ture. M ke n incision over the spleen nd remove it to 60 mm Petr
i dish cont ining
out 5 mL of medium. 4. Fl me 2 frosted microscope slides. Pi
ck up the spleen with one of the slides nd use the second to express the cells.
St rt t one end nd work tow rds the middle. Then st rt t the other end until
ll the cells re expressed. Frequently dip the slides in the medium in the pet
ri dish to tr nsfer the cells. Disc rd the rem ining connective tissue. 5. Tr ns
fer cells to 15 mL conic l tu
e, m ke up to 10 mL tot l volume with medium. Al
low l rge chunks to settle out for 2 min. C refully remove cell suspension to
new tu
e nd centrifuge 5 min t 400g to pellet the cells. Remove supern t nt n
d resuspend cells in RBC lysis
uffer. Use 5 mL per spleen. Pellet the cells, 5
min, 400g. Resuspend in medium nd pellet. Repe t 2 times to thoroughly remove l
ysis
uffer. Resuspend in 10 mL complete medium nd count cells with hem cytom
eter. Yield should
e 0.71.5 108 cells/spleen for most mouse str ins. 6. Adjust c
ell concentr tion for prolifer tion studies to 25 106/mL. nd for cytokine ss ys
to 25 107/mL. Tr nsfer cells to 96-well pl tes, 100 L/well. 7. Prep re DNA nd con
trol mitogens/cytokine inducers t 2 times the fin l concentr tion in complete m
edium. Most stimul tory DNAs give m ximum response t fin l concentr tion of
pproxim tely 50 g/mL. LPS will give m ximum response t 110 g/mL nd ConA 15 g/mL
(see Note 4). Allow 100 L/well. Prep re triplic te wells. Pipette onto cells nd
mix. Pl ce pl tes in humidified incu
tor, 37C, 5% (v/v) CO2. 8. Incu
tion time
s for optimum response v ry. V rious cytokines re t their m ximum from 448 h. P
rolifer tion is gener lly gre test t 48 h
ut time courses should
e est
lishe
d.
3.2. Prep r tion of Hum n Peripher l Blood Cells
1. Dilute nti-co gul nt tre ted
lood with n equ l volume of RPMI 1640. 2. C r
efully pipet 15 mL Ficoll-P que into the
ottom of 50 mL conic l centrifuge tu

e. 3. C refully l yer the diluted
lood on top of the Ficoll-P que.
180
Reich nd Pisetsky
4. Spin t 400g for 30 min t 20C. 5. Inspect tu
es. There should
e pink upper
l yer, n interf ce of cells, cle r or slightly cloudy l yer of Ficoll-P que
nd pellet of red
lood cells. If sep r tion is not dequ te, spin for n ddi
tion l 20 min. 6. C refully remove the interf ce of cells with pipet trying no
t to remove ny Ficoll-P que nd dilute with 4 volumes of RPMI. 7. Spin t 400g
for 5 min. Disc rd supern t nt nd suspend cells in fresh RPMI. Centrifuge. Repe
t twice to ensure cells re thoroughly w shed. 8. Remove s mple nd count wit
h hem cytometer. 9. Centrifuge cells nd resuspend in RPMI-1640/10% (v/v) FBS
to ppropri te concentr tion nd tr nsfer to 96 well pl tes, 100 L/well.
3.3. Triti ted Thymidine Incorpor tion

1. Prep re 2 mL of diluted l
el for e ch 96 well pl te
y dding 40 L of [3H]thy

midine stock to 1.96 mL serum free RPMI 1640. Add 20 L to e ch well (0.5 Ci), mix
gently, l
el pl te to denote th t it cont ins r dio ctive m teri l nd return i
t to the incu
tor for 6 h. 2. At the end of the incu
tion period, h rvest cell
s on gl ss fi
er strips. W sh with distilled w ter for t le st 10 pulses of the
h rvester rocker v lve. 3. Remove filters nd llow to dry overnight. 4. With g
loved h nds nd p ir of forceps remove individu l filter disks from the gl ss
fi
er strip nd pl ce them in scintill tion vi ls. (Vi ls pl ced in r ck in n
8 12 rr y simplifies this procedure.) 5. Add 3 mL of scintill tion fluid to e
ch vi l nd c p vi l. 6. Count in scintill tion counter using the st nd rd progr
m for tritium customized to give the me ns of three successive vi ls.
3.4. An lysis of Secreted Cytokines
1. C refully remove 150 L of fluid from e ch well using multi-ch nnel pipettor,

eing c reful not to distur
the cell l yer. Tr nsfer to polypropylene 96 well
pl tes, cover nd freeze t 20C until ss yed. 2. Dilute c pture nti
ody in PBS p
H 8.5 to concentr tion of 0.55.0 g/mL. The optimum concentr tion should
e est

lished for e ch lot
ut s tisf ctory results c n usu lly
e o
t ined t 1 g/mL. P
l ce 100 L of diluted nti
ody in e ch well, wr p pl tes in luminum foil or cove
r with pl te covers nd pl ce in the refriger tor (4C) overnight. 3. The next mor
ning prep re st nd rds in PBSBSAT. The st nd rds will occupy two rows of eleven we
lls on e ch pl te. The twelfth wells re left s
l nks. Include these st nd rds
on every pl te. Dilutions re m de in polypropylene tu
es r cked in the s me sp
cing s the wells of the pl tes. 4. M ke 1:2 dilutions. St rt with 0.5 mL of st
nd rd diluted in the first tu
e nd 0.25 mL PBSBSAT in e ch successive tu
e. Sugg
ested st nd rd r nges re indic ted in T
le 2. 5. W sh the pl tes 3 times with
PBS with the pl te w sher.

In Vitro Ass y of Pl smid Vectors

T
le 2 Detection R nge of Cytokine Ass ys mIL2 mIL4 mIL6 mIL10 mIL12p40 mIL12p70 mIFN
0002 p/mL 50005 p/mL 20002 p/mL 01000.1 n/mL 50005 p/mL 50005 p/mL 10001 U/mL hI
hIL12p40 hIL12p70 hTNF hIFN
181
1,50005 p/mL 10,00010 p/mL 1,50005 p/mL 1,50005 p/mL 1,50005 p/mL
6. Place 75 L PBSBSAT in each sample well and transfer 25 L of each sample from the
storae plate to its correspondin position on the assay plate. Mix well
y pipe
ttin. Transfer the standards to desinated positions on plate, 0.1 mL/ well. In
cu
ate 2 h at room temperature. 7. Wash plates 3 times and add 100 L
iotinylated
detection anti
ody diluted in PBSBSAT to each well. The optimum concentration of

iotinylated detection anti
ody must
e determined for each lot,
ut in eneral
will
e in the rane of 140 /mL. Incu
ate the plates for 2 h at room temperature,
then wash them 3 times with PBS with the plate washer. 8. Add 0.1 mL avidin/per
oxidase diluted 1:5000 in PBSBSAT and incu
ate for 30 min at room temperature. 9.
Wash the plate 3 times with PBS, reverse the plate and wash it 3 times more with
the plate washer. 10. Add 0.1 mL TMB solution to each well (1 mL TMB stock, 17 L
H2O2/50 mL citrate
uffer pH 4.0). This should
e made fresh just
efore use. 1
1. Incu
ate the plate at room temperature 530 min. Timin must
e determined for
each assay. Read the OD380 with the plate reader. 12. Plot the lo of the concen
tration of standards vs. OD380 and use the curve to determine the sample concent
rations.
3.5. Determination of Total Murine I
1. Coat 96 well Microtiter plates overniht with oat anti-mouse polyvalent immu
nolo
ulins, diluted to 5 /mL in PBS pH 7.4. Use 100 L of diluted anti
ody/ well.
Set up a plate to measure IG and another to measure IM. 2. Next mornin, wash
the plates 3 times with PBS in the plate washer and add 200 L PBSBSA to each well
. Incu
ate the plate for 1 h at room temperature. 3. Make standards dilutions. S

tart at 1.0 /mL and make 10 twofold dilutions in PBSBSAT. Dilute samples 1:5 in PB
SBSAT and make four 1:5 dilutions. Make adequate volumes to run in triplicate on t
wo plates (at least 600 L of each) 4. Wash the plates and add the diluted standar
ds and supernatants, 100 L/well. Incu
ate at room temperature for 1 h. 5. Wash th
e plates and add diluted anti-IG peroxidase to one plate of the set and anti-I
M peroxidase to the second. Each anti
ody should
e titrated
ut ener182
Reich and Pisetsky
ally 1:1000 in PBSBSAT is adequate. Use 0.1 mL per well. Incu
ate for 1 h at room
temperature. Wash the plates, reverse and wash them aain. Add 0.2 mL TMB/H2O2/c
itrate solution to each well and incu
ate for 30 min at room temperature (see Su

headin 3.4.10.) Read the OD380 on the plate reader. Plot the lo concentration
of the standards vs OD380. Pick a dilution of sample which ives an OD that fal
ls in the straiht portion of the curve and calculate the initial sample concent
ration.
6. 7. 8. 9.
3.6. Control for Possi
le Endotoxin Contamination
1. Esta
lish the endotoxin concentrations in olionucleotides and DNA samples us
in the Limulus ame
ocyte lysate assay (see Note 5). 2. Set up cytokine/prolifer
ation assay cultures and stimulate them with serial dilutions of endotoxin,
rac
ketin the concentration detected (if any) in the DNA sample in its rane of sti
mulatory concentrations (see Note 6).
3.6.1. DNase Control for Endotoxin Contamination
1. Dilute DNA to 100 /mL and DNase I to 200 Kunitz units/mL in complete medium.
Also set up controls which include medium and DNase
ut no DNA and medium and LP
S plus DNase. Incu
ate them for 2 h at 37C (see Note 7). 2. Set up cytokine/proli
feration assay cultures and stimulate them with DNA and with DNA that has
een t
reated with DNase. 3. A response that is still o
tained after DNase treatment su
ests endotoxin contamination.
4. Notes
1. Althouh any strain of mouse may
e used, the C3H/HeJ strain is recommended a
s these mice have reduced responses to endotoxin. While the use of these mice ma
y eliminate confusion with contaminatin endotoxin, it does not prevent possi
le
immunostimulatory effects of other
acterial products. It is useful to confirm
results with other mouse strains usin Polymixin B as an inhi
itor of endotoxin.
2. Commercial DNA preparations often have residual RNA and protein and should

e further purified
y conventional methods. DNA may
e sterilized
y ethanol pre
cipitation. The precipitated DNA is then redissolved in sterile
uffer. Olionuc
leotide solutions are conveniently sterilized
y filterin throuh a 0.22 micron
Millex-GV low
indin filter unit (Millipore). 3. Hydroen peroxide should
e s
tored at 4C,
ut has a limited shelf life, and should
e replaced every 6 mo. 4.
It is recommended that dilution curves are prepared for all mitoens and inducer
s of cytokine as well as for controls. 5. The Limulus ame
ocyte assay is a conve
nient and well-accepted method of measurin endotoxin contamination. Some olioor polynucleotides, however, may
In Vitro Assay of Plasmid Vectors
183

e scored as positive in this assay. If a false positive is suspected, an immuno

assay for endotoxin can
e used as an alternative. 6. If the presence of endotox
in is detected
y the Limulus assay, it is important to determine whether the le
vel measured can account for the
ioloical effects o
served on cells. Dilution

curves should therefore
e esta
lished to include the concentration of endotoxin

detected. Curves should also
e constructed usin endotoxin plus a DNA to rule
out syneristic effects. 7. Conventional aarose el electrophoresis may
e used
to confirm that diestion was complete.
References
1. Voel, F. R. and Sarver, N. (1995) Nucleic acid vaccines. Clin. Micro
iol. Re
v. 8, 406410. 2. Pardoll, D. M. and Beckerle, A. M. (1995) Exposin the immunolo
y of naked DNA vaccines. Immunity 3, 165169. 3. Corr, M., Lee, D. J., Carson, D.
A., and Tihe, H. (1996) Gene vaccination with naked plasmid DNA: mechanism of
CTL primin. J. Exp. Med. 184, 15551560. 4. Pisetsky, D. S. (1996) The immunoloi
c properties of DNA. J. Immunol. 156, 421423. 5. Pisetsky, D. S. (1996) Immune ac
tivation
y
acterial DNA: a new enetic code. Immunity 5, 303310. 6. Yamamoto, S
., Kuramoto, E., Shimada, S., and Tokunaa, T. (1988) In vitro aumentation of n
atural killer cell activity and production of interferon-/ nd - with deoxyri
onucl
eic acid fraction from Myco
acterium
ovis BCG. Jpn. J. Cancer Res. 79, 866873. 7
. Yamamoto, S., Yamamoto, T., Shimada, S., Kuramoto, E., Yano, O., Kataoka, T.,
and Tokunaa, T. (1992) DNA from
acteria,
ut not from verte
rates, induces int
erferons, activates natural killer cells and inhi
its tumor rowth. Micro
iol. I
mmunol. 36, 983997. 8. Messina, J. P., Gilkeson, G. S., and Pisetsky, D. S. (1991
) Stimulation of in vitro murine lymphocyte proliferation
y
acterial DNA. J. I
mmunol. 147, 17591764. 9. Krie, A. M., Yi, A.-K., Matson, S., Waldschmidt, T. J.
, Bishop, G. A., Teasdale, R., et al. (1995) CpG motifs in
acterial DNA trier
direct B-cell activation. Nature 374, 546549. 10. Klinman, D. M., Yi, A.-K., Bea
ucae, S. L., Conover, J., and Krie, A. M. (1996) CpG motifs present in
acteri
al DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and
interferon . Proc. Natl. Acad. Sci. USA 93, 28792883. 11. Halpern, M. D., Kurlande
r, R. J., and Pisetsky, D. S. (1996) Bacterial DNA induces murine interferon- pro
duction
y stimulation of interleukin-12 and tumor necrosis factor-. Cell. Immuno
l. 167, 7278. 12. St cey, K. J., Sweet, M. J., nd Hume, D. A. (1996) M croph ges
ingest nd re ctiv ted
y
cteri l DNA. J. Immunol. 157, 21162122. 13. Yi, A.
- K., Klinm n, D. M., M rtin, T. L., M tson, S., nd Krieg, A. M. (1996) R pid
immune ctiv tion
y CpG motifs in
cteri l DNA. Systemic induction of
184
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14.
15. 16.
17. 18.
19.
20.
21. 22.
IL-6 tr nscription through n ntioxid nt-sensitive p thw y. J. Immunol. 157, 53
945402. K t ok , T., Y m moto, S., Y m moto, T., Kur moto, E., Kimur , Y., Y no,
O., nd Tokun g , T. (1992) Antitumor ctivity of synthetic oligonucleotides wit
h sequences from cDNA encoding proteins of Myco
cterium
ovis BCG. Jpn. J. C nc
er Res. 83, 244247. Krieg, A. M. (1995) CpG DNA: p thogenic f ctor in systemic
lupus erythem tosus? J. Clin. Immunol. 15, 284292. Messin , J. P., Gilkeson, G. S
., nd Pisetsky, D. S. (1993) The influence of DNA structure on the in vitro sti
mul tion of murine lymphocytes
y n tur l nd synthetic polynucleotide ntigens.
Cell. Immunol. 147, 148157. Pisetsky, D. S. nd Reich, C. (1993) Stimul tion of
in vitro prolifer tion of murine lymphocytes
y synthetic oligodeoxynucleotides.

Molec. Biol. Rep. 18, 217221. Kimur , Y., Soneh r , K., Kur moto, E., M kino, T.
, Y m moto, S., Y m moto, T., et l. (1994) Binding of oligogu nyl te to sc veng
er receptors is required for oligonucleotides to ugment NK cell ctivity nd in
duce IFN. J. Biochem. 116, 991994. Krieger, M. nd Herz, J. (1994) Structures nd
functions of multilig nd lipoprotein receptors: m croph ge sc venger receptors
nd LDL receptor-rel ted protein (LRP). Annu. Rev. Biochem. 63, 604637. S to, Y.,
Rom n, M., Tighe, H., Lee, D., Corr, M., Nguyen, M.-D., Silverm n, G. J., Lotz,
M., C rson, D. A., nd R z, E. (1996) Immunostimul tory DNA sequences necess ry
for effective intr derm l gene immuniz tion. Science 273, 352354. Klinm n, D. M.
, Y mshchikov, G., nd Ishig tsu
o, Y. (1997) Contri
ution of CpG motifs to the
immunogenicity of DNA v ccines. J. Immunol. 158, 36353639. R z, E., Tighe, H., S
to, Y., Corr, M., Dudler, J. A., Rom n, M., et l. (1996) Preferenti l induction
of Th1 immune response nd inhi
ition of specific IgE nti
ody form tion
y p
l smid DNA immuniz tion. Proc. N tl. Ac d. Sci. USA 93, 51415145.
17
Adjuv nts for Pl smid DNA V ccines
Jon Norm n, Jukk H rtikk , P mel Str uch, nd M rston M nthorpe 1. Introductio
n In the l te 1980s, Jon Wolff of the University of Wisconsin nd Phil Felgner h
ere t Vic l were screening c tionic lipids for their
ility to enc psul te nd
deliver purified pl smid DNA into mouse tissues. They discovered th t direct in
jection of lipid-DNA complexes into muscle resulted in me sur
le protein expres
sion. A
el ted control experiment without lipid led to the serendipitous discov
ery th t n ked pl smid DNA w s t ken up nd expressed in muscle to gre ter exten
t th n DNA-lipid complexes (1). This key o
serv tion led to the demonstr tion th
t i.m. injection in mice of st nd rd 50 g of pl smid DNA encoding reporter g
ene
ecomes re dily expressed exclusively in myofi
er cells t 180 pg of gene pr
oduct per muscle (2). More recently, pl smid DNA expression vectors were improve
d such th t n ver ge of 300 ng of gene product could
e produced from single i
ntr muscul r (i.m.) injections of pl smid DNA, nd up to 40 g of gene product cou
ld
e produced fter multiple injections (3 nd J. H rtikk , unpu
lished o
serv
tions). One of the first pplic tions of pl smid DNA injection technology w s th
e induction of n immune response. Since tr nsduction of cells with pl smid DNA
injection would le d to intr cellul r synthesis of ntigen, it w s expected th t
ntigen could
e processed for Cl ss I nd Cl ss II MHC present tion, which wou
ld le d to su
sequent stimul tion of cell-medi ted immunity. The first v lid t
ion of this concept w s pu
lished in J nu ry 1991
y Felgner nd Rhodes (4) who
reported prelimin ry experiments indic ting th t mice could
e immunized g inst
HIV gp120
y i.m. v ccin tion with gp120 pl smid DNA. Extending this concept to
n nim l model of infectious dise se, Ulmer nd colle gues (5) showed th t mic
e could
e protected from leth l
From: Methods in Molecul r Medicine, vol. 29, DNA V ccines: Methods nd Protocol
s Edited
y: D. B. Lowrie nd R. G. Wh len Hum n Press Inc., Totow , NJ
185
186
Norm n et l.
ch llenge of influenz virus fter DNA immuniz tion with influenz nucleoprotein
(NP) ntigen. Protection o
t ined fter immuniz tion with the highly conserved
NP ntigen extended cross 2 different vir l str ins, nd protection required CD
8+ CTL (6). Sever l hum n clinic l tri ls re in progress to ev lu te DNA v ccin
tion. In the p st sever l ye rs, gents h ve
een descri
ed th t f cilit te pl
smid DNA expression or ct s djuv nts to DNA v ccin tion (see Note 1). One of
these gents,
upiv c ine (BP), is loc l nesthetic which is lso myotoxin (
711). Injected BP destroys myofi
er cells le ding to m croph gic cle r nce of cel
l de
ris nd prolifer tion of muscle precursor cells (s tellite cells or myo
l s
ts) which then fuse to one nother nd to the rem ining vi
le myofi
er syncytiu

m, ll s the norm l p rt of skelet l muscle regener tion. When injected into mu

scle prior to injection of pl smid DNA, BP w s shown to incre se pl smid DNA exp
ression in muscle nd presum
ly stimul te the resulting immune response. Anothe
r gent which h s
een reported to stimul te lucifer se expression is polyvinyl
pyrollidone (PVP) which is formul ted nd co-injected with pl smid DNA (12). Add
ition lly, neutr l, nionic nd c tionic lipids re reported to enh nce pl smid
DNA expression in non-muscle tissues (1315). Only recently h ve lipids
een shown
to enh nce pl smid DNA expression nd immunity fter injection into muscle (16)
. Although the
ove studies using f cilit ting gents with pl smid DNAs re encou
r ging, there rem ins need to system tic lly est
lish the comp r tive potency
of these gents in enh ncing gene expression nd immuniz tion. In this study,
qu ntit tive comp rison of the effects of the co-injection of BP, PVP or DMRIE:
DOPE c tionic liposomes on pl smid DNA-medi ted lucifer se gene expression nd
nti-NP nti
ody responses is performed.
1.1. Effects of Bupiv c ine on Lucifer se Expression in Mouse Skelet l Muscle
Two pl smids, RSV-Lux nd VR1223, e ch encoding the lucifer se enzyme were chose
n for study. Previous work h d shown th t VR1223 expressed over 70-fold higher t
h n RSV-Lux in st nd rd in vivo muscle expression ss ys (3). Here, the pl smids
were individu lly injected s 50 g DNA/50 L s line into mouse rectus femoris musc
le, either (i) lone, (ii) lone,
ut 5 d fter injection of 0.75% BP, or (iii)
mixed with 0.75% BP. Muscles were collected 7 d l ter nd ss yed for lucifer se
enzyme ctivity. The chosen BP dose corresponded to th t used
y others (17,18)
. The results re shown in Fig. 1A,B. The DNAs injected lone (white
rs in Fig
. 1) yielded expression levels of 0.74 (RSVLux) nd 60 (VR1223) ng of lucifer se
/muscle, representing n 80-fold higher expression from VR1223. When injected 5
d fter BP tre tment (gr y
rs in Fig. 1), the RSV-Lux nd VR1223 DNAs yielded
5.1 nd 99 ng lucifer se/
Adjuv nts for Pl smid DNA V ccines
187
Fig. 1. Adult BALB/c mice were e ch injected i.m. with 50 mg of the indic ted lu
cifer se pl smid DNA either lone (None, white
rs) or formul ted with 0.75% Bu
piv c ine (BP co-inj,
l ck
rs) or lone
ut 5 d fter injection of 0.75% BP w
ithout DNA (BP5d, sh ded
rs) (Su
he dings 2. nd 3.). The pl smid DNAs were pre
viously reported to yield low (RSV-Lux) or high (VR1223) expression levels fter
injection into mouse muscle (3). At 7 d post-DNA injection, muscles were extr c
ted nd lucifer se enzyme ctivity w s me sured. V lues shown
ove e ch
r rep
resent the ver ges of 20 muscles (2 experiments, 10 muscles e ch) nd error
r
s re St nd rd Errors of the Me n.
muscle, respectively. Thus, the lower expressing RSV pl smid expressed
out sev
en-fold more lucifer se when injected into BP-tre ted muscle. This f cilit ted RSV
-Lux expression w s still n order of m gnitude less th n the injection of VR122
3 in BP untre ted muscle (5.1 vs 60). Pre-injection with 0.75% BP enh nced VR122
3 expression
y less th n twofold from 6099 ng/muscle,
ut with the norm l v ri t
ion in expression from different muscle, the BPtre ted muscle w s not st tistic
lly different (P = 0.53; n = 20). Co-injection of
188
Norm n et l.
pl smid nd 0.75% BP decre sed expression with
oth vectors
y 7.4-fold (RSV-Lux
: from 0.740.1) nd 6.7-fold (VR1223: 608.9 ng lucifer se/ muscle), respectively.
In summ ry, 0.75% BP pre-injection enh nced the expression of poor expression
vector,
ut h d no effect on good expression vector nd BP co-injection with e
ither vector decre sed expression
y ne rly n order of m gnitude. Thus, the d t

 confirm previous reports (711,17,18) th t the pre-injection of mouse skelet l m

uscle with
upiv c ine myotoxin followed 5 d l ter
y pl smid DNA injection resu
lts in n incre se in the expression of the DNA. However, this enh ncement only
occurs with pl smid construct th t expresses in muscle t rel tively low level
s. The high efficiency muscle expression vector used in these experiments w s no
t signific ntly enh nced
y BP preinjection. Furthermore, the injection of hig
h-efficiency pl smid like the CMV-driven VR1223 c n express 10-fold more gene pr
oduct th n the BPenh nced low-efficiency-expressing RSV-driven pl smid. Thus, th
e preferred method for intr muscul r expression of n i.m. tr nsgene is the inje
ction of high-expressing n ked DNA without the use of
upiv c ine.
1.2. Effects of PVP nd C tionic Lipids on Lucifer se Expression in Mouse Skelet
l Muscle
Lucifer se expression fter intr muscul r injection of VR1223 formul ted with 5%
PVP s reported (12) or DMRIE:DOPE c tionic lipid w s ev lu ted. The results r
e shown in Fig. 2A,B. VR1223 expression 7 d fter injection w s decre sed
y 5.0
- nd 8.8-fold (from 387.6 or 374.2) respectively,
y PVP or DMRIE:DOPE. Thus, PVP
or lipid co-injection, like BP co-injection, gre tly decre sed pl smid DNA expr
ession in muscle. These results with PVP contr dict those of Mumper nd colle gu
es (12), who showed th t co-injection of PVP with pl smid DNA in r t ti
i lis mu
scle c n enh nce tr nsgene expression up to fivefold. A v riety of conditions us
ing different DNAs nd dos ges were ttempted
ut with the s me result (see Note
2). This discrep ncy could
e due to differences in nim l species (mouse vs r
t), muscle type (rectus vs ti
i lis), reporter gene (lucifer se vs CAT), pl smid
vector (CMV-CAT vs VR1223) or other less defined experiment l conditions.
1.3. Comp r tive Adjuv nt Effects of Bupiv c ine, PVP nd C tionic Lipid on DNA
V ccin tion
A direct comp rison of BP, PVP nd DMRIE:DOPE w s m de
y co-injecting these ge
nts i.m. with DNA to f cilit te the gener tion of nti
odies to the encoded gene
product, influenz nucleoprotein (NP). To minimize nim l toxicity to BP (see N
ote 3) the co-injected BP dose w s decre sed from 0.75% to 0.25% ccording to th
e liter ture (19). The results re shown in Fig. 3. AntiAdjuv nts for Pl smid DNA V ccines
189
Fig. 2. Adult BALB/c mice were e ch injected i.m. with 10 g of VR1223 lucifer se
pl smid DNA lone (None, white
rs) or formul ted with 5% PVP (PVP, A.
l ck

r) or 50% DMRIE : 50%DOPE t 2:1 mol r/1:1 m ss r tio of DNA:DMRIE (DM:DP, B.

l ck
r) (Su
he dings 2. nd 3.). At 7 d post-DNA injection, muscles were extr
cted nd lucifer se enzyme ctivity w s me sured. V lues shown
ove e ch
r r
epresent the ver ges of 20 muscles (2 experiments, 10 muscles e ch) nd error

rs re st nd rd errors of the me n.
NP nti
ody titers t one month post-i.m. injection of VR4700 (Fig. 3A) were dec
re sed
y BP nd PVP
y 12-fold (from 1312107) nd 17-fold (from 166496), respecti
vely. In contr st, DMRIE:DOPE co-injection with VR4700 resulted in 3.3-fold in
cre se (from 16645632) in ver ge nti-NP titer. Although BP, PVP, nd DMRIE:DOPE
ll decre sed reporter gene expression levels, only DMRIE:DOPE incre sed the hu
mor l immune response resulting from gene expression. These results were the s m
e using the less potent vector, CMV-NP (Fig. 3B). BP nd PVP decre sed nti-NP t
iters
y 4.6-fold (from 1392304) nd 3.2-fold (from 24064), respectively, where s
DMRIE:DOPE incre sed
190
Norm n et l.

Fig. 3. Adult BALB/c mice were e ch injected i.m. once with 10 g of VR4700 or CMV
-NP DNAs encoding influenz nucleoprotein (NP). DNAs were injected either lone
(None, white
rs) or formul ted with 5% Polyvinyl pyrrolidone or DMRIE:DOPE (PV
P or DM:DP,
l ck
rs) (Su
he dings 2. nd 3.). The corresponding lucifer se DN
A to VR4700 NP (VR1223) is
out 27 times more potent th n the corresponding luc
ifer se DNA to CMV-NP (VR1205) vi the muscle expression ss y (3). At 28 d post
-DNA injection, ser were collected nd diluted seri lly for ntiNP IgG nti
ody
ss y using ELISA. V lues represent ver ge reciproc l nti-NP titers defined
s the dilution giving n O.D. v lue twice the ver ge
ckground (n = 5 mouse se
r ; error
rs re st nd rd errors of the me n). Note th t for
oth vectors, BP
nd PVP decre se,
ut DM:DP incre se the nti
ody response.

nti-NP titers 26-fold (from 2406160). Simil r results were chieved using v riou
s other conditions (see Note 2). This unique ctivity of DMRIE:DOPE m y
e due t
o its
ility to slowly rele se pl smid DNA for upt ke
y muscle. This possi
ili
ty could
e tested
y
Adjuv nts for Pl smid DNA V ccines
191
conducting n expression kinetic study. DMRIE:DOPE m y lso ct s n djuv nt t
h t induces infl mm tion, resulting in the de th of m ny tr nsduced muscle fi
er
s,
ut t the s me time stimul ting the immune response to the ntigen. This cou
ld
e ex mined
y ch r cterizing the immune response in more det il with respect
to the n ture of infl mm tory intr muscul r infiltr ting cells, Th1 or Th2 resp
onses, CTL ctivities, etc. Another potenti l expl n tion of the
ility of DMRI
E:DOPE to decre se tr nsgene expression in muscle, nd, t the s me time, enh nc
e immunity to the tr nsgene product, m y rel te to the n ture of the tr nsduced
cells. It is well documented th t i.m. injection of n ked pl smid DNA results in
the exclusive tr nsduction of myofi
er cells (2,7,17,20). This restricted tr ns
duction of myofi
er cells occurs even when BP is pre-injected
efore DNA injecti
on (8,21). The DMRIE:DOPE/DNA complex, which is quite efficient in tr nsducing n
on-muscle cells, m y
e more c p
le th n n ked DNA in tr nsducing other cells w
ithin the muscle (22,23). Altern tively, the DNA-lipid complex m y
e
etter
l
e to exit the muscle nd tr nsduce dist l tissues (13,16,24). Since intr muscul
r myofi
er cells do not express Cl ss I or Cl ss II MHC ntigens or B7 costimul
tory molecules, it is unlikely th t myofi
er cells tr nsduced with foreign gen
e would
e
le to present ntigen nd stimul te humor l immunity, or eng ge or
ctiv te T cells nd induce cellul r immunity (25). Studies with h plotypic chim
eric mice with reconstituted
one m rrow h ve provided evidence th t n ked DNA-t
r nsduced myofi
er cells rele se ntigen th t is picked up
y resident dendritic
cells, which then process the ntigen for present tion to the immune system (262
9). Since pl smid DNA is r pidly degr ded once injected into muscle (30),
one m
rrow reconstitution w s c rried out 3 wk fter i.m. injection of DNA to dis llo
w loc l tr nsduction of intr muscul r dendritic cells
y residu l DNA (27). In t
his c se, immunity to the tr nsgene epitopes were still ch r cteristic of the tr
nspl nted dendritic cell h plotype nd not the host muscle h plotype. These res
ults support the ide th t tr nsduced muscle provides ntigen to dendritic cells
, which then processes it for present tion to the immune response. This mech nis
m, where
y myofi
er cells synthesize nd provide ntigen to dendritic cells, mig
ht
e gre tly distur
ed if DNA is provided s liposome complex. Tr dition lly,
liposomes re viewed s very effective systems for delivery of proteins or drug
s to the reticuloendotheli l system, in p rticul r to dendritic nd m croph gic
cells (3134). Thus DMRIE:DOPE m y directly nd efficiently deliver i.m. pl smid D
NA to profession l ntigen-presenting cells. This m y
e tested experiment lly

y following the f te of DNA tr nsduction fter i.m. injection of lipid-DNA compl
exes. In summ ry, we report th t the i.m. injection of DMRIE:DOPE/pl smid DNA co
mplexes induce
etter humor l immunity th n injection with n ked

192
Norm n et l.
DNA. Future experiments re imed t
etter ch r cteriz tion of such lipid ind
uced djuv ncy, nd the testing of
ttery of other c tionic lipids for their
inn te
ility to incre se immune responses when complexed with pl smid DNA. 2.
M teri ls
1. Lucifer se su
str te nd cell culture lysis re gent re v il
le from Promeg
(M dison, WI). 2. Firefly lucifer se enzyme re gent is v il
le from An lytic
l Luminescence L
s (S n Diego, CA). 3. 0.9% s line is v il
le from R dix L

s (E u Cl ire, WI). 4. Euth n si -5 solution is v il
le from H. Schein Inc. (P
ort W shington, NY). 5. Fem le 812-wk-old BALB/c mice re v il
le from H rl n S
pr gue D wley (Indi n polis, IN). 6. Bupiv c ine hydrochloride (M rc ine) is v
il
le from A

ott L
s (N. Chic go, IL). 7. PVP (Pl sdoneC30; MW 50 kD ) is v il

le from ISP Technologies, W yne, NJ. 8. DMRIE (()-N-(2-hydroxyethyl)-N,N-dimeth
yl-2,3-
is(tetr decyloxy)-1-prop n minium
romide) is synthesized nd mixed with
co-lipid s descri
ed (20). 9. The lucifer se VR1223 pl smid DNA is constructed
s outlined in det il previously (3). The CMV-NP nd VR4700 re constructed
y
repl cing the lucifer se genes in, respectively, VR1205 nd VR1223, with the inf
luenz /A NP gene (3). 10. Dispos
le, sterile, pl stic insulin syringes nd 28G
1 / 2 needle (BectonDickenson, Rutherford, NJ, C t. No. BD9430) fitted with pl
stic coll r cut from micropipet tip. 11. Micropl te luminometer (Dyn tech, Ch
ntilly, VA, model ML2250). 12. Firefly lucifer se enzyme re gent is o
t ined fr
om An lytic l Luminescence L
s (C t. No. 2400; e.g. 1.69 1013 Rel tive Light Un
its/mg protein). 13. Alk line phosph t se-conjug ted, Fc-specific, go t nti-mou
se IgG is o
t ined from J ckson Immuno Res L
s ( B r H r
or, ME). 14. ELISA pl
te re der is v il
le from Molecul r Devices (Menlo P rk, CA).
3. Methods 3.1. Pl smid DNA Purific tion
1. Tr nsform pl smid DNA into Escherichi coli DH5 or DH10B competent cells nd
grow in Terrific Broth (35) complemented with 100 g/mL mpicillin or 50 g/mL k n
mycin in 5-L fermentor (Applikon, Foster City, CA). Control temper ture nd pH
t 30  0.5C nd 7.0  0.5, respectively. Set the stirring speed t 600 RPM  50 nd th
e ir flow t 5L/L/min. 2. H rvest cells
y centrifug tion t the end of the exp
onenti l growth ph se. Isol te cov lently closed circul r pl smid DNA
y modif
ied lysis procedure (36) followed
y st nd rd dou
le CsCl-ethidium
romide gr di
ent ultr centrifug tion. 3. Determine th t the endotoxin content
y the Limulus
Ame
ocyte Lys te (LAL, Associ tes of C pe Cod, F lmouth, MA) ss y is less th n
0.6 Endotoxin Units/g of pl smid DNA.
Adjuv nts for Pl smid DNA V ccines
193
4. Determine protein content using the
icinchoninic cid ss y (Pierce Chem. Co
., Rockford, IL). The spectrophotometric A260/A280 r tio should
e
etween 1.752.
0. 5. Ensure ll pl smid prep r tions re free of detect
le chromosom l DNA, RN
A nd protein impurities
sed on gel n lysis nd the
icinchoninic ss y, resp
ectively. 6. Eth nol precipit te pl smids nd re-solu
ilize in USP s line t 4C.
Store the DNA t 20C.
3.2. DNA Injections
1. Equili
r te injection fluids nd syringes to room temper ture nd inject sing
le 50 or 100 L volumes in 12 s. 2. Inject the qu driceps muscle of restr ined, w
ke mice with 50 L of DNA in s line, using the syringe nd needle fitted with co
ll r, into the centr l p rt of the rectus femoris muscle. Extensive histologic l
n lyses of muscle injected with L cZ DNA shows th t the rectus femoris is the
prim ry t rget of DNA tr nsfection (20) (see Note 4).

3.3. Extr ction nd Ass y of Lucifer se

1. Add 100 L of lucifer se su
str te to 20 L of muscle extr ct (3). 2. Record s mp
le Light Units sever l times within 5 s fter ddition. 3. C lcul te the lucifer
se content of the s mples from Rel tive Light Units using st nd rd curve of p
urified firefly lucifer se, which is diluted in pooled extr ct from uninjected m
uscles to control for quenching (38). Lucifer se v lues re expressed s ng luci
fer se per mL of muscle extr ct (see Note 5).
3.4. ELISA for Anti-NP Anti
odies
1. Levels of nti-NP IgG A
in mouse ser re determined
y st nd rd ELISA ss y
. Co t ELISA pl tes with recom
in nt NP protein (expressed in
culovirus) t 0.
125 g/well overnight t 4C. 2. W sh,
lock with 5% nonf t milk, nd incu
te with
ser for 2 h t room temper ture (r.t.). 3. W sh, nd then incu
te pl tes with
lk line phosph t se-conjug ted go t ntimouse IgG for 2 h t r.t. 4. W sh g in
, nd incu
te with su
str te (p-nitrophenylphosph te) for 2 h t r.t. T ke O.
D. re dings t 405 nm using n ELISA pl te re der. Titers re determined to
e t
h t serum dilution yielding n O.D. twice th t for
ckground nonimmune serum.
4. Notes
1. Over the p st 7 y, we h ve performed i.m. lucifer se pl smid DNA injections i
nto over 20,000 mouse muscles. An lyses of the expression d t reve l th t the l
evels of expression of given lucifer se pl smid DNA in the mouse rectus femori
s muscle re not st tistic lly different
sed on the nim ls sex, ge (4 wk1 yr),
str in (BALB/c vs C57/Bl), size (2050 gm), or the level of ero
ic ctivity, or
194
Norm n et l.
ffected
y the se son, time of d y, temper ture of injected solution or even co
-injection of the pl smid with wide v riety of ph rm cologic l gents (36 nd
J. H rtikk , unpu
lished o
serv tions). Simil r experiments were repe ted using
medium doses of BP (0.5%), lower or higher DNA doses (10 g nd up to 150 g) with B
P nd PVP, nd with different c tionic lipids. In ll c ses, BP nd PVP nd the
lipids decre sed lucifer se expression, the BP nd PVP decre sed nti-NP titers
nd the lipids incre sed nti-NP titers (d t not shown). In ddition nti-NP ti
ters were me sured t 2 wk nd 2 mo with the s me result (d t not shown). Preli
min ry experiments with mice showed th t st nd rd i.m. injection of 0.75% BP
s reported in the liter ture w s leth l in mice. The
ove results, therefore, c
ould only
e o
t ined using slow (over sever l minutes) injection of the BP th
t ultim tely resulted in the loss of only
out 40% of the nim ls, with the re
st of the surviving mice undergoing v rious degrees of pp rent c rdi c stress.
Thus, BP is severely toxic, even t doses used consistently in the liter ture to
enh nce the expression of i.m. injected pl smid DNA. However, unlike injections
with BP, neither PVP nor DMRIE:DOPE c used visi
ly-untow rd effects on the mice
. Anim l c re throughout the study is in compli nce with the Guide for the Use n
d C re of L
or tory Anim ls, U.S. Dep rtment of He lth nd Hum n Services, N tio
n l Institutes of He lth (NIH Pu
lic tion No. 86-23, revised 1985) s well s wi
th loc l regul tions (e.g., Vic ls Institution l Anim l C re nd Use Committee).
The lucifer se ss y yields <5% v ri tion in light units within triplic te s mpl
es. In control experiments, represent tive muscle extr cts cont ining high, medi
um nd low levels of lucifer se ctivity were stored t 78C in replic te liquots
nd periodic lly ss yed over 3-yr-interv l, yielding <10% v ri tion in light
units.
2.
3.
4.

5.
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in mice. Hum. Gen. Ther. 4, 419431.
18
Cytokine nd Costimul tory F ctor-Encoding Pl smids s Adjuv nts for DNA V ccin
tion
Kenji Okud , Susumu K w moto, nd Jun Fukushim 1. Introduction The induction of
potent nd long-l sting immunity is one of the most import nt elements to con
sider in developing n effective v ccine. DNA v ccines induce m rkedly stronger
CD8+ cytotoxic T lymphocyte (CTL) ctivity th n do tr dition l peptide v ccines
through their p rticul r mech nism of ntigen present tion medi ted
y MHC cl ss
I molecules. Induction of CTL specific to p thogenic viruses is thought to prov
ide reli
le me ns of protecting host from infection nd h lting dise se pro
gression, s these cells c n directly recognize nd lyse infected cells. However

, in most of the e rly studies showing induction of p thogen-specific CTL, ntig

en-encoding immunogenic DNA lone w s used nd DNA vectors encoding immunomodul
ting molecules were not considered. It now ppe rs th t v rious types of immunom
odul tory molecules such s cytokines (IL-1 [1], IL-2 [2], IL-12 [3], IFN- [4], I
L-7 [57], and GM-CSF [8,9]), chemokines (TCA-3 [10], RANTES [11], MIP-1 [11]), an
d costimulatory molecules (CD40L [12], B7-1 [13] and B7-2 [14]) could enhance or
modify the specific immune responses elicited
y DNA immunization (see Ta
le 1)
. In this chapter, we descri
e an approach for achievin more effective DNA vacc
ination throuh the use of plasmids expressin such immunostimulatory molecules.
These plasmids have unique immunomodulatory properties, and coinoculation of
o
th immunoenic and adjuvant plasmids can result in aumentation of an antien-sp
ecific humoral and/or cell-mediated immune response. Several roups have recentl
y reported that coadministration of plasmids expressin cytokines can have sini
ficantly enhanced vaccine-specific immune responses. Althouh the precise mechan
ism responsi
le for their
From: Methods in Molecular Medicine, vol. 29, DNA Vaccines: Methods and Protocol
s Edited
y: D. B. Lowrie and R. G. Whalen Humana Press Inc., Totowa, NJ
197
198
Okuda, Kawamoto, and Fukushima
Ta
le 1 Summary of Effects of Cytokines after Conventional Vaccination and of Ex
pression Plasmids followin DNA Immunization Immunomodulatory Molecules A. Cytok
ine proteins IL-1 IL-2 IL-12 IFN- GM-CSF B. Expression plasmids IL-12 Effect Anti

ody (A
) A
TH1(DTH) A
, DTH A
Ref. (23,24) (2,25,26) (3) (4,25,27) (28,29) (1
5,21,22,30) (15,21) (15,21,22) (9,18,22) (18) (9) (31) (31) (31) (19) (19) (19)
(19) (19) (19) (A. Ihata et al., unpu
lished data) (A. Ihata et al., unpu
lished
data)
GM-CSF
TCA3
B7-1
B7-2

CTL (i.m. and i.n.) DTH (i.m. and i.n.) A
(i.m. and i.n.) A
(i.m.) CTL (i.m.)
3H-TdR uptake (i.m.) CTL (i.m.) DTH (i.m.) A
(i.m.) CTL (i.m.) DTH (i.m.) A
(
.m.) CTL (i.m.) DTH (i.m.) A
(i.m.) A
CTL (i.m.) (i.m.)
CD40(L)
i.m., intramuscular administration; i.n., intranasal administration; A
, anti
od
y production; DTH, delayed type hypersensitivity; 3H-TdR, incorporation of 3H-Th
ymidine; , activated immune response.
remarka
le enhancement of immunity is not yet clear, it is possi
le that these e
xpression plasmids can induce continuous cytokine production for more than sever
al weeks (15). Generally speakin, the
ioloical half-life of recom
inant cytok
ines is short followin injection, compared with that of cytokines continuously
synthesized after injection of expression plasmids. This simple and effective ap
proach for enhancin DNA vaccination may allow new ways of
Cytokine and Costimulatory Plasmids
199

controllin infectious diseases. Several reports have also shown that
acterial
plasmids containin the CpG motif enhance the Th1-type immune response (16,17).
We have focused here on the use of cytokine-expression plasmids as well as certa
in costimulatory molecules as a means of enhancin the immunoenicity of protein
antiens derived from plasmid DNA. For the systematic development of vaccinatio
n strateies usin cytokines or their expression plasmids as adjuvants, it is es
sential to understand the precise involvement of Th1 and Th2 cells in protective
immunity aainst specific micro
ial infections. Usin Th1 (IL-2, IFN-, and IL-12
) and Th2 (IL-4, and IL-10) type cytokines, we can also define the respective ro
les of Th1 and Th2 cells at the various staes of each infection. Recent reports
have indicated that certain costimulatory molecules also enhance the immunity c
onferred with plasmid DNA immunoens (18,19). In Ta
le 1, we have summarized the
immunomodulatory effects o
served with co-inoculation of various plasmids. 2. M
aterials 2.1. Expression Plasmids
1. HIV-1IIIB p160 (pCMV160IIIB) and Rev (pcREV) (20): each injection mixture co
ntains 20  pCMV160IIIB and 510  of pcREV. 2. Interleukin-12 (pCAGGSIL-12) (15): d
onated
y Dr. J. Miyazaki (Osaka University, Japan) (Fi. 1). We use 1020  per mo
use of this plasmid. The pCAGGS vector ives hiher expression levels than the p
CMV vector (15). 3. Interleukin-2 (pBCMGNeo-mIL2): donated
y Dr. H. Karasuyama
(Tokyo Metropolitan Institute of Medical Science, Japan). This plasmid enhances
the Th1 type immune response. We use 1020  per mouse as cytokine adjuvant. 4. Int
erleukin-4 (pCAGGS/IL-4): donated
y Dr. J. Miyazaki (Osaka University, Japan) (
Fi. 1). This plasmid enhances Th2 type immune responses at a dose of a
out 1020 
per mouse. 5. Interferon- (nkCMVintMuIFN): donated
y Dr. H. Kohsaka (Tokyo Medic
al and Dental University, Tokyo, Japan). This cytokine enhances the Th1 type res
ponse. 6. TCA3 (pPGKX
aI/Neo/TCA3): donated
y Dr. M. E. Dorf (Harvard Medical S
chool, Boston, MA) (10). TCA3 enhances Th1 type immune responses at a dose of 120
 per mouse. 7. B7-1 and B7-2: donated
y Dr. G. J. Freeman (Dana-Far
er Cancer
Institute, Boston, MA) (13,14). B7-2 enhances Th1-type immune responses (18) whe
n used at around 10100  per mouse. 8. GM-CSF (VR-1701): donated
y Dr. R. H. Zau
 (VICAL Inc., San Dieo, CA). It stronly enhances Th2-type immune responses, a
nd su
stantial activation of the Th2-type response occurs with around 520  per mo
use. There are other reports in which 50100  of GM-CSF plasmid were used (9).
200
Okuda, Kawamoto, and Fukushima
Fi. 1. Constructs of cytokine expression plasmids. (A) Expression plasmid of IL
-12; (B), Expression plasmid of IL-4. 9. CD40L: donated
y Dr. J. Inoue (Institu
te of Medical Science, University of Tokyo, Japan). This plasmid enhances
oth T
h1 and Th2 type immune responses when 1050  per mouse is administered.
2.2. Other Materials
1. 2. 3. 4. 5. 610-wk-old BALB/c mice. 27 aue needle syrine. Hematocrit capill
ary tu
e. 100 L pipettor. 70% (v/v) ethanol in a spray
ottle.
Cytokine and Costimulatory Plasmids
201
3. Methods We enerally use two routes for vaccine delivery. Intramuscular immun
ization enhances the Th1-type immune response. The intranasal route of immunizat
ion activates the production of intestinal and vainal IA (21), an important fe
ature for sexually-transmitted diseases.
3.1. Immunization and Sample Collection
The
iceps femoris muscle of BALB/c mice is inoculated with naked DNA constructs
. In our early experiments, to enhance muscle cell uptake of plasmid DNA, muscle

was injected with 100 L of 25% (w/v) sucrose in PBS 1530 min
efore DNA inoculati
on. However, our recent data show that the injection of 25% sucrose does not si
nificantly modify immune responses. A total of 100 L of the DNA mixture, containi
n 530  of pCMV160IIIB and pcREV and expression plasmids of cytokine (150 ) or cos
timulatory su
stance (150 ), is injected into the muscle usin a 27-aue needle
syrine. Blood is collected
y retro-or
ital puncture usin a capillary tu
e, an
d sera are o
tained
y centrifuation. Sera are stored at 80C until ELISA assays a
re performed.
3.2. Intramuscular Immunization (see Note 1)
1. Pour a
out 2030 mL of diethyl ether into a jar with a volume of a
out 500 mL.
Gauze should
e spread on the
ottom. 2. After the diethyl ether has fully evapo
rated, introduce the mice into the jar. 3. When the mouse is no loner active (a
fter a
out 20 s), allow several more seconds to pass. When the animal
eins
re
athin deeply, remove it from the anesthetic jar. When
reathin
ecomes deep, t
he mouse should not
e kept in the anesthetic jar any loner than 10 s since dea
th may ensue at this stae of anesthesia (see Notes 2 and 3). 4. Place the mouse
in the prone position and slowly inject the vaccine preparation into the
iceps
femoris muscle (see Notes 4 and 5). 5. Withdraw the needle from the muscle appr
ox 5 s after completin the injection. This is thouht to prevent the injected s
olution in the muscle from leakin throuh the needle pore.
3.3. Intranasal Administration (see Notes 15)
1. Anesthetize mice
y inhalation of diethyl ether and spray the injection site
with 70% (v/v) ethanol for disinfection. 2. Grasp the mouse ently with one hand
and turn it face upward (see Note 6). 3. Drop the mixture of DNA vaccine and cy
tokine plasmids into the nasal cavity with a micropipetter. Allow the mouse to i
nhale the DNA as it
reaths (see Note 7). 4. Allow the mouse to inhale one drop
(57 L)
efore another drop is introduced (see Note 8). 5. Repeat steps 5 and 6.
202
4. Notes
Okuda, Kawamoto, and Fukushima
1. We used 150  of DNA per mouse whereas other roups (9,22) use 20100  per mouse.
We have not o
tained a sinificant
ody of data reardin the use of over 50  o
f DNA. 2. Too deep or too lon a period of anesthesia will often kill mice. 3. I
t is important to ventilate the room or to perform the anesthesia step in a vent
ilated hood since inhalation of ether as represents a health risk. 4. Injectin
hypertonic sucrose solution into muscle results in muscular edema that is thou
ht to help to retain the DNA solution. However, results of previous work on the
effect of this treatment on reporter ene expression are am
iuous; one showed t
hat it was hihly useful whereas another found that it had no effect. Moreover,
there has
een no pu
lished study showin conclusively that this treatment enhan
ces antien-specific immune responses. Our current thinkin is that sucrose-pret
reatment of muscle does not reatly enhance DNA-
ased immunity. 5. For ease and
convenience, lare muscle is preferred when choosin the site for the DNA inject
ion. Previous work has demonstrated that quadriceps femoris muscle is superior t
o astrocnemius muscle for reporter ene expression. However, injection into qua
driceps requires that the mouse
e placed in a supine position, which is difficu
lt to maintain once the animal awakens from anesthesia. Since it is easier to ke
ep a mouse in the prone position, we usually use the
iceps femoris muscle. 6. T
he neck should not
e tihtened or pressed for more than a few seconds as this w
ill easily kill the mouse. 7. It is important not to wet the face of the mouse w
ith alcohol or ether since moisture on the hair around the nasal cavity will oft
en cause the vaccine solution to leak away
y widenin the drops of the DNA vacc
ine. 8. It is easy to drop the DNA solution into only one nasal cavity
ecause t
he mouse can inhale freely throuh the other nostril without distress. A 30-L vol
ume of the DNA mixture is the maximum administered in one i.n. immunization. If

the mouse starts to dischare the DNA solution from its nose, it is safe to stop
the inhalation process. If additional inhalation steps are desired usin the sa
me animal, it is
etter to wait 30 min
efore repeatin the procedure. If the to
tal volume employed is over 30 L, i.n. administration should
e carried out in se
veral treatments spaced 45 h apart. If the volume inhaled is too reat, the mouse
will die
y asphyxiation.
References
1. Nencioni, L., Villa, L., Talia
ue, A., Antoni, G., Presentini, R., Perin, F.
, et al. (1987) In vivo immunostimulatin activity of the 163171 peptide of human
IL1
eta. J. Immunol. 139, 800804. 2. Xim, K. Q., Hamajima, K., Saski, S., Honsho
, A., Tsuji, T., Ishii, N., Cao, X.-R., Lu, Y., Fukushima, J., Kawamoto, S., and
Okuda, K., (1998) Intranasal co-inoculation of IL-2 expression plasmid with AID
S DNA vaccine enhances all-mediated immunity aainst HIV-1. Immunoloy 94, 438444
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, and Scott, P. (1994) The adjuvant effect of interleukin-12 in a vaccine aains
t Leishmania major. Science 263, 235237. 4. Playfair, J. H., and De, S. J. (1987)
Recom
inant amma interferon is a potent adjuvant for a malaria vaccine in mice
. Clin. Exp. Immunol. 67, 5-10. 5. Bui, T., Faltynek, C. R., and Ho, R. J. (1994
) Bioloic response of recom
inant interleukin-7 on herpes simplex virus infecti
on in uinea pis. Vaccine 12, 646652. 6. Bui, T., Faltynek, C., and Ho, R. J. (1
994) Differential disposition of solu
le a liposomes-formulated human recom
inan
t interleukin-7: Effect on
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s. 11, 633641. 7. Bui, T., Dykers, T., Hu, S.-L., Faltynek, C. R., and J., H. R.
(1994) Effect of MTPPE liposomes and interleukin-7 on induction of anti
ody and
cell-mediated immune responses to a recom
inant HIV-envelope protein. J. AIDS 7,
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y, A., Golum
ek, P., Levitsky, H., Bro
se, K., et al. (1993) Vaccination with irradiated tumor cells enineered to secr
ete murine ranulocyte-macrophae colony-stimulatin factor stimulates potent, s
pecific, and lonlastin anti-tumor immunity. Proc. Natl. Acad. Sci. USA 90, 353
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onse to a plasmid-encoded viral antien
y coinoculation with plasmids expressin
 cytokines. Immunity 2, 129135. 10. Lanin, J., Kawasaki, H., Tanaka, E., Luo, Y
., and Dorf, M. E. (1994) Inhi
ition of in vivo tumor rowth
y the
eta chemoki
ne, TCA3. J. Immunol. 153, 46254635. 11. Schrum, S., Pro
st, P., Fleischer, B., a
nd Zipfer, P. F. (1996) Synthesis of the cc-chemokines Mip-1a, Mip-1
, and RANTE
S is associated with a type 1 immune response. J. Immunol. 157, 35983604. 12. Yan
, Y., and Wilson, J. M. (1996) CD40 liand-dependent T cell activation: require
ment of B7-CD28 sinalin throuh CD40. Science 273, 18621864. 13. Freeman, G. J.
, Gray, G. S., Gimmi, C. D., Lom
ard, D. B., Zhou, L.-J., White, M., et al. (199
1) Structure, expression, and T cell costimulatory activity of the murine homolo
ue of the human B lymphocyte activation antien B7. J. Exp. Med. 174, 625631. 14
. Freeman, G. J., Borriello, F., Hodes, R. J., Reiser, H., Gri

en, J. G., N, J
. W., et al. (1993) Murine B7-2. An alternative CTLA4 counter-receptor that cost
imulates T cell proliferation and interleukin 2 production. J. Exp. Med. 178, 21
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I., et al. (1997) Enhancement of cell-mediated immunity aainst HIV-1 induced
y
coinoculation of plasmid-encoded HIV-1 antien with plasmid expressin IL-12. J
. Immunol. 158, 40084014. 16. Krie, A. M., Yi, A.-K., Matson, S., Waldschmidt, T
. J., Bishop, G. A., Teasdale, R., et al. (1995) CpG motifs in
acterial DNA tri
er direct B-cell activation. Nature 374, 546549. 17. Krie, A. M. (1996) Lympho
cyte activation
y CpG dinucleotide motifs in prokaryotic DNA. Trends Micro
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4, 7377. 18. Iwasaki, A., Stiernholm, B. J. N., Chan, A. K., Bernstein, N. L., a
nd Bar
er, B. H. (1997) Enhanced CTL responses mediated
y plasmid DNA immunoen

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al. (1997) Immunomodulatory effects of a plasmid expressin B7-2 on human immuno
deficiency virus-1-specific cell-mediated immunity induced
y a plasmid encodin
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ima, K., Kawamoto, S., Sekiawa, K., Yamada, Y., et al. (1995) Induction of pote
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anulocytemacrophae colony-stimulatin factor (GM-CSF) expressin plasmids in li
posomes induce stron mucosal and cell-mediated immune response aainst HIV-1 an
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Chatteroon, M. A., Dan, K., Wan, B., et al. (1997) In vivo enineerin of a
cellular immune response
y coadministration of IL-12 expression vector with a D
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, Pihl, D. L., Conlon, P. J., and Gra
stein, K. H. (1989) IL-1 as adjuvant. Role
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ody production
y recom
inant h
uman IL-1 alpha. J. Immunol. 142, 31293133. 25. A
raham, E., and Shah, S. (1992)
Intranasal immunization with liposomes containin IL-2 enhances
acterial polysa
ccharide antien-specific pulmonary secretory anti
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, 37193726. 26. Wein
er, A., and Merian, T. C. (1988) Recom
inant interleukin 2
as an adjuvant for vaccine-induced protection. Immunization of uinea pis with
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unit vaccines. J. Immunol. 140, 294299. 27. Heath, A. W.
, and Playfair, J. H. L. (1993) Cytokines as immunoloical adjuvants. Vaccine 7,
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., Huse
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: an effective adjuvant for protein and peptide-
ased vaccine. Blood 88, 202210.
29. Tao, M. H., and Levy, R. (1993) Idiotype GM-CSF fusion protein as a vaccine
for B cell lymphoma. Nature 362, 755758. 30. Kim, J. J., Ayyavoo, V., Baarazzi,
M. L., Chatteroon, M. A., Dan, K., Wan, B., et al. (1997) Enhancement of cell
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y coinoculation of plasmid-encoded HIV
-1 antien with plasmid expressin IL-12. J. Immunol. 158, 816826. 31. Tsuji, T.,
Fukushima, J., Hamajima, K., Ishii, N., Aoki, I., Bukawa, H., Ishiatsu
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Tani, K., Oku
o, T., Dorf, M. E., and Okuda, K. (1997) HIV-1-specific cellmediat
ed immunity is enhanced
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ccine. Immunoloy 90, 16.
19
Optimization of DNA Vaccines Throuh the Use of Molecular Adjuvants
Jon J. Kim and David B. Weiner 1. Introduction Althouh the injection of DNA in
to tissues was oriinally reported in the 1950s, the technoloy has ained more
attention in recent years as a safe means of mimickin in vivo protein productio
n normally associated with natural infection (13). Nucleic acid or DNA inoculatio
n is an important vaccination technique that delivers DNA constructs encodin sp
ecific immunoens directly into the host (411). These expression cassettes transf
ect the host cells, which
ecome the in vivo protein source for the production o
f antien. This antien then is the focus of the resultin immune response. This
vaccination technique is
ein explored as an immunization stratey aainst a v
ariety of infectious diseases as well as cancer. Nucleic acid immunization may a
fford several potential advantaes over traditional vaccination strateies such
as whole killed or live attenuated virus and recom
inant protein-
ased vaccines.
Since DNA vaccines are nonreplicatin and the vaccine components are produced w
ithin the host cells, they can
e constructed to function safely with the specif

icity of a su
unit vaccine. However, DNA vaccine cassettes should produce immuno
loical responses that are more similar to live vaccine preparations. By directl
y introducin DNA into the host cell, the host cell is essentially directed to p
roduce the antienic protein, mimickin viral replication or tumor cell marker p
resentation in the host. This process has
een reported to enerate
oth anti
od
y and cell-mediated, particularly cytotoxic T-cell-mediated, immunity. Unlike a
live attenuated vaccine, conceptually there is little risk from reversion to a d
isease-causin pathoen from the injected DNA, and there is no risk for secondar
y infection as the material injected is not livin and non-infectious. In
From: Methods in Molecular Medicine, vol. 29, DNA Vaccines: Methods and Protocol
s Edited
y: D. B. Lowrie and R. G. Whalen Humana Press Inc., Totowa, NJ
205
206
Kim and Weiner
Fi. 1. Cytokines as immune modulators. Cytokines play critical roles in the imm
une and inflammatory responses. Based upon their specific function in the immune
system, these cytokines can
e further rouped as Th1 and Th2 cytokines. Alon
with costimulatory molecules, these cytokines also play important roles in the a
ctivation and proliferation of T and B cells.
addition, enes that lead to undesired immunoloic inhi
ition or cross-reactivit
y (autoimmunity) may
e either altered or deleted altoether. Finally, DNA vacci
nes can
e manipulated to present a particular enome of the pathoen or display
specific tumor antiens in non-replicatin vectors. The oal of the first ener
ation DNA immunization was to demonstrate the DNA vaccines a
ility to elicit humo
ral and cellular responses in vivo in a safe and well-tolerated manner in model
systems. As we explore the next eneration of DNA vaccines, our oal is to refin
e the current stratey to elicit more clinically efficacious immune responses. T
he next eneration immunoens, for example, may require finer control of the ma
nitude and direction (humoral or cellular) of the immune responses induced. Such
refinement could
e accomplished
y co-deliverin enes for immunoloically imp
ortant molecules, such as costimulatory molecules and cytokines that play critic
al reulatory and sinalin roles in immunity (12) (Fi. 1). There have
een sev
eral reports of immune modulation
y protein delivered cytokines. However, the r
esults in eneral appeared marinal. More recently, we and others have focused o
n analyzin immune responses induced to such ene delivery. Raz et al. o
served
that intramuscular injection of plasmids
Optimization Usin Molecular Adjuvants
207
encodin IL-2, IL-4, or TGF-1 modestly modulated immune responses to transferrin
protein delivered at a separate site (13). IL-2 immunization resulted in an enha
ncement of anti
ody and T helper proliferative responses while TGF-1 immunization
reduced anti-transferrin responses. Xian and Ertl reported that intramuscular
co-inoculation of plasmid expressin the lycoprotein of ra
ies virus and plasmi
d-encodin mouse GM-CSF enhanced the B and T helper cell activity to ra
ies viru
s, while co-inoculation with plasmid-expressin IFN- resulted in a decrease of th
ese responses (14). Similarly, Kim et al. reported that co-immunization of GM-CS
F enes with DNA vaccine constructs increased antien-specific anti
ody and T he
lper cell proliferation responses while co-immunization with IL-12 enes resulte
d in weaker anti
ody responses and enhanced T helper cell proliferation (15). In
addition, IL-12 co-immunization resulted in a sinificant enhancement of CTL re
sponses. Kim et al. also reported on the effects of costimulatory molecules, ano
ther set of immunoloically relevant molecules, as DNA vaccine adjuvants (16). C

o-immunization of DNA vaccines with CD86 results in increased T helper cell prol
iferation and CTL (8). Iwasaki et al. more recently reported that GM-CSF and IL12 co-delivery, with DNA immunoen encodin for influenza NP, resulted in enhanc
ed cellular immune responses. Furthermore, two other reports in addition to Kims
independently confirmed that co-delivery of CD86, and not CD80, with a DNA immun
oen-enhanced T cell mediated immune responses (8,17,18). In addition to these r
eports, Chow et al. reported that either injection of plasmid co-expressin hepa
titis B surface antien (HBsA) and IL-2 or co-injection of IL-2 enes with plas
mid expressin HBsA resulted in the enhancement of
oth anti
ody and T helper c
ell responses (19). More recently, we investiated the induction and reulation
of immune responses from the co-delivery of proinflammatory cytokines (IL-1, TNF-,
nd TNF-), Th1 cytokines (IL-2, IL-15, nd IL-18), nd Th2 cytokines (IL-4, IL-5
, nd IL-10) (20). We o
served enh ncement of ntigen-specific humor l response
with the co-delivery of Th2 cytokines IL-4, IL-5, nd IL-10, s well s th t of
IL-2 nd IL-18. A dr m tic incre se in ntigen-specific T helper cell prolifer t
ion w s seen with IL-2 nd TNF- co-injections. In ddition, we o
served signifi
c nt enh ncement of the cytotoxic response with the co- dministr tion of TNF- nd
IL-15 genes with HIV-1 DNA immunogens. These incre ses in CTL response were
ot
h MHC cl ss I-restricted nd CD8+ T cell-dependent. These results collectively d
emonstr te th t ntigen-specific immune responses c n
e modul ted
y the co-inj
ection of costimul tory molecule nd cytokine genes with DNA immunogen c ssettes
. More gener lly, they demonstr te the potenti l of this str tegy of co-deliveri
ng immunologic lly import nt genes s vehicle for the development of import nt
immunogens nd the investig tion of immunologic lly import nt molecules in vivo
.
208
Kim nd Weiner
Fig. 2. Flow di gr m for using molecul r djuv nts.
1.1. Prep r tion of Molecul r Adjuv nts
The first step is to clone t rgeted genes into m mm li n expression vector (Fi
g. 2) (21). Such genes c n
e cloned using PCR with set of primers nd cDNA
templ te. For inst nce, CD80 nd CD86 genes were cloned from hum n B cell cDNA l
i
r ry (Clontech, Inc., P lo Alto, CA) nd pl ced into n expression vector unde
r the control of CMV promoter (8). In cloning molecul r- djuv nt gene into
n expression vector, two different ppro ches c n
e utilized. One str tegy is c
loning directly sever l different inserts into single vector under the control
of one or more independent promoter (18,19). The second str tegy is cloning int
o sep r te construct for e ch insert nd then mixing together the desired com

in tions t the time of injection (8,15). The dv nt ge of cloning different ins
erts into vector with single
ck
one is gre ter likelihood of co-expressi
ng the v rious proteins in the s me cell. In ddition, the prep r tion of sing
le pl smid m y s ve time nd m teri ls. The m jor dis dv nt ge of this method is
th t e ch ntigen/ djuv nt pl smid must
e independently cloned for e ch com
i
n tion. Such specific utiliz tion of n ntigen/ djuv nt com
in tion is inflex
i
le for investiOptimiz tion Using Molecul r Adjuv nts
209
g ting v rious ntigen nd djuv nt com
in tions. Although cloning sep r te cons
tructs ffords v ri
ility in constructs, the
ility to express ll of these in
the s me cells is still re son
le concern (8,15). We h ve shown, however,
o
th in vivo nd in vitro th t the mixture of pl smids prior to tr nsfection le ds
to co-expression of different pl smids within the s me cell (22). Once gene i
nsert is cloned into n expression vector, it c n then
e sequenced to test the

fidelity of the insert. If the sequencing result is s tisf ctory, the pl smid ex
pression c ssette c n
e tested for the correct protein expression
y v riety
of methods. The test of expression in vitro requires tr nsfection of cell line
with the desired pl smid construct. Tr nsfection of m mm li n cells c n
e done
with the liposom l or electropor tion methods. The liposom l method involves en
c psul tion of the pl smid DNA into liposom l rtifici l mem
r ne vesicle. The
liposomes fuse with the t rget cell mem
r ne nd introduce DNA to the cytopl sm
of the cell. Some tr nsfected DNA will loc lize in the nucleus through n unch
r cterized process nd this nucle r-loc lized DNA gener tes the RNA tr nscripts
th t re tr nsported to the endopl smic reticulum for tr nsl tion into ntigenic
proteins. In the electropor tion method,
rief, high volt ge electric pulse c
re tes n nometersized pores in the cell mem
r ne. DNA is t ken directly into the
cell cytopl sm either through these pores, or s consequence of the redistri

ution of mem
r ne components th t ccomp nies closure of the pores. The tr nsfec
ted cells c n then
e tested for expression of the protein encoded through the c
onstruct
y ny of the following methods:
1.1.1. Immunoprecipit tion
The expression of protein produced
y the tr nsfected cells c n
e ss yed
y im
munoprecipit tion. This involves r diol
eling the tr nsfected cells. Following
l
eling, the cells re lysed, nd specific nti
odies re dded to precipit te
the desired protein. The ntigen- nti
ody complex is then n lyzed
y electropho
resis nd visu lized
y r diogr phy. Altern tively, following electrophoresis, u
nl
elled protein c n
e tr nsferred to nitrocellulose. The specific ntigen det
ection then c n
e ccomplished
y the immuno
lotting technique (Western
lottin
g).
1.1.2. Flow Cytometer
The expression of proteins c n
e tested on the tr nsfected cells with fluores
cence- ctiv ted cell sorter (FACS) ss y. FACS uses the
ility of flow cytomete
r to detect nd count individu l cells p ssing in stre m in front of it. These
instruments re used to study the properties of cell su
sets identified with mo
noclon l nti
odies to cell-surf ce proteins. Individu l cells within mixed po
pul tion re first t gged
y tre tment with specific monoclon l nti210
Kim nd Weiner
Fig. 3. Detection of murine IL-12
y ELISA. IL-12 genes (encoding
oth the p35
nd the p40 ch ins) were cloned into expression pl smids under the control of C
MV promoter. The resulting pl smid w s tr nsfected in vitro into RD cells. Expre
ssion of IL-12 in the culture supern t nt w s identified using ELISA.

odies l
eled with fluorescent dyes. The mixture of l
eled cells is then force
d with much l rger volume of s line through nozzle, vi
r ted t high frequen
cy in order to
re k the stre m into droplets, some of which will cont in cells.
As e ch droplet with cell p sses in front of l ser
e m, it sc tters the l
ser light, nd dye molecules
ound to the cell will
e excited nd, if ppropri
tely t gged, will fluoresce. Sensitive photomultiplier tu
es detect
oth the sc
ttered light, which gives inform tion on the size nd gr nul rity of the cell,
nd the fluorescent emissions, which give inform tion on the specific
inding of
the l
eled nti
odies nd hence on the expression of cell-surf ce proteins
y t
r nsfected cells.
1.1.3. ELISA (Enzyme-Linked Immunosor
ent Ass y)
Detection of solu
le protein such s cytokines c n
e conducted using ELISA (Fig
. 3). Numerous vendors offer cytokine ELISA kits. These ss ys involve c pturing
the protein of interest
y monoclon l nti
ody co ted onto polystyrene micr
otiter pl te. A second nti
ody conjug ted to n enzyme re cts with the c ptured

molecule, immo
ilizing the enzyme in the microtiter well. Su
str te is dded to

the well, nd the enzyme gener tes color proportion l to the mount of molecu
le. One dv nt ge of this ss y is th t the mount of protein expression c n
e
qu ntified.
Optimiz tion Using Molecul r Adjuv nts 1.1.4. Immunohistochemistry/Immunofluores
cence
211
Immunohistochemistry or immunofluorescence c n
e used to n lyze the tr nsfecte
d cells. Immunohistochemic l n lysis involves the
inding of specific nti
odie
s to protein expressed on cells nd detecting them
y microscopy. Immunofluore
scence is technique in which molecules re detected using nti
odies l
eled w
ith fluorescent dyes. The
ound fluorescent nti
odies c n
e detected
y micros
copy. In vivo detection of proteins
y immunohistochemistry or immunofluorescenc
e techniques re simil r to those of in vitro methods. Anim ls re immunized wit
h cDNA constructs nd the tissues t the site of expression re removed. For int
r muscul r injection, the expression of pl smid-encoded protein c n
e detected
s e rly s 72 h post-injection (8). The fresh tissue is then frozen, nd the sl
ides prep red
y sectioning the tissues (Fig. 4).
1.2. An lyzing Immune Responses Following Immuniz tion.
Once the ntigenic nd djuv nt DNA v ccine constructs re prep red, they c n
e
dministered to nim ls to study the induction of immune responses. The nim ls
c n
e immunized vi different routes (intr muscul r, intr derm l, or mucos l),
or different delivery mech nisms (p rticle gun, or syringe nd needle). The co dministr tion of v rious gene expression c ssettes involves the mixing of chose
n pl smids prior to injection. To
etter study the modul tion effect of the mole
cul r djuv nt constructs, su
-optim l dose of ntigenic gene construct should

e utilized. The host immune system responds to infection or immuniz tion vi i
nduction of specific nd regul ted humor l nd cellul r immune responses. The im
mune responses induced
y immuniz tion using DNA v ccine constructs c n
e n ly
zed.
1.2.1. Humor l Response
B cells medi te the humor l immune response. M ture B cells c rry surf ce immuno
glo
ulins th t ct s their ntigen receptor. They then move through the circul
tion to second ry lymphoid tissues, where they respond to ntigenic stimuli
y d
ividing nd differenti ting into pl sm cells under control of cytokines produce
d
y T cells. Pl sm cells re termin lly differenti ted B cells th t re entire
ly devoted to the production of ntigen-specific nti
odies. Anti
odies re seru
m proteins secreted
y pl sm cells th t re induced following cont ct with nti
gen, nd they
ind specific lly to the inducing ntigen. The n lysis of the nt
i
ody responses c n
e done
y ELISA with specific ntigen to determine the pr
esence of ntigen-specific nti
odies in the serum of the immunized nim ls (Fig
. 5).
212
Kim nd Weiner
Fig. 4. Co-expression of HIV-1 envelope gp120 protein with CD80 or CD86 on muscl
e cells. Frozen muscle sections were prep red from DNA-injected nim ls nd st i
ned with FITC-l
eled (green) -CD86 nti
odies nd Tex s Red-l
eled (red) -gp120
nti
odies. A) A slide from leg immunized with pcDNA3 (control vector) w s st
ined with -CD86 nd -gp120 nti
odies. B) A slide from leg immunized with pCEnv+
pCD86 w s st ined with -CD86 nd -gp120 nti
odies.
1.2.2. T Helper Cell Prolifer tive Response

Activ tion nd prolifer tion of T helper (Th) lymphocytes pl y critic l roles in

inducing nd exp nding
oth humor l nd cellul r immune responses
y ctiv ting

oth B nd cytotoxic T cells, respectively. T cells from immunized nim ls prol
ifer te when exposed to specific ntigens,
ut not to unrel ted protein. Prolife
r tion c n
e determined
y me suring the level of incorpor tion of 3 H-thymidin
e into the DNA of ctively dividing cells. We c n n lyze the v ccines
ility to
elicit CD4 + Th cell immunity
y me suring the ntigen-specific prolifer tion i
n this m nner (Fig. 6).
Optimiz tion Using Molecul r Adjuv nts
213
Fig. 5. HIV-1 envelope-specific nti
ody response following co-immuniz tion with
IL-12 or GM-CSF genes. Fifty g of IL-12 or GM-CSF genes were co- dministered wit
h pCEnv cDNA expression c ssettes intr muscul rly t d y 0. Prior to injection
nd t d ys 14 nd 28, the mice were
led nd the ser collected. The mouse ser
were tested for envelope-specific nti
ody responses, with the ELISA s descri
e
d using HIV-1 gp120 protein.
1.2.3. Cytotoxic T Lymphocyte (CTL) Response
CD8+ cytotoxic cells or CTLs kill t rget cells th t displ y ntigenic fr gments
of cytosolic p thogens, most not
ly viruses,
ound to MHC cl ss I molecules t
the cell surf ce. Specific recognition of peptide:MHC complexes on t rget cell

y cytotoxic CD8 T cell c n le d to the killing of the t rget cell through tw
o different mech nisms. First, the killer T cells rele se gr nzymes, which re t
oxic su
st nces th t puncture holes in the mem
r nes of t rget cells. Second, th
e cytotoxic T cells sign l the t rget cells to poptosis vi the f s-f s lig nd
inter ction. Cytotoxic T cells c n recycle to kill multiple t rgets. CD8 T-cell
function c n
e determined
y T-cell
io ss y me suring the killing of t rge
t cell
y cytotoxic T cell. This is usu lly detected in 51Cr-rele se ss y.
Live cells will t ke up,
ut do not spont neously rele se, r dio ctively-l
eled
sodium chrom te, N 251CrO4. T rget cells re l
eled with r dio ctive N 251CrO4
nd exposed to cytotoxic T cells. When these l
eled cells re killed
y either
mech nism of CTL induced killing, the r dio214
Kim nd Weiner
Fig. 6. T-helper cell prolifer tion responses following co-immuniz tion with IL12 or GM-CSF genes. Two weeks fter the first DNA immuniz tion with pCEnv, the m
ice were
oosted with s me mount of pl smid. After one ddition l week, spleens
were collected from immunized mice nd the lymphocytes were isol ted. Recom
in
nt gp120 protein (5 g/mL fin l concentr tion) w s dded to e ch well to stimul te
the prolifer tion of T helper cells.

ctive chrom te is rele sed nd its presence in the supern t nt of mixtures of t

rget cells nd cytotoxic T cells c n
e me sured. Cell destruction is me sured

y the rele se of r dio ctive chromium into the medium, detect
le within four h
ours of mixing t rget cells with T cells (Fig. 7).
1.2.4. Ch llenge Models
The over ll go l of n immuniz tion str tegy is to induce specific immune respon
ses th t confer lifetime protection from the p thogen of interest. Thus, n impo
rt nt test of v ccines immunogenicity is the
ility to protect the host from ch
llenge. For infectious dise ses, it would
e optim l to ch llenge the immunized
nim ls with the ctu l p thogen of interest in the host of interest. However, f
or s fety nd pr ctic l re sons, this is usu lly not done. Accordingly nim l mo
del system of infection c n provide us with import nt evidence of v ccine utilit

y. Sever l DNA v ccine constructs h ve
een reported to protect sm ll nim ls fr

om vir l or
cteri l p thogens such s influenz , m l ri , nd TB p thogens (7,
10,23). On the other h nd, not ll models re
Optimiz tion Using Molecul r Adjuv nts
215
Fig. 7. HIV-1 envelope-specific CTL responses in mice immunized with pcDNA3, pCE
nv, pCEnv+IL-12, or pCEnv+GM-CSF. CTLs were n lyzed g inst v ccini infected t
rgets within vitro stimul tion of effector cells.
relev nt for different infectious gents. Bec use HIV-1 does not infect mice nd
other sm ll rodents, prim tes represent the most relev nt ch llenge system for
HIV v ccine ev lu tion. Specific lly, there re currently three different prim t
e models for HIV v ccine studies. They re the SIV nd chimeric (SIV)/ HIV-1 (SH
IV-1) ch llenge models in m c ques nd HIV ch llenge model in chimp nzees. Our l

or tory h s o
served protection of m c ques from SHIV-1 ch llenge s well s
protection of chimp nzees from n HIV-1 ch llenge (24,25). Ch llenging of immun
ized nim ls with tumor cells expressing relev nt p thogenic or tumor ntigens
lso represents relev nt protection model (26,27). 2. M teri ls 2.1. Immunoprec
ipit tion
1. RIPA
uffer: 50 mM Tris-HCl pH 7.6; 150 mM N Cl; 0.2% (v/v) Triton X-100; 0.2
% (w/v) Deoxycholic cid; 0.1% (w/v) SDS nd 1 mM PMSF. 2. 35S protein l
eling
mix is v il
le from NEN/DuPont (Boston, MA). 3. SDS-PAGE m teri ls.
216 2.2. Flow Cytometer
Kim nd Weiner
1. FACS
uffer: PBS cont ining 1% (w/v) BSA nd 0.1% (w/v) sodium zide. 2. Anti

odies.
2.3. Immunohistochemistry/Immunofluorescence
1. 2. 3. 4. 5. 6. Leic 1800 Cryost t (Leic Inc., Deerfield, IL). Pro
eOn Plus
Slides (Fisher Biotech, Pitts
urgh, PA). 1.5% (v/v) go t serum (Vector L
s, Bur
ling me, CA). Strept vidin-Biotin HRP (Rese rch Genetics, Huntsville, AL). DAB (
Vent n Biotech, Tucson, AZ). Microscope.
2.4. Detecting Humor l Response Using ELISA
1. 2. 3. 4. 5. 6. 0.1M c r
on te-
ic r
on te
uffer (pH 9.5). PBS-0.05% (v/v) Tw
een-20. 3% (w/v) BSA in PBS with 0.05% (v/v) Tween-20. HRP-conjug ted go t ntimouse IgG (Sigm , St. Louis, MO). 3355 TMB (Sigm ). Pl te re der.
2.5. T Helper Cell Prolifer tion Ass y
1. H rvested splenocytes. 2. Culture medi : RPMI (Gi
co-BRL, Gr nd Isl nd, NY),
10% (v/v) fet l c lf serum (Gi
co-BRL). 3. 96-well microtiter fl t
ottom pl te.
4. Triti ted (3H) thymidine. 5. Pl te re der.
2.6. CTL Ass y
1. H rvested splenocytes. 2. CTL culture medi : RPMI (Gi
co-BRL), 10% (v/v) fet
l c lf serum (Gi
coBRL), nd 10% (v/v) RAT-T-STIM without Con A (Becton Dickinso
n L
w re, Bedford, MA). 3. N 251CrO4. 4. G mm counter. 5. 5% (v/v) Triton X-100
.
3. Methods 3.1. Immunoprecipit tion
1. Tr nsfect cells with the pl smid construct of interest. 2. W sh the cells twi
ce with PBS, st rve them for one hour in DMEM l cking serum, methionine nd cyst
eine, nd then l
el them with 200 Ci/mL (1200 Ci/mmole) of 35S protein l
eling
mix.

Optimiz tion Using Molecul r Adjuv nts

217
3. Lyse the l
eled cells in 0.5 mL of RIPA
uffer on ice nd then cl rify the l
ys te
y centrifug tion t top speed in microcentrifuge for 10 min. 4. Incu
t
e the cl rified lys tes with specific nti
ody for 90 min on ice. 5. Add Protein
A-Seph rose to the result nt ntigen- nti
ody complexes nd mix
y sh king t 4C
for 90 min. 6. Resuspend the protein pellet in 50 L of s mple
uffer, extensivel
y w shing it in
uffers cont ining high s lt nd BSA nd he t the fin l suspensi
on t 100C for 35 min fter. 7. An lyze the protein s mple
y 12% (w/v) SDS-PAGE.
3.2. Flow Cytometer
1. W sh cells (1 105) three times with FACS
uffer nd incu
te with FITC- or PE
-conjug ted mA
s t s tur ting conditions for 30 min on ice. 2. W sh cells thr
ee times with FACS
uffer nd n lyze them using FACSc n.
3.3. Immunohistochemistry/Immunofluorescence
1. Inocul te mouse qu driceps muscles with the pl smid construct of interest (8)
. 2. Remove the qu driceps muscles 72 h l ter. 3. Cut 6 m cryost t sections using
Leic 1800 cryost t nd pl ce on Pro
eOn Plus slides (Fisher Biotech). 4. Fix
the slides in cetone, w sh, nd ir dry. 5. Block the slides with 1.5% (v/v) g
o t serum nd incu
te in prim ry nti
ody t room temper ture. 6. Incu
te the
slides with
iotinyl ted second ry nti
ody followed
y incu
tion with strept
vidin-
iotin HRP. 7. Tre t the slides with DAB nd counter-st in them. 8. View
the slides with microscope.
3.4. Detecting Humor l Response Using ELISA
1. Dilute the protein ntigen to 2 g/mL diluted in 0.1M c r
on te-
ic r
on te
uf
fer (pH 9.5). 2. Adsor
50 L of the diluted protein onto microtiter wells overnig
ht t 4C. 3. W sh the pl te with PBS-0.05% (v/v) Tween-20 nd
lock with 3% (w/v)
BSA in PBS with 0.05% (v/v) Tween-20 for 1 h t 37C. 4. Add mouse ntiser dilut
ed with 0.05% (v/v) Tween-20 nd incu
te the pl te for 1 h t 37C. 5. Add HRP-co
njug ted go t nti-mouse IgG nd incu
te the pl te for 1 h t 37C. 6. W sh the p
l te nd develop it with 3355 TMB
uffer solution. 7. Re d the pl te on pl
te re der with the optic l density t 450 nm.
3.5. T Helper Cell Prolifer tion Ass y
1. H rvest lymphocytes from the spleen nd remove the erythrocytes. 2. Resuspend
the isol ted cell suspensions to concentr tion of 5 106 cells/mL.
218
Kim nd Weiner
3. Add 100 L liquot cont ining 5 105 cells to e ch well of 96-well microtite
r fl t-
ottom pl te cont ining specific protein t v rious concentr tions. 4.
Incu
te the pl te t 37C in 5% (v/v) CO2 for 3 d. 5. Add 1 Ci of triti ted thymid
ine to e ch well nd incu
te the pl te for ddition l 1218 h t 37C. 6. H rvest t
he pl te nd me sure the mount of incorpor ted triti ted thymidine on Bet Pl
te re der. 7. The stimul tion index w s determined from the formul : Stimul tio
n Index (SI) = (experiment l count/spont neous count)
3.6. Cytotoxic T Lymphocyte Ass y
1. H rvest lymphocytes from spleens nd remove the erythrocytes. 2. Stimul te th
e effector cells with stimul tor cells for 45 d in CTL culture medi t 5 106 cel
ls per mL. 3. CTL culture medi consists of RPMI, 10% (v/v) fet l c lf serum, n
d 10% (v/v) RAT-T-STIM without Con A. 4. Perform st nd rd chromium-rele se ss
y in which the t rget cells re l
eled with 100 Ci/mL N 251CrO4 for 60120 min n

d used to incu
te with the stimul ted effector splenocytes for 46 h t 37C. 5. De
termine CTL lysis t effector:t rget (E:T) r tios r nging from 50:1 to 12.5:1. 6
. Following incu
tion, h rvest supern t nts nd count on LKB CliniG mm g mm
-counter. 7. Percent specific lysis w s determined from the formul : 100 experim
ent l spont neous rele se (m ximumrele se) rele se spont n
8. M ximum rele se is determined
y lysis of t rget cells in 5% (v/v) Triton X-1
00 cont ining medium. An ss y should not
e considered v lid if the v lues for
the spont neous rele se counts re in excess of 20% of the m ximum rele se.
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. Press, Cold Spring H r
or, NY. 22. Kim, J.
J., Ag dj ny n, M. G., Trivedi, N., Wilson, D. M., Morrison, L. D., Nottingh m,
L. K., Ts i, A., D ng, K., et l. (1999) CD86 (B7-2) expression on non-
one m r
row derived cells c n induce ntigen-specific cytotoxic T lymphnodes in vivo. J.
Immunol., in press. 23. Dool n, D., Sedeg h, M., Hedstrom, R., Ho
rt, P., Ch r
oenvit, nd Hoffm n, S. (1996) Circumventing genetic restriction of protection
g inst m l ri with multigene DNA immuniz tio: CD8+ cell-; interferon g mm -, n
d nitric oxidedependent immunity. J. Exp. Med. 183, 17391746. 24. Boyer, J. D., W
ng, B., Ugen, K., Ag dj ny n, M. G., J v di n, M. A., nd Frost, P., et l. (19
96) Protective nti-HIV immune responses in non-hum n prim tes through DNA immun
iz tion. J. Med. Prim tol. 25, 242250. 25. Boyer, J. D., Ugen, K. E., W ng, B., A
g dj ny n, M. G., Gil
ert, L., B g r zzi, M., et l. (1997) Protection of chimp
nzees from high-dose heterologous HIV-1 ch llenge
y DNA v ccin tion. N t. Med.
3, 526532. 26. W ng, B., Merv , M., D ng, K., Ugen, K. E., Boyer, J. D., Willi ms
, W. V., nd Weiner, D. B. (1994) DNA inocul tion induces protective in vivo imm
une responses g inst cellul r ch llenge with HIV-1 ntigen-expressing cells. AI
DS Res. Hum. Retroviruses 10, S35S41. 27. W ng, B., Merv , M., D ng, K., Ugen, K.
E., Willi ms, W. V., nd Weiner, D. B. (1995) Immuniz tion
y direct DNA inocul
tion induces rejection of tumor cell ch llenge. Hum. Gene Ther. 6, 407418.
20
Cytokine Fusion Constructs s DNA V ccines Ag inst Tumors
Holden T. M ecker, Ath n si Syrengel s, nd Ron ld Levy 1. Introduction 1.1. Tu
mor Antigens
1.1.1. Overview of Types of Tumor Antigens
V rious studies h ve used DNA v ccin tion s method of immunizing g inst tumo
rs (112). As with ny tumor v ccine, one ch llenge is to find truly tumor-speci
fic ntigen (13,14). The m jority of immunologic lly t rgeted tumor ntigens re
lso expressed on su
set of norm l host cells. Ex mples of such ntigens incl
ude prost te-specific ntigen, nd CD20, B cell m rker. Some tumor ntigens r
e specific for ctiv ted cells of cert in types, such s c rcinoem
ryonic ntige
n (CEA) or the IL-2 receptor. These re often found on em
ryonic or fet l cells
s well s tumor cells. The c r
ohydr te ntigens of mel nom s nd the immunoglo

ulin (Ig) idiotype of B cell lymphom s represent tumor-specific ntigens (TSA).
Unfortun tely, TSA h ve not
een identified in more common m lign ncies. Furthe
rmore, the ntigenic determin nts of known TSA m y differ
etween p tients; for
ex mple, the tumor idiotype (Id) of B cell lymphom is highly p tient-specific
nd must
e determined for e ch c se. In ddition to protein nd c r
ohydr te nt
igens, peptide determin nts h ve lso
een exploited s tumor-specific v ccines.
These include the MAGE ntigens of cert in mel nom s s well s peptides derive
d from ctiv ted oncogenes such s r s or myc. Using this ppro ch, present tion
of peptide in the context of host MHC le ds to ctiv tion of ntigen-specific T
cells. Peptide sequences h ve
een used successfully s DNA v ccines (3). Howev
er, the polymorphic n ture of MHC complic tes the design of peptide v ccines, n
d
From: Methods in Molecul r Medicine, vol. 29, DNA V ccines: Methods nd Protocol

s Edited
y: D. B. Lowrie nd R. G. Wh len Hum n Press Inc., Totow , NJ

221
222
M ecker, Syrengel s, nd Levy
the
sence of the n tive protein from which the peptide w s derived most likely
precludes the induction of humor l immunity. In n effort to further explore DN
A immuniz tion g inst TSA, work in our l
or tory h s focused on DNA v ccin tio
n g inst Id using the murine B cell lymphom , 38C13. Immuniz tion with pl smids
encoding the he vy nd light ch in v ri
le regions of 38C13, which cont in the
tumor Id, c n protect mice g inst su
sequent tumor ch llenge (11,12). Inductio
n of nti-Id nti
odies
y DNA v ccin tion,
ut not tumor protection, h s
een d
emonstr ted in nother mouse B lymphom model, BCL-1 (9,10). In ddition to the
38C13 model, we h ve used ov l
umin. s n ntigen for DNA v ccin tion. Although
it is not true tumor ntigen, ov l
umin. is dv nt geous in th t prolifer tiv
e nd cytotoxic T cell responses g inst it c n
e re dily me sured.
1.1.2. Pro
lems of Immune Responses to Tumor Antigens
Ig idiotypes, s well s m ny of the other tumor ntigens descri
ed
ove, re onl
y we kly immunogenic. The ntigenic differences th t confer specificity for tumo
r re su
tle when comp red to simil r proteins norm lly found in host tissue. In
ddition, there m y
e need to overcome host toler nce to these ntigens. For
ex mple, B cell lymphom -
e ring host will h ve lre dy
een exposed to l rge
qu ntities of the tumor idiotype. Thus, potenti lly responsive host cells m y h
ve
een tolerized
y deletion or nergy (15,16). The host m y, therefore, not r
espond to these ntigens unless they re presented in novel, immunostimul tory
context. This m y consist of foreign c rrier protein such s keyhole limpet h
emocy nin, or foreign immunoglo
ulin (Ig) const nt region. Altern tely, cytoki
ne sequences, whose products c n provide stimul tory sign ls to the proper immun
e cells, c n
e used to m ke fusion constructs. In the c se of the 38C13 system,
foreign const nt region sequences re required for tumor protection when v ccin
ting with DNA encoding whole Ig (12). A single-ch in Fv (scFv) construct, which
cont ins the V regions of he vy nd light ch ins connected
y flexi
le linker
, is poorly immunogenic on its own. However, ddition of sequence derived from
IL-1 to the scFv DNA construct renders it immunogenic nd results in good tumor
protection (11).
1.2. Adv nt ges of DNA V ccin tion 1.2.1. No Need to Purify Protein
Unlike protein immuniz tion, DNA immuniz tion elimin tes the need to produce r
ecom
in nt protein in su
st nti l qu ntities. Ig production is often limiting
step when m king customized v ccines for p tients with B cell lymphom . The simp
licity of cloning the necess ry sequences nd the e se of producing l rge qu nti
ties of pl smid m ke production of DNA v ccine much f ster in such c ses.
Cytokine Fusion Constructs 1.2.2. A
ility to Induce Cellul r Immunity
223
DNA v ccin tion lso h s the dv nt ge of inducing cellul r immune responses. Si
nce DNA immuniz tion results in endogenous production of ntigen
y host cells,
the t rget ntigen c n
e presented on
oth cl ss I nd cl ss II MHC. In f ct, D
NA immuniz tion h s
een shown to induce strong cytotoxic T cell (CTL) responses
in num
er of systems (1732). By contr st, immuniz tion with solu
le protein c
n sometimes induce CTL responses,
ut gener lly only in the presence of strong
djuv nts (33,34). This is presum
ly due to the difficulty of getting solu
le pr
oteins introduced into the intr cellul r p thw y for present tion
y cl ss I MHC
, requirement for CD8-restricted CTL responses.

1.3. Construction of Pl smids 1.3.1. Immunostimul tory Sequences

The immunostimul tory qu lities of cert in CpG-cont ining sequences derived from

cteri l DNA h ve
een widely reported (3537). It is therefore dv nt geous to
choose n expression pl smid for DNA v ccin tion th t cont ins optim l immunosti
mul tory sequences (ISS). We h ve chosen to work with the pl smid pId (12,38) (F
ig. 1), which cont ins n mpicillin-resist nce gene th t h s
een shown in othe
r systems to encode powerful ISS (36).
1.3.2. Foreign Const nt Regions for Immunoglo
ulins
When constructing n idiotype-specific DNA v ccine, it is possi
le to com
ine th
e he vy nd light ch in V regions of the tumor Ig in n scFv construct. This eli
min tes the need for pl smid expressing two sep r te proteins for he vy nd li
ght ch ins. However,
ec use of the immunostimul tory effect of foreign Ig const
nt regions mentioned
ove, it m y
e more desir
le to construct vectors th t
encode whole Ig molecules. For this purpose,
icistronic pl smid th t independ
ently codes for Ig he vy nd light ch ins w s designed (38). This pl smid cont i
ns ppropri te restriction sites into which Ig he vy nd light ch in V regions c
n
e cloned (Fig. 1).
1.3.3. Du l Promoter vs IRES for Multiple Ch in Constructs
In lieu of vectors encoding two promoters, one for he vy nd one for light ch in
, it is lso possi
le to link the he vy nd light ch in coding regions with n i
ntern l ri
osom l entry site (IRES). The IRES llows for tr nsl tion of two codi
ng regions (he vy nd light ch in in this c se) from the s me RNA mess ge. In th
eory, this should coordin te the expression levels of the two ch ins most precis
ely, since ri
osomes will
e
le to initi te tr nsl tion of
oth ch ins with eq
u l efficiency. In pr ctice, we h ve found no dv nt ge in tumor protection in v
ivo, when comp ring du l promoter pl smid to the s me pl smid modified to cont
in single promoter with n IRES (unpu
lished d t ).
224
M ecker, Syrengel s, nd Levy
Fig. 1. Schem tic of the pId pl smid used for DNA immuniz tion of 38C13 Igcytoki
ne constructs, nd pOVA pl smid used for DNA immuniz tion with ov l
umin.
1.3.4. Le der Sequences for Secretion of Protein Le der sequences, found t the
5 end of the coding region of mem
r ne nd secreted proteins, ensure th t the s
ynthesized protein is t rgeted to the endopl smic reticulum nd thus enters the
secretory p thw y. Ig molecules h ve endogenous le ders, which c n
e cloned fro
m tumor cDNA together with the v ri
le region sequences, s we h ve done for 38
C13. We h ve lso used exogenous le ders, such s the hum n k pp -ch in le der s
equence, with nonIg coding regions, such s ov l
umin. (32). Is secreted produ
ct necess ry for efficient DNA v ccin tion? If DNA is tr nsfected into resident
host tissue, such s muscle or skin cells, these cells
Cytokine Fusion Constructs
225
c n secrete the protein for upt ke
y host APC. However, this m y not
e necess
ry for DNA v ccin tion to work, since resection of the t rget tissue even minute
s fter DNA injection did not lter nti
ody or CTL responses in two different s
ystems (39). In f ct, direct tr nsfection of APC in the tissue nd/ or dr ining
lymph nodes h s
een implic ted s critic l to the effic cy of DNA v ccin tion (
40). However, we h ve comp red DNA v ccin tion g inst 38C13 idiotype using two
simil r pl smids, one of which led to efficient secretion of the encoded protein
, while the other did not. In this system, nti
ody responses nd tumor protecti

on were not detected in the nim ls th t received the pl smid encoding the nonse
creted version of 38C13 (our unpu
lished d t ). Thus, it m y
e helpful in t le
st some systems to ensure th t cells t king up the pl smid DNA secrete the t rg
et ntigen.
1.3.5. Choice of Cytokines
The use of cytokine gene linked to the coding region for the t rget ntigen h
s
een shown to
e n effective w y of inducing nti-tumor immune responses when
using solu
le protein v ccin tion (41,42). In the first c se, the GM-CSF coding
region w s linked to the 3 end of the he vy ch in const nt region. This result
ed in production of protein th t consisted of the entire Ig molecule with two
GM-CSF molecules, one tt ched to e ch of the he vy ch in const nt regions. The
tumor protection o
served with 38C13-GM-CSF fusion protein w s dependent upon
the
io ctivity of the cytokine, s hum n GM-CSF, which is in ctive in mice, cou
ld not induce protection (41). GM-CSF w s chosen for its
ility to enh nce nti
gen present tion
y dendritic cells. However, other cytokines, including IL-2 n
d IL-4, h ve lso
een shown to
e effective in the s me system (42). The cytoki
nes th t we h ve tested in fusion constructs with t rget ntigens c n
e divided
into three groups: (i) those th t c n potenti lly enh nce ntigen present tion,
such s GM-CSF nd IL-1
; (ii) those th t c n enh nce Th1 immunity, such s IL2, IFN, and IL-12; and (iii) those that can enhance anti
ody production or Th2 im
munity, such as IL-4. The most extensive comparison of these cytokines has
een
done in the oval
umin system (32). In this system, anti
ody production was relat
ively unchaned reardless of the choice of cytokine, while sinificant CTL acti
vity could
e found with all constructs. However, IL-12 and a nonapeptide sequen
ce derived from IL-1 appeared to induce the stronest and most rapid CTL response
s (32). In the 38C13 system, the IL-1 peptide construct was a
le to induce tumor
protection in a scFv system, whereas scFv alone was not protective (11). In prot
ein vaccination with cytokine fusion constructs, vaccine efficacy was dependent
upon covalent linkae of the antien and cytokine (41,43). This was
226
Maecker, Syrenelas, and Levy
true when measurin tumor protection in the 38C13 system (41) as well as when me
asurin the conversion of a Th2-dominated immune response to a Th1-dominated imm
une response in the oval
umin. system (43). However, this was not the case for D
NA vaccination. No difference could
e seen in anti
ody responses when usin a 3
8C13-GMCSF fusion construct versus coinjection of 38C13 and GM-CSF on separate p
lasmids (our unpu
lished data). In either case, the addition of GM-CSF resulted
in a hiher proportion of immunized animals (12). Similarly, others have shown t
hat coinjection of a DNA vaccine with a cytokine protein (5) or with a cytokineencodin plasmid (9,44) could enhance anti
ody responses to tumor or viral anti
ens. The difference
etween protein and DNA vaccines reardin the need for cyto
kine fusion may
e explained
ecause coinjection of two DNA plasmids results in
cotransfection of the same host antien-presentin cells. This would result in t
he cytokine actin on the same cells as are processin the antien. Alternativel
y, it may
e sufficient to express the antien and cytokine in the same microenv
ironment even if not
y the same cells.
1.3.6. Linkers
If antien and cytokine are to
e fused in a recom
inant construct, native anti
enic conformation as well as cytokine
ioactivity must
e preserved. We have use
d a linker consistin of two lycine residues to connect the antien and cytokin
e. While this almost always works,
ioactivity of the cytokine is diminished in
some cases, possi
ly due to conformational pro
lems. A loner linker, [(Gly)4Ser
]3, was used to fuse the heavy and liht chain V reions in scFv constructs.
1.3.7. Tas: His, Myc

A variety of ta sequences can
e added to recom
inant constructs to facilitate pu

rification or identification of the protein product. Two tas studied in our la

oratory are the hexahistidine ta (his ta) and a sequence of six amino acids fr
om the myc oncoprotein (myc ta). The his ta can
ind to nickel chelate resins,
while the myc ta encodes an epitope reconized
y the 9E10 antimyc anti
ody. T
hese tas have
een added to the 3 end of a construct, althouh alternative pla
cements may also
e feasi
le. Althouh DNA vaccination does not require a ta, s
ince there is no need to purify protein, it may
e convenient to introduce one s
o that the construct can
e easily tested for secretion in vitro. Followin tran
sient transfection into COS-7 cells, for example, the cell supernatant can
e as
sayed
y ELISA or Western
lot for production of the proper protein. The myc ta
is useful when the protein product is to
e detected
y Western
lottin, as th
e 9E10 anti
ody is a superior reaent for Westerns. In
Cytokine Fusion Constructs
227
certain cases, we have found these tas to fail in that the recom
inant proteins
are not detected in such assays, althouh the constructs are detected
y anti
o
dies to the antien itself. This may occur throuh maskin of the ta due to the
conformation of the protein. Thus, the efficacy of such tas must
e tested on
a case-
y-case
asis. The presence of forein ta sequences may also introduce n
ew antienic epitopes into the recom
inant protein or DNA vaccine. Althouh we h
ave never demonstrated this definitively, the possi
ility cannot
e discounted.
We therefore recommend that appropriate controls
e included when comparin vacc
ines that contain tas. In the majority of our experiments, mice were immunized
intramuscularly with 100  of naked DNA. When compared to intradermal immunizatio
n, we found that equivalent levels of specific anti
ody could
e raised
y eithe
r method,
ut that intramuscular injection yielded a hiher ratio of IG2a to I
G1 anti
odies (12). This is consistent with a Th1-dominated immune response for
intramuscular injection, and a Th2-dominated response for intradermal injection.
This has
een found for
oth 38C13 and oval
umin. antiens (our unpu
lished dat
a). However, in other systems, investiators have reported that the isotype rati
o and cytokine profile of immunized animals was unaffected
y the route or dose
of immunization
ut instead corresponded to the method of vaccination (ene un
versus injection) (45). Other investiators have found that the injected muscle
cells can
e resected after DNA vaccination without detrimental effect, suesti
n that the taret tissue is not important in inducin the immune response (39).
2. Materials 2.1. Plasmid Construction
2.1.1. Expression Plasmids
The mammalian expression plasmids pId (12,38) and pOVA (32) are depicted in Fi.
1. The pId plasmid is desined for clonin of heavy and liht chain V reions (
human or mouse) into cassettes which contain human I constant reions. Appropri
ate restriction sites have
een included in order to clone I V reions as well
as cytokines into this plasmid. A sinle promoter version of this plasmid contai
nin an IRES (see Su
headin 1.3.3.) has also
een made. The pOVA plasmid is a m
odified version of pId desined for expression of oval
umin or other non-immuno
lo
ulin enes (32).
2.2.2. I V Reions
The primers shown in Ta
le 1 were used to clone the V reions of 38C13 from cDNA
of a hy
ridoma expressin 38C13 I. The heavy chain primers contain SalI and Nh
eI sites at their 5 and 3 ends, respectively, to allow in228
Maecker, Syrenelas, and Levy

Ta
le 1 Primers for Clonin 38C13 V Reions, Oval
umin, and Cytokine enesa Gene
38C13 VK Restriction sites Forward primer (restriction sites) Reverse primer (r
estriction sites) 5 GTAGATCTCTCACCATGGGACCGTCT 3 5 TCTACGTACGTTTTATTTCCAACTTG
GTCCC 3 5 ACAGTCGACATGGAGTTGTGG 3 5 AGGTGCTAGCTGAGGAGACGGTGA 3 5 GGCCCACGA
TGTGGCTCCATCGGCGCAG 3 5 CCTCTCGAGGGGGGAAACACATCT 3 5 CCTCTCGAGGGGGAAACACATCT
3 5 GTCATCGATGGCGGAGCACCCACCCGC 3 5 CCGGATCCTCATTTTTGGACTGGTTT 3 5 GTCATC
GATAGTTCAGGGTGAAGAAAGTAA CGATAAATAAG 3 5 GGGGATCCTTTATCGTTACTTTCTTCACCCTG AACT
CCGC 3 5 CTTATCGATGGCGGAGCACCCACTTCAAG 3 5 CCGGATCCTTATTGAGGGCTTGTTG 3 5 G
TCATCGATGGCGGATGTTACTGCCACGGC 3 5 CCCGGGGATCCTCAGCAGCGACTCCTTTTC 3 5 GTCATCG
ATGGCGGAATGTGGGAGCTGGAG 3 5 GGGGATCCCTAGGATCGGACCCTGCAG 3 5 GTCATCGATGGCGGAC
ATATCCACGGATG 3 5 CCCGGGGATCCCTACGAGTAATCCATTTG 3
BlII BsiW1 38C13 VH SalI NheI oval
umin DraIII XhoI
XhoIc mGM-CSF ClaI BamHI h
IL-1pep ClaI BamHI mIL-2 ClaI BamHI mIFN ClaI BamHI mIL-12p40 ClaI BamHI mIL-4 Cla
I BamHI
aFor
With
cytokine primers used for the pOVA plasmid, see (32). stop codon. cWithout stop
codon, for linkin to cytokine ene.
frame clonin of the pId plasmid with the human IG1 constant reion. The liht
chain primers contain BlII and BsiW1 sites at their 5 and 3 ends, respectivel
y, to allow in-frame clonin with the human kappa constant reion.
2.2.3. Cytokine Genes The primers shown in Ta
le 1 were used to clone the enes
for murine GMCSF, IL-2, IFN, and IL-4
y PCR from Concanavalin A-stimulated spl
een cells. IL-12 p35 and p40 enes were amplified in a similar way from previous
ly cloned plasmids (a enerous ift of Steven Wolf, Genetics Institute, Cam
rid
e, MA), usin the primers shown in Ta
le 1. For the IL-1
nonapeptide sequence (
46), the primers shown in Ta
le 1 were annealed toether and extended usin Pfu
polymerase without the need for an exoenous template. All cytokine primers cont
ain ClaI and BamHI restriction sites at their 5 and 3 ends, respectively, to a
llow clonin into the pId vector downstream of and in
Cytokine Fusion Constructs
229
frame with the codin sequence of the heavy chain constant reion. In the oval
u
min system, cytokine enes were cloned into XhoI and BamHI sites so as to
e dow
nstream of and in frame with the oval
umin. codin sequence. All I and OVA sequ
ences were recloned without stop codons, which were then incorporated into the c
ytokine reverse primers just upstream of the BamHI site.
1. RNAzol B reaent is availa
le as from TelTest B Inc. (Friendswood, TX). 2. Prom
ea RT kit is availa
le from Promea Corp. (Madison, WI). 3. Cloned Pfu polymera
se Pfu
uffer is availa
le from New Enland Biola
s (Beverly, MA). 4. Restrictio
n enzymes are availa
le from New Enland Biola
s. 5. Concanavalin A is availa
le
from Sima Chemical Co. (St. Louis, MO). 6. MeaPrep column is availa
le from P
romea.
2.2. Vaccination 2.2.1. Tumor Model The murine B lymphoma cell line 38C13, deriv
ed in C3H/eB mice, has
een previously descri
ed (47).
1. Culture the cells in RPMI-1640 medium with 10% (v/v) fetal calf serum, 100 U/
mL penicillin, 100 /mL streptomycin, 2 mM lutamine, and 50 M 2mercaptoethanol (c
RPMI). 2. Thaw the cells from a common stock 3 d prior to tumor challene and r
ow them in lo phase.
2.2.2. Chemotherapeutics
1. Dissolve cyclophosphamide (Sima) in sterile saline and inject it intraperito
neally (i.p.) at 100 m/k.

2.3. Readouts 2.3.1. Direct Readouts of Tumor Plot survival usin Kaplan-Meier p
lots, with sinificance determined
y a lo-rank or Gehan score (48). 2.3.2. Rea
douts of Immune Response
2.3.2.1. ELISA OF SERUM ANTIBODIES 1. 38C13 IM: purify this from ascites of a h
y
ridoma-rescue fusion secretin 38C13 I (49). 2. Microtainer serum separator t
u
es, Cat. No. 36-5960, are availa
le from Becton Dickinson (Rutherford, NJ). 3.
Coat microtiter plates, Maxisor
, are availa
le from Nunc, Inc. (Naperville, IL
). 4. Anti-mouse IG-peroxidase, or su
class specific anti
odies are availa
le f
rom Southern Biotechnoloy Associates (Birminham, AL) or Calta La
oratories (S
outh San Francisco, CA).
230
Maecker, Syrenelas, and Levy
5. Anti-38C13 monoclonal anti
odies: mix S4C8 (IG1), S1C5 (IG2a), and S5A8 (I
G2
) (49) at a 2:1:1 ratio and use as a standard. 6. Microplate reader is availa

le from Molecular Devices (Sunnyvale, CA).
2.3.2.2. T CELL PROLIFERATION ASSAYS 1. [3H]-Thymidine is availa
le from Amersha
m Corp. (Arlinton Heihts, IL). 2. Lympholyte M is availa
le from Accurate Chem
ical Co. (West
ury, NY). 3. 96-well cell harvester and scintillation counter for
96-well format are availa
le from Wallac (Turku, Finland). 2.3.2.3. CTL ASSAYS
1. Oval
umin-transfected cell line EG.7-OVA (a ift of M. Bevan, University of W
ashinton, Seattle, WA): row the cells in DMEM medium with 10% (v/v) fetal calf
serum and 200 /mL G418 (Gi
co-BRL, Grand Island, NY). 2. Parental cell line EL4 for use as a neative control taret is availa
le from the American Type Cultu
re Collection: row it in DMEM medium with 10% (v/v) fetal calf serum. 3. Recom

inant human IL-2 is availa
le from Cetus Corporation (Emeryville, CA). 4. [51Cr]
-sodium chromate is availale from Amersham. 5. Gamma counter is availa
le from P
ackard Instruments (Meriden, CT).
3. Methods 3.1. Plasmid Construction 3.1.1. Clonin of I V Reions
1. Extract RNA
y centrifuin up to 107 hy
ridoma cells expressin the desired
I and resuspendin them in 1 mL of RNAzol B reaent. Follow the manufacturers di
rections. 2. Use 1  of total RNA in a total volume of 20 L to produce cDNA usin
the Promea RT kit. Incu
ate the reaction at 42C for 60 min, then keep it at 4C or
frozen. 3. Use 1 L of cDNA, or approximately 1  of cDNA, in a PCR reaction conta
inin 84 L water, 10 L of cloned Pfu
uffer, 4 L of 10 mM dNTP mix, 2 L of a pair of
50 M primers as shown in Ta
le 1, and 1 L of Pfu polymerase. Overlay the PCR reac
tion with mineral oil and cycle as follows: 1 cycle: 5 min at 94C, 5 min at 50C, 5
min at 72C. 35 cycles: 30 s at 94C, 30 s at 50C, 1 min at 72C. 1 cycle: 7 min at 72C
. 4. Extract the PCR product with chloroform, precipitate with 0.1 volume of 3 M
sodium acetate and 2 volumes of ethanol, spin, wash in 70% (v/v) ethanol, and r
esuspended in 10 L water. Diest with restriction enzymes as shown in Ta
le 1, us
in the manufacturers recommended conditions. 5. Purify the diested PCR product
on a el and liate it into the pId vector; cut the vector with matchin enzymes
to allow directional liation of the V reion. 6. Transform the liated DNA int
o
acteria
y conventional methods. Screen colonies
y restriction diests of mi
niprep DNA to identify those that contain a correct insert. Verify the identity

y DNA sequencin
efore proceedin with a iven clone.
Cytokine Fusion Constructs
231
7. Repeat the restriction diestion, liation, and transformation to allow cloni
n of the remainin (heavy or liht chain) V reion. When clonin new V reions
into the plasmid, note whether the heavy or liht chain V reions contain either

of the restriction sites used to clone the opposite chain. This will determine
the order in which the V reions must
e cloned into the plasmid.
3.1.2. Clonin of Cytokine Genes
1. For clonin most cytokine enes, harvest C3H.HeN mouse spleen cells as descri

ed
elow. Resuspend the lymphocytes at 5 106 cells per mL in cRPMI medium and a
dd 5 /mL Concanavalin A. Incu
ate the cells for 24 h at 37C. 2. Extract RNA
y ce
ntrifuin the cells and then resuspendin them in 1 mL of RNAzol B reaent. Car
ry out RNA purification, cDNA synthesis, and PCR as descri
ed a
ove for clonin
of V reions. 3. Once a clone containin the proper insert is o
tained, row the
plasmid in larescale culture, and purify
y either cesium chloride
andin or

y use of a MeaPrep column (see Notes 1 and 2).
3.1.3. Testin for Cytokine Bioactivity Before usin a new construct for DNA vac
cination, it is prudent to test it for expression and cytokine
ioactivity.
1. 2. 3. 4. Transfect COS-7 cells. Two to three days after transfection, harvest
the supernatant. Test for protein
y immunoprecipitation, Western
lottin, and
ELISA. Check the
ioactivity of the cytokine
y usin an indicator cell assay (
see ref. 32 for cell lines and procedure) (see Note 3).
3.2. Vaccination 3.2.1. Tumor Challene Experiments
1. Vaccinate mice intramuscularly, with 100  of DNA in 100 L of 0.9% (w/v) saline
iven
ilaterally, 50 L into each quadriceps muscle, 23 times at 13 wk intervals,
then challened with tumor cells 23 wk after the last vaccination. 2. Draw
lood
approximately 18 d after each vaccination, and assay the serum for specific anti

odies
y ELISA (see Su
headin 3.3.2.1.). 3. A
out two wk after the last vaccin
ation, wash 38C13 cells in lo phase rowth three times and resuspend them in RP
MI-1640 medium without serum. 4. Serially dilute the cells to 400 cells per mL a
nd inject 0.5 mL (200 cells) i.p. or s.c. per mouse (see Notes 46).
3.2.2. Use of Chemotherapy and Vaccination A more riorous test of vaccine effic
acy is the a
ility to induce an antitumor immune response in animals with alread
y esta
lished tumors. However, this is difficult
ecause of the fast rowth rate
of experimental tumors such as 38C13. To more closely mimic the human clinical
situation, as well as contain tumor rowth, we have performed chemotherapy and v
accination experiments.
232
Maecker, Syrenelas, and Levy
1. Inject 200 38C13 cells s.c. 2. Vaccinate with DNA as descri
ed,
einnin sev
eral hours to days post tumor challene. 3. When palpa
le tumors are evident on
all mice (usually a
out 1012 d), inject the animals i.p. with a non-curative dose
of 100 m/k cyclophosphamide (Sima). 4. Record mouse survival as a
ove.
3.3. Readouts 3.3.1. Direct Readouts of Tumor
The endpoint of all tumor challene experiments is survival.
1. Record the date of death of each animal. 2. Plot survival on a Kaplan-Meier p
lot (Fi. 2). 3. Calculate the statistical sinificance of differences
etween 
roups
y the Gehan or lo rank alorithm (48) which takes into account
oth prol
onation of survival and the appearance of lon-term survivors (see Notes 7 and
8).
3.3.2. Readouts of Immune Response
3.3.2.1. ANTIBODY RESPONSE: SERUM ELISA (see Notes 9 and 10)
1. Bleed mice from the tail vein into Microtainer serum separator tu
es. Spin th
e tu
es for 30 s in a microfue to separate serum from
lood cells, and freeze t
he sera for use in later assays. 2. Coat microtiter plates with 38C13 or a simil
arly purified control IM at 2.5 /mL in PBS for 24 h at 4C. Store coated plates w

rapped in parafilm at 4C for up to several months. 3. Wash the coated plates thre
e times
y flickin out the contents of the wells over a sink, and immerse the p
lates in a
ucket of ELISA wash
uffer (0.9% (w/v) saline with 0.5% (v/v) Triton
-X100). 4. Saturate the washed plates with 100 L per well of 5% (w/v) nonfat milk
in PBS and incu
ate at room temperature for 30 min, and flick out nonfat milk/P
BS without washin. 5. Prepare serial dilutions of the sera from vaccinated mice
in 2% (w/v)
ovine serum al
umin. (BSA) in PBS, startin at 1:201:200 dilution,
with serial 1:2 dilutions in a final volume of 50 L, over 816 wells. Include a sta
ndard anti38C13 anti
ody cocktail containin IG1, IG2a, and IG2
isotypes in
a 2:1:1 ratio over 8 wells on each assay plate, startin at 1 /mL. Incu
ate the
plates an additional 1 h at room temperature. 6. Wash the plates as in step 3, t
hen add 50 L per well of anti-mouse IG (amma chain specific) at a 1:3000 diluti
on in 2% (w/v) BSA in PBS. Incu
ate the plates for 1 h at room temperature. Note
that IM anti
odies are not detected
y this assay, since an IM-
indin detect
or would cross-react with the 38C13 IM used to coat the ELISA plate. 7. Wash th
e plates as in step 3, then add 100 L per well of a freshly made su
strate soluti
on (100 L of 15 m/mL ABTS (Sima), 3.3 L of 30% (v/v) hydroen
Cytokine Fusion Constructs
233
Fi. 2. Representative Kaplan-Meier survival plot comparin protein and DNA vacc
ination with idiotype or idiotype-GM-CSF fusion constructs. Reprinted from (12).
peroxide, and 10 mL of 0.1 M citrate
uffer, pH 4.0). O
serve the plates for co
lor development, and read them on a microplate reader at a test wavelenth of 40
5 nm and a reference wavelenth of 490 nm. Maximum color usually develops after
1530 min incu
ation at room temperature. Use SOFTmax software (Molecular Devices)
to calculate anti
ody responses in each animal
ased upon the standard curve.
3.3.2.2. T CELL PROLIFERATION ASSAYS
1. Harvest the spleens of representative vaccinated mice from each roup 2 wk af
ter the last immunization for cellular assays of immune response. Remove spleens
usin sterile technique and place them in a tu
e containin 56 mL of cRPMI mediu
m. 2. Prepare a sinle cell suspension
y ently squeezin the spleen in a frost
ed lass tissue homoenizer. 3. Allow de
ris to settle for 12 min, remove the sup
ernatant containin the spleen cells and pipet it into a fresh polypropylene 15
mL conical tu
e. 4. Underlay 35 mL of Lympholyte M into each tu
e with a 5 mL pip
et, there
y creatin a step radient. Spin the tu
es at 400 for 20 min, and col
lect the lymphocytes from the interface with a sterile 5 mL pipet. 5. Wash the c
ollected lymphocytes two times with RPMI-1640 medium without serum, then resuspe
nd at 5 106 cells per mL in RPMI-1640 containin 1% (v/v) fresh normal mouse ser
um. 6. Add 100 L of cells in each well of a microtiter plate, alon with 100 L per
well of medium containin titered amounts of 38C13 I or a control I.
234
Maecker, Syrenelas, and Levy
7. Use I doses of 100 /mL, 10 /mL, and 1 /mL (final concentrations) to esta
lis
h a dose-response curve. Use a class-matched control I and purify it in a manne
r similar to that used for the test 38C13 I (see Note 11). 8. Incu
ate the plat
e at 37C for 3 d and add 1 Ci/well of 3H-thymidine in ~25 L of medium to each well
on d 3. 9. Approximately 1620 h later, harvest the wells onto filters usin a cel
l harvester and determine the proliferation
y quantifyin [3H]-thymidine incorp
oration into DNA. Dry the filters and measure the
ound radioactivity in a scint
illation counter. Calculate specific proliferation as cpm of 38C13-stimulated we
lls with reference to cpm of irrelevant I-stimulated wells.
3.3.2.3. CTL ASSAYS

1. Harvest spleen cells from immunized mice as for T-cell proliferation assays (
steps 14 a
ove). 2. Wash the cells twice after recoverin them from Lympholyte M
radients, then resuspend them at 5 106 cells per mL in cRPMI containin 1015 U/m
L recom
inant human IL-2. Add up to 8 mL per well of a 6-well plate. 3. Harvest
EG.7-OVA cells and irradiate them (6000 rad) in a cesium irradiator. Resuspend t
he cells at 5 10 6 cells per mL, and add one-tenth volume to each spleen cell cu
lture (10:1 responder:stimulator cell ratio). Incu
ate the cultures at 37C for 56
d, addin fresh medium to the wells as needed (usually every 2 d). 4. On the day
of assay, recover 106 cells of
oth EG.7-OVA and EL-4 cells
y centrifuation.
Resuspend them in approximately 100 L residual volume of medium, and add 200 Ci [5
1Cr]-sodium chromate. Incu
ate the cells at 37C for 12 h, shakin them occasionall
y. 5. While the taret cells are incu
atin with 51Cr, transfer the stimulated s
pleen cells to 15 mL tu
es
y entle pipettin, leavin adherent cells
ehind. C
ount the harvested cells and resuspend them at 5 106 cells per mL in cRPMI mediu
m. Prepare two serial 1:5 dilutions to allow effector:taret ratios of 50:1, 10:
1, and 2:1. Add 100 L of cells from each dilution in triplicate in each of two 96
-well round-
ottom microtiter plates (one for EL-4 and one for EG.7-OVA). Prepar
e triplicate wells for each plate containin 100 L medium (for spontaneous lysis)
and 100 L 0.1% (v/v) Triton-X (for maximum lysis). 6. Wash the la
eled taret ce
lls three times with cRPMI medium and resuspend them at 105 per mL. Add 100 L of
cells to each well of the microtiter plates containin the spleen cells. Spin th
e plates
riefly and incu
ate them for 4 h at 37C. 7. Followin the 4 h incu
atio
n, spin the plates
riefly to pellet cells, then remove 100 L of supernatant from
each well and place it in a tu
e for countin on a amma counter. Use 2 mL tu
e
s that are racked in a 96-well format for convenient transfer from plates to tu

es. After countin each tu
e for 12 min, calculate the mean cpm of triplicate tu

es. Calculate specific lysis for each sample as: [cpm (sample) cpm (spontaneous
lysis)]/[cpm(maximum lysis) cpm (spontaneous lysis)].
Cytokine Fusion Constructs
4. Notes
235
1. DNA made from
acterial cultures contains endotoxin, which can act as an adju
vant for DNA immunization. Different methods of DNA preparation, as well as diff
erent
atches made
y the same method, can vary in their endotoxin content. Alth
ouh endotoxin effects have not
een detecta
le in our system, it would
e prude
nt to test
atches of DNA that are to
e used for vaccination to ensure that the
ir endotoxin content is low. We have found that cesium chloride
andin enerall
y yields low levels of endotoxin (<100 U/m DNA). Alternately, low-endotoxin DNA
purification columns (Qiaen Corp., Chatsworth, CA) may
e used. It is recommen
ded that a lare
atch of each DNA
e prepared and frozen for repetitive use. 2.
Test for endotoxin usin a Limulus amoe
ocyte lysate assay (Associates of Cape
Cod, Woods Hole, MA). Follow the manufacturers instructions. This assay relies up
on the elation activity of a lysate of amoe
ocytes from the Limulus cra
. By ma
kin several dilutions of each DNA sample
ein tested, the hihest dilution tha
t still causes ellin can
e found. Record the endotoxin concentration as a ran
e
etween the calculated amount of DNA in this dilution and the amount in the n
ext hiher dilution. Run all samples in duplicate. 3. It is possi
le to quantify
the amount of fusion protein in the supernatant
y comparison with a standard i
n an ELISA assay. When the supernatant is used in an indicator cell assay alons
ide a standard of free recom
inant cytokine, one can then calculate the
ioactiv
ity of the cytokine fusion protein on a molar
asis. 4. Like other cell lines, 3
8C13 cells can vary from
atch to
atch with reard to their tumorienicity in v
ivo. Prepare and titer frozen stocks prior to use in tumor challene experiments
. Avoid proloned in vitro culture. We recommend freezin multiple aliquots of c
ells, then thawin a vial, rowin the cells for 3 d, and testin for tumorieni
city in a titration experiment. 5. Tumorienicity is also dependent upon the hea
lth and rowth phase of the cell culture. Harvest cells durin loarithmic rowt

h (e.., 3 d after thawin), then wash three times in cold RPMI-1640 medium with
out serum to avoid any carrier effect of fetal calf serum. Count and dilute the
cells and keep them on ice prior to injection to avoid loss of via
ility. 6. To
allow for statistically sinificant comparisons
etween roups, at least 10 mice
per treatment are used for tumor challene experiments. Mice vaccinated with sa
line or an irrelevant DNA construct should die
etween d 15 and 25 postchallene
. 7. The two statistical analyses commonly used for survival data are lo-rank a
nd Gehan scores (48). Both methods will yield p values for pairwise comparisons
of survival curves. However, they may yield slihtly different p values and thus
different levels of sinificance for the same comparisons, due to differences i
n the alorithms. In eneral, the Gehan alorithm places reater sinificance on
differences at the top of the survival curve, that is, when animals first start
to die. Thus, two survival curves that are similar in the initial part of the c
urve may not
e sinificantly different
y Gehan score, even thouh the
ase of
the two curves
236
Maecker, Syrenelas, and Levy
may appear quite different. By contrast, the lo-rank alorithm places equal wei
ht on all parts of the survival curve, so that such roups may yield a more si
nificant p value
y lo-rank than
y Gehan score. It is important to titer the t
umor-cell doses so that control animals die within a reasona
le period of time (
1525 d has proven optimal for 38C13). On the other hand, injectin too many tumor
cells will make it difficult to show vaccinespecific effects, as the tumor may
overwhelm the immune response. Experiments as descri
ed can
e terminated 60 d p
ost-tumor challene, with no chane in lon-term survival. With 38C13, as with o
ther systems studied in our la
oratory, the immune response as measured
y serum
anti
odies can vary
etween individual mice, despite usin an in
red strain suc
h as C3H/HeN. It is therefore important to use lare roups of mice (10 or more
mice per roup) to o
tain meaninful results for comparison. It is possi
le to c
ompare the anti
ody titer and survival data from individual mice
y ear-punchin
or toe-clippin the mice in each roup. When this has
een done, we have found
that varia
ility in anti
ody titers exists within mice that were all equally pro
tected from tumor challene. Mice that do not make detecta
le anti
ody are almos
t never protected; however, it is difficult to determine a precise threshold of
anti
ody level required for tumor protection. The isotypes of the specific anti

odies have
een measured (42),
ut a clear correlation of isotype profile with p
rotection cannot
e esta
lished. Contaminants that the two preparations may have
in common can lead to nonspecific proliferation, or hih
ackrounds, which may
o
scure a weak idiotypespecific proliferative response. Use normal mouse serum
instead of fetal calf serum to avoid the hih
ackround reactivity o
served wit
h fetal calf serum.
8.
9.
10.
11.
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Eur. J. Immunol. 7, 413417. 48. Stuschke, M., Budach, V., Bam
er, M., and Budach
, W. (1990) Methods for analysis of censored tumor rowth delay data. Radiat. Re
s. 122, 172180. 49. Maloney, D. G., Kaminski, M. S., Burowski, D., Haimovich, J.,
and Levy, R. (1985) Monoclonal anti-idiotype anti
odies aainst the murine B ce
ll lymphoma 38C13: characterization and use as pro
es for the
ioloy of the tum
or in vivo and in vitro. Hy
ridoma 4, 191209.
21
The Use of Conventional Immunoloic Adjuvants in DNA Vaccine Preparations
Shin Sasaki and Kenji Okuda 1. Introduction The term adjuvant oriinates from the
Latin word adjuvare, which means to help or aid. An immunoloic adjuvant is defi
ned as any su
stance that acts to accelerate, prolon, or enhance antien-specif
ic immune responses when used in com
ination with specific vaccine antiens (1).
In a
road sense, immunoloic adjuvants include certain cytokines and other imm
unomodulatory molecules (i.e. chemokines and costimulatory factors) or their exp
ression vectors. Since these adjuvants are descri
ed elsewhere in this
ook, her
e, we are concerned only with adjuvants that are derived from microoranisms and
plants or are synthesized chemically. Our roup has attempted to
oost the immu
ne response to a DNA vaccine for HIV-1 usin several immunoloic adjuvants and r
esults have
een promisin. The search for adjuvants for enhancin DNA-derived i
mmunity has just started and knowlede on this topic is
einnin to accumulate.
In this chapter, we
riefly characterize the immunoloic adjuvants evaluated in
our DNA vaccine study and summarize the findins of others. We also ive an acc
ount of the preparation and usae of these adjuvant-DNA vaccines. This section d
oes not aim at providin an all-inclusive review of the current status of adjuva
nts; for this readers can refer to various excellent review articles and
ooks (
14). There are a num
er of candidate adjuvants for DNA vaccines other than those
referred to here.
1.1. A CompendiumClassification of Immunoloic Adjuvants
The immunoloic adjuvants include various chemical compounds with unique immunol
oical or physicochemical properties. They exert their adjuvanticity throuh a p
lurality of mechanisms. In addition, many have overFrom: Methods in Molecular Me
dicine, vol. 29, DNA Vaccines: Methods and Protocols Edited
y: D. B. Lowrie and
R. G. Whalen Humana Press Inc., Totowa, NJ
241
242
Sasaki and Okuda
lappin mechanistic features toether with unique physicochemical properties, an
d are therefore not easily classified. As an expedient measure, we have chosen h
ere to follow the classification system of Voel (1). The efficacy of adjuvants
in DNA vaccination does not always correlate with their usefulness in convention
al polypeptide vaccination as Ta
le 1 shows. This is clearly
ecause DNA vaccina
tion involves a unique mode of antien presentation distinct from that of peptid
e-
ased vaccination. We present the method for preparin our adjuvant-DNA com
in
ation usin the adjuvants indicated in
old style in the Ta
le.

1.2. Mechanism of Adjuvant Action in DNA Vaccination

The mechanisms responsi
le for elicitin specific responses to a DNA vaccine rem
ain conjectural. As attempts to use immunoloic adjuvants with DNA vaccines have
just
eun, it is impossi
le to present here the precise adjuvant mechanism ope
rative in DNA vaccination. However, a few findins afford insiht into this issu
e and we now know of three mechanisms
y which adjuvants enhance DNAderived immu
nity. These are: (i) recruitment of macrophaes or other lymphoid cells, (ii) in
duction of cytokines which can amplify responses enerated
y immunoenic DNA, a
nd (iii) facilitation of DNA entry into host cells. Antien-presentin cells (AP
C), such as macrophaes or dendritic cells, have
een shown to prime the cytolyt
ic response to DNA-derived antiens (5,6). Our adjuvant studies on DNA vaccines
demonstrated that mononuclear cell recruitment in muscles injected with DNA and
adjuvants was associated with su
stantial enhancement of DNA-derived immunity (79
). Interferon amma and IL-2 synthesis
y antien-restimulated splenocytes was o

served with a QS-21 adjuvant-DNA com
ination (9), and enhanced IL-4 and IL-5 pr
oduction was reported usin cholera toxin adjuvant-DNA preparation (10). Concurr
ent administration of cytokine expression plasmids and immunoenic DNA is report
ed to
e effective for enhancin the response to an encoded antien (1114). These
findins suest that APC recruitment at the DNA-injection site as well as cyto
kine induction, help to enhance antien-specific immune responses. Carriers such
as cationic liposomes are reported to
e useful as adjuvants for DNA vaccinatio
n (15,16). Their mechanistic utility is thouht to arise from the incorporation
of DNA into the liposomal vesicle, and the su
sequent facilitation of DNA entry
into host cells. Further studies are necessary to clarify the mechanism for adju
vant-mediated enhancement of the immunity induced
y DNA vaccination. 2. Materia
ls 2.1. Gel Type Adjuvants
1. Special equipment: spectrophotometer to measure DNA adsorption onto aluminum
or calcium els.
Use of Immunoloic Adjuvants
Ta
le 1 Immunoloic AdjuvantClassification and Efficacy in DNA Vaccination Cateo
ry Gel-type adjuvant Adjuvant name Aluminum hydroxide (alum) Effectiveness in DN
A vaccination Injection prior to DNA is effective Reference Saski and Okuda, unp
u
lished o
servation Saski and Okuda, unpu
lished o
servation 8 10
Calcium phosphate (CPA)
Injection prior to DNA is effective
Bacterial adjuvant
Monophosphoryl lipid A (MPL) Cholera toxin (CT) Muramyl peptides (e.., MDP, MTP
-PE) Immunostimulatory complexes (ISCOMS) Cationic liposomes Mannan-coated lipos
omes Bioderada
le polymers
Particulate adjuvant
Emulsifier-
ased adjuvant
Incomplete Freunds adjuvant (IFA) QS-21
Effective in intramuscular and intranasal immunization Effective Not evaluated N
ot evaluated Effective in intramuscular and intranasal immunization Effective in
intramuscular and intranasal immunication Effective in oral and nasal immunizat
ion Not evaluated
15,16,23 18,19 23
Synthetic adjuvants

Nonionic
lock copolymers U
enimex (UBX)

Effective in intramuscular 9,21 immunization and particle
om
ardment Not evalua
ted Effective in intramuscular immunization 22
243
The adjuvants indicated in
old are referred to in the text for preparin a com

ined adjuvant-DNA vaccine.
244
Sasaki and Okuda
2. Aluminum hydroxide (alum) is availa
le from Sima (St. Louis, MO). 3. Calcium
phosphate adjuvant (CPA) is availa
le from Superfos Biosector a/s (Ved
aek, Den
mark). 4. Sterile phosphate
uffered saline (PBS), pH 7.4.
2.2. Liposome-Associated Adjuvants 2.2.1. DC-Chol:DOPE Liposome Adjuvant
1. DC-Chol:DOPE liposome, prepared as descri
ed previously (17). 2. Sterile PBS,
pH 7.4. 3. Sterile 10 mM HEPES
uffer, pH 7.8.
2.2.2. diC14-Amidine Adjuvant (Vectamidine
1. N-t-
utyl-N-tetradecyl-3-tetradecylaminopropionamidine (diC14-amidine) is av
aila
le from ULB-Interface (Brussels, Belium). 2. Sterile PBS, pH 7.4. 3. Steri
le 10 mM HEPES
uffer, pH 7.8.
2.3. Mannan-Coated Cationic Lipids
1. {N-[2-(Cholesterylcar
oxyamino)-ethyl]car
amoylmethyl}mannan (CholAECM-Mannan
) is availa
le from Wako Chemicals (Osaka, Japan). 2. DC-Chol:DOPE liposome adju
vant. 3. diC14-amidine (Vectamidine).
2.4. Ready-to-use Adjuvant Products
1. MPL-SE (Monophosphoryl lipid A in a sta
le emulsion) is availa
le from Ri
i I
mmunoChem Research Inc. (Hamilton, MT). 2. MPL-AF (Monophosphoryl lipid A in an
aqueous formulation) is availa
le from Ri
i ImmunoChem Research Inc. (Hamilton,
MT). 3. QS-21 is availa
le from Aquila Biopharmaceuticals (Worcester, MA). 4. U

enimex is availa
le from Nippon Kayaku Co. Ltd. (Tokyo, Japan).
3. Methods Our standard inoculation procedures for intramuscular and intranasal
delivery are descri
ed in detail in Chapter 18. We descri
e here only the method
s for adjuvant-DNA vaccine preparations.
3.1. Gel Type Adjuvants (Aluminum and Calcium Gels)
This class of adjuvants are known to act in polypeptide-
ased vaccination
y ads
or
in antien onto their surfaces. Althouh the extent of their activity in DNA
vaccination has not
een esta
lished, they consistently adsor
plasmid DNA. The
method for preparin adjuvant-adsor
ed plasmid DNA is presented here.
Use of Immunoloic Adjuvants 3.1.1. Adsorption of Plasmid DNA onto Mineral Gels
245
Experience has shown that approximately 8090% of 10 to 100  of plasmid DNA can
e
adsor
ed
y over 30300 L of alum or CPA irrespective of the DNA-encoded antien.
We descri
e here the procedure for adsor
in 10  of plasmid DNA onto adjuvant e
ls.
1. Transfer 30 L of alum or CPA to a 1.5 mL-microcentrifue tu
e. 2. Add 10  of T
E-dissolved DNA into the tu
e, then fill the tu
e with an appropriate volume of

PBS to o
tain the desired volume for inoculation. 3. Briefly touch the tu
e to a
vortex mixer to ensure a homoenous el suspension. 4. Incu
ate the mixture at
4C for 30 min. Time-dependent adsorption kinetic analysis has shown that the opti
mum incu
ation time is 30 min or 24 h.
3.1.2. Measurement of DNA Adsor
ed to the Gels
1. After incu
ation, centrifue the el at 10,000 rpm (1000) for 3 min to o
tai
n a el-free supernatant (see Note 1). 2. Measure the a
sor
ance of the supernat
ant
y spectrophotometry at 260 nm. 3. The percent adsorption efficiency is calc
ulated as follows: [1 (10DNA  in the supernatant)/10] 100. 4. Before inoculation,
resuspend the precipitated els
y
riefly touchin to the vortex mixer.
3.2. Cationic Liposome Species
The liposome/DNA ratio employed in synthesizin liposome-DNA complex should
e s
uch that the effect of the adjuvant is optimized. Our experience has shown that
the appropriate liposome/DNA ratio for enhancin a DNAderived immune response is
4:1 to 6:1.
3.2.1. Synthesis of the DC-Chol:DOPE Liposome-DNA Complex
1. Transfer the required amount of TE-dissolved DNA for immunization into a 1.5
mLmicrocentrifue tu
e. 2. Add an appropriate amount of DC-Chol:DOPE liposomes i
n 10 mM HEPES
uffer (pH 7.8) into the tu
e. The recommended liposome dose is 46
times the DNA dose. Mix the solution ently
y pipettin several times (see Note
2), then fill the tu
e with an appropriate volume of PBS to o
tain the desired
liposomeDNA complex concentration. 3. Stand the tu
e at room temperature for at
least 1 h for complex formation, overniht incu
ation may
e necessary. This pre
paration is now ready for inoculation.
3.2.2. Synthesis of the diC14-Amidine-DNA Complex
1. The diC14-amidine is in crystalline form and should
e hydrated with 10 mM of
the HEPES
uffer in the kit accordin to the manufacturers instruction. 2. Trans
fer the amount of TE-dissolved DNA that is required for immunization to a 1.5 mL
-microcentrifue tu
e.
246
Sasaki and Okuda
3. Add an appropriate amount of diC14-amidine in 10 mM HEPES
uffer (pH 7.3) to
the tu
e. The recommended liposome dose is 46 times the DNA dose. Mix the solutio
n ently
y pipettin several times (see Note 2), then fill the tu
e with an app
ropriate volume of PBS to yield the desired liposome-DNA complex concentration.
4. Stand the tu
e on ice for 15 to 30 min to allow for complex formation. This p
reparation is now ready for inoculation.
3.3. Mannan-Coated Cationic Lipids
Althouh we have used Chol-AECM-Mannan to modify the adjuvant effect of DC-Chol:
DOPE and diC14-amidine liposomes (18,19), other neolycolipids are thouht to ha
ve su
stantial immunomodulatory effects as descri
ed elsewhere (20). The procedu
re for coatin mannan onto liposomes is the same, irrespective of type of liposo
me used. The term liposome in this section is applied to
oth DC-Chol:DOPE and d
iC14-amidine liposomes.
3.3.1. Coatin Mannan onto Liposomes
1. Dissolve Chol-AECM-Mannan with PBS to a concentration of 1 m/mL, then transf
er 25 L into a 1.5 mL-microcentrifue tu
e. 2. Add 75 L of liposomes in HEPES
uff
er (1 m/mL), and mix the solution ently
y pipettin (see Note 2). 3. Add 400 L
of PBS to the tu
e and mix it
y inversion, then stand the tu
e at 4C overniht
to allow the mannan to attach to the liposomes. The use of a rollin mixer for t
his incu
ation is recommended. 4. Centrifue the tu
e at 3,000 rpm for 5 min and

discard 250 L of the supernatant. 5. Resuspend the pellet
y pipettin. This pre
paration can
e used as the stock solution of mannan-coated liposomes.
3.3.2. Synthesis of the Mannan-Coated Liposome-DNA Complex
1. Transfer the amount of TE-dissolved DNA required for immunization to a 1.5 mL
microcentrifue tu
e. 2. Add an appropriate amount of the mannan-coated liposome
preparation to the tu
e. The recommended liposome dose is 46 times the DNA dose.
Mix the solution ently
y pipettin several times, then fill the tu
e with an
appropriate volume of PBS to yield the desired mannan-coated liposome-DNA comple
x concentration. 3. The incu
ation time and temperature required for complex for
mation are the same as those descri
ed in Su
headins 3.2.1. and 3.2.2.
3.4. Monophosphoryl Lipid A (MPL)
We have used two preparations of MPL: MPL-SE and MPL-AF. Both preparations have
exhi
ited similar adjuvant activity in our murine DNA vaccination system (8). MP
L adjuvant-DNA vaccine can
e prepared simply
y mixin
Use of Immunoloic Adjuvants
247
the adjuvant solution with immunoenic DNA dissolved in TE. No incu
ation and em
ulsification are necessary. The optimum MPL dose for aumentin DNA-derived immu
nity is 50 m per mouse in our experience.
3.5. QS-21
QS-21 adjuvant-DNA vaccine can also
e prepared simply
y mixin the adjuvant so
lution with immunoenic DNA without incu
ation and emulsification. However, we h
ave found that there are different optimum QS-21 doses for antien-specific cyto
lytic activity and anti
ody production; a 5  and a 25  QS-21 dose are recommende
d for the cytolytic and the anti
ody response, respectively. To enhance
oth the
antien-specific cytolysis and humoral immunity, 10  QS-21 per mouse is recomme
nded (9). Use of QS-21 as an adjuvant for DNA vaccination
y particle
om
ardmen
t is also reported (21). Althouh some peptide-
ased vaccination studies have sh
own that alumadsor
ed antien can
e formulated with QS-21, our experience sue
sts that concurrent administration of alum-adsor
ed DNA mixed with QS-21 does no
t aument an antien-specific immune response.
3.6. U
enimex (UBX)
UBX as supplied
y the manufacturer is a powder that is difficult to dissolve in
an aqueous solution. Usin ultrasonication and heatin, a maximum 4.5 m of UBX
can
e dissolved with 1 mL of water or PBS. The UBX solution can
e filter-ster
ilized throuh a 0.2 micron filter and can
e frozen and thawed without denatura
tion. In our study, 100500  of UBX per mouse had an adjuvant effect on antien-sp
ecific humoral and cell-mediated immune responses (22). Incu
ation and emulsific
ation are not necessary, and UBX adjuvant-DNA vaccine can
e prepared simply
y
mixin the immunoen and UBX solution. 4. Notes
1. Aluminum or calcium els that have precipitated naturally
y ravity can
e e
asily resuspended with a vortex mixer. However, a tiht el pellet formed
y cen
trifuation is harder to resuspend. We therefore used only part of the solution
to measure A260. 2. Gentle mixin of the DNA-liposome(-mannan) mixture is import
ant for consistent synthesis of the complex. Rouh mixin with the vortex mixer
seems to reduce the effect of the adjuvant in our experience.
References
1. Voel, F. R. (1995) Immunoloic adjuvants for modern vaccine formulations, in
Com
ined Vaccine and Simultaneous AdministrationCurrent Issues and Perspectives,
Ann. NY Acad. Sci., vol. 754 (Williams, J. C., Goldenthal, K. L., Burns, D. L.,
and Lewis, B. P. J., eds.) N Y Academy of Sciences, New York. pp. 153160.

248
Sasaki and Okuda
2. Powell, M. F. and Newman, M. J., eds. (1995) Vaccine Desin: The Su
unit and
Adjuvant Approach, Plenum Press, New York. 3. Goldin, B. and Scott, D. E. (1995
) Vaccine Strateies: Tarettin helper T cell responses, in Com
ined Vaccine an
d Simultaneous AdministrationCurrent Issues and Perspectives, Ann. NY Acad. Sci.
vol. 754 (Williams, J. C., Goldenthal, K. L., Burns, D. L., and Lewis, B. P. J.,
eds.) NY Academy of Sciences, New York. pp. 126137. 4. Gupta, R. K. and Si
er, G
. R. (1995) Adjuvants for human vaccines-current status, pro
lems and future pro
spects. Vaccine 13, 12631276. 5. Corr, M., Lee, D. J., Carson, D. A., and Tihe,
H. (1996) Gene vaccination with naked plasmid DNA: Mechanism of CTL primin. J.
Exp. Med. 184, 15551560. 6. Doe, B., Sel
y, M., Barnett, S., Baenzier, J., and W
alker, C. M. (1996) Induction of cytotoxic T lymphocytes
y intramuscular immuni
zation with plasmid DNA is facilitated
y
one marrow-derived cells. Proc. Natl.
Acad. Sci. USA 93, 85788583. 7. Tsuji, T., Fukushima, J., Hamajima, K., Ishii, N
., Aoki, I., Xin, K.-Q., et al. (1997) HIV-1-specific cell-mediated immunity is
enhanced
y co-inoculation of TCA3 expression plasmid with DNA vaccine. Immunolo
y 90, 16. 8. Sasaki, S., Tsuji, T., Hamajima, K., Fukushima, J., Ishii, N., Kane
ko, T., et al. (1997) Monophosphoryl lipid A enhances
oth humoral and cell-medi
ated immune responses to DNA vaccination aainst Human Immunodeficiency Virus Ty
pe 1. Infect. Immun. 65, 35203528. 9. Sasaki, S., Sumino, K., Hamajima, K., Fukus
hima, J., Ishii, N., Kawamoto, S., et al. (1998) Induction of systemic and mucos
al immune responsees to human immunodeficiency virus type 1
y a DNA vaccine for
mulated with QS-21 saponin adjuvant via intramuscular and intranasal routes. J.
Virol. 72, 49314939. 10. Kuklin, N., Daheshia, M., Karem, K., Manickan, E., and R
ouse, B. T. (1997) Induction of mucosal immunity aainst herpes simplex virus
y
plasmid DNA immunization. J. Virol. 71, 31383145. 11. Watana
e, A., Raz, E., Koh
saka, H., Tihe, H., Baird, S. M., Kipps, T. J., and Carson, D. A. (1993) Induct
ion of anti
odies to a kappa V reion
y ene immunization. J . Immunol. 151, 28
712876. 12. Xian, Z. Q., and Ertl, H. C. J. (1995) Manipulation of the immune re
sponse to a plasmid-encoded viral antien
y coinoculation with plasmids express
in cytokines. Immunity 2, 129135. 13. Tsuji, T., Hamajima, K., Fukushima, J., Xi
n, K.-Q., Ishii, N., Aoki, I. (1997) Enhancement of cell-mediated immunity aain
st HIV-1 induced
y coinoculation of plasmid-encoded HIV-1 antien with plasmid
expressin IL-12. J. Immunol. 158, 40084014. 14. Iwasaki, A., Stiernholm, B. J. N
., Chan, A. K., Berinstein, N. L., and Bar
er, B. H. (1997) Enhanced CTL respons
es mediated
y plasmid DNA immunoens encodin costimulatory molecules and cytok
ines. J. Immunol. 158, 45914601. 15. Greoriadis, G., Saffie, R., and de Souza, J
. B. (1997) Liposome-mediated DNA vaccination. FEBS Lett. 402, 107110.
Use of Immunoloic Adjuvants
249
16. Ishii, N., Fukushima, J., Kaneko, T., Okada, E., Tani, K., Tanaka, S.-I., et
al. (1997) Cationic liposomes are a stron adjuvant of DNA vaccine for a Human
Immunodeficiency Virus Type-1 (HIV-1). AIDS Res. Hum. Retroviruses 13, 14211428.
17. Gao, X. and Huan, L. (1991) A novel cationic liposome reaent for efficient
transfection of mammalian cells. Biochem. Biophys. Res. Comm. 179, 280285. 18. T
oda, S., Ishii, N., Okada, E., Kusaka
e, K.-I., Arai, H., Hamajima, K., et al. (
1997) HIV-1 specific cell-mediated immune responses induced
y DNA vaccination a
re enhanced
y mannan-coated liposomes and inhi
ited
y anti-interferon (IFN)-a
mma anti
ody. Immunoloy 92, 111117. 19. Sasaki, S., Fukushima, J., Arai, H., Kus
aka
e, K., Hamajima, K., Ishii, N., et al. (1997) Human immunodeficiency virus t
ype 1 specific immune response induced
y DNA vaccination are reatly enhanced

y mannan-coated diC14-amidine. Eur. J. Immunol. 27, 31213129. 20. Suimoto, M., O
hishi, K., Fukasawa, M., Shikata, K., Kawai, H., Itakura, H., et al. (1995) Oli
omannose-coated liposomes as an adjuvant for the induction of cell-mediated immu

nity. FEBS Lett. 363, 5356. 21. Wan, S., Mannin, S., Kensil, C. R., and Lu, S.
(1997) QS-21 is effective in
oostin anti-HIV-1 Env anti
ody responses induced

y DNA immunization. 9th Annual Meetin of The National Cooperative Vaccine Deve
lopment Groups for AIDS, poster a
stract 15. Bethesda, MD, USA. 22. Sasaki, S.,
Hamajima, K., Fukushima, J., Tsuji, T., Xin, K.-Q., Ishii, N., et al. (1997) Adj
uvant effect of u
enimex on a DNA vaccine for human immunodeficiency virus type1. Clin. Exp. Immunol. 111, 3035. 23. Hamajima, K., Sasaki, S., Fukushima, J., Ka
neko, T., Xin, K.-Q., Kudo, I., and Okuda, K. (1998) Intranasal administration o
f HIV-DNA vaccine formulated with a polymer, car
oxymethylcellulose aument muco
sal anti
ody production and cellmediated immune response. Clin. Immunol. Immunop
athol. 80, 205210.
22
Genetic Adjuvants
Hildeund C. J. Ertl, Susanna Pasquini, Zhennin He, Honyin Den, Louise Showe
, Wynetta Giles-Davis, Yijie Wan, InSu O, Hilary Marston, Madalena BlaszczykThurin, and Zhiquan Xian 1. Introduction In 1992, the era of DNA vaccines
ean
with the report of anti
ody production upon intradermal injection of mice with
a plasmid vector expressin a forein antien (1). A rapid succession of su
sequ
ent manuscripts showed stimulation of immune responses, includin cytolytic T ce
lls, upon inoculation of expression-vectors specific for antiens derived from v
iruses,
acteria, protozoa and tumor-associated antiens (27). Plasmid DNA can
e
applied throuh various routes of injection includin: intradermal, intramuscul
ar, su
cutaneous, intravenous, or directly on mucosal mem
ranes (1,2,8,9). The m
ost commonly used methods of inoculation involve the use of DNA-coated old
ead
s propelled into the skin
y a ene un or intramuscular inoculation of the vect
or in saline solution. Both T and B cell-mediated immunity can
e elicited to di
fferent forms of antien, includin secreted and cell surface antiens, as well
as antiens with nuclear taretin domains (2,9). Most manuscripts report succes
sful immunization with DNA vaccines showin potent, lon-lastin immunity and, w
hen the appropriate animal models are availa
le, protection to su
sequent challe
ne (2,4,68). Nevertheless, in some models includin our own, which uses DNA vacc
ines expressin ra
ies virus antiens, immunization with vector DNA resulted in
an immune response that was
y no means as potent as the one seen upon immunizat
ion with traditional inactivated or recom
inant viral vaccines (10). We therefor
e investiated avenues to enhance the immune response
y usin enetic adjuvants
, i.e., vectors expressin cytokines (11,12) or chemokines.
From: Methods in Molecular Medicine, vol. 29, DNA Vaccines: Methods and Protocol
s Edited
y: D. B. Lowrie and R. G. Whalen Humana Press Inc., Totowa, NJ
251
252 1.1. A Ra
ies Virus Model
Ertl et al.
Ra
ies virus infects and causes a potentially fatal disease in all warm
looded m
ammals includin mice, the host of our experiments. The ra
ies virus strain used
in our challene studies, i.e., CVS-24, is mouse-adapted
y serial passaes thr
ouh mouse
rain; it is therefore hihly virulent for mice, i.e., a small num
er
of infectious particles can induce an encephalitic fatal disease. Anti
odies me
diate vaccine-induced protection to ra
ies virus and cytolytic T cells do not pl
ay a role; CD4+ T cells are only needed to promote B cell responses (13). Only l
ow levels of anti
odies are needed to neutralize the small dose of challene vir
us, thus makin this system hihly amena
le for the demonstration of protection
even when usin comparatively weak vaccines includin anti-idiotypic anti
odies
(14) or peptide vaccines (15). 2. Materials 2.1. Mice
1. O
tain in
red mice from Jackson La
oratories (Bar Har
or, ME). Immunize them
at 612 wk of ae. 2. Purchase Lewis rats from Harlan Spraue Dawley Inc. (Indiana
polis, IN). Use at 68 wk of ae.

2.2. Bacteria
1. Grow plasmid vectors in the E. coli strain DH5. 2. Purch se competent
cteri
from Gi
co-BRL (G ithers
urg, MD). 3. Prop g te tr nsformed
cteri in LB medi
um supplemented with nti
iotics (see Note 1).
2.3. Vectors
1. Purch se the pcDNA3 vector from Invitrogen (S n Diego, CA). 2. Purch se the p
SG5 vector from Str t gene (L Joll , CA). 3. O
t in the vectors cont ining the I
L-2 nd IL-4 genes from the ATCC (Americ n Tissue Culture Collection, Rockville,
MD).
2.4. Cells
1. Grow

y h mster kidney (BHK)-21 cells, WEHI cells, L929 mouse fi
ro
l sts
nd murine hy
ridom cell lines 11-B-11 (secreting nti-mouse IL-4) nd S4B6 (sec
reting nti-mouse IL-2) in Dul
eccos modified E gles medium (DMEM), high glucose v
ersion, supplemented with 10% (v/v) he t-in ctiv ted fet l
ovine serum (FBS). 2
. Gener te nti
ody-cont ining scitic fluid in pristine-primed SCID mice
y ino
cul tion of 5 106 hy
ridom cells. 3. Grow the IL-2- nd IL-4-sensitive HT-2 cel
l line in DMEM supplemented with 10% (v/v) FBS nd 10% (v/v) r t Conc n v lin A
supern t nt (RCAS).
Genetic Adjuv nts
253
4. M int in the IL-4-dependent CT4S cell line (origin lly o
t ined from Dr. W. P
ul, NIH, Bethesd , MD) in DMEM supplemented with 10% (v/v) FBS nd 10 units of
recom
in nt mouse IL-4 (Becton Dickinson L
w re Coll
or tive, Bedford, MA). 5.
Grow the IL-3 nd Gr nulocyte M croph ge-Colony Stimul ting F ctor (GMCSF) depe
ndent 32DC13G/GM cell line (16) in DMEM supplemented with 10% (v/v) FBS nd 10%
(v/v) WEHI supern t nt (see Note 2).
2.5. Cytokines
1. Gener te WEHI supern t nt, source of mouse IL-3 nd GM-CSF,
y culturing WE
HI cells t 5 105 cell/mL for 48 h in DMEM supplemented with 10% (v/v) FBS. Coll
ect cell-free supern t nt, titr te on the 32DC13G/GM cell line, nd then liquot
nd store t 20C (see Note 3). 2. Gener te RCAS, source of IL-2,
y culturing r
t splenocytes t 1.25 106 cells/ mL for 48 h in 2.5 g/mL of Conc n v lin A (Ph r
m ci Fine Chemic ls, Pisc t w y, NJ) (17). H rvest cell-free supern t nts, dso
r
twice to Seph dex G-25
e ds (Ph rm ci Fine Chemic ls) t 0.2 g/100 mL of su
pern t nt to remove ConA, titr te using HT-2 cells, nd then liquot nd store
t 20C. Sterile-filter the
tch
efore e ch use nd supplement with -methyl m nno
side to in ctiv te ny residu l ConA. 3. Purch se recom
in nt hum n GM-CSF nd I
L-4 from Genzyme Di gnostics (C m
ridge, MA). 4. Purch se recom
in nt mouse GM-C
SF nd IL-4 from Sigm Chemic l Co. (St. Louis, MO) (see Note 4).
3. Methods 3.1. Construction of Expression Vectors
1. Clone cytokine genes into the multicloning site of n expression vector such
s pSG5 or pcDNA3. 2. Excise the cDNA of the cytokines from the origin l pl smid

y use of ppropri te restriction enzymes. Select the restriction enzymes ccor
ding to the following criteri : enzymes th t do not cut within the coding region
, enzymes th t cut upstre m of the ATG st rt codon nd downstre m of the stop co
don, nd, if v il
le, enzymes th t llow unidirection l cloning into the lter
n te vector
ck
one. 3. Purify the excised DNA fr gments
y gel electrophoresis
using 1% (w/v) g rose gel supplemented with ethidium
romide. Visu lize the
DNA
nd of the ppropri te size
y longw ve UV light nd cut it from the gel. I
sol te the DNA fr gment using the Gene Cle n Kit (Bio 101, Vist , CA) ccording
to the m nuf cturers protocol. 4. Cut the expression pl smid within the multiclon
ing site downstre m of the promoter using, when possi
le, the s me enzyme(s) use

d for excision of the insert. 5. After dephosphoryl tion nd purific tion
y the
Gene Cle n Kit, lig te the
ck
one nd insert, using direct lig tion or
lunt
end lig tion. For
lunt end lig tion,
254
Ertl et l.
fill in the overh ngs of
oth insert nd fr gment using the l rge fr gment of Kl
enow enzyme prior to dephosphoryl tion of the
ck
one. 6. C rry out the lig tio
ns overnight using T4 lig se enzyme t 14C (18). 7. Tr nsfect the lig tion mixtur
e into competent DH5 cells nd pl te onto g rose pl tes supplemented with the p
propri te nti
iotics. Isol te colonies the next d y nd mplify them in 2 mL li
quid cultures (LB medium + nti
iotic) (see Note 5).
3.1.1. Construction of Vector Expressing Chemokine Whose Sequence W s Recent
ly Pu
lished (19)
1. Isol te hum n peripher l
lood mononucle r cells
y purific tion of hep riniz
ed whole hum n
lood over Ficoll-Isop que gr dient. 2. W sh the cells nd then
suspend them in RPMI 1640 medium supplemented with 10% (v/v) FBS in Petri dishe
s. 3. Remove non- dherent cells 1 h l ter. Incu
te the dherent cells for 37 d w
ith RPMI 1640 medium supplemented with recom
in nt hum n GM-CSF (50 ng/mL) nd r
ecom
in nt hum n IL-4 (1000 units/mL). 4. H rvest the cells nd isol te tot l RN
A using Ultr spec RNA (Biotecx L
or tories, Houston, TX). Reverse tr nscri
e nd
mplify the resulting cDNA using oligonucleotide primers deline ted from the 5
nd 3 non-coding regions of the pu
lished sequence in 40-cycle polymer se ch
in re ction. 5. Sequence the PCR product, which should ppe r s single
nd
of the expected size upon gel electrophoreses, in n utom ted DNA sequencer to
ensure f ithful mplific tion. Clone the insert into the pcR2.1 vector (Str t ge
ne Cloning Systems) (see Note 6).
3.2. In Vitro Biologic l Ch r cteriz tion of Vectors 3.2.1. Tr nsient Tr nsfecti
ons
1. Tr nsiently tr nsfect BHK-21 cells to ensure th t vectors express function ll
y ctive cytokines. Gener te st
le tr nsfect nts to further qu ntit te expressi
on. 2. Pl te 5 105 trypsinized BHK-21 cells in 3 mL of DMEM supplemented with 10
% (v/v) FBS in tissue-culture gr de culture dishes. 3. The next d y, precipit te
1 g of pl smid DNA (qu ntit ted
y g rose gel electrophoresis nd optic l densi
ty re ding g inst known st nd rd) with Lipofectin (Gi
co-BRL) with some modif
ic tions of the m nuf cturers protocol. 4. Dilute 10 L of Lipofectin in 100 L of se
rum-free DMEM nd mix with vector DNA lso diluted to 100 L in DMEM. Use s con
trol vector expressing n unrel ted sequence. 5. Precipit te DNA for 3060 min
t room temper ture. During this time, w sh the dherent BHK-21 cells 45 times wit
h 5 mL of serum-free DMEM. 6. Add n ddition l 0.8 mL of serum-free medium to t
he precipit ted DNA nd then dd this mixture to the BHK-21 cells for 1216 h t 3
7C. 7. Remove the supern t nt nd resuspend the cells in DMEM supplemented with 1
0% (v/v) FBS. H rvest the supern t nts 48 h l ter nd test for cytokines.
Genetic Adjuv nts 3.2.2. St
le Tr nsfections
255
1. Incu
te 1 105 BHK-21 cells with mixture of 1 g of cytokine expressing vector
nd 0.2 g of pSV2neo (20). Co-precipit te the two vectors with Lipofectin s des
cri
ed
ove. 2. Add G418 (1 mg/mL) to the cultures two d ys fter the tr nsfect
ion. Repl ce medium cont ining G418 in 45 d interv ls until colonies
ecome visi

le. 3. Remove the medium once the colonies re ch size of >1 mm2. W sh the pl t
es once in serum-free DMEM nd dd 2 mL of trypsin (0.25 % (w/v) trypsin) in Ver
sene (BioWhitt ker, W lkersville, MD). H rvest individu l colonies nd tr nsfer
them to 24-well pl tes (Cost r, Thom s Scientific, Swedes
oro, NJ). 4. Exp nd th

e cells in 1.6 mL of DMEM. H rvest supern t nts once the cells h ve
ecome confl
uent (see Note 7).
3.3. Cytokine Ass ys
1. Ass y cytokines on ppropri te indic tor cell lines, i.e., GM-CSF on 32DC13G/
GM cells, nd IL-2 nd IL-4 on HT-2 cells. For other cytokine vectors, use eithe
r indic tor cells (IL-12), other
iologic l ss ys (reduction of virus-induced p
l que form tion on L929 cell monol yers for IFN-) or sandwich ELISAs usin specif
ic MA
s (IL-5, IL-10). 2. To test for GM-CSF, dilute 2 103 32DC13G/GM cells in 7
5 L of DMEM supplemented with 10% (v/v) FBS. Incu
ate with 75 L of serial dilution
s of cell-free supernatants from transfected cells in 96-well round
ottom micro
titer plate wells (Lim
ro, Thomas Scientific). Use serial dilutions of WEHI supe
rnatant or recom
inant purified mouse GM-CSF as a positive control; use supernat
ants from sham-transfectants as a neative control. 3. Pulse the cells 48 h late
r for 6 h with 0.5 Ci of [3H]thymidine, harvest onto filtermats, and analyze in a
-counter. 4. Consider as positive those supernatants that induce proliferation (
i.e., [3H] thymidine incorporation) three times a
ove
ackround (see Note 8).
3.4. Immunization of Mice
1. Use a GM-CSF expressin vector to assess the a
ility of cytokines to serve as
enetic adjuvants in the initial experiments. Inoculate mice intramuscularly wi
th a mixture of 50  of an antien-expressin vector, such as the pSG5ra
.p vect
or, expressin the ra
ies virus lycoprotein, with various amounts of the GM-CSF
expressin vector (10250 ) in a total volume of 100 L of saline
uffer. Alternati
vely, inject mice with the cytokine-expressin vector, then inject the pSG5ra
.
p plasmid 24 or 48 h later into the same muscle. In some experiments, you can
o
ost the mice either once or twice at 14 d intervals with an additional 50  of pS
G5ra
.p. 2. Analyze 23 different mouse strains (e.., C3H/He, C57Bl/6 and Bal
/c
) to determine the effect of a cytokine. 3. Test the cytokine vectors at three d
ifferent doses (e.., 10, 50 and 250 ). 4. For some cytokines, test different ve
ctor constructs expressin the cytokine ene under the control of the SV40 promo
ter (IL-4, IL-2), the Rous sarcoma LTR (GM-CSF) or the CMV promoter (IL-2, IL-4
and GM-CSF) (see Note 9).
256 3.5. Analysis of the Immune Response
Ertl et al.
3.5.1. Anti
ody Titers to the Antien Measured
y an ELISA (21)
1. Bleed the mice at varied intervals and analyze serum anti
ody titers to the a
ntien such as ra
ies virus. To determine potential shifts in the kinetic of the
anti
ody response,
leed the mice
y retro-or
ital puncture early in the time c
ourse, e.., 2 wk after immunization, and then in monthly or
imonthly intervals
for at least 46 mo. 2. Coat plates with antien, such as ERA-BPL virus (i.e., pu
rified inactivated ra
ies virus) and incu
ate with duplicate or triplicate sampl
es of serial dilutions of pooled sera usin sera of naive mice for comparison. 3
. Use an alkaline phosphatase-conjuated oat anti-mouse I as a second anti
ody
. 4. Test the su
isotype profile of anti
odies to the antien (ra
ies virus) usi
n the Cal
iochem Hy
ridoma Su
isotypin Kit (La Jolla, CA) with some modificati
ons of the manufacturers protocol as follows. Coat microtiter plate wells with an
tien (ERA-BPL) virus overniht and then
lock with a 2% (w/v) BSAPBS solution.
After washin, add a 1:200 dilution of serum diluted in the
uffer provided with
the kit. Incu
ate the plates at room temperature for 1 h. Next, add 100 L of eac
h of the typin antisera to the different wells. Incu
ate plates for 1 h at room
temperature. Add the peroxidase conjuate and incu
ate for 1 h at room temperat
ure. Then add TMB su
strate and stop color development after 510 min
y the addit
ion of 50 L of 1 M HCL. Read plates at 450 nm (see Note 10).
3.5.2. T-Cell Cytokine Secretion in Response to Restimulation in Vitro
1. Assay sinle cell suspensions of lymphocytes, isolated from drainin lymph no

des or spleens, either for cytokine release or cytolytic activity. The
est resu
lts are o
tained in mice tested 24 wk after the last immunization. 2. To test for
cytokine release (23), culture cells at 6 106 splenocytes or 2 106 lymph node l
ymphocytes in 1.6 mL DMEM, supplemented with 106 M 2mercaptoethanol and 2% (v/v)
FBS. Use 24-well Costar plates without antien or with 5 /well of antien, such
as purified, inactivated ra
ies virus. 3. Harvest supernatants 24 h later and te
st for IL-2 and IL-4 on HT-2 cells. 4. To distinuish
etween the two cytokines,
retest supernatants that score positive in the initial assay
y addition of a 1
:200 and 1:1000 dilution of ascitic fluid containin the 11-B-11 monoclonal anti

ody to IL-4 or the S4B6 anti
ody to IL-2. Alternatively, test supernatants on t
he IL-4-sensitive CT4S cell line usin a proliferation assay. Test GM-CSF and IL
-3 on the 32DC13G/GM cell line in a proliferation assay. Test IFN- for plaque red
uction on equine encephalitis virus infected L929 cells (see Note 11).
3.5.3. Testin for Cytolytic T Cell Activity
1. Restimulate the same sets of splenocytes used for cytokine release with a liv
e, non-cytopathic virus expressin the taret antien, such as with 0.1 pfu of a
n adenoviral recom
inant expressin the ra
ies virus lycoprotein (25).
Genetic Adjuvants
257
2. Alternatively, harvest cells early after immunization and restimulate with 1 
/mL of inactivated virus such as ERA-BPL virus. The inactivated preparation is n
ot appropriately presented for activation of memory CD8+ cells
ut it stimulates
cytokine secretion for expansion of activated cytolytic effector T cells. 3. Ha
rvest cells after 5 d and test at various effector:taret cell ratios (100:112:1)
on 51Cr-la
eled H-2 compati
le cells expressin the same antien (i.e., ra
ies
virus). Prepare these cells
y sta
le transfection with a plasmid vector or infe
ct with a viral recom
inant in 100 L of DMEM supplemented with 10% (v/v) FBS in V
-
ottom microtiter plate wells. 4. Incu
ate control taret cells in medium to de
termine spontaneous lysis, or add 1% (w/v) SDS to measure maximal release. 5. Ha
rvest supernatants 6 hrs later and assay for 51Cr (4).
3.6. Discussion of the Results
We have, to date, analyzed nine different cytokines for their a
ility to serve a
s enetic adjuvants for DNA vaccines. GM-CSF consistently ives the
est results
(11). This cytokine enhances the anti
ody and T-cell response to
oth the pSG5r
a
.p and the pVR1012ra
.np vector. The pVR1012 ra
ies vector expressed the ra
i
es virus nucleoprotein under the control of the CMV promoter; Vical Inc. (San Di
eo, CA) kindly provided the vector
ack
one.The type of the immune response, as
measured
y anti
ody isotype mappin and cytokine secretion pattern, is not aff
ected
y GM-CSF. We only o
tain an increase in the immune response if the GM-CSF
-expressin vector and the antien-expressin vector are injected simultaneously
as a mixture. In some experiments where the GM-CSF vector was injected prior to
the antien expressin vector we saw a dramatic reduction of the response. This
miht reflect a depletion of the muscle of professional antien-presentin cell
s, miratin to lymphatic tissue upon activation
y GM-CSF (and the CpG sequence
s present in the vector), however, this is speculation. The most impressive resu
lts were o
tained usin either low doses of the antien-expressin vector or a v
ector expressin a poorly immunoenic protein. We tested two different vectors e
xpressin GM-CSF under the control of either the Rous Sarcoma virus LTR (pRJB-GM
) or the CMV promoter (pcDNA3GM-CSF). Both vectors enhanced the early B- and T-c
ell immune response. The effect of GM-CSF as a enetic adjuvant was transient, w
hile the immune response was only enhanced when tested early after immunization.
Mice inoculated with the antien-expressin vector alone had slihtly
etter an
ti
ody titers after several months. The effect of GM-CSF was dose-dependent and
the
est results were o
tained with 250  of DNA,
ut 50  also ave a clear incre
ase. IL-4 causes a shift in the kinetics of the immune response, at least in C3H

/ He mice. The response is reduced when tested early after immunization
ut,
258
Ertl et al.
after several months, a sinificant increase for anti
odies, T-helper and cytoly
tic T cells is o
served. IL-4 expressin vectors do not affect the phenotype of
the T-helper response. IFN- normally reulates the expression of class I and II M
HC molecules as well as that of the co-stimulatory molecules, proteasomes, and t
ransporters involved in antien processin. It consistently reduced the B- and T
-cell response to the DNA vaccine (12). In summary, cytokine-secretin vectors c
an serve as enetic adjuvants for DNA vaccines. Due to the localized effects of
vector-encoded cytokines at the site of antien presentation and due to the rela
tive ease of constructin such vectors, they provide an attractive alternative t
o systemic use of the often toxic, and always expensive recom
inant cytokines. N
evertheless, extensive safety studies are needed to assess their potential usefu
lness as vaccine adjuvants in humans. 4. Notes
1. Stock
acteria are stored frozen at 80C in LB supplemented with 30% (v/v) lyce
rol. 2. Most of these cell lines are maintained as frozen stocks in liquid nitro
en. The HT-2 cell line is difficult to recover from frozen aliquots; we have no
t yet
een a
le to successfully recover the CT4S cell line upon freezin; this c
ell line has therefore
een kept in continuous culture. 3. Batches in use can
e
kept for up to 4 mo at 4C. 4. Lyophilized cytokines are supplemented accordin t
o the manufacturers specifications, aliquoted, and stored at 20C. 5. For small-scal
e amplification of
acteria (15 mL), plasmid DNA is purified usin the Promea Wi
zard Minipreps DNA Purification System (Promea Corporation, Madison, WI) accordi
n to the manufacturers specifications. The miniprep plasmid DNA is cut with the
appropriate restriction enzymes to ensure the presence and appropriate orientati
on of the insert. Bacteria carryin the vector with the correct orientation of t
he cytokine cDNA are expanded into a 1:10 dilution and isolated usin the Prome
a Wizard Maxipreps DNA Purification System. 6. From this point on, the further p
rocedures to construct an expression vector are identical to those descri
ed a
o
ve. 7. In eneral, 1020 colonies are analyzed for each sta
le transfection. Cells
that are shown to secrete cytokine are then plated at a defined cell num
er; af
ter 48 h the supernatants are retested for
etter quantification of cytokine sec
retion. 8. Units are determined
y comparison to a defined standard. IL-2 and IL
-4 are tested on HT-2 cells usin the same procedure. 9. In eneral, roups of f
ive mice are used for anti
ody and T cell assays. In most experiments, control m
ice are immunized with empty vector to account for an effect of additional plasmid
. Initially we used a lacZ-expressin construct that
Genetic Adjuvants
259

ave consistently hih
ackround, possi
ly due to
acterial CpG sequences prese
nt in the insert. 10. We initially tested this kit
y usin an immunolo
ulin co
ntrol kit provided
y Cal
iochem Co. to show that it has compara
le sensitivity
for the different immunolo
ulin isotypes (22). In addition, sera from pSG5ra
.
p immune mice are tested
y a neutralization assay (21) that enerally ives res
ults compara
le to those of the ELISA. 11. We have on occasion used ELISPOT assa
ys (24) to determine cytokine secretion patterns. The assay is too la
or-intensi
ve for routine use. Alternatively, sandwich ELISAs are availa
le for cytokines;
these tests are sinificantly more expensive than the relatively simple indicato
r cell assays outlined a
ove. Nevertheless, for la
oratories with limited tissue
culture experience or a reluctance to su
culture CT4S and HT-2 cells every 34 d,
indicator cell assays miht not
e suita
le.
References

1. Tan, D., DeVit, M., and Johnston, S. A. (1992) Genetic immunization is a sim
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r, J. B., Donnelly, J. J., Parker, S. E., Rhodes, G. H., Felner, P. L., Dwarki,
V. J., et al. (1993) Heteroloous protection aainst influenza
y injection of
DNA encodin a viral protein. Science 259, 17451749. 3. Wan, B., Uen, K. I., Sr
ikantan, V., Aadjanyan, M. G., Dan, K., Refaeli, et al. (1993) Gene inoculatio
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 the ra
ies virus lycoprotein ene induces protective immunity aainst ra
ies
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Silva, C. L. (1994) Towards a DNA vaccine aainst tu
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91533. 6. Doolan, D. L., Sedeah, M., Hedstrom, R. C., Ho
art, P., Charoenvit, Y.
, and Hoffman, S. L. (1996) Circumventin enetic restriction of protection aai
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nitric oxide-dependent immunity. J. Exp. Med. 183, 17391746. 7. Schirm
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ohm, W., and Reimann, J. (1996) DNA vaccination primes MHC class I-restricted, s
imian virus 40 lare tumor antien-specific CTL in H-2d mice that reject synene
ic tumors. J. Immunol. 157, 35503558. 8. Fynan, E. F., We
ster, R. G., Fuller, D.
H., Haynes, J. R., Santoro, J. C., and Ro
inson, H. L. (1993) DNA vaccines: pro
tective immunizations
y parenteral, mucosal and ene-un inoculations. Proc. Na
tl. Acad. Sci. USA 90, 11,47811,482. 9. Xian, Z. Q., Spitalnik, S. L., Chen, J.
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ies virus. Viroloy 209, 569579. 10. Xian, Z. Q., Pasquini
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11. Xian, Z. Q. and Ertl, H. C. J. (1995) Manipulation of the immune response t
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ian, Z. Q., Knowles, B. B., McCarrick, J. W., and Ertl, H. C. J. (1995) Immune
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ies virus. Viroloy 214, 398404
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ies virus lycoprotein
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ies virus nucleoprotein defined
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ies virus-specific T-helper c
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23
DNA Immunization in Com
ination with the Immunostimulant Monophosphoryl Lipid A
Donald L. Lodmell, Nancy B. Ray, and Larry C. Ewalt 1. Introduction The use of t
he MPL immunostimulant, a monophosphoryl lipid A preparation derived from the lip
opolysaccharide (LPS) of Salmonella minnesota R595,
ean with the studies of Jo
hnson et al. (1). It was shown that LPS was a potent adjuvant for protein antie
ns, even if administered at a different site and a different time than the anti
en (2,3). Nonetheless, the toxicity of the LPS precluded its usefulness as a pra
ctical adjuvant. Studies
y Ri
i and co-workers (46) and others (7) resulted in t
he attenuation of the toxicity throuh exposure to mild acid hydrolytic conditio
ns. The resultin acid hydrolysate was shown to
e the 4-monophosphoryl derivat
ive of the lipid A moiety (8). Numerous
ioloical studies confirmed that this 4
-monophosphoryl lipid A derivative was a potent immunostimulant which lacked ma
ny toxic properties of the parent LPS. Su
sequent studies determined that mild a
lkaline treatment resulted in removal of one fatty acid from the MPL, resultin
in additional attenuation of toxicity without chanin the immunostimulatin act
ivity (9). These o
servations led to the development of the product MPL which is
presently underoin trials as an adjuvant for several human vaccines. The manu
facture, chemical composition and structure of MPL has
een detailed
y Ulrich a
nd Meyers (10). We will descri
e our techniques for usin MPL as an immunostimul
ant in mice with the aim of enhancin the manitude and duration of the protecti
ve neutralizin anti
ody response elicited
y a DNA vaccine encodin the lycopr
otein of the CVS ra
ies virus.
From: Methods in Molecular Medicine, vol. 29, DNA Vaccines: Methods and Protocol
s Edited
y: D. B. Lowrie and R. G. Whalen Humana Press Inc., Totowa, NJ
261
262
2. Materials 2.1. Plasmid Construction
Lodmell, Ray, and Ewalt
1. TA clonin vector PCRII is availa
le from Invitroen (San Dieo, CA). 2. Gene
clean II is availa
le from Bio 101 (Vista, CA). 3. Plasmid pRABpreG is a constru
ct from Dr. Joseph Esposito (Centers for Disease Control and Prevention, Atlanta
, GA); plasmid pCMV4 is a construct from Dr. David Russell (University of Texas
Southwestern Medical Center, Dallas, TX,); plasmid pcDNA3 is availa
le from Invi
troen (see Note 1).
2.2. DNA Purification
1. Luria
roth (LB): make 10  Bacto tryptone (Difco, Detroit, Ml), 5  Bacto ye
ast extract (Difco), 10  NaCI and 1  lucose, up to 1 L with distilled water,
adjust pH to 7.2 and autoclave. 2. Terrific
roth (TB): make 12  Bacto tryptone
and 24  Bacto yeast extract up to 896 mL with distilled water, sterilize
y au
toclavin, add 100 mL of 0.17 M KH2PO4, 0.72 M K2HPO4 sterile solution and 4 mL
sterile lycerol. 3. Make stock solution of ampicillin (e.., 25 m/mL), filter
sterilize and store at 20C. Dilute in media to appropriate concentration (e.., 10

0 u/mL). 4. Grow plasmids in hih-efficiency competent cells. These should
e r

ecA () end () such as Max Efficiency DH5 cells v il
le from Life Technologies (Gr
nd, Isl nd, NY). 5. M ke Tris-EDTA (TE)
uffer: 0.16g Tris-HCL (10 mM), 0.37 g
EDTA (1 mM) in 100 mL distilled w ter, djust pH to 8.0 with N OH nd utocl ve.
6. DNA purific tion
uffers nd columns re v il
le from Qi gen (S nt Cl rit
, CA) (see Note 2).
2.3. MPL These products m y
e o
t ined from Dr. Terry Ulrich (Ri
i Immunochemic
ls, H milton, MT). 2.4. Accell
1. The Accell-gene delivery system (genegun) is v il
le from powderject V ccin
es, Inc. (M dison, Wl). 2. Spermidine (free
se) nd polyvin l-pyrrolidone (PVP
-360) re v il
le from Sigm (St. Louis, MO). 3. Eth nol (dehydr ted, punctili
ous) is v il
le from Ph rmco, Inc. (Brookfield, CT). 4. Gold
e ds, (0.95 or 2
.6 ) re v il
le from Powderject V ccines. 5. M sterflex tu
ing L/S (size 16) i
s v il
le from Cole P lmer (Vernon Hills, IL). 6. S lve emulsions re v il
l
e from Ri
i Immunochemic ls. 7. Intr medic Tygon tu
ing (PE-60, ID 0.76 mm, OD 1
.22 mm) is v il
le from Cole P lmer (Vernon Hills, IL). 8. Cy no cryl te (supe
r glue) is v il
le from Chemence, Inc. (Alph rett , GA). 9. Metof ne (methoxyf
lur ne) is v il
le from M llinckrodt Veterin ry (Mundelein, IL).
Monophosphoryl Lipid A
263
10. 1 cc tu
erculin syringes nd hypodermic needles [263/8 (9.5 mm) with intr de
rm l
evel] re v il
le from Becton Dickinson (Bedford, MA). 11. Sm ll nim l
clippers, Model A-2, re v il
le from Oster (Milw ukee, WI).
3. Methods 3.1. DNA Meg -Prep Pl smid Prep r tion
1. Pick one colony from pl te of freshly tr nsformed high-efficiency competent
cells (see Note 3) nd inocul te st rter culture of 3 mL of LB or TB medium (
see Note 4) cont ining n ppropri te concentr tion of selective nti
iotic (see
Note 5). Incu
te for 8 h t 37C in sh king incu
tor set t 200300 rpm. 2. Dil
ute st rter culture into 500 mL of selective LB or TB medium in 1 L sterile cu
lture fl sk nd incu
te culture t 37C in sh king incu
tor t 200-300 rpm ove
rnight. 3. Pellet the cells t 6000 g for 10 min nd resuspend the pellet comple
tely
y pipetting up nd down in 50 mL of resuspension
uffer cont ining RN se A
(P1-Qi gen). 4. Add 50 mL of lysis
uffer (P2-Qi gen), mix
y inverting 23 times
nd incu
te t room temper ture for 5 min (see Note 6). 5. Add 50 mL of pre-ch
illed (4C) precipit tion
uffer (P3-Qi gen), mix
y inverting 45 times nd incu
t
e on ice for 30 min (see Note 7). 6. Split the solution cont ining the precipit
te into 50 mL conic l F lcon tu
es, nd centrifuge t pproxim tely 20,000g for
30 min t 4C. Equili
r te DNA
inding column with 35 mL equili
r tion
uffer (t
ip 2500 column, Qi gen, QBT
uffer, Qi gen). 7. Filter supern t nt fluid from ce
ntrifug tion through sterile g uze to remove flo ting precipit te nd pply to c
olumn
y gr vity flow. 8. W sh the column with sever l column vol of w sh
uffer
(200 mL QC
uffer-Qi gen) nd elute DNA from the column with elution
uffer (35
mL QF
uffer-Qi gen). Precipit te DNA
y dding 0.7 vol of isoprop nol, mix
y
inversion nd split the volume into 15 mL Corex tu
es (no more th n 12 mL/tu
e)
nd centrifuge t m ximum rpm in swinging
ucket rotor for 30 min t 4C (see No
te 8). 9. Resuspend the pellets in 200500 L of TE
uffer (depending upon the pelle
ts), m ke ppropri te dilutions in distilled w ter nd qu ntit te
y UV spectrop
hotometry t 260 nm. 10. An lyze purity of DNA
y A260/A280 r tio (see Note 9),
enzyme digestion nd g rose gel electrophoresis. 11. Store DNA t 4C in TE
uffe
r (see Note 10) nd dilute s needed for v ccin tions in sterile dou
le-distille
d w ter or sterile PBS (e.g., 1 mg/mL).
3.2. Intr muscul r V ccin tion
1. Fit 26 g uge 3/8 in. (intr derm l
evel) needles with Tygon tu
ing sleeves. 2
. Sh ve the re r legs of Metof ne nesthetized mice with nim l clippers.

264
Lodmell, R y, nd Ew lt
3. Use tu
erculin syringe nd needle fitted with tygon tu
ing sleeve (see No
te 11) to inocul te e ch ti
i lis nterior muscle with 50 g of MPL in 50 L (stock
MPL solution supplied
y Ri
i Immunochem) or with 50 L of PBS without MPL (see No
te 12). 4. Forty-eight hours l ter, inject 1100 g of DNA in 50 L of PBS into the pr
eviously tre ted muscle. Booster v ccin tions, if given, re dministered using
the identic l procedure.
3.3. Intr derm l V ccin tion
1. Sh ve the re over the dors l surf ce of e ch hindqu rter of metof ne nesth
etized mice. 2. Com
ine 50 g of MPL nd v rying concentr tions (1100 g) of pl smid
DNA per 50 L of PBS nd inocul te the sh ved re ID (see Note 13). If
ooster v
ccin tions re given use n identic l protocol. 3. Inocul te control mice simil
rly,
ut without the MPL.
3.4. Genegun V ccin tion
1. Prep re 2.6 m gold
e ds co ted with 2 g of DNA ccording to the specific instr
uctions provided
y Powderject V ccines. 2. Sh ve the
domen of Metof ne nesth
etized mice with nim l clippers. 3. With gloved finger, m ss ge 40 L of s lv
e emulsion cont ining 48 g of MPL within micell r n no-p rticles into 1.5 cm2
re of the sh ved
domen. Use s lve without the MPL to simil rly tre t control
mice. 4. Immedi tely shoot the DNA-co ted
e ds through the s lve-tre ted skin i
nto the epidermis using the Accell gene delivery system t helium pressure set
ting of 400 psi. A helium pressure setting of 300 psi is used for mice 24 h to 1
0 d of ge.
4. Notes
1. Pl smid Constructions: cDNA encoding the glycoprotein (G) gene of the CVS str
in of r
ies virus w s mplified
y polymer se ch in re ction (PCR) with T q DN
A polymer se. The cDNA h d previously
een cloned into pBR322 resulting in the r
ecom
in nt pl smid pRABpreG. The 28
se 5 primer used for mplific tion w s de
signed to include B mHI site for cloning nd, for improved expression, Koz k
sequence more closely m tching the Koz k consensus sequence. The 22
se 3 pri
mer included pre-existing B mHI site loc ted 3 of the termin tion codon. Tenf
old dilutions of solution cont ining 10 ng of pRABpreG were mplified. One mic
roliter of PCR mplified DNA encoding G w s used to clone into the TA cloning ve
ctor PCRII. Resulting clones were sequenced
y DNA dou
le-str nd dideoxy sequenc
ing. One mut tion-free clone w s used for su
sequent su
cloning. The 1.6 k
fr g
ment cont ining the entire coding sequence for G w s excised from PCRII with B m
HI, purified with Genecle n II methodology, nd su
cloned into the Bgl11 site of
pCMV4, or the B mHI site of pcDNA3. Recom
in nt clones were sequenced nd deter
mined to
e mut tion-free. Both pl smids cont ined the G insert downstre m from
the
Monophosphoryl Lipid A
265
2.
3. 4. 5. 6. 7. 8. 9. 10. 11.
12.
13.

CMV m jor immedi te-e rly promoter. Al1 recom
in nt DNA w s tr nsformed into com
petent DH5 cells. Recom
in nt pl smid DNA w s isol ted from cultures grown in eit
her LB or TB cont ining 100 g/mL of mpicillin. Wiz rd minipreps nd restriction
enzyme n lyses were done to screen potenti l clones. Recom
in nt DNA used for s
equencing nd v ccin tion w s prep red using Qi gen M xi nd Meg kits. Qi gen P
l smid Meg Kits, which cont in ll the necess ry
uffers, yield pproxim tely 12
mg of highly pure DNA. Low copy num
er pl smids give lower yields. Endo-free ki
ts re v il
le from Qi gen to prep re endotoxin-free DNA. Continuous p ss ging
of tr nsformed cells m y result in lower yields with cert in pl smids. Some TB
cultures produce too much protein, which c n interfere with pl smid purific tion
. In such c ses use LB. Anti
iotic choice depends on selective resist nce of the
pl smid. Solution should
ecome cle r nd viscous, do not exceed 5 min. A white
floccul nt precipit te is formed th t cont ins genomic DNA, protein nd SDS. A
cle r to white pellet should
e concentr ted t the
ottom of the Corex tu
e. Pu
re pl smid DNA should h ve n A260/A280 r tio of pproxim tely 2.0. Pl smid DNA
is more st
le in TE
uffer since it cont ins EDTA, which chel tes Mg2+ ions w
y from nucle ses, inhi
iting their ctivity. The tu
ing is pulled w y from the
hu
of the needle (2 mm), cut with scissors nd then slipped
ck to the needle
hu
where it is firmly tt ched with super glue. The tu
ing
elow the unshe thed
tip of the needle controls the depth of the IM inocul tion to 2 mm. Tu
ing with
sm ller ID will fit tightly to the needle, elimin ting the need for super glu
e. Anim ls must
e nesthetized to st te in which there is tot l rel x tion of
the muscles. If the muscle flexes t the time of the injection, there is the po
ssi
ility th t some of the inoculum will
e squeezed from the site. Use 26 g uge
3/8 in. needles with ID
evels th t re not she thed with Tygon tu
ing. It is v
ery import nt th t needles with ID
evels re used for this inocul tion. Without
the ID
evel it is very difficult to see the tip of the needle
elow the skin T
he needles re inserted through the skin nd then upw rd tow rd the skin surf ce
until the
evel is o
served just
elow the dermis. Expulsion of the 50 L t this
position le ves typic l ID
le
in the skin. ID injections re more difficult
nd t ke more time th n IM injections. Thus, e ch mouse must
e nesthetized well
enough to rem in
solutely quiet for 3045 s.
References
1. Johnson, A. G., G ins, S., nd L ndy, M. (1956) Studies on the O ntigen of S
lmonell typhos . V. Enh ncement of nti
ody responses to protein ntigens
y p
urified lipopolys cch ride. J. Exp. Med. 103, 225246. 2. Munoz, J. (1964) Effects
of
cteri nd
cteri l products on nti
ody response, in Adv nces in Immunol
ogy (Dixon, F. J. nd Kunkel, H. G., eds), Ac demic Press, New York, pp. 397440.
266
Lodmell, R y, nd Ew lt
3. Johnson, A. J. (1964) Adjuv nt ction of
cteri l endotoxins on the prim ry
nti
ody response, in B cteri l Endotoxins (L ndy, M. nd Br un,W., eds), Rutger
s University Press, New Brunswick, pp. 252262. 4. Ri
i, E., P rker, R., Str in, S
. M., Mizuno, Y., Nowotony, A., Von Eschen, K. B., C ntrell, J. L., McL ughlin,
C. A., Hw ng, K. M., nd Goren, M.B. (1979) Peptides s requirement for immunoth
er py of the guine -pig Line 10 tumor with endotoxins. C ncer Immunol. Immunothe
r. 7, 4358. 5. Ri
i, E., Am no, K., C ntrell, J., Schw rtzm n, S., P rker, R., n
d T k y m , K. (1982) Prep r tion nd ntitumor ctivity of nontoxic lipid A. C
ncer lmmunol. Immunother. 12, 9196. 6. Ri
i, E. (1984) Benefici l modific tion of
the endotoxin molecule. J. Biol. Response Mod. 3, 19. 7. T k y m , K., Ri
i, E.,
nd C ntrell, J. L. (1981) Isol tion of nontoxic fr ction cont ining tumor re
gression ctivity. C ncer Res. 41, 26542657. 8. Qureshi, N., T k y m , K., nd Ri

i, E. (1982) Purific tion nd structur l determin tion of nontoxic lipid A o
t
ined from lipopolys cch ride of S lmonell typhimurium. J. Biol. Chem. 257, 11,8
0811,815. 9. Meyers, K. R., Truchot, A. T., W rd, J., Hudson, Y., nd Ulrich, J.T
. (1990) A critic l determin nt of lipid A endotoxic ctivity, in Cellul r nd M

olecul r Aspects of Endotoxin Re ctions (Nowotony, A., Spitzer, J. J., nd Ziegl

er, E. J., eds.) Elsevier, Amsterd m, pp.145156. 10. Ulrich, J. T. nd Myers, K.
R.(1995). Monophosphoryl lipid A s n djuv nt. P st experiences nd new direct
ions. Ph rm. Biotechnol. 6, 495524.
24
Controlled Pl smid Delivery nd Gene Expression
Applic tions for Nucleic Acid-B sed V ccines
Russell J. Mumper, H rry C. Lede
ur, Jr., Al in P. Roll nd, nd Eric Tomlinson 1
. Introduction After the concept of genetic immuniz tion w s first demonstr ted

y Johnstons group in 1992 (1), numerous studies h ve reported the potenti l prop
hyl ctic nd ther peutic use of nucleic cid-
sed v ccines for com
ting v riou
s infectious dise ses (24). V ccines of this composition ppe r to
e
oth effic
cious in the short term, nd
le to elicit prolonged n mnestic response c p

le of preventing or resolving infection when ch llenged t up to one ye r fter
v ccin tion (5). Nucleic cid-
sed v ccines elicit
ro der immune response t
h n do su
unit v ccines, inducing
oth cellul r nd humor l responses th t re r
eminiscent of ttenu ted nd whole-killed vir l v ccines. Further, nucleic cid
sed v ccines c n
e prep red with rel tive e se of synthesis nd production. E
xpression pl smids c n
e gener ted quickly once the ntigens coding sequence is
known nd sm ll- nd l rge-sc le purific tion methods re well est
lished. Nucl
eic cid-
sed v ccines lso void some of the s fety concerns of convention l v
ccines in th t there is no ch nce of dise se due to co-purific tion of cont min
ting virus or reversion of the ttenu ted str in in the p tient. This is not to
cl im th t the s fety issues surrounding nucleic cid-
sed v ccines re minim
l. The m jor theoretic l concerns surrounding the s fety of this technology incl
ude pl smid integr tion into the host genome, tr nsform tion of som tic or stem
cells, nd toler
ility. However, there is no pu
lished evidence th t dministr
tion of unformul ted or n ked pl smid produces severe short or long term deleter
ious effect (6).
From: Methods in Molecul r Medicine, vol. 29, DNA V ccines: Methods nd Protocol
s Edited
y: D. B. Lowrie nd R. G. Wh len Hum n Press Inc., Totow , NJ
267
268
Mumper et l.
Even though there is tremendous mount of expect tion surrounding the use of n
ucleic cid-
sed v ccines for the prevention nd tre tment of infectious dise s
es, sever l spects of the technology need to
e signific ntly improved
efore s
uch ppro ches will h ve widespre d use. One re centers on the delivery of pl
smid to control precisely its deposition in the
ody. The second re focuses on
control of gene expression to limit the cell types c p
le of producing the gen
e product. With the heightened focus on the s fety of nucleic cid-
sed v ccine
s
y the FDA (7), control of pl smid delivery nd gene expression is p r mount n
ot only to enh nce the over ll effic cy of these v ccines
ut lso to elimin te
v rious s fety concerns. In Fig. 1, we h ve summ rized the six key product requi
rements for nucleic cid-
sed v ccine. Essenti lly five critic l f ctors need
to
e considered to chieve these product requirements. This p per discusses ho
w the critic l f ctors of formul ted nucleic cid-
sed v ccines c n
e controll
ed
y using well-defined pl smid delivery nd gene expression systems.
2. Mech nism of Action
Sever l different routes h ve
een investig ted for the dministr tion of nuclei
c cid-
sed v ccines. These routes include su
cut neous, intr venous, intr derm
l, n s l, or l nd intr muscul r dministr tion. Of these routes, the most wide
ly used route for the dministr tion of nucleic cid-
sed v ccine in nim l s
tudies nd hum n clinic l tri ls is direct intr muscul r dministr tion of pl sm

id. Initi lly, it w s hypothesized th t tr nsfected muscle cells expressing the

ntigen fter intr muscul r dministr tion served s n ntigen depot or f ctory
, c p
le of initi ting
oth the cellul r nd humor l responses to p rticul r
ntigen. Antigen rele sed from tr nsfected myocytes (
y secretion or cell de th)
into the circul tion or Iymph nodes w s supposedly t ken up
y m croph ges or B
cells nd used to initi te T-helper dependent nti
ody response. CTL priming
w s
elieved to occur vi endogenous prote some-dependent processing of the nti
gen nd present tion of the ntigen on the surf ce in ssoci tion with MHC cl ss
I molecules. In ccord nce with this model, tr nspl nt tion of tr nsfected myo

l sts into syngeneic mice led to the gener tion of
oth n ntigen-specific nti

ody nd CTL response (8). However, su
sequent doptive tr nsfer studies in mi
ce with mix-m tched nd m tched h plotypes demonstr ted th t the cells ctu lly
presenting cl ss I restricted ntigens were derived solely from the
one m rrow
(910). In simil r studies, Doe et l. reported th t
one m rrow reconstitution co
uld occur sever l d ys to weeks fter v ccin tion, thus demonstr ting th t
one
m rrow derived ntigen presenting cells (APCs) could present ntigens synthesize
d t other sites in the
ody nd not from ntigen synthesized endogenously (11).
From these studies, it h s
een postul ted th t tr nsfected myocytes serve s
n
Controlled Pl smid Delivery
269
Fig. 1. Elements of formul ted nucleic cid-
sed v ccine.
endogenous f ctory producing ntigen th t is picked up nd processed
y the
one
m rrow derived APCs. Interestingly, Torres et l. h ve recently demon270
Mumper et l.
str ted th t ntigen expression in the muscle is not required for the elicit tio
n of n immune response to n intr muscul rly dministered nucleic cid-
sed v
ccine (12). This experiment involved removing the injected muscle within minutes
fter dministr tion nd demonstr ting th t this h d no effect on the immune re
sponse to the encoded ntigen. If these studies ccur tely reflect wh t is h ppe
ning in vivo, it is quite possi
le th t there m y
e redund nt p thw ys for the
elicit tion of n immune response fter intr muscul r dministr tion of nuclei
c cid-
sed v ccine. For ex mple, gene expression in the muscle ppe rs to
e s
ufficient for the elicit tion of the immune response,
ut is not required. Promi
scuous deposition of pl smid ensures th t other cells or tissues in the
ody re
c p
le of producing the ntigen nd stimul ting the immune response, even when
the prim ry site of expression is removed. One possi
le expl n tion for the res
ults o
served
y Torres et l. is direct tr nsfection of APCs fter intr muscul
r dministr tion of pl smid. Theoretic lly, APCs could express the ntigen endog
enously for present tion on their surf ce r ther th n relying on ntigen produce
d
y tr nsfected cells in the periphery (i.e., muscle cells). This hypothesis is
supported
y studies using gene gun to dminister pl smid. After intr derm l
dministr tion of pl smid with gene gun, pl smid directly tr nsfects the resid
ent APCs of the skin (L ngerh ns cells), resulting in the su
sequent migr tion o
f these cells to the dr ining Iymph node, where ntigen is expressed (13). All c
urrent evidence suggests th t direct tr nsfection of APCs using the gene gun is
responsi
le for the initi tion of the ntigen-specific immune response to the nu
cleic cid-
sed v ccine. 3. Appro ches to the Control of Pl smid Delivery A key
issue for the development of effective nucleic cid-
sed v ccines is the ident
ific tion of those tissues or cells where pl smid needs to
e delivered in order
to elicit the desired response. If pl smid could ctively nd specific lly
e t
rgeted to critic l sites r ther th n relying on the r ndom ph rm cokinetics of r

 ked pl smid or pl smid-l den gold p rticles, one could theoretic lly reduce the
dose of pl smid required for prophyl ctic or ther peutic immune response. Thes
e dose reductions could tr nsl te into more cost-effective product. In dditio
n to reducing the effective dose for nucleic cid-
sed v ccines, t rgeted deliv
ery of pl smid would elimin te cert in s fety concerns
out the distri
ution of
pl smid fter dministr tion. D t suggest th t pl smid c n
e detected in nume
rous tissues fter v rious routes of dministr tion. For ex mple, pl smid DNA c
n enter the
loodstre m or Iymph tic system fter intr muscul r dministr tion
nd tr ffic to the spleen, liver, kidney, dr ining Iymph nodes, nd
one m rrow (
14). The deposition of pl smid in the
ody c n
e controlled
y use of t rgeted
delivery systems. For
Controlled Pl smid Delivery
271
successful t rgeting of pl smid within the
ody, one must
e
le to effectively
t rget pl smid to the ppropri te cell type
y controlling pl smid st
ility, c
olloid l size, nd surf ce ch rge/morphology (see Fig. 1). This str tegy m y pot
enti lly llevi te s fety concerns de ling with r ndom distri
ution of pl smid i
n vivo. Presently, it is uncle r whether production of ntigen in muscle h s uni
que properties with respect to the elicit tion of prolonged immune response or
whether expression in ny tissue in the periphery is sufficient for the inducti
on of n ntigen-specific immune response. It is conceiv
le th t one m y circum
vent the need for peripher l expression of ntigen nd directly t rget pl smid
nd su
sequent gene expression to APCs s me ns of inducing the desired immune
response. Although the ex ct identity of which APC should
e t rgeted h s yet to

e determined, in vitro nd in vivo evidence suggests th t the ide l cells to t
rget re dendritic cells (DCs). DCs h ve
een demonstr ted to
e potent initi t
ors of immune responses nd possess the co-stimul tory nd dhesion molecules re
quired for T cell ctiv tion (15). In ddition, DCs possess unique
ility to
process nd present extr cellul r ntigen in the context of
oth cl ss I nd cl
ss II molecules (16). If pl smid were to tr nsfect these cells directly, it is l
ikely th t these cells would elicit
oth the cellul r nd humor l response o
ser
ved (i.e., gener tion of CTL response, ntigen production for ctiv tion of B
cells nd the T cell help necess ry for humor l response). Systems th t c n ef
ficiently t rget nd deliver pl smid to these cells m y provide the necess ry te
chnology le p to provide effective nd low cost v ccines (see Fig. 1). In the ne
xt sections, the use of protective inter ctive non-condensing (PINC) systems, pe
ptide-
sed systems, nd lipid-
sed systems will
e discussed s potenti l deli
very systems to control the in vivo deposition of pl smid nd potenti lly t rget
pl smid to DCs (see T
le 1).
3.1. PINC Systems
It is estim ted th t less th n 0.01% of pl smid injected into muscle is t ken up

y muscle cells nd expressed (14,1720). The m jority of injected n ked pl smid p
pe rs to
e r pidly degr ded
y extr cellul r nucle ses nd/or removed from the
muscle vi the lymph tic system. To incre se the
io v il
ility of int ct pl sm
id to muscle cells nd protect it from nucle se digestion, protective, inter cti
ve, non-condensing (PINC; GENE MEDICINE INC., The Woodl nds, TX) polymers h ve

een developed to inter ct with pl smids without condens tion into comp ct p rtic
les (2123) (see T
le 1). Recently, PINC polymer/pl smid complex formul tion w
s pproved
y the FDA for Ph se I hum n clinic l tri l for direct intr muscul
r dministr tion of hum n insulin-like growth f ctor (hIGF-I) expression pl sm
id (24). Unlike condensed pl smid p rticles, these PINC complexes re
le to di
ffuse throughout the
272
T
le 1 Potenti l Appro ches to the Control of Pl smid Delivery for Nucleic Acid
V ccines PINCTM Systems

Mumper et l.
Non-condensing mphip thic polymers Approved
y the FDA in Ph se I hum n clinic
l tri ls for non-vir l gene ther py Inter ct with pl smid to form polymer/pl smi
d complexes Protect pl smid from r pid extr cellul r nucle se degr d tion Design
ed for intr muscul r, su
cut neous, nd intr derm l routes of v ccin tion Incre
se
oth the levels nd extent of gene expression in rodent muscle F cilit te the
upt ke of pl smid into cells Cell-specific t rgeting (using lig nd-PINC conju
g te) m y
e possi
le using PINC Peptide-B sed Systems Short c tionic peptides s
ynthesized
y solid ph se chemistr Highly pure nd homogeneous; e sy to ch r cter
ize Controlled colloid l nd surf ce properties of peptide/pl smid complexes Des
igned for multiple routes of v ccin tion Lipophilic moieties h ve
een tt ched
to provide dded colloid l st
ility Improved intr cellul r delivery of pl smids
h s
een chieved using endosomolytic peptides Structur l motifs th t potenti t
e immune responses c n
e included Cell-specific t rgeting h s
een chieved Lip
id-B sed Systems Amphiphilic c tionic lipids/
iodegr d
le c tionic lipids Appro
ved
y the FDA in Ph se I hum n clinic l tri ls for non-vir l gene ther py Contr
olled colloid l nd surf ce properties of lipid/pl smid complexes Designed for m
ultiple routes of v ccin tion Improved intr cellul r delivery of pl smids using
fusogenic co-lipids nd/or endosomolytic peptides Demonstr ted potenti tion of i
mmune responses Cell-specific t rgeting chiev
le
tight l ttice of muscle to incre se the extent of pl smid deposition in the musc
le. In ddition, the inter ction of PINC polymers with pl smid results in incre
sed resist nce to nucle se degr d tion. Complex tion of the polymers nd pl smid
s m y occur
y ny one or sever l mech nisms including hydrogen
onding ( ccepti
ng or don ting), V n der W ls, ionic inter ction, dipole-dipole (or induci
le d
ipoles), ionic-dipole, or hydropho
ic inter ctions. PINC polymers h ve common
fe ture in th t they re mphip thic in n ture, h ving function l group th t c
n
ind to pl smid nd
ck
one th t provides hydroControlled Pl smid Delivery
273
pho
ic surf ce on the polymer/pl smid colloid. We hypothesize th t the hydropho

ic surf ce of the polymer/pl smid complex f cilit tes the cellul r upt ke of pl
smid through incre sed hydropho
ic inter ction of the polymer/ pl smid complex w
ith cell mem
r nes. There is simil r phenomenon reported
y K
nov et l. whe
re
y upt ke of pl smid complexes
y cells h s
een shown to
e incre sed
y the
use of c tionic polyvinyl
sed polymers th t condense pl smid (25). These conden
sing c tionic polymers of K
nov et l. result in polymer/pl smid complex wit
h hydropho
ic surf ce due to the presence of the polyvinyl
ck
one. Prototype
PINC polymers, such s polyvinyl pyrrolidone (PVP; 50 kD ) nd polyvinyl lcoho
l (PVA; 18 kD ),
ind to pl smid vi hydrogen
ond ccepting nd don ting mech n
isms, respectively. Optimized stoichiometry for complex tion of these polymers w
ith pl smids h s
een shown (
y microtitr tion c lorimetry, zet potenti l modul
tion, nd ethidium
romide fluorescence) to
e 1:17 w/w (pl smid/PVP) nd 1:10
w/w (pl smid/PVA). Injection of these complexes into r t muscle results in up to
10-fold incre se in the
oth the level nd extent of gene expression in r t m
uscle. Using series of co-polymers consisting of polyvinyl-pyrrolidone nd covinyl cet te, we h ve shown th t polymer structure, independent of ll other v
ri
les, ffects the level of reporter gene expression in r t muscle. This struc
ture- ctivity rel tionship is
eing used s the
sis for designing dv nced PIN
C polymers th t
ind to pl smid with incre sed ffinity. It is thought th t the
incre sed
inding
etween the polymer nd pl smid will result in incre sed resis
t nce of the pl smid to nucle se degr d tion. In ddition, the improved st
ilit
y m y result in further incre ses in the levels nd extent of gene expression in
muscle s result of the enh nced ccess of int ct pl smid to gre ter num
er

of muscle cells. Incre sed
inding ffinity to micromol r r nge or lower m y l

so fford the possi
ility of tt ching cellspecific lig nds to novel PINC polyme
rs for t rgeting cells with enh nced immune function. such s DCs. PINC systems
h ve
een tested s delivery systems for nucleic cid-
sed v ccines
y dminist
r tion of PINC polymer/pl smid complexes vi intr muscul r, su
cut neous, nd in
tr derm l routes. These studies h ve included the use of needle-free injection d
evices to reproduci
ly deposit nucle se-protected pl smid t these sites. We h v
e shown th t pl smid complexed to PINC polymers g ins ccess to the dr ining lym
ph nodes fter intr muscul r nd su
cut neous dministr tion (unpu
lished result
s). Further, we h ve shown th t complex
etween PVP nd n expression pl smid
for hum n growth hormone (hGH) formul ted in 150 mM N C1 nd injected using ne
edle-free injection device (Medi-Ject Corpor tion; Minne polis, MN) into dog mus
cle results in up to five-fold incre se in nti-hGH nti
ody titers t wk 8 s
comp red to the s me dose of pl smid formul ted s n ked pl smid in 150 mM N C1
274
Mumper et l.
(26). Further, nti-hGH nti
ody titers using the needle-free injection device w
ere 20-fold gre ter th n with needle injection. The mech nism for this incre se
in nti-hGH nti
ody titers using PVP/pl smid complex is currently under inves
tig tion in our l
or tory. PINC polymer/pl smid complexes h ve promise in the f
ield of nucleic cid-
sed v ccines since they dd to the usefulness of n ked pl s
mid technology th t h s
een shown to work in multiple nim l models s prophy
l ctic v ccine (24). Potenti lly, second gener tion, more inter ctive PINC polyme
rs will en
le the dose of pl smid to
e gre tly reduced s result of incre se
d
io v il
ility of pl smid dministered
y these routes. It is lso fe si
le t
h t PINC polymers will en
le the t rgeting of DCs vi conjug tion of the PINC p
olymers with DC-specific lig nds.
3.2. Peptide-B sed Systems
Proteins, such s higher molecul r weight poly-L-lysine (PLL), h ve
een investi
g ted to condense pl smid into comp ct structure. For m ny pplic tions, v rio
us cell-specific t rgeting lig nds h ve
een cov lently tt ched to PLL, includi
ng tr nsferrin, fol te, CD3 nti
ody, si loorosomucoid, l ctose, g l ctose, nd
m nnose (2734). These ppro ches h ve shown th t cell-specific t rgeting c n
e
chieved in vitro nd, in some c ses, in vivo. PLL h s lso
een used to enh nce
the upt ke of peptide ntigens into ntigen presenting cells in vitro, which su
ggests th t polyc tionic c rriers m y lso
e utilized to t rget pl smid to impo
rt nt cells of the immune system (35). M jor limit tions of PLL-
sed systems in
clude the heterogeneity of the PLL used, cytotoxicity, nd the irreproduci
ility
in
oth pl smid complex tion nd
iologic l effect. As result, sever l ttemp
ts h ve
een m de to utilize shorter nd more welldefined peptides for pl smid d
elivery. We h ve developed propriet ry peptide-
sed systems derived from the or
igin l work reported
y Smith nd colle gues (3638) (see T
le 1). For ex mple, t
he peptide
ck
one, YKAKnWK, h s
een extensively modified nd conjug ted to o

t in multiple f milies of lipophilic condensing peptides. Other lipophilic PLL or
peptide deriv tives for pl smid delivery h ve
een descri
ed in the liter ture (
3941). These short lipophilic condensing peptides re synthesized
y solid ph se
chemistry, highly pure, nd unlike PLL, e sy to ch r cterize. These peptides h v
e
een shown to h ve signific ntly less cytotoxicity comp red to PLL. These lipo
philic condensing peptides h ve lso
een cov lently modified to o
t in condensi
ng peptides with cell-specific t rgeting lig nds such s mono- or multi- ntenn r
y g l ctose or m nnose. For ex mple, we h ve developed pl smid complexed with
tri- ntenn ry g l ctose modified-lipophilic condensing peptide, com
ined with
lipophilic lytic peptide, which c n selectively t rget mouse hep tocytes fter
t il vein injection
Controlled Pl smid Delivery

275
(unpu
lished results). This pl smid complex, with n ver ge p rticle size
elow
100 nm, h s shown to
e st
le to
oth s lt nd s lt/serum ch llenge, ch r ct
eristic th t is critic l for successful in vivo receptor-medi ted endocytosis. L
ipophilic lytic peptides, which promote pH specific lysis of endosom l vesicles,
re lso
eing developed s me ns to enh nce cytopl smic entry of pl smid. Th
e colloid l nd surf ce properties of condensed systems, nd the st
ility in
i
ologic l milieu, c n
e controlled to ultim tely ffect the
iodistri
ution of t
hese systems fter in vivo dministr tion vi v rious routes. Control of the col
loid l properties is m de possi
le
y the inclusion of st
ilizing moieties incl
uding: (i) peptide sequences th t re more resist nt to prote se degr d tion, (i
i) sp cer link ges
etween the condensing group of the peptide nd the cell-spec
ific t rgeting moiety th t provide for steric st
iliz tion, nd (iii) lipophili
c moieties th t provide st
iliz tion vi hydropho
ic contri
ution. For use in d
elivery of nucleic cid-
sed v ccines, we envision the possi
ility of including
in the lipophilic condensing peptides motifs th t potenti te immune responses,
ttr ct DCs, nd/or th t selectively
ind DCs, s descri
ed
y B rry et l. (42)
.
3.3. Lipid-B sed Systems
Of the fifteen clinic l protocols pproved
y the FDA for non-vir l gene ther py
s of Septem
er 1996, twelve involved the use of c tionic lipid formul tions of
pl smid (43). Initi l hum n clinic l tri ls (4446) using sever l different c tio
nic lipids h ve demonstr ted th t no signific nt toxicity results from lipid-
s
ed formul tions, even for protocols involving repe ted dministr tion. Thus, it
is fe si
le th t c tionic lipid-
sed systems h ve immedi te pplic
ility s de
livery systems for nucleic cid-
sed v ccines if these systems re shown to mee
t other product requirements (see Fig. 1) . C tionic lipids m y serve m ny funct
ions including: (i) protection of the pl smid from nucle se degr d tion, (ii) mo
dific tion of the size nd surf ce ch rge of the pl smid, (iii) enh ncement of t
he cell upt ke of pl smid, (iv) enh ncement of the rele se of pl smid from the e
ndosomes, nd v) enh ncement of the entry of int ct pl smid into the nucleus (ei
ther directly or indirectly) (37). Sever l ltern tive lipid-
sed systems, incl
uding cochle tes (4748) nd emulsion-
sed systems (4951) for gene ther py, nd nu
cleic cid-
sed v ccines, h ve lso
een descri
ed. These novel systems m y ls
o possess simil r functions s mentioned
ove. T rgeting motifs m y lso
e inc
luded in lipid-
sed systems to enh nce cell-specific upt ke of
iomolecules. Fo
r ex mple, nti
odies (5253), si lofetuin (5456), tr nsferrin (57), l ctosylcer m
ide (58), N- cetyl glucos mine (59), fucose (59), nd m nnose (5964) h ve
een t
t ched to lipids nd shown to incre se the efficiency of cell upt ke.
276
Mumper et l.
Specific t rgeting of dendritic cells residing in the lymph nodes m y
e vi
l
e str tegy for nucleic cid-
sed v ccines. Lipid-
sed systems (c tionic nd no
n-c tionic) h ve shown preferenti l (non-specific) t rgeting to the dr ining lym
ph nodes fter su
cut neous dministr tion. These liposomes were found to
e t k
en up l rgely
y m croph ges (65) nd possi
ly other ntigen-presenting cells (6
6). Sever l p r meters c n govern the efficiency of Iymph dr in ge fter intr mu
scul r nd su
cut neous dministr tion of colloid l formul tions, including the
p rticles size, surf ce ch rge, nd surf ce hydropho
icity/hydrophilicity
l nce
(6770). M ximum lymph tic upt ke
y colloid l system fter intr muscul r nd s
u
cut neous dministr tion h s
een reported to
e potenti lly up to 40% of the
dministered dose, depending on the colloid l properties, s mentioned
ove (70
). Liposomes, especi lly those cont ining c tionic lipids, h ve lso
een shown
to h ve strong immunopotenti ting properties th t m y prove suit
le for their u

se s djuv nts (7172). For ex mple, the use of c tionic lipids to deliver pl s
mid encoding for the S (sm ll) region of hep titis B surf ce ntigen (HBsAg) res
ulted in 100-fold gre ter IgG1 nti
ody titers nd 10-fold gre ter IgG2 nd IgG
2
nti
ody titers when delivered vi c tionic lipid-
sed system s comp red
to n ked pl smid (66). In ddition to elev ted immunoglo
ulin titers, pl smid form
ul ted with c tionic lipid-
sed delivery system signific ntly incre sed the l
evels of cert in cytokines comp red to n ked pl smid, most not
ly INF and IL-4. Li
pid-
ased systems, as delivery systems for nucleic acid-
ased vaccines, may serv
e many diverse functions includin controllin the surface and colloidal propert
ies of plasmid, plasmid deposition, cell-specific taretin, and immunopotentiat
ion. It is expected that a considera
le amount of effort will continue to
e dev
oted to the development of these delivery systems for nucleic acid-
ased vaccine
s. 4. Approaches to the Control of Gene Expression Presently it is unknown wheth
er the sites of plasmid deposition har
or plasmid framents or intact plasmid ca
pa
le of producin the viral/
acterial antien. Potentially, uncontrolled antie
n expression could have toxic effects dependin on the nature of antien (e..,

acterial toxins or viral oncoenes) or lead to the destruction of non-professio
nal APCs presentin the ene product in the context of class I
y antien-specif
ic CTLs. The second area for improvement in the development of nucleic acid-
ase
d vaccines centers on the control of ene expression. Two different aspects of 
ene expression will
e discussed. The first deals with tissue-specific expressio
n to limit antien production to a particular cell type. The second focuses on t
he expression of multiple antiens from a sinle plasmid.
Controlled Plasmid Delivery
277
Fi. 2: Muscle-specific expression system. The expression cassette was derived f
rom the chicken skeletal - ctin gene nd cont ins the following components: the c
hicken skelet l ot ctin promoter (411 to +1), the first exon [5 untr nsl ted reg
ion (UTR)] of the skelet l (- ctin gene (+1 to +196), the first intron of the ske
let l - ctin gene, restriction enzyme cle v ge sites for insertion of gene coding
sequence nd 2 kilo
ses of the skelet l - ctin gene 3 (UTR) (79).
4.1. Tissue-Specific Expression
All current nucleic cid-
sed v ccines descri
ed in the liter ture rely on vir
l promoters for the expression of gene product. These genes not only include t
he ntigen of choice,
ut genetic djuv nts
eing tested s w ys to potenti te t
he immune response like CD80, CD86, IL2, GM-CSF, IL-4 nd IL-12 (7378). Since pl
smid c n potenti lly ccess sever l tissues throughout the
ody, uncontrolled ex
pression of ntigen from promiscuous vir l promoters m y result in serious sideeffects. These side-effects could r nge from n utoimmune-like re ction (i.e.,
destruction of the expressing tissue) to expression of n oncogenic vir l protei
n. In theory, restricting the site of expression of genes through the use of tis
sue-specific regul tory elements would minimize or elimin te risks rel ted to

err nt expression of gene product. Cell-specific expression systems h ve yet t
o
e exploited for use in nucleic cid-
sed v ccines,
ut re presently
eing d
eveloped for the expression of ther peutic gene products. These systems re cent
ered on tissue-specific regul tory elements th t l y outside the coding region o
f gene nd, s such, c n
e used to restrict the expression of v rious genes,
reg rdless of their intended purpose. We h ve developed one such system for the
expression of hIGF-I in skelet l muscle fter intr muscul r dministr tion (7980)
. This novel muscle-specific expression system cont ins the promoter nd portion
s of the 5 nd 3 untr nsl ted region (UTR) of the chicken skelet l - ctin gene
(see Fig. 2). This system m y h ve pplic
ility to nucleic cid-
sed v ccines,
s ntigen expressed solely in muscle is c p
le of eliciting prolonged proph
yl ctic nd ther peutic immune response. In ddition, other systems th t restric
t expression to sites in the periphery (i.e., sites of dministr tion) would h v
e
enefit s other routes
ecome suit
le for the delivery of nucleic cid-
s

ed v c278
Mumper et l.
cine. For ex mple, restriction of expression to irw y epithelium m y
e of impo
rt nce for intr n s l delivery, s portions of the dose m y end up in the digest
ive tr ct. Reg rdless of the route of dministr tion, the critic l cell types to
t rget with nucleic cid-
sed v ccine m y
e DCs. If specific t rgeting of p
l smid to DCs proves to
e difficult, one m y w nt to limit expression of ntige
n to these cells s n dded s fety me sure. Dendritic cell-specific expression
systems h ve not yet
een developed,
ut inform tion is
eing gener ted s to wh
t proteins re restricted to these cell types. To d te, only few proteins h v
e
een identified whose expression is limited to DCs. These include DEC-205, S-1
00, p55, nd CD83 (8182). Regul tory elements of these genes re now
eing identi
fied nd ch r cterized to determine their role in limiting expression to these c
ell types. As
etter underst nding of the tissue-specific expression of protei
ns in DCs is o
t ined, novel expression c ssettes will
e gener ted to restrict
the expression of ntigen to these cells.
4.2. Expression of Multiple Antigens: Multiv lent Systems
It is conceiv
le th t for some p thogens expression of single gene will not s
uffice for n effective nucleic cid-
sed v ccine. Addition l p thogen ntigens
m y
e needed to provide
ro der immunologic l cover ge or cytokine/co-stimul t
ory molecules to function s genetic djuv nts. Single pl smids encoding for the
tr nscription of e ch gene c n
e cre ted,
ut this could signific ntly incre s
e production costs ssoci ted with the v ccine. In ddition, it would
e difficu
lt to reproduci
ly formul te sever l pl smids s single product for dministr
tion or tr nsfect single cell with multiple pl smids in vivo. One ltern tive
ppro ch to multiple expression pl smids is single pl smid producing ll the d
esired gene products. Sever l str tegies c n
e employed to ccomplish this fe t
(see Fig. 3). The first str tegy uses multiple independent tr nscription l unit
s on single pl smid, e ch defined
y its own promoter nd poly denyl tion sign
l (Poly A). E ch tr nscription l unit functions independently of the other to p
roduce high levels of
oth gene products. The promoters driving the tr nscriptio
n of e ch unit do not h ve to
e identic l to one nother nd c n
e used to dir
ect expression in tissue-specific or non-specific m nner. The second str tegy
m kes use of the intern l ri
osome entry site (IRKS) of picorn viruses. Pl cemen
t of n IRKS sequence
etween two coding regions on single tr nscript f cilit
tes the entry of ri
osomes t this site nd tr nsl tion of the downstre m coding
sequence. IRKS sequence elements elimin te the need for 5 c p structure or
free 5 end on the mess ge for tr nsl tion (83,84). This str tegy h s
een empl
oyed for the production of IL-12 from
Controlled Pl smid Delivery
279
Fig. 3. Multiv lent expression systems. Two pl smids: two coding sequences re e
ncoded s single tr nscription l units on individu l pl smids. Two genes: two co
ding sequences re encoded s independent tr nscription l units on single pl s
mid. IRKS: two coding sequences re encoded s single tr nscription l unit wit
h n IRKS sequence loc ting
etween the two coding sequences. Altern tive splici
ng: two coding sequences re encoded s single tr nscription l unit with n in
tron (5 ss/3 ss) pl ced upstre m of the first coding sequence nd n ltern ti
ve 3 splice site [3 ss (2)] loc ted upstre m of the second coding sequence.
retrovir l vector (85) nd n denovir l vector (86) nd h s
een tested in muri
ne tumor models (87). The third str tegy utilizes ltern tive splicing of sing

le tr nscript to produce the individu l gene products. In this system, n intron

cont ining consensus 5 nd 3 splice site is pl ced upstre m of the first co
ding sequence. A second 3 splice site is pl ced fter the stop codon of the fir
st coding sequence, upstre m of the second coding sequence. A single promoter di
rects the tr nscription of this unit nd produces single pre-mRNA species cont
ining
oth coding sequences. Depending on the strength of the individu l 3 spl
ice sites, the endogenous splicing m chinery will either excise the sm ller upst
re m intron, producing tr nscript with two coding sequences, or excise l rge
r intron th t deletes the upstre m coding sequence. Tr nsl tion of tr nscripts
280
Mumper et l.
th t result from the first splicing scen rio will only gener te gene product fro
m the first coding sequence, s only th t coding sequence which is proxim l to t
he 5 end of the mRNA is efficiently recognized
y the tr nsl tion m chinery. Re
mov l of the first coding sequence in the second splicing scen rio pl ces the se
cond coding sequence proxim l to the 5 end of the mRNA. Su
sequent tr nsl tion
of this tr nscript produces only gene product from the second coding sequence. I
n ddition to the production of multiple gene products, the ltern tive splicing
construct c n
e used to gener te different r tios of the gene products. By v r
ying the strength of the 3 splice sites, one c n regul te the levels of gene ex
pression with respect to one nother
y controlling the splicing preference of t
he tr nscript. This would
e dv nt geous for the synthesis of multi-su
unit gen
e products where there is n uneven stoichiometry of the su
units. Unpu
lished i
n vitro nd in vivo d t from our group h s shown th t single pl smid h r
orin
g multiple independent tr nscription l units is c p
le of producing the desired
gene products t levels equ l to or gre ter th n those o
served when individu l
pl smids with single tr nscription l units re utilized. In ddition, we h ve d
emonstr ted th t the IRKS nd ltern tive splicing systems re effective me ns o
f producing gene products t different r tios. Presently, ll of these systems c
ould
e re dily used in nucleic cid-
sed v ccines to gener te multiple gene
products from single pl smid. The choice of which system to use will ultim tel
y depend on the n ture of the selected genes, the desired r tio of gene products
nd how they perform nd inter ct with the expression elements th t comprise e
ch system. 5. Concluding Rem rks Adv nces in the field of nucleic cid-
sed v c
cines will stem from the delivery nd expression technologies currently
eing de
veloped
y those in the non-vir l gene ther py field for the production of ther
peutic gene products in vivo. First nd foremost, the use of dv nced pl smid de
livery technologies llows one to control the site of pl smid deposition in the

ody nd ccur tely t rget gene expression to the most ppropri te cells. For nu
cleic cid
sed-v ccines this m y
e profession l ntigen presenting cells like
DCs. Second, the use of tissue-specific expression c ssettes llows one to rest
rict expression of gene product to specific cell popul tion. This m y
ecome
of p r mount import nce if pl smid deposition rem ins uncontroll
le nd promis
cuous gene expression is concern. In ddition, the
ility to encode multiple
gene products on single pl smid m y prove to
e of tremendous
enefit s sev
er l gene products m y need to
e expressed for the elicit tion of n effective
prophyl ctic or ther peutic immune response. It is likely th t the com
in tion o
f these technologies will gener te s fer nd more potent v ccines in ccord nce
with the criteri put forth in Fig. 1.
Controlled Pl smid Delivery
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281
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25
Mucos l Immuniz tion with DNA V ccines
Mich el J. McCluskie nd He ther L. D vis 1. Introduction 1.1. Adv nt ges of Muc
os l Immuniz tion The mucos l surf ce re of the g strointestin l, genitourin r
y nd respir tory tr cts is more th n 200 times gre ter th n th t of the skin n
d is the prim ry site of tr nsmission of numerous dise ses. The entry of p thoge
nic org nisms t mucos l surf ces c n
e prevented
y mucos l,
ut not systemic
immunity. V ccines which re delivered
y intr muscul r (IM) or su
cut neous (SC
) injection induce strong systemic responses
ut gener lly no mucos l immunity.
In contr st, v ccines delivered t mucos l surf ces trigger
oth mucos l ( t loc
l nd dist nt sites) nd systemic responses (1,2). Other dv nt ges of mucos l
immuniz tion include
ro der ge r nge of recipients, the v ccines re e sy n
d non-inv sive to dminister nd there is no risk of needle stick injury nd cro
ss cont min tion (3). There h ve
een only limited num
er of reported c ses of
DNA-
sed immuniz tion t mucos l surf ces. Intr n s l (IN) dministr tion of p
ure pl smid DNA (often referred to s n ked DNA) expressing influenz virus hem gg
lutinin glycoproteins protected
oth mice nd chickens g inst leth l influenz
ch llenge lthough mucos l-specific responses were not me sured (4). In nother
study,
oth systemic nd mucos l immunity were gener ted following IN immuniz ti
on with n ked pl smid DNA expressing herpes simplex virus type 1 glycoprotein B,

ut not enough to protect g inst v gin l ch llenge with HSV (5).
1.2. Direct Gene Tr nsfer in the Lung
Numerous studies h ve indic ted th t gene tr nsfer m y
e c rried out in the lun
g using pl smid DNA either lone or formul ted with c tionic liposomes,
From: Methods in Molecul r Medicine, vol. 29, DNA V ccines: Methods nd Protocol
s Edited
y: D. B. Lowrie nd R. G. Wh len Hum n Press Inc., Totow , NJ
287
288
McCluskie nd D vis
however reports often conflict s to their rel tive efficiencies. Some studies r
eport th t liposome-formul ted
ut not n ked DNA c n tr nsfect lung tissue (68).
Others st te th t tr nsfection is possi
le with n ked DNA
ut this is less effic
ient th n with lipids (911). Yet other studies report th t n ked DNA nd lipid s
soci ted DNA re equ lly efficient (12,13). We h ve shown th t the efficiency of
tr nsfection of lipid-formul ted DNA depends very much on the n ture of the lip
id (i.e., neutr l nd c tionic lipid components, length of c r
on ch ins [14]),
nd this likely expl ins the discrep nt findings reported heretofore. Numerous
ppro ches for delivering DNA (formul ted or not) to the lung h ve
een reported.
The most common methods of dministr tion re IN (inh led or instilled), which
re indirect
ut non-inv sive, or intr tr che l (IT) (injected or vi c nnul )
which is more direct
ut inv sive. E ch of these techniques is descri
ed
elow.
In order to determine the effectiveness of mucos lly delivered DNA v ccine, m
ucos l w shes c n
e collected ( s descri
ed in Su
he ding 2.2.) nd nti
odies
specific for the expressed protein me sured
y ELISA, the h llm rk of mucos l im
munity
eing the loc l production of secretory immunoglo
ulin A (S-IgA) nti
odi
es. 2. M teri ls 2.1. Direct Gene Tr nsfer to the Murine Lung
2.1.1. Intr n s l Instill tion
1. Appropri te nesthesi for mice (e.g., H loth ne, H loc r
on L
or tories, Ri
ver Edge, NJ) (see Note 1). 2. Micropipettor with suit
le tips (see Note 2). 3.
Pl smid DNA v ccine dissolved in endotoxin-free s line t ppropri te concentr
tion (see Note 3). 4. Liposome formul tion, if pplic
le, t ppropri te concen

tr tion (see Note 4).

2.1.2. Intr n s l Inh l tion
1. Appropri te nesthesi for mice (e.g., H loth ne, H loc r
on L
or tories) (s
ee Note 1). 2. Micropipettor with suit
le tips (see Note 2). 3. Pl smid DNA v c
cine dissolved in endotoxin-free s line t ppropri te concentr tion (see Note 3
). 4. Liposome formul tion, if pplic
le, t ppropri te concentr tion (see Not
e 4).
2.1.3. Intr tr che l Injection
1. Appropri te nesthesi for mice (e.g., Somnotol, 75 mg/kg IP; MTC Ph rm ceuti
c ls, C m
ridge, ON) (see Note 1). 2. Fine-tipped surgic l scissors (Fine Scienc
e Tools Inc., North V ncouver, BC).
Mucos l Immuniz tion
289
3. Insulin syringe, 0.33 cc with 29G1/2 needle tt ched (Becton Dickinson, Fr
nklin L kes, NJ). 4. Pl smid DNA v ccine dissolved in endotoxin-free s line t
ppropri te concentr tion (see Note 3). 5. Liposome formul tion, if pplic
le,
t ppropri te concentr tion (see Note 4). 6. Fine degr d
le surgic l sutures.
2.1.4. Intr tr che l C nnul tion
1. Appropri te nesthesi for mice (e.g., Somnotol, 75 mg/kg IP) (see Note 1). 2
. Fine-tipped surgic l scissors. 3. A 20-g uge olive tip steel feeding tu
e (Fin
e Science Tools Inc.) tt ched to 1 cc tu
erculin syringe (Becton Dickinson).
4. Pl smid DNA v ccine dissolved in endotoxin-free s line t ppropri te concent
r tion (see Note 3). 5. Liposome formul tion, if pplic
le, t ppropri te conc
entr tion (see Note 4). 6. Fine degr d
le surgic l sutures.
2.2. Collection of Mucos l Secretions 2.2.1. V gin l W sh
1. 2. 3. 4. 5. Micropipettor with st nd rd 200 L tip tt ched. Appropri te nesth
esi for mice (e.g., H loth ne). Phosph te-
uffered s line (PBS) or Tris-
uffere
d s line (TBS) (see Note 6). Microcentrifuge tu
es for s mple collection. Centri
fuge (15,690g).
2.2.2. Fec l Pellets
1. 2. 3. 4. 5. 6. Individu l c ges for mice (see Note 7). Micropipettor with st
nd rd 200 L tip tt ched. PBS or TBS (see Note 6). 50 mL Centrifuge tu
es for s m
ple collection (one for e ch mouse). Sodium zide (10 L per 0.1 mg fec l m teri l
). Centrifuge (3340g).
2.2.3. Lung W sh
1. 2. 3. 4. 5. 6. 7. 8. Appropri te nesthesi for mice (see Note 8). Fine-tippe
d surgic l scissors. Two surgic l microcl mps (Fine Science Tools Inc.) (see Not
e 9). Insulin syringe, 1 cc with 29G1/2 needle tt ched. Polyethylene tu
ing (
PE 20, ID = 0.38 mm). PBS or TBS (see Note 6). Microcentrifuge tu
es for s mple
collection. Centrifuge (15,690g).
290 2.2.4. N s l W sh
1. 2. 3. 4. 5. 6. 7. 8.
McCluskie nd D vis
Appropri te nesthesi for mice (see Note 8). Fine-tipped surgic l scissors. Two
surgic l microcl mps. Insulin syringe, 1 cc with 29G1/2 needle tt ched. Poly
ethylene tu
ing (PE20, ID = 0.38 mm) (Becton Dickinson). PBS or TBS (see Note 6)
. Microcentrifuge tu
es for s mple collection. Centrifuge (15,690g).

3. Methods 3.1. Direct Gene Tr nsfer to the Murine Lung

3.1.1. Intr n s l Instill tion
1. Prep re DNA nd liposome solution (see Notes 3, 4, 10, nd 11). 2. Dr w 25 L D
NA solution into pipet tip nd ensure there re no ir
u

les (see Note 2). 3.
Anesthetize mice (see Note 1). 4. Hold mouse on its
ck nd c refully insert th
e tip of the gel lo ding tip few mm into the n s l c vity, so s not to d m ge
n s l epithelium, nd slowly instill DNA solution. Immedi tely repe t instill t
ion into second nostril (see Note 12). 5. Apply pressure to the lower m ndi
le t
o reduce sw llowing nd hold the mouse in this position for 10 s to ensure th t
the fluid is not expelled (see Notes 13 nd 14).
3.1.2. Intr n s l Inh l tion
1. Prep re DNA nd liposome solution (see Notes 3, 4, 10, nd 11). 2. Dr w DNA s
olution into pipet tip nd ensure there re no ir
u

les (see Note 2). A volum
e of 25 to 200 L c n
e dministered t one time (see Note 15). 3. Anesthetize mi
ce (see Note 1). 4. Deposit the DNA solution s droplets pplied
il ter lly dir
ectly over the extern l n res of mice during period of pproxim tely 5 s. Appl
y pressure to the lower m ndi
le to reduce sw llowing during dministr tion (see
Note 14). Although the DNA solution is delivered
y pipet tip, this is not pl
ced inside the n s l c vity. 5. Hold mouse for further 10 s to ensure th t fl
uid is not expelled.
3.1.3. Intr tr che l Injection
1. Prep re DNA nd liposome solution (see Notes 3, 4, 10, nd 11). 2. Anesthetiz
e mice (see Note 5). 3. Dr w desired volume of DNA solution (100200 L) into the sy
ringe nd ensure there re no ir
u

les. Set syringe side. 4. Using fine-tipp
ed surgic l scissors expose the mouse tr che through n nterior midline incisi
on.
Mucos l Immuniz tion
291
5. Inject the DNA solution through the nterior w ll of the tr che t king c re
not to puncture posterior w ll (see Note 14). 6. Suture the skin incision nd pl
ce the mouse in n incu
tor until fully recovered from the nesthetic ( pprox
45 min).
3.1.4. Intr tr che l C nnul tion
1. 2. 3. 4. 5. Prep re DNA nd liposome solution (see Notes 3, 4, 10, nd 11). A
nesthetize mice (see Note 5). Att ch feeding tu
e to syringe. Dr w desired volum
e of DNA solution (100200 L) into the syringe nd ensure there re no ir
u

les.
Set syringe side. Using fine-tipped surgic l scissors expose the mouse tr che
through n nterior midline incision. This incision is used to visu lize the in
sertion of the c nnul into the tr che . Gently p ss the feeding tu
e through th
e or l c vity nd into the tr che . Slowly inject the DNA solution directly into
the lungs (see Note 14). Suture the skin incision nd pl ce the mouse in n inc
u
tor until fully recovered from the nesthetic ( pprox 45 min).
6. 7. 8.
3.2. Collection of Mucos l Secretions 3.2.1. V gin l W sh
1. Dr w 30 L PBS or TBS (see Note 6) into pipet tip nd ensure there re no ir

u

les. 2. Anesthetize mice. 3. Hold mouse
y the
ck of neck, stom ch upw rd.
C refully insert pipet tip few mm into the v gin l c vity. 4. Instill nd with
dr w solution into the v gin l c vity 10 times. 5. Centrifuge s mples t 15,690g
for 7 min to remove v gin l l v ge sediments. 6. Remove supern t nt nd store
t 20C. 7. Repe t procedure over 5 consecutive d (see Note 16). 8. Pool supern t nt
s nd store t 20C until ss yed
y ELISA (see Note 17).

3.2.2. Fec l Pellets

1. 2. 3. 4. 5. 6. Isol te mice in individu l c ges (see Note 7) for 24 h perio
d. Collect nd weigh fec l pellets. Add 1 mL TBS or PBS (see Note 6) nd 10 L sod
ium zide per 0.1 mg of fec l m teri l. Rehydr te s mples for 30 min t RT. Cent
rifuged t 3340g for 15 min to remove fec l de
ris. Collect supern t nt nd stor
e t 20C until ss yed for S-IgA.
3.2.3. Lung W sh
1. Dr w 1 mL TBS or PBS (see Note 6) into the syringe nd ensure there re no i
r
u

les. Att ch PE tu
ing to needle. Use length of tu
ing 1 cm longer th n nee
dle. Set syringe side.
292
McCluskie nd D vis
2. Kill mouse
y cervic l disloc tion or nesthesi overdose (see Note 8). 3. Us
ing fine-tipped surgic l scissors expose the mouse tr che through n nterior m
idline incision. 4. M ke sm ll incision in tr che nd pl ce cl mp
ove it
(see Note 9). 5. P ss PE tu
ing few mm through incision in direction of the lu
ngs nd pl ce second cl mp
elow incision to hold PE tu
ing in tr che . 6. Slo
wly instill nd withdr w solution into lungs three times (80% recovery expected)
. 7. Centrifuge s mples t 15,690g for 7 min, collect supern t nt nd store t 2
0C until ss yed for S-IgA.
3.2.4. N s l W sh
1. Dr w 0.5 mL TBS or PBS (see Note 6) into the syringe nd ensure there re no
ir
u

les. Att ch PE tu
ing to needle. Use length of tu
ing 1 cm longer th n n
eedle. Set syringe side. 2. Kill mouse
y cervic l disloc tion or nesthesi ov
erdose (see Note 8). 3. Using fine-tipped surgic l scissors expose the mouse tr
che through n nterior midline incision. 4. M ke sm ll incision in tr che
nd pl ce cl mp
elow it (see Note 9). 5. P ss PE tu
ing few mm through incis
ion in direction of n res nd pl ce second cl mp
ove incision to hold PE tu

ing in upper tr che . 6. Invert mouse nd slowly inject solution into the tr che
. Allow solution to flow through n res nd collect in microtu
e. 7. Centrifug
e s mples t 15,690g for 7 min, collect supern t nt nd store t 20C until ss ye
d for S-IgA.
4. Notes
1. Mice must
e nesthetized s they will sw llow the instilled m teri l if they
re w ke. H loth ne is suit
le nesthetic s recovery time is short nd thu
s the risk of choking is reduced. 2. We h ve found th t fine-tipped gel lo din
g tip is
est s it h s sm ller tip to
e inserted into the n s l c vity for I
N instill tion nd it llows very sm ll droplets for the IN inh l tion method. 3
. Pl smid DNA m y
e purified
y cesium chloride (CsCl) or nion-exch nge chrom
togr phy. The qu lity of the DNA
y these two methods is equiv lent for DNA immu
niz tion, however, nion-exch nge methods re consider
ly quicker, e sier to pe
rform nd sc le-up th n CsCl methods nd void the use of h z rdous chemic ls (1
5). In our l
or tory, DNA is purified on Qi gen nion-exch nge chrom togr phy c
olumns (Qi gen Gm
H, Hilden, Germ ny) nd resuspended in sterile s line (0.15 M
N Cl, Sigm ). An endotoxin remov l kit is lso v il
le from Qi gen which yield
s DNA with extremely low levels of endotoxin. The concentr tion of DNA is c lcul
ted
sed on
sor
nce of ultr violet light (OD 260) with fin l concentr tions
usu lly
eing 510 mg/mL, purity determined t OD260/280, nd DNA verified
y res
triction enzyme digest nd g rose gel elecMucos l Immuniz tion
293

trophoresis. For mucos l delivery, it is import nt to use concentr ted DNA solut
ions to minimize dministr tion volume. DNA solutions c n
e stored t 20C until r
equired for in vivo delivery. 4. V rious c tionic liposome formul tions re comm
erci lly v il
le. We routinely use Cellfectin (Life Technologies, Inc., G ithe
rs
urg, MD) t DNA:lipid w/w r tio of 1:1 which we h ve found to
e efficient
for gene tr nsfer to lungs. However, other liposome formul tions, such s Lipofe
ctin or Lipofect mine (Life Technologies, Inc.), resulted in lower levels of exp
ression th n pl smid DNA lone (14). It is import nt to llow time for DNA/lipid
complex form tion
efore dministr tion (usu lly 3060 min) nd there fter to use
s quickly s possi
le. 5. For intr tr che l dministr tions where surgery is i
nvolved, it is necess ry to use long l sting gener l nesthetic. We h ve found
Somnotol (75 mg/kg IP) to
e suit
le for this procedure. During recovery time
( pprox 45 min) mice c n
e pl ced in 37C incu
tor to m int in
ody temper tur
e. 6. Phosph te c n ct s n inhi
itor in cert in commerci l kits th t re used
to detect low levels of nti
ody in w shes (e.g., ELISA Amplific tion System, L
ife Technologies, Inc., G ithers
urg, MD). Phosph te cont ining
uffers should

e voided when these kits re used. 7. Individu l c ges re required to prevent
mixing of fec l m teri l from different mice. We h ve found th t if we repl ce t
he regul r
edding m teri l (s wdust) with thick p per towels fec l pellet colle
ction is gre tly f cilit ted. 8. Mice c n
e killed
y cervic l disloc tion whil
e under H loth ne nesthesi ; however to void d m ge to the tr che , n overdos
e with suit
le nesthesi (e.g., Somnotol) m y
e prefer
le. 9. We h ve foun
d th t surgic l microcl mp is n efficient nd e sy w y to close the tr che . Al
tern tively, surgic l suture m y
e used to tie the tr che off t the desired
pl ce. 10. When DNA is formul ted with c tionic liposomes, it is desir
le to d
etermine the lowest DNA:lipid r tio t which ll the DNA is complexed, s we h v
e found this optim l for gene tr nsfer in the lung. Free pl smid DNA c n
e visu
lized on 0.5% g rose gel st ined with 0.04 g/mL ethidium
romide s migr te
d
nd t the expected dist nce (
sed on molecul r size) where s DNA-lipid comp
lexes f il to enter the gel. 11. Indi ink, tryp n
lue or methylene
lue m y
e
used initi lly to visu lize the distri
ution of fluid in the irw ys. In this c
se, mice re killed nd lungs h rvested 1 or 2 min fter dministr tion. The su
ccess of direct gene tr nsfer in the lung c n
e ev lu ted using ny of the rout
ine reporter genes, such s
et g l ctosid se (l cZ), lucifer se (luc), chlor mp
henicol cetyl-tr nsfer se (CAT) or green fluorescent protein (GFP). 12. The gel
-lo ding tip must
e c refully inserted to ensure the n s l epitheli l l yer is
not d m ged otherwise systemic immuniz tion, r ther th n mucos l, m y result.
13. The instill tion procedure c n
e repe ted s necess ry
ut it is
est to le
ve 15 min interv ls
etween dministr tions to llow time for the nim ls to re
cover.
294
McCluskie nd D vis
14. We h ve shown essenti lly no differences
etween IN inh l tion, IN instill t
ion nd IT c nnul tion for reporter gene expression, except for higher v ri
ili
ty with the l tter. IT injection provides somewh t lower tr nsfection efficiency
th n the other three methods, possi
ly due to DNA le k ge (14). Thus our method
of choice is IN inh l tion since it is the e siest to perform nd le st inv siv
e. Other investig tors h ve descri
ed d pt tions on these techniques; for ex mp
le, the tongue c n
e pulled out with IN dministr tion to limit sw llowing (16)
, sm ll volume of ir c n
e injected fter IT dministr tion to disperse the
fluid throughout lung (12), or n erosol or ne
ulizer system c n
e used for de
livery of mist r ther th n liquid (17). 15. For multiple dministr tions, t
le st 15 min should
e llowed
etween dministr tions p rticul rly when l rger
volumes (e.g., 200 L) re used. 16. This is necess ry in order to ccount for v
ri tions in S-IgA levels during the murine estrus cycle (18). 17. Mucos l nti
o
dy levels c n
e me sured
y ELISA using prim ry nti
ody specific for the exp

ressed protein nd n nti-IgA second ry nti
ody. Since IgA levels re typic ll

y low, more sensitive nti
ody ss y is the ELISA mplific tion system (EIA) (
Gi
co-BRL, G ithers
urg MD, USA). Altern tively, the presence of nti
ody-secret
ing cells (ASC) m y
e determined in single cell suspensions o
t ined from lung
tissue or lymph nodes using the ELISPOT technique (5), nd ntigen-specific prol
ifer tive T-cell responses or the induction of cytotoxic T lymphocytes m y lso

e me sured.
References
1. Mestecky, J. nd McGhee, J. R. (1992) Prospects for hum n mucos l v ccines, i
n Genetic lly Engineered V ccines (Ci rdi, J. E., ed.) Plenum Press, New York, p
p. 1323. 2. G llich n, W. S. nd Rosenth l, K. L. (1995) Specific secretory immun
e responses in the fem le genit l tr ct following intr n s l immuniz tion with
recom
in nt denovirus expressing glycoprotein B of herpes simplex virus. V cci
ne 13, 15891595. 3. OH g n, D. T. (1994) Or l immuniz tion nd the common mucos l
immune system, in Novel Delivery Systems for Or l V ccines (OH g n, D. T., ed.) C
RC Press, Boc R ton, pp. 124. 4. Fyn n, E. F., We
ster, R. G., Fuller, D. H., H
ynes, J. R., S ntoro, J. C., nd Ro
inson, H. L. (1993) DNA v ccines: protective
immuniz tions
y p renter l, mucos l, nd gene-gun inocul tions. Proc. N tl. Ac
d. Sci. USA 90, 11,47811,482. 5. Kuklin, N., D heshi , M., K rem, K., M nick n,
E., nd Rouse, B. T. (1997) Induction of mucos l immunity g inst Herpes simples
virus
y pl smid DNA immuniz tion. J. Virol. 71, 31383145. 6. Oudrhiri, N., Vign
eron, J.-P., Peuchm ur, M., Leclerc, T., Lehn, J. M., nd Lehn, P. (1997) Gene t
r nsfer
y gu nidinium-cholesterol c tionic lipids into irw y epitheli l cells
in vitro nd in vivo. Proc. N tl. Ac d. Sci. USA 94, 16511656. 7. H zinski, T. A.
, L dd, P. A., nd DeM tteo, C. A. (1991) Loc liz tion nd induced expression of
fusion genes in the r t lung. Am. J. Respir. Cell Mol. Biol. 4, 206209.
Mucos l Immuniz tion
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8. Stri
ling, R., Brunette, E., Liggitt, D., G ensler, K., nd De
s, R. (1992) A
erosol gene delivery in vivo. Proc. N tl. Ac d. Sci. USA 89, 11,27711,281. 9. Yos
himur , K., Rosenfeld, M., N k mur , H., Scherer, E., P vir ni, A., Lecocq, J.-P
., nd Cryst l, R. (1992) Expression of the hum n cystic fi
rosis tr nsmem
r ne
conduct nce regul tor gene in the mouse lung fter in vivo intr tr che l pl smid
-medi ted gene tr nsfer. Nuc. Acids Res. 20, 32333240. 10. Wheeler, C. J., Felgne
r, P., Ts i, Y. J., M rsh ll, J., Sukhu, L., Doh, S., H rtikk , J., Nietupski, J
., M nthorpe, M., Nichols, M., Plewe, M., Li ng, X., Norm n, J., Smith, A., nd
Cheng, S. (1996) A novel c tionic lipid gre tly enh nces pl smid DNA delivery n
d expression in mouse lung. Proc. N tl. Ac d. Sci. USA 93, 11,45411,459. 11. Felg
ner, P. L., Ts i, Y. J., Sukhu, L., Wheeler, C. J., M nthorpe, M., M rsh ll, J.,
nd Cheng, S. H. (1995) Improved c tionic lipid formul tions for in vivo gene t
her py. Ann. NY Ac d. Sci. 772, 126139. 12. Ts n, M.-F., White, J. E., nd Shep r
d, B. (1995) Lung-specific direct in vivo gene tr nsfer with recom
in nt pl smid
DNA. Am. J. Physiol. 268, L1052-L1056. 13. Meyer, K., Thompson, M., Levy, M., B
rron, L., nd Szok , F. J. (1995) Intr tr che l gene delivery to the mouse irw
y: ch r cteriz tion of pl smid DNA expression nd ph rm cokinetics. Gene Ther p
y 2, 450460. 14. McCluskie, M. J., Chu, Y., Xi , J. L., Jessee, J., Ge
yehu, G.,
nd D vis, H. L. (1998) Direct gene tr nsfer to the respir tory tr ct of mice wi
th pure pl smid nd lipid-formul ted DNA. Antisense Nucleic Acid Drug Dev. 8, 40
1414. 15. D vis, H. L., Schleef, M., Moritz, P., M ncini, M., Schorr, J., nd Wh
len, R. G. (1996) Comp rison of pl smid DNA prep r tion methods for direct gene
tr nsfer nd genetic immuniz tion. Biotechniques 21, 9299. 16. Li, X.-M., Chopr ,
R. K., Chou, T.-Y., Schofield, B. H., Wills-K rp, M., nd Hu ng, S-K., H. (1996
) Mucos l IFN-g mm gene tr nsfer inhi
its pulmon ry llergic responses in mice.
J. Immunol. 157, 32163219. 17. Alton, E. W. F. W., Middleton, P. G., C plen, N.
J., Smith, S. N., Steel, D. M., Munkonge, F. M., et l. (1993) Non-inv sive lipo
some-medi ted gene delivery c n correct the ion tr nsport defect in cystic fi
ro

sis mut nt mice. N ture Genetics 5, 135142. 18. G llich n, W. S. nd Rosenth l, K

. L. (1996) Effects of the estrous cycle on loc l humor l immune responses nd p
rotection of intr n s lly immunized fem le mice g inst herpes simplex virus typ
e 2 infection in the genit l tr ct. Virology 224, 487497.
26
Prep r tions for P rticle-Medi ted Gene Tr nsfer Using the Accell Gene Gun
Mich el D. M cklin, Ro
ert J. Dr pe, nd Willi m F. Sw in 1. Introduction P rtic
le-medi ted delivery involves co ting m teri ls onto the surf ce of dense su
-ce
llul r sized (0.55 mm) p rticles nd cceler ting the p rticles to sufficient vel
ocity to penetr te t rget cells. The technique w s invented
y S nford nd Wolf
t Cornell University (1) to tr nsfer DNA into int ct pl nt cells (2), nd w s f
urther developed into n effective process for producing genetic lly engineered
crop pl nts
y sever l groups (reviewed in 3). Su
sequent work h s shown th t th
is method is gener lly pplic
le for tr nsferring m teri ls including DNA, RNA,
proteins, peptides nd ph rm cologic l compounds into wide v riety of tissue
nd cell types in vivo, ex vivo, or in vitro (reviewed in 4). The topic of p rti
cle-medi ted gene tr nsfer to epidermis s me ns of nucleic cid immuniz tion
is specific lly ddressed in n ccomp nying ch pter. This ch pter descri
es pro
cedures for prep r tion of the necess ry m teri ls (see Note 1). The procedures
re divided into two sections:
e d prep r tion nd tu
e prep r tion. The first
section descri
es procedures for m king slurry of DNA-co ted gold p rticles,
nd the second section procedures for lo ding the DNA-co ted p rticles into c rtri
dges. P rticle-medi ted gene tr nsfer h s prim rily employed gold p rticles s c
rriers. P rticles in the su
-cellul r size r nge must
e dense to chieve the mo
mentum necess ry for dequ te penetr tion. Among the m teri ls with sufficient d
ensity (e.g., gold, iridium, pl tinum, tungsten) gold is preferred
ec use of it
s low chemic l re ctivity nd low toxicity. Moreover, high purity gold powders i
n the desired size r nge re commerci lly v il
le.
From: Methods in Molecul r Medicine, vol. 29, DNA V ccines: Methods nd Protocol
s Edited
y: D. B. Lowrie nd R. G. Wh len Hum n Press Inc., Totow , NJ
297
298
M cklin, Dr pe, nd Sw in
Powder injection is conceptu lly distinct technology th t is, in sever l spec
ts, complement ry to p rticle-medi ted delivery. R ther th n using dense su
-cel
lul r sized c rrier p rticles, s in p rticle-medi ted delivery, powder injectio
n employs l rger low-density p rticles th t c n
e formul ted without dense c
rrier. S rphie et l. (5) demonstr ted the systemic distri
ution of inulin dmin
istered derm lly in the form of low-density (1.5 g/cm3) p rticles. P rticles in
the 3050 m size r nge were required, thus delivery w s likely extr -cellul r; howe
ver, signific nt systemic
io- v il
ility w s chieved. These results suggest t
h t powder injection is suit
le for delivery of convention l ph rm ceutic ls, c
onvention l v ccines, nd other su
st nces where extr -cellul r delivery is effe
ctive nd cellul r upt ke of the delivered m teri l is efficient. P rticle-medi
ted delivery, on the other h nd, is n effective me ns of direct intr -cellul r
delivery. The use of powder injection for nucleic cid immuniz tion is under inv
estig tion. A num
er of p r meters th t influence p rticle-medi ted gene deliver
y c n
e re dily djusted to optimize perform nce in p rticul r system. These
p r meters include the cceler ting force, the size of the p rticles, the num
er
of p rticles per t rget site, the distri
ution of p rticles within the t rget,
nd the mount of DNA lo ded onto the p rticles. M nipul tion of these p r meter
s provides vers tility, llowing d pt tion of the method to t rget tissues with
widely differing physic l ch r cteristics. A num
er of conveniently ss yed rep
orter genes (e.g., hum n growth hormone, -g l ctosid se, green fluorescent protei
n) re v il
le th t c n
e used to optimize p r meters for specific system.

Reporter genes th t c n
e ss yed histologic lly re especi lly v lu
le for id

entific tion of tr nsfected cells within tissue s well s me surements of tr
nsfection efficiency. Sever l devices h ve
een developed to cceler te the co t
ed p rticles; these include the origin l Biolistic Device of S nford nd Wolf (2
), the PDS-1000/ He device nd Helios Gene Gun m nuf ctured
y Bio-R d L
or tor
ies Inc. (Hercules, CA), h nd-held helium-driven device designed
y John S nfo
rd nd Stephen Johnston (6), nd the Accell electric-disch rge (7) nd heliumpow
ered (8) devices designed
y Dennis McC
e nd co-workers t Agr cetus, Inc. A d
ispos
le device (PowderJect), descri
ed
y S rphie et l. (5) for powder inject
ion, offers sever l ttr ctive fe tures in terms of pr ctic l clinic l pplic ti
on of the technology. A device th t com
ines these fe tures of the PowderJect de
vice with the delivery c p
ilities of the Accell helium device is currently und
er development. This ch pter descri
es procedures for using the helium-driven Ac
cell device (8). A simil r device, the Helios Gene Gun, nd ssoci ted equipment
nd supplies re commerci lly v il
le from Bio-R d. The Helios device w s dev
eloped in coll
or tion with PowderJect V ccines Inc. (M dison, WI) to provide
P rticle-Medi ted Gene Tr nsfer
299
devices for the life sciences rese rch community. P rticle-medi ted gene tr nsfe
r is propriet ry technology covered
y p tents held
y PowderJect V ccines, In
c. (previous n mes or ffili tes include Geniv Inc., Aur gen Inc., nd Agr cetu
s Inc.) nd E. I. DuPont de Nemours & Co. (Wilmington, DE). Devices from Bio-R d
th t implement this technology re licensed for rese rch purposes only, nd re
not intended or pproved for clinic l use. 2. M teri ls 2.1. Be d Prep r tion
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. An lytic l
l nce c p
le of ccur t
ely weighing milligr m qu ntities of gold. 0.54 m gold p rticles. 1.5 mL Eppendorf
tu
es. Micro-pipettors. 0.05 M spermidine (Sigm ). Vortex mixer. Ultr sonic cle
ner (Model FS3, Fisher Scientific, Pitts
urgh, PA). Pl smid DNA solution in TE
(DNA concentr tion should
e >1 g/L). Microcentrifuge. Dehydr ted lcohol, 200 pro
of (ET107, Spectrum, New Brunswick, NJ). 1 mg/mL eth nolic polyvinylpyrrolidone
K-90 (PVP) (P1416, Spectrum). 5, 10 nd 25 mL pipettes nd pipette- id. 22 mL gl
ss scintill tion vi ls nd Teflon c ps. Tefzel tu
ing: ID 0.09375  0.002 in., OD
0.127  0.001 in. 5 mL syringe with M sterflex size 14 silicon tu
ing d pter. St
opw tch. Tu
ing Prep Unit (Bio-R d). Compressed helium g s gr de 4.5 (99.995%) o
r higher. Compressed nitrogen g s gr de 4.8 (99.998%) or higher. Helium pressure
regul tor (delivery pressure r nge 10800 psi). Nitrogen pressure regul tor (deli
very pressure r nge 01000 psi). G s m nifold. Perist ltic pump c p
le of pumping
110 mL/min. Cutting
o rd with ruler. Sc lpels. Humi-c p desicc nt pellets (Unit
ed Desicc nts, Belen, NM).
2.2. Tu
e Prep r tion
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
3. Methods 3.1. Be d Prep r tion
1. Weigh the ppropri te mount of gold into 1.5-mL Eppendorf tu
e. For
e d
lo ding r te (BLR) (see Note 2) of 0.5 mg of gold/c rtridge, weigh ~20 mg gold
for 3540 c rtridges.
300
M cklin, Dr pe, nd Sw in
2. Pipette 100 L of 0.05 M spermidine into the Eppendorf tu
e cont ining the gold
. 3. Vortex the spermidine/gold mixture for 23 s to suspend the gold in the sperm
idine solution. 4. Sonic te the spermidine/gold mixture for 10 s to disperse ny
ggreg ted gold p rticles. 5. Add the volume of DNA required to chieve the des
ired DNA lo ding r te (DLR) (see Note 3) to the spermidine gold mixture. The vol

ume of DNA should not exceed the volume of spermidine (100 L). If gre ter volum
e of DNA is required, incre se the volume of spermidine to equ l th t of the DNA
solution (see Note 4). 6. Invert the DNA/spermidine/gold mixture sever l times
to thoroughly mix. 7. Quickly dd 100 L of 1 M C Cl2, dropwise, to the DNA/spermi
dine/gold mixture while gently vortexing. Close c p nd vortex vigorously for 23
s fter the completion of ddition of the C Cl2. The DNA/gold precipit te should
r pidly sediment to the
ottom of the tu
e when vortexing is stopped. The volum
e of the 1 M C Cl2 should
e equ l to the volume of spermidine dded; i.e., if t
he volume of spermidine w s djusted in step 5, the volume of C Cl2 should
e d
justed ccordingly. 8. Allow the mixture to continue to precipit te t room temp
er ture for 510 min. 9. Centrifuge the
e d prep for 10 s to pellet the gold nd
c refully remove the supern t nt. 10. Disrupt the DNA/gold pellet
y r king the

ottom of the tu
e cross the holes of n Eppendorf r ck. 11. W sh the DNA/gold
pellet
y vortexing in 700 L of dehydr ted lcohol (see Note 5). Pellet the gold

y centrifug tion for 10 s nd remove the supern t nt with pipette. Repe t the
w sh two times. 12. Prep re 10 L of fresh 1 mg/mL eth nolic PVP stock solution (
see Note 6). Weigh 10 mg of PVP in gl ss scintill tion vi l. Add 10 mL of dehy
dr ted lcohol. Sonic te the suspension until the PVP is dissolved (see Note 5).
13. Mix the ppropri te mount of dehydr ted lcohol nd eth nolic PVP stock to
give the desired fin l PVP concentr tion (see Note 6). Pipet the tot l volume o
f the fin l PVP solution needed for the desired BLR (see Note 2) into 22-mL gl
ss scintill tion vi l with Teflon c p. Add 500 L of this solution to the DNA/
gold pellet nd resuspend the p rticles thoroughly. Tr nsfer this suspension to
the
ottom of the scintill tion vi l. Using nother 500 L of the solution from th
e vi l repe t the suspension, c refully rinsing the sides of the Eppendorf tu
e,
until ll of the DNA/gold h s
een tr nsferred from the Eppendorf tu
e into the
scintill tion vi l. 14. Tightly screw-on the Teflon c p nd se l with p r film.
The
e d prep is re dy for tu
e prep r tion, or it c n
e stored in the d rk t
20C.
3.2. Tu
e Prep r tion
1. Purge the Tu
ing Prep Unit overnight
y flushing with dry nitrogen g s t f
low r te of 0.1 L/min (LPM). If tu
es re m de frequently the tu
e turner should

e
P rticle-Medi ted Gene Tr nsfer
301
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
15. 16. 17.
left purging with nitrogen t flow r te of 0.1 LPM. In ddition, the tefzel tu

ing should
e purged overnight with nitrogen t 0.01 LPM. Cut the tefzel tu
ing
to length th t is pproxim tely 2.5 cm longer th n the Tu
ing Prep Unit. Vort
ex the DNA/gold suspension for 3 s nd
riefly sonic te to disrupt ny ggreg te
s. Turn off the nitrogen flow to the Tu
ing Prep Unit. Att ch the cut tefzel tu

e to the 5-mL syringe vi the M sterflex d pter. Vortex the DNA/gold suspension
until the DNA/gold is completely, homogeneously suspended. Quickly remove the c
p from the vi l nd dr w the DNA/gold slurry into the length of the tu
e. Quick
ly slide the tu
e into Tu
ing Prep Unit then st rt the stop w tch. Settle for 15
s then rot te the tu
e pproxim tely 90
y twisting the end (use the syringe m r
kings to indic te the degree of rot tion). Settle for 30 s then rot te the tu
e
n ddition l 90 in the s me direction. Settle for nother 45 s then rot te the t
u
e nother 90 in the s me direction. Allow the tu
e to settle for 2 min fter th
is rot tion. Rot te the tu
e 45, remove the syringe, tt ch the end of the tu
e t
o perist ltic pump nd dr w off the liquid t pproxim tely 3.5 mL/min. Det ch
the tu
e from the perist ltic pump nd turn on the Tu
ing Prep Unit t r te o
f 20 rpm (0.05g). After 30 s st rt N2 flow t 0.4 LPM. After 3 min, remove the t

u
e from the Tu
ing Prep Unit nd tt ch it to g s m nifold th t is tt ched t

o nitrogen t nk. Flush with dry N2 for t le st 1 h. Cut the tu
e t e ch end,
4 cm in from the edge of the gold
e d co ting, nd disc rd the end pieces. Cut
into 1.27 cm (0.5 in) lengths. Test segments from e ch end of tu
e (see Note 7)
. Pool tu
es nd store in se led cont iner with desicc nt pellet. A tightly
c pped gl ss scintill tion vi l wr pped with p r film m kes good stor ge cont
iner.
4. Notes
1. DNA for p rticle-medi ted gene tr nsfer h s most frequently
een in the form
of pl smid molecules in the 515 kilo
se (k
) size r nge, however recom
in nt l m

d ph ge clones nd cosmid clones ( 50 k
), s well s defective vir l genomes
nd uncloned genomic DNA fr gments in the r nge of 150200 k
h ve lso
een succes
sfully tr nsferred
y this method. The use of very l rge DNA molecules requires
th t gre t c re
e t ken in the m nipul tions to void she ring. Mixtures of pl
smids c n lso
e co ted onto the gold p rticles (9). 2. The
e d-lo ding r te (
BLR) is the mount of gold in e ch c rtridge. In most DNA v ccin tion experiment
s the BLRs re 0.25 mg to 0.5 mg of gold per c rtridge. However, the BLRs m y r
nge
etween 0.0625 to 2 mg per c rtridge in tre tment p r meter studies. The BLR
is determined
y c lcul ting the volume of the inside of the tefzel tu
ing used
to prep re the c rtridges.
302
BLR 0.25 mg/shot 0.5 mg/shot 1.0 mg/shot
M cklin, Dr pe, nd Sw in
DNA/gold suspension 4.42 mg of gold/mL of EtOH/PVP solution 8.84 mg of gold/mL o
f EtOH/PVP solution 17.68 mg of gold/mL of EtOH/PVP solution
3. The DNA lo ding r te (DLR) is the mount of DNA, usu lly in microgr ms, per m
illigr m of gold. For DNA v ccin tion experiments the DLRs usu lly r nge
etween
12.5 g of pl smid DNA per milligr m of gold nd c n r nge
etween 5 ng to 5 g of p
l smid DNA per milligr m of gold using the st nd rd protocol. At the lower DLRs
the ddition of c rrier DNA is helpful to efficiently precipit te the pl smid on
to the gold
e ds. Aggreg tion of the gold p rticles c n
ecome pro
lem t DLR
s gre ter th n 3 g of DNA per milligr m of gold. Incre sing the over ll volume of
the prep r tion m y reduce ggreg tion somewh t. Higher DLRs c n lso
e chiev
ed using ltern tive formul tion procedures. 4. The volume of DNA solution neede
d for
e d prep c n
e c lcul ted s follows: Volume of DNA Stock = [(milligr
ms of gold in prep) (DLR)] / (pl smid concentr tion) 5. After the first eth nol
w sh speci l c re should
e used to ensure th t the eth nol nd
e d prep is not
exposed to moisture. Be d nd tu
e preps re extremely sensitive to humidity. G
ener lly,
e d nd tu
e preps re not performed if the rel tive humidity is gre
ter th n 60%. 6. PVP is used s
inding gent to prevent prem ture rele se of
the DNA/gold p rticles during the rise-time of the helium jet. The PVP concentr
tion for most pplic tions is 0.05 mg/mL, however, djustment of the PVP concent
r tion in the r nge 0.010.1 mg/mL m y
e helpful if the tu
es re not rele sing p
roperly. The effect of PVP on perform nce is gre test in situ tions th t require
m ximum penetr tion. 7. The c rtridges c n
e tested for proper rele se
y shoo
ting the DNA/gold into p r film t the ppropri te pressure. A 0.5 mg shot shoul
d produce circul r p ttern of p rticles pproxim tely 1 cm in di meter. The p
ttern from 0.25 mg shot is slightly sm ller. In ddition, the spent c rtridge
c n
e ex mined for complete rele se of DNA/gold. Penetr tion c n
e tested
y s
hooting the p rticles into w ter- g r nd visu lizing the p rticles microscopic
lly, or
y shooting the p rticles through
rrier th t pproxim tes the intend
ed t rget tissue. The qu lity of the DNA precipit tion nd tu
e prep r tion c n

e ss yed
y eluting the DNA
ound to the gold in the c rtridge nd n lyzing t
he eluted DNA
y g rose gel electrophoresis. The
iologic l ctivity of the pre
p c n
e determined
y n lyzing tr nsient gene expression fter tr nsfection of
tissue culture cells or n ppropri te in vivo model.

References
1. S nford, J. C. (1988) The
iolistic process. TIBtech. 6, 299302. 2. Klein, T.,
Wolf, E., Wu, R., nd S nford, J. (1987) High-velocity microprojectiles for del
ivering nucleic cids into living cells. N ture 327, 7073.
P rticle-Medi ted Gene Tr nsfer
303
3. Christou, P. (1994) Gene tr nsfer into pl nts using p rticle
om
rdment, in
P rticle Bom
rdment Technology for Gene Tr nsfer. Oxford University Press, New
York. 4. Y ng, N.-S. nd Christou, P., eds. (1994) P rticle Bom
rdment Technolo
gy for Gene Tr nsfer. Oxford University Press, New York. 5. S rphie, D. F., John
son, B., Cormier, M., Burkoth, T. L., nd Bellhouse, B. J. (1997) Bio v il
ilit
y following tr nsderm l powdered delivery (TPD) of r diol
eled inulin to h irle
ss guine pigs. J. Cont. Rele se 47, 6169. 6. S nford, J. C., Devit, M. J., Russe
ll, J. A., Smith, F. D., H rpending, P. R., Roy, M. K., nd Johnston, S. A. (199
1) An improved, helium-driven
iolistic device. Technique 3, 316. 7. McC
e, D.
nd M rtinell, B. (1992) App r tus for genetic tr nsform tion. U.S. P tent #51496
55. 8. McC
e, D. (1995) G s driven gene delivery instrument. PCT P tent WO 95/
19799. 9. Christou, P. nd Sw in, W. F. (1990) Cotr nsform tion frequencies of f
oreign genes in soy
e n cell cultures. Theor. Appl. Genet. 79, 337341. 10. B rry,
M. A., L i, W. C., nd Johnston, S. A. (1995) Protection g inst mycopl sm inf
ection using expression-li
r ry immuniz tion. N ture 377, 632635.
27
Entr pment of Pl smid DNA V ccines into Liposomes
y Dehydr tion/Rehydr tion
Gregory Gregori dis, Brend McCorm ck, Mi O
renovich, nd Yvonne Perrie 1. Intr
oduction Intr muscul r injection of n ked pl smid DNA is known (13) to elicit hum
or l nd cell-medi ted immune responses g inst the encoded ntigen. It is thoug
ht (2,3) th t immunity follows DNA upt ke
y muscle cells, le ding to the expres
sion nd extr cellul r rele se of the ntigen which is then t ken up
y ntigen
presenting cells (APC). In ddition, it is fe si
le th t some of the injected DN
A is t ken up directly
y APC. Dis dv nt ges (13) of n ked DNA v ccin tion includ
e: upt ke of DNA
y only minor fr ction of muscle cells, exposure of DNA to de
oxyri
onucle se in the interstiti l fluid thus necessit ting the use of rel tive
ly l rge qu ntities of DNA, nd, in some c ses, injection into regener ting musc
le in order to enh nce immunity. We h ve recently proposed (1,4) th t DNA immuni
z tion vi liposomes (phospholipid vesicles) could circumvent the need of muscle
involvement nd inste d f cilit te (5) upt ke of DNA
y APC infiltr ting the si
te of injection or in the lymph tics, t the s me time protecting DNA from nucle
se tt ck (6). Moreover, tr nsfection of APC with liposom l DNA could
e promot
ed
y the judici l choice of vesicle surf ce ch rge, size nd lipid composition,
or
y the co-entr pment, together with DNA, of pl smids expressing ppropri te
cytokines (e.g., interleukin 2), or immunostimul tory sequences. To th t end,
method h s
een developed (6) which llows for the qu ntit tive entr pment of pl
smid DNAs into neutr l, nionic nd c tionic liposomes th t re c p
le of tr n
sfecting cells in vitro with v rying efficiency (6). Moreover, it w s shown (1,4
) th t immuniz tion of B l
/c nd out
red (T.O.) mice
From: Methods in Molecul r Medicine, vol. 29, DNA V ccines: Methods nd Protocol
s Edited
y: D. B. Lowrie nd R. G. Wh len Hum n Press Inc., Totow , NJ
305
306
Gregori dis et l.

y v riety of routes with (c tionic) liposom l DNA le ds to much gre ter humor

 l immune responses ( s indic ted
y splenocyte IL-4 production nd circul ting

IgG su
cl sses) nd cell-medi ted immune responses (splenocyte IFN- production) t
han those o
tained with naked DNA or DNA complexed to similar pre-formed liposom
es. We have o
served (1,4,7) that a cationic lipid is essential for the promotio
n of stron responses and that PE and DOPE are equally effective in inducin imm
une responses in enetic vaccination. Here we descri
e methodoloy for the incor
poration of plasmid DNA into liposomes of varyin lipid composition, vesicle siz
e and surface chare. The dehydration-rehydration procedure is characterized
y
its mildness and is thus compati
le with most la
ile materials. It can also
e a
pplied for the entrapment of DNA into non-phospholipid liposomes in which PC (or
DSPC) is replaced
y non-ionic surfactants (8). Furthermore, as already shown (
9,10) for other entrapped solutes, DNA-containin liposomal suspensions as prepa
red here can
e freeze-dried (for storae) in the presence of a cryoprotectant w
ithout sinificant loss of material from within the vesicles on reconstitution w
ith 0.9% NaCl. 2. Materials
1. E phosphatidylcholine (PC), e phosphatidylethanolamine (PE), phosphatidic
acid (PA), phosphatidyl lycerol (PG), and phosphatidylserine (PS) (more than 9
9% pure) are availa
le from Lipid Products (Nutfield, Surrey, UK), or Lipoid GmB
H (Ludwishaten, Germany). 2. Dioleoyl phosphatidylcholine (DOPE), distearoyl ph
osphatidylcholine (DSPC) and stearylamine (SA) are availa
le from Sima Chemical
Co. (Poole, Dorset, UK). 3. 1,2-dioleyloxy-3-(trimethylamonium propane) (DOTAP)
and 1,2-dioleyl-3-dimethyl- ammonium propane (DODAP) are availa
le from Avanti
Polar Lipids Inc. (Ala
aster, AL). 4. 1,2-Bis (hexadecylcycloxy)-3-trimethylamin
o propane (BisHOP) was a ift from Syntex Corp. (Palo Alto, CA). 5. N[1-(2,3-dio
leyloxy) propyl]-N,N,N-triethylammonium (DOTMA) was a ift from GeneMedicine (Ho
uston, TX). 6. 3(N,N,-dimethylaminoethane)-car
amyl cholesterol (DC-CHOL) was a 
ift from Dr. C. Kir
y. 7. Sepharose (CL) 4B and polyethylenelycol 6000 are avai
la
le from Pharmacia (Hertfordshire, UK) Ltd. 8. Lipid solutions: dissolve 16 mol
es of PC and 8 moles of DOPE (or PE) in ~25 mL chloroform. For neatively chared
(anionic) liposomes, add 4 moles of PA, PG or PS. For positively chared (cationi
c) liposomes add 4 moles of SA, BisHOP, DOTMA, DOTAP, DODAB or DC-CHOL. Greater a
mounts of chared lipids can
e added, dependin on the amount of vesicle surfac
e chare required.
Entrapment into Liposomes
307
9. DNA solution: dissolve up to 500  of plasmid DNA in 2 mL distilled water or 1
0 mM sodium phosphate
uffer pH 7.2 (PB) if needed. The nature of
uffer in resp
ect to composition, pH, and molarity can
e varied as lon as this does not inte
rfere with liposome formation or entrapment yield. Amounts of added DNA can
e i
ncreased in proportion to the total amount of lipid used.
3. Methods The procedure for the entrapment of plasmid DNA into liposomes entail
s the preparation of a lipid film from which multilamellar vesicles (MLV) and, e
ventually, small unilamellar vesicles (SUV) are produced. SUV are then mixed wit
h the plasmid DNA destined for entrapment and dehydrated. The dry powder is su
s
equently rehydrated to enerate multilamellar dehydrationrehydration vesicles (DRV
) (11) containin the plasmid DNA. On centrifuation, liposome-entrapped DNA is
separated from non-entrapped DNA. When required, DNA-containin DRV are reduced
in size
y microfluidization in the presence or a
sence of non-entrapped DNA.
3.1. Preparation of the Lipid Film
1. Place the chloroform solution of lipids in a 50-mL round-
ottomed spherical Q
uick-fit flask and remove the solvent in a rotary evaporator at a
out 37C to leav
e a thin lipid film on the walls of the flask. 2. Flush the film for a
out 60 s
with oxyen-free nitroen (N2) to ensure complete solvent removal and to replace
the air.

3.2. Preparation of MLV

1. Pre-warm some distilled water and the flask containin the lipid film plus a
few lass
eads to a temperature a
ove the liquid-crystalline transition tempera
ture (Tc) of the phospholipid (>Tc). 2. Add 2 mL of the warm distilled water int
o the flask, replace the stopper and shake viorously
y hand or mechanically wh
ile maintainin the temperature >Tc until the lipid film has
een transformed in
to a milky suspension. 3. Allow the suspension to stand at >Tc for a
out 12 h whe
reupon multilamellar liposomes of diverse sizes are formed.
3.3. Preparation of SUV
1. Remove the lass
eads and sonicate the milky suspension at >Tc (with frequen
t intervals), usin a titanium pro
e slihtly immersed into the suspension. Keep
the flask flushed with N2 (achieved
y the continuous delivery of a entle stre
am of N2 throuh thin plastic tu
in). This step (see Note 1) should produce a s
lihtly opaque to clear suspension of SUV of up to 80 nm in diameter. 2. Centrif
ue the sonicated suspension of SUV for 2 min at 3000 rpm to remove titanium fra
ments, and allow the supernatant to stand at >Tc for a
out 12 h.
308 3.4. Dehydration of SUV Mixed with Plasmid DNA
Greoriadis et al.
1. Mix the SUV with the DNA solution and rapidly freeze the flask in liquid nitr
oen while rotatin it. 2. Freeze-dry overniht under vacuum (<0.1 torr).
3.5. Rehydration of the Powder
1. Add H2O, pre-warmed at >Tc, (0.1 mL per 16 moles of PC) and swirl the mixture
viorously at >Tc (see Note 2). Keep the sample at >Tc for a
out 30 min. 2. Repe
at the process with a further 0.1 mL H2O. 3. After 30 min at >Tc, repeat with 0.
8 mL PB (pre-warmed at >Tc) and allow the sample to stand for 30 min at >Tc. It
now contains multilamellar DRV (size rane a
out 0.55.0 m in diameter) with entrap
ped and non-entrapped plasmid DNA
3.6. Separation of DRV Liposome-Entrapped from Non-Entrapped DNA
1. Centrifue the suspension at 40,000 for 60 min at 4C and resuspend the pellet
(DNA- containin DRV) in H2O (or PB). 2. Repeat the process at least once to re
move the remainder of the non-entrapped material and resuspend the final pellet
in an appropriate volume (e.., 2 mL) of H2O or PB (see Note 3).
3.7. Measurement of DNA Entrapment
The extent of DNA entrapment in DRV liposomes is monitored
y measurin the DNA
in the suspended pellet and com
ined supernatants. The easiest way to monitor en
trapment is
y usin radiola
elled (32P or 35S) DNA (e.., see Ta
le 1). If a ra
diola
el is not availa
le or cannot
e used, DNA can
e li
erated from liposomes

y addin Triton X-100 (up to 5% final concentration) or, prefera
ly, isopropan
ol (1:1 volume ratio). If Triton X-100 or the solu
ilized liposomal lipids inter
fere with the assay of the DNA, it must
e extracted usin appropriate technique
s (6). Entrapment values rane
etween a
out 40100%, dependin on the amounts of
lipid and DNA used and the presence or a
sence of a cationic chare. Hihest val
ues are achieved when DNA is entrapped in cationic DRV (Ta
le 1) (see Note 4).
3.8. Microfluidization of DNA-Containin DRV
This step and those followin are needed when DNA-containin DRV liposomes are t
o
e converted to smaller vesicles (down to a
out 100 nm averae mean diameter)
while still retainin a su
stantial proportion of the DNA.
1. Take the rehydrated liposome preparation from 3.5 or a
ove (prior to separati
on of trapped from non-trapped DNA) and dilute it to 10 mL with H2O.
Ta
le 1 Incorporation of Plasmid DNA into Liposomes
y the Dehydration-rehydrati
on Method Liposomes pGL2 PC, DOPEa PC, DOPE
PC, DOPE, PSa PC, DOPE, PS
PC, DOP

E, PGa PC, DOPE, PG
PC, DOPE, SAa PC, DOPE, SA
PC, DOPE, BisHOPa PC, DOPE, DOT
MAa PC, DOPE, DC-Chola PC, DOPE, DC-Chol
PC, DOPE, DOTAPa PC, DOPE, DOTAP
PC,
DOPE, DODAPa PC, DOPE, DODAP

35S-la
eled
Entrapment into Liposomes
Incorporated plasmid DNA (% of used) pRc/CMV HBS 55.4 pRSVGH 45.6 11.3 pCMV4.65
28.6 pCMV4. EGFP VR1020
44.2 12.1 57.3 12.6
53.5 10.2 74.8 48.3 69.3 86.8 87.1 80.1 88.6 76.9 77.2 79.8 80.6 57.4 64.8
52.7 67.7
71.9
89.6 81.6
plasmid DNA (10-500 ) was incorporated into, (a) or mixed (
) with neutral (PC,
DOPE), anionic (PC, DOPE, PS or PG), or cationic (PC, DOPE, SA, BisHOP, DOTMA, D
C-Chol, DOTAP or DODAP) dehydration-rehydration vesicles (DRV). Incorporation va
lues for the different amounts of DNA used for each of the liposomal formulation
s did not differ sinificantly and were therefore pooled (values shown are means
of values o
tained from 35 experiments). PC (16moles) was used in molar ratios of
1:0.5 (neutral), and 1:0.5:0.25 (anionic and cationic liposomes). Plasmid DNAs
used encoded luciferase (pGL2), hepatitis B surface antien (HBsA, su
type ayw)
(pRc/CMV HBS, availa
le from Dr. R. Whalen), human rowth hormone (pRSVGH), Myc
o
acterium leprosy protein (pCMV 4.65, availa
le from Dr R. Tascon), fluorescent
reen protein (pCMV 4.EGFP) and Schistosome protein (VR1020) (from [1] with permi
ssion).
309
310
Greoriadis et al.
2. Cycle the suspension several times throuh a Microfluidizer 110S (Microfluidi
cs, Newton, MA). The pressure aue is set at 60 psi throuhout the procedure to
ive a flow rate of 35 mL per min (see Note 5).
3.9. Separation of DNA Entrapped in Microfluidized DRV from Non-entrapped DNA
1. Reduce the volume of the microfluidized sample
y placin it in dialysis tu
i
n which is then covered with polyethylenelycol 6000 in a flat container (see N
ote 6). 2. When the required volume has
een reached, separate entrapped from fr
ee DNA
y molecular sieve chromatoraphy usin a Sepharose CL 4B column, or
y c
entrifuation as in Su
headin 3.6. 3. Estimate the DNA within liposomes as % of
DNA relative to the oriinal amount as descri
ed a
ove (see Note 7). Liposome s
ize measurements can
e carried out
y photon correlation spectroscopy as descri

ed elsewhere (6,12). The DNAcontainin cationic liposomes can also
e su
jected
to microelectrophoresis in a Zetasizer to determine their net surface chare.
4. Notes
1. The time required to produce SUV varies, dependin on the amount of lipid use
d and the diameter of the pro
e. For the amounts of lipid mentioned a
ove, a cle
ar or slihtly opaque suspension is usually o
tained within up to four sonicatio
n cycles, each lastin 30 s, with 30 s rest intervals in
etween, usin a pro
e
of 0.75 inch diameter. The process of sonication is considered successful when a

djustment of the settins in the sonicator is such that the suspension is aitat
ed viorously. 2. The volume of H2O added must
e kept at a minimum, with just e
nouh H2O to ensure complete hydration of the powder under viorous swirlin. 3.
When the liposomal suspension is destined for in vivo use (e. intramuscular in
jection), NaCl is added to a final concentration of 0.9%. 4. As part of the lipo
some-associated DNA may have interacted with the liposomal surface (e.., cation
ic roups) durin the entrapment procedure, it may
e necessary to determine act
ual entrapment of the DNA (as opposed to surface-
ound DNA). This can
e achieve
d
y the use of deoxyri
onuclease which will derade most of the external materi
al (6). Our experience (6) shows that most of the DNA entrapped within liposomes
remains intact on exposure to ri
onuclease, presuma
ly protected
y the surroun
din
ilayers. 5. The num
er of cycles used depends on the vesicle size required
(6) or on the sensitivity of the plasmid DNA. In the case of pGL2 (see Ta
le 1)
, microfluidization for more than three cycles resulted in proressive smearin
of the DNA and failure to transfect cells in vitro (6). It is likely that other
plasmid DNAs will
ehave similarly on extensive microfluidization. Microfluidiza
tion of the sample can also
e carried out after the removal of non-entrapped DN
A as in Su
headin 3.6. (washed liposomes), althouh DNA retention in this case ma
y
e reduced: the presence of non-entrapped DNA durin microfluidization (a proEntrapment into Liposomes
311
cess that desta
ilizes liposomes, which then reform as smaller vesicles) is expe
cted (6) to diminish DNA leakae, perhaps
y reducin the osmotic rupture of ves
icles (11). 6. Removal of excess H2O from the tu
in is relatively rapid and it
is therefore essential that the sample is inspected reularly. 7. When the sampl
e is microfluidized followin Su
headin 3.5., i.e.,
efore the estimation of en
trapment, it is necessary that a small portion of the sample which is to
e micr
ofluidized,
e held aside for the estimation of entrapment, accordin to Su
head
in 3.7.
References
1. Greoriadis, G. (1998) Genetic vaccines: strateies for optimization. Pharm.
Res. 15, 661670. 2. Davis, H. L., Whalen, R. G., and Demeneix, B. A. (1993) Direc
t ene transfer in skeletal muscle in vivo: factors influencin efficiency of tr
ansfer and sta
ility of expression, Hum. Gene Ther. 4, 151156. 3. Manickan, E., K
arem, K. L., and Rouse, B. T. (1997) DNA vaccinesa modern immick or a
oon to va
ccinoloy? Crit. Rev. Immunol. 17, 139154. 4. Greoriadis, G., Saffie, R., and de
Souza, J. B. (1997) Liposome-mediated DNA vaccination. FEBS Lett. 402, 107110. 5
. Greoriadis, G. (1995) Enineerin tareted liposomes: Proress and pro
lems.
Trends Biotechnol. 13, 527537. 6. Greoriadis, G., Saffie, R., and Hart, S. L. (1
996) Hih yield incorporation of plasmid DNA within liposomes: Effect on DNA int
erity and transfection efficiency. J. Dru Taretin 3, 469475. 7. Perrie, Y. an
d Greoriadis, G. (1998) Liposome-mediated vaccination: the effect of vesicle co
mposition. J. Liposome Res. 8, 95,96. 8. O
renovic, M., Perrie, Y., and Greoria
dis, G. (1998) Entrapment of plasmid DNA into niosomes: characterization studies
. J. Pharm. Pharmacol. 50, 155. 9. Greoriadis, G., Davis, D., and Davies, A. (1
987) Liposomes as immunoloical adjuvants: Antien incorporation studies. Vaccin
e 5, 143149. 10. Greoriadis, G., ed. (1993) Liposome Technoloy, 2nd ed., Vols II
II, CRC Press, Boca Raton, FL. 11. Kir
y, C. and Greoriadis, G. (1984) Dehydrat
ion-rehydration vesicles (DRV): A new method for hih yield dru entrapment in l
iposomes. Biotechnoloy 2, 979984. 12. Skalko, N., Bouwstra, J., Spies, F., and G
reoriadis, G. (1996) The effect of microfluidization of protein-coated liposome
s on protein distri
ution on the surface of enerated small vesicles. Biochim. B
iophys. Acta 1301, 249254.
28
DNA-Based Vaccination with Polycistronic Expression Plasmids

Reinhold Schirm
eck, Jan von Kampen, Karin Metzer, Jens Wild, Beate Grner, Marti
n Schleef, Andrea Krer, Hansjr Hauser, and Jr Reimann 1. Introduction DNA-
ased
vaccination is a potent technique to prime cellular (T-cell mediated) immune res
ponses (reviewed in 1). Many details of the primin of T-cell precursors
y anti
en translated from injected expression plasmid DNA are unknown. The relevant ce
ll that is transfected in situ after DNA vaccination and that can process and pr
esent the protein in an immunoenic form has not yet
een identified. Alternativ
ely, the transfected cell may initiate crossprimin in vivo
y transferrin proces
sed antien to a professional antienpresentin cell (APC). Normally only a sin
le protein is translated from a eukaryotic mRNA. However, efficient internal ri

osomal entry sites (IRES) that support cap-independent initiation of translation
al have
een identified in certain viruses (e.., picornaviruses) (see Notes 1,2
). This o
servation has
een used to construct polycistronic expression plasmids
in which different proteins are translated from a sinle mRNA species. The tran
slational efficiency of individual cistrons in multicistronic mRNAs is influence
d
y the IRES element used and
y the RNA sequences located
etween the IRES ele
ments (or the cap) and the AUG initiation codon (see Note 3). Expression of diff
erent cistrons in polycistronic expression plasmids is strictly coupled. Althou
h the cotransfer of different enes into a cell can
e achieved
y mixin differ
ent plasmids, ene expression from such mixtures of plasmids is never
alanced.
Protein expression of the different enes, and thereFrom: Methods in Molecular M
edicine, vol. 29, DNA Vaccines: Methods and Protocols Edited
y: D. B. Lowrie an
d R. G. Whalen Humana Press Inc., Totowa, NJ
313
314
Schirm
eck et al.
fore antien presentation, will vary sinificantly (even if the same promoter/ e
nhancers are used to control expression). To circumvent these pro
lems, we used
polycistronic plasmids to deliver different antiens
y injection of a sinle DN
A species (see Notes 4 and 5). As model antiens, we used the surface (HBsA) an
d core (HBcA) antien of the hepatitis B virus (HBV). We have characterized the
murine cellular (CTL), and humoral (serum anti
ody), response that can
e elici
ted
y DNA-
ased vaccination in H-2d and H-2
mice (25). Polycistronic expression
plasmids contain a promoter/enhancer element, an IRES element, selected IRES-fl
ankin sequences, different sets of multiple clonin sites, intron-encodin sequ
ences, and/or a polyA sinal (see Note 3). To facilitate construction of polycis
tronic plasmids, we desined different monocistronic expression plasmids from wh
ich elements can
e taken to construct di- and tricistronic plasmids (see Note 6
). 2. Materials
2.1. Construction of Monocistronic pCI-1 and pCI-2 Expression Plasmids
1. pCI-1: The hih copy num
er plasmid pCI (Promea, cataloue no. E1731) was di
ested with EcoRI and NotI. A 675
p NotIEcoRI insert derived from pVBC3, contain
in the IRES and approximately 80
p of flankin sequence, was cloned into pCI,
resultin in the pCI-1 plasmid (Fi. 1). 2. pCI-2: The pCI plasmid was diested
with SalI and XhoI and reliated, resultin in the pCI plasmid. A 687 EaI frame
nt of the pVBC2 vector containin the IRES sequence and a
out 50
p of flankin
reions was cloned into the NotI site of pCI, resultin in plasmid pCI-2 (Fi. 1)
.
2.2. Construction of Dicistronic Expression Plasmids Containin Two Different An
tiens
1. The pCI-1 plasmid contains a unique NotI-site followin the IRES sequence. In
to this site, the EaI frament from pCI-2 (containin the ene of interest foll
owed
y another IRES element) was inserted. This eliminates the upstream NotI si
te,
ut leaves the downstream NotI site intact. This stratey can therefore
e u

sed to construct tri- or polycistronic plasmids. Fis. 2A and B demonstrate repr

esentative examples of the clonin stratey. 2. The SmaI/HindIII frament from p
lasmid pCMV-2/C (6) encodes the complete core antien (HBcA) sequence. 3. This
frament was inserted into the SmaI site of pCI-1 to enerate the vector pCI-1/C
. 4. The SalI/XhoI frament from plasmid pCMV-S is derived from pSBC-1 (6) into
which the StuI frament encodin the HBsA of HBV was cloned. The SV40 promoter
of this plasmid was replaced
y the HCMV promoter of plasmid BCMGneo (a enerous
ift of Dr. H. Karasuyama, Basel, Switzerland).
Polycistronic Expression Plasmids
315
Fi. 1. Monocistronic expression plasmids pCI-1 and pCI-2. 5. This frament was
cloned into the SalI-site of pCI-2 to enerate the pCI-2/S vector. 6. The EaI f
rament of pCI-2/S (containin the HBsA-encodin reion and the IRES) was inser
ted into the NotI site of pCI-1/C to enerate the dicistronic expression plasmid
pCI/C-S.
2.3. Special Materials
1. Taret cells: monkey COS-7 kidney cells (ATCC; CRL-1651), murine L929 fi
ro
l
asts (ATCC, CCL-1), chicken LMH hepatoma cells. 2. Antisera: polyclonal ra

it a
nti-HBsA antiserum (a enerous ift of the Behrin AG, Mar
ur, Germany), polyc
lonal ra

it anti-HBcA antiserum Hc-1 (a enerous ift of Dr. H. J. Schlicht).
3. Protein-A Sepharose (cat. no. 17-0780-01, Pharmacia, Frei
ur, Germany). 4. U
ltrapure 100 anion exchane chromatoraphy columns (Qiaen, Hilden, Germany). 5.
Cardiotoxin solution (Latoxan, cataloue no. L8102, Rosans, France). 6. Anti-HB
sA IMxAUSAB test kit (cataloue no. 2262-20, A

ott, Wies
aden, Germany) 7. Ant
i-HBcA CORE anti-HBc test kit (cataloue no. 2259-20, A

ott). 8. A reference s
ample from the Paul-Ehrlich-Institute (Lanen, Germany).
3. Methods 3.1. Coexpression of HBsA and HBcA from Dicistronic Expression Plas
mids Coexpression of HBsA and HBcA from the pCI/S-C and pCI/C-S can
e analyze
d in transient transfection assays, usin COS-7, L929, or LMH cells. Antiens cl
oned into the first cassette of the
icistronic vectors are expressed at levels
compara
le to those in monocistronic constructs. In contrast, antiens cloned in
to the second cassette are expressed at 320-fold lower levels than o
served in mo
nocistronic vectors.
1. Grow cells to a density of 104-105 cells/mL in 100 mm tissue culture dishes i
n Dul
eccos modified Eale medium (DMEM) supplemented with 10% (v/v) FCS.
316
Schirm
eck et al.
Fi. 2. [Construction of dicistronic expression plasmids encodin surface (HBsA
) and core (HBcA) antien of HBV.] (A) The dicistronic plasmid pCI/S-C was cons
tructed
y insertin the HBcA/IRES-containin EaI frament from pCI-2/C into t
he NotI-diested pCI-1/S plasmid.
2. Transfect 1  plasmid DNA per dish usin the CaPO4-method. 3. Test transient e
xpression of HBsA and HBcA
y [35S]-methionine la
elin of cells at 48-72 h po
st transfection. 4. Lyse cells and remove cell de
ris
y centrifuation for 30 m
in, 20,000 at 4C. 5. Immunoprecipitate HBsA
y addin 5  of a polyclonal ra

it
anti-HBsA antiserum. Precipitate HBcA
y addin 5  of a polyclonal ra

it ant
i-HBcA and 50 L protein-A dissolved in PBS. 6. Process the immunoprecipitates fo
r SDS-PAGE and fluororaphy.
3.2. Preparation of Plasmid DNA Used for DNA-
ased Vaccination
Plasmid DNA to
e used for DNA vaccination is purified
y anion exchane chromat

oraphy.
Polycistronic Expression Plasmids
317
Fi. 2. [Construction of dicistronic expression plasmids encodin surface (HBsA
) and core (HBcA) antien of HBV.] (B) The dicistronic plasmid pCI/C-S was cons
tructed
y insertin the HBsA/IRES-containin EaI frament from pCI-2/C into t
he NotI-diested pCI-1/C plasmid.
1. Harvest a
out 60  of wet weiht of
iomass from 5 L cultures of cloned E. co
li DH5 cells har
orin the plasmid DNA; su
mit the
acteria to alkaline lysis. 2
. Isolate a
out 100 m plasmid DNA from the lysate
y use of ultrapure anion exc
hane columns. 3. Suspend plasmid DNA at 10 /L in 10x TE
uffer (100 mM Tris-HCl,
10 mM EDTA, pH 7.4) and store at 20C (see Note 7). 4. Dilute this solution 1:10 w
ith Ca++/M++ -free PBS, within 30 min
efore injection, to o
tain a 1 /L DNA sol
ution.
3.3. Intramuscular Nucleic Acid Inoculation
1. Inject 100 L of a 0.01 mM cardiotoxin solution into each ti
ialis anterior or
quadriceps muscle of the mice.
318
Schirm
eck et al.
2. Five days later, inject 50 L of the 1 /L DNA solution into each reeneratin ti

ialis anterior or quadriceps muscle. 3. Use non-injected mice or mice injected
with plasmid DNA without insert as neative controls.
3.4. Cytotoxic T Cell Assay (see Note 8)
1. O
tain spleens from DNA-vaccinated BALB/c (H-2d) mice. 2. Prepare sinle cell
suspensions from these spleens in -MEM tissue culture medium supplemented with 1
0 mM HEPES
uffer, 5 105 M 2-merc ptoeth nol, nti
iotics nd 10% (v/v) of sele
cted
tch of fet l c lf serum (FCS). 3. Co-culture 3 107cells with 1.5 106 irr
di ted (20,000 r d) stimul tor cells in mixed tumor cell-lymphocyte culture (M
TLC). M int in 10 mL volume liquots in 25 cm2 upright tissue culture fl sks in
humidified tmosphere of 5% (v/v) CO2 t 37C. Use stimul tor cells t ken from s
yngeneic P815 m stocytom tr nsfect nts expressing either the sm ll hep titis B
ntigen (HBsAg), or the hep titis core ntigen (HBcAg). 4. H rvest in vivo-prime
d nd in vitro-restimul ted spleen cell popul tions from the 5-d MTLC. W sh the
cells twice nd coculture in 200 L round-
ottom wells for 4 h t 37C t titr ted d
ensities with 2 103 [51Cr]-l
eled t rgets. Use effector/t rget r tios in the r
nge of 120. 5. Collect 100 L of the supern t nt for -radiation countin. Calculate
the percent specific release as [(experimental release spontaneous release)/(tot
al release spontaneous release)] 100. Measure total counts
y resuspendin tare
t cells. Measure spontaneously released counts usin taret cell cultures withou
t cytolytic effector cell populations. Plot the data as the mean of triplicate c
ultures. The SEM of triplicate data was usually less than 10% of the mean. Fis.
3C,D show representative examples of the anti-HBsA and anti-HBcA cytotoxic T
lymphocyte (CTL) response of BALB/c (H-2d) mice immunized with mono- or dicistro
nic expression plasmid DNA.
3.5. Serum Anti
ody Assays
Use the IMxAUSAB test to measure anti-HBsA titers and the CORE kit to measure a
nti-HBcA in sera of immunized mice at different time points postvaccination as
follows
elow.
3.5.1. Serum Anti
odies Aainst HBsA

1. Mix microparticles coated with HBsA with mouse serum samples to allow specif
ic
indin of anti-HBsA anti
odies. 2. Bind the microparticles coated with anti
en-anti
ody complexes to the lass fi
re matrix. 3. Add
iotinylated recom
inan
t HBsA. 4. Quantify the
indin usin an alkaline phosphatase-conjuated anti-

iotin anti
ody followed
y the su
strate 4-methyl-lum
elliferyl-phosphate.
Polycistronic Expression Plasmids
319
Fi. 3. [Polyvalent induction of humoral and cellular immune responses usin dic
istronic plasmid DNA.] BALB/c mice were intramuscularly injected once with 100 
monocistronic pCI-1/C or pCI-1/S plasmid DNA, dicistronic pCI/S-C plasmid DNA, d
icistronic pCI/C-S plasmid DNA, or pCI plasmid DNA without insert. Five wk postvaccination, the serum anti
ody titers aainst HBsA (A) and HBcA (B), and the
MHC-I-restricted cytotoxic T lymphocyte (CTL) response aainst HBsA (C) and HBc
A (D) were measured. 5. Quantify the anti
ody levels usin six standard serum d
ilutions (01000 mIU/mL); dilute the test sera so that the measured OD values fall
within the standard curve, and calculate values as mIU/mL.
3.5.2. Serum Anti
odies Aainst HBcA
1. Follow the instructions supplied with the CORE kit. 2. Standardize the concen
trations of anti-HBc aainst the reference sample from the Paul-Ehrlich Institut
e (Lanen, Germany) and express the results as PaulEhrlich units (PEU)/mL.
Fis. 3A and B show representative examples of anti-HBsA and antiHBcA serum an
ti
ody responses of BALB/c (H-2d) mice immunized with the mono- or dicistronic e
xpression plasmid DNA descri
ed. 4. Notes
1. The use of IRES elements allows the co-expression of two or more cistrons fro
m one promoter. Tricistronic expression has
een successful (R. Schirm
eck, unpu

lished results). The promoter/enhancer element defines the strenth of
320
Schirm
eck et al.
expression, which also depends on the cell type in which the enes are to
e exp
ressed. The use of a promoter that is active in most cell types, such as the HCM
V promoter in pCI, is advisa
le. Different IRES elements are availa
le today. Ho
wever, only few of them show stron translational enhancement in different tissu
es. Currently, the IRES element from the poliovirus (as it is used in this work)
, and the IRES element from Encephalomyelocarditis Virus (EMCV), are the most un
iversal and stronest sequences known. Since the promoter/enhancer, as well as t
he IRES element, have an influence on the expression of the individual cistrons
in the tissue, the strenth and ratio of expression miht vary to some extent. T
his has to
e considered if experiments carried out in primary or continuous cel
l lines are transferred to animals in particular, as it is not known in which ti
ssue the immunoenic response is initiated. In usin IRES elements, the followin
 points have to
e considered: (i) The first cistron, usually a cDNA, should no
t have a poly (A) sinal. Otherwise, premature termination would result in monoc
istronic expression; and (ii) In usin the polio virus IRES element, the distanc
e
etween the IRES element and the AUG of the second cistron is varia
le. Howeve
r, in usin the EMCV IRES, the AUG has to
e exactly positioned in order to et
stron translational re-initiation. Coexpression of two or more distinct ene pr
oducts at stochiometrically defined ratios from a sinle transcript within the s
ame APC allows novel options in DNA
ased vaccination. Polycistronic expression p
lasmids can deliver a multivalent vaccine with a sinle injection, i.e., it can
stimulate the eneration of specific immune reactivities aainst two or more ant
iens from the same or different pathoens. Coexpression of different antiens

y the same APC is expected to provide an immune response-enhancin helper effect.
We have demonstrated this usin HBsA and HBcA (7): (i) polycistronic expressio

n plasmids can codeliver an antien and a cytokine to the same APC; and (ii) pol
ycistronic expression constructs allow the coexpression of the antien and an in
duci
le suicide ene in the in situ transfected cell. This can restrict the pres
ence of in vivo transfected cells in the vaccinated oranism to a limited period
of time (sufficient to prime an immune response). Su
sequent elimination of all
transfected (
ioloically relevant or
ystander) cells can
e achieved
y deliv
erin an external stimulus. This excludes persistent enetic alteration of the i
mmunized host as a result of DNA-
ased vaccination. Major o
jectives in the desi
n of DNA vaccines are the enhancement of the immunoenicity of antiens express
ed from expression plasmids, and the selective modulation of the spectrum of eff
ector functions that the elicited T cell responses mediate. Two major o
servatio
ns stress the relevance of these o
jectives: (i) Only particular functional su
c
lasses of T cells are a
le to confer protective immunity aainst certain pathoe
ns while others mediate the immunopatholoy accompanyin persistin infections.
To prime protective responses, and to avoid complications of immunopatholoy, DN
A vaccination will have to achieve efficient, selective, and sta
le primin of t
he appropriate T cell reactivity; and (ii)
2.
3.
4.
5.
Polycistronic Expression Plasmids
321
DNA vaccination may
e useful to convert an esta
lished inefficient, potentially
pathoenic T cell response into a protective immune response. This is relevant
in the immunotherapy of cancer and allery, and in the desin of protective, the
rapeutic vaccines for chronic persistent infections. Here DNA vaccination will h
ave to overcome an esta
lished, inappropriately polarized T cell response and co
nvert it into T cell reactivity with an alternative functional phenotype. 6. Adj
uvants have played a prominent role in formulatin vaccines. Naked, nonmethylated
plasmid DNA has stron adjuvanticity for T-cell responses shiftin them towards
the TH1 phenotype as has
een known for a lon time for poly I:C [reviewed in (8
)]. The mechanism of action of adjuvants is supposed to
e mediated
y inducin
and/or amplifyin the in situ cytokine milieu. A rational approach to adjuvants
would
e the codelivery of selected cytokines with antien. Codelivery of cytoki
nes with antien in DNA-
ased vaccination has
een achieved
y different means.
Current strateies in DNA-
ased vaccinations use the followin approaches: (i) v
accination with a mixture of expression plasmids that encode either an antien,
or a cytokine; (ii) vaccination with complex expression plasmids that express th
e antien and a cytokine under separate promoter controls; (iii) vaccination wit
h expression plasmids encodin a fusion protein composed of the antien (or an i
mmunoenic domain of the antien) and a cytokine; and (iv) vaccination with poly
cistronic expression constructs encodin the antien and cytokine(s). We have de
scri
ed the latter stratey. The potential of this approach, and the advantaes
and disadvantaes of this approach relative to alternative strateies, will have
to
e investiated in different animal models and different antien systems. 7.
Quality controls showed that this plasmid DNA typically contains <100 endotoxin
units/m DNA, >90% supercoiled DNA, and <1% (w/w) residual protein content. 8.
T-cell responses have a strenth and a functional phenotype. A specific T cell react
ivity can
e stron or weak reflectin (amon other parameters), the num
er of T cel
l clones specifically activated and their avidity for antien. Tcell responses a
re usually polarized, i.e., the primed T cell clones mediate distinct sets of ef
fector functions. The main functional su
classes of CD4+ T cells desinated Th1

and Th2 show differences in the profile of secreted cytokines, in the suscepti
i
lity to activation-induced cell death, in CD95/CD95L-dependent cytolytic reactivit
y, and in cell contact-dependent APC activation. Furthermore, Tc1 and Tc2 su
set
s have
een identified in primed CD8+ cytotoxic T lymphocyte (CTL) populations t
hat are less well characterized.
References
1. Donnelly, J. J., Ulmer, J. B., Shiver, J. W., and Liu, M.A. (1997) DNA vaccin
es. Annu. Rev. Immunol. 15, 617648. 2. Kuhr
er, A., Wild, J., Pudollek, H.-P., Chi
sari, F. V., and Reimann, J. (1997) DNA vaccination with plasmids encodin the i
ntracellular (HBcA) or secreted (HBeA) form of the core protein of hepatitis B
virus primes T cell responses to two overlappin K
- and Kd-restricted epitope
s. Int. Immunol. 9, 12031210.
322
Schirm
eck et al.
3. Kuhr
er, A., Pudollek, H.-P., Reifen
er, K., Chisari, F. V., Schlicht, H. J.,
Reimann, J., and Schirm
eck, R. (1996) DNA immunization induces anti
ody and cy
totoxic T cell responses to hepatitis B core antien in H-2
mice. J. Immunol.
156, 36873695. 4. Davis, H. L., Schirm
eck, R., Reimann, J., and Whalen, R. G. (1
995) DNA-mediated immunization in mice induces a potent MHC class I-restricted c
ytotoxic T lymphocyte response to Hepatitis B virus surface antien. Hum. Gene T
her. 6, 14471456. 5. Schirm
eck, R., Bhm, W., Ando, K., Chisari, F. V., and Reiman
n, J. (1995) Nucleic acid vaccination primes hepatitis B surface antien-specifi
c cytotoxic T lymphocytes in nonresponder mice. J. Virol. 69, 59295934. 6. Dirks,
W., Wirth, M., and Hauser, H. (1993) Dicistronic transcription units for ene e
xpression in mammalian cells. Gene 128, 247249. 7. Wild, J., Grner, B., Metzer, K
., Kuhr
er, A., Pudollek, H.-P., Hauser, H., et al. (1997) Polyvalent vaccination
aainst hepatitis B surface and core antien usin dicistronic expression plasm
ids. Vaccine 16, 16,35316,360. 8. Pisetsky, D. S. (1997) DNA and the immune syste
m. Ann. Intern. Med. 126, 169171.
29
A Nonviral Cytoplasmic T7 Autoene System and Its Applications in DNA Vaccinatio
n
Franck G. Sturtz, Yunshen Li, Janine Shulok, H. Ralph Snodrass, and Xiao-zhuo
Chen 1. Introduction The use of DNA vectors to elicit an immune response has pro
duced a lot of interest. Unfortunately, one of the limitin factors has
een the
pro
lem of ene expression. In order to o
tain a stron expression of the vacci
natin ene, several steps are necessary. The vector has to
e delivered in such
a way that it is not
ein deraded
y the immune nor
y the hepatic system; it
has also to enter efficiently the tareted cells; and it must
e expressed in t
he appropriate compartment of the cells at a hih level. For these reasons, we h
ave developed a ene expression vector that contains a T7 autoene and is
ein
expressed in the cytoplasm of the cells (1,2). We will descri
e this system and
two possi
le applications: infectious disease vaccination and tumor a
lation. Th
e latter application may
e com
ined with DNA vaccination aainst cancer cells.
1.1. Description of the T7 Autoene System
Traditionally, a nonviral mammalian ene expression vector consists of a circula
r plasmid DNA encodin a ene driven
y a eukaryotic promoter. Since the eukaryo
tic promoter functions only in the nuclei of mammalian cells, the plasmid DNA ha
s to
e transported into the nuclei for ene expression. In contrast, the T7 vec
tor has a cytoplasmic expression
ecause it contains a
acteriophae T7 promoter
that is neither reconized nor used
y the mammalian transcriptional machinery.
This T7 promoter is functional in the cytoplasm of the transfected mammalian ce
lls when coinjected with T7 RNA polymerase (RNAP)
ecause the T7 RNAP is a
acte
rial protein and it localizes in the cytoplasm when introduced into mammalian ce

lls (3). The T7 vector has
een

From: Methods in Molecular Medicine, vol. 29, DNA Vaccines: Methods and Protocol
s Edited
y: D. B. Lowrie and R. G. Whalen Humana Press Inc., Totowa, NJ
323
324
Sturtz et al.
Fi. 1. Schematic structure of the T7 vaccination vector. A eneral T7 vaccinati
on ene expression vector contains a T7 autoene (a T7 RNAP ene driven
y a T7
promoter (PT7)) as shown in the top half of the vector, and a vaccine ene (in t
his study, it can
e either a HA, or a HIV p120 enes) driven
y a second PT7 a
s shown in the
ottom half of the vector. EMC sequences provide ri
osome interna
l entry sites (IRES) for efficient translation of uncapped mRNAs enerated in th
e cytoplasm, 1 and 1. When the vector is cotransfected with T7 RNAP into a tar
et cell, the T7 RNAP initiates the transcription from the two PT7 in the cytopla
sm of the transfected cells. 2 and 2, cytoplasmically transcri
ed T7 RNAP mRNA
will
e translated into T7 RNAP protein which
ind to the two PT7 for 3 and 3 m
ore transcriptions of
oth the T7 RNAP and vaccine enes. The ratio
etween T7 a
utoene and the vaccine ene can
e further adjusted
y separatin the two seque
nces into two plasmids (pT7T7 and pT7-vaccine), as in the case for the pT7a.
constructed to express hih levels of mRNA and proteins
y includin a T7 autoe
ne. The T7 autoene is defined as a T7 RNAP ene driven
y its own conate T7 pr
omoter (1,4). With such a enetic arranement, a positive feed
ack loop is forme
d. The T7 RNAP enzyme produced from the T7 RNAP ene can return to the T7 promot
er, makin more and more T7 transcripts (1) (Fi. 1). Another T7 promoter leadin
 the expression of the ene of interest can
enefit from this stron T7 RNAP pr
oduction and allow for the expression of this ene. This T7 autoene system have

een found to rapidly express hih levels of proteins in cultured cells (1,2) a
nd in various animal tissues (2).
1.2. Humoral Immunity Induced
y the T7 System
When the pT7T7/T7HA, a plasmid vector containin
oth a T7 autoene and an influ
enza viral hemalutinin ene driven
y a second T7 promoter,
T7 Autoene System
325
Fi. 2. Detection of anti-HA anti
odies in BALB/c mice immunized with pT7T7/ T7H
A. Immunocompetent BALB/c mice were immunized with pT7T7/T7HA as descri
ed in Me
thods. ELISA results from the second
leedin were shown in this fiure. NC-1 =
sera from three uninjected mice from the site where the ELISA was performed; NC2 = sera from 5 uninjected mice from the site where the plasmid injections were
performed; T7Luc = sera from 5 mice injected with pT7T7/T7Luc, a luciferase- (ir
relavant-) ene encodin plasmid; T7HA = sera from 5 mice injected with pT7T7/T7
HA; and PC = sera from three mice infected with influenza virus PR8.
was repeatedly coinjected with T7 RNAP into muscles and tail connective tissues
of immunocompetent BALB/c mice, specific anti-HA anti
odies were detected in thr
ee out of the five injected mice (Fi. 2).
1.3. Cellular Response Induced
y the T7 System
Specific CTL response aainst HIV-1 p55a was induced
y intramuscular immuniza
tion with a T7 vaccine system (Ta
le 1) (5). The T7 vaccine system consists of t
hree components: a pT7a plasmid encodin a p55a ene driven
y a T7 promoter
, a second plasmid pT7T7, and T7 RNAP. This result demonstrates for the first ti

me that reulated expression of vaccine enes in vivo could
e accomplished usin

 this T7 system. This system may
e useful in vaccine settins where short-term
cytoplasmic expression of protein in antien presentin cells is desira
le.
326
Ta
le 1 HIV-1 p24a-specific CTL Activity in Mice Immunized with T7 Vaccine Sys
tem
Sturtz et al.
Percent specific lysis of tarets SV Bal
pT7 aa pT7T7 50  50  T7 RNAP 2,500 U
E:T ratio
50:1 10:1 2:1 50:1 10:1 2:1 50:1 10:1 2:1 50  50  10,000 U 50:1 10:1
2:1 50:1 10:1 2:1 50:1 10:1 2:1
aBal
/c
MC57
Untreated p24(199207) p24(199207) <1 c <1 <1 <1 <1 <1 <1 <1 <1 <1 1 2 3 3 2 4 3 1
13 8 3 51 27 17 30 16 6 31 25 11 38 19 19 44 27 13 <1 2 2 2 1 <1 <1 <1 <1 <1 <1
<1 1 2 2 5 3 2
mice were immunized i.m. with pT7a, pT7T7, and T7 RNAP in DOTAP. Results are s
hown for 3 individual mice per roup. Results are representative of two replicat
e experiments.
E:T ratio is the effector:taret cell ratio. cValues represent p
ercent specific 51Cr release in a 4 h assay.
1.4. Tumor A
lation
DNA vaccination aainst cancer cells is a tantalizin application of DNA vaccina
tion. The existence of tumor-specific antiens com
ined with the use of cytokine
s to enhance the immune response of the patient seems to
e an appealin path fo
r treatin metastatic cancers. Althouh the T7 vector has not
een used yet to i
nduce a vaccination aainst cancer cells, it has
een used to a
late tumors raf
ted in nude mice (6). A human osteosarcoma cell line (143B)
T7 Autoene System
327
Fi. 3. Tumor rowth inhi
ition
y direct tumor injections of pT7T7/T7TK DNA. 14
3B tumors are enerated in the flank of nude mice
y intradermal injection of 10
6 cells. Ten days after the tumor cells injection, the tumors are
ein injected
with 20  of DNA pre
ound with 1000 units of T7 RNAP with tu
erculin syrines mo
unted with 28G needles. The tumor injections are performed every three days for
21 d. GCV is injected i.p. twice a day for 28 d. The tumor sizes are monitored w
ith a caliper twice a week. The tumor rowths are represented in relative volume
s with the initial tumor volumes of all treatment roups at d 0 (the day the tum
or-
earin nude mice started to receive T7 DNA and GCV injections) assined as 1
00.
was rafted in the flank of nude mice. The tumors were thereafter injected with
a T7 vector
earin the thymidine kinase ene. The prodru anciclovir was injec
ted intraperitoneally and tumor sizes were monitored with a caliper. The tumors
treated with the T7-TK vectors exhi
ited a much slower rowth pattern (66%) compa
red to the control animals (Fi. 3). 2. Materials 2.1. Plasmids and Humoral Immu
nity
2.1.1. Plasmid
1. Escherichia
paation of T7
) for pl smids

Constructions
coli strains: HMS174(DE3)pLysE from Novaen (Madison, WI) for pro
autoene-containin plasmids, and DH5 (Gi
co-BRL, G ithers
urg, MD
th t do not cont in the T7 utogene (see Note 1). 2. Restriction

enzymes nd T7 RNAP (New Engl nd BioL
s, Beverly, MA). 3. LipofectAMINE s tr n

sfection gent (Gi
co-BRL).
2.1.2. Immunochemic l St ining nd Hem dsorption
1. C6 cells or ny other dherent m mm li n cells th t c n
e e sily tr nsfected

y LipofectAMINE.
328
Sturtz et l.
2. Slide ch m
ers for tr nsfection (Nunc Intern tion l, Rochester, NY). 3. AntiHA nti
odies o
t ined from Dr. W lter Gerh rd of Wist r Institute, University o
f Pennsylv ni , Phil delphi . 4. Vect st in Elite ABC Ig G kit nd peroxid se AE
C kit from Vector L
or tories (Burling me, CA) for immunochemic l st ining. 5.
15 cc hum n red
lood cells. 6. PBS for hem dsorption.
2.1.3. DNA Injections (see Note 2)
1. Five milliliter sterile polystyrene tu
es, P20, P200, nd P1000 pipets, steri
le tips, nd tu
e-holding r ck nd PBS for prep ring the DNA solution. 2. One
cu
ic centimeter tu
erculin pl stic syringes nd 28G needles for injection. 3. H
igh qu lity (Cesium chloride or Qi gen purified) pT7T7/T7HA DNA resuspended in e
ndotoxin-free w ter. 4. T7 RNAP (1000 units/L) from New Engl nd BioL
s. 5. Ice

ucket for tempor ry stor ge of DNA nd T7 RNAP.
2.2. Cellul r Immunity (see Note 3) 2.2.1. Pl smids, Synthetic Peptides, nd Rec
om
in nt Proteins
1. Pl smids pT7g g nd pT7T7, T7 RNAP s three components of the T7 v ccine. 2.
Synthetic epitope peptide HIV-1 p24g g (p24(199-207)) synthesized with free min
e N-termin l nd free cid C-termin l using Fmoc solid ph se methods
y Rese rch
Genetics (Huntsville, AL), nd p24 g g protein purified from ye st cells.
2.2.2. Anim ls nd Immuniz tions
1. B l
/c (H-2d) 46 wk-old mice from the Ch rles River L
or tories, the N tion l
Institute of He lth) 2. Injection m teri ls re the s me s in Su
he ding 2.1.3
.
2.2.3. Lymphocyte Cultures
1. 24-Well cell culture pl tes. 2. RPMI 1640 nd -MEM medi (Gi
co-BRL), fet l c
lf serum, merc ptoeth nol, nti
iotics. 3. IL-2 (R t T-Stim, Coll
or tive Biome
dic l Products, Bedford, MA).
2.2.4. Cytotoxic Cell Ass ys
1. R dioisotope 51Cr (1 Ci = 37 G
q). 2. 96-Well round
ottom tissue culture pl
tes.
2.3. Tumor A
l tion
1. High qu lity pT7-TK pl smid DNA. 2. The hum n osteos rcom 143B cell line c n

e o
t ined from the Americ n Tissue Culture Collection (ATCC).
T7 Autogene System
3. 4. 5. 6. 7. 8. 9.
329
DMEM plus 10% fet l
ovine serum, trypsin, nd PBS. Fem le in
red Nu-nu mice o
t
ined from Ch rles River L
or tories. Injection m teri ls for DNA: 28G needles
nd tu
erculin syringes. Injection m teri ls for GCV: 28G needles nd tu
erculin
syringes. Stock solution of g nciclovir (Cytovene, Syntex L
or tories, P lo Al
to, CA). Anesthetic solution. C liper.

3. Methods 3.1. Humor l Immunity

3.1.1. Construction of the T7 V ccin tion Vector In order to ev lu te the T7 sys
tem in DNA v ccin tion pplic tion, hum n hem gglutinin (HA) gene from influen
z virus str in Mount Sin i PR8 (7) w s chosen s model system for humor l imm
unity study. First, the HA gene w s removed from the origin l pl smid vector pT3
PR8HA (8) using NheI/X
I restrictions. The 1.8 k
HA fr gment w s then inserted
into the SpeI site of pT7-2, deriv tive of pTM-1 (1). Then, for efficient gen
e expression in the T7 vector,
oth of the 5 nd 3 untr nsl ted regions of the
HA gene were deleted using ph gemid mut genesis ppro ch (9). The result nt p
l smid vector w s n med s pT7HA. To explore single T7 vector system, NotI D
NA fr gment cont ining
oth the T7 promoter nd the HA gene w s removed from the
pT7HA nd inserted into the NotI site of pT7T7-N, deriv tive of pT7T7 (1). Th
e result nt fin l pl smid w s design ted pT7T7/T7HA. The correct deletions nd t
he junction sequences
etween the intern l ri
osome entry site sequence EMC (1),
the 5 end of the HA gene, nd
etween the 3 end of the HA gene nd the T7 ter
min tion sequence (1), were verified
y DNA sequencing n lysis (10). 3.1.2. Hem
dsorption nd Immunochemic l St ining of Cells Tr nsfected with pT7T7/T7HA Vect
or To test whether the vector pT7T7/T7HA w s function l nd the HA protein could

e produced, properly processed, nd presented on the cell surf ce, pT7T7/T7HA
w s introduced into r t gliom C6 cells with T7 RNAP
y lipofection (1,2). Twent
y-four hours fter tr nsfection, the tr nsfected cells were n lyzed for the exp
ression of the HA genes
y either hem dsorption using hum n red
lood cells (RBC
) or immunochemic l st ining using ntiHA nti
odies. The immunochemic l st inin
g reve led th t s high s 30% of the tr nsfected C6-Bu cells expressed the HA p
rotein nd the hem dsorption ss y showed th t the HA protein molecules were pro
perly processed
y the tr nsfected C6 cells nd presented on the cell surf ce s
demonstr ted
y the strong hem dsportion of hum n RBC on proportion of tr nsf
ected C6 cells.
330
Sturtz et l.
3.1.3. Induction of Humor l Immune Response Ag inst HA in Mice
y Injection of p
T7T7/T7HA Vector
1. Once the function lity of the gene expression vector pT7T7/T7HA w s confirmed

y ss ys using cultured cells, n nim l study w s c rried out to test if pT7T
7/ T7HA could induce the humor l immune response in nim ls. 2. BALB/c 24 mo-old
mice, 5 per group, were injected with 20 g of pT7T7/T7HA plus 1,000 units of T7 R
NAP in PBS solution in tot l volume of 50 L into the mouse t il
se (connectiv
e tissue) nd leg muscle, 25 L for e ch t il nd muscle, respectively. The pT7T7/
T7HA DNA nd T7 RNAP were first mixed in 5 mL sterile polystyrene tu
e t room
temper ture for 10 min, then tr nsferred to 1 cc tu
erculin pl stic syringe w
ith n tt ched 28 G needle. The DNA w s then slowly (~1020 s) injected into the
t rget tissue. 3. As control group for the
seline re ding, nother group of
mice were identic lly injected with 20 g of pT7T7/T7Luc DNA plus 1,000 units of T
7 RNAP. The pT7T7/T7luc is lucifer se-encoding pl smid (1) constructed simil r
ly to the pT7T7/T7HA. The DNA injections were performed three times t two wk in
terv ls. The leg muscles were injected in n ltern te f shion (leftrightleft). Th
e injected mice were
led 3 d
efore nd 1 wk fter the third injections. 4. The
presence of the nti-HA nti
odies in the mouse serum w s detected
y ELISA usi
ng purified virus s solid ph se. E ch serum s mple w s tested in triplic te t
dilutions of 1/20, 1/100, 1/500, nd 1/2500. The ss y w s st nd rdized with p
urified nti-HA mA
(H36-4). Ser from BALB/c mice t 7, 14, nd 21 d fter pulm
on ry infection with influenz virus str in PR8, were used s positive controls.
3.2. Cell Medi ted Immunity 3.2.1. Pl smid Construction nd Prep r tion
pT7g g w s constructed
y insertion of p55g g gene, PCR mplified from HIV-1

SF2 genomic clone, into pTM-1 (1). The pl smid pT7T7 (1) is the s me s descri

ed in Su
he ding 3.3.1. Pl smids pT7g g nd pT7T7 were tr nsformed into E. coli
str ins DH5 nd HMS174(DE3)pLysE, respectively. The pl smid DNA were prep red usi
ng Qi gen M xiprep kits.
3.2.2. Anim l Immuniz tion
Mice were infected i.p. with 1 107 pfu of vTF7-3. At 2 nd 18 h l ter, pT7g g n
d pT7T7 (T
le 1) were mixed with T7 RNAP in PBS, nd injected into the ti
i lis
nterior muscle. Control mice were infected intr peritone lly with 1 107 pfu of
the VVg g/pol viruses. Spleen cells pooled from 23 mice per group were ssessed
46 wk l ter for HIV-1 g g-specific CTL ctivity.
T7 Autogene System 3.2.3. Lymphocyte Culture nd Cytotoxic Cell Ass y
331
1. Spleen cells from the immunized mice were cultured in 24-well dishes t 5 106
cells/well. Of those cells, 1 106 were sensitized with synthetic epitope peptid
e p24 g g protein t concentr tion of 1 M for 1 h t 37C, w shed, nd co-culture
d with the rem ining 4 106 untre ted spleen cells in 2 mL of culture medium (50%
RPMI 1640 nd 50% -MEM, supplemented with 10% he t-in ctiv ted FCS, 5 105 M 2-mer
c ptoeth nol, nti
iotics, nd 2% IL-2). Cells were fed with 1 mL of fresh mediu
m on d 3 nd 5, nd cytotoxic ctivity w s ssessed on d 6. 2. Histocomp ti
le S
VB l
(H-2d) nd histoincomp ti
le MC57 (H-2
) t rget cells used in the 51Cr rel
e se ss ys express cl ss I
ut not cl ss II MHC molecules. Approxim tely 1 106
t rget cells were incu
ted in 200 L cont ining 50 Ci of 51Cr nd synthetic HIV-1
or HSV-2 peptides (1 M) for 60 min nd w shed three times. Effector (E) cells wer
e cultured with 5 103 t rget (T) cells t v rious E:T r tios in 200 L of culture
medium in 96-well pl tes for 4 h. The ver ge cpm from duplic te wells w s used
to c lcul te percent specific rele se (11).
3.3. Tumor A
l tion
1. One million cells from the hum n osteos rcom cell line (143B) re gr fted in
tr derm lly in the fl nk of nude mice (see Note 4). 2. Seven to 10 d fter gr ft
ing, when the tumor volumes re
etween 50 to 150 mm3, the tumors re injected w
ith 20 g of pT7T7/T7TK DNA pre
ound with 1000 units of T7 RNAP, every 3 d for 21
d. The intr tumor l injections re performed slowly (~20 s) with 28G needles on
nesthetized nim ls (see Notes 5 nd 6). 3. Twenty-five mg/kg
ody weight of g
nciclovir re injected I.P. twice d y for 28 d. 4. Tumor sizes re me sured wi
th c liper twice week during the entire experiment. The formul used to c lc
ul te the tumor volume is: Vol. = L H W 0.52.
4. Notes
1. B sed on our experience with the T7 pl smids, the success of n experiment wi
th the T7 utogene cont ining vectors depends prim rily on the qu lity of the T7
pl smids produced. Bec use of the extr ordin ry efficiency of the T7 utogene,
the E. coli host cell th t h r
ors T7 utogene-cont ining pl smid will
e kill
ed
y the toxicity gener ted
y the T7 utogene if ppropri te protective me sur
es re not t ken (1,4). Those clones th t m n ged to grow up h ve
een found to
h r
or mut ted nd in ctiv ted pl smids (unpu
lished o
serv tions). In order to
produce fully function l T7 utogene-cont ining pl smids, the T7 utogenecont in
ing pl smids h ve to
e tr nsformed into nd prop g ted in specific str in of
E. coli HMS174(DE3)pLysE which c n
e purch sed from Nov gen. This E. coli host
provides protective pl smid pLysE encoding T7 lysozyme protein th t inhi
its
T7 RNAP ctivity in the
cteri (12). Since the T7 lysozyme lso we kens the E
. coli cell mem
r ne, E. coli hosts with pLysE pl smid tend to lyse fter long p
eriod of sh king incu
tion. In order to produce high yield of T7
332

Sturtz et l.
utogene-cont ining pl smids in l rge sc le prep r tions, the sh king incu
tion
s were usu lly done t 2150 rpm, nd the incu
tion time should
e shorter th n
217 h (most often = 16 h). Although the repe ted pT7T7/T7HA injections gener ted
the nti-HA nti
odies t level comp r
le to th t gener ted
y direct vir l
infection (Fig. 2), the A
levels in e ch individu l injected mice re found mor
e v ri
le th n those in the virus infected mice (Fig. 2). One re son for this v
ri
ility is th t the mech nism for DNA upt ke
y the nim l cells is l rgely u
nknown. However, we found th t, to incre se consistency of gene expression level
s, 3 10 min room temper ture incu
tion of T7 DNA nd the T7 RNAP in PBS w s i
mport nt. Secondly, consistency should
e m int ined for injection site, depth,
nd speed. CTL ctivity specific to p55g g c n
e induced with the T7 v ccine sy
stem when the v ccine is delivered intr muscul rly, with or without c tionic l
ipid such s DOTAP. In contr st, DOTAP will
e strictly required for CTL priming
when pT7g g nd T7 RNAP re delivered i.p. into mice infected with vTF7-3
y th
e s me route. CTL response specific to p55g g c n
e induced
y even twocompon
ent T7 v ccine system: either (pT7g g + T7 RNAP), or (pT7g g + pT7T7) (5). Simil
r CTL response g inst HIV-1 gp120 w s o
served when pT7gp120 pl smid w s use
d in the T7 v ccine system (5). Reproduci
ility in tumor volumes seems to
e c
ritic l element in this kind of experiment. We suggest to get used to the intr d
erm l injections
y pr cticing on extr nim ls
efore the experiment
egins nd

y checking tumor volume v ri
ility there fter. A reli
le nesthesi is lso
key element of this ppro ch. Bec use mouse movements m y provoke DNA mish ndl
ing, nesthesi is recommended during DNA injections. The DNA/T7 RNAP pre
inding
must
e performed t room temper ture in order for the pre
inding to
e complet
ed in
out 10 min. Pre
inding on ice m y result in low expression r tes.
2.
3.
4.
5.
6.
References
1. Chen, X., Li, Y., Xiong, K., nd W gner, T. (1994) A self-initi ting euk ryot
ic tr nsient gene expression system
sed on cotr nsfection of
cterioph ge T7
RNA polymer se nd DNA vectors cont ining T7 utogene. Nucleic Acids Res. 22,
21142120. 2. Chen, X., Li, Y., Xiong, K., Xie, Y., Aizicovici, S., Snodgr ss, R.,
et l. (1995). A novel nonvir l cytopl smic gene expression system nd its impl
ic tions in c ncer gene ther py. C ncer Gene Ther. 2, 281289. 3. Dunn, J. J., Kri
ppl, B., Bernstein, K. E., Westph l, H., nd Studier, F. W. (1988) T rgeting
c
terioph ge T7 RNA polymer se to the m mm li n cell nucleus. Gene 68, 259266. 4. D
u
endorff, J. W. nd Studier, F. W. (1991) Controlling
s l expression in n in
duci
le T7 expression system
y
locking the t rget T7 promoter with l c repress
or. J. Mol. Biol. 219, 6168.
T7 Autogene System
333
5. Sel
y, M. J., Doe, B., nd W lker, C. M. (1997) Virus-specific cytotoxic T-ly
mphocyte ctivity elicited
y coimmuniz tion with hum n immunodeficiency virus t
ype-1 genes regul ted
y the
cterioph ge T7 promoter nd T7 RNA polymer se pro
tein. J. Virol. 71, 78277831. 6. Chen, X., Li, Y., Xiong, K., Aizicovici, S., Xie
, Y., Zhu, Q., et l. (1998) C ncer gene ther py
y direct injection of nonvir
l T7 vector encoding thymidine kin se gene. Hum. Gene Ther. 9, 729736. 7. Wint

er, G., Fields, S., nd Brownlee, G. (1981) Nucleotide sequence of the hem gglut
inin gene of hum n influenz virus H1 su
type. N ture 292, 7275. 8. C ton, A. J
., Brownlee, G. G., Yewdell, J. W., nd Gerh rd, W. (1982) The ntigenic structu
re of the influenz virus A/PR/8/34 hem gglutinin (H1 su
type). Cell 31, 417427.
9. Kunkel, T. A. (1985) R pid nd efficient site-specific mut genesis without ph
enotypic selection. Proc. N tl. Ac d. Sci. USA 82, 488492. 10. S nger, F., Nickle
n, S., nd Coulson, A. R. (1977) DNA sequencing with ch intermin ting inhi
itors
. Proc. N tl. Ac d. Sci. USA 74, 54635467. 11. Doe, B., Steimer, K. S., nd W lke
r, C. M. (1994) Induction of HIV-1 envelope (gp120)-specific cytotoxic T lymphoc
yte responses in mice
y recom
in nt CHO cell-derived gp120 is enh nced
y enzym
tic remov l of N-linked glyc n. Eur. J. Immunol. 24, 23692376. 12. Studier, F. W
. (1991) Use of
cterioph ge T7 lysozyme to improve n induci
le T7 expression
system. J. Mol. Biol. 219, 3744.
30
Immuniz tion with N ked DNA Coexpressing Antigen nd Cytokine vi IRES
Jochen Heinrich, Bettin Str ck, Mich el N wr th, Jov n P vlovic, nd K rin Moel
ling 1. Introduction Inocul tion of pl smid DNA vectors encoding immunogenic pro
teins induce humor l s well s cell-medi ted immune responses. Protection g in
st ch llenge with p thogens h s provided protective immunity in sever l inst nce
s in nim l models. (1,2). DNA v ccines llow the simult neous expression of nt
igens nd immune-stimul tory cytokines vi n intern l ri
osom l entry site (IRE
S). Here we descri
e the construction of DNA v ccine g inst m lign nt mel nom
s using: (i) the tumor- ssoci ted ntigen gp100 (or pmel17), known to
e over-e
xpressed in m ny m lign nt mel nom s (3,4), nd (ii) the gr nulocyte m croph ge
stimul ting f ctor (GM-CSF) which h s
een shown to h ve stimul tory effect on
humor l nd cellul r immune responses (5). Vectors for DNA immuniz tion in cl
inic l context should llow high levels of expression of the t rget protein in m
mm li n cells
ut should not
e
le to replic te or to integr te into the host
chromosome. Therefore vectors re not llowed to cont in: (i) origins of replic
tion for pl smids in m mm li n cells, e.g., from SV40 DNA,
ec use proteins th
t re
le to function lly repl ce the T ntigen of SV40 might
e ctive in the
recipient cells; (ii) retrovir l long termin l repe ts s promoter/enh ncer sequ
ences,
ec use they might le d to integr tion; nd (iii) n Ampicillin-resist nc
e gene for selection in
cteri ,
ec use this might confer resist nce to nti
i
otics th t re used ther peutic lly. Estim tes of integr tion frequencies indic
te
out 1 in 1016 per DNA molecule to ffect gene expression
y n integr tion e
vent. However 200 g of 6-k
DNA pl smid cont ins pproxim tely 3 1013 molecules
, which is therefore
out 1000-fold
elow the s fety m rgin (6).
From: Methods in Molecul r Medicine, vol. 29, DNA V ccines: Methods nd Protocol
s Edited
y: D. B. Lowrie nd R. G. Wh len Hum n Press Inc., Totow , NJ
335
336
Heinrich et l.
For DNA v ccines, we use the vector pVR1012 (Fig. 1A) which origin ted from Vic
l Co., S n Diego. pVR1012 is very simil r to the vector V1J constructed
y Montg
omery et l. (7),
ut cont ins the Km-resist nce gene inste d of the Ampicillin
resist nce gene for selection in
cteri . The origin of replic tion of the high
copy-num
er pl smid pUC18 in
cteri llows the production of l rge mounts of
pVR1012 DNA in Escherichi coli. The expression of the t rget gene is directed

y com
in tion of the hum n cytomeg lovirus (CMV)-immedi te-e rly (IE-1) promo
ter/enh ncer region, including the intron A sequence which incre ses mRNA st
il
ity, with the
ovine growth hormone (BGH) poly denyl tion sign l. This com
in ti
on turned out to
e very efficient for gene expression in mouse muscle cells (7,
8). In order to clone two genes in w y th t llows
oth genes to
e under the
control of the s me promoter, nd re tr nsl ted from
icistronic mRNA,
oth g

enes re cloned together with n intern l ri
osom l entry site (IRES) yielding t
he structure: promoter-gene A-IRES-gene B. For th t purpose pl smid pIRES 2 (Fig
. 1B) is used, which cont ins 650nt of the IRES of enceph lomyoc rditis virus (E
MCV) inserted into pBSKS deriv tive. At le st 450 nt of the IRES sequence re
required to form t the RNA level, the second ry structures th t encomp ss some
less-known recognition motifs. Only one of sever l st rt codons, n mely the 11th
ATG codon (Fig. 1B) in EMCV str in R is used for c p-independent initi tion of
tr nsl tion (9,10). Therefore it is import nt to insert the st rt codon of gene
B in this ATG codon. The origin l IRES sequence h s
een modified in order to cr
e te NcoI site CCATGG overl pping the 11th st rt codon, there
y le ding to inc
re sed initi tion of tr nsl tion due to the higher homology to the Koz k sequenc
e (11). Genes h r
oring corresponding NcoI site c n directly
e fused to the I
RES. However, usu lly the gene of interest does not possess such n NcoI site. I
n this c se either the NcoI site, or second restriction site recognized
y Bsm
FI loc lized 20 nucleotides upstre m of NcoI, c n
e used together with oligodeo
xynucleotide linkers, especi lly when the second mino cid of the gene of inter
est must not
e ch nged. Altern tively, n NcoI site overl pping the st rt codon
of the gene of interest might
e cre ted
y oligonucleotide directed mut genesi
s or
y PCR mplific tion using modified upstre m primer h r
oring the NcoI si
te. The construction of the DNA pl smids pVR1012.gp100, pVR1012.mGMCSF, nd pVR1
012.gp100-IRES-mGM-CSF for the expression of gp100/ pmel17, mGM-CSF nd of
oth
genes, respectively, is shown in Fig. 1C. In the Methods section we descri
e how
oligodeoxynucleotides re used in order to fuse correctly the IRES sequence wit
h the mGM-CSF sequence in the intermedi te pl smid pVR1012.IRES-mGM-CSF. For one
tumor model, K1735M2 mel nom cells re st
ly tr nsfected with pl smid pVR1012
.gp100 in
Immuniz tion with N ked DNA
337
Fig. 1. Construction of vectors for DNA v ccin tion. (A) Pl smid pVR1012 cont in
s the CMV immedi te e rly promoter, n intron A sequence, multicloning site, t
he termin tor of
ovine growth hormone (BGH Term.), nd k n mycin resist nce g
ene (KmR) on pUC 18 origin of replic tion. (B) Pl smid pIRES 2 is deriv tive
of pl smid pBSKS.IRES (kindly provided
y Silvio Hemmi), the multicloning sites
of which h ve
een modified to fit to the multicloning site of the retrovir l p
BABE vectors. It served s source of n IRES sequence th t origin ted from enc
eph lomyoc rditis virus (EMCV). (C) Construction of DNA v ccine pl smids. The hu
m n gp100/pmel17 cDNA nd the murine mGM-CSF cDNA were inserted into the S lI/ N
otI nd B mHI sites of the pVR1012 multiple cloning site, yielding pl smids pVR1
012.gp100 nd pVR1012.mGM-CSF, respectively. The intermedi te pl smid pVR1012.IR
ES-mGM-CSF results from the lig tion of the IRES sequence s NotI/ BsmFI-fr gm
ent nd the indic ted oligodeoxynucleotide linker into the BseRI/NotIdigested pV
R1012.mGM-CSF. This pl smid w s used to clone the gp100/pmel17 gene s S lI/No
tI fr gment in front of the IRES-mGM-CSF c ssette. The expression of gp100/pmel1
7 nd mGM-CSF is verified in cell-culture experiments
y indirect immunofluoresc
ence n lysis, using the monoclon l nti
ody HMB45 directed g inst gp100/pmel17
(kindly provided
y S. W gner, Essen, Germ ny [4]), nd commerci lly v il
l
e ELISA for mGM-CSF.
338
Heinrich et l.
order to overexpress gp100/pmel17. Mice re v ccin ted following est
lished pro
tocols (12)
y intr muscul r (i.m.) injection of 2 25 g of e ch pl smid DNA into
the right nd left qu driceps. Usu lly 510 nim ls per point re tested. The mice
re
oosted one time 3 wk fter the initi l immuniz tion. Tumor ch llenge is pe
rformed fter n ddition l 2 wk
y su
cut neous (s.c.) injection of 1 106 synge

neic mel nom cells: (i) the murine mel nom cellline B16-F0 nd its su
clone B1
6-F10,
oth expressing high levels of the murine homologue of gp100/pmel17, re
used in syngeneic C57B1/6 mice (13), nd (ii) K1735-M2/gp100+ cells re used in
C3H mice syngeneic to the p rent l K1735-M2 cells (14). 2. M teri ls 2.1. Gener
tion of the IRES-mGM-CSF Fusion Using n Oligodeoxynucleotide Linker
1. Pl smid DNAs: pVR1012.mGM-CSF, pIRES 2 (see Fig. 1). 2. Oligodeoxynucleotidelinkers re v il
le from Microsynth (B lg ch, Switzerl nd): (IRES-mGM-CSF) 5TTTGAAAAACACGATAATACCATGTGGCTG-3 (IRES-mGM-CSF reverse) 5-GCCACATGGTATTATCGTGT
TTTTC-3 3. 10X nne ling
uffer: 10 mM Tris-HCl pH 7.8, 10 mM MgCl2, 50 mM N Cl
. 4. T4-DNA lig se nd 5X lig tion
uffer re v il
le from Gi
co-BRL. 5. K n m
ycin sulf te: 40 mg/mL stock in H2O, sterile-filtered. Store t 20C. At 4C the solu
tion is st
le for sever l weeks. 6. St nd rd cloning m teri l: Restriction endo
nucle ses, 10X re ction
uffers, g rose nd low-melting g rose re v il
le f
rom m ny suppliers. A gel extr ction kit nd pl smid purific tion kits re v il

le from Qi gen (Ch tsworth, CA).
2.2. Cotr nsfection of K1735-M2 Cells
1. Pl smids: pVR1012.gp100 nd pX343(kindly provided
y H. Diggelm nn). 2. Murin
e m lign nt mel nom cells K1735-M2. 3. Stock cell culture re gents: Dul
eccos mo
dified E gle medium cont ining 0.11 g/L sodium-pyruv te nd pyridoxine (DMEM), 2
00 mM L-glut mine, penicillinstreptomycin cont ining 10,000 IU/mL penicillin nd
10,000 g/mL streptomycin re v il
le from Gi
co-BRL; fet l c lf serum (FCS) is
v il
le from Ser -Tech Zell
iologische Produkte Gm
H (Aiden
ch, Germ ny); tr
ypsin EDTA Solution B 0.25% in Pucks s line A is v il
le from Biologic l Indust
ries (Ki

utz Beit H emek, Isr el). 4. Lipofect mine re gent, 2 mg/mL is v il

le from Gi
co BRL. 5. Hygromycin B is v il
le from Boehringer M nnheim (M nnhe
im, Germ ny). 6. Growth medium: DMEM supplemented with 0.1 vol FCS, 0.01 vol glu
t mine, nd 0.01 vol penicillin/streptomycin. 7. Tr nsfection medium: DMEM suppl
emented with 0.01 vol glut mine. 8. Selection medium: growth medium supplemented
with 200 g/mL hygromycin.
Immuniz tion with N ked DNA 2.3. V ccin tion of Mice nd Tumor Ch llenge
339
1. Mice (68-wk-old with n v r ge
ody weight of 30 g). 2. Syngeneic tumor cell
line (here we used B16-F0, B16-F10 or K1735-M2/gp100+). 3. 1.1mg purified DNA of
e ch pl smid: pVR1012, pVR1012.gp100, pVR1012.mGMCSF, pVR1012.gp100-IRES-mGM-CS
F. 4. Dul
eccos PBS without c lcium nd m gnesium, nd without
ic r
on te, is v
il
le from Gi
co-BRL. 5. Metof ne is v il
le from M llinckrodt Veterin ry, I
nc. (Mundelein, IL). 6. 1-litre vessel, or simil r size, for n rcotizing mice. 7
. P per towels. 8. Precision Glide 0.5-mL tu
erculin syringes with 27G1/2 needle
s for i.m. injection of DNA re v il
le from Becton Dickinson (Fr nklin L kes,
NJ). 9. 1-mL syringes (Pl stip k) with 25G5/8 needles (Microl nce 3) for s.c. i
njection of tumor cells re v il
le from Becton Dickinson. 10. Neu
uer cell-c
ounting ch m
er.
3. Methods 3.1. Gener tion of the IRES-mGM-CSF Fusion Using n Oligodeoxynucleot
ide Linker
1. For the vector DNA, digest 5 g pVR1012mGM-CSF with BseRI nd NotI, nd for the
insert DNA, digest 5 g of the pl smid pIRES2 with BsmFI ( t 65C) nd NotI (see No
te 1). Do not dephosphoryl te the vector (see Note 2). Sep r te the DNA fr gment
s
y 1.2% g rose-gelelectrophoresis, using low-melting g rose nd purify the v
ector- nd insert-fr gments using the QIAquick gel extr ction kit. Elute the DNA
with 30 L of twice-distilled H2O (see Note 3). 2. For the nne ling, mix
oth ol
igodeoxynucleotide linkers (see Note 4) to fin l concentr tion of 10 M e ch in
20 L 1X nne ling
uffer. Den ture for 3 min t 95C nd then let equili
r te to RT
during 2 min to 3 h (see Note 5). 3. Mix 2.5 L vector pVR1012mGM-CSF(BseRI/NotI)
with 5 L NotI/BsmFI-fr gment (pIRES2), 0.5 L of the nne led oligodeoxynucleotide
-linkers, 3 L 5X lig tion
uffer, nd 1 L T4-DNA lig se to fin l volume of 15 L,

 nd incu
te overnight t 16C for lig tion (see Note 6). 4. Tr nsform competent

cteri nd select tr nsform nts on LB-pl tes cont ining 50 g/mL k n mycin. From
10 single colonies, grow 2.5 mL over night cultures. Use 1.5 mL for n lytic l p
rep r tions of the pl smid DNA nd test for correct recom
in tion
y restriction
n lysis. Use the rem ining 1 mL to inocul te over night cultures for glycerol
stocks nd prep r tion of the pl smid DNA. Confirm the structure of the pl smid

y dideoxy sequencing.
3.2. Cotr nsfection of K1735-M2 Cells
1. Pl te K1735-M2 cells in growth medium on 10cm dish nd grow them for 2 d t
37C, in n tmosphere of 5% CO2 nd 90% humidity, so th t the cells will h ve
confluency of 70% t the time of tr nsfection (see Note 7).
340
Heinrich et l.
2. Prep re: (i) in 1.5-mL Eppendorf tu
e DNA solution m de
y mixing 5 g pVR1
012.gp100, 3 g pX343 (see Note 8), nd 800 L tr nsfection medium; (ii) in 2.2-mL
Eppendorf tu
e prep re lipofect mine solution
y mixing 30 L lipofect mine nd
800 L tr nsfection medium; nd (iii) pl ce 6.4 mL of tr nsfection medium in 15
-mL F lcon tu
e. 3. Pipet the DNA solution into the lipofect mine solution, mix

y inverting the tu
e sever l times, then let the tu
e sit t room temper ture f
or 45 min.. 4. Pipet the DNA/lipofect mine mixture with serologic l 2-mL pipet
into the F lcon tu
e nd mix gently
y inverting the tu
e three to four times.
5. Remove the growth medium from the cells nd c refully pipet or pour the tr ns
fection mixture onto the cells. 6. Incu
te the cells for 16 h. 7. Repl ce the t
r nsfection mixture with growth medium nd incu
te the cells for ddition l 24
h. 8. Split the cells 1:10 on nine 10-cm dishes nd one 6-cm dish nd let them g
row for 1216 h (see Note 9). 9. Use the 6-cm dish to test for the expression of g
p100/pmel17
y immunofluorescence n lysis using the monoclon l nti
ody HMB45 (
see Note 10). 10. Use the 10-cm dishes for the selection of st
ly tr nsfected c
ells s follows. Repl ce the growth medium
y selection medium. After pproxim t
ely 2 d, m ssive cell de th occurs. As long s m ny cells die nd det ch from th
e surf ce, ch nge the medium every d y. After sever l d ys, the num
er of dying
cells decre ses. Then ch nge the medium every 3 or 4 d until the surviving cells
form visi
le colonies nd cells no longer die. 11. Use sterile sc lpel to scr
tch single colonies long with thin l yer of the pl stic from the surf ce of
the dish nd tr nsfer e ch into well of 24-well pl te th t h s
een pre-fill
ed with 1 mL of selection medium. Pipet the content of e ch well 56 times with
p steur pipet up nd down in order to rinse cells from the surf ce of the tr nsf
erred pl stic (see Note 11). 12. Let the cells grow in selection medium until th
ey c n
e split for: (i) further cultiv tion; (ii) n lysis of the protein expre
ssion; (iii) cryo-conserv tion of cells expressing the protein; nd (iv) tumor c
h llenge.
3.3. V ccin tion of Mice nd Tumor Ch llenge
1. When v ccin ting, for e ch 10 nim ls, prep re DNA solutions
y mixing 550 g p
l smid DNA with PBS to fin l volume of 550 L. 2. Prep re to n rcotize the mice

y putting 35 drops of metof ne on p per towel in suit
le vessel (see Note 1
2). Pl ce second dry p per towel on top of the first one in order to protect t
he mice from direct cont ct to metof ne. Cover the vessel with lid to gener te
s tur ted tmosphere,
ut llow ir supply. 3. Set one mouse into the vessel
for few min until it is n esthesized. This is when the mouse no longer moves
nd
re thing
ecomes c lm. Then t ke the mouse out nd l y it on its
ck.
Immuniz tion with N ked DNA
341

4. Inject 25 L of the DNA solution into the left, nd 25 L into the right qu drice
ps muscle (see Note 13). 5. After injection, m rk the nim l, nd proceed with t
he next mouse with steps 24. 6. 21 d fter the initi l immuniz tion,
oost the mi
ce once
y repe ting steps 15. 7. After further 14 d, use the mice for tumor ch
llenge. 8. Grow K1735/pVR1012.gp100 cells on 10-cm dishes, so th t they will h
ve confluency of pproxim tely 90% (12 107 cells/10-cm dish) t the d y of the
tumor ch llenge. Remove the growth medium nd w sh the cells once with 1.5 mL of
trypsin EDTA solution for few sec. Incu
te the pl te for 3 min. t 37C nd re
suspend the cells in 5 mL of growth medium
y pipetting up nd down 510 times. Co
unt the cells using Neu
uer ch m
er. 9. Centrifuge the cell suspension for 5
min t 150g. Aspir te the supern t nt nd resuspend the cells in 5mL DMEM. Adjus
t the suspension to fin l cell density of 3.3 106/mL. Store the cells on ice f
or up to 2 h. 10. An esthetize mice s descri
ed
ove nd inject 300L of the cel
l suspension (1 106 cells) su
cut nously in the right fl nk using 1mL syringe
with 25 g uge needle (see Note 14). 11. Monitor the growth of tumors
y determ
ining the tumor volume (16) every fifth d y. Me sure the l rgest di meter (d1)
nd the perpendicul r di meter (d2) nd c lcul te the tumor volume, ccording to
the formul V = d1 d22 0.5.
4. Notes
1. The oligodeoxynucleotides re designed to cre te the fusion
etween the BsmFI
gener ted 3end of IRES nd unique BseRI site in pVR1012.mGM-CSF loc lized t
the extreme 5-end of the mGM-CSF coding sequence (see Fig. 1). This llows the
lig tion of the IRES sequence s NotI/BsmFI-fr gment into BseRI/ NotI-digested
pVR1012.mGM-CSF vi the linker which is comp ti
le to the ends gener ted
y Bsm
FI nd BseRI. 2. The oligonucleotides re not phosphoryl ted, to void multimeri
z tion th t might
e difficult to detect
y restriction n lysis of the recom
in
nt pl smid products. Therefore, the vector must not
e dephosphoryl ted. In ord
er to limit oligomeriz tion of vector- nd insert-DNA-fr gments, n excess of ol
igodeoxynucleotides is used in the lig tion re ction, which cont ins vector:inse
rt:linker in mol r r tio of pprox 1:3:40. The mol rity of the purified fr gme
nts is c lcul ted ccording to the formul : x = pl smid [g]M/(elution volume [L] p
l smid length [
p] 660), e.g., here for the vector, x = 47nM. 3. We usu lly chec
k for the presence of the correct DNA fr gments fter their purific tion
y su
j
ecting 0.10.2 vol of the eluted DNA to g rose-gel electrophoresis. The concentr
tions of the fr gments c n
e roughly estim ted from the intensities of the
nd
s in order to set up the lig tion with correct r tio of vector nd insert. 4.
In the c se of longer oligonucleotides (>30 nucleotides), it is recommended to u
se gel-purified oligodeoxynucleotides.
342
Heinrich et l.
5. The nne ling c n
e done in sever l w ys. Usu lly the oligodeoxynucleotides
re den tured in w ter
th t 9095 nd then the tu
e is tr nsfered together with
1020 mL of the hot w ter into sm ll vessel to cool slowly down during 1015 min.
Altern tively, thermo
lock might
e switched off to cool slowly for
out 3 h
. The
uffer conditions re not stringent. For the nne ling, some s lt should

e included,
ut void using high s lt concentr tions, since T4 DNA lig se requir
es low s lt conditions. 6. The lig tion c n
e monitored
y g rose gel electrop
horesis. For sm ll insert DNA fr gments, the lig tion products re slightly shif
ted, comp red to nonlig ted control fr gment. Altern tively, it is possi
le to
set up positive control with ll components,
ut using 0.5 g of DNA-fr gments
th t h ve ends th t re non-comp ti
le to the linker (e.g., X174-HaeIII blunt-end
ed fragments), and to test for ligated products. urthermore, the incorporation
of the linker is often indicated by the inhibition of the re-ligation of DNA fra
gments compared to a control without linker. 7. In our experience, the efficacy
of transfection depends highly on the confluency of the cells, which should be 6
080%. urthermore transfection might be more efficient when cells have grown for

2 d rather than only overnight. It is a good idea to test the efficacy of transf
ection using a reporter plasmid, expressing for example LacZ, in order to optimi
ze the transfection procedure. 8. Plasmid pVR1012.gp100 does not confer any resi
stance to mammalian cells. Therefore a cotransfection is performed using plasmid
pX343, which harbors a hygromycin B resistance gene under the control of the SV
40 early promoter, to allow the selection of stably transfected cells. It is imp
ortant to determine the sensitivity of the cells against hygromycin B before tra
nsfection. This is done by growing the cells in 6 wells of a 6-well plate to a c
onfluency of 80%. Subsequently the growth medium is changed and hygromycin B is
added to wells in a range of concentrations, e.g., 20, 50, 100, 200, 400, 800 g/m
L. After a lag phase of 12 d, cells will die dependent on the concentration of hy
gromycin B in the growth medium. or the selection of transfectants use the lowe
st concentration of hygromycin B that causes complete elimination of all non-tra
nsfected cells. 9. The cells are split 1:10 in order to have single cells instea
d of a compact cell layer (since single cells are more sensitive to hygromycin B
), and to limit the final number of colonies on one dish. When the cells are not
split, the selection procedure will last longer and might select for resistance
to hygromycin in some nontransfected cells. It can be helpful to make a range o
f cell dilutions, e.g., 1:20, 1:10, 1:5, 1:2.5. 10. Protein expression should be
detected by immunofluorescence analysis in order to estimate the ratio of trans
fected and non-transfected cells. The result allows estimation of how many cells
will die or survive during the selection procedure. 11. There are several metho
ds for subcloning described in the literature, but this method worked best in ou
r hands. Make certain that the scalpels and pipets have equilibrated to room tem
perature before use. Additionally, the cells of one colony can be trypsinized by
putting a 25 L spot of trypsin EDTA solution for 2060 s
Immunization with Naked DNA
343
onto the colony immediately before it is transfered to the 24-well plate. Usuall
y a fraction of the colonies will not grow further after the transfer procedure.
Therefore it is recommended to pick about 50 colonies to finally have at least
10 independent clones. 12. A simple way to narcotize the mice is to use a 1-L ve
ssel, which can be covered by aluminium foil. However, a glass cylinder (depth =
10 cm, height = 10 cm) with a glass lid is more comfortable. Use 1 drop of meto
fane for approximately 200 cm3. When the vessel stays covered, every 10 min add
the same amount of metofane to maintain the saturated atmosphere. 13. At the fle
xed hind leg, start to prick directly above the patella parallel to the femur an
d penetrate through the whole muscle. Then start to inject. During the injection
, very slowly pull the syringe back in order to distribute the DNA along the who
le muscle. Repeat this procedure with the other quadriceps. 14. or s.c. injecti
on, take the skin with two fingers and lift it up. The needle is directed parall
el to the surface of the body so that it penetrates the lifted skin. Stop penetr
ating after resistance is lost. Then inject the cell suspension all at once (bol
us injection). Transiently (35 min), the injected suspension forms a visible bolu
s under the skin.
References
1. Hassett, D. E. and Whitton, J. L. (1996) DNA immunization. Trends in Microbio
l. 4, 307312. 2. Ulmer, J. B., Sadoff, J. C., and Liu, M. A. (1996) DNA vaccines.
Curr. Opin. in Immunol. 8, 531536. 3. Wagner, S. N., Wagner, C., Hfler, H., Atkin
son, M. J., and Goos, M. (1995) Expression cloning of the cDNA encoding a melano
ma-associated Ag recognized by mAb HMB-45Identification as melanocyte-specific pm
el17 cDNA. Lab. Invest. 73, 229235. 4. Moelling, K., Strack, B., Nawrath, M., Hei
nrich, J., Dhring, C., Wagner, S. N., and Pavlovic, J. (1997) Development of a DN
A vaccine against malignant melanoma, in Strategies for Immunointerventions in D
ermatology (Burg, G. and Dummer, R., eds.), Springer Verlag, pp. 195206. 5. Ziang
, Z. and Ertl, H. C. (1995) Manipulation of the immune response to a plasmid-enc

oded viral antigen by coinoculation with plasmids expressing cytokines. Immunity

2, 129135. 6. Nichols, W. W., Ledwith, B. J., Manam, S. V., and Troilo, P. J. (1
995) Potential DNA vacchine integration into host cell genome, in Annals of the
New York Academy of Sciences 772 (Liu, M. A., Hillemann, M. R., and Kurth, R., e
ds.), pp 3038 7. Montgomery, D. L., Shiver, J. W., Leander, K. R., Perry, H. C.,
riedman, A., Martinez, D., et al. (1993) Heterologous and homologous protection
against influenza a by DNA vaccination: Optimization of DNA Vectors. DNA Cell B
iol. 9, 777783. 8. Manthorpe, M., Cornefert-Jensen, ., Hartikka, J., elgner, J.
, Rundell, A., Margalith, M., and Dwarki, V. (1993) Gene therapy by intramuscula
r injection
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9. 10. 11. 12. 13. 14.
of plasmid DNA: Studies on firefly luciferase gene expression in mice. Hum. Gene
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by internal ribosomal entry. Curr. Top. Microbiol. Immunol. 203, 3163. Kozak, M.
(1984) Compilation and analysis of sequences upstream from the translational sta
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31
Genetic Subunit Vaccines
A Novel Approach for Genetic Immunization
I. rank Ciernik and David P. Carbone 1. Introduction Induction of epitope-speci
fic cytotoxic T lymphocyte (CTL) responses can improve the outcome of disease in
a variety of model systems. However, natural immunity or that arising by immuni
zation with whole proteins or organisms with multiple potential epitopes, may no
t always result in the desired effect. or example, antigenic variation of CTL e
pitopes can result in immune escape in HIV disease (1,2), and malaria (3). This
could potentially be avoided if it were possible to direct the immunity to selec
ted regions of proteins that are highly conserved and required for function. The
immunization with specific CTL epitopes rather than whole proteins also avoids
induction of unwanted responses such as enhancing antibodies in HIV, which may a
ctually promote infection (4). By analogy to the use of purified proteins and mi
xtures of proteins, genetic immunization has been shown to allow induction of ce
llular as well humoral immune responses (5,6). There is increasing evidence that
genetic immunization might be even more efficient than regular immune approache
s using protein in adjuvant (7). We have explored the ability of genetic immuniz
ation to specifically target defined CTL epitopes. Such epitope-targeted immunit
y may be especially desirable when the epitope is altered self, as when targeting
the mutant site of endogenous oncoproteins. The ability to direct a cellular imm
une response to a defined epitope allows increased precision in studies of cellm
ediated immunity and immunotherapy. Synthetic peptides are a useful approach to
the induction of epitope-specific immunity. The use of peptides as immunogens is
complicated, however, by
rom: Methods in Molecular Medicine, vol. 29, DNA Vaccines: Methods and Protocol
s Edited by: D. B. Lowrie and R. G. Whalen Humana Press Inc., Totowa, NJ
345

346
Ciernik and Carbone
their weak inherent immunogenicity and variable chemical and physical properties
. Some peptides are nearly impossible to synthesize for chemical reasons. A vari
ety of strategies have been employed to enhance the immunological efficacy of pe
ptide-based vaccines, including chemical, bacterial, and mammalian cell-based ad
juvants. The chemical and physical problems of protein or peptide-based vaccines
can be avoided by the use of genetic vaccines. Several studies have shown that pl
asmid DNA expression vectors encoding the entire cloned open reading frame of pr
oteins may generate substantial humoral and cellular immunity when directly intr
oduced into living animals (5,811). Strategies for increasing the efficacy of gen
etic immunization are in constant demand. or instance, the presence of immunomo
dulatory sequences, such as CpG sequences, in the expression vector may dramatic
ally improve immunogenicity (12). Others have observed enhanced effective immuni
ty using multigene DNA immunization (13). Additional co-expression with immunomo
dulatory cytokines may further increase the efficacy of genetic vaccines (14). W
e have designed a vector using the adenovirus E3 leader sequence, which facilita
tes the transport of a single epitope encoded in a short open reading frame into
the endoplasmic reticulum (ER). This leader is designed to enhance the concentr
ation of the peptide in the ER and thus binding to class I MHC molecules, and to
bypass the need for transport by the TAP transporter. We show that this leader
sequence can improve CTL induction and lead to enhanced protective immunity. ig
. 1 indicates the strategy used to obtain the appropriate expression vector cont
aining a short open reading frame driven by a CMV promoter coding for a model pe
ptide derived from mutant p53. 2. Materials
1. Special equipment: Gene Pulser, available from Bio-Rad (Hercules, CA); PE Bio
systems (oster City, CA) 430A peptide synthesizer; harvesting system, available
from Skatron (Lier, Norway); gamma counter, available from LKB (Gaithersburg, M
D). 2. Purchase female BALB/c mice (age: 1016 wk) from Harlan Inc. (Indianapolis,
IN). 3. Obtain BALB/c 3T3 (H2d) fibroblasts and DBA/2 P815 (H2d) mastocytoma ce
lls from ATCC (Rockville, MD). Grow cells in flasks with 10% (v/v) CS in RPMI 1
630 medium supplemented with gentamycin and penicillin in a 5% CO2 incubator at
37C. 4. Obtain the pRc/CMV expression vector from Invitrogen (San Diego, CA). Thi
s 5.4 Kb plasmid includes a multiple cloning site flanked by a T7 and SP6 promot
er. A human cytomegalovirus promoter/enhancer element allows expression of the c
loned sequence in mammalian cells. 5. DH5 competent cells re v il
le from Life
Technologies, Inc. (G ithers
urg, MD) (C t. No. 18265-017).
Genetic Su
unit V ccines
347
Fig. 1. Structure of the whole open re ding fr me nd genetic epitope expression
vectors. The c ssette vector llows convenient insertion of synthetic oligonucl
eotide sequences in-fr me with n ER t rgeting sequence in n expression vector.
See Su
he ding 3 for det ils of construction. 6. Oligonucleotides re purch sed
from vendors speci lizing in their synthesis (see Note 1). 7. Prep re synthetic
peptides using t-Boc solid-ph se peptide synthesis on n peptide synthesizer. C
ustom m de peptides c n lso
e o
t ined
y commerci l me ns. Store the peptides
t 70C fter dilution to 100 M fin l concentr tion in RPMI 1640. 8. O
t in monocl
on l nti
ody 536.7 (r t nti-mouse CD8 IgG2 ) nd H129.19 (r t nti-mouse CD4 Ig
G2 ) from Boehringer M nnheim Biochemic ls (Indi n polis, IN). 9. O
t in R t-T S
tim l cking conc n v lin A from Coll
or tive Biomedic l Products (Bedford, MA).
348
3. Methods 3.1. Gene Construction
Ciernik nd C r
one

1. Amplify the full open re ding fr me of the p53 mRNA
y cDNA PCR using RNA fro
m the hum n non-sm ll cell lung c ncer T1272. The mino cid sequence of this pr
otein cont ins the point mut tion C to Y t position 135. 2. Clone the cDNA
etw
een HindIII nd X
I sites in the m mm li n expression vector pRc/CMV using st n
d rd m teri ls nd molecul r
iology techniques. 3. Sequence the full open re di
ng fr me in
oth directions to exclude rtif ctu l polymer se ch in re ction-der
ived mut tions.
3.2. Construction of the Minigene Expression Vector
1. Verify the purity of the oligonucleotides on convention l sequencing gel (s
ee Note 2). 2. The oligonucleotide AGCTA TGAGG TACAT GATTT TAGGC TTGCT CGCCC TTG
CG GCAGTC TGCAG CGC coding for the E3 le der sequence of Adenovirus (including t
he ATG st rt codon) is inserted
etween the restriction sites HindIII nd NotI.
Then the oligonucleotide GGCCC TCAAC AAGAT GTTTT ACCAA CTGGC CAAGA CCTGC CCTGT G
CAGCT coding for the mino cids 128145 of p53, with tyrosine t codon 135, is
inserted
etween NotI nd X
I following the E3 le der sequence, or is inserted
s AGCTA TGCTC AACAA GATGT TTTAC CAACT GGCCA AGACC TGCCC TGT GCAGCT into pRc/CMV

etween HindIII nd X
I without the E3 le der sequence. The vector X
I site g
ener tes n engineered stop codon. 3. As control, insert the oligonucleotide G
GCCC TCAAC AAGAT GTTTT GCCAA CTGGC CAAGA CCTGC CCTGT GCAGCT from mino cids 1281
45 of p53 coding for the wild-type sequence
etween NotI nd X
I following the
E3 le der sequence. 4. Insert scr m
led Adenovirus E3-le der sequence AGCTA TG
TGC CTTGC AAGGG GCGCC TACGC GATGT TGATT AGCTT AGCAG TCGC
etween the restriction
sites HindIII nd NotI, followed
y the oligonucleotide coding for the mino c
ids 128145 of p53, with tyrosine t codon 135
etween NotI nd X
I. 5. Insert
the sequence coding for the T cell epitope p18IIIB of HIV gp160
etween HindIII
nd X
I, without the E3 le der sequence (AGTCA TGCGT ATACA ACGCG GGCAA GGTCG CG
CGT TCGTA ACGAT AGGTA AGTAG), or following the Adenovirus E3 le der sequence (GG
CCC GTATA CAACG CGGAC CAGGT CGCGC TTTCG TAACG ATAGG TAAG TAG). 6. After lig tion
, tr nsform the vectors into DH5 competent cells.
3.3. Gener tion of Tumor Cells
1. Grow BALB/c 3T3 fi
ro
l sts nd DBA/2 P815 in RPMI 1640 medium supplemented w
ith 10% (v/v) FCS, gent mycin nd penicillin in 5% CO2 incu
tor t 37C.
Genetic Su
unit V ccines
349
2. Suspend P815 cells in serum-free RPMI 1640. Electropor te the pl smid DNA int
o the cells using Bio-R d Gene Pulser using conditions recommended
y the m nu
f cturer. 3. Grow the cells under G418 selection (600 g/mL) nd use the surviving
, st
ly tr nsfected cells for experiments.
3.4. Construction of the Genetic V ccine
1. Construct expression vectors coding only for the p53 or HIV epitopes with or
without the E3 le der sequence
y lig ting dou
le-str nded oligonucleotides into
the pRc/CMV vectors. 2. Gener te the dou
le-str nded oligonucleotides
y nne l
ing the oligonucleotides with their complements with longer or shorter ends s n
eeded to gener te the ppropri te restriction site overh ng (5 HindIII or NotI
nd 3 NotI or X
I). See Fig. 1. 3. Digest 1 L of stock of vector t concentr
tion of 1 mg/mL with HindIII nd NotI in re ction volume of 10 L t 37C for 1 h.
Use 13 L in the lig tion re ction. 4. Determine the concentr tion of dou
le-str n
ded oligonucleotide using the
sorption of UV light t w velength of 260 (OD2
60). The extinction coefficient needs to
e djusted for the length of the oligo
nucleotide in question. 5. Dissolve the complement ry oligonucleotides in TE
uf
fer (pH 8.0) nd nne l t mol r r tio of 1:1 in w ter
th gr du lly coolin
g from 60C to 4C. 6. Test for proper nne ling on high percent ge g rose gel. 7
. Lig te the nne led oligonucleotide into the digested vector under conditions

recommended
y the m nuf cturer. 8. Th w competent cells on wet ice. Add 1 L of t

he lig tion re ction to 50100 L of cells nd incu
te on ice for 30 min. He t-shoc
k the cells for 2060 s t 3742. Put them
ck on ice for 2 min. Add
cteri l growt
h medium, such s TB or LB without Ampicillin nd sh ke the culture gently for 1
h t 37C. Pl te the cells on mpicillin-cont ining pl tes overnight. 9. Pick 612
colonies nd m ke minipreps of pl smid DNA. 10. Perform restriction digest usi
ng restriction enzymes th t cut in the insert nd not in the origin l vector to
screen for c ndid te clones. 11. Completely sequence ll of the inserts to verif
y their structure (see Note 3). Use the UBS sequencing kit. Den ture the pl smid
nd nne l with T7 nd SP6 primers; sequence in
oth directions. Perform sequen
cing on n 8% cryl mide gel. Use CsCl gr dient for pl smid purific tion. Alte
rn tively, use Qi gen columns. Both methods yield pl smid suit
le for genetic i
mmuniz tion.
3.5. Gene Gun-
sed Immuniz tion
1. Prep re the DNA for
llistic gene tr nsfer (see Note 4) s follows. Suspend
2 mg of gold
e ds in H2O to o
t in concentr tion of 2 mg/mL nd store t 20C in
1.5 mL Eppendorf tu
es.
350
Ciernik nd C r
one
2. Att ch the DNA to micron-sized gold p rticles with c lcium nd spermidine (10
g DNA / 5 L gold
e d suspension t 2 g microp rticles per L). Dilute DNA with 2.5
M C Cl2 to yield fin l concentr tion of t le st 1 M C Cl2. Att ch DNA to the
gold p rticles with 1/10 of the volume 1 M spermidine (Sigm , St. Louis, MO) (se
e Note 5). 3. W sh the pl smid DNA nd gold p rticles with 70% eth nol first nd
g in with 100% eth nol in volume of 0.5 mL. 4. Resuspend the gold
e ds in 4
0 L of 100% eth nol nd lo d 8 L onto m croc rrier th t is ppropri te for the g
un used (K pt n disk). The m croc rrier is thin pl stic disk onto which the DN
A-lo ded gold p rticles re co ted. Prep re five disks for e ch mouse to
e v cc
in ted. 5. Lo d the disk onto the gene gun nd shoot t 12002000 psi into the dor
s l p rt of the e rs of nesthetized mice.
3.6. Restimul tion of CTL
1. Use peptide T1272 (TSPALNKMFYQLAKTCPVQL) for in vitro restimul tion of effect
or cells t concentr tion of 1 to 5 M (see Note 6). 2. Pulse t rget cells with
peptide V10 (FYQLAKTCPV) for CTL ss ys. 3. Use the wild-type counterp rt (wtV10
) (FCQLAKTCPV) or peptide VSV-8 (RGYVYQGL) s controls for the pulsing of t rget
cells. 4. Restimul te 3 106 immune spleen cells in vitro for 56 d three wk fter
immunizing mice in 50:50 mixture of IMDM nd E gle-H nks mino cid medium co
nt ining 10% (v/v) fet l c lf serum, 2 mM L-glut mine, 5 105 M -merc ptoeth nol,
nd nti
iotics. 5. Restimul te with or without T1272 peptide (5 M) in 10% R t-T S
tim l cking conc n v lin A.
3.7. CTL Phenotype nd MHC Restriction
1. Use monoclon l r t nti-mouse CD8 nd r t nti-mouse CD4 t 30 g/mL in the pre
sence of 1:5 diluted r

it complement for 1 h t 37C. 2. For the CTL ss y, puls
e t rget cells with peptide V10. 3. As controls for the pulsing of t rget cells,
use the wild-type counterp rt wtV10 or peptide VSV-8. 4. Incu
te peptide t 1 M
with 1 to 5 106 cells for 1 h t 37C. W sh un
ound peptide twice using RPMI 1640
with 2% FCS in polyethylene 15 mL
lue-c p tu
es. 5. Spin t 1200200 rpm for 510
min.
3.8. CTL Ass y
1. Use round-
ottom 96-well pl tes with covers. 2. Pl te cells in volume of 10
0 L in duplic tes t different concentr tions, st rting with concentr tion of 1
06 cells /mL. Seri lly diluted t le st four times with n equ l volume of cellfree medium. 3. L
el t rget cells with 51Cr in sm ll volume, e.g., 106 cells

in 0.3 mL, in order to llow dequ te upt ke of 51Cr. Use 15 mL
lue-c p F lcon
tu
es. Slight sh kGenetic Su
unit V ccines
351
4. 5. 6. 7. 8.
ing during incu
tion t 37C is prefer
le,
ut regul r resuspension of the cells
every 1020 min is dequ te. Incu
te for 12 h. Remove un
ound r dio ctivity
y w
shing with RPMI 1640 cont ining 2% (v/v) FCS. Bring t rgets to concentr tion o
f 104/mL nd overl y the t rget cell suspension onto CTL lo ded wells in 100 L. T
o llow optim l cont ct
etween t rget cells nd effector cells, spin the pl tes
t room temper ture t 1500g in t
letop centrifuge. Incu
te the pl tes t 3
7C for 46 h. H rvest the supern t nt with Sk tron H rvesting System. Count the r
dio ctivity in g mm counter. An lyze e ch nim l independently. Aver ge the
results within groups nd determine the st nd rd error. C lcul te percent specif
ic lysis using the equ tion: 100 [(experiment l rele se spont neous rele se) / (
m xim l rele se spont neous rele se)].
3.9. Tumor Protection
1. Immunize mice with 20 g of DNA on the
ck of their e rs (e ch e r, 10 g) using
the
llistic pp r tus. 2. Ch llenge mice with 9 105 P815 m stocytom cells ex
pressing minigene coding for the ppropri te T-cell epitope. 3. For tumor prot
ection experiments with the HIV-gp160 p18IIIB epitope, use fi
ros rcom s express
ing gp160. Gener te these cells
y tr nsfection of murine fi
ro
l sts th t endog
enously express gp160 of isol te IIIB with EJ-r s nd c-myc (15). These cells ex
press HIV gp160 nd h ve
een shown to signific ntly present the epitope to p18I
IIB specific CTL (16). 4. Immunize mice s descri
ed
ove for protection experi
ments t rgeting the mut nt p53 epitope nd ch llenge with su
cut neous tumor 14
d l ter. Me sure tumor-protective immunity nd p53-specific CTL (17).
4. Notes
1. When designing oligonucleotide encoding the desired epitopes, keep in mind th
e redund ncy of the genetic code. You c n design restriction site without ch n
ging the tr nsl tion product. 2. Before using the oligonucleotide, it should
e
run on high percent ge g rose gel to
etter estim te the purity, or prefer
l
y n 8% cryl mide gel. 3. Ch r cteriz tion of the constructs should
e done wit
h 23 restriction enzymes
efore sequencing to s ve time. 4. We use custom-
uilt
helium-driven
llistic device. The K pt n disk lo ded with the DNA-co ted micr
op rticles is pl ced in v cuum ch m
er f cing stopping screen. The skin of t
he mouse se ls n outlet. A mem
r ne restr ins pressure
uilt up with helium g
inst the v cuum ch m
er. The mem
r ne is su
sequently ruptured
y l nce nd l
lows the disk to
e propelled tow rd the t rget tissue
y the resulting shock w
ve. The stopping screen stops the disk while the microp rticles continue to pene
tr te the tissue. Commerci l units work on simil r principles.
352
Ciernik nd C r
one
5. Addition of the spermidine is done while keeping the suspension of gold p rti
cles on the vortex in order to prevent ggreg tion of the
e ds. 6. Avoid peptid
e concentr tions over 5 M for restimul tion. Cert in peptides re only slightly s
olu
le t low or high pH m king them difficult to h ndle nd potenti lly toxic t
o cells.
References
1. Rowl nd-Jones, S. L., Phillips, R. E., Nixon, D. F., Gotch, F. M., Edw rds, J

. P., Ogunlesi, A. O., et l. (1992) Hum n immunodeficiency virus v ri nts th t

esc pe cytotoxic T-cell recognition. AIDS Res. Hum. Retroviruses 8, 13531354. 2.
Phillips, R. E. nd McMich el, A. J. (1993) How does the HIV esc pe cytotoxic T
cell immunity? Chem. Immunol. 56, 150164,. 3. Udh y kum r, V., Shi, Y. P., Kum r,
S., Jue, D. L., Wohlhueter, R. M. nd L l, A. A. (1994) Antigenic diversity in
the circumsporozoite protein of Pl smodium f lcip rum
rog tes cytotoxic-T-cell
recognition. Infect. Immun. 62, 14101413. 4. Levy, J. A. (1993) P thogenesis of
Hum n Immunodeficiency Virus Infection. Micro
iol. Rev. 57, 183289. 5. T ng, D.,
DeVit, M., nd Johnston, S. A. (1992) Genetic immuniz tion: simple method for
eliciting n immune response. N ture 356, 152154. 6. B rry, M. A., L i, W. C., n
d Johnston, S.A. (1995) Protection g inst mycopl sm infection using expression
li
r ry immuniz tion. N ture 377, 632635. 7. Donnelly, J. J., Friedm n, A., M rt
inez, D., Montgomery, D. L., Shiver, J. W., Motzel, S. L., et l. (1995) A. Prec
linic l effic cy of prototype DNA v ccine: Enh nced protection g inst ntigen
ic drift in influenz virus. N t. Med. 1, 583587. 8. Ulmer, J. B., Donnelly, J. J
., P rker, S. E., Rhodes, G. H., Feigner, P. L., Dw rki, V. J., et l. (1993) He
terologous protection g inst influenz
y injection of DNA encoding vir l pro
tein. Science 259, 17451749. 9. Eisen
r un, M. D., Heyden
urg Fuller, D., nd H y
nes, J. R. (1993) Ex min tion of p r meters ffecting the eliction of humor l im
mune response
y p rticle
om
rdment-medi ted genetic immuniz tion. DNA Cell Bi
ol. 12, 791797. 10. W ng, B., Ugen, K. E., Srik nt n, V., Ag dj ny n, M. G., D ng
, K., Ref eli, Y., et l. (1993) Gene inocul tion gener tes immune response g i
nst hum n immunodeficiency virus type 1. Proc. N tl. Ac d. Sci. USA 90, 41564160.
11. Fyn n, E. F., We
ster, R. G., Fuller, D. H., H ynes, J. R., S ntoro, J. C.,
nd Ro
inson, H. L. (1993) DNA v ccines: Protective immuniz tion
y p rent l, m
ucos l nd gene-gun inocul tion. Proc. N tl. Ac d. Sci. USA 90, 11,47811,482. 12.
S to, Y., Rom n, M., Tighe, H., Lee, D., Corr, M., Nguyen, M.-D., et l. (1996)
Immunostimul tory DNA sequences necess ry for effective intr derm l gene immuni
z tion. Science 273, 352354. 13. Dool n, D. L., Sedeg h, M., Hedstrom, R. C., Ho

rt, P., Ch roenvit, Y., nd Hoffm n, S. L. (1996) Circumventing genetic restric
tion of protection g inst m l ri with mulitgene DNA immuniz tion: CD8+ T Cell, Interferon g mm -, nd nitric oxide-dependent immunity. J. Exp. Med. 183, 17391
746.
Genetic Su
unit V ccines
353
14. Irvine, K. R., R o, J. B., Rosen
erg, S. A., nd Restifo, N. P. (1996) Cytok
ine enh ncement of DNA immuniz tion le ds to effective tre tment of est
lished
pulmon ry met st ses. J. Immunol. 156, 238245. 15. T k h shi, H., Cohen, J., Hosm
lin, A., Ce se, K. B., Houghten, R., Cornette, J. L., et l. (1988) An immunodo
min nt epitope of the hum n immunodeficiency virus envelope glycoprotein gp160 r
ecognized
y cl ss I m jor histocomp ti
ility complex molecule-restricted murine
cytotoxic T lymphocytes. Proc. N tl. Ac d. Sci. USA 85, 31053109. 16. T k h shi,
H., N k g w , Y., Yokomuro, K., nd Berzofsky, J. A. (1993) Induction of CD8+ C
TL
y immuniz tion with syngeneic irr di ted HIV-1 envelope derived peptide-puls
ed dendritic cells. Int. Immunol. 5, 849857. 17. Ciernik, I. F., Berzofsky, J. A.
, nd C r
one, D. P. (1996) Induction of cytotoxic T lymphocytes nd nti-tumor
immunity with DNA v ccines expressing single T cell epitopes. J. Immunol. 156, 2
3692375.
32
Antigen Engineering in DNA Immuniz tion
Sh n Lu, Steve M nning, nd J mes Arthos 1. Introduction J. A. Wolff nd colle g
ues demonstr ted in 1990 (1) th t n ked, non-infectious DNA pl smids c n
e tr n
sferred into muscle cells of living mice to express the encoded protein in vivo.
Further studies h ve shown th t proteins expressed in vivo vi such DNA inocul
tion process c n serve s immunogens to effectively stimul te the
odys immune sy
stem to produce potenti lly strong, longl sting, ntigen-specific, humor l nd c

ell-medi ted immune responses (28). The technique of DNA immuniz tion is simple.
It c n
e used to develop DNA-
sed v ccines, nd it c n lso serve s tool to
gener te nim l ntiser s rese rch re gents. DNA immuniz tion ppro ch is qui
cker, less expensive, nd more likely to preserve
etter ntigen conform tion
th n the recom
in nt protein v ccine ppro ch. In ddition, DNA immuniz tion ll
ows in vivo p ck ging of newly synthesized proteins, which m y
e critic l for
ntigens with multiple su
units. DNA immuniz tion is n extension of the origin l
su
unit immuniz tion ppro ch,
ut with its unique dv nt ges. However, it is f
requently neglected th t the over ll immunogenicity of ny given protein ntigen
still pl ys n import nt role in DNA immuniz tion even though the ntigen is no
w delivered in the form of DNA pl smid. If p rticul r protein ntigen is not
immunogenic, it is unlikely th t immuniz tion with DNA v ccine encoding for thi
s ntigen will induce high immune responses. But DNA immuniz tion is n ide l p
pro ch to modify the sequences of protein ntigens so th t the immunogenicity of
protein ntigens c n
e improved. The convention l recom
in nt su
unit ppro ch
requires ddition l steps for in vitro protein expression nd purific tion whil
e DNA immuniz tion c n test the immunogenicity of modified protein ntigens with
direct inocul tion of ntigen-expressing
From: Methods in Molecul r Medicine, vol. 29, DNA V ccines: Methods nd Protocol
s Edited
y: D. B. Lowrie nd R. G. Wh len Hum n Press Inc., Totow , NJ
355
356
Lu, M nning, nd Arthos
DNA pl smids. Therefore, well-thought protein ntigen engineering process m y
improve the ch nce to chieve successful DNA immuniz tion. Hum n immunodeficie
ncy virus-1 (HIV-1) DNA v ccines re good ex mples of how the principles of prot
ein chemistry, cell
iology nd virology h ve to
e considered to design n effe
ctive AIDS DNA v ccine. In this ch pter, some of the HIV-1 DNA v ccines re n l
yzed to demonstr te the key conceptu l components of ntigen engineering for e c
h of these DNA v ccines. Currently most
iomedic l rese rch l
or tories re c p

le of doing the routine molecul r
iology nd protein chemistry experiments, t
echnic l det ils in this ch pter re provided only for steps unique to DNA immun
iz tion. The emph sis in this ch pter is to void the potenti l pitf lls of DNA
immuniz tion. 2. M teri ls 2.1. HIV-1 DNA Templ tes
1. HIV-1 molecul r clone NL4-3 (GenB nk Accession no.: M19921). 2. HIV-1 molecul
r clone HXB2 (GenB nk Accession no.: K03455, M38432). 3. pSVIIIenv encoding for
HIV-1 envelope protein with m jor deletions in V1/2 nd V3 dom ins (9).
2.2. Expression Vectors
1. pBC12/CMV/IL-2 vector, provided
y Dr. B. Cullen, Duke University Medic l Cen
ter, Durh m, NC, USA (10). 2. pJW4303 vector, provided
y Dr. J. Mullins l
or to
ry, University of W shington, Se ttle, WA, USA (1113).
2.3. PCR Oligonucleotide Primers
1. JApcr503: (cont ining n X
I site, TCTAGA). 5-GTCGCTCCTCTAGATTGTGGGTCACAGTC
TATTATGGGTACC-3. 2. JApcr502: (cont ining B mHI site, GGATCC , nd stop cod
on, TTA). 5-CGACGGATCCtt TGTTATGTCAAACCAATTCCAC-3. 3. JApcr504: (cont ining
B mHI site, GGATCC , nd stop codon, TTA). 5-GGTCGGATCCtt CTGCACCACTCTTCTCTTT
GCC-3.
2.4. Speci l Re gents nd Equipment
1. Qi gens QIAEX II kit (C t no. 20021) is v il
le from Qi gen Inc. (S nt Cl r
it , CA). 2. T4 DNA lig se is v il
le from Boehringer M nnheim (Indi n polis,
IN). (1 U/L, C t. no. 4812201 or 5 U/L, C t. no. 799009). 3. Gene Pulser Electropo
r tor is v il
le from Bio-R d L
or tories (Hercules, CA). 4. Electropor tion

cuvettes (C t. no.165-2088) is v il
le from Bio-R d L
or tories. 5. TEN
uffe

r (0.1 M N Cl, 10 mM Tris-HCl/pH 8.0, 1 mM EDTA/pH 8.0). 6. Helios Gene Gun Syst
em (C t. no. 165-2431) is v il
le from Bio-R d L
or tories. 7. Protein concen
tr tion spin columns, selected on the
sis of volume nd molecul r weight cut-o
ff, re v il
le from Amicon, Inc. (Beverly, MA).
Antigen Engineering in DNA Immuniz tion
357
Fig. 1. HIV-1 DNA v ccine inserts expressing full-length envelope proteins.
3. Methods 3.1. Construction of HIV-1 env-Expressing DNA V ccine Inserts
3.1.1. pNL4-3.dpol nd pNL4-3/HXB2.dpol DNA V ccines
1. Digest HIV-1 NL4-3 (9709
p) with B lI (=MscI) t sites 2621 nd 4553 (see re
f. 14 nd Fig. 1) (see Note 1). Remove the sm ller 1932-
p fr gment (including m
ost of the pol gene) nd re-lig te the rem ining NL4-3 sequences to produce ne
w single B lI site t 2621. 2. Digest the NL4-3 sequence t the H eII (641) nd
the B nII site following the nef region (now t nt 7556 fter pol deletion) to r
emove
oth LTRs. This produces the fin l NL4-3.dpol insert with tot l length o
f 6915
p. 3. Su
clone the NL4-3.dpol insert into the pBC/CMV/IL-2 (-Kpn) vector
(see Su
he ding 3.2.) t the HindIII (756) nd B mHI (1439) sites to give pNL
4-3.dpol DNA v ccine construct of 10,962
p. 4. Construct the chimeric pNL4-3/HX
B2.dopl DNA v ccine pl smid. Repl ce the KpnI (4532)B mHI (6650) sequences in pNL
4-3.dpol DNA v ccine with DNA fr gment sp nning from Kpn I (6350) to B mHI (84
74) in the HXB2 genome. The resulting construct will express the chimeric HXB2/N
L4-3 Env on n otherwise m inly NL4-3-
sed vir l p rticles (Fig. 1).
358
Lu, M nning, nd Arthos
3.1.2. pHXB2.env nd pNL4-3.env DNA V ccines pHXB2.env (15) (Fig. 1)
1. Digest the full-length HIV-1 HXB2 cDNA with S lI (5787) nd XhoI (8898) to pr
oduce 3111-
p fr gment th t encodes
oth the rev nd env genes (see Note 2). 2
. Su
clone the fr gment into pBC/CMV/IL-2 (-Kpn) vector t the HindIII (756) nd
B mHI (1439) sites to give pHXB2.env DNA v ccine of 7158
p.
3.1.3. pNL4-3/HXB2.env
1. Gener te 2118-
p, KpnI (6347) to B mHI (8465) fr gment from the full-length
HIV-1 NL4-3 genome (see ref. 14 nd Fig. 1). 2. Use the foregoing fr gment to s
u
stitute the KpnI (1323)-B mHI (3447) sequences within the pHXB2.env construct
(see step 1). The resulting pNL4-3/ HXB2.env DNA v ccine expresses chimeric HI
V-1 Env with the most of the env sequences coming from the NL4-3 str in.
3.1.4. pHXB2. gp140
1. Produce 1929
p of HXB2 DNA sequences (nt 6323-8251) encoding for gp140 f
orm of env
y PCR using primers JApcr503 nd JApcr502 nd HIV-1 HXB2 genome DNA
s templ tes (see refs. 1517 nd Fig. 2) (see Note 3). The 5 primer JApcr503 con
t ins n X
I site to clone the fr gment in fr me with the tPA le der sequences
in vector pJW4303. The 3 ntisense primer JApcr502 introduced stop codon prec
eding the tr nsmem
r ne region of gp41. There is B mHI site t the 3 end for
use in cloning with the pJW4303 vector. 2. Cut the vector pJW4303 t the NheI si
te (nt 2102) nd B mHI site (nt 2119). 3. Disc rd the sm ll fr gment nd insert
the gp140-expressing segment into the NheI/B mHI sites to produce 7051
p pHXB
2.gp140 DNA v ccine construct.
3.1.5. pHXB2. gp120
1. Produce 1422
p of HXB2 DNA sequences (nt 6323-7744) encoding for gp120 f

orm of env
y PCR using primers JApcr503 nd JApcr504 nd HIV-1 HXB2 genome DNA
s templ te (see refs. 1517 nd Fig. 2). The 5 primer JApcr503 cont ins n X
I
site to clone the fr gment in fr me with the tPA le der sequences in the vector
pJW4303. The 3 ntisense primer JApcr504 introduced stop codon ne r the junct
ion of gp120 nd gp41. There is B mHI site t the 3 end for use in cloning wi
th the pJW4303 vector. 2. Cut the vector pJW4303 t the NheI site (2102) nd B m
HI site (2119). 3. Disc rd the sm ll fr gment nd insert the gp120-expressing se
gment into the NheI/B mHI sites to produce 6544
p pHXB2.gp120 DNA v ccine con
struct.
3.1.6. pHXB2.dV123.gp140 nd pHXB2.dV123.gp120
1. Produce the pHXB2.dV123.gp140 nd pHXB2.dV123.gp120 pl smids in the s me w y
s for pHXB2.gp140 nd pHXB2.gp120 using the pSVIIIenv pl smid (6) s templ te f
or the PCR re ctions (see ref. 15 nd Fig. 2) (see Notes 4 nd 5).
Antigen Engineering in DNA Immuniz tion
359
Fig. 2. HIV-1 DNA v ccine inserts expressing gp120, gp140, nd the v ri
le dom
in-deleted forms of envelope proteins.
The pSVIIIenv pl smid encodes the HIV-1 Env proteins with deletions of the m jor
v ri
le regions V1/2 ( mino cids 121203) nd V3 ( mino cids 297 329). A 3- min
o cid sequence (Gly-Al -Gly) repl ced e ch of the two deletions. 2. Produce the
pHXB2.dV123.gp140 insert PCR primers JApcr503 nd JApcr502. 3. Produce the pHXB
2.dV123.gp120 insert with PCR primers JApcr503 nd JApcr504. 4. Su
clone the ins
erts into the pJW4303 vector t the NheI nd B mHI sites s for the pHXB2.gp120
nd pHXB2.gp140 DNA v ccines (see Su
he dings 3.1.3. nd 3.1.4.).
3.2. Su
cloning of DNA V ccine Inserts into Expression Vectors 3.2.1. pBC12/CMV
Vector
The pBC12/CMV/IL-2 (4732
p) vector (Fig. 3A) cont ins the following elements (s
ee Note 6):

p 1-755: CMV immedi te e rly promoter
p 756-1439: hum n IL-2 sequences (to
e
repl ced
y ntigen inserts)
p 1440-2407: r t preproinsulin II gene
p 1631-211
3: intron
p 2313: polyA site
p 2408-4619: pXF3
ck
one ( mp R)
p 4620-4732:
SV40 Ori
360
Lu, M nning, nd Arthos
Fig. 3. DNA v ccine vectors: (A) the origin l pBC12/CMV, (B) modified pBC12/ CMV
with the remov l of KpnI site, nd (C) pJW4303 with the ddition l CMV intron A
nd the tissue pl sminogen ctiv tor sequences.
Use the pBC12/CMV/IL-2 vector to su
clone DNA v ccine inserts s follows:
1. Digest the p rent l pBC12/CMV/IL-2 t HindIII (756) nd B mHI (1439) sites. T
wo fr gments will
e gener ted: the 685
p hum n IL-2 insert nd the 4047
p pBC
12/CMV vector. 2. Purify the 4047
p pBC12/CMV vector (e.g., Qi gens QIAEX II kit
, West Sussex, UK). Disc rd the 685-
p hum n IL-2 fr gment.
Antigen Engineering in DNA Immuniz tion
361
3. (Option l) Blunt one or
oth of the two ends on pBC12/CMV vector
y Klenow tr
e tment if the ends of DNA v ccine insert do not m tch these two sites. 4. Lig t
e DNA v ccine insert into the pBC12/CMV vector.

3.2.2. pBC12/CMV (Kpn I) Vector Remove the single-KpnI site (nt 1845) in pBC12/CM
V/IL-2 vector (Fig. 3B) to produce pBC12/CMV (Kpn) vector. This llows for the
e sy use of KpnI site(s) in ntigen inserts. This w s done in some of our HIV-1
DNA v ccines to f cilit te the HIV-1 env cloning mong different str ins of HIV1 virus:
1. Digest the p rent l pBC12/CMV/IL-2 with KpnI to gener te line r form of pBC
12/CMV/IL-2. 2. Tre t this line r form with T4 DNA polymer se to
lunt the 3 ov
erh ngs gener ted
y KpnI digestion. This removes nt 18411844 (GTAC). 3. Re-lig t
e the pl smid to produce the pBC12/CMV/IL-2 (-Kpn) pl smid vector. 4. Su
clone D
NA v ccine inserts into this vector t the HindIII nd B mHI sites, s descri
ed
for the pBC12/CMV/IL-2 vector in Su
he ding 3.2.1.
3.2.3. The pJW4303 Vector The pJW4303 vector (5139
p) (Fig. 3C) (see Note 7) co
nt ins the following:

p 1- 1194: CMV IE (immedi te e rly) promoter
p 1195-2027: CMV IE promoter intr
on A
p 2027-2102: tissue pl sminogen ctiv tor (tPA)
p 2119-2419: BGH (
ovine
growth hormone) polyA
p 2419-4794: pBR vector
ck
one ( mp R)
p 4794-5139: SV
40 Ori
The complete nucleotide sequences of pJW4303 h s recently
een confirmed. Due to
the wide distri
ution of pJW4303, its sequences nd the list of its restriction
endonucle se sites re included in Appendices A nd B. Su
clone DNA v ccine ins
erts into pJW4303 vector
y either of the following two w ys. To use HindIII nd
B mHI s the cloning sites:
1. Digest the pJW4303 with HindIII (2027) nd B mHI (2119). Purify the l rge fr
gment (5047
p) nd disc rd the sm ll one (92
p). 2. (Option l) Blunt one or
o
th of these two ends
y Klenow tre tment if the ends on DNA v ccine insert do no
t m tch these two sites in pJW4303. 3. Lig te the DNA v ccine insert into the pJ
W4303 vector.
To use NheI nd B mHI s the cloning sites: Sequences
etween HindIII nd NheI s
ites in pJW4303 encode for the le der sequences from tissue pl sminogen ctiv to
r (tPA). Using the NheI site would require n in-fr me su
cloning of the DNA v c
cine insert to utilize the tPA le der, therefore the origin l le der sequences f
rom the encoded protein ntigen must
e removed from the DNA insert. By c reful
design of n oligo primer, n NheI site t the 5 end of the v ccine insert c n

e cre ted
y polymer se ch in re ctions (PCR).
362
Lu, M nning, nd Arthos
The su
cloning steps re simil r to those listed in the HindIII - B mHI ppro ch
(see
ove) except the NheI site is used inste d of the HindIII site:
1. Digest pJW4303 t NheI (2102) nd B mHI (2119) sites. 2. Purify the l rge fr
gment (5122
p) nd disc rd the sm ll one (17
p). 3. (Option l) Blunt one or
o
th of these two ends
y Klenow tre tment if the ends of the DNA v ccine insert d
o not m tch these two sites. 4. Lig te the DNA v ccine insert into the pJW4303 v
ector.
3.3. Verific tion of Antigen Expression in COS Cells
1. Grow COS cells (see Notes 811) two to three p ss ges from frozen cells in RPMI
medium nd 10% fet l
ovine serum in 100 mm culture dishes. 2. Split the cells
1:2 into new dishes the night
efore tr nsfection when cells re ch 90% confluenc
e. 3. The next d y, disc rd the tissue culture medi . Collect the cell l yer
y
tre ting the dish with 5 mL of Trypsin-EDTA solution, nd incu
ting it for 510 m
in until the cells
ecome round. 4. Collect the cell suspension, nd pellet the
cells t 1500 rpm for 5 min; disc rd the supern t nt. 5. Resuspend the cells in
0.51 mL of RPMI medium without fet l
ovine serum. Count the cells nd resuspend

the cells t 2 106 cells/mL. 6. Tr nsfer 0.70.8 mL of cell suspension into spec
i l cuvette for electropor tion. 7. Add 210 mg pl smid DNA to the cuvette nd mix
it with the cells. 8. T p the cuvette to suspend the cells
efore electropor ti
on. 9. Pl ce the cuvette in the electropor tion ch m
er nd expose it in the pp
lied electric field: 250 V nd 960 mF when using the Bio-R d Gene Pulser II. 10.
Keep the cells in the cuvette for
out 10 min. 11. Tr nsfer the cells to 100
-mm tissue culture dish, dd 610 mL medium nd incu
te for 2478 h t 37C in 5% (v/
v) CO2. 12. H rvest the tr nsfected cells in 4872 h s follows. Collect the super
n t nt; tre t the confluent cells with TEN
uffer for 510 min; collect the cells
nd microfuge them t 1500 rpm for 5 min; sep r te the pellet from the supern t
nt. 13. Store the supern t nt t 70C for future use or concentr te it
y using pro
tein concentr tion spin columns. 14. Lyse the cells s follows. Resuspend the ce
ll pellet in 100200 L of cell lysis
uffer with selected prote se inhi
itors nd i
ncu
te for 510 minutes on ice; centrifuge the lysed cell suspension for 1 h t 1
2000 rpm in cold room; collect the supern t nt nd disc rd the pellet; store t
he cell lysis t 70C for future use.
3.4. Anim l Immuniz tion (see Notes 12,13) 3.4.1. Routes nd Doses Intr muscul r
route (see Notes 1416) with DNA concentr tion of ~1 mg/mL:
B l
/C mice: 50100 g for e ch of the qu driceps NZW r

its: ~100 g for e ch of the
qu driceps M c ques: 100250 g for e ch of the qu driceps
Antigen Engineering in DNA Immuniz tion
Gene gun (see Note 17):
B l
/C mice: 0.20.5 g per gun shot, 46 shots per nim l, NZW r

its: 0.5 g per gun
shot, 636 shots per nim l. M c ques: 0.57.2 g per gun shot, 420 shots per nim l
363
3.4.2. Immuniz tion Schedule The immunogenicity of encoded ntigens dict tes the
need nd the num
er of
oost immuniz tions (see Note 18). In gener l, series
of two to three immuniz tions sep r ted
y 48 wk is re son
le initi l tri l.
4. Notes 4.1. Construction of HIV-1 Env-Expressing DNA V ccine Inserts
1. Remov l of the gene sequence for pol from the full length HIV vir l genome pr
oduced non-replic ting vir l p rticles encoded
y .dpol-like constructs. Env prote
ins re present on the surf ce of vir l p rticles, therefore mimicking the ctu
l viruses (14). Virus-like p rticles c n lso include more th n one vir l protei
n. One c ve t is th t the effective dose for Env in this type of pl smid m y
e
less th n in those DNA v ccines encoding for only one single Env protein if equ
l m sses of these two types of DNA pl smid constructs re used for immuniz tion.
2. Full length Env-expressing DNA v ccines produce complete envelope glycopro
tein. Once
sic Env pl smid is constructed, DNA sequences encoding for Env fr
om different str ins of HIV-1 c n
e exch nged into this construct t KpnI nd B
mHI sites to produce m ny chimeric Env-expressing pl smids. This m y
e quick
w y to test the ntigen specificity of Env sequences from the different HIV-1 p
tient isol tes. However, the full-length gp160-expressing DNA v ccine w s shown
to
e less immunogenic th n the gp140 nd gp120-expressing ones in r ising nti
-Env nti
ody responses (15). 3. HIV-1 gp120- nd gp140-expressing constructs fu
rther demonstr te the potenti l of DNA immuniz tion to m ke su
unit-
sed v ccin
es. The gp120 form is the n tur l proteolytic product of the HIV-1 Env gp160 pre
cursor protein. It is highly immunogenic, presum
ly due to its monomer conform
tion nd high level of secretion of gp120 from the cells. The gp140 form of en
v encodes for the entire extr cellul r portion of Env, including gp120 plus the
ectodom in of gp41. This construct is used to produce solu
le form of Env yet
m int ining Envs oligomeric conform tion. 4. Remov l of the v ri
le dom ins V1,
V2, nd V3 from the Env protein would further expose cert in hidden ntigenic de
termin nts to improve the over ll immunogenicity of Env. The e se of the DNA imm
uniz tion technique offers unique opportunity to eventu lly find n Env design
th t is very immunogenic yet preserves the critic l conform tion of its origin
l protein structure. 5. These constructs demonstr te th t one ntigenic protein

(such s the HIV-1 envelope) c n
e expressed in m ny forms. The over ll go l is

to find the most immunogenic ones th t lso h ve conform tion most closely re
sem
ling the n tive
364
Lu, M nning, nd Arthos
ntigens. Judging from the results of expressing ntigens r nging from ne rly co
mplete vir l p rticles down to only cert in segments of one vir l protein, the

ove model constructs suggested th t it is extremely import nt to design the rig
ht DNA inserts for the right purpose. If protein is highly hydropho
ic or m ny
of its epitopes re in ccessi
le due to complex protein conform tion, c reful
ntigen engineering is essenti l.
4.2. Su
cloning of DNA V ccine Inserts into Expression Vectors
6. Except for few very immunogenic ntigens, high in vivo protein ntigen ex
pression is required for most DNA v ccine inserts. Therefore, it is critic l to
select m mm li n expression vectors th t h ve high efficiency nd lso m tch wit
h the design of DNA v ccine inserts. M ny DNA v ccine studies h ve employed the
CMV immedi te e rly promoter s the key component in vectors, simil r to the two
descri
ed in this ch pter. For
eginners in DNA immuniz tion, it is wise to try
vectors th t re known to
e effective, r ther th n to simply put DNA v ccine i
nserts into ny v il
le pl smid vector in the initi l tri l. 7. In comp rison
with the cl ssic l pBC12/CMV vector, the pJW4303 vector h s two m jor dditions:
the intron A sequences just downstre m of the CMV promoter nd tPA le der seq
uences. By including the intron A sequences in the vector, the over ll expressio
n level w s improved (13). The tPA le der sequences in pJW4303 provide the choic
e of m king secreted protein ntigens (13). It is desir
le to use the tPA le de
r sequences to produce solu
le proteins. On the other h nd, the tPA le der m y

ecome less helpful if the go l is to h ve high expression of m inly intr cellu
l r proteins, such s to m inly induce the cytotoxic lymphocyte responses.
4.3. Verific tion of Antigen Expression in COS Cells
8. Verific tion of the uthenticity for p rticul r DNA v ccine is necess ry
e
fore gre t effort is pl ced on the immuniz tion process. The in vitro tr nsient
expression in tissue cultured cells is quick nd reli
le qu lity control step
to detect the encoded ntigens in DNA v ccines. 9. COS cells re descri
ed here
s n ex mple. The most import nt element for successful tr nsfection is the
condition of cells. Only he lthy cells in log rithmic growth ph se c n produce h
igh expression. 10. Electropor tion nd the liposome tr nsfection re gents re e
qu lly effective. Electropor tion is simple
ut needs speci l equipment (such s
one descri
ed in this ch pter). The settings of electropor tion need to
e fine
-tuned for e ch type of t rget cell. Ide lly, killing curve should
e est
lishe
d to find the highest electric l pulsing under which 5080% cells c n still recove
r nd grow in 23 d ys. 11. Enzyme-linked immunosor
ent ss y (ELISA), Western
lo
t, or indirect immunofluorescent st ining of the tr nsfected cells c n
e used t
o detect the expression of encoded ntigens, if there is specific serum or nt
i
ody v il
le to identify the ntigen. If there is no nti
ody v il
le, t g
sequence c n
e dded to the v ccine insert sequences, nd then n ntiserum g
inst the t g c n
Antigen Engineering in DNA Immuniz tion
365

e used to detect the expression of the whole fusion proteins. In the worst situ
tion, the expression study c n
e postponed until post-immuniz tion. Ser from
immunized nim ls c n
e used t th t time to verify ntigen expression in tr ns
fected cells.

4.4. Anim l Immuniz tion

12. Ide lly, it will
e most useful to demonstr te in vivo expression of DNA v c
cines. However, due to the minute mount of protein expressed in the loc l tissu
e of living nim l, ntigen expression m y frequently f ll
elow the level of
detection in n ELISA ss y. One ltern tive w y is to do immunohistochemistry s
t ining on tissue sections. T king
iopsy s mples t ~24 h post-immuniz tion pp
roxim tely coincides with the pe k ntigen expression t the site of inocul tion
. 13. Using DNA v ccine encoding for m rker protein, such s lucifer se (18)
, m y incre se the sensitivity of detecting ntigen in vivo,
ut c n only
e use
d s n indirect reference for the expression of specific ntigens. 14. Intr mus
cul r inocul tion with convention l syringe nd epiderm l inocul tion using th
e gene gun h ve
ecome the domin nt methods of delivering DNA v ccines. However,
the following routes c n lso
e used to dminister DNA v ccines: intr venous,
intr derm l, su
cut neous, or peritone l routes
y syringe, or vi mucos l sur
f ce
y gene gun (5,1920). 15. Both intr muscul r nd gene gun routes re effec
tive in delivering DNA v ccines. From our experience, for given ntigen insert
, gene gun delivery m y require less DNA nd m y induce more homogenous respon
se for group of nim ls th n the intr muscul r route. On the other h nd, the g
ene gun ppro ch is more expensive due to the cost of gene gun, gold
e ds, nd
other ncill ry equipment ssoci ted with gene gun use. 16. The DNA doses descri

ed in this ch pter c n only
e used s gener l reference. There is gre t v ri
tion in the immunogenicity of different ntigens. For
eginners in DNA immuniz
tion, higher dose of DNA pl smids c n
e used t first to t le st induce some
level of desired immune responses
efore eventu lly titr ting down the dose-res
ponse curve. 17. With the m rketing of commerci l Helios Gene Gun, more st nd
rdized gene gun use is nticip ted nd the m nuf cturer will provide post-s le s
upport including det iled protocol. However, two critic l points re emph size
d here for interested users: (i) Alw ys give enough time to dry the pl stic tu
i
ng with nitrogen g s so th t the potenti l moisture c n
e removed; (ii) Alw ys
test the delivery depth of gene gun
efore st rting on new nim l species. T
his c n
e chieved
y t king
iopsy s mple from gene gun-inocul ted skin to e
x mine the gold
e d distri
ution in H & E histology sections. 18. The num
er of
required immuniz tions is dependent on individu l ntigens nd m y lso
e depe
ndent on nim l species. Gener lly, very close-sp ced immuniz tion schedule is
not helpful. Sp cing the immuniz tions to h ve t le st 4 wk resting period is
recommended. For some ntigens, one immuniz tion is sufficient nd the nti
ody
responses m y t ke 810 wk to pe k (11).
366
Appendix A. pJW4303 DNA Sequences
Lu, M nning, nd Arthos
Antigen Engineering in DNA Immuniz tion
367
368
Lu, M nning, nd Arthos
Antigen Engineering in DNA Immuniz tion
369
370
Lu, M nning, nd Arthos
Antigen Engineering in DNA Immuniz tion

Appendix B. The Potenti l Enzyme Sites on pJW4303

371
372
Lu, M nning, nd Arthos
Antigen Engineering in DNA Immuniz tion
References
373
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irus
oosting. Virology 230, 265274. 18. B rry, M. A. nd Johnston, S. A. (1997)
Biologic l fe tures of genetic immuniz tion. V ccine 15, 788791. 19. R z, E., C r
son, D. A., P rker, S. E., P rr, T. B., A
i, A. M., Y nk uck s, M. A., B iro, S
. M., nd Rhodes, G. H. (1994) Intr derm l gene immuniz tion: the possi
le role
of DNA upt ke in the induction of cellul r immunity to viruses. Proc. N tl. Ac d
. Sci. USA 91, 95199523. 20. Livingston, J. B., Lu, S., Ro
inson, H., nd Anderso
n, D. J. (1998) The use of DNA immuniz tion to induce nti
ody responses in the
genit l tr ct of fem le r ts. Infection Immunity 66, 322329.
33
Genetic V ccin tion T rgeting T-Cell Receptors
Alexis P. Godillot, Qiong F ng, Terry Higgins, C therine P chuk, Je n Boyer, D v
id B. Weiner, Stu rt Lessin, nd Willi m V. Willi ms 1. Introduction T-cell nti
gen receptor (TCR) genes (which consist of v ri
le (V), diversity (D), joining
(J) nd const nt (C) segments) undergo re rr ngement during T-cell development
nd result in the expression of disulfide linked heterodimer ( nd ch ins) on th
e surf ce of m ture T-cells (1,2). The TCR confers specificity to e ch T-cell fo
r ntigen recognition (in the context of m jor histocomp ti
ility (MHC) molecule
s (1,3). Clon l TCR- ch in gene re rr ngements h ve
een demonstr ted in DNA s mp
les derived from cut neous tumors, peripher l
lood lymphocytes nd lymph nodes
of p tients with cut neous T cell lymphom s (CTCL) (46). Together with immunohist
ologic d t (7), these findings indic te th t CTCL is clon l dise se of m lign
nt T cells th t express /-TCR. The TCR, like surf ce immunoglo
ulin (sIg) on B-ce
lls, is cell specific idiotype nd c n serve s t rget for nti-idiotype the
r py. Anti-idiotype ther py (8) h s
een reported in B-cell m lign ncies (9) nd
T-cell medi ted utoimmune dise se (1012). A v riety of ppro ches h ve
een tt
empted in murine nd hum n B-cell lymphom s nd include: monoclon l nti
ody (mA

) directed t surf ce Ig (13), immuniz tion with sIg protein (9) nd sIg conjug
ted to cytokine/growth f ctors (14). In the rodent model of multiple sclerosisexperiment l utoimmune enceph lomyelitis (EAE), reports demonstr te th t the di
se se c n
e modul ted
y immunizing the nim ls to TCR peptides on p thogenic C
D4+ T-cell clones re ctive to myelin
sic protein nd responsi
le for inducing
EAE (1012).
From: Methods in Molecul r Medicine, vol. 29, DNA V ccines: Methods nd Protocol
s Edited
y: D. B. Lowrie nd R. G. Wh len Hum n Press Inc., Totow , NJ
375
376
Godillot et l.
Recently, direct in vivo inocul tion with pl smid DNA h s
een shown to result i
n the intr cellul r synthesis of pl smid encoded protein which, in turn, produce
s t rgeted immune response (DNA v ccin tion) (1517). DNA constructs encoding Tcell receptor (TCR) nd surf ce immunoglo
ulin (sIg) h ve
een utilized in v cci
n tion protocols, producing protective immunity in murine models of T-cell medi
ted utoimmune dise se nd B-cell lymphom (18,19). B nd T cell neopl sms provi
de likely t rget for genetic immuniz tion in th t e ch of these tumors comes w
ith its own tumor ntigen in the form of unique ntigen receptor. Recom
in nt

DNA technology llows us to
orrow from the ntigen receptor the coding sequence f
or the v ri
le region. This region cont ins the structures most unique to the m
olecule. Here we focus on the
et ch in of the T-cell receptor (TCR) s the sou
rce of our immunogen, lthough n logous str tegies could
e useful for nti
ody

sed immunogens, nd thus B-cells. The pplic tion of this technology to tumor
immunity m y confer the unique dv nt ge th t lthough the product immunogen pr
otein derives from n tive structures, the synthetic n ture of the product m y
e
su
ject to novel immunologic l surveill nce.
1.1. Identific tion nd Cloning of P thologic l TCR
The experiments performed to d te h ve involved cloning p rticul r T cell recept
or v ri
le region sequences with no reg rd to the clonotype. In other words, we
re v ccin ting with v ri
le region coding sequence representing the f mily
of the p thogenic clone
ut not necess rily the idiotype. Ultim tely, we would l
ike to use v ccines th t would gener te specific response to the TCR idiotype
(third hyperv ri
le region) s well s specificity for the entire f mily. The s
uccessful immuniz tion g inst T-cell receptors m y eventu lly llow us to not o
nly elimin te the o
vious t rget of T-cell derived c ncers
ut lso to com
t T
cell-medi ted utoimmunity. Our experience h s
een with chronic T-cell lympho
m . Two methods were used to determine the TCR f mily of the tumor. First, the p
tients peripher l
lood lymphocytes were st ined with
ttery of nti
odies s
pecific for the v rious TCR V-
et f mily gene products. Second, cDNA reverse tr
nscri
ed from tot l cellul r RNA of peripher l
lood mononucle r cells (PBMC) w
s su
jected to semi-qu ntit tive PCR method which utilizes
ttery of TCR

et specific primers. These two independent methods should expose domin nt sig
n l for one TCR-V
et gene. Due to sp ce limit tions, neither of these techniqu
es c n
e discussed here in det il.
TCR Genetic V ccin tion
377
Once the p rticul r f mily of V
et gene is identified, we c n specific lly mp
lify clone
e ring such segment. The ssumption th t the clone mplified in
these experiments dequ tely represents the lymphom is re son
le for two re so
ns: first, the clone in question is likely to h ve derived from domin nt clone
(tumor) within the origin l s mple cell popul tion, nd second, if non-tumor
clonotype from the correct f mily is used, it should provide immunity to the ent
ire T-cell repertoire derived from V genes of th t f mily. The mplified coding
sequence sp nning the
et ch in v ri
le region through the second
et const n
t region is then su
cloned into our expression vector. The result nt clone is se
quenced to confirm pl usi
le open re ding fr me of the correct V
et gene f m
ily. 2. M teri ls 2.1. Cloning V13.1 T Cell Receptor (TCR) V ri
le Regions nd D
NA Inocul tion (see Note 1) 2.1.1. PBMC Isol tion
1. Ficoll-Hyp que is v il
le from Ph rm ci Biotech (Upps l , Sweden). 2. Dul

eccos Phosph te Buffered S line, C t. No. 14040-059, is v il
le from Life Techn
ologies Inc. (G ithers
urg, MD). 3. Sterile Eppendorf tu
es. 4. Freezing medium:
90% (v/v) hum n AB serum, C t. No. H2520, which is v il
le from Sigm (St. Lo
uis, MO) nd 10% (v/v) dimethylsulfoxide (DMSO), C t. No. D5879, Sigm . 5. Cryog
enic vi ls: Dispos
le Sterile Vi ls, C t. No., 25704, re v il
le from Cornin
g (Corning, NY). 6. Prop nol
th: Cryo Freezing Cont iner, C t. No. 5100-001, i
s v il
le from N lgene (Rochester, NY).
2.1.2. RNA Isol tion nd Reverse Tr nscription
1. 2. 3. 4. 5. RNAse-free pipet tips. RNAse-free microfuge tu
es. TRIzol re gent
is v il
le from Life Technologies. 70 % (v/v) eth nol. M loney Murine Leukemi
Virus Reverse Tr nscript se (M-MLV) RT nd 5X RT
uffer re v il
le from Life
Technologies. 6. R ndom hex mer RT primers. 7. dNTPs, 10 mM: Av il
le from Boe
hringer M nnheim Biochemic ls (Indi n polis, IN), mix 20 L of e ch 100 mM stock (
G, A, T, C) into 120 L of sterile w ter.

2.1.3. PCR nd Su
cloning

1. Speci l equipment: Thermocycler. 2. V13.1 5/NotI primer (g g ggcggccgc ATGAGC
ATCGGCCTCCTGTG).
378
Godillot et l.
3. C2/MluI primer (cgtctc cgcgtCTATGGGAACACGTTTTTCAGGTCCTC). 4. T q polymer se is
v il
le from Boehringer M nnheim. 5. dNTPs, 1.25 mM: Mix 12.5 L of e ch 100 mM
stock (G,A,T,C) into 950 L of sterile w ter. 6. 10X PCR
uffer: 500 mM KCl, 100
mM Tris-HCl pH 8.3, 15 mM MgCl2, 0.1% (w/v) Gel tin. 7. Se Pl que low-melting-te
mper ture g rose is v il
le from FMC Inc. (Rockl nd, ME). 8. C lf intestin l
lk line phosph t se is v il
le from Boehringer M nnheim Biochemic ls. 9. Dye
Termin tor Cycle Sequencing Kit is v il
le from Perkin-Elmer/Cetus (Norw lk, C
T). 10. T4 Lig se is v il
le from Life Technologies. 11. APL400-004 vector is
propriet ry m teri l; cont ct Apollon, Inc. (M lvern, PA).
2.1.4. In Vitro Tr nsform tion nd RNA Expression
1. 2. 3. 4. Lipofect mine is v il
le from Gi
co-BRL (G ithers
urg, MD). Ultr s
pec Re gent is v il
le from Biotecx (Houston, TX). DNA se is v il
le from Wo
rthington (Freehold, NJ). Meg prime Kit, C t. No. RPN 1606, nd Hyperfilm-MP re
v il
le from Amersh m Life Sciences (Arlington Heights, IL).
2.2 Immuniz tion
1. 1 cm3 Syringe nd 25 g uge needles re v il
le from Becton Dickinson (Fr nk
lin L kes, NJ).
2.3. Immune Response Assessment 2.3.1. Prolifer tion Ass ys
1. B95-8 cell line producing the cell-tr nsforming Epstein-B rr virus (EBV). 2.
Complete medium: RPMI 1640 with penicillin/streptomycin, L-glut mine, sodium pyr
uv te, non-essenti l mino cids, 10 mM HEPES
uffer pH 8.0, 5 105 M 2-merc ptoet
h nol with either 4% or 10% (v/v) hum n serum (HS). 3. Round-
ottom 96-well tiss
ue culture pl tes, F lcon C t. No. 3077, re v il
le from Becton Dickinson. 4.
Triti ted thymidine is v il
le from NEN Life Science Products (Boston, MA). 5
. Peptides: CDR I, CDR II, CDR III, CDR IV dissolved t 1 mg/mL in deionized dis
tilled w ter. We h d set of peptides m de which represent the complement rity
determining regions of our p rticul r V-
et gene. These were designed to e ch i
ncorpor te one of the surf ce hyperv ri
le loops (complement rity determining r
egions or CDR) of the t rgeted TCR
et ch in. 6. Peptide mixture: Mix equ l g m
ounts of CDR I, CDR II, CDR III, CDR IV nd dilute in 10% (v/v) HS for use. 7. S
mple
gs, C t. No. 1450-432, re v il
le from W ll c (Turku, Finl nd).
TCR Genetic V ccin tion 2.3.2. CTL Ass ys
379
1. Complete medium cont ining 4% or 10% (v/v) HS. 2. B95-8 EBV tr nsforming cell
line. 3. Peptides: CDR I, CDR II, CDR III, CDR IV dissolved t 1 mg/mL in deion
ized distilled w ter. 4. Peptide mixture: Mix equ l microgr m mounts of CDR I,
CDR II, CDR III, CDR IV nd dilute in medium. 5. Round-
ottom 96-well tissue cul
ture pl tes, F lcon C t. No. 3077, re v il
le from Becton Dickinson. 6. Apopt
osis lysis
uffer (10 mM EDTA/50 mM Tris-HCl, pH 8.0/0.5% [w/v] S rkosyl [Sigm ]
) (20). 7. Optiph se Supermix, 96-well s mpler pl te nd se ling t pe, C t. No.
1450-461, re v il
le from W ll c. 8. 51Cr-N 2CrO4 (NEN).
2.3.3. DNA Enzyme-Linked Immunosor
ent Ass y (ELISA) Micropl te re der: Molecul
r Devices E m x Precision Micropl te Re der MR5000, is v il
le from Dyn tech (
Ch ntilly, VA).

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 2M H2SO4. TMB, T-3405, is v il
le from Sigm

. W sh
uffer: PBS cont ining 0.05% (v/v) Tween-20. Peroxid se-conjug ted ntihum n IgG, C t. No. A-8400, is v il
le from Sigm . Blocking
uffer: Phosph te
uffered s line (PBS) cont ining 0.05% (v/v) Tween20 nd 2% (w/v)
ovine serum
l
umin (BSA). CBC
uffer: 0.1M c r
on te-
ic r
on te
uffer. Microtiter pl tes,
C t. No. 0110103455, re v il
le from Dyn tech. Prot mine sulf te: 0.1% (w/v)
stock. St
le for 30 d t 4C. DNA diluent: 15 mM N Citr te, 150 mM N Cl, 0.02% (
w/v) N N3, pH 8.0). Trieth nol mine-
uffered s line (TBS): 50 mM Tris-HCl, 150 m
M N Cl, pH 7.5. Su
str te
uffer: dissolve one c psule of phosph te-citr te
uff
er with sodium per
or te in 100 mL deionized w ter to yield 0.05M
uffer pH 5.
0 / 0.03% (w/v) sodium per
or te.
2.3.4. Anti-V Bet ELISA
1. 50 mM NH4HCO3 2. Blocking
uffer: PBS cont ining 0.05% (v/v) Tween-20 nd 2%
(w/v) BSA. 3. Biotin-conjug ted nti-hum n IgG1, 2, 3, 4, or IgM, with respectiv
e C t. Nos. B-6775, B-3398, B-3523, B-3523, B-3648 nd B-1265, re v il
le fro
m Sigm . Dilute them in
locking
uffer s follows: nti-H IgG1, 1:1,000; nti-H
IgG2, 1:20,000; nti-H IgG3, 1:3,000; nti-H IgG4, 1:10,000; nti-H IgM, 1:10,0
00. 4. Avidin-HRP, 5 mg/mL, C t. No. 29994, is v il
le from Pierce (Rockford,
IL). Dilute in
locking
uffer to 10 mg/mL. Store 1 mL liquots s stocks.
380
Godillot et l.
3. Methods 3.1. Cloning V13.1 T Cell Receptor (TCR) V ri
le Regions nd DNA Inoc
ul tion O
t in peripher l
lood mononucle r cells (PBMCs) from p tient with pr
eviously documented V13-expressing CTCL
y Ficoll-Hyp que gr dient sep r tion. Ex
tr ct RNA from the PBMCs nd reverse tr nscri
e it. Amplify the V13.1 segment wit
h specific PCR primers, digest it with restriction enzymes nd su
clone it into
the v ccine vector. Sequence the vector nd produce milligr m qu ntities of the
v ccine. Formul te it with 0.25% (w/v) Bupivic ne nd s line nd dminister. The
steps of v ccine production re presented in Fig. 1.
3.1.1. PBMC Isol tion
1. Collect
lood into green c p (hep rinized) tu
es nd dilute it 1:3 with Dul
e
ccos PBS (DPBS) (see Note 2). 2. L yer 30 mL of diluted
lood s mple over 15 mL
of Ficoll-Hyp que in sterile 50-mL conic l centrifuge tu
e t room temper tur
e (see Note 3). 3. Centrifuge t 360g t room temper ture in swinging-
ucket c
entrifuge for 20 min with no
r ke (see Note 4). 4. Collect the lymphocytes t t
he interf ce of the PBS nd Ficoll then w sh them in 30 mL DPBS, count the cells
nd centrifuge the suspension t 280g (see Note 5). 5. W sh the cells once more
in DPBS nd resuspend them in freezing medium (see Note 6). 6. Prep re liquots
of the cells in cryogenic vi ls t concentr tion of 1 x 107 cells per vi l (s
ee Note 7). 7. Pl ce the vi ls in room-temper ture prop nol
th, tr nsfer it
to the 80C freezer for more th n 4 h nd fin lly pl ce the vi ls in liquid nitroge
n (see Note 8).
3.1.2. RNA Isol tion nd Reverse Tr nscription
Extr ct RNA from the PBMCs using TRIzol re gent nd reverse tr nscri
e the tot l
cellul r RNA with nonspecific r ndom-hex mer priming.
1. Suspend e ch 5 106 cells in 500 L of TRIzol re gent without llowing ir
u

l
es to form (see Note 9). 2. Tr nsfer the suspension to n RNAse free microfuge t
u
e nd vortex it to ensure complete mixing (see Note 10). 3. Let st nd for 5 mi
n t room temper ture. 4. Add 100 L chloroform for every 500 L TRIzol used nd vor
tex thoroughly. 5. Microfuge t <12,000g for 15 min t 4C. 6. Tr nsfer the queou
s ph se to fresh RNAse-free microfuge tu
e nd precipit te the RNA
y dding 2
50 L isoprop nol for e ch 500 L TRIzol used, vortexing nd llowing to st nd for 1
0 min t room temper ture. 7. Microfuge t <12,000g for 15 min t 4C.

TCR Genetic V ccin tion

381
Fig. 1. Steps in v ccine development. 8. C refully remove the supern t nt withou
t distur
ing the pellet of RNA nd then w sh with 1 mL ice cold 75% (v/v) eth no
l. 9. Microfuge t <7,500g for 5 min t 4C. 10. Air dry the RNA for 10 min nd re
dissolve in the RNA in 24 L RNAse-free w ter (see Note 11). 11. Set up reverse tr
nscription re ctions. E ch re ction cont ins 10 L of tot l cellul r RNA, 0.5 L of
pl cent l RNAse inhi
itor, 2 L r ndom hex mers, 5 L of 10 mM dNTPs, 5 L of 5X RT

uffer, nd 2 L M-MLV RT (see Note 12). 12. Incu
te the mixture for 1 h t 37oC.
13. Store the cDNA (i.e., the completed RT re ctions) t 20C.

3.1.3. PCR nd Fr gment Prep r tion Amplify the p rticul r V gene segment of inte
rest with specific primers using high stringency PCR progr m (see Note 13). De
sign the primers to introduce restriction sites into the product to f cilit te c
loning (see Note 14). Digest the product nd purify it on gel.
1. Set up the PCR re ction with V-
et specific primers (in our c se V13 5/Not I
nd C2 3/Mlu I primers). E ch re ction cont ins 5 L of cDNA,16 L dNTPs, 10 L 10X P
CR
uffer, 63 L dou
le-distilled w ter, 5 L of e ch primer nd 1 L of T q polymer s
e 2. Cover the queous re ction mixture with miner l oil nd pl ce it in therm
ocycler (40 cycles of 95C 1 min 55C 1 min 72C 1 min) (2123) (see Note 15). 3. Tre t
the PCR products with protein se K (24) to void interference of the T q polymer
se with restriction digestion. Com
ine 80 L of PCR re ction (without oil) with 1
0 L 10X PK
uffer nd 10 L of 5 mg/mL protein se K. 4. He t in ctiv te the protein
se K for 10 min t 68C (see Note 16). 5. Extr ct with phenol:chloroform:iso myl
lcohol (25:24:1), then with chloroform: iso myl (24:1) (see Note 17).
382
Godillot et l.
6. Precipit te fin l queous ph se
y dding one-tenth volume of 3M sodium cet
te, pH 5.0, nd 2 volumes of ice-cold 100% (v/v) eth nol (see Note 18). Redissol
ve the DNA in 50 L deionized distilled w ter. 7. Restriction digest with NotI nd
MluI: digest 50 L of PK-tre ted PCR product with 30 units (3 L) of e ch restricti
on enzyme (RE) nd 6 L of 10X RE
uffer (see Note 19). 8. At the s me time, diges
t 10 g of the vector using t le st 10 units of e ch enzyme. 9. Dephosphoryl te t
he vector (not the insert V) with c lf intestin l lk line phosph t se (CIAP, see
Note 20). Add 1 L of CIAP nd one-tenth volume of 10 CIAP
uffer to the complete
d restriction digestion nd incu
te the mixture t 37C for 15 min. 10. Lo d the
entirety of the digestion products nd insert on 1% (w/v) low-melting temper t
ure g rose sl
gel cont ining 100 ng/mL ethidium
romide (see Note 21). 11. Ru
n the gel t 75 V for t le st 2 h to resolve the individu l fr gments (see Note
22). 12. Visu lize the fr gments with h ndheld UV light source nd excise the
m with single-edged r zor
l de (see Note 23). The gel fr gments c n
e stored
for sever l d ys t 4C
efore use. 13. Completely melt the fr gments cont ining

nds t 68C for 515 min nd extr ct the DNA extr cted with phenol, phenol:chlorof
orm:iso myl lcohol (25:24:1) nd then chloroform:iso myl (24:1) (see Notes 17
nd 24). 14. Add one-tenth volume of 2M sodium cet te pH 5.0 nd 2 vol of ice-co
ld 100% (v/v) eth nol to precipit te the queous ph se from the fin l extr ction
(see Note 25). 15. Incu
te these precipit tions t 80C or on dry ice for t le s
t 20 min nd microfuge to pellet the DNA (see Note 26). 16. W sh the pellet with
500 L ice cold 70% (v/v) eth nol to remove s lt nd ir dry the pellet (see Note
27). 17. Redissolve the pellets in 20 L deionized w ter. 18. Perform fr gment qu
ntit tion
y lo ding 2 L of e ch product onto 1% (w/v) g rose gel long with
500 ng l m
d HindIII m rkers. Comp rison of the ethidium
romide st ining inten
sities nd the knowledge of the size (thus intensity) distri
ution in the l dder
will llow rough qu ntit tion of the ng mount of product in 2 L (see Note 28)
.

3.1.4. Lig tion, Tr nsform tion nd Sequencing

1. For e ch t rget molecule, com
ine 50 ng of dephosphoryl ted vector with 0.3,
1, or 3M equiv lents of insert nd 1 L T4 lig se nd 2 L 5X lig se
uffer (see Not
e 29). 2. Also set up vector only re ction cont ining no insert (see Note 30).
3. Incu
te the lig tion re ctions t 16C overnight nd store for less th n 1 wk
t 4C (see Note 31). 4. Tr nsform 2 L e ch lig tion into su
cloning efficiency DH
5- E. coli nd pl te on LB g r continuing the ppropri te nti
iotic or selectin
g gent. Follow the m nuf cturers directions or st nd rd protocol.
TCR Genetic V ccin tion
383
5. Pick re son
le num
er (>10) of colonies from the pl te(s) nd grow 3 mL
overnight culture (see Note 32). Freeze 450 L of e ch culture with 50 L glycerol i
n sterile microfuge tu
e nd store t 80C. 6. Perform Qi gen minipreps on the cl
on l cultures following the m nuf cturers protocol. 7. Digest e ch DNA with cloni
ng restriction enzymes to confirm insert lig tion. 8. Sequence clones with corre
ct insert with ppropri te primer(s) using the PerkinElmer/Cetus Dye Termin tor
Cycle Sequencing Kit to confirm n open re ding fr me from the expected V-
et g
ene f mily (follow m nuf cturers protocol). 9. L rge-sc le prep r tion of v ccine
for hum n use is
eyond the scope of this ch pter, nd requires the v il
ilit
y of gu nosine monophosph te (GMP) f cility with extensive s fety profiles, sh
elf st
ility, nd other ev lu tions. V ccines destined for use in mice re prep
red with Qi gen tip-500 kit (Qi gen, V lenci , CA).
An ex mple of TCR DNA v ccine is shown in Fig. 2.
3.1.5. Assessment of Expression
y Northern Blot An ex mple of expression n lys
is
y Northern Blot is shown in Fig. 3.
1. Tr nsfect RD cells (25) with pl smid DNA using lipofect mine ccording to the
m nuf cturers protocol. 2. H rvest the cells 48 h post-tr nsfection nd isol te
RNA using Ultr -Spec Re gent ccording to the m nuf cturers protocol (Biotecx L

or tories, Houston, TX). 3. Digest 20 g of RNA with DNAse nd n lyze it
y North
ern
lot n lysis using the form ldehyde-gel procedure of M ni tis et l. (26).
4. L
el pro
e comprised of the cloned TCR-V13.1 insert (excised
y restriction
digestion nd low melting temper ture gel purific tion) with 32P using the Meg
prime Kit ccording to the instructions; hy
ridize the
lot t 65C for 16 h. 5. W
sh the
lots s descri
ed (26) nd expose them to Hyperfilm-MP (Amersh mPh rm c
i Biotech, Pisc t w y, NJ) for 224 h.
3.2. Immuniz tion
1. Inject clinic l-tri l p tients intr muscul rly every 24 wk with DNA v ccine fo
rmul ted in
uffer with
upiv c ine (see Note 33).
3.3 Immune Response Assessment 3.3.1. Prolifer tion Ass y for TCR DNA V ccine To
ch r cterize the lymphocyte prolifer tion response induced
y genetic v ccin ti
on, use n ss y with PBMCs collected
y the Ficoll-Hyp que purific tion protoco
l (Ph rm ci , Pisc t w y, NJ). Expose PBMCs to homologous EBVtr nsformed B cells
th t h ve
een lo ded with v rious peptides. These peptides represent p rticul
r epitopes found in the specific V-
et ntigen. Assess prolifer tion in respons
e to these epitopes
y incorpor tion of triti ted thymidine.
384
Godillot et l.
Fig. 2. Design of the GENEVAX-TCR-V13.1 vector. The sequence of the V13.1 insert cl
oned from p tient JS is shown
elow the di gr m of the vector.

TCR Genetic V ccin tion

385
Fig. 3. Detection of V13 mRNA in GENEVAX-TCR-V13.1 tr nsfected cells. Northern
lot
n lysis w s performed on RNA isol ted from RD cells tr nsfected with GENEVAX-TC
R-V3 (l nes 1 nd 2), GENEVAX-TCR-V13.1 (l nes 3 nd 4), or mock tr nsfected (l ne
5). The positions of the RNA molecul r weight m rkers re shown. The RNA w s hy

ridized to V13 intern l pro
e.
1. Prep re EBV-immort lized B cells from the p tient nd m int in them for sever
l weeks
efore doing the cellul r immune response ss ys (see Note 34). Resuspe
nd 5 106 PBMCs in 5 mL medium nd n equ l volume of cell-free supern t nt from
B95-8 cells. M int in this culture until cell growth is pp rent; feed the cells
with medium lone through t le st three p ss ges. Resuspend these tr nsformed
B cells (XX EBV) t 7.5 106/mL nd incu
te them with 100 g/mL of the peptide mix
ture, or with individu l peptides, or no peptide for 46 h. Irr di te with 10,000
r ds (see Note 35). 2. Isol te PBMCs
y Ficoll-Hyp que sep r tion s descri
ed
nd freeze them in 10% (v/v) DMSO/HS in liquid nitrogen until needed. 3. Th w the
PBMCs, w sh them 2 times with 4% (v/v) HS, resuspend them t concentr tion of
106/mL nd prep re liquots of 100 L/well in 96-well round
ottom pl te (see No
te 36). 4. Prep re liquots of EBV-immort lized B cells (EBV B cells) t 106/mL
t 100 L/well in 96-well round-
ottom pl te then tr nsfer them to the PBMC pl te.
For neg tive nd positive controls, dd 100 L/well of medium or 20 U/mL IL2/4 re
spectively.
386
Godillot et l.
5. Incu
te pl tes for 3 d in CO2 incu
tor t 37oC nd dd 1 Ci 3H-thymidine i
n 20 L 10% (v/v) HS to e ch well (see Note 37). Incu
te the pl tes overnight (182
0 h). 6. H rvest the pl tes the next morning with n utom tic 96-well h rvester
nd me sure the mount of incorpor ted triti ted thymidine in liquid scintill
tion counter (see Note 38).
3.3.2. Cytotoxic T-Lymphocyte Ass y for TCR DNA V ccine Clinic l Tri l
Ch r cterize the cytotoxic T lymphocyte (CTL) response induced
y genetic v ccin
tion using CTL ss y with PBMCs collected
y Ficoll-Hyp que gr dient centrifu
g tion nd previously derived EBV-tr nsformed B cells from the p tient. Use se
t of peptides to ssess CTL ctivity g inst the V gene. An ex mple of d t resu
lting from CTL ss ys is shown in Fig. 4.
3.3.2.1. PREPARATION OF EFFECTOR CELLS
1. Isol te PBMC
y Ficoll-Hyp que gr dient centrifug tion s
ove (Su
he ding 3
.1.1.) nd freeze them in 10% (v/v) DMSO/HS in liquid nitrogen. 2. R pidly th w
the effector cells (~2 107 PBMCs) in 37C w ter
th, w sh them twice with 10 mL
medium to remove DMSO, recount the cells with hem cytometer nd culture them
t 5 105/mL with 5 g/mL PHA for 48 h (see Note 39). 3. Prep re Stimul tors: on d
y two, fter PBMCs re th wed, incu
te EBVimmort lized B cells from the p tient
(XX EBV) t 7.5 106/mL with 100 g/mL of peptide mixture for 46 h, then irr di te
with 10,000 r ds
efore using them s stimul tor cells (see Note 40). Count the
cells in the effector culture lso. 4. W sh the effector cells twice with medium
nd resuspend in medium cont ining 10 U/mL IL2 nd 10 g/mL peptide mixture. Mix
the stimul tors with the effectors t r tio of 1:20 (stimul tor:effector) nd
incu
te the mixture for
out 10 d nd then count the cells. Exp nd the cells

y feeding them with medium cont ining 10 U/mL IL2 nd 10 g/mL peptide mixture unt
il sufficient cells re v il
le for the lysis re ctions. Sep r te the he lthy
restimul ted cells
y FicollHyp que centrifug tion, count them nd then use in
CTL ss y (see Note 41).

3.3.2.2. LYSIS REACTIONS

1. In the morning of the d y on which the lysis re ction will
e run, resuspend
XX EBV cells in 7.5 106/mL cont ining 100 g/mL of peptide mixture, or individu
l peptides, or no peptide, nd incu
te t 37C for 2 h. Spin down the cells, pour
off the supern t nt nd resuspend the cells
y gentle pipetting up nd down. (D
o not exceed volume of 500 L t this point). Add 20 Ci of 51Cr to the cells, inc
u
te nother 1.5 h t room temper ture, w sh 3 times, nd resuspend the 51Cr-lo
ded t rget cells t 105/mL in medium (see Note 42). Do not w sh these t rget ce
lls until it is time to use them.
TCR Genetic V ccin tion
387
Fig. 4. Induction of cytotoxic T-cells
y DNA-
sed v ccin tion. The % specific
lysis (me n  SD of triplic te wells) is shown for sever l effector:t rget r tios.
(A) Preimmune PBMC re comp red with those o
t ined t week 5 following the ini
ti l v ccin tion. The t rgets were EBV-APC co ted with mixture of ll four V13d
erived peptides. (B) Preimmune PBMC re comp red with those o
t ined t week 5
nd week 22 following the initi l v ccin tion. The t rgets were EBV-APC co ted wi
th the CDR III peptide. 2. Resuspend the effector cells t concentr tion of 1
107/mL nd put 100 L medium into columns 412 of 96-well round-
ottom pl te (see
Note 43). 3. Put 100 L liquots of cells in columns 1-6 effecting 2-fold diluti
on of cells in columns 4,5 nd 6; wells 13, 46, 79, nd 1012 re triplic tes (see No
te 44). E ch row cont ins different effector s mples. Devote one row for e ch ty
pe of t rget cell for estim tes of minimum spont neous rele se nd m ximum rele
se. To me sure minimum rele se, dd 100 L of medi to wells 16 of one row. To me s
ure m ximum rele se, dd 100 L of poptosis lysis
uffer to wells 712 of th t row
(see Note 45). 4. M ke su
sequent 1:2 dilutions of effectors
y mixing columns 4
-6 nd tr nsferring 100 L to columns 7-9. Avoid the minimum/m ximum row in these
effector dilution steps. 5. Repe t step 4 from wells 79 to 1012 nd then disc rd 1
00 L from the mixed wells 1012. Thus, ll wells h ve volume of 100 L. 6. Add 100 L
of t rget cells (105/mL) to ll wells including those for minimum nd m ximum r
ele se estim tes. The result nt effector:t rget r tios re 100:1, 50:1, 25:1, 12
.5:1. Centrifuge the pl te t 800 rpm (160200g) for 2 min (see Note 46). 7. Incu

te the effector nd t rget cells t 37C in CO2 incu
tor, remove 50 L liquot
of supern t nt t the time-points 5 h nd 16 h respectively (see Note 47).
388
Godillot et l.
8. Mix 150 L of Optiph se Supermix (W ll c, UK) with 50 L supern t nt in W ll c
96-well s mple pl te nd cover the pl te with se ling t pe. Count the pl te(s) i
n liquid scintill tion counter (W ll c Inc., G ithers
urg, MD) (see Note 48).
9. Percent specific lysis equ ls 100 [(experiment l rele sespont neous rele se)/
(m ximum rele sespont neous rele se)].
3.3.3. Anti-DNA ELISA
To ch r cterize the immune response to DNA induced
y genetic v ccin tion, colle
ct the serum s mples from su
jects inocul ted with V-expressing DNA pl smids nd
n lyze nti-DNA nti
odies
y ELISA.
1. Tre t microtiter pl tes t room temper ture for 90 min with 200 L/well of 0.00
01% (w/v) prot mine sulf te solution. Prep re this solution just
efore use
y d
ilution from 0.1% (w/v) stock into dou
le-distilled H2O. W sh 3X under the dei
onized w ter t p nd empty nd sl p dry on p per towel (see Note 49). 2. Co t mi
crotiter pl tes with 50 L/well of pl smid DNA t concentr tion of 3 g/mL in DNA
diluent. Co t pl te with 1% (w/v) BSA in DNA diluent s neg tive control. Pl
ce the pl tes t 4C in se led moist cont iner t le st overnight or until re d

y for use (see Note 50). W sh the pl tes 3 times with TBS0.05% (v/v) Tween-20. 3
. Add
locking
uffer, 200 L per well (see Note 51). Incu
te t 37C for 1 h. Empt
y nd sl p dry on p per towel. 4. Add 50 L of diluted serum (diluted in
lockin
g
uffer) to e ch well (see Note 52). Incu
te t 4C O/N (or t 37C for 1 h). W sh
pl te 6 times with TBS0.05% (v/v) Tween-20. 5. Add 50 L/well of nti-hum n Ig co
njug ted to peroxid se diluted 1:8,000 in
locking
uffer. Incu
te t 37C for 1.
5 h. W sh pl te 8 times with TBS-0.05% (v/v) Tween-20. 6. Dissolve 1 mg of TMB i
n 10 mL of su
str te
uffer. Pl ce 100 L liquots in e ch well nd incu
te in th
e d rk t room temper ture until the
lue color develops (see Note 53). 7. Add 2
0 L of 2M H2SO4 to e ch well to stop the re ction (see Note 54) nd re d the pl t
e t 450 nm on micropl te re der.
3.3.4. V-Bet ELISA
To ch r cterize the immune response induced
y genetic v ccin tion, perform enzy
me-linked immuno ss y n lysis (ELISA) of ser s mples collected from su
jects i
nocul ted with V-expressing DNA pl smids. An ex mple of d t resulting from ntiV ELISA n lysis is shown in Fig. 5.
1. Co t microtiter pl tes with 50 L/well of peptide ntigen t concentr tion of
10 g/mL in 50 mM NH4HCO3. Co t one pl te with 50 mM NH4HCO3 s control (see No
te 55). Pl ce the pl tes in 37C dry incu
tor to dry for 4 h or overnight.
TCR Genetic V ccin tion
389
Fig. 5. Humor l response to DNA-
sed v ccin tion. ELISA wells were co ted with
peptides sp nning the CDR1 region of the V13 T-cell receptor. Serum w s ss yed f
or
inding to this peptide
y ELISA ss y. The OD 450 nm refers to the
inding t
o the peptide on ELISA minus the
inding to control wells with no peptide (me n
of duplic te wells). The times of v ccin tion re indic ted with rrows. One of
sever l ss ys, ll showing simil r trends, is shown.
2. Add 200 L per well of
locking
uffer. Incu
te the pl te t 37C for 1 h. Pour
off the
locking
uffer. 3. Add 50 L of diluted serum (diluted in
locking
uffer
) to e ch well (see Note 56). Incu
te the pl tes t 4C overnight (or t 37C for 1
h). W sh the pl tes 6 times with PBS-0.05% (v/v) Tween-20 (see Note 57). 4. Add
100 L/well of peroxid se-conjug ted nti-hum n IgG diluted 1:8,000 in
locking

uffer s recommended. Incu
te the pl tes t 37C for 1 h. W sh the pl tes 8 times
with PBS-0.05% (v/v) Tween-20. 5. For isotype-specific responses dd 100 L/well
of
iotin-conjug ted nti-hum n IgG1, 2, 3, 4, or IgM diluted in
locking
uffer
. Incu
te the pl tes t 37C for 1 h then w sh them 6 times with PBS-0.05% (v/v)
Tween-20. 6. Ass y of isotype-specific responses (skip this step for non-isotype
-specific ELISA): dd 100 L/well of 0.1 g/mL Avidin-HRP. Incu
te the pl tes t 37C
for 1 h, w sh them 8 times with PBS-0.05% (v/v) Tween-20.
390
Godillot et l.
7. Dissolve 1 mg of TMB in 10 mL of su
str te
uffer. Aliquot 100 L to e ch well
nd incu
te in the d rk t room temper ture until the
lue color developed (see
Note 53). 8. Add 20 L of 2M H2SO4 to e ch well to stop the re ction (see Note 54
) nd re d the pl te t 450 nm on micropl te re der (Dyn tech MR5000).
4. Notes
1. Figs. 1 nd 2 show the over ll design of the DNA v ccine vector. 2. Blood dil
ution is necess ry for good yields of PBMCs. 3. Ficoll-Hyp que is stored t 4C
u
t should
e
rought to room temper ture prior to use. Overl ying should
e done
gently to void mixing with Ficoll. Underl ying of the Ficoll lso works well. D
o not git te the tu
es upon tr nsport to nd from the centrifuge s this m y re

duce the yield of PBMCs. 4. The centrifug tion step must
e done t room temper
ture to void cooling of the Ficoll. T ke time to w rm up refriger ted unit if
th t is ll th t is v il
le. (RCF(g) = 1.12r (rpm/1000)2 where r is the r diu
s of the rotor expressed in millimeters). 5. The l yer of PBMCs t the gr dient
interf ce should
e slowly nd thoroughly pipetted out of the tu
e. If too gre t
n mount of Ficoll is collected with the cells, the volume of the w sh should

e djusted to insure pelleting of cells. 6. The tre tment of cell s mples t th
is step v ries depending on their intended use. For cloning purposes, dry pellet
s of 510 million cells should
e stored t 80C. 7. For immune response ss ys cells
should
e resuspended t 107/mL in freezing medi nd frozen in 1 mL liquots.
8. The prop nol
th insul tes the cryovi ls nd thus f cilit tes slow ste dy
cooling to 80C. This enh nces cell vi
ility upon th wing. The prop nol
th shoul
d
e left in the 80C for t le st 4 h nd c n
e left t 80C for up to 2 d nd then
tr nsferred to liquid nitrogen. 9. Use enough cells! We find th t 5 106 cells yi
eld consistently high-qu lity cDNA. More re even
etter
ut
e sure to sc le up
the RNA prep r tion re gents. 10. Use st nd rd RNAse-free prec utions. Mix comp
letely to ensure th t ll cells re lysed. As with ll viscous solutions of m cr
omolecules,
u

les or fo ming tend to diminish product yield. 11. Do not overdr
y the product RNA s it
ecomes very difficult to dissolve. 12. Add reverse tr n
script se re gents in the order listed to protect the RNA from RNAse nd to prov
ide
uffered environment for the M-MLV-RT. 13. The polymer se ch in re ction
mplific tion of ny p rticul r DNA segment should
e optimized to yield produc
t th t cont ins minim l
ckground mplific tion products
ut is lso sufficient
in qu ntity. Optimize the nne ling temper ture first s this ffects the purit
y of the product gre tly. We recommend trying 40, 45, 50, 55, 60, nd 65C. Higher
temper tures gener lly yield less
ckground mplific tion. 14. Primer design i
s
sed on simple principles using the le der peptide sequence or the m ture pro
tein mino terminus nd going through to the end of the coding
TCR Genetic V ccin tion
391
15.
16. 17. 18. 19. 20.
21. 22. 23.
24. 25. 26.
27. 28. 29. 30.
31. 32. 33.
region or (if the CDR III is going to
e included), using the 5 end of the cons
t nt region. The complete sequence of the hum n V gene locus h s
een pu
lished
nd is v il
le from GenB nk (27). Another good source of sequence inform tion c
omes from K
t (28). Miner l oil is required to prevent ev por tion of the re c
tion during prolonged he ting. Avoid collecting miner l oil with your queous PC
R product
y t king only 80 out of 100 L nd lso
y wiping the tip with cle n
p per towel. He t in ctiv tion protects the restriction enzymes th t re used in
the su
sequent step from protein se K. Microfuge the phenol/chloroform/iso myl
lcohol extr ction for 5 min t full speed. Only 1 min is required for the chlor
oform/iso myl extr ction. Precipit tion should
e
y incu
tion t 80C for t le s
t 20 min. The restriction enzymes will v ry depending on the vector used. Dephos
phoryl tion reduces the ch nce th t incompletely digested comp ti
le sticky ends
on the vector will medi te self-lig tion nd contri
ute to f lse positive sig
n l following tr nsform tion. C re should
e t ken when h nding low-melting temp
er ture (LMT) gels s they re more fr gile th n regul r g rose gels. Allow mor

e time for LMT gel to solidify. Do not exceed 75 V s the gel m y
e he ted

ove its melting temper ture. T ke prec utions to void cont min ting the gel sli
ces. Use f ce shield for UV protection. Long-w ve UV is less d m ging to DNA
nd molecul r
iologists th n short w ve. C refully inspect the tu
es to insure t
h t the slices re completely melted. Me sure the volume of the fin l queous ph
se with n ppropri te pipet tip to c lcul te precipit ting gents. Orient te t
he tu
es in the microfuge so th t the pellet (if visi
le) will
e expected t
cert in spot within the tu
e. For ex mple; the pellet will
e found
elow the hi
nge of the tu
e if the hinge is oriented tow rd the outside in the rotor. If sol
ids re seen suspended in the w sh it will
e necess ry to microfuge g in
efor
e removing the w sh fluid. A n nogr m qu ntity c n
e ssigned to e ch of the l
dder
nds ccording to the fr ction of the whole l m
d molecule e ch em
odies.
One mol r equiv lent of 50 ng of 3000-
p vector is 16.66 ng of 1000-
p inse
rt. Approxim tions re ccept
le here. This vector-only re ction is n import n
t control for the lig tion/tr nsform tion experiment. The num
er of
ckground c
olonies derived from this re ction upon lig tion re su
tr cted from the num
er
of colonies from n insert-cont ining lig tion to determine on co rse level wh
ether the lig tion/tr nsform tion worked. Immedi te tr nsform tion of the lig ti
on re ctions (i.e., within one d y) seems to work
est. Ten to 20 colonies per t
rget molecule should yield suit
le clone. Too few hum n experiments re v i
l
le to provide ny useful hints. We lw ys formul te our v ccines with
upiv c
ine s this enh nces muscle cell upt ke nd
392
Godillot et l.
expression of the encoded gene. Injections m y
e delivered into the deltoid or
glute l muscles following sw

ing the skin with n lcohol wipe. 34. EBV-immort
lized B cells from the p tient must
e prep red sever l weeks
efore doing the
cellul r immune response ss ys. The sequences of the peptides we used were: CDR
I: CDR II: HV IV: CDR III: NAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSC RQDPGMGLRLIHYSVGA
GITDQGEVC PNGYNVRSTTEDFPLRLLSAAPSQTSVC YFCASSFPRQPSYNEQFFGPGTRLTVLC
35.
36.
37.
38. 39.
40.
41.
42.
43.
44. 45.
These peptides h ve n dded C-termin l cysteine th t is not p rt of the n tive
sequence. 10,000 r ds will require different mounts of time depending on the ir
r di tor source. Consult ppropri te sources. If no irr di tor is v il
le, one
c n tre t the cells (106/mL) with 25 g/mL mitomycin C (Sigm C t. No. M0503). Mi
tomycin must
e completely w shed w y (3 w shes)
efore the cells c n
e used.
Determine the num
er of pl tes needed nd therefore the num
er of cells nd ll
other re gent qu ntities
efore st rting this experiment. Proper pl nning will m
ke it much e sier. Add the triti ted thymidine c refully in work
le volume (

20 L) with multich nnel pipettor. Use proper s fety techniques for h ndling 3Hthymidine. Tritium cont min tion of the work re c n only
e detected
y wipe t
ests followed
y scintill tion counting. H rvesters nd counters will v ry. When
first ttempting CTL ss y 2 107 of e ch PBMC s mple should
e th wed s effe
ctors for e ch intended t rget. R pidly remove the DMSO
y w shing
ec use it is
toxic to the cells. E ch stimul tion culture will only require 34 million stimul
tor cells. When first doing the ss y, use 3.75 million (0.5 mL) EBV-tr nsforme
d cells with e ch peptide. Experience will llow you to reduce this mount nd t
hus conserve peptide. Remov l of de d cells from the stimul tor culture is neces
s ry to prevent the de d stimul tors from competing with t rgets for the inter c
tion with cytotoxic effectors. It lso m kes counting much e sier. C lcul te the
num
er of t rget cells necess ry for your ss y nd lo d 2550% more cells th n y
ou need with peptide nd 51Cr. This will minimize the w ste of peptide. E ch s m
ple of effector (PBMC) will
e used for one row of pl te for e ch t rget used.
E ch full pl te will get 9.6 105 t rgets. Be cert in th t ny cells to
e liqu
otted into pl te re thoroughly mixed. Minimum spont neous rele se nd m ximum
rele se should
e done in triplic te or more. Often we do six wells of e ch s
this t kes one complete row of the pl te. We usu lly dilute the effectors in col
umn 4 into column 7, 5 to 8, nd 6 to 9 s convention. By the s me convention
dilute 7 to 10, 8 to 11, nd 9 to 12 with the 8-ch nnel pipettor.
TCR Genetic V ccin tion
393
46. Centrifug tion enh nces the requisite cell-cell cont ct. A 200:1 r tio m y p
rovide useful d t if the v il
le num
er of cells llows. 47. S mpling of the
CTL supern t nt must
e done with ste dy h nd. The multich nnel pipettor is he
ld t 45 ngle nd tips re immersed
out h lfw y into the liquid in the well.
Remove supern t nts slowly. If ir is withdr wn do not mix the wells
y dding n
y of the solution
ck to the wells for more even s mpling. Note the
d s mpl
e nd move on. Pr ctice with non-r dio ctive w ter (200 L/well). 48. The scintill
tion fluid is dded to the supern t nts nd then mixed
y sh king fter the pl
te is covered with the se ling t pe. Counting pp r tus v ry. 49. The prot mine
sulf te solution confers positive ch rge to the pl te for eventu l DNA dhesio
n. Poly-L lysine is sometimes used inste d. 50. DNA/BSA pl tes c n
e stored t
4C for up to one month provided they do not dry out. 51. This step
locks nonspec
ific sites. 52. Dilution of ser c n
e done in the pl te. Triplic tes of diluti
on from 25: to 200:1 cross the pl te re often used. 53. W tch closely for the

lue color to develop (usu lly 560 min). 54. If the
lue color is f int, llow mo
re time for its development. However, eventu lly ddition l incu
tion will o
sc
ure me ningful differences
etween wells. 55. The pl te without peptide is use
ful control to confirm the specificity of ntiV
et ctivity in the ser . This p
l te should
e used in p r llel with the specific peptide cont ining pl te(s). 5
6. We find th t dilutions r nging from 10:1 to 1000:1 work well in screening
ss y. Endpoint titer is determined in su
sequent ss y
y seri l two-fold dilu
tions st rting t dilution th t gives positive result in the screening ss y
. 57. Disc rding m teri l from ELISA pl tes is done with flick of the wrist in
to w ste sink followed
y sever l sl ps of the pl te into p per towel. This
procedure ssures th t su
sequent ddition to the pl te will not
e unduly dilut
ed nd thus incre ses the effectiveness of w shes.
Acknowledgments Dr. Lessin w s supported
y n NIH gr nt CA 55017 nd VA c reer
development w rd RA1710. Dr. Willi ms nd this m nuscript were supported
y n
FDA orph n gr nt FD-R-001149-01. We would like to cknowledge the excellent tech
nic l nd intellectu l support from Bernice Benoit, Wei C i, D n McC llus, Felic
i W tson, Henry Klepser, Leslie Coney, nd Apollon Inc. References
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issel, M. S. (1992) V(D)J recom
in tion: Molecul r
iology nd regul tion. Ann.

Rev. Immunol. 10, 359383.

394
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nce 246, 668670. 12. V nden
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in s v ccine for B-cell lymphom . N ture 362, 755758. 15. McDonnell, W. M. nd
Ask ri, F. K. (1996) DNA v ccines. N. Engl. J. Med. 334, 4245. 16. W ng, B., Uge
n, K. E., Srik nt n, V., Ag dj ny n, M. G., D ng, K., S to, A. I., Ref eli, Y.,
Boyer, J., Willi ms, W. V., nd Weiner, D. B. (1993) Gene inocul tion gener tes
immune responses g inst hum n immunodeficiency virus type 1. Proc. N tl. Ac d.
Sci. USA 90, 41564160. 17. P rdoll, D. M. nd Beckckerleg, A. M. (1995) Exposing
the immunology of n ked DNA v ccines. Immunity 3, 165169. 18. W ism n, A., Ruiz,
P. J., Hirsch
erg, D. L., Gelm n, A., Oksen
erg, J. R., Brocke, S., Mor, F., Coh
en, I. R., nd Steinm n, L. (1996) Suppressive v ccin tion with
TCR Genetic V ccin tion
395
19. 20.
21.
22.
23.
24.
25. 26. 27. 28.
DNA encoding v ri
le region gene of the T-cell receptor prevents utoimmune e

nceph lomyelitis nd ctiv tes Th2 immunity. N t. Med. 2, 899905. Syrengel s, A.

D., Chen, T. T., nd Levy, R. (1996) DNA immuniz tion induces protective immunit
y g inst B-cell lymphom . N ture Genetics 2, 10381040. R jotte, D., H dd d, P.,
H m n, A., Cr goe, E. J., Jr., nd Ho ng, T. (1992) Role of protein kin se C nd
the N +/H+ ntiporter in suppression of poptosis
y gr nulocyte m croph ge col
ony-stimul ting f ctor nd interleukin-3. J. Biol. Chem. 267, 99809987. Zwillich,
S., F ng, Q., Kie
er-Emmons, T., VonFeldt, J. M., Monos, D., R m nuj m, T., W n
g, B., Weiner, D. B., nd Willi ms, W. V. (1994) V lph gene us ge in rheum toi
d comp red with osteo rthritic synovi l T cells. DNA Cell Biol. 13, 923931. Willi
ms, W. V., Kie
er-Emmons, T., F ng, Q., Von Feldt, J., W ng, B., R m nuj m, T.,
nd Weiner, D. B. (1993) Conserved motifs in rheum toid rthritis synovi l tiss
ue T cell receptor
et ch ins. DNA Cell Biol. 12, 425434. Willi ms, W. V., F ng,
Q., Dem rco, D., VonFeldt, J., Zurier, R. B., nd Weiner, D. B. (1992) Restrict
ed heterogeneity of T cell receptor tr nscripts in rheum toid synovium. J. Clin.
Invest. 90, 326333. Crowe, J. S., Cooper, H. J., Smith, M. A., Sims, M. J., P rk
er, D., nd Gewert, D. (1991) Improved cloning efficiency of polymer se ch in re
ction (PCR) products fter protein se K digestion. Nucleic Acids Res. 19, 184.
Levy, D. N., Fern ndes, L. S., Willi ms, W. V., nd Weiner, D. B. (1993) Inducti
on of cell differenti tion
y HIV-1 vpr. Cell 72, 541550. S m
rook, J., Fritsch,
E. nd M ni tis, T., eds., (1989) Molecul r Cloning. A L
or tory M nu l. Cold S
pring H r
or L
or tory Press, Cold Spring H r
or, NY. Rowen, L., Koop, B. F.,
nd Hood, L. (1996) The complete 685-kilo
se DNA sequence of the hum n T cell re
ceptor locus. Science 272, 17551762. K
t, E. A., ed. (1991) Sequences of protei
ns of immunologic l interest. U.S. Dep rtment of He lth nd Hum n Services, Beth
esd , MD.
34
Immunity to T-Cell Receptor
Suppressive V ccin tion with DNA Encoding V ri
le Region Gene of the T-Cell R
eceptor
Ari W ism n 1. Introduction A common theme found in studies with mouse models of
utoimmune dise ses is th t p thogenic T cells re prim rily responsi
le for th
e p thology. Such models include di
etes in non-o
ese di
etic (NOD) mice, expe
riment l utoimmune enceph lomyelitis (EAE) model of multiple sclerosis (MS), co
ll gen II (CII)-induced rthritis (CIA) model of rheum toid rthritis (RA). P th
ogenic T cells in EAE utilize restricted repertoire of genes encoding the T-ce
ll receptor (TCR) (1). For ex mple, upon immuniz tion of H-2u mice with either m
yelin
sic protein, or its immunodomin nt fr gment, peptide Ac1-20, the V8.2 TCR
gene product is expressed in the m jority of p thogenic T cells (24). The restri
cted us ge of the V8.2 TCR gene product h s lso
een found in r ts in which EAE
w s induced
y peptide of myelin
sic protein (5). Simil rly, restriction i
n the TCR us ge w s found lso in CIIinduced rthritis in mice (6,7), nd in the
TCR lph ch in us ge in non-o
ese di
etic (NOD) mice (8). Restriction in the
TCR gene us ge
y T cells isol ted from hum n MS p tients h s lso
een determin
ed (9). To lesser extent, some TCR
et genes were expressed in T cells implic
ted in p thogenesis of type 1 di
etes (10) nd RA (11). Two str tegies h ve
e
en utilized to ther peutic lly t rget these p thogenic T cells in highly speci
fic m nner. The dministr tion of monoclon l nti
odies directed to p thogenic V
gene products (3,12) nd T cell v ccin tion with peptides from the second or th
ird complement rity determining regions of the
From: Methods in Molecul r Medicine, vol. 29, DNA V ccines: Methods nd Protocol
s Edited
y: D. B. Lowrie nd R. G. Wh len Hum n Press Inc., Totow , NJ
397
398
W ism n
p thogenic TCR V region h ve
oth proven successful in the ther py of EAE (13,14

). A simil r ppro ch h s lso proved to
e successful in CII induced rthritis

(15,16). We h ve descri
ed the prevention of EAE
y injection of pl smid DNA enc
oding the V8.2 region of T-cell receptor th t is critic l for the p thogenesis
of the dise se (17). This is novel demonstr tion of suppressive v ccin tion, n
ppro ch th t is in contr st to wh t is norm lly desired when v ccin ting for im
munity to micro
es. We h ve shown th t the mech nism of DNA v ccin tion g inst
the p thogenic TCR V gene segment, does not involve the depletion of these p tho
genic T cells, nor does it push them into nergy. Inste d, we found th t DNA v c
cin tion promoted shift in the p ttern of cytokines produced
y the p thogenic
T cells. The T cell popul tions no longer produced p thogenic cytokines like -in
terferon and IL-2, cytokines that define a Th1-type response (18,19). Instead, t
he suppressive cytokine IL-4, which characterizes a Th2 response, was produced.
This shift towards Th2 immunity had not
een seen previously with DNA vaccinatio
n for a micro
ial protein (20). This novel mechanism, induced
y DNA vaccination
to a ene encodin a TCR varia
le reion polypeptide, may have potential effect
for the treatment of autoimmune diseases triered
y specific Th1 T cells, suc
h as dia
etes and RA. 2. Materials 2.1. Plasmid Construction
1. We used pcDNA3 from Invitroen (Carls
ad, CA). It is important that the vecto
r has a stron non-specific promoter if the DNA is injected into muscle tissue.
Alternatively, if a specific cell type is the taret of the vaccination (e.., B
cells), a specific promoter can
e employed (e.., a promoter of a B cell speci
fic ene). In addition, the vector should allow the transcription of a mature an
d sta
le mRNA,
y providin polyadenylation and transcription termination sinal
s. 2. Mea-prep kit (Qiaen, Chatsworth, CA). 3. Mini-prep kit (Qiaen). 4. QIAq
uick el extraction kit (Qiaen). 5. Poly (dT) (Boehriner Mannheim, Mannheim, G
ermany).
2.2. DNA Vaccination
1. Dissolve one milliram of cardiotoxin (Sima, St. Louis, MO) in 14.7 mL steri
le saline (0.9% (w/v) NaCl solution), filter and store in aliquots at 20C. 2. TRIz
ol: use accordin to the manufacturers instructions (Life Technoloies, Gaithers

ur, MD). 3. ABTS: use pre-prepared ABTS solution (2,2-azino-di-[3-ethyl
enzthi
azoline sulfonate) (Kirkeaard & Perry La
oratories, Gaithers
ur, MD).
Immunity to T-Cell Receptor 2.3. Detection of Anti-TCR Anti
odies
399
1. Monoclonal anti
odies directed aainst the T-cell receptor conjuated to phen
ylephrine (PE) or fluorescein isothiocyanate (FITC) (Pharminen, San Dieo, CA).
2. Goat anti-mouse immunolo
ulin conjuated to FITC (Jackson ImmunoResearch La

oratories, West Grove, PA). 3. PBS/BSA: PBS/0.1% (w/v) BSA/0.05% (w/v) azide.
2.4. Quantification of Cytokines
1. Enriched RPMI: RPMI 1640 medium supplemented with 2 mM lutamine, nonessentia
l amino acids, 1mM sodium pyruvate, 100 U/mL penicillin, 100 /mL streptomycin, 0
.25 /mL funizone (BioLa
, Jerusalem, Israel), 5 105M -mercaptoethanol (Fluka AG,
Buchs, Switzerland) and 10 mM HEPES
uffer (Sima). 2. Cytokines levels: use ant
i
ody pairs from Pharminen (La Jolla, CA).
2.5. Determinin Anti
odies Isotype
1. Maxisorp microtiter plates (Nunc, Naperville, IL). 2. Goat anti-murine IG1 o
r IG2a (Fc frament specific) conjuated to alkaline phosphatase (Southern Biot
echnoloy Associates, Birminham, AL).
3. Methods 3.1. Plasmid Construction
1. Isolate enomic DNA or cellular RNA from the spleen or peripheral
lood lymph
ocytes (see Notes 1 and 2). Synthesize first strand cDNA usin poly(dT) as a prim
er. 2. Genomic DNA or cDNA serves as template for polymerase chain reaction (PCR
) (see Note 3), with Taq polymerase and TCR specific primers (see Note 4). 3. Pu

rify the PCR products from the el, liate it with the expression vector, and tr
ansform the
acteria (see Notes 5 and 6). 4. Sequence the DNA from a
acterial c
olony with an insert in the expected lenth, to verify the insertion of the rih
t ene with an appropriate open readin frame. 5. Prepare lare quantities of pl
asmid DNA with a Mea-prep kit.
3.2. DNA Injection
1. Inject into the ti
ialis anterior muscle of the mouse 50 L of cardiotoxin solu
tion per le. The injection is done via the anterior surface of the muscle usin
a 27aue needle with a collar to limit penetration to 2 mm. One week later the
mice are ready for DNA vaccination. 2. Inject the DNA three times with weekly i
ntervals in the same manner as in step 1. Inject 50 L DNA per le (stock solution
of 1 m/mL DNA in sterile phosphate
uffered saline [PBS]). 3. Verify expression
of the injected DNA: Remove the injected muscle, and extract RNA (see Note 1).
Synthesize cDNA from the RNA, and use it as a template for PCR amplification (se
e Note 3). Sequence PCR products of the correct lenth, to confirm expression.
400 3.3. FACS Stainin
Waisman
In order to detect the presence of anti
odies that reconize the product of the
TCR used for immunization, cells that express the desired TCR can
e used (see N
otes 7 and 8).
1. Incu
ate the cells (5 105 cells/tu
e) with the sera of the immunized mice (di
luted 1:100 in PBS/BSA). Keep the cells on ice and in the dark throuhout the st
ainin. A monoclonal anti
ody that reconizes the same TCR should
e used as a p
ositive control (see Note 9). 2. Pellet the cells and wash them with cold PBS. 3
. Incu
ate the cells with oat anti-mouse I coupled to FITC for 30 min, wash an
d analyze them with a FACScan (Becton Dickinson, Rutherford, NJ).
3.4 Cytokines from Lymph Node Cells
1. Incu
ate lymph node cells (1 107) in enriched RPMI 1640 with 1% (v/v) synene
ic sera and 10  of the desired peptides (see Note 10). 2. Collect the medium 24
and 48 h later and test it for cytokines. Use the assay kits accordin to the ma
nufacturers instructions.
3.5. Anti
ody Isotype
IG1 secretion is triered
y IL-4, whereas IG2a secretion is induced
y -inter
feron. These are the main cytokines of Th2 and Th1, respectively. It is therefor
e important to assess the cytokine function in vivo
y determinin the isotype o
f the anti
odies produced
y the mice as a response to immunization.
1. Bleed the mice a
out two weeks after antien immunization. For example, we in
duced EAE
y a peptide, and tested the anti
odies directed to that peptide two w
eeks after immunization. 2. Coat Maxisorp microtiter plates with the antien (se
e Note 11). 3. Wash and
lock over-niht with 10% (v/v) FCS in PBS. 4. Incu
ate
for 90 min with the sera of the mice, diluted serially in duplicates from 1:10 t
o 1:1000. 5. Wash and incu
ate for 75 min with oat anti-mouse IG1 or IG2a con
juated to alkaline phosphatase. 6. Wash and incu
ate with ABTS. 7. Read at 405
nm usin an ELISA reader.
4. Notes
1. For extraction of enomic DNA or total RNA, we used TRIzol reaent. The reae
nt contains phenol, and therefore should
e used under a chemical hood. 2. Care
should
e taken when selectin the mouse from which the DNA is to
e prepared si
nce some mouse strains contain lare deletions in their chromosomes in the reio
n that contains the TCR enes.
Immunity to T-Cell Receptor

401
3. We amplified only the V ene-codin reion, not includin the D and the J ele
ments. In case the whole varia
le reion is needed, it is important to amplify t
he DNA from cDNA, and not from enomic DNA. 4. It is important to include an ini
tiation codon in the 5 primer that will code for methionine, in frame with the
TCR amplification product. Similarly, it is essential to introduce an in-frame t
ermination codon in the 3 primer. In addition, restriction sites should
e intr
oduced in the same primers to simplify the su
clonin process. The restriction e
nzyme sites chosen should exist in the expression vector to allow for clonin. I
n addition, it is important to verify that the selected sites are not found in t
he PCR product. As an example, we used the followin primers for the TCR V
8.2 c
lonin: 5-CCGGAATTCATGGAGGCTGCAGTCACCCAAAGC-3 and 5-TGCTCTAGATTAGCTGGCACAGAAG
TACACTGATGT-3. These primers cover the complete V reion (a
out 310
p) and inc
lude EcoRI and X
aI sites used for clonin. 5. After PCR, the DNA product of the
riht lenth should
e extracted from the aarose el (we used the Qiaen QIAqu
ick extraction kit). Prior to loadin the preparative el, cleave the DNA
y the
restriction enzymes selected. Cleave the DNA vector with the same enzymes, and
then treat with alkaline phosphatase prior to el purification. It is useful to
treat the vector with CIP
efore loadin the el, since it saves a step of pheno
l extraction. 6. After liation and transformation (21) row colonies for mini-p
reparations of DNA. This step should
e undertaken only if the
ackround (i.e.,
the num
er of colonies after self-liation of the vector) is very low. Otherwis
e it may
e hard to find a positive colony. 7. Positive T-cell populations that
express the desired TCR are sometimes hard to find. For example, we used a T-cel
l clone that was rown in the la
oratory and that expresses TCR V8.2. Another pos
si
ility is to isolate the desired cell population with a FACS sorter. 8. It is
advisa
le to work with pure T cell populations, since B cells and macrophaes wi
ll
ind immunolo
ulin in a non-specific manner throuh their Fc receptor. 9. Wh
en analyzin the sera, use normal mouse sera (from non-immunized mice) as a nea
tive control. These sera may
ind with low affinity to the T cells, and this
in
din should
e considered as the
ackround level. 10. Drainin lymph node cells
are easily visualized after immunization. In addition, spleen cells can
e used
. In some cases we have found that some cytokines (such as IL-4) are easier to d
etect from supernatants of activated spleen cells than from lymph node cells. 11
. We used 10 /mL of peptide in PBS, and coated the plates for 90 min. Different
peptides may adhere differently to the plate, and some will need to
e conjuate
d to a carrier such as BSA
efore coatin.
Acknowledments I would like to thank Dr. S. Casola and A. Gaur for comments on
the manuscript. I especially would like to thank Prof. L. Steinman for his suppo
rt.
402
References
Waisman
1. Zamvil, S. and Steinman, L. (1990) The T lymphocyte in autoimmune encephalomy
elitis. Ann. Rev. Immunol. 8, 579621. 2. Zamvil, S. S., Mitchell, D. J., Lee, N.
E., Moore, A. C., Waldor, M. K., Sakai, K., Roth
ard, J. B., McDevitt, H. O., St
einman, L., and Acha-Or
ea, H. (1988) Predominant expression of a T cell recepto
r V
eta ene su
family in autoimmune encephalomyelitis. J. Exp. Med. 167, 158615
96. 3. Acha-Or
ea, H., Mitchell, D. J., Timmermann, L., Wraith, D. C., Tausch, G
. S., Waldor, M. K., Zamvil, S. S., McDevitt, H. O., and Steinman, L. (1988) Lim
ited heteroeneity of T cell receptors from lymphocytes mediatin autoimmune enc
ephalomyelitis allows specific immune intervention. Cell 54, 263273. 4. Ur
an, J.
L., Kumar, V., Kono, D. H., Gomez, C., Horvath, S. J., Clayton, J., Ando, D. G.
, Sercarz, E. E., and H ood, L. (1988) Restricted use of T cell receptor V enes
in murine autoimmune encephalomyelitis raises possi
ilities for anti
ody therap

y. Cell 54, 577592. 5. Gold, D. P., Offner, H., Sun, D., Wiley, S., Vanden
ark, A
. A., and Wilson, D. B. (1991) Analysis of T cell receptor
eta chains in Lewis
rats with experimental alleric encephalomyelitis: Conserved complementarity det
erminin reion 3. J. Exp. Med. 174, 14671476. 6. Osman, G. E., Toda, M., Kanaaw
a, O., and Hood, L. E. (1993) Characterization of the T cell receptor repertoire
causin collaen arthritis in mice. J. Exp. Med. 177, 387395. 7. Banerjee, S., H
aqqi, T. M., Luthra, H. S., Stuart, J. M., and David, C. S. (1988) Possi
le role
of V
eta T cell receptor enes in suscepti
ility to collaen-induced arthritis
in mice. J. Exp. Med. 167, 832839. 8. Simone, E., Daniel, D., Schloot, N., Gottl
ie
, P., Ba
u, S., Kawasaki, E., Wemann, D., and Eisen
arth, G. S. (1997) T cel
l receptor restriction of dia
etoenic autoimmune NOD T cells. Proc. Natl. Acad.
Sci. USA 94, 25182521. 9. Oksen
er, J. R., Panzara, M. A., Beovich, A. B., Mit
chell, D., Erlich, H. A., Murray, R. S., Shimonkevitz, R., Sherritt, M., Roth
ar
d, J., Bernard, C. C., and Steinman, L. (1993) Selection for T-cell receptor V-D-J
ene rearranements with specificity for a myelin
asic protein peptide in
rain
lesions of multiple sclerosis. Nature 362, 6870. 10. Naserke, H. E., Durinovic-B
ello, I., Seidel, D., and Zieler, A. G. (1996) The Tcell receptor
eta chain CD
R3 reion of BV8S1/BJ1S5 transcripts in type 1 dia
etes. Immunoenetics 45, 8796.
11. Waase, I., Kayser, C., Carlson, P. J., Goronzy, J. J., and Weyand, C. M. (1
996) Olioclonal T cell proliferation in patients with rheumatoid arthritis and
their unaffected si
lins. Arthritis Rheum. 39, 904913. 12. Sakai, K., Sinha, A.
A., Mitchell, D. J., Zamvil, S. S., Roth
ard, J. B., McDevitt, H. O., and Steinm
an, L. (1988) Involvement of distinct murine T-cell receptors in the autoimmune
encephalitoenic response to nested epitopes of myelin
asic protein. Proc. Natl
. Acad. Sci. USA 85, 86088612.
Immunity to T-Cell Receptor
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13. Vanden
ark, A. A., Hashim, G., and Offner, H. (1989) Immunization with a syn
thetic T-cell receptor V-reion peptide protects aainst experimental autoimmune
encephalomyelitis. Nature 341, 541544. 14. Howell, M. D., Winters, S. T., Olee,
T., Powell, H. C., Carlo, D. J., and Brostoff, S. W. (1989) Vaccination aainst
experimental alleric encephalomyelitis with T cell receptor peptides. Science 2
46, 668670. 15. Moder, K. G., Luthra, H. S., Griffiths, M., and David, C. S. (199
3) Prevention of collaen induced arthritis in mice
y deletion of T cell recept
or V
eta 8
earin T cells with monoclonal anti
odies. Br. J. Rheumatol. 32, 263
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urine collaen-induced arthritis with T cell receptor V
eta-specific anti
odies
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Gelman, A., Oksen
er, J. R., Brocke, S., Mor, F., Cohen, I. R., and Steinman,
L. (1996) Suppressive vaccination with DNA encodin a varia
le reion ene of th
e T-cell receptor prevents autoimmune encephalomyelitis and activates Th2 immuni
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cells: different patterns of lymphokine secretion lead to different functional
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rook, J
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35
DNA Fusion Vaccines Aainst B-Cell Tumors
Delin Zhu, Myfanwy B. Speller
er, Catherine A. Kin, Jason Rice, Andrew R. Thom
psett, and Freda K. Stevenson 1. Introduction The a
ility of naked DNA to induce
immune responses aainst encoded antien has
een clearly demonstrated for infe
ctious diseases (1). In many cases, the induced immunity is a
le to protect aai

nst infection, and can approach the efficacy of exoenous antien (2). For cance
r, the pro
lems are reater since tumor antiens often represent small structura
l modifications of self proteins, and may therefore
e poor at primin the immun
e system. Also, immunity has to
e induced in patients already
earin tumor. No
vel methods of presentin antien in a potentially immunoenic manner must
e de
vised, and DNA vaccines may
e ideal for this. A further consideration is that l
on-term exposure to potential tumor antiens may have deleted or enerized T ce
lls a
le to reconize tumor (3), and it is not clear if these can recover or
e
replaced. Inclusion of known epitopes to activate additional T-cell help may pro
vide a way to circumvent this pro
lem (4). If these added epitopes are common to
all patients vaccines, the immune response aainst them can act as an indicator
of immune status. This could
e invalua
le for patients who may have varia
le im
mune capacity followin disease or treatment. Finally, cytokine enes can
e use
d to promote and direct the immune response to attack tumor (5). For our DNA vac
cines, we have focused on the idiotypic determinants of immunolo
ulin that repr
esent defined tumor antiens of neoplastic B cells (6). These determinants are k
nown to induce specific protective anti-idiotypic immunity in mouse lymphoma mod
els when injected as IM protein antiens with adjuvant (7,8). However, they are
individual to each tumor, and preparation of idiotypic IM proteins for patient
application is expensive and
From: Methods in Molecular Medicine, vol. 29, DNA Vaccines: Methods and Protocol
s Edited
y: D. B. Lowrie and R. G. Whalen Humana Press Inc., Totowa, NJ
405
406
Zhu et al.
difficult. This has iven added impetus to the development of DNA vaccines since
the varia
le reion ene sequences (VH and VL) encodin the determinants can
e
readily identified
y PCR (9). There are several options for assem
in these e
nes in the vaccine plasmid; we have chosen the sinle chain Fv (scFv) format, wh
ich includes the minimal sequence known to fold into a conformation resem
lin t
hat of the varia
le reion of whole I (10). Foldin is likely to
e important i
n inducin protection aainst B-cell lymphoma, since anti-idiotypic anti
ody has

een found to
e a crucial component of protective immunity (1). Assem
ly as sc
Fv is rapid, convenient and economical, and is therefore applica
le to vaccine p
roduction on a patient-specific
asis. In this chapter we descri
e the preparati
on of scFv DNA vaccines from
iopsy material of patients with B-cell tumors. In
mouse models we have found that scFv sequence alone is capa
le of inducin only
a low level of anti-idiotypic immunity. However, a small clinical trial of this
preliminary desin was undertaken in patients with advanced disease, larely to
assess potential toxicity of DNA injection into muscle. Further vaccine developm
ent in mice showed that the anti-idiotypic response can
e dramatically promoted

y fusin a ene encodin the Frament C (FrC) portion of tetanus toxin (TT) to
the scFv ene sequence (4). The desin and testin of this modified vaccine wil
l
e descri
ed. The fused TT sequence has the additional advantae of inducin a
n anti-TT response, which can
e used as an indicator of immune capacity in pati
ents. Althouh we have chosen idiotypic antien as a taret in B-cell tumors, th
e principles of vaccine desin are applica
le to other tumor antiens.
1.1. Identification of Tumor Varia
le Reion Genes
Ideally, startin material should
e a fresh
iopsy, or frozen via
le cells, so
that RNA may
e prepared. B cells can have two rearraned VH enes, one of which
will
e non-functional, and tends to
e transcri
ed at a lower level, therefore
use of RNA favors identification of the functional allele. cDNA is then usually
prepared
y reverse transcription usin an olio dT primer, althouh constant r
eion primers can
e used (11). If fresh material is unavaila
le, enomic DNA ca
n
e used as a source,
ut the sequence must
e carefully scanned for mutations

or frameshifts in case the non-functional allele is amplified. The VH and VL en

es used to encode the tumor idiotypic I are o
tained
y PCR usin mixes of fami
ly-specific 5-primers in the V-ene toether with mixes of primers complementar
y to JH sequences. The 5-primers can
e
ased in either the leader sequence or
the first framework (FR1) reion. The nucleotide sequences are shown in Ta
le 1.
The positions of the primers in the VH sequence are shown in Fi. 1 with the si
ze of product expected. A similar PCR method is applied to o
tain VL product, wi
th either or primers used depending on the phenotype of the tumor, which is usua
y known.
DNA Fusion Vaccines
Tabe 1 Primers Used for Identification and Construction of scFv and svFv-Frag C
Fusiona Human VH eader primary PCR primers VH1 Ldr 5'-CAC ACC ATG CAC VH2 Ldr
5'-ATG CAC ATA CTT VH3 Ldr 5'-CCA TGG AGT TTG VH4 Ldr 5'-ACA TGA AACAYC VH5 Ldr
5'-ATG GGG TCA ACC VH6 Ldr 5'-ATG TCT CTC TCC Human VH eader scFv assemby prim
ers scVH1 Ldr 5'-TAT AAG CTT GCC scVH2 Ldr 5'-TAT AAG CTT GCC scVH3 Ldr 5'-TAT A
AG CIT GCC scVH4 Ldr 5'-TAT AAG CTT GCC scVH5 Ldr 5'-TAT AAG CTT GCC scVH6 Ldr 5
'-TAT AAG CTT GCC scFv-Frag C fusion assemby primers A31 scVH Ldr 5'-TAT AAG CT
T GCC A31-FCrev 5'-TTT CAT ACC TCC YJ-FCrev 5'-TTT CAT AGGTCC FCfor 5-CCA CCC GG
ACCT FCrev 5'-TAA TGC GGCCCC FC (SacII)rev 5'-TTG TCC GGCAGG TGG ACC TGT TCC GGC
TGA TGT CGT CCC ATC TTC CTC GCC GCC CCC GCC GCC GCC ACC ACC ACC ACC ACC ACC TGG
ACC OCT TCT CTC ATC ATG ATG ATG ATG ATG ATG AG-3' CTC-3' GG-3' TCC-3' G-3' TTC3'
407
GAC TGG ACC TGG AG-3' GAC ATA CTT TGT TCC-3' GAC TTT GGG CTG AGC-3' AAA CAY CTG
TGG TTC-3' GGG TCA ACC GCC ATC-3' TCT GTC TCC TTC CTC-3' AAG TTG TGG CTG ACC-3'
TTT GAT CTC CAC CTT-3' TAG GAC CGT CAG CTT-3' CTT GAT TGT TG-3' GGT CCA ACC TTC3' G-3'
GCC ACC ACC GGG TCC ACC GGG TCC ACC ATG AAA AAC TGA GTC CTT TCC CGG AAA
aHuman VH FR1, V/V FR1, JH, and J/J primary and assemby primers have been described
previousy (9).
Fig. 1. Detection of tumor-reated VH-genes by PCR. Ampification of cDNA or gen
omic DNA from a tumor biopsy using the 5'-primers (Ldr mix or FR1 mix) together
with 3'-Jmix primers is foowed by separation of product by ge eectrophoresis
.
408
Zhu et a.
The ampified V-genes are then coned into a pGEM-T vector and pasmids from ~10
randomy seected bacteria coonies are sequenced. Since the CDR3 sequence of
VH is unique to each B ce, identity of repeated simiar CDR3 stretches in the
coned VH product reveas the tumor-reated sequence. In our hands the success r
ate in B-ce ymphoma is ~90%, with faiure sometimes due to somatic mutations
in the V-genes at the primer sites. Identity between repeated simiar sequences
can be confirmed by comparison of VH with the cosest germ ine gene in the data
bases, which indicates sites of somatic mutation (Fig. 2). A simiar approach is
used to identify VL sequence, which, athough VL has a ess unique CDR3, is gen
eray ceary reveaed. In some B-ce tumors, such as foicuar ymphoma, a d
egree of nuceotide variation between sequences which are ceary derived from t
he same origina B ce may be evident (12). This does not affect construction o
f the scFv vaccine since there is usuay a predominant sequence, and minor vari
ation in amino acid sequence shoud be insufficient to aow escape from a poyc
ona anti-idiotypic response (1).

1.2. Assemby and Coning of ScFv Construct

The tumor-reated VH and VL are then assembed as scFv by a two-step PCR procedu
re using overapping primers (Fig. 3). A inker encoding 15 amino acids, (GyGy
GyGySer) 3 is incorporated, and the fu ength scFv is cut and coned into th
e pcDNA3 vaccine vector for vaccination (9). When using the 5'-VH eader sequenc
e primer, the natura eader sequence of the scFv is incuded in the vaccine, an
d this is preferred. If the primer is based in the first framework region (FR1),
a human VH1 eader is used (9).
1.3. Assemby and Coning of ScFv-Fragment C Fusion Construct
FrC is a non-toxic component of tetanus toxin which carries epitopes abe to ind
uce protective immunity against infection by Costridium tetani (13). It has as
o been used in mice as a DNA vaccine construct, and found to be effective (14).
The FrC sequence was ampified from pTech2 as tempate (kindy provided by Dr. S
tephen Chatfied, Medeva pc, Leatherhead, Surrey, UK). For the first scFv-FrC c
onstruct, the whoe FrC sequence was ampified and assembed with scFv by a twostep assemby process using the primers in Tabe 1. In order to minimize PCR err
or, subsequent scFv-FrC constructs were made using a 3'-primer cose to the uniq
ue SacII site near the 5'-end of FrC, so that ony partia sequence was ampifie
d (Tabe 1). The scFv-FrC partia sequence coud then be coned as the HindIII-S
acII fragment into the vaccine vector aready containing the FrC gene, and diges
ted with the same enzymes for coning. The procedure introduces a peptide of Gy
ProGyPro as a spacer between the scFv and FrC. For a constructs, sequences we
re checked after coning to ensure fideity. Constructs were again coned into p
cDNA3 (Fig. 4).
DNA Fusion Vaccines
Fig. 2. Identification of tumor-reated VH sequences. Ampified VH products are
coned in bacteria and random cones are sequenced. Deduced amino acid sequences
are shown using the singe etter code, and are compared with the cosest germ
ine VH gene. Dashes indicate identity, and amino acid repacements are identifi
ed. Repeated sequences with identica or cosey simiar CDR3s are ikey to be
derived from tumor ces. Further individua sequences are from norma B ces i
n the biopsy.
409
410
Zhu et a.
Fig. 3. Assemby procedure for scFv. Tumor-reated VH and VL sequences are inke
d by a sequence encoding (GyGyGyGySer) 3 prior to coning into the vaccine v
ector.
1.4. Expression of ScFv or Fragment C Genes In Vitro
Athough the sequences are aways checked for mutations, we aso assess the abi
ity of each set of encoded genes in the vaccine vectors to express protein in vi
tro. This is especiay important for the arge scFv-FrC constructs where sequen
cing is more difficut. We have found that the simpest expression method is Pro
megas TNTR (Madison, WI) coupled in vitro system, which uses rabbit reticulocyte
lysate to incorporate biotinylated lysine residues into protein, with detection
by streptavidin-labeled alkaline phosphatase, following fractionation by SDS-PAG
E.
DNA usion Vaccines
411

ig. 4. DNA plasmid designed for vaccination. The pcDNA3-based vector contains f
used scv-rC genes with expression driven from the CMV promoter.
1.5. Plasmid Preparation
or large scale purification of vaccine plasmids, the Qiagen Plasmid Giga Kit (Q
iagen, Chatsworth, CA) is used. Aliquots can then be stored at 20C as ethanol prec
ipitates. Before use, the DNA precipitate is washed with 70% ethanol and redisso
lved in normal saline at 1 mg/mL. Vaccination procedure for mouse models will be
described. We have done one small clinical trial in patients using an earlier p
lasmid containing only scv, with the RSV LTR to drive gene expression. The prot
ocol for this has been published (15). Subsequently we decided on the basis of o
ur own unpublished data, and on that from other groups (16), that the CMV promot
er in pcDNA3 was superior, and the new vaccines all use this vector.
1.6. Vaccination Protocol
In order to establish the procedure for optimizing the immune response to a scv
DNA vaccine, we first used a human scv sequence either alone, or fused to the
rC gene (4). However, preliminary experiments showed that scv alone was a weak
immunogen (9), and all further experiments to optimize protocols have been carr
ied out with the scv-rC fusion construct. This construct allows assessment of
response against both scv and rC. or scv, the objective has been to induce a
ntibody against the patients idiotypic IgM, the molecule expressed by the tumor c
ells. By using IgM as the test molecule, only the therapeutically relevant antib
odies, which recognize idiotypic determinants expressed in a manner similar to t
hose on tumor cells, are being measured. Generally, we have used the intramuscul
ar route for vaccination. With our vaccines, we have found that intramuscular an
d intradermal sites induce rather
412
Zhu et al.
similar immune responses, although we have not explored the use of the gene gun.
Mice are injected in two sites in the quadriceps muscles with a total dose of 5
0 g of plasmid DNA in 100 L saline. We have found that a total of three injections
of DNA vaccine, spaced at 3 wk intervals, induces high levels of antibodies aga
inst both rC and tumor IgM (see below). Attempts to boost antibody levels by in
creasing the amount of DNA, or by a further injection after d 63, did not signif
icantly improve antibody levels. Similar protocols appear applicable in both C57
BL/6 and BALB/c mice.
1.7. Assessment of Effect of Co-injection of Cytokine Gene Plasmids
To assess the effects of co-delivery of cytokine genes with the vaccine vector,
we use a minimal dose (10 g) of DNA vaccine containing scv-rC fusion vector ass
embled from a patient YJ with lymphoma (4). A pVAC.CYTO vector (50 g) containing
the gene for murine GM-CS, with expression driven by the CMV promoter (kind gif
t of Professor R. E. Hawkins, University of Bristol, Bristol, UK), is mixed with
the vaccine vector and co-injected (5). The protocol is as for the vaccine vect
or, with co-injection of cytokine vector at each boost. As a control for the eff
ect of injecting additional DNA, a vaccine plasmid with no scv incorporated is
used at the same dose as the cytokine vector. Outcome is measured as antibody le
vels against the two encoded proteins at d 63 (see Subheading 1.8.). Other cytok
ine genes can be investigated by the same procedure.
1.8. Measurement of Antibody Tesponses
Bleeds are taken at intervals by tail tipping, or from the cardiac site at termi
nation of the experiment. The serial bleeds are taken one day prior to injection
. Measurement of antibody against rC is by ELISA using purified recombinant rC
(kind gift from Dr. Stephen Chatfield, Medeva plc). Antibodies against idiotypi
c IgM are measured also by ELISA using IgM obtained from heterohybridoma rescue fu

sions between tumor cells and the mouse myeloma cell line OURI-A, a subline of t
he X63-Ag8.653 line (17). Purification of IgM proteins is by precipitation of eu
globulin following extensive dialysis against water (18). In preparation for app
lication to patients, several human scv constructs have been tested in mice, pr
imarily to assess the ability of a range of patients scv molecules to induce ant
i-idiotypic antibodies when fused with rC. Specificity of antibodies is tested
by measuring reactivity of sera with autologous and control idiotypic IgM protei
ns, using ELISA. In three out of three cases, induced antibodies were largely di
rected against autologous IgM, indicative of efficient folding of the encoded sc
v in the fusion protein. The ability of antibodies to recognize idiotypic IgM o
n the surface of tumor cells is assessed by ACS-SCAN.
DNA usion Vaccines
413
The antibody levels obtained using a scv gene from patient YJ in fusion with th
e rC gene, in C57BL/6 mice, are shown in ig. 5. The effects of vaccinating wit
h two different doses of DNA, and of repeated booster doses of DNA have been inv
estigated. In general, responses against the two encoded antigens, scv and rC,
are fairly similar, and allow conclusions to be drawn for planning protocols. I
t is clear that 10 g of DNA is insufficient to obtain a strong antibody response,
and that 50 g improves both levels and consistency of antibody production. Howev
er, there is considerable heterogeneity among mice in the levels attained, and i
ncreasing the dose to 100 g does not appear to raise the level further or reduce
heterogeneity (data not shown). At least two injections of DNA are required, and
we have found that a third injection improves response (ig. 5). However, furth
er boosts do not appear to increase the antibody titre significantly (data not s
hown). Co-injection of a GM-CS-encoding vector together with the 10 g dose of sc
v-rC vaccine from patient YJ significantly increased the levels of antibodies
against both proteins (ig. 6). Interestingly, empty vector DNA also increased t
he response to the low dose of vaccine vector, presumably by the immunostimulato
ry effects of bacterial DNA (19). This effect emphasizes the need to include thi
s control in all experiments, especially when using low doses of vaccine vector.
No significant promotion of antibody responses above this background was detect
ed using co-delivery of genes encoding IL-2, IL-4 or INg (data not shown).
1.9. Mouse Lymphoma Model
To assess the ability of scv DNA constructs to induce immunity in a syngeneic m
odel, and to allow testing of protection against tumor challenge, the A31 murine
lymphoma is being used (20). The VH and VL genes of mouse tumors can be identif
ied by PCR/cloning as for human genes, using primers listed in Table 1. Assembly
can be carried out similarly and the vaccination protocol is as for the human s
cv. or measurement of antibodies induced by the mouse scv, the IgM rescued from
the A31 tumor can be used. Results are similar to those obtained with human sc
v constructs, in that the scv gene alone is ineffective as a vaccine, but that
fusion with rC markedly promotes the antibody response. The immunity generated
by the fusion vaccine is also able to protect against tumor challenge (21). 2. M
aterials Special equipment needed throughout this work: thermal cycler, agarose
gel electrophoresis apparatus, DNA sequencing equipment, and suitable power supp
lies. Materials for preparing agarose and polyacrylamide sequencing gels and for
doing ELISA tests are required.
414
Zhu et al.
DNA usion Vaccines 2.1. Identification of Tumor-Related VH and VL Genes
415

1. Oligonucleotide primers for PCR amplification are listed in Table 1. All olig
onucleotides are diluted to a working concentration of 10 pmol/L. 2. RNAzolB: Cinn
a Biotecxlabs, Houston, Texas. 3. cDNA synthesis: irst-Strand cDNA Synthesis Ki
t (Pharmacia Biotech, Uppsala, Sweden). 4. Taq DNA polymerase, Boehringer Mannhe
im, Mannheim, Germany. 5. Taq buffer: 1.5 mM MgCl2, 10 mM Tris-HCl, 50 mM KCl (p
H 8.3). 6. K buffer: 1X Taq buffer plus 0.5% Tween 20. 7. Proteinase K: make a s
tock solution at 20 mg/mL and store at 20C. Use within 6 mo. 8. GENECLEAN II kit:
BIO 101, Vista, CA. 9. pGEM-T vector: Promega, Madison, WI. 10. DNA sequencing:
T7 Sequenase version 2.0 DNA sequencing kit, Amersham Life Science, Buckinghamsh
ire, UK.
2.2 . Assembly and Cloning of scv and scv-ragment C usion Construct
1. Primer list: see Table 1. 2. Pfu DNA polymerase: Stratagene, La Jolla, CA. 3.
Pfu Pol buffer: 10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-Cl (pH 8.75), 2 mM MgSO4
, 1% Triton X-100, 100 mg/mL BSA. 4. Vectors (pcDNA3): Invitrogen BV, The Nether
lands. 5. PCR template for rC amplification: pTech2 plasmid containing the frag
ment C gene, kindly provided by Medeva plc.
3. Methods A number of commercial reagent kits are used throughout this work. Re
fer to the manufacturers manuals for instructions. or general molecular biology
techniques, see Sambrook et al. (22).
3.1. Identification of Tumor-Related VH and VL Genes 3.1.1. Isolation of Genomic
(g) DNA (see Note 1)
1. Wash 106 cells (peripheral blood mononuclear cells or lymphoid tissues) with
1 mL of PBS, and then 1 mL of 1 X Taq buffer. 2. Resuspend the cells in 1 mL K b
uffer. 3. Add 5 L of Proteinase K and incubate at 56C for 1 h. ig. 5. Induction o
f antibodies by intramuscular injections of DNA vaccine plasmid containing the s
cv-rC fusion gene. Scv sequence was derived from tumor cells of patient YJ. S
erum antibodies recognizing YJ tumor IgM or rC were measured by ELISA following
vaccination of mice at day 0, 21 and 42, using two different doses of DNA. Each
point represents the antibody level in a single mouse.
416
Zhu et al.
DNA usion Vaccines
417
4. Inactivate Proteinase K by heating to 95C for 1530 min. 5. Centrifuge for 5 min
in a microfuge at maximum speed (13,000 rpm). 6. Transfer the supernatant to a
fresh tube. Store the gDNA at 20C if not used immediately.
3.1.2. Isolation of Total RNA
1. To lyse cells, add 0.2 mL of RNAzolB per 106 cells and mix by inverting the t
ube several times (see Note 2). 2. Add 1/10 vol. of chloroform and shake vigorou
sly for 15 s. Leave on ice for 5 min. 3. Centrifuge for 15 min in a microfuge at
maximum speed. 4. Carefully transfer the upper aqueous phase containing RNA in
a fresh tube. 5. Add 1 vol. of isopropanol, mix well and leave on ice for 15 min
. 6. Centrifuge at maximum speed for 15 min. 7. Wash the pellet with 0.5 mL of 7
5% ethanol by vortexing and subsequent centrifugation for 8 min. 8. Dry the pell
et for 10 min at room temperature and redissolve in RNase-free water.
3.1.3. Preparation of cDNA
1. Heat 15 g total RNA in 20 L of water at 65C for 5 min to denature RNA. Chill the
tube on ice. 2. Add 1 L of DTT, 1 L of oligo (dT)18 (0.2 g) and 11 L of Bulk irst S
trand Reaction Mix containing RNase inhibitor, dNTPs and M-MuLV reverse transrip

tase. 3. Incubate at 37C for 1 h.

3.1.4. PCR Amplification of VH and VL Genes

1. Prepare a PCR reaction mixture that contains all the necessary components for
the reaction except for the enzyme (see Note 3). If there is more than one samp
le, a master mix without DNA and enzyme should be made to reduce the steps and e
rrors of pipetting. A typical reaction contains: 10X Taq Pol buffer 2.5 mM dNTPs
VH or Vk/V primer JH or J/J primer Genomic DNA or cDNA H2 O 5 L 5 L 2 L 2 L 15 L (~
g) 15 L to 49.5 L
Fig. 6. Effect of co-deivery of a pasmid containing the gene encoding GM-CSF o
n induction of antibodies against a ow dose of DNA scFv-FrC vaccine. Mice were
injected with 10 g of vaccine pasmid either aone, with added 50 g empty vector,
or with added 50 g of GM-CSF pasmid. ScFv sequence was as for Fig. 5, and sera w
ere taken at day 63 for ELISA.
418
Zhu et a.
2. Perform the ampification in a therma cycer as foows: Incubate for 5 min
at 94C. Add 0.5 L (2.5 units) of Taq DNA poymerase and then repeat 30 cyces of 3
0 s at 94C, 30 s at 65C, and 45 s at 72C. Finay, incubate for 7 min at 72C. 3. Ana
yze the PCR reaction by eectrophoresis on an 1.5% agarose ge aongside a moe
cuar weight size standard. 4. Excise and purify the bands of appropriate sizes
(Fig. 1). 5. Cone the ampified V genes into the pGEM-T vector. 6. Isoate pas
mid from randomy seected bacteria coonies. 7. Sequence 610 positive cones. 8
. Identify tumor-reated VH and VL genes.
3.2. Assemby and Coning of scFv The tumor VH and VL genes are assembed as a s
inge-chain Fv (scFv) by a two-step PCR procedure as iustrated in Fig. 3. The
scFv assemby primers are isted in Tabe 1 (see Note 4). In the first step, the
VH and VL genes are ampified separatey from the pasmids containing tumor-re
ated VH and VL genes with the appropriate assemby primers to incorporate the re
striction sites and the (GyGyGyGySer) 3 scFv inker. In the second step, the
secondary PCR products are combined and the fu-ength scFv is assembed by PC
R using the outer primers ony. The fuy assembed scFv is cut and coned into
a vaccine vector for genetic vaccination. 3.2.1. Secondary PCR
1. Prepare two PCR reaction mixes (see Note 5) by adding: 10X Pfu Po buffer 2.5
mM dNTPs scVH primer scJH scV/V scJ/J VH pasmid VL pasmid H2 O Tube A (VH) 5 L 5 L
2 L 2 L 1 L (100ng) 34.5 L Tube B (VL) 5 L 5 L 2 L 2 L 1 L (100ng) 34.5 L
2. Carry out the ampification in a therma cycer as foows: Incubate for 5 mi
n at 94C. Add 0.5 L (2.5 units) of Pfu DNA poymerase, repeat 5 cyces of 30 s at
94C, 30 s at 45C, and 1.5 min at 72C, and then 15 cyces of 30 s at 94C, 30 s at 65C,
and 1.5 min at 72C. Finay, incubate for 7 min at 72C. 3. Anayze the PCR reacti
on on an 1.5% agarose ge with a moecuar weight size standard. 4. Excise and p
urify the bands of appropriate sizes. 5. Resuspend the DNA fragments in 25 L of s
terie ddH2O as scVH and scVL.
DNA Fusion Vaccines 3.2.2. Assemby PCR
1. Prepare a PCR reaction mix by adding: 10X Pfu Po buffer 2.5 mM dNTPs scVH pr
imer scJ/J scVH scVL H2 O 5 L 5 L 2 L 2 L 5 L 5 L 25.5 L
419
2. Carry out the ampification in a therma cycer as in the
pt with a onger extension step of 3 min at 72C. 3. Anayze
n 1.5% agarose ge with a moecuar weight size standard. 4.
he band of ~800bp. 5. Digest the purified scFv fragment with

secondary PCR, exce

the PCR reaction on a
Excise and purify t
appropriate enzymes

(see Note 6). 6. Cone the scFv into the pcDNA3 expression vector.
3.3. Construction of scFv-Fragment C Fusion 3.3.1. Pre-assemby PCR
1. Prepare 2 PCR reaction mixes by adding: Tube A (scFv) 5 L 5 L 2 L 2 L 1 L (100ng)
34.5 L Tube B (Frag C) 5 L 5 L 2 L 2 L 1 L (100ng) 34.5 L
10 X Pfu Po buffer 2.5 mM dNTPs scVH eader primer scFv-FrCrev FrCfor FrCrev sc
Fv pasmid pTech 2 pasmid H2 O
2. Carry out the ampification in a therma cycer as foows: Incubate for 5 mi
n at 94C. Add 0.5 L (2.5 units) of Pfu DNA poymerase, repeat 5 cyces of 30 s at
94C, 30 s at 45C, and 3 min at 72C, and then 15 cyces of 30 s at 94C, 30 s at 65C, a
nd 3 min at 72C. Finay, incubate for 10 min at 72C. 3. Anayze the PCR reaction
on an 1.2% agarose ge aongside a moecuar weight size standard. 4. Excise and
purify the bands of appropriate sizes. 5. Resuspend the DNA fragments in 25 L of
water as scFv and FrC.
420 3.3.2. Assemby PCR
1. Prepare a PCR reaction mix by adding: 10X Pfu Po buffer 2.5 mM dNTPs scVH e
ader primer FrCrev primer scFv FrC H2 O 5 L 5 L 2 L 2 L 5 L 5 L 25.5 L
Zhu et a.
2. Carry out the ampification in a therma cycer as in the pre-assemby PCR, e
xcept with a onger extension step of 5 min at 72C. 3. Anayze the PCR reaction o
n an 1.2% agarose ge aongside a moecuar weight size standard. 4. Excise and
purify the scFv-FrC band (~2.2 kb). 5. Digest the purified scFv-FrC fragment wit
h HindIII and NotI restriction enzymes. 6. Cone the scFv-FrC into the pcDNA3 ex
pression vector.
3.4. Assessment of Antibody Production Antibodies induced foowing injection of
the DNA vaccines are measured by ELISA. Antibodies raised via encoded scFv moe
cues can be detected by reactivity with the tumor-derived IgM proteins, thus en
suring that they are directed against idiotypic determinants expressed by the tu
mor. For this anaysis, IgM has to be obtained from heterohybridomas between tum
or ces and a mouse myeoma ine. 3.4.1. Preparation of Idiotypic IgM
1. Fuse tumor ces from each patient (or mouse donor) with the mouse myeoma ce
 ine OURI-A, an ouabain-resistant subine of the X63-Ag8.653 ine, using PEG
(17). 2. Foowing fusion, assess production of IgM by ELISA and cone, with reseection for IgM of tumor ight chain type. 3. Sequence the V-genes of secretin
g cones to ensure that the tumor ces have been rescued. 4. Prepare IgM from c
uture supernatants by eugobuin precipitation foowing diaysis into distie
d water (18). Check purity by ge eectrophoresis.
3.4.2. Measurement of Serum Antibody Leves Antibody eves in sera of vaccinate
d mice are measured by ELISA.
3.4.2.1. ANTI-FRAGMENT C ANTIBODIES 1. Coat ELISA pates overnight at 4C with rec
ombinant Fragment C at 1 g/mL in PBS. Treat washed pates then with PBS/0.5% BSA
to bock non-specific binding sites.
DNA Fusion Vaccines
421
2. After washing, diute test sera in PBS/TWEEN20 containing 0.1% BSA and incuba
te in the coated pates for 1.5 h at 37C. 3. After washing, detect bound mouse Ig
G by incubation with HRP-goat anti-mouse Fcg (Serotec, Kidington, UK; 1/1000 di
ution) for 1 h at 37C. Standardize the assay for comparative purposes by prepari
ng a poo of positive sera, and assigning to it an arbitrary vaue of 200U/mL.

3.4.2.2. ANTI-SCFV ANTIBODIES

1. Coat ELISA pates overnight at 4C with purified tumor-derived idiotypic IgM at
0.5g/mL in sodium carbonate buffer at pH 9.5. Bock washed pates with PBS/ 0.5%
BSA as above. 2. After incubation with diuted contro or test sera, bound mous
e IgG is detected as for anti-FrC.
3.4.2.3. FACS ANALYSIS TO TEST REACTIVITY OF SERA WITH TUMOR CELLS
1. Mononucear ces from patients blood samples or lymphoid tissue are prepared
by centrifugation through a icoll-Hypaque gradient. 2. After washing, cells are
incubated with diluted control or test sera for 20 min on ice. After washing, b
ound IgG is then detected following incubation with ITC-labeled goat anti-mouse
cg, using the ACS-SCAN with LYSISII software.
4. Notes
1. The DNA can usually be used directly for PCR amplification. However, DNA can
be further purified by phenol extraction and ethanol precipitation to give clean
er PCR products. Amplification of a house-keeping gene, such as -actin, can also
be included as a control for the template quality. 2. Various RNA isolation kits
which are commercially available also can be used. 3. Care must be taken to min
imize contamination when performing PCR amplification, and a negative control re
action in which no DNA template has been added should always be included. 4. The
scVH primers contain a HindIII (Leader) or an SfiI (R1) restriction site and h
ybridize to the original set of VH primers. The scJH and scV/V primers hybridize t
o their respective initia primers, but aso incude the scFv inker sequences c
ompementary to each other to aow production of an scFv. The scJ/ J primers simi
ary hybridize to their respective initia primers, but aso incude the NotI r
estriction site. 5. In the secondary and assemby PCR reactions, the high-fidei
ty Pfu DNA poymerase is used to minimize possibe PCR errors. Due to the ow pr
ocessivity of the enzyme, the extention step of the PCR reactions has been proo
nged. Refer to the manufacturers instructions for full description of the enzyme.
6. If an scv leader primer is used, digest the scv fragment with HindIII and
NotI enzymes. If an scv R1 primer is used, digest the scv fragment with SfiI
and NotI and then clone into the vector already containing a human VH 1 leader a
nd SfiI site (9).
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Zhu et al.
7. or scv, the 5-primer is the VH leader scv assembly primer. or the 3 end
, a J/J reverse primer which aso contains the GyProGyPro (anti-sense) inker se
quence is used. The 5'-FrC primer aso contains the GyProGyPro (sense) inker
sequence and the 3'-primer incorporates a NotI site.
Acknowedgments This work was supported by the Leukaemia Research Fund, the Canc
er Research Campaign and Tenovus, UK. References
1. Umer, J. B., Donney, J. J., Parker, S. E., Rhodes, G. H., Fegner, P. L.,
Dwarki, V. J., Gromkowski, S. H., Deck, R. R., DeWitt, C. M., Friedman, A., Hawe
, L. A., Leander K. R., Martinez, D., Perry, H. C., Shiver, J. W., Montgomery, D
. L., and Liu, M. A. (1993) Heteroogous protection against infuenza by injecti
on of DNA encoding a vira protein. Science 259, 17451749. 2. Davis, H. L. and Wh
aen, R. G. (1995) DNA-based immunization. In Moecuar and Ce Bioogy of Huma
n Gene Therapeutics. (Dickson, G., ed.) Chapman & Ha, New York, p. 368. 3. Bog
en, B. (1996) Periphera T ce toerance as a tumor escape mechanism: deetion
of CD4+ T ces specific for a monocona immunogobuin idiotype secreted by a
pasmacytoma. Eur. J. Immuno. 26, 26712679. 4. Speerberg, M. B., Zhu, D., Thom
psett, A., King, C. A., Hambin, T. J., and Stevenson, F. K. (1997) DNA vaccines
against ymphoma: Promotion of antiidiotypic antibody responses induced by sing
e chain Fv gene by fusion to tetanus toxin fragment C. J. Immuno. 159, 18851892
. 5. Stevenson, F. K., Zhu, D., King, C. A., Ashworth, L. J., Kumar, S., and Haw

kins, R. E. (1995) Idiotypic vaccines against B-ce ymphoma. Immuno. Rev. 145
, 211228. 6. George, A. J. T., and Stevenson, F. K. (1989) Prospect for the treat
ment of B ce tumors using idiotypic vaccination. Int. Rev. Immuno. 4, 271310.
7. George, A. J. T. Tutt, A. L., and Stevenson, F. K. (1987) Anti-idiotypic mech
anisms invoved in suppression of a mouse B ce ymphoma, BCL1. J. Immuno. 138
, 628634. 8. Kaminski, M. S., Kitamura, K., Maoney, D. G., and Levy, R. (1987) I
diotypic vaccination against a murine B ce ymphoma: inhibition of tumor immun
ity by free idiotypic protein. J. Immuno. 138, 12891296. 9. Hawkins, R. E., Zhu,
D., Ovecka, M., Winter, G., Hambin, T. J. Long, A., and Stevenson, F. K. (1994
) Idiotypic vaccination against human B-ce ymphoma: rescue of variabe region
gene sequences from biopsy materia for assemby as singe-chain Fv persona va
ccines. Bood 83, 32793288. 10. Winter, G., Griffiths, A. D., Hawkins, R. E., and
Hoogenboom, H. R. (1994) Making antibodies by phage dispay technoogy. Ann. Re
v. Immuno.12, 433455. 11. Sahota, S. S., Leo, R., Hambin, T. J., and Stevenson,
F. K. (1996) IgVH gene mutationa patterns indicate different tumor ce status
in human myeoma and monocona gammopathy of undetermined significance. Bood
87, 746755.
DNA Fusion Vaccines
423
12. Levy, R., Levy, S., Ceary, M. L., Carro, W., Kon, S., Bird, J., and Skar
, J. (1987) Somatic mutation in human B-ce tumors. Immuno. Rev. 96, 4358. 13.
Panina-Bordignon, P., Tan, A., Termitjeen, A., Demotz, S., Corradin, G., and La
nzavecchia, A. (1989) Universay immunogenic T ce epitopes: promiscuous bindi
ng to human MHC cass II and promiscuous recognition by T ces. Eur. J. Immuno
. 19, 22372242. 14. Anderson, R., Gao, X.-M., Papakonstantinopouou, A., Roberts,
M., and Dougan, ca Protoco: A piot study of idiotypic vaccination for foic
uar B-ce ymphoma using a genetic approach. Hum. Gene Ther. 8, 12871299. 16. M
anthorpe, M., Cornefert-Jensen, F., Hartikka, J., Fegner, J., Runde, A., Marg
aith, M., and Dwarki, V. (1993) Gene therapy by intramuscuar injection of pas
mid DNA: studies on firefy uciferase gene expression in mice. Hum. Gene Ther.
4, 419431. 17. Pascua, V., Victor, K., Speerberg, M., Hambin, T. J., Stevenso
n, F. K., and Capra, J. D. (1992) VH restriction among human cod aggutinins. T
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Immuno. 149, 23372344. 18. Vomers, H. P., Wozniak, E., Stepien-Btsch, E., Zimme
rmann, U., and MerHermeink, H.-K. (1996) A rapid method for purification of mo
nocona human IgM from mass cuture. Hum. Antibodies Hybridomas 7, 3741. 19. Sat
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Lotz, M., Carson, D. A., and Raz, E. (1996) Immunostimuatory DNA sequences nece
ssary for effective intraderma gene immunization. Science 273, 352354. 20. Dyke,
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1) Idiotypic vaccination against B-ce ymphoma eads to dormant tumour. Ce I
mmuno. 132, 7083. 21. King, C. A., Speerberg, M. B., Zhu, D., Rice, J., Sahota
, S. S., Thompsett, A. R., Hambin, T. J., Rad, J., and Stevenson, F. K. (1998)
DNA vaccines with singechain Fv fused to fragment C of tetanus toxin induce pr
otective immunity against ymphoma and myeoma. Nat. Med. 4, 12811286. 22. Sambro
ok, J., Fritch, E. F., and Maniatis, T. (1989) Moecuar Coning: A aboratory m
anua. Cod Spring Harbor Laboratory, Cod Spring Harbor, NY.
36
DNA-Based Vaccination Primes Tumor-Rejecting T-Ce Responses
Watraud Bhm, Martin Scheef, Stefan Thoma, Reinhod Schirmbeck, and Jrg Reimann I
ntroduction DNA-based vaccination efficienty primes MHC-restricted T-ce respo
nses. This technique specificay stimuates MHC-II-restricted CD4+ T-ce respo
nses and MHC-I-restricted CD8 + T-ce responses against strong (immunodominant) o
r weak (subdominant or cryptic) epitopes of intraceuar, secreted or membrane-as
sociated protein antigens. In many experimenta systems, T-ce-mediated effecto
r functions have the potentia to contro tumor growth. In particuar MHC-I-rest

ricted cytotoxic T ymphocytes (CTL) can reject tumors. This has been shown usin
g either defined tumor-associated antigens (TAA), or vira antigens containing w
e-defined, MHC-binding and CTLstimuating epitopes that are expressed by trans
fected tumor ces.
1.1. T-Ces Primed By DNA Vaccination Have the Potentia to Reject Tumor Ces
We have studied two murine tumor modes, i.e. the mastocytoma ce ine P815 (H2d) and the meanoma ce ine B16.F10 (H-2b). We have generated stabe transfec
tants of these tumorigenic ce ines that express either the sma hepatitis B
surface antigen (HBsAg), or the arge tumor antigen (T-Ag) of simian virus 40. C
es of the non-transfected parenta ine and the transfected subines form aggr
essivey growing tumors when 103 to 104 ces of these ines are subcutaneousy
injected into adoptive, syngeneic hosts. Both tumor ce ines are deficient in
T-ce co-stimuating activity. P815 ces constitutivey express high eves of
MHC-Ia (Kd, Dd, Ld) moecues but no MHC-II moecues on the ce surface. B16
ces constitutivey express ony ow eves of MHC-Ia (Kb,
From: Methods in Moecuar Medicine, vo. 29, DNA Vaccines: Methods and Protoco
s Edited by: D. B. Lowrie and R. G. Whaen Humana Press Inc., Totowa, NJ
425
426
Bhm et a.
D b) moecues on the ce surface, but MHC-I expression is strikingy upreguat
ed and MHC-II expression is induced on the surface of these ces in response to
interferon- (IFN-) stimulation. A fraction (1040%) of B16 cells express the TNF-li
ke CD95L (FasL) on the surface, which is supposed to paralyze potentially reject
in effector CTL. DNA-
ased vaccination efficiently primes potent MHC-I-restrict
ed CTL responses to HBsA or T-A in hih and low responder (H-2d or H-2
) mouse
strains (13). The MHC-I-restricted, HBsA-specific or T-A-specific CTL primed

y DNA vaccination can reject P815 or B16 tumor cell rafts expressin the respec
tive viral antiens when 100fold hiher num
ers than the minimal tumorienic raf
t size are transferred (4, data not shown). Representative examples are shown in
Fi. 1. The T cell-mediated, specific rejection of B16 melanomas was thus not o
verridden
y CD95L expression of the taret cell. The time point of DNA vaccinat
ion relative to tumor cell enraftment is critical. Inoculation of antien-encod
in plasmid DNA up to 4 d
efore tumor cell transfer confers protection. In cont
rast, immunizations 1 to 3 d
efore, at the same time of, or after tumor cell en
raftment are not protective. Alternative treatment protocols were therefore req
uired to taret rejectin CTL to rowin tumors.
1.2. Taretin Anti-viral CTL Reactivity to a Growin Tumor Facilitates its Reje
ction, and Cross-primes Rejectin Immune Responses Aainst TAA
We found an effective taretin protocol that involved the followin three steps
: A potent anti-viral CTL reactivity was induced
y DNA-
ased vaccination; A su

cutaneously rowin tumor was esta
lished; The esta
lished CTL reactivity was ta
reted to the tumor
y repeated intra-tumor injections of DNA expression constru
cts that encode the relevant antien. Usin this protocol, we o
tained sta
le re
jection of the P815 mastocytoma tumors in 50-70% of the treated, tumor-
earin H
-2d DBA/2 mice (4, Fi. 2). Most encourain was the o
servation that all mice t
hat had rejected the mastocytoma followin this therapeutic protocol showed sta

le resistance aainst a challene with nonmodified tumor cell rafts for >4 mont
hs post-rejection. Hence, the tumor rejection process initiated
y in vivo resti
mulation of primed MHC-I-restricted, anti-viral CTL within the tumor (followin
intra-tumor DNA injections) facilitated cross-primin of a TAA-specific, rejecti
n immune response. This rejectin immune response could not control metastases

ecause reression of a treated P815 mastocytoma on one flank of an adoptive hos
t did not coincide with reression of a contralateral, non-treated, proressivel

y rowin P815 mastocytoma in the same host (unpu
lished data).

Tumor-Rejectin T-Cell Responses
Fi. 1. T-cell responses to different viral antiens elicited
y DNA-
ased vacci
nation can protect mice aainst rowth of different types of malinant tumors. D
BA/2 (H-2d) or C57BL/6 (H-2
) mice were vaccinated with HBsA- or T-A-encodin
DNA constructs (S-immune or T-immune host). Vaccinated mice or non-pretreated co
ntrol mice (non-immune host) were su
cutaneously transplanted with 106 tumor cel
ls (10 mice/roup); the tumor cells were non-transfected (P815 or B16.F10), or t
ransfected expressin the HBsA (P815/S, B16.F10/S) or the T-A (P815/T, B16.F10
/T). The fraction of tumor-free mice was monitored for 50 d.
427
428
Bhm et al.
Fi. 2. Taretin anti-viral T-cell reactivity to rowin tumors can facilitate
their rejection. DBA/2 or C57BL/6 mice were immunized aainst HBsA usin DNA-
a
sed vaccination. The mice were su
cutaneously transplanted with 5 105 non-transfe
cted tumor cells (P815 or B16.F10) 4 weeks post-vaccination. When su
cutaneous t
umor nodules reached a diameter of >2 mm, HBsA-encodin DNA was injected into t
he tumors every 23 d for a maximum of 3 wk. The size of the tumor was measured ev
ery 34 d.
In contrast to the P815 mastocytoma model, we o
served only transient control of
B16 melanoma rowth for 3 to 8 wk in 1030% of tumor-
earin H-2
C57BL/6 mice us
in this protocol (Fi. 2, Waltraud Bhm, unpu
lished o
servations). In this model
, restimulation of a potent, primed T-cell response to an immunodominant viral a
ntien within the tumor apparently did not facilitate cross-primin of a rejecti
n, TAA-specific immune response under the experimental conditions applied.
1.3. Potential for Specific Immunotherapy of Cancer Usin DNA Vaccines
Because DNA-
ased vaccination is one of the most potent, currently availa
le tec
hniques to prime T-cell responses, it is of interest to test in vivo the tumor-r
ejectin potential of T-cell reactivities induci
le
y this novel type of vaccin
ation. Our data set provides
oth encourain and discourain findins concerni
n the specific immunotherapy of cancer
ased on DNA vaccination.
Tumor-Rejectin T-Cell Responses
429
1.3.1. T-Cells Primed
y DNA Vaccination Can Potentially Control Growth of Solid
Malinant Tumors We used two viral model antiens (surface antien HBsA of HBV
, and the nucleoprotein T-A of the papovavirus SV40) that are immunoenic for M
HCI-restricted, murine CTL precursors, elicitin CTL responses of well-character
ized epitope and restriction specificities. T-cell responses aainst these anti
ens elicited
y DNA-
ased vaccination could protect a syneneic host aainst a
ressively rowin tumors (Fi. 1). The specific immune response conferred resist
ance aainst an up to 100-fold tumorienic dose of tumor cells,
ut failed at hi
her doses. Tumors escapin after transfer of >106 transfected tumor cells into
the immune host still expressed and presented the viral antien, i.e. tumor row
th was not the result of selection of antien loss escape variants. Hence, there s
eems to
e a limit to the tumor cell
urden with which an efficient T-cell respo
nse can cope. This limit is well
elow averae tumor cell
urdens present durin
aressive rowth of tumors in vivo. Neither the type of antien, nor the histo
type of the tumor seemed to have a major influence on the T-cell-mediated reject
ion induci
le
y DNA-
ased vaccination. In H-2d mice, HBsA is a stron antien an

d T-A is a weak antien for CTL; the inverse is true for H-2
mice, in which HBsA
 is a weak antien and T-A is a stron antien for CTL. In
oth strains,
oth anti
-viral CTL responses apparently mediate compara
le levels of CTL-mediated protec
tion aainst tumors. Mastocytomas and melanomas are very different types of tumo
rs. The DNA vaccination-induced T cell responses conferred compara
le levels of
protection aainst
oth types of tumors in the two antien systems. A su
set of
B16 melanoma cells expresses CD95L (FasL) that has
een shown to paralyze or to
eliminate cytotoxic effector cells. Anti-viral CTL induced
y DNA vaccination co
uld apparently override this protective mechanism of tumor cells aainst immune at
tack. 1.3.2. DNA Vaccination Protocols Can Be Desined to Facilitate Cross-Primi
n of Rejectin, TAA-Specific T-Cell Responses Once a tumor (expressin a viral
antien) is rowin in vivo, it seems larely resistant to attack
y CTL. We the
refore desined the taretin technique to: facilitate cross-primin of CTL prec
ursors to TAA, and to deliver cytokines to tumors in situ to recruit rejectin,
specific and non-specific immune mechanisms. This protocol was unexpectedly succ
essful with P815 mastocytomas
ut controlled only transiently the rowth of B16
melanomas in vivo (Fi. 2). The mechanism of protection aainst P815 cells stimu
lated
y the CTL taretin protocol remains to
e elucidated. Rejectin mice are
sta
ly resistant aainst challene with non-modified P815 cells, and har
or P81
5-specific CTL reactivity. The eneration of TAA-specific CTL aainst this tumor
may there430
Bhm et al.
fore play a prominent role in this tumor-specific resistance. If this is the cas
e, it would
e a hih priority to elucidate the mechanism that facilitates CTL c
rossprimin to TAA in this protocol. 2. Materials 2.1. Expression Vectors Used f
or DNA-
ased Vaccination The CMV promoter controls the expression of the small H
BsA (su
type ayw) in plasmid pCI/S (Fi. 3A) and the SV40 lare tumor antien (
T-A) in the plasmid pCMV-1/T (Fi. 3B). The construction of these expression pl
asmids has
een descri
ed (3,4).
1. Plasmid pTKTHBV2: A ift of Dr. M. Meyer, Munich Germany; contains the comple
te HBV enome (su
type ayw). 2. Plasmid pEARLY: Provided
y Dr. von Hoyniner-Hu
ene, Institute for Viroloy, University of Wrz
ur, Germany; encodes the complete
wild-type T-A sequence of SV40 (5). A BlII site replaces the HindIII site at
position 5147 of the SV40 enome. 3. pCI vector, Cataloue No. E1731, is availa

le from promea (Mannheim, Germany). 4. 3T3 fi
rolasts, CCL-92, are availa
le fr
om ATCC. 5. Dul
eccos modified Eale medium (DMEM), Cataloue No. Q41-01885, is
availa
le from Gi
co-BRL (Eenstein, Germany). 6. HBS
uffer: 40 mM HEPES, 280
mM NaCl, 1.5 mM Na2HPO4 2H2O, pH 7.1.
2.2. Expression of Viral Antiens from Constructs Used for DNA Vaccination
1. [35S]-methionine, Cataloue No. SJ1015, is availa
le from Amersham (Braunschw
ei, Germany). 2. Methionine-free RPMI-1640 medium, Cataloue No. F1243, is avai
la
le from Seromed (Berlin, Germany). 3. Polyclonal ra

it anti-HBsA antiserum:
may
e availa
le as a ift of the Behrin AG (Mar
ur, Germany). 4. Monoclonal
anti-T-A anti
ody PA
108, directed aainst the N-terminal extremity of the prot
ein: a ift of Dr. W. Deppert, Ham
ur, Germany. 5. Protein-A Sepharose, Catalo
ue No. 17-0780-01, is availa
le from Pharmacia (Frei
ur, Germany). 6. Fetal cal
f serum (FCS), amino acid-free: dialyze FCS aainst a lare volume of Ca++/M++free PBS for 2 d. 7. Lysis
uffer: 120 mM NaCl, 1% (w/v) aprotinin (Trasylol, Ca
taloue No. 48764; Bayer, Leverkusen, Germany), 50 M leupeptin, 0.5% (v/v) Nonide
t P-40 (NP40), 10% (v/v) lycerol, 50 mM Tris/HCl, pH 8.0. 8. Wash
uffer: 0.5 M
LiCl, 1% (v/v) NP-40 and 0.1 M Tris/HCl, pH 9.0. 9. Elution
uffer: 1.5% (w/v)
SDS, 5% (v/v) 2-mercaptoethanol (2-ME) and 7 mM Tris/HCl, pH 6.8.
Tumor-Rejectin T-Cell Responses

431
Fi. 3. Maps of the plasmids descri
ed.
2.3. Preparation of Plasmid DNA Used for Immunization
1. Ultrapure 100 anion exchane chromatoraphy columns are availa
le from Qiaen
, (Hilden, Germany). 2. Endo-free
uffer system is availa
le from Qiaen.
2.4. Tumor Cell Lines
1. The B16 melanoma cell line oriinated in an H-2
C57BL/6 mouse, the B16 su
li
nes B16.F0 and B16.F1 lines are availa
le from the ATCC as Cataloue Nos. CRL-63
22 and CRL-6323. In most experiments we used the B16.F10 line, which we o
tained
from Dr. P. Antonsson, Lund, Sweden. The mastocytoma cell line P815 (TIB64) ori
inated in an H-2d DBA/2 mouse. 2. Bovine papilloma virus-
ased vector BMGneo: a
ift from Drs. Y. Karasuyama and F. Melchers (Basel, Switzerland). This vector
was used to construct the BMG/HBS expression plasmid and the BMG/T-A.1 expressi
on plasmid (Fi. 3C,D).
432
Bhm et al.
3. DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate):
liposomal transfection reaent, Cataloue No. 1202375, is availa
le from Boehrin
er-Mannheim (Mannheim, Germany). 4. RPMI-1640/Clicks medium, Cataloue No. 9000
53, is availa
le from Serva, Boehriner-Inelheim. 5. G418 sulfate (Geneticin),
Cataloue No. 11811-064, is availa
le from Gi
coBRL. A stock solution of 125 m/
mL in PBS was stored at 20oC.
2.5. Nucleic Acid Immunization
1. Cardiotoxin, Cataloue No. L8102, is availa
le from Latoxan (Rosans, France).
Prepare a 0.01 mM stock solution in PBS. 2. Metofane is availa
le from Janssen
Gm
H (Neuss, Germany).
2.6. Preparation of Tumor Cells for Transplantation UltraCulture serum-free cult
ure medium, Cataloue No. 12-725F, is availa
le from Boehriner-Inelheim. 2.7.
Intratumor Injections of Recom
inant DNA
Syrine needle, 0.4 19 mm 28G: Microlance 3, Cataloue No. E0896F07, is availa
l
e from Becton-Dickinson (Heidel
er, Germany).
2.8. Cytotoxic T Cell Assay MEM tissue culture medium, Cataloue No. 22561-021,
is availa
le from Gi
co-BRL .
3. Methods 3.1. Expression Vectors Used for DNA-
ased Vaccination 3.1.1. Express
ion Plasmid pCI/S
1. Prepare the XhoI/BlII frament (containin the small HBsA codin reion and
the HBV 3 non-codin reion and polyA sinal) from pTKTHBV2. 2. Liate the Xho
I/BlII frament into the pCI vector cut with XhoI/BamHI to enerate the plasmid
pCI/S.
3.1.2. Expression Plasmid pCMV-1/T
1. Diest pEARLY with BlII and BamHI. 2. Cut pCMV-1 (6) with BamHI and treat it
with shrimp alkaline phosphatase. 3. Clone the T-A encodin BlII/BamHI frame
nt into the BamHI-diested pCMV-1 plasmid to enerate the plasmid pCMV-1/T.
3.1.3. Transient Transfection of Recom
inant DNA
1. Grow 3T3 fi
ro
last cells to a density of 104105 cells/mL in 100 mm tissue cul
ture dishes in DMEM supplemented with 10% (v/v) FCS. 2. Introduce plasmid DNA (1
0  DNA/dish) into the cells
y the CaPO4-method.
Tumor-Rejectin T-Cell Responses

433
3. Prepare Ca/DNA solution as follows: (i) pipet 62 L of a 2 M CaCl2 solution int
o a polystyrene vial; (ii) add 10  DNA; (iii)
rin the final volume to 500 L wit
h H2O; (iv) vortex the mixture ently and pipet it slowly into 500 L 2 HBS
uffer,
which is
ein ently vortexed; (v) incu
ate the mixture for 90 s at room tempe
rature. 4. Add the suspension (1 mL) to the cells in 5 mL of culture medium and
incu
ate it for 16 h at 37C. 5. Chane the culture medium 16 h after the transfec
tion and culture the cells for a further 48 h. 6. Test the cells for antien exp
ression.
3.2. Expression of Viral Antiens from the Constructs to
e Used for DNA Vaccina
tion Expression of the viral proteins (HBsA or T-A) is tested in meta
olically
la
eled cells.
1. La
el the transfected cells with 400 Ci [35S]-methionine for 1218 h at 37oC in
methionine-free RPMI-1640 medium supplemented with lutamine and 10% (v/ v) amin
o acid-free FCS. 2. Wash the la
eled cells twice in Ca2+/M2+-free PBS. 3. Lyse
the cells with 1 mL lysis
uffer for 30 min at 4C. 4. Remove cell de
ris
y centr
ifuation (30 min, 20.000, 4C). 5. Immunoprecipitate HBsA
y addin 5  of polyc
lonal ra

it anti-HBsA antiserum. 6. Immunoprecipitate T-A protein usin 5  of
the monoclonal anti-T-A anti
ody PA
108. 7. Incu
ate the lysate/anti
ody mixtu
res for 24 h at 4oC. 8. Dissolve 50 L protein-A Sepharose in PBS, add it to the ly
sates, and incu
ate the mixture for 1 h at 4C with entle shakin. 9. Wash the im
munoprecipitates four times in 1 mL wash
uffer, two times in 1 PBS, and once in
0.1 PBS. 10. Resuspend the protein-A Sepharose pellets
y extensive vortexin. 11
. Recover the immunoprecipitates from the protein-A Sepharose
y a 30-min incu
a
tion at 37C in 400 L elution
uffer. 12. Lyophilize the SDS-denatured eluates and
redissolve them in 30 L aqueous solution of 7% (v/v) 2-ME, 10% (v/v) lycerol and

romophenol
lue. 13. Boil the solution for 2 min. 14. Analyze 5-10 L samples
y
SDS-PAGE (usin the Laemmli
uffer system). 15. Visualize the
ands of la
eled
protein on X-ray film.
3.3. Preparation of the Plasmid DNA to
e Used for Immunization
1. Transform plasmid DNA into E. coli DH5 cells and plate the cells under anti
i
otic selection (7). 2. Select sinle colonies of transformants and row up in 5
L fermentation cultures in modified LB medium at 37oC overniht and pH 7.5, with
pH control and maximum aeration (Schleef, M., unpu
lished data).
434
Bhm et al.
3. Harvest the cells and use 60  of the wet weiht
iomass for alkaline lysis.
4. Isolate plasmid DNA usin ultrapure 100 anion exchane chromatoraphy columns
. A
out 100 m plasmid DNA can
e o
tained from a 5-L culture of transformants.
5. Remove endotoxin contamination usin the Endo-Free
uffer system. 6. Su
ject
the DNA to quality controls to ascertain that it meets the appropriate quality c
riteria (7). The preparations should contain <100 endotoxin units per 1 m DNA,
>90% (w/w) supercoiled plasmid DNA, and <1% (w/w) residual protein content. Meas
ure the concentration and the UV a
sor
ance spectrum
y spectrophotometric analy
sis
etween 220 nm and 320 nm. These quality controls are descri
ed
y Schorr et
al. (7). 7. Suspend the plasmid DNA at 10 /L in 10 TE
uffer (100 mM Tris-HCl, 10
mM EDTA, pH 7.4) and store it at 20oC. 8. Dilute the DNA solution 1:10 with Ca2+
/M2+ -free PBS to o
tain a 1 /L DNA solution within the 30 min
efore injection
of the plasmid DNA into mice.
3.4. Tumor Cell Transfectants
1. Construct the T A-expressin vector BMG/T-A.1 from the vector BMGneo (810) a
nd the plasmid pEARLY. 2. Diest pEARLY with BlII and BamHI. 3. Partially fill

in the T A-encodin BlII/BamHI frament with A and G and fill in the XhoI-line
arized vector BMGneo with C and T usin Klenow DNA polymerase. 4. Liate the com
pati
le 5 ends to yield the T-A-expression vector BMG/T-A.1. 5. Construct the
HBsA-expressin vector BMG/HBS from the vector BMGneo and the plasmid TKTHBV2.
6. Diest BMGneo with the restriction enzymes XhoI and BamHI to delete the poly
A sinal of ra

it lo
in. 7. Liate the XhoI/BlII-frament of HBV with the Xho
I/BamHI diested BMGneo vector to yield the HBsA-expression vector BMG/HBS. 8.
Transfect BMG/HBS and BMG/T-A.1 vector DNA, or BMGneo vector DNA (control witho
ut insert), into B16.F10 or P815 cells usin DOTAP followin the manufacturers in
structions. 9. Culture the transfected cells in RPMI-1640/Clicks medium suppleme
nted with penicillin and streptomycin, 5 10-5 M 2-ME, 5% (v/v) FCS and 2 mM lut
amine. 10. Start selection 48 h after the transfection
y addin 125 /mL G418. 1
1. Chane the medium every 23 d. Slowly increase the G418 concentration to 1 m/m
L durin a 46 wk selection period. 12. Test G418-resistant clones for sta
le expr
ession of HBsA or T-A as descri
ed in Su
headin 3.2.
3.5. Nucleic Acid Immunization
1. Anesthetize mice with Metofane. 2. Shave their hind les. 3. Inject 100 L of t
he 0.01 mM cardiotoxin solution into each ti
ialis anterior muscle.
Tumor-Rejectin T-Cell Responses
435
4. Inject 50 L of 1  DNA/L into each reeneratin ti
ialis anterior muscle at 5 d
after the cardiotoxin injection. 5. Use non-injected mice or mice injected with
a plasmid DNA without insert as neative controls.
3.6. Preparation of Tumor Cells for Transplantation
To avoid false immunoenicity of in vitro cultured tumor cells presentin heterolo
ous serum components from tissue culture medium, we either adapted transfected
or non-transfected tumor cells to in vivo rowth in mice, or rew tumor cells in
serum-free medium.
3.6.1. Adaptation of Tumor Cell Lines to Growth in Mice
1. Inject P815 cells (non-transfected or transfected lines) intraperitoneally in
to immunodeficient C.B-17 scid/scid (SCID) mice usin 105106 cells per mouse. 2.
Recover the cells 6 d later
y rinsin the peritoneal cavity with 10 mL PBS. 3.
Wash the cell suspensions three times in serum-free UltraCulture medium. 4. Use
these cells directly for transplantation experiments.
3.6.2. Adaptation of Tumor Cells to Growth in Serum-Free Medium
B16 melanoma cells could not
e recovered from the peritoneal cavity of SCID mic
e after injection
ecause these mice efficiently reject alloeneic cells. These
cells were adapted to rowth in serum-free medium.
1. Transfer B16 cells and transfected su
lines of this tumor cell line into seru
m-free UltraCulture medium. 2. Grow the cells for 2-3 wk prior to testin their
in vivo immunoenicity.
3.7. Su
cutaneous Transplantation of Tumor Cells into Mice
1. Su
cutaneously inject titrated num
ers of cells (102 to 106) in 50 L PBS into
the shaved left lateral flank of mice; use 5 or 10 animals per roup. 2. Measure
tumor rowth every second or third day. 3. Kill mice
earin tumors with a diam
eter >1 cm.
3.8. Intratumor Injections of Recom
inant DNA
1. Select mice
earin su
cutaneous tumors with a diameter of 0.30.5 mm for treat
ment. 2. Add 25 L DOTAP to 20  DNA dissolved in 25 L PBS to enerate DNA/ DOTAP li
posomes. 3. Use a 0.4 19 mm 28G needle to inject 50 L PBS/DOTAP (control roup) o
r 50 L DNA/DOTAP (experimental roup) into the tumors every second day. 4. Follow

tumor rowth until the tumor diameter reaches 1 cm or the tumor has reressed t
o a macroscopically undetecta
le size.
436 3.9. Cytotoxic T Cell Assay
Bhm et al.
1. O
tain spleens or lymph nodes from the DNA vaccinated mice. 2. Prepare sinle
spleen cell or lymph node cell suspensions. 3. Suspend cells in MEM tissue cult
ure medium supplemented with 10 mM Hepes
uffer, 5 10-5 M 2-ME, anti
iotics and
10% (v/v) of a selected
atch of FCS and 2 mM lutamine. 4. Coculture responder
or primed effector cells (3 x 107) with irradiated (20,000 rad) stimulator cells
(1.5 106 cells) expressin the antien of interest. Use 10 mL aliquots in upri
ht 25 cm2 tissue culture flasks in a humidified atmosphere containin 5% (v/v) C
O2 at 37C. 5. Harvest the in vivo-primed and in vitro-restimulated effector cell
populations after 5 d of culture and wash twice. 6. Coculture serial dilutions o
f the effector cells with 2 103 of 51Cr-la
eled tarets in 200 L round-
ottom wel
ls for 4 h at 37C. Use effector/taret ratios in the rane of 120. 7. Collect 50 L
of supernatant for -radiation countin followin the 4-h assay period. 8. Calcula
te the percentae of specific release as [(experimental release-spontaneous rele
ase)/(total release-spontaneous release)] 100. Measure the total radioactivity c
ounts
y resuspendin the taret cells; measure the spontaneously released count
s usin taret cell cultures without cytolytic effector cell populations. 9. Plo
t the data as the mean of triplicate cultures. The standard deviation of triplic
ate data is usually less than 10% of the mean.
References
1. Schirm
eck, R., Bhm, W., Ando, K., Chisari, F. V., and Reimann, J. (1995) Nucl
eic acid vaccination primes hepatitis B surface antien-specific cytotoxic T lym
phocytes in nonresponder mice. J. Virol. 69, 59295934. 2. Davis, H. L., Schirm
ec
k, R., Reimann, J., and Whalen, R. G. (1995) DNA-mediated immunization in mice i
nduces a potent MHC class I-restricted cytotoxic T lymphocyte response to Hepati
tis B virus surface antien. Hum. Gene Ther. 6, 14471456. 3. Schirm
eck, R., Bhm,
W., and Reimann, J. (1996) DNA vaccination primes MHC class I-restricted, simian
virus 40 lare tumor antien-specific cytotoxic T lymphocytes in H-2d mice that
reject syneneic tumors. J. Immunol. 157, 35503558. 4. Bhm, W., Schirm
eck, R., a
nd Reimann, J. (1997) Taretin an anti-viral cytotoxic T cell response to a ro
win tumor facilitates its rejection. Cancer Immunol. Immunother. 44, 230238. 5.
von Hoyninen-Huene, V., Kurth, M., and Deppert, W. (1992) Selection aainst lar
e T-antien expression in cells transformed
y lymphotropic papova virus. Virol
oy 190, 155167 6. Wild, J., Grner, B., Metzer, K., Kuhr
er, A., Pudollek, H. -P.,
Hauser, H., Schirm
eck, R., and Reimann, J. (1997) Polyvalent vaccination aain
st hepatitis B surface and core antien usin dicistronic expression plasmids. V
accine 16, 353360.
Tumor-Rejectin T-Cell Responses
437
7. Schorr, J., Moritz, P., Seddon, T., and Schleef, M. (1995). Plasmid DNA for h
uman ene therapy and DNA vaccines. Ann. NY Acad. Sci. 772, 271274. 8. Karasuyama
, H. and Melchers, F. (1988) Esta
lishment of mouse cell lines which constitutiv
ely secrete lare quantities of interleukin 2, 3, 4 and 5, usin modified cDNA e
xpression vectors. Eur. J. Immunol. 18, 97104. 9. Schirm
eck, R., Mel
er, K., Kuh
r
er, A., Janowicz, Z. A., and Reimann, J. (1994) Immunization with solu
le hepat
itis B virus surface (S) protein particles elicits murine H-2 class I-restricted
CD8+ cytotoxic T lymphocyte responses in vivo. J. Immunol. 152, 11101119. 10. Sc
hirm
eck, R., Zerrahn, J., Kuhr
er, A., Kury, E., Deppert, W., and Reimann, J. (1
992) Immunization with solu
le simian virus 40 lare T antien induces a specifi
c response of CD3+ CD4-CD8+ cytotoxic T lymphocytes in mice. Eur. J. Immunol. 22

, 759766.
37
Development of Female Contraceptive Vaccine Throuh DNA Inoculation of Human Cho
rionic Gonadotropin Beta Su
unit (hCG)
Fu-chun Zhan, Ni Wan, Don-mei Liu, Youli Jian, You-zhen Chen, Xiao-zhou Shen,
Yon-qin Cao, and Bin Wan 1. Introduction Human chorionic onadotropin (hCG)
has
een considered as a primary taret molecule for a contraceptive vaccine
y
the World Health Oranization
ecause of its physioloical and temporal specific
ity. hCG is an essential factor for the successful implantation and esta
lishmen
t of early prenancy. For a decade, the most advanced works in development of a
contraceptive vaccine have
een concentrated on polypeptide
ased vaccines tare
ted directly aainst hCG. Althouh many studies have reached the stae of clinic
al testin usin a num
er of prototype vaccines
ased on different parts of hCG
molecule, their efficacy has
een disappointin (14). Nucleic acid vaccination is
a novel technique that has many advantaes over other methodoloies. One of the
advantaes is to enerate humoral responses as well as cell-mediated immune res
ponses (58). Our la
oratory has considered usin this technoloy for development
of a potent contraceptive vaccine. There are several reasons that hCG could
e t
he ood taret for the DNA inoculation approach: (i) hCG is produced
y the fert
ilized e and is an essential factor for the implantation in the uterus, (ii) D
NA immunization can enerate specific anti
ody that could
lock the hCG function
, and (iii) DNA immunization can enerate specific cell mediated immunities that
could attack the fertilized e or expel the implanted e. Furthermore, hCG is
a mem
er of lycoprotein hormone family includin lutenizin hormone (LH), foll
icle-stimulatin hormone (FSH), and thyroid stimulatin hormone (TSH). They are
enetically,
iochemically, and immuFrom: Methods in Molecular Medicine, vol. 29
, DNA Vaccines: Methods and Protocols Edited
y: D. B. Lowrie and R. G. Whalen H
umana Press Inc., Totowa, NJ
439
440
Zhan et al.
noloically closely related. All four hormones are heterodimeric proteins that s
hare the same alpha su
unit
ut differ in their hormone-specific
eta su
unit, t
hus, the unique specificity to hCG can
e determined
y the hCG
eta su
unit (hC
G). hCG vaccines have
een shown to have a profound anti-implantation effect in cl
inical trials (2). In the methods descri
ed here, the specific hCG su
unit ene i
s selected as the taret and cloned into the plasmid vectorpCMV4 to construct a
prototype DNA vaccine (9). This prototype hCG vaccine can
e examined in the ani
mal system descri
ed and miht
e developed into a new and potent contraceptive
vaccine. 2. Materials 2.1. Construction of pCMV-hCG Plasmid DNA
1. Vector: pCMV4 (10). 2. Taret ene: hCG (pUC-hCG). 3. Enzymes: HindIII, SmaI an
d T4 DNA liase are availa
le from Promea (Madison, WI). 4. 1.5% (w/v) low melt
in temperature (LMT) aarose el. 5. TBE electrophoresis
uffer, 10X stock solu
tion: com
ine 108  Tris ( availa
le from Bio-Rad, Hercules, CA), 55 
oric aci
d, 40 mL of 0.5M ethylenediaminetetraacetic acide (EDTA), pH 8.0, distilled wate
r to 1000 mL.
2.2. Isolation and Purification of Plasmid
1. Host
acteria: E. coli DH5. 2. LB-
roth: com
ine 10 g B cto-tryptone (Difco, D
etroit, MI), 5 g B cto-ye st extr ct (Difco), 10 g N Cl in 1000 mL distilled w t
er; utocl ve. 3. LB-
roth/Ag rose: 1.4 g g rose in 100 mL LB-
roth medium. 4.
Cle n
uffer 3.0: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5% (v/v) Triton X-100, 3
M N Cl (Sino-Americ n Co., Beijing, Chin ). 5. TE
uffer: 10 mM Tris-HCl, pH 7.5
, 1 mM EDTA. 6. Lysozyme (Sino-Americ n Co., Beijing): 10 mg/mL, freshly prep re
d in distilled w ter. 7. RN se (Promeg ): 10 mg/mL, prep red in distilled w ter,


oiled for 15 min nd stored t 20C.

2.3. Tr nsfection of pCMV-hCG Using Lipofect mine
1. Lipofect mine re gent: 1 mL t 2 mg/mL (Gi
co-BRL, Gr nd Isl nd, NY). 2. DMEM
culture medium (serum-free): com
ine 10.0 g DMEM (Gi
co), 1 mL penicillin (100,
000 U/mL; Northern Chin ph rm ceutic l f ctory, Shigi zhu ng), 1 mL Streptomyci
n (100,000 g/mL; Northern Chin ph rm ceutic l f ctory), 0.843 g HEPES (Sigm , St
. Louis, MO), 1.2 g N HCO3, djust the fin l volume to 1000 mL with distilled w
ter nd sterilize the medium
y filtr tion using 0.22-m filter. 3. Cell line: m
int in Hel cells in DMEM medium with 10% (v/v) fet l c lf serum (FCS; v il
l
e from Ti n Ji n Biochemic l Products F ctory, Beijing).
Fem le Contr ceptive V ccine 2.4. An lysis of Tr nsient Expression of hCG
441
1. 96-well cell culture pl tes re v il
le from Nunc (Roskilde, Denm rk). 2. D
MEM + 20% (v/v) FCS: com
ine 20 mL FCS nd 80 mL DMEM culture medium. 3. DMEM +
10% (v/v) FCS: com
ine 10 mL FCS nd 90 mL DMEM culture medium.
2.5. DNA Immuniz tion
1. Anim ls: p thogen-free BALB/C fem le mice, ged 810 wk,
odyweight
out 20 g,
re v il
le from the Center of Experiment l Anim l, Institute of Genetics, th
e Chinese Ac demy of Sciences (CAS), Beijing. House the mice in temper ture co
ntrolled light-cycled room. 2. Adjuv nt: 0.5% (w/v)
upiv c ine-HCl (Injection U
SP) is v il
le from Astr Ph rm ceutic l Products Inc. (West
orough, MA). 3. V
ccines: pCMV-hCG t 1 mg/mL; pCMV4; hCG-TT protein v ccine is v il
le from Pro
f. Liu Xue-G o (Jinn n University, Sh ntou, Chin ). 4. S line solution: dissolve
0.9 g N Cl in 100 mL distilled w ter nd utocl ve the solution.
2.6. Antigen nd Anti
ody Detection
y ELISA
1. Mouse nti-hCG monoclon l nti
ody is v il
le from Sino-Americ n. 2. Anti-hC
G r

it serum nd purified hCG
is v il
le from the L
or tory of Endocrinolog
y, Institute of Zoology, CAS. 3. Bic r
on te
uffer c psules re v il
le from
Sigm (St. Louis, MO). 4. 0.01 M PBS (pH 7.4): 2.90 g N HPO412H2O, 0.30 g NaH2PO42
H2O, 8.5 g NaCl, distilled water 1000 mL. 5. PBST (pH 7.4): add 0.5 mL Tween-20
(Merck) to 1000 mL of PBS. 6. Horseradish peroxidase-conjugated goat anti-mouse
IgG: this is available from Boehringer-Mannheim (Indianapolis, IN). 7. Phosphate
-citrate buffer with sodium perborate: capsules are available from Sigma. 8. TMB
: 3,3,5,5-tetramethylbenzidine dihydrochloride tablets, 10 mg, are available f
rom Sigma. 9. 2 M H2SO4: slowly add 11 mL 98% (v/v) H2SO4 to 89 mL distilled wat
er. 10. Microtiter plates are available from Corning, NY. 11. Plate reader: a su
itable instrument is available from Bio-Rad (Hercules, CA).
2.7. Antigen-Specific Lymphocyte Proliferative Responses
1. RPMI-1640 culture medium: 10.4 g RPMI-1640 (Gibco-BRL), 5.95 g HEPES (Sigma),
3.4 mL 2-mercaptoethanol (Bio-Rad), 1 mL penicillin (100,000 U/mL; Northern Chi
na pharmaceutical factory), 1 mL Streptomycin (100,000 g/mL; Northern China pharm
aceutical factory), 2 g NaHCO3, dissolved in distilled water to final volume of
1000 mL, sterilized with a 0.22-m filter. 2. [3H]-thymidine: 28 Ci/mmol, availabl
e from Amersham Ltd. (Piscataway, NJ). 3. Beckman LS Analyzer, Beckman Instrumen
ts (ullerton, CA).
442 2.8. Analysis of Th1 and Th2 Immune Responses 2.8.1. Analysis of IgG1 and Ig
G2a
Zhang et al.
1. Anti-hCG anti serum and hCG are available from the Laboratory of Endocrinology,
Institute of Zoology, CAS. 2. Bicarbonate buffer capsules (0.05 M) and phosphat

e-citrate buffer with sodium perborate capsules (0.05 M) are available from Sigm
a. 3. Corning microtiter plates. 4. Goat anti-mouse IgG1 and goat anti-mouse IgG
2a (both horseradish peroxidaselabeled) are available from Southern Biotechnolog
y Associates, Inc. (Birmingham, AL). 5. Mouse IgG1 and mouse IgG2a are available
from Sigma.
2.8.2. Analysis of Cytokine Expression by RT-PCR
1. RNAgents Total RNA Isolation System, AMV reverse transcriptase and dNTPs are a
vailable from Promega. 2. Bio-Rad Thermal Cycler. 3. 10  PCR buffer: 15 mM MgCl2,
100 mM Tris-HCl (pH 9.0), 500 mM KCl, 1% (v/v) Triton X-100. 4. Plasmid pPQRS (
11).
2.9. Antifertility Trial
1. Animals: 810 wk old male and female Kunming mice are available from the Center
of Experiment Animal, Institute of Zoology, CAS, Beijing. 2. Superovulation rea
gents: 5 IU hCG and 5 IU PMSG are available from the Laboratory of Endocrinology
, Institute of Zoology, CAS.
3. Methods
3.1. The Construction and Transformation of pCMV-hCG Plasmid DNA 3.1.1. The Cloni
ng Strategy of pCMV-hCG Plasmid DNA
1. 2. 3. 4. Digest pCMV4 with HindIII. Digest the complete hCG gene fragment from
pUC-hCG with HindIII. Isolate the resulting hCG fragment and purify it on a 1.5%
(w/v) LMT agarose gel. Ligate the hCGb fragment and the HindIII-digested pCMV4 w
ith T4 DNA ligase to yield pCMV-hCG (ig. 1).
3.1.2. Transformation
1. Aliquot 100 L of CaCl2-treated competent cells into a 1.5 mL pre-chilled steri
le tube. 2. Add 5 L of plasmid, swirl, and place the tube on ice for 25 min. 3. H
eat-shock the cells by placing the tube tube in a 42C water bath for 90 s, then p
lace it on ice for 2 min. 4. Add 0.9 mL LB medium to each tube.

emale Contraceptive Vaccine

443

ig. 1. The cloning strategy for HCG subunit. 5. Incubate the tubes for1 h at 37C
on air-bath shaker (250 rpm). 6. Plate several dilutions on ampicillin-containin
g plates, and incubate 1216 h at 37C.
3.2. Isolation and Purification of Plasmid 3.2.1. Miniprep of Plasmid DNA by Alk
aline Lysis
1. Transfer 1 mL of LB culture medium into each of two 1.5 mL Eppendorf tubes, m
icrocentrifuge at top speed for 30 s, and discard the supernatant (see ref. 12).
2. Add 100 L of prechilled solution I to each tube and vortex. 3. Add 200 L of fr
esh solution II, mix, and place the tubes on ice for 5 min. 4. Add 150 of prechi
lled solution III, vortex the tubes for 2 sec., and place them on ice for 5 min.
444
Zhang et al.
5. Microcentrifuge at top speed for 5 min and transfer the supernatant to a new
tube. 6. Add to the supernatant an equal volume of a 1:1 phenol-chloroform mixtu
re, vortex, and microcentrifuge for 2 min. Transfer the supernatant into another
tube. 7. Add 2 volumes of ethanol, vortex, and let the mixture sit for 2 min at
room temperature, then place it on ice for 5 min. 8. Microcentrifuge at 12,000g
for 10 min to precipitate plasmid DNA. 9. Wash the pellets twice with cold 70%
(v/v) ethanol. Airdry the pellets for 15 min, redissolve the pellet in 50 L of TE

buffer and store the DNA solution at 20C. 10. Digest 10 L aliquots with appropriat
e restriction enzymes, and run the digested product on an agarose gel to analyze
the recombinant plasmid.
3.2.2. Isolation and Large-Scale Plasmid Purification
1. Pick one colony from the plate or take 10 L of frozen stock, inoculate 50 mL o
f LB and incubate it at 37C overnight. 2. Centrifuge the overnight culture at 7,0
00 rpm for 5 min and discard the supernatant. 3. Resuspend the pellet with 10 mL
of buffer 3.0. 4. Add 100 L of lysozyme (freshly prepared at 10 mg/mL) and 20 L o
f RNase (10 mg/mL). 5. Incubate the suspension at 50C for 10 min or until bacteri
al lysis visible. 6. Centrifuge the cell lysate for 20 min at 15,000 rpm at 4C. 7
. Transfer the supernatant into a clean tube and mix it thoroughly with 0.6 vol
of isopropanol. 8. Centrifuge the pellet for 10 min at 12,000g at 4C and discard
the supernatant. 9. Wash the pellet with 70% (v/v) ethanol. 10. Dry the pellet a
nd add 1 mL of TE into the tube to dissolve the DNA. 11. Centrifuge to remove an
y undissolved material, transfer the supernatant into a new tube. 12. Determine
the OD260/OD280 ratio and perform gel analysis.
3.3. Transfection of pCMV-hCG Using Lipofectamine
1. Seed 105 Hela cells per well in 2 mL DMEM media plus 10% (v/v) CS. 2. Incuba
te the cells at 37C in a 5% (v/v) CO2 in air atmosphere until they reach 70% conf
luence. 3. Dilute 12 g of the hCG construct or the control vector DNA into 100 L ser
um-free medium (solution A) and dilute 10 L of lipofectamine reagent into a furth
er 100 L serum-free medium (solution B). 4. Combine the two solutions A and B, mi
x them gently, and incubate the mixture at room temperature for 30 min to allow
DNA-liposome complexes to form. 5. Rinse the Hela cells once with 2 mL of serumfree medium. 6. or each transfection, add 0.8 mL of serum-free medium to the tu
be containing the complexes, mix gently and overlay the diluted solution onto th
e rinsed cells. 7. Incubate the cells with the complexes for 5 h at 37C in a CO2
incubator. 8. ollowing incubation, add 1 mL growth medium containing 20% (v/v)
CS without removing the transfection mixture.

emale Contraceptive Vaccine 3.4. Analysis of Transient Expression of hCG Subunit

3.4.1. Sample Collection
445
1. Collect the culture medium from the transfected cells after 24 h. 2. Replace
the DMEM medium with fresh medium containing 10% (v/v) CS. 3. Continue to incub
ate the transfected cells for another 48 h, then collect the culture medium and
cells. 4. Analyze the collected media and cell lysates for expression of the hCG
subunit gene (see Note 1).
3.4.2. ELISA for the Detection of hCG Protein In Vitro.
1. Coat 96-well microtiter plates at 4C overnight with 100 L per well of a monoclo
nal antibody against native hCG at a 1:500 dilution in 0.05M bicarbonate buffer (
pH 9.6). 2. Block each well with 3% (w/v) BSA-PBST at 37C for 1h. 3. Use twofold
serial dilutions from 0.1 g to 15 ng to establish a standard antigen curve. 4. Ad
d 100 L of culture medium from transfected cultures and the supernatant of cell l
ysates. 5. Incubate the plate for 1 h at 37C. 6. Add rabbit anti-hCG serum at a 1:
500 dilution. 7. Incubate at 37C for 1 h with a secondary goat anti-rabbit antise
rum conjugated with horseradish peroxidase at 1:1000. 8. Dissolve a 10 mg TMB ta
blet in 0.025 M phosphate-citrate buffer and add the solution to each well for c
olor development. 9. Stop the color development by adding 2M H2SO4. 10. Read the
light absorption with a Bio-Rad plate reader at 450 and 655 nm.
3.5. DNA Immunization
1. Inject BALB/C mice in at least three sites of the leg muscles with 50 L of 0.5
% (w/v) bupivacaine-HCl per site using a syringe equipped with a 27-gauge needle
(see Note 2). 2. After 24 h, inject 50 g or 100 g of the pCMV-hCG construct into t

he same regions as the bupivacaine-HCl injections. 3. Boost the immune response

with the same amount of DNA construct at wk 3 and 5. 4. Use the same amount of t
he pCMV4 vector as a negative control. 5. Inject 5 g hCG-TT protein as a positive
immunization control. 6. Collect sera from the postorbital vein after each immun
ization. 7. Analyze the sera by ELISA (1315).
3.6. Antibody Detection by ELISA
1. Coat 96-well microtiter plates overnight at 4C using 100 L per well of 2 g/mL of
hCG antigen in 0.05 M bicarbonate buffer (pH 9.6).
446
Zhang et al.
2. Add to each well 100 L of two-fold serial dilutions of sera starting with 1:10
0 from immunized mice (see Note 3); incubate at 37C for 1 h. 3. Add horseradish-p
eroxidase conjugated goat anti-mouse IgG at 1:10000 in PBST; incubate at 37C for
1 h. 4. Dissolve a 10 mg TMB tablet in 0.025 M phosphate-citrate buffer and add
the solution to each well for color development. 5. Stop the color development b
y adding 2 M H2SO4. 6. Read the plates using a Bio-Rad plate reader at 450 and 6
55 nm.
3.7. Analysis of Th1 and Th2 Immune Responses 3.7.1. Analysis of IgG1 and IgG2a
Detection of IgG1 and IgG2a is performed using an ELISA as described in Subheadi
ng 3.6. The only difference is the use of secondary antibodies specific for IgG1
or IgG2a isotypes instead of against whole IgG molecules.
3.7.2. Analysis of Cytokines Expression by RT-PCR (10)
1. Extract total RNA from spleens of immunized or control BALB/C mice using the
Promega Total RNA Isolation System (see ref. 10). 2. Homogenize tissue in denatu
ring solution for 5 min. 3. Add 2 M pH 4.0 sodium acetate and phenol:chloroform:
isoamyl alcohol and keep the mixture on ice for 15 min. 4. Transfer the mixture
to a fresh tube and centrifuge it for 20 min at a speed of 10,000 rpm. 5. Remove
the aqueous phrase and precipitate it with isopropanol for 5 min. 6. Wash the p
elleted RNA with ethanol and centrifuge it for 30 min at 10,000 rpm. 7. Dry the
pellet and dissolve in nuclease-free water for 15 min. 8. Add 4 g of total RNA in
10 L of diethyl pyrocarbonate (DEPC)-treated water to 2 L of random-hexamer prime
rs (0.5 mg/mL), and 0.5 L RNasin (Promega); heat the mixture at 65C for 5 min. 9.
Cool the tube on ice and spin it briefly in a microfuge at 4C to bring the conden
sation water to the bottom of the tube. 10. Add 5 L of 1.25 mM dNTPs, 4 L of 5X RT
buffer and 1 L AMV reverse transcriptase; incubate the 20.5 L reaction at 37C for
1.5 h. 11. Stop the reaction by heating the tube to 95C for 5 min. 12. Bring the
volume to 200 L using TE buffer (pH 8.0). 13. Add 10 L of the diluted first-strand
cDNA to a reaction containing 10 L of 10X PCR buffer, 2 L each of 20 M sense and a
nti-sense primers, 1 L of 20 mM dNTPs, 74.5 L of distilled water and 0.5 L of Taq p
olymerase. 14. Amplify for 35 cycles in a thermal cycler using 94C for 40 s, 60C f
or 20 s, and 72C for 40 s per cycle, with a final extension of 72C for 10 min. 15.
Use the polycompetitor pPQRS and first-strand cDNA to detect the mRNA expressio
n level of different cytokines in immunized mice, including IL-2, IL-4, IL-5, IL
-10, IL-12, IN-, TNF-, TNF-, iNOS nd HPRT.
Fem le Contr ceptive V ccine
T
le 1 Effects of pCMV-hCG DNA V ccine on Fertility in Mice Group I I-1 I-2 I-3
I-4 I-5 I-6 I-7 I-8 I-9 I-10 OD450 0.138  0.005 0.097  0.011 0.159  0.003 0.137  0.0
03 0.217  0.016 0.191  0.010 0.143  0.005 0.100  0.005 0.141  0.004 0.121  0.006 No. o
f Em
ryos 28 27 32 33 0 6 23 36 19 33 Group II II-1 II-2 II-3 II-4 II-5 II-6 II7 II-8 II-9 II-10 OD450 0.253  0.036 0.175  0.010 0.120  0.019 0.205  0.021 0.223  0.
011 0.155  0.020 0.126  0.024 0.103  0.012 0.146  0.003 0.242  0.003
447

No. of Em
ryos 0 18 24* 24* 7 32* 25 18 33* 0

Fem les in the control group cont in 30-40 em
ryos per uterus. E ch experiment l
group cont ins 10 mice num
ered I-1 to I-10 nd II-1 to II-10. Asterisks indic
te the presence of de d em
ryos. The OD450 v lues represent the levels of nti
o
dies to hCG in ser diluted 1:100; the
ckground v lue in the control groups is
0.049  0.003.
3.8. Antigen-Specific Lymphocyte Prolifer tive Responses
1. Inject five mice per group intr muscul rly with 100 g/mL of pCMV-hCG construct.
2. Boost some groups of mice t wk 3 nd wk 5. 3. Prep re single splenocyte sus
pensions from immunized nd non-immunized (control) mice one week fter the l st
injection. 4. Grow the cells in RPMI 1640 culture medium. 5. Dilute n ntigen
in RPMI-1640 to the following concentr tions: 10, 1, 0.1, nd 0.01 g/mL. 6. Add 3
0 L of protein ntigen or 100 L for cellul r ntigens per well to 96-well fl t-
ot
tom micropl tes. 7. Use three replic te wells nd include control wells with med
ium only. 8. Dilute splenocytes to 106 cells/mL with RPMI-1640. 9. Pl ce 100 L in
to e ch well nd incu
te the pl tes t 37 C in 5% (v/v) CO2 for 56 h. 10. Add 1 C
i of [3H]-thymidine to e ch well nd incu
te the pl tes for ddition l 16 h. 11
. H rvest cells nd count the incorpor ted r dio ctivity in Beckm n LS n lyze
r.
3.9. Anti-fertility Tri ls To ex mine the effic cy of pCMV-hCG DNA v ccine in mic
e, perform nti-fertility tri ls s follows, see the results in T
le 1. 3.9.1.
Group I
1. Immunize fem le mice once with pCMV-hCG DNA v ccine. 2. On d 3, tre t the immu
nized mice with 5 IU of PMSG in 200 L i.p. nd 5 IU of hCG in 200 L i.p. for 54 h
to induce superovul tion.
448
Zh ng et l.
3. M te the tre ted nim ls with m le p rtners. 4. Ex mine the fem le mice for v
gin l plugs the next morning to determine if they re pregn nt (see Note 4). 5.
Collect ntiser on d 9 fter pregn ncy nd n lyze for the presence of specifi
c nti-hCG nti
ody.
3.9.2. Group II
1. Immunize fem le mice once with pCMV-hCG DNA v ccine. 2. On d 17, tre t the imm
unized mice with 5 IU of PMSG intr peritone lly nd 5 IU of hCG intr peritone ll
y for 54 h to induce superovul tion. 3. M te the tre ted nim ls with m le p rtn
ers. 4. Ex mine the fem le mice for v gin l plugs the next morning to determine
if they re pregn nt (see Note 4). 5. Collect ntiser on d 9 fter pregn ncy n
d n lyze for the presence of specific nti-hCG nti
ody.
3.9.3. Group Control Use group of non-immunized fem le mice.
4. Notes
1. The concentr tion of hCG protein is 6.78 ng/mL nd 15.2 ng/mL t 24 nd 48 h
fter tr nsfection, respectively. There is no detect
le hCG in cell lys tes. No h
CG c n
e detected in the groups of mice receiving the control vector or the nti
-sense pCMV-hCG
construct. 2. Multiple injection sites in the leg muscle is hig
hly recommended. 3. We h ve found th t mouse serum t less th n 1:100 dilution r
e cts with TMB nd cre tes high
ckground. Thus, we suggest th t 1:100 dilu
tion should
e the st rting point for ELISA. 4. Mice with no sign of v gin l plu
gs should
e excluded from the group in ntifertility tri ls.
References
1. Ji ng, L., Xu, Y., De, S., nd Zh ng, Z. (1985) Antifertility effect of hCG

nd its ,
su
units nti
odies. Act . Zoologic . Sinic 4, 301307. 2. T lw r, G.

P., Singh, O. M., P l, R., Ch tterjee, N., S h i, P., Dh ll, K., K ur, J., et
l. (1994) A v ccine th t prevents pregn ncy in women Proc. N tl. Ac d. Sci. USA
91, 85328536. 3. Stevens V, C, (1981) Prep r tion nd formul tion of hCG ntife
rtility v ccine. Selection of peptide immunogen. Am. J. Reprod. Immunol. 6, 30731
1. 4. Stevens V. C. (1996) Progress in the development of hum n chorionic gon do
tropin ntifertility v ccines. Am. J. Reprod. Immunol. 35, 148155. 5. Wolff, J. A
., M lone, R. W., Willi ms, P., Chong, W., Acs di, G., J ni, A., nd Felgner, P.
L. (1990) Direct gene tr nsfer into mouse muscle in vitro. Science 247, 14651468
.
Fem le Contr ceptive V ccine
449
6. T ng, D. C., DeVit, M., nd Johnston, S. A. (1992) DNA inocul tion is simpl
e method for eliciting n immune response. N ture 356, 152154. 7. W ng, B., Boyer
, J., Srik nt n, V., Coney, L., C rr no, R., Ph n, C., Merv , M., D ng, K., Ag d
j ny n, M. G., Gil
ert, L., Ugen, K., Willi ms, W. V., nd Weiner, D. B. (1993 )
DNA inocul tion induces neutr lizing immune responses g inst HIV-1 in mice nd
nonhum n prim te. DNA Cell Biol. 12, 799805. 8. W ng, B., Ugen, K., Srik nt n, V
., Ag dj ny n, M., D ng, K., Fef eli, Y., S to, A. I., Boyer, J., Willi ms, W. V
., nd Weiner, D. B. (1993
) DNA inocul tion gener tes immune responses g inst
hum n immunodeficiency virus type 1. Proc. N tl. Ac d. Sci. USA 90, 41564160. 9.
Zh ng, F., W ng, N., nd Liu, D. (1996) DNA V ccine of hum n Chorionic gon dotro
pin (hCG)
et -su
unit induces immune responses in mouse (
str ct). J. Reprod.
Med. 5, 192. 10. Anderson, S., D vis, D. L., D hl
ck, H., Jornv ll, H., nd Rus
sell, D. W. (1989) Cloning, structure, nd expression of the mitochondri l cytoc
hrome P-450 sterol 26-hydroxyl se,
ile cid
iosynthetic enzyme. J. Biol. Che
m. 264, 82228229. 11. Reiner, S. L., Shichun, Z., Corry, D. B., nd Locksley, R.
M. (1993) Constructing polycompetitor cDNAs for qu ntit tive PCR. J. Immunol. Me
thods 165, 3746. 12. Ausu
el, F.M. (1995) Short Protocols in Molecul r Biology (t
hird edition), John Wiley & Sons, Inc., pp. 116. 13. W ng, B., Boyer, J., Srik nt
n, V., Ugen, K. E. , Gil
ert, L., Ph n, C., D ng, K., Merv , M., Ag dj ny n, M.
G., Newm n, M., C rr no, R., Mcc llus, D., Coney, L., Willi ms, W. V., nd Wein
er, D. B. (1994) Induction of humor l nd cellul r immune responses to the hum n
immunodeficiency type 1 virus in nonhum n prim tes
y in vivo DNA inocul tion.
Virology 211, 102112. 14. W ng, B., Boyer, J. D., Srik nt n, V., Ugen, K. E., Ayy
roo, V., Ag dj ny n, M. G., Willi ms, W. V, Newm n, M., Coney, L., C rr no, R.,
nd Weiner, D. B. (1995) Nucleic cid-
sed immuniz tion g inst HIV-1 inductio
n of protective in vivo immune responses. AIDS 9, S159S170. 15. W ng, B., Merv ,
M., D ng, K., Ugen, K. E., Willi ms, W. V., nd Weiner, D. B. (1995) Immuniz tio
n
y DNA inocul tion induces tumor rejection. Hum. Gene Ther. 6, 407418.
38
Genetic Immuniz tion for Allergy Immunother py
M rk Rom n, Helen Tighe, H ns L. Spiegel
erg, D vid Broide, nd Ey l R z 1. Intr
oduction 1.1. Allergy, A M nifest tion of n Enh nced Th2 Response to Allergens
Allergic disorders re ch r cterized
y the prev lence of immunoglo
ulin (Ig) is
otype E nti
odies, nd re considered to result from enh nced T helper type-2 (
Th2) responses to llergens. A Th2 response is ch r cterized
y enh nced humor l
responses nd the production of IL-4 nd IL-5
y CD4+ T cells (Th2) in response
to ntigen (1,2). These Th2 cytokines enh nce the llergic response
y inducing B
cell isotype switching to IgE (35),
y inducing undifferenti ted Th0 cells to fu
rther differenti te into Th2 cells, nd
y inducing eosinophil growth, nd diffe
renti tion (5). In ddition, the Th2 cytokines inhi
it Th0 differenti tion into
Th1 cells, there
y reducing the recruitment of interferon g mm (IFN-) producin
Th1 cells that could then down-reulate or modulate the Th2 responses (3,5). Con
sequently, the a
ility of a Th2 response to allerens to exert a positive feed
a
ck effect leads to a vicious cycle and may explain the exacer
ation of alleric

responses that follows continued exposure to allerens in atopic humans (2,6). S

ince the
einnin of the 20th century, alleric disorders have
een treated
y
immunotherapy. This involves the su
cutaneous injection of small,
ut radually
increasin amounts of alleren. The administration of low levels of alleren may
result in induction of Th1 cells and hih levels of IG anti
odies that
lock a
lleren
indin to IE (5). Another possi
le mechanism of action is that low lev
els of antien induce tolerance (anery) of the activated Th2 cells (5). Thouh
partially successful, the injection of allerens involves the risk of anaphylaxi
s. Over the last 15 years the use of new pharmacoloical aents for
From: Methods in Molecular Medicine, vol. 29, DNA Vaccines: Methods and Protocol
s Edited
y: D. B. Lowrie and R. G. Whalen Humana Press Inc., Totowa, NJ
451
452
Roman et al.
alleric disorders allowed the control of alleric symptoms at a lower risk to

enefit ratio than was o
tained with immunotherapy (7), which has led to a declin
e in the practice of immunotherapy. The model a
ove suests that interventions
that reduce or inhi
it allerenspecific Th2 responses would
e therapeutic in th
e treatment of alleries. As previously mentioned, immunotherapy most likely ach
ieved this result
y inducin partial anery in the activated Th2 cells, or
y

lockin the allerens from
indin to the IE on the mast cells (5). A different
approach for the treatment of alleries or asthma would
e to induce antien (a
lleren)-specific Th1 responses (813) that su
sequently could inhi
it the Th2 (al
leric) cycle due to the inhi
itory effects of IFN- (3,5). The induction of aller
en-specific Th1 responses would enhance the differentiation of Th0 cells to Th1
cells, result in a B cell isotype switch to IG2a (in the mouse), and the produ
ction of IFN- from Th1 cells upon exposure to alleren (Fi. 1). The production o
f IFN- could then suppress Th2 and eosinophil cell recruitment, Th2 cytokine and
IE production, and su
sequently mast cell deranulation. The induction of IG2a
anti
odies could also act to neutralize alleren. Gene vaccination, either intr
adermal (i.d.), or intramuscular (i.m.) characteristically induces a Th1 type re
sponse. This antien-specific Th1 response has
een shown to result in
oth the
prevention of IE induction (8,9,13), the down-reulation of existin IE anti
o
dy levels (8,10,13), and decreased eosinophil infiltration into a taret oran (
i.e. lun) (14,15) after alleren inhalation challene. A proposed model for the
induction of a Th1 immune response
y pDNA immunization (Fi. 1) includes the i
nduction of type 1 cytokines
y immunostimulatory sequences (ISS) contained with
in the plasmid
ack
one (11,18,19) causin the Th1-
ased induction. In addition,
immunostimulatory sequences can induce B-cell proliferation and isotype switchi
n to IM and IG2a without IE induction. The details of ISS effects are review
ed elsewhere in this
ook.
Fi. 1. (facin pae) (A) Th1 induction
y immunostimulatory sequence-containin
plasmid DNA (ISS-pDNA) immunization. This is the proposed mechanism
y which pD
NA immunization induces a Th1-
iased immune response. Note that in this system T
h1-
iased immunization may
e achieved
y vaccination with ISS-containin pDNA t
hat encodes an antien (alleren), or
y coinjection of a protein antien with I
SS-pDNA or ISS-phosphorothioate olio DNA. In this model, antien-presentin cel
ls (APCs) and natural killer (NK) cells produce type 1 cytokines in response to
ISS as part of an innate immune response, which leads to the differentiation of
Th0 cells to Th1 cells. APCs process and present antien from ene expression of
the pDNA or from phaocytosis of a protein antien, resultin in the eneration
of antien-specific Th1 cells. The su
sequent exposure or challene of these Th
1 calls with protein antien (alleren) results in the production and secretion
of interferon amma (IFN-), which is inhi
itory to the
Allery Immunotherapy

453
Th2 processes that mediate alleric reactions. Not shown,
ut relevant, is the I
SS-stimulated proliferation of B cells with a concomitant anti
ody isotype switc
h to the IG2a su
type. Increased levels of IG2a anti
odies help to
lock aller
en contact with IE on mast cells, there
y reducin alleric responses to aller
en exposure. (B) The inhi
itory effect of IFN- on Th2 cell recruitment and type
2 cytokine secretion, part of a positive feed
ack cycle in alleric reactions. I
FN- produced as part of the innate immune response
y NK cells and
y antien-spe
cific Th1 cells stimulated with antien acts to inhi
it the differentiation of T
h0 cells to Th2 cells, there
y inhi
itin the recruitment of new antien-specifi
c Th2 cells. In addition, IFN- inhi
its the secretion of type 2 cytokines, there

y inhi
itin eosinophil rowth and differentiation, as well as inhi
itin the pr
oduction of IE from B cells. The inhi
ition of IE and eosinophils ultimately r
esults in reduced levels of inflammatory secretion (IL-4, IL-5, histamine, trypt
ase, leukotrienes, major
asic protein, eosinophil cationic protein, and other m
ediators of inflammation)
y mast cells and eosinophils on exposure to alleren.
Taken toether, induction of Th1 immune responses help to
reak the cycle of al
leric reactions.
454
Roman et al.
Fi. 2. Primary IG su
class and IE immune response of BALB/c mice after intrad
ermal injection of 100  LacZ-pDNA (pCMV-LacZ) ( ), 10  -al in saline ( ) or 1  -
al in alum ( ).
1.2. Gene Immunization: The Primary Immune Responses to Gene or Protein Immuniza
tions The immunization of mice (i.d. or i.m.) with plasmid (p) DNA encodin an a
ntien results in the induction of a Th1 immune response. This Th1 phenotype res
ponse is enerally characterized
y the induction of an IG2a anti
ody response
and IFN- production from CD4+ splenocytes (8,16,17), whereas protein in alum (alu
minum hydroxide) immunization mainly induces IG1 and IE anti
ody responses and
IL-4 and IL-5 production from CD4+ splenocytes (8,18). The different I isotype
s induced
y E. coli -alactosidase (-al) plus alum immunization or ene immuniza
tion with pDNA expressin LacZ (LacZ-pDNA, plasmid-encodin -al) are illustrated
in Fi. 2. The cytokine responses of splenocytes stimulation in vitro from thes
e mice are shown in Ta
le 1. 1.3. The Th1 Response Induced
y Gene Vaccination I
s Dominant over the Th2 Response Induced
y Protein Vaccination
As mentioned a
ove, protein in alum induces a Th2-type immune response. However,
if mice were first immunized with pDNA encodin -al, -al/alum
oostin resulted
in an increase of the pre-existin IG2a anti
ody response, an increase in the
IFN- production in vitro
y antien stimulated splenocytes, and prevented the ind
uction of antien-specific IE anti
odies (8,9). When LacZpDNA immunized mice we
re
oosted with an unrelated antien such as hen e oval
umin (OVA) in alum, I
E anti
odies to OVA were induced, thus demonstratin that the IE anti
ody inhi

ition
y pDNA-immunization is antien-speAllery Immunotherapy
455
Ta
le 1 Lymphokine Secretion
y -Gal-Activated CD4+ Splenic T Cells from pDNA and
/or Protein Immunized Mice Exp. A B C D E F G Primin None LacZ-pDNA -Gal/saline Gal/alum LacZ-pDNA -Gal/saline -Gal/alum Boostin None None None None -Gal/alum -Gal
/alum LacZ-pDNA IFN p/mL <10 741170 <10 <10 1050314 5348 730193 IL-4 p/mL <2 <2 122
35124 42 12643 307128 IL-5 Units/mL <2 <2 154 64251 4610 16554 18370

Lymphokine secretion
y -al-activated CD4+ splenic T cells from pDNA and/or prot
einimmunized mice. The LacZ-pDNA mice were injected with 100  pDNA (AmpR) encodi
n -al. The -al/alum mice received 1  of -al protein in 3 m alum. The -al in sa
line mice were injected with 10  -al in normal saline. Cytokines were measured

y ELISA usin kits or reaents from Biosource (Camarillo, CA). Data represent me
an  SE of spleens of four mice per roup.
cific. In contrast, when mice primed with -al protein were
oosted with -al in a
lum or saline, the levels of
oth -al-specific IG1 and IE increased. Therefore
, the Th1 immune response, which is initiated
y ene vaccination, is
oosted
y
a vaccination reimen that would induce a Th2 response in naive mice or protein
-primed mice. The Th1 response to ene vaccination also prevents the su
sequent
induction of antien-specific IE anti
odies (8,9) (Fi. 3). These data demonstr
ate the potential of prophylactic immunization for the prevention of an alleric
IE response in suscepti
le individuals. This could
e useful
ecause the devel
opment of alleric disorders has a stron enetic component. Healthy individuals
from families with a history of alleries and/or asthma could potentially
e im
munized with ene vaccines in order to initiate a protective Th1 response to all
erens
efore the alleren could induce a Th2 response.
1.4. Down-Reulation of IE Anti
odies
y pDNA Immunization
In order to investiate the potential of pDNA immunization for the treatment of
an existin IE response, we first induced anti--al IE anti
odies
y immunizati
on with -al protein in alum. Six weeks after primin, these mice were immunized
with a LacZ-pDNA. This resulted in a 75% reduction of anti-al IE anti
odies wit
hin 6 wk and in an increase in anti--al IG2a anti
odies (8) (Fi. 4). The decre
ase in IE anti
ody levels was antien specific, as pDNA immunization with an ir
relevant oval
umin (Ova)-pDNA did not result in decreases in anti--al IE.
456
Roman et al.
Fi. 3. IG2a and IE response of BALB/c mice primed with 100  LacZ-pDNA (pCMV-L
acZ) and
oosted with 1  -al in alum ( ). Control mice were primed with 10  -al
in saline ( ). For the antien specificity control, mice primed with LacZ-pDNA w
ere injected with 2  oval
umin (Ova) in alum ( ).
Fi. 4. IG2a and IE response of BALB/c mice primed with 1  -al in alum and
oo
sted with 100  LacZ-pDNA (pCMV-LacZ) ( ). Control mice were primed with 1  -al i
n saline ( ). For antien specificity control, mice were
oosted with OvapDNA (
).
Allery Immunotherapy
457
1.5. Gene Immunization Suppresses Antien-Induced Pulmonary Eosinophilic Inflamm
ation
The inhalation of allerens results in alleren
indin to specific IE anti
odi
es attached via IE receptors to the surface of mast cells and
asophils. This

indin results in the release of histamine, leukotrienes, IL-4, IL-5 and other m
ediators of inflammation. This IE-mediated response is known as the immediate (
hypersensitivity) reaction (within seconds to min. of alleren exposure) (6). Th
e late-phase response occurs 424 h later, as a result of the initial mediator rel
ease, and is characterized
y the infiltration of eosinophils into the site of a
lleren exposure. There, the eosinophils deranulate in response to immune compl
exes and mast cell mediators, resultin in the release of toxic su
stances such
as major
asic protein (MBP), which causes tissue damae (19). The release of pr
oinflammatory mediators from
oth the immediate and late phase responses then le

ads to pulmonary hyperreactivity. In order to enerate a model for the developme

nt of pulmonary alleric inflammation, Bal
/c mice were made hyper-reactive to o
val
umin
y multiple injections of the antien and alum, delivered either intrap
eritoneally or su
cutaneously. Mice were then exposed to aerosolized protein wit
hin a cham
er. This inhalation challene induced eosinophil infiltration into th
e lun tissue and
ronchioli, as well as increasin eosinophil num
ers in the
o
ne marrow and
lood stream (indicative of production and traffickin of eosinoph
ils to the luns). Usin this model of an alleric reaction, we investiated the
effect of ene immunization on the late phase reaction. We found that allerenene immunization not only inhi
ited IE anti
ody formation in mice
ut also inh
i
ited the eosinophil infiltration into the lun that occurred after Ova inhalat
ion challene (15). Mice immunized with Ova-pDNA and challened
y Ova inhalatio
n showed a sinificantly reduced proportion of cells as eosinophils in the
ronc
hial airway lavae (BAL) fluid (43% to 3.5%), in the lun tissue and also in the

one marrow (10.3% to 1.4%) as compared to control mice (15) (Ta
le 2). This de
monstrated that ene vaccination could also inhi
it the late phase alleric reac
tion. Splenocytes from these ene-vaccinated mice secreted IFN-
ut no IL-5 upon
Ova stimulation, indicatin that antien-specific Th1 cells, rather than Th2 cel
ls were enerated
y the pDNA immunization. Because IL-5 influences eosinophil d
ifferentiation and survival, it is likely that the o
served decrease in eosinoph
il num
ers mentioned a
ove, was the result of the Th1 response induced
y Ova-pD
NA ene immunization, and occurred
ecause of the lack of IL-5 secretion
y anti
en-specific Th cells (Ta
le 1).
458
Ta
le 2 Percent Inhi
ition of Eosinophils
y pDNA Immunization Primin Saline Ov
a-pDNA pUC19 Naive Sensitization Challene Blood + + + + + + 5.0+2.4 4.0+0.8 8.1
+0.7 7.4+2.0 BM 10.2+1.8 13.1+0.7 18.4+0.5 13.7+0.5
Roman et al.
BALF 39.5+5.0 36.2+1.8 32.2+7.2 30.2+0
Lun 40.3+15.8 12.0+1.4 38.5+1.7 30.2+0
Eosinophil counts in taret orans: The num
ers are the percent of eosinophils (
Eos) present in various tissues after Ova inhalation. Oval
umin ene immunizatio
n (50  Ova-pDNA administered intradermally) iven prior to repeated Ova/alum adm
inistration (Corry protocol, [26]), inhi
ited the su
sequent Eos num
ers in the

one marrow [BM] and
lood (an indication of Eos rowth), and inhi
ited the su
s
equent Eos num
ers in the
ronchial airway lavae fluid (BALF) and in the lun t
issue (Lun) (an indication of Eos infiltration) after inhalation challene with
Ova. There were 8 mice per roup. The Corry protocol was not applied on the nai
ve animals. Eos counts were performed
y a
linded investiator on slides coded wi
th num
ers. Five hundred WBCs were counted in the BALF of each mouse and 10 rand
om microscope fields of lun sections were analyzed for determination of the Eos
in the lun tissue. N/S denotes normal saline. Results are means  S.E.
1.6. A Potential Mechanism of IE Down-Reulation
y pDNA-Immunization
Analysis of the lymphokine secretion profiles of CD4+ splenic T cells stimulated
in vitro with antien ives us insiht into what may
e occurrin in vivo. Sple
nocytes o
tained after LacZ-pDNA immunization of either naive mice or mice that
had
een primed
y vaccination with -al in alum produced similar levels of IFN- a
fter antien stimulation in vitro (Ta
le 1). The lack of effect of pDNA immuniza
tion on IL-4 secretion
y protein-in-alum primed mice indicates that the down-re
ulation of IE levels caused
y pDNA immunization was not mediated
y a down-re
ulation of IL-4. It seems, therefore, that the IFN- production
y the newly indu
ced Th1 cells acts to inhi
it B cell synthesis of IE, and that this is responsi

le for the 75% decrease of the IE anti
ody levels, despite the continued IL-4
production (Fi. 3). Plasmid DNA immunization results in proloned antien expre

ssion
y the cells transfected in vivo (20). Therefore, Th1 stimulation and recr
uitment may
e a continual process that leads to increasin amounts of Th1-B cel
l interactions (Fi. 4). Durin this time there are increased levels of antienspecific IG2a anti
odies. Increases in
oth protective IG, and CD4+ Th1 memory
cells capa
le of secretin IFN- could explain the decreasin levels of anti-al I
E after injections of LacZ-pDNA. It remains to
e shown whether a preexistin T
h2 response and IE anti
ody formation can
e completely eliminated over a lone
r time period
y multiple pDNA injections, and to what extent the IE-down-reul
ation will have a therapeutic effect.
Allery Immunotherapy 1.7. The Role of Immunostimulatory DNA Sequences (ISS) in
the Induction of a Th1 Response to pDNA Immunization
459
As we were characterizin the immune responses induced
y pDNA immunization, we
found that the presence of specific DNA sequences contri
uted to the induction o
f a Th1 response (21). The Th1-inducin effect of ISS-DNA occurred when these se
quences were within pDNA expression vectors, within non-codin pDNA vectors co-i
njected with pDNA expression vectors, or when ISS-DNA (ISS-pDNA or ISS-olionucl
eotides) were co-injected with protein antiens (22). Details of the
ioloical
activities of the ISS are reviewed elsewhere in this
ook.
1.8. Conclusions
The recent findins descri
ed in this review suest that alleren ene-vaccinat
ion has potential as a novel form of safe and effective immunotherapy. Gene vacc
ination does not result in inflammation when injected intradermally and has
een
shown to prevent (813) and down-reulate (8) existin antienspecific IE anti
o
dies in mice and rats, and to result in the reduction of eosinophil infiltration
of the lun in a model of the late phase alleric response (14,15). The Th1 adj
uvant effect of the ISS-containin DNA, whether within the pDNA expression vecto
rs, or co-injected with pDNA expression vectors or protein, is important for the
induction of IFN- and further promotion of Th1 cell differentiation. This respon
se results in inhi
ition of IL-5 and IE production. To date, all in vivo experi
ments showin IE inhi
ition have
een performed in mice and rats. However, pDNA
is taken up
y human skin cells transplanted onto nude mice (23), and ISS-conta
inin DNA has
een shown to induce Th1promotin cytokines from human PBMCs (22).
This suests that the ISSDNA induced Th1 responses o
served in rodents are lik
ely to also occur in human. It has yet to
e determined what reimen of enetic
vaccination or ISSDNA/protein mixture immunotherapy will
e needed to decrease I
E levels and to reduce the num
ers of alleren-specific Th2 cells to achieve cl
inical efficacy in humans. As for safety considerations, in vitro data indicate
that pDNA-transfected cells secrete low levels of antien that are unlikely to i
nduce the anaphylactic reactions o
served in traditional immunotherapy. Furtherm
ore, allerens encoded
y pDNA constructs can
e desined to include a transmem

rane domain and anchor, there
y preventin the secretion of alleren and the pos
si
le induction of anaphylactic reactions. It is likely that alleren ene vacci
nation can
e performed with a lower frequency of immunizations than the current
mode of immunotherapy,
ecause of the lonevity of ene expression, the Th1 adj
uvant effect of ISS-DNA, and a mechanism of action different from
460
Roman et al.
alleren desensitization. In addition, ISS-DNA/protein co-immunization should
e
effective at low doses of allerens, without the necessity for increasin amoun
ts as in desensitization immunotherapy. 2. Materials 2.1. General Materials
1. Triton X-114; availa
le from Sima Chemical Co. (St. Louis, MO). 2. QCL-1000
LAL Test kit, 50-648U; availa
le from BioWhittaker Inc. (Walkersville, MD). 3. L

AL water, W50-500; BioWhittaker Inc. 4. Endo Free Plasmid Maxi kit, 12362; avail
a
le from Qiaen, Inc. (Valencia, CA). 5. Ficoll 400, Bromophenol
lue, B 392; a
vaila
le from Fisher Scientific (Fairlawn, NJ). 6. RNase A; from Sima. 7. 10X T
ris
orate EDTA
uffer (TBE), 1666-703; availa
le from Boehriner Mannheim (Indi
anapolis, IN). 8. Maic cloth, 475855; availa
le from CalBiochem (San Dieo, CA)
.
2.2. Vaccination with a Tyne Device
1. Allery skin test applicator (Tyne device), Connauht La
oratories (Toronto,
Canada). 2. NaOH, 0.1 M. 3. Sodium laurel sulfate (SDS), 0.5% (w/v); Sima (St.
Louis, MO). 4. Normal saline, (hospital rade, 0.9% (w/v) NaCl in sterile water)
. 5. Hair trimmer (animal or other). 6. pDNA (prepared as in Su
headin 3.2.). 7
. Aluminum hydroxide (alum).
2.3. ELISA for Antien-Specific Anti
odies
1. 2. 3. 4. 5. 6. Car
onate
uffer (for 1 L): 1.59  Na2CO3, 2.93  NaHCO3, pH t
o 9.6. Tween-20; Sima. Phosphate-
uffered saline (PBS)-1% (w/v)
ovine serum al

umin (BSA), pH 7.4. BSA, rade 5, A-9647; Sima. Nonfat milk (any supermarket

rand). 96-Well flat
ottom ELISA plates, EIR/RIA, #3690; availa
le from Cornin
Costar Corp. (Cam
ride, MA). 7. Alkaline phosphate la
eled oat anti-mouse IG1
, IG2a, IG2
, or IG3; availa
le from Southern Biotechnoloy Associates (Birmi
nham, AL). 8. p-Nitrophenyl phosphate, pNPP ta
lets N-2770; Sima.
2.4 Radio-AlleroSor
ent test (RAST) for Specific-IE Anti
odies
1. PBS, 1/10 dilution of 10X DPBS, 17-515F; BioWhittaker Inc. 2. Sample to
e io
dinated in PBS, pH 7.0-7.2 at a concentration of 1-2 m/mL (no thiocyanate or az
ide).
Allery Immunotherapy
461
3. 0.025M phosphate
uffer pH 7.0-7.4 (NaH2PO4/Na2HPO4, pH 7.0, no chloride). 4.
Freshly prepared: chloramine-T at 1 m/mL in 0.25M PO4
uffer. 5. Freshly prepa
red: sodium meta
isulfite 1 m/mL in 0.25M PO4
uffer. 6. 125I-Na, carrier-free
(ICN #63037; usual specific activity 17 Ci/m I, 10 mCi in 100 L, i.e., 1 mCi/10 L
), ICN Biochemicals (Costa Mesa, CA). 7. 1% (w/v) BSA-PBS or PBS-5% (w/v) nonfat
milk. 8. 12 75 mm polypropylene tu
es with cap. 9. Dialysis tu
in (appropriate
to retain protein antien), tied and tested for leaks. 10. Dialysis tu
e closur
es. 11. Gloves (use dou
le loves for 125I work). 12. Plastic disposa
le 1 mL pi
pets. 13. Adjusta
le pipettors (desinated for radioactive use) and pipet tips.
14. Disposa
le plastic 1 L
ottle with a cap, filled with PBS and with a maneti
c stirrer. 15. Scissors and forceps. 16. Borate-
uffered saline (BBS), 0.1 M, pH
8.4 (18 liters): 171.65  Na2B4O7 10 H2O (0.025 M), 111.31  H3BO3 (0.1 M), 78.
91  NaCl (0.075 M). 17. Blotto stock (5X) (1 L): 50  non-fat dry milk (any sup
ermarket
rand), 0.1 mL antifoam A, (Sima), 10 mL of 1% (w/v) thimerosal (as a
preservative) (see Note 1). Brin the volume up to 1 L with BBS. Dilute 1: 5 wit
h BBS for workin stock. 18. Monoclonal rat anti-mouse IE; availa
le from Pharm
inen (San Dieo, CA). 19. Monoclonal mouse IE anti-dinitrophenol (DNP) anti
od
y IGELa2; availa
le from American Type Culture Collection, Rockville, MD. 20. Go
at polyclonal anti-mouse IE anti
odies; availa
le from Nordic Immunoloical La

s (San Clemente, CA). 21. Purified antien of interest for coatin plates. 22. P
rotein-G Sepharose, #17-0618-02; Pharmacia (Piscataway, NJ). 23. Radioimmunoassa
y (RIA) plates, 96-well polyvinyl chloride (PVC); Dynatech (Chantilly, VA).
2.5. Induction of Antien or Alleren Sensitization
1. Camco Quickstain-2
uffered differential Wriht-Geimsa stain (VWR Scientific,
San Dieo, CA)). 2. PBS: 1/10 dilution of 10X DPBS, 17-515F; BioWhittaker Inc.
3. Sterile saline (hospital rade, 0.9% (w/v) NaCl). 4. Inhalation Cham
er (4" 8
" plexilass cham
er fitted with inflow and outflow lines). 5. DeVil
iss UltraNe


-99 ne
ulizer; availa
le from Sunrise Medical (Somerset, PA). 6. Cytospin appar
atus; availa
le from Shandon Southern Inc. (Pitts
urh, PA). 7. Tissue Tech OCT
medium for tissue freezin; Sakura Finetek (availa
le from VWR Scientific). 8. F
reezin cryostat. 9. Chicken oval
umin, rade 5, A-5503; Sima. 10. Red
lood ce
ll lysin solution: potassium car
onate (100 mM, ammonium chloride, 1.5M).
462
11. 12. 13. 14. 27-Gaue mm silicon tu
in. 23-Gaue 1 mL tu
erculin syrines. A
luminum hydroxide. Purified antien of interest.
Roman et al.
3. Methods 3.1. Gene Vaccination, a General Introduction pDNA used in ene vacci
nation should
e endotoxin free. Plasmid preparations must
e performed in such
a manner to remove most endotoxin. Our procedure yields pDNA with endotoxin leve
ls in the rane of <12 n/m of pDNA. Slihtly hiher levels of endotoxin may
e
within the accepta
le rane for use,
ut care should
e taken for endotoxin may
induce IG2 and IE (25).
3.2. Preparation of Low Endotoxin Plasmid DNA (Modified Qiaen Maxi Prep Protoco
l) Plasmid DNA preparation or Maxiprep procedure may
e performed as outlined in
the Qiaen protocol
y usin Qiaens low endotoxin kit (0.10.5 / m DNA). This is
enerally sufficient for most applications. However, we find that our modified
procedure (with 2 Triton X-114 steps), thouh more time consumin, enerates pla
smid DNA with endotoxin levels of <15 n/m DNA. Endotoxin may also
e removed
y
Triton extraction (see Su
headin 3.3., Endotoxin Removal from pDNA preparation
).
1. Streak plasmid-containin
acteria onto an L
roth (LB) aar plate containin
the appropriate anti
iotic, i.e., 25  /mL kanamycin or 50 /mL ampicillin, in or
der to select sinle colonies, and allow colonies to row overniht at 37C. 2. In
oculate 5 mL LB with a sinle colony from the a
ove plate, and incu
ate in a 37C
shaker for 68 h, in order to o
tain a lo phase culture. 3. Inoculate one liter o
f LB (containin appropriate anti
iotic) usin a 1:10,000 dilution of the 8-h lo
 phase culture and then incu
ate it for16 h in a 37C shaker. 4. Centrifue the c
ultures at 40C at 6000 for 15 min (6000 rpm in Sorvall GSA or GS3 rotors [Kendro
La
oratory Products, Newtown, CT]). 5. Pour off the medium, resuspend the
acte
rial pellet in 50 mL P1
uffer (Qiaen) containin 200 /mL RNAase A (prepare
y
addin 2 mL of 100 m/mL RNAase A to 1 L P1
uffer) and transfer the supernatant
to 500 mL Nalene
ottles. 6. Add 50 mL P2
uffer (Qiaen), rotate the
ottles
ently 68 times to mix and incu
ate them at room temperature for no more than 5 m
in. 7. Add 50 mL P3
uffer (Qiaen), rotate the
ottles ently 68 times to mix. C
entrifue the
ottles for 30 min at 40C at 20,000 (13,000 rpm in a Sorvall SS-34
rotor). 8. Filter the supernatant
y pourin it throuh maic cloth in a funnel
into clean 500 mL Nalene
ottles, and centrifue them for 15 min at 10,000 rpm
. 9. Pour the supernatant over maic cloth into clean 500 mL Nalene
ottles. Ad
d 0.1 volumes 10% (v/v) Triton X-114, incu
ate the mixture on ice for 30 min.
Allery Immunotherapy
463
10. Precipitate DNA
y addin an equal volume of isopropanol. Centrifue the
ot
tles at 40C for 30 min at 15,000 (11,000 rpm in a Sorvall SS-34 rotor [Kendro]).
11. Wash the DNA pellet with 70% (v/v) ethanol, dry it in air and redissolve it
in 5 mL TE, pH 7.0 at room temperature. 12. Run 1.0 L of the DNA solution on a 1
% (w/v) aarose el to check for enomic DNA or RNA contamination and estimate a
plasmid concentration
y comparin the plasmid
and aainst a DNA mass ladder o
r known quantities of purified plasmid (determined from the optical density at 2
60 nm). Plasmid DNA contaminated with enomic DNA is discarded; plasmid DNA with
RNA contamination can
e treated with additional RNAase A. 13. After estimatin

the plasmid concentration, place 1 m of plasmid (equivalent volume) into a 50mL conical tu
e. Add an equal amount of 2X QBT (made 2X usin Qiaens recipe) and
a su
sequent volume of 1X QBT to o
tain a final concentration of 41.6  /mL (1 m
/24 mL). Add 0.1 volumes (2.4 mL) of 10% (v/v) Triton X-114 and incu
ate the mi
xture on ice for 30 min. 14. Equili
rate a Qiaen 500-tip with 10 mL QBT (Qiaen
). 15. Load 13.2 mL (or a 0.5 m DNA equivalent) onto a Maxiprep column. Collect
the flow-throuh in a 50 mL conical tu
e. 16. Wash the column twice with 30 mL
of QC
uffer (Qiaen). 17. Elute the plasmid with 15 mL of pyroen-free QF (made
usin Qiaen recipe, usin pyroen-free water) into a 50-mL conical tu
e. 18. R
e-equili
rate the column with 10 mL QBT. 19. Re-load the column with the flow-th
rouh and repeat steps 15 and 16. 20. Repeat steps 1418 as needed. 21. Precipitat
e the plasmid DNA
y addin 0.7 vol. of isopropanol. For maximal precipitation,
place the mixture at 20C for at least 1 h. 22. Centrifue for 30 min at 40C at 15,0
00 (11,000 rpm in a Sorvall SS-34 rotor [Kendro]). 23. Pour off the supernatant
, add 10 mL 70% (v/v) ethanol, and swirl it ently to wash the plasmid DNA pelle
t. 24. Centrifue for 30 min at 40C at 15,000 (11,000 rpm in a Sorvall SS-34 rot
or [Kendro]). 25. Pour off the supernatant and dry the pellet in air. 26. Dissol
ve the plasmid DNA pellet in 300 L of pyroen-free LAL water. 27. Quantitate the
plasmid DNA
y measurin the a
sor
ance at 260 nm, and check for RNA or enomic
DNA contamination
y aarose el electrophoresis.
3.3. Endotoxin Removal from pDNA Preparation If plasmid endotoxin levels are sti
ll hih after isolation (or if a different method of isolation was used) endotox
in may
e removed
y the followin method.
1. Dilute the pDNA preparation up to 40 mL in a 50-mL Falcon tu
e (Falcon, Los A
neles, CA) with (limulus ame
ecyte lysate) LAL water; use one tu
e per liter of
culture.
464
Roman et al.
2. Add 10 L of 100% (v/v) Triton X-114 per mL of diluted DNA prep (400 L/40 mL). 3
. Place the tu
es on a rotator in a cold room for 15 min. Check for complete dis
solution
efore continuin. 4. Incu
ate the tu
es on ice for 20 min. 5. Place tu

es in 55C water
ath for 25 min. A
iphasic separation should occur with a lare
, cloudy top aqueous layer, and a clear lower Triton layer (approx 5% volume). 6
. Centrifue at 37C for 20 min at 4000 (2000 rpm in a ta
letop centrifue, radiu
s 4 in.). 7. Transfer the upper phase to a fresh 50-mL Falcon tu
e. 8. Repeat st
eps 27 two more times. 9. Transfer the DNA solution to a 50-mL Falcon tu
e. Put o
nly 12 mL of solution in each tu
e to accommodate the EtOH/NaOAc precipitate mix
ture. 10. Add 0.1 volume of 3M sodium acetate, pH 5.2 and 2.5 vol. of 100% (v/v)
EtOH. 11. Store over niht at 20C for complete precipitation. 12. Centrifue the
tu
es at 4C and 4000 rpm for 30 min. 13. Wash the pellet twice with 25 mL 70% (v/
v) ethanol, then centrifue it at 4000 rpm for 10 min. 14. Air dry the pellet an
d dissolve it in an appropriate volume of saline.
3.4. Limulus Ame
ocyte Lysate Assay This assay may
e performed usin a Pyrotell
LAL kit or the BioWhittaker LAL kit. The latter has an increased sensitivity of
endotoxin determination. Special care with plasmid preparation and endotoxin det
ermination is required when experimental plasmids (or olionucleotides) are
ein
 tested or compared for their a
ility to induce, enhance, or
ias the immune re
sponse (contaminatin endotoxin may induce artifactual immune responses) (25). W
e have found that synthetic olionucleotides must also
e tested for endotoxin i
n order to allow clear interpretations of their immunostimulatory effects, as we
have found that an occasional olio synthesis could have hih endotoxin levels.
3.5. Animals
Pre-
leeds are taken prior to immunizations. Perhaps
ecause of diet or other en
vironmental factors, we have found that some mice show
ackround titers of anti

odies to common allerens or antiens. It is therefore advisa
le to screen anim

als prior to use in experiments where allerens will
e used as experimental ant
iens. You can then choose truly naive animals for comparisons of IE induction
and inhi
itions. Recommended vendors include Harlan Spraue Dawley (Indianapolis
, IN) and Jackson La
s (Bar Har
or, ME). Adult mice (16 wk of ae), sex-matched a
re used for immunizations.
Allery Immunotherapy 3.6. Intradermal Immunizations
465
Vaccinations are performed
y injectin 50 L volumes (50 ) of pDNAs or ODNs, in 0
.9% (w/v) NaCl, intradermally at the
ase of the tail. For protein in saline, co
ntrols, or protein-DNA co-immunizations, 1050  of protein are injected (dependin
on the antien; we use 10  of -al, and 50  of Ova), alone or with 50  of DNAs.
Durin injection a
le
forms under the surface of the skin. Immunizations are 
enerally iven at 13 wk intervals. Mice are
led at 2-wk intervals, startin at 2
-wk post-immunization. Injections are enerally performed with tu
erculin syrin
es with 25 aue needles.
3.7. Immunization with Protein
In order to induce a primary IE anti
ody response or
oost a secondary response
, mice are immunized either i.p. or s.c. with 125  of protein (dependin on the a
ntien, some are less immunoenic and require hiher doses) and 15 m of alum in
either 500 L or 200 L of saline (intraperitoneally and su
cutaneously, respectivel
y) (see Note 2). For
oostin, 125  of protein in 0.5 mL of saline containin 310
m of alum, is injected intraperitoneally.
3.8. Vaccination with a Tyne Device
Intradermal immunization may
e performed usin a Tyne device (tu
erculosis, or
allery skin test applicator, supplied
y Connauht La
oratories) to deliver pDN
A into the skin (9). This delivery, achieved
y puncturin the shaved skin, resu
lts in the eneration of immune responses compara
le to those achieved
y intrad
ermal injection of pDNA at the
ase of the tail (Fi. 5). At the time of our tes
tin, the Tyne devices were not commercially availa
le in the uncoated form.
1. Wash the Tyne applicators extensively in distilled water, soak them overniht
in 0.5% (w/v) sodium dodecyl sulfate (SDS), rinse with water, soak overniht in
0.1M NaOH, rinse with water, and dry at 37C for 8 h. 2. Dissolve 50  of pDNA in
6 L of saline and drip the viscous solution onto the spikes of the Tyne applicato
r. 3. Immunize mice with two applications of the Tyne applicator (100 /immunizat
ion) onto the shaved lower
ack area. 4. Immunize once a week for 3 wk.
3.9. ELISA for Antien-Specific Anti
odies
1. Coat microtiter plates overniht or for 4 h with 5  of -al per mL in car
onat
e
uffer. 2. Wash the wells with BBS, 0.05% (v/v) Tween-20, and
lock non-specif
ic
indin sites with PBS with 1% (w/v) BSA or 5% (w/v) nonfat milk for a least
2 h.
466
Roman et al.
Allery Immunotherapy
3. 4. 5. 6. 7.
467
8. 9. 10.
Wash the wells twice with BBS, 0.05% (v/v) Tween-20. Dilute serum samples 1:40 a
nd then 1:4 for 8 steps in PBS-1% (w/v) BSA, pH 7.4. Use dilutions of a hih-tit

er anti--al serum as positive controls. Add 50 L volumes to the wells, in duplica

te, and incu
ate the plates overniht at 4C. Wash the plates with BBS, 0.05% (v/v
) Tween-20 and incu
ate with alkaline phosphate-la
eled oat anti-mouse IG1, I
G2a, or IG2
for 2 h at room temperature. Wash the plates 3 times with BBS, 0.0
5% (v/v) Tween-20, and then incu
ate with developin
uffer (1 m/mL p-nitrophen
yl phosphate). Read the a
sor
ency of the solutions in the wells at 405 nm on a
Elisa reader after 1 h. Express the results as units per milliliter,
ased on ar

itrary units per milliliter of the standard serum.
3.10. Radioallerosor
ent Test for Specific-IE Anti
odies
Specific IE anti
odies are measured
y a radioallerosor
ent assay (RAST) (15,2
1). La
el proper anti
ody with 125I
y the chloramine-T method (see Note 3) at o
ne week to one day
efore settin up the RAST. The percent uptake of 125I should

e reater than 30%.
3.10.1. Protein Iodination Usin Chloramine-T
1. Place 10 L of protein at 12 m/mL concentration in a 12 75 mm polypropylene tu

e, stoppered and with a tape la
el. 2. Add 25 L of 0.25M PO4
uffer. 3. Usin a p
ipettor desinated for radioactive use, dispense 10 L of Na 125I (1 mCi). 4. Add
10 L of chloramine-T solution. Stopper the tu
e and mix rapidly,
ut without spla
shin onto the wall of the tu
e. 5. Let the reaction proceed for 5 min with inte
rmittent mixin. 6. Stop the reaction
y addin 10 L of meta
isulfite solution. 7
. Add 1.5 mL of PBS-1% (w/v) BSA (or PBS-NFM) with a 1-mL plastic pipette and tr
ansfer the contents of the tu
e to a dialysis
a, snap on the
a closure, tran
sfer the tape la
el to the
a closure and put the
a in a 1-L
ottle of PBS (t
he
ottle must
e capped). 8. Dialyze with stirrin overniht at 4C
ehind a lead
shield to remove un
ound 125I. 9. Remove the iodinated sample from the dialysis

a and put it into a 15 mL conical tu
e usin a 1-mL plastic pipet. Be careful
to keep the outside of the tu
e free from iodinated material. Fi. 5. IG1, IG
2a and IE anti
ody response to -al encodin pDNA administered via intradermal i
njection, or
y pressin the points of a pDNA-coated Tyne device into the skin.
The manitude and characteristic responses are similar with
oth intradermal adm
inistrations, illustratin that pDNA vaccinations may
e performed easily
y usi
n skin test applicators.
468
Roman et al.
10. Determine the 125I uptake
y countin a small aliquot in a amma counter. 11
. If desired, dilute the iodinated protein up to 5 mL with PBS-1% (w/v) BSA or P
BS-NFM. 12. Store the iodinated protein at 4C in a lead container or aliquoted an
d frozen at 20 to 70C (see Note 4)).
3.10.2. RAST Method
1. Num
er the polyvinyl chloride plates on the
ottom of the wells from riht to
left. 2. Coat the wells with 5 to 10 /mL of -al (or other antien of interest)
in
orate
uffered saline (BBS) (pH 8.4). 3. Incu
ate the plates for 1 h at room
temperature. 4. Aspirate the solution and wash the plates 5 times with BBS 5% (w
/v) non-fat milk (BBS-NFM). 5. Incu
ate for 1 h minimum or overniht at 4C with B
BS-NFM in order to
lock non-specific
indin sites. 6. Aspirate the
lockin
u
ffer and pat the plates dry aainst paper towels. 7. For the
est quantitation o
f specific IE anti
odies, pass the sera over protein G-Sepharose (Pharmacia) in
order to remove essentially all of the IG2a, IG2
and IG3, and a
out 90% of
the IG1 (see Note 5). Use a pool of 0.5 mL of 1:10 diluted serum of 4 mice per
roup. Add 100 L of protein G slurry (50 L of
eads + 50 L BBS), to 15 L serum, and
85 L BBS = 150 L of 1:10 serum dilution + 50 L
eads. Rotate the
eads and serum fo
r 12 h at room temperature, then overniht at 4C. 8. Dilute the serum samples 1:10
and 1:20 in BBS-NFM. 9. Add 100 L of serum dilutions and standards to the wells
in duplicate and incu
ate for 16 h or overniht at 4C. 10. Wash the plates 5 time

s with BBS-NFM. 11. Add 100 L of 125I-radiola
eled purified oat anti-IE anti
od
ies (diluted to 46 106 cpm/100 L) for 4 h at 4C. 12. Wash the plates 5 times with B
BS-NFM, and pat the plates dry. 13. Cut off the plastic wells into la
eled plast
ic tu
es and count the radioactivity with a amma scintillation counter. Use app
ropriate precautions for the use of radioactivity and storae of radioactive was
te. 14. Use serial dilutions made from anti-antien-containin serum (from anti
en/alum immunized mice) as a standard curve and positive control, and normalize
the cpm from different plates (see Note 6).
3.11. RIA Method for Total IE The RIA for total IE is performed identically to
the aforementioned RIA IE RAST assay except that the assay plates are coated w
ith a monoclonal rat anti-mouse IE anti
ody at 10 /mL and a mouse IE myeloma p
rotein is used for the standard curve (15). Make antien standard solutions ran
in from 0.098200 n/mL in BBS-NFM. The standard curve is usually linear in the 0
.39 n/mL to 100 n/mL rane.
Allery Immunotherapy 3.12. Induction of Antien or Alleren Sensitization (Mode
l of Late-Phase Pulmonary Alleric Response) 3.12.1. Method of Antien Sensitiza
tion: Corry Protocol (26)
469
For sensitization (induction of IE anti
odies) mice receive 4 s.c. injections o
f 25  protein antien (oval
umin) + 200 L PBS + 1 m aluminum hydroxide (alum) on
d 1, 7, 14, and 21.
3.12.2. Method of Antien Inhalation Challene The inhalation challene consists
of three 30-min inhalations in an inhalation cham
er, separated
y 30-min inter
vals. An antien concentration of 10 m/mL is used in the ne
ulizer, set up to a
erosolize 80100 mL of protein solution in the 30-min inhalation period. The outfl
ow of the inhalation cham
er is attached to a vacuum line, and adjusted to a min
imal suction rate that is enouh to prevent excessive condensation from occurrin
 in the cham
er. 3.13. Method for the Harvest of Tissues for Eosinophil Counts
At 24 h after the antien inhalation challene, kill the experimental mice
y ce
rvical dislocation. 3.13.1. Blood
1. Collect
lood from the carotid artery. 2. Lyse red
lood cells usin a 1:10 s
olution of 100 mM potassium car
onate, 1.5M ammonium chloride. 3. Attach the cel
ls from this fluid to microscope slides usin a cytospin 3 min at 28 or 500 rpm
(in a Shandon Cytospin 3 centrifue) and dry the slides in air at room temperat
ure.
3.13.2. Bronchial Airway Lavae Fluid (BALF)
1. Canulize the tracheas of the mice with 27 aue mm silicon tu
in attached to
a 23-aue needle on a 1-mL tu
erculin syrine. 2. Pump 600 L of sterile saline
into the luns. 3. After massain the luns, withdraw the fluid. 4. Attach the
cells from this fluid to microscope slides usin a cytospin 3 min. at 28 or 500
rpm (in a Shandon Cytospin 3 centrifue) and let them dry in air (see Note 7).
3.13.3. Bone Marrow (BM)
1. Extract
one marrow cells from femurs and rinse twice in PBS. 2. Attach the c
ells to microscope slides
y cytospin 28 or 500 rpm (in a Shandon Cytospin 3 ce
ntrifue)and stain them with Wriht-Geimsa stain for cell differential counts.
470 3.13.4. Lun
Roman et al.
1. Freeze unfixed lun tissue in OCT medium at 80C. 2. Prepare thin sections (10 M)
of frozen lun tissue, attach them to a microscope slide, fix them with acetone
for 5 min, and dry them in air.

3.14. Eosinophil Cell Stainin for Differential Cell Counts Evaluate the eosinop
hil num
ers
y stainin and microscopic examination as follows.
1. 2. 3. 4. 5. 6. Esta
lish a key code for the roups of mice, and assin slides
colors. La
el color-coded slides to desinate mouse num
ers within roups (i.e.
, 16). Apply Camco quickstain-2
uffered differential Wriht-Geimsa stain for 10
s. Rinse the slides twice with distilled water and dry them in air. Mount lass
no. 1 coverslips onto the slides with permount Examine the slides under hih ma
nification, usin someone unfamiliar with the key code. 7. Select random fields
and count the eosinophils (cells containin lare dark redstainin ranules) (27
) and the total cells until >500 cells have
een counted. 8. Express eosinophil
counts as a percent of total cells.
4. Notes
1. Do not use azide or thiocyanate. 2. Protein in saline ives only a transient
increase in IE in mice. 3. All steps involvin use of 125I are performed in fum
e hood with appropriate precautions. 4. 125I-la
eled anti
odies can
e used up t
o 3 wk. 5. A
sorption of IG from the sera eliminates competition for antien on
the plate, which may vary dependin on the IG titer, and it increases the cpm
of the specific IE anti
odies. 6. To quantitate the relative amount of anti
ody
, compare titration curves for individual sera to a standard curve on each plate
usin DeltaSOFT II v. 3.66 software (BioMetallics, Princeton, NJ). 7. If the BA
L fluid is
loody, RBCs may
e lysed
efore attachin the cells to the microscop
e slides.
Acknowledment Fiures 24 are reproduced with the permission of Proc. Natl. Acad.
Sci. USA (1996), 93, 51415145. This work was supported
y U.S. National Institut
es of Health rants AI40682 and
y Dynavax Technoloies (Berkeley, CA).. Referen
ces
1. Mosmann, T. R. and Coffman, R. L. (1989) The Th1 and Th2 cells: different pat
terns of lymphokine secretion lead to different functional properties. Annu. Rev
. Immunol. 7, 145173.
Allery Immunotherapy
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2. Coffman, R. L. and Carty, J. (1986) A T cell activity that enhances polyclona
l IE production and its inhi
ition
y interferon-amma. J. Immunol. 136, 949954.
3. Le
run, P., Lucas, A. H., McKenzie, D. T., and Spieel
er, H. L. (1989) Int
erleukin 4 acts on
oth hih- and low-density murine B cell su
population to ind
uce IE and IG1 synthesis in vitro. Intl. Arch. Allery Appl. Immunol. 88, 10811
0. 4. Snapper, C. M. and Paul, W. E. (1987) Interferon- and B cell stimulatory fa
ctor-1 reciprocally reulate I isotype production. Science 236, 944947. 5. Wiere
na, E. A., Snock, M., de Groot, C., Chretien, I., Bos, J. D., Jansen, H. M. and
Kapsen
er, M. (1990) Evidence for compartmentalization of functional su
sets o
f CD4+ T lymphocytes in atopic patients. J. Immunol. 144, 46514656. 6. Myind, N.
, Dahl, R., Pederson, S., and Thestrup-Pederson, K. (1996) Essential Allery, 2n
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., Eney, D., Goldstein, E. O., Schu
erth, K. C., Bacon, J. R., Hamilton, R. G.,
Weiss, M. E., Arshad, H., Meinert, C. L., Tanascia, J., and Wheeler, B. (1997) A
controlled trial of immunotherapy for asthma in alleric children. New Enland
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A., Roman, M., Swain, S. L., Spieel
er, H. L., and Carson, D. A. (1996) Prefer
ential induction of a Th1 immune response and inhi
ition of specific IE anti
od
y formation
y plasmid DNA immunization. Proc. Natl. Acad. Sci. USA 93, 51415145.
9. Hsu, C. H., Chua, K. Y., Tao, M. H., Lai, Y. L., Wu, H. D., Huan, S. K., an
d Hsieh, K. H. (1996) Immunoprophylaxis of alleren-induced immunolo
ulin E syn
thesis and airway hyper responsiveness in vivo
y enetic immunization. Nature M
edicine 2, 540544. 10. Hsu, C. H., Chua, K. Y., Tao, M. H., Lai, Y. L., Wu, H. D.

, Huan, S. K., and Hsieh, K. H. (1996) Inhi
ition of specific IE response in v

ivo
y alleren-ene transfer. Int. Immunol. 8, 14051411. 11. Hartl, A., Kiesslic
h, J., Weiss, R., Bernhaupt, A., Most
ock, S., Schei
lhofer, S., E
ner, C., Ferr
eira, F., and Thalhamer, J. (1999) Immune responses after immunization with plas
mid DNA encodin Bet v 1, the major alleren of
irch pollen. J. Allery Clin. I
mmunol. 103(1 Pt 1), 107113. 12. Li, X. M., Huan, C. K., Schofield, B. H., Burks
, A. W., Bannon, G. A., Kim, K. H., Huan, S. K., and Sampson, H. A. (1999) Stra
in-dependent induction of alleric sensitization caused
y peanut alleren DNA i
mmunization in mice. J. Immunol. 162, 30453052. 13. Slater, J. E., Zhan, Y. J.,
Athur-Smith, A. and Col
er-Poley, A. (1997) A DNA vaccine inhi
its IE response
s to the latex alleren Hev
5 in mice. J. Allery Clin. Immunol. 99, S504. 14.
Raz, E., Roman, M., Orozco, E. M., and Carson, D. A. (1997) Immunostimulatory D
NA sequences (ISS) are a Th1 promotin adjuvant. J. Allery Clin. Immunol. 99, S
365. 15. Broide, D., Orozco, E. M., Roman, M., Carson, D. A., and Raz, E. (1997)
Intradermal ene vaccination down-reulates
oth arms of the alleric response.
J. Allery Clin. Immunol. 99, S129.
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16. Ulmer, J. B., Donnelly, J. J., Parker, S. E., Rhodes, G. H., Felner, P. L.,
Dwarki, V. J., Gromkowski, S. H., Randall-Deck, R. R., DeWitt, C. M., Friedman,
A., Hawe, L. A., Leander, K. R., Martinez, D., Perry, H. C., Shiver, J. W., Mon
tomery, D. L., and Liu, M. A. (1993) Heteroloous protection aainst influenza

y injection of DNA encodin a viral protein. Science 259, 17451749. 17. Coutelie
r, J. P., van der Lot, J. T. M., Heessen, W. A., Waarner, G., and vanSnick, J.
(1987) IG2a restriction of murine anti
odies elicited
y viral infections. J. E
xp. Med. 165, 23732378. 18. Beck, L. and Spieel
er, H. L. (1989) The polyclonal
and antien-specific IE and IG su
class response of mice injected with oval
u
min in alum or complete Freunds adjuvant. Cellular Immunol. 123, 18. 19. Frias, E
., Motojima, S., and Gleich, G. J. (1991) The eosinophilic injury to the mucosa
of the airways in the pathoenesis of
ronchial asthma. European Respiratory J.
123s135s. 20. Raz, E., Carson, D. A., Parker, S. E., Parr, T. B., A
ai, A. M., Ai
chiner, G., Gromkowski, S. H., Sinh, M., Lew, D., Yankauckas, M. A., Baird, S.
and Rhodes, G. H.. (1994) Intradermal ene immunization: The possi
le role of D
NA uptake in the induction of cellular immunity to viruses. Proc. Natl. Acad. Sc
i. USA 91, 95199523. 21. Sato, Y., Roman, M., Tihe, H., Lee, D., Corr, M. P., N
uyen, M. D., Silverman, G. J., Lotz, M., Carson, D. A., and Raz, E. (1996) Immun
o-stimulatory DNA sequences necessary for effective intradermal ene immunizatio
n. Science 273, 352354. 22. Roman, M., Orozco, E. M., Goodman, J., Nuyen, M. D.,
Sato, Y., Ronahy, A., Korn
luth, R. S., Richman, D. D., Carson, D. A., and Raz
, E. (1997) Immunostimulatory DNA sequences function as T helper-1-promotin adj
uvants. Nature Medicine 3, 849854. 23. Hene, U. R., Walker, P. S., and Voel, J
. C. (1996) Expression of naked DNA in human, pi, and mouse skin. J. Clin. Inve
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run, P. and Spieel
er, H. L. (1987) Concomitant immuno
lo
ulin E and immunolo
ulin G1 formation in Nippostronylus
rasiliensis-infec
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5. J. Allery Clin. Immunol. 102, 977983.
26. Corry, D. B., Folkesson, H. G., Warnock, M. L., Erle, D. J., Matthay, M. A.,
Wiener Kronish, J. P., and Locksley, R. M. (1996) Interleukin 4,
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eukin 5 or eosinophils, is required in a murine model of acute airway hyperreact
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The Morpholoy of Human Blood Cells, in Wriht Stained Smears of peripheral
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ott Park, IL.
39
Preclinical Safety of DNA Vaccines
A Method to Analyze the Distri
ution of Plasmid DNA in Animal Models

Richard B. Ciccarelli, Catherine J. Pachuk, Manoj Samuel, Laurie A. Winter, and

C. Satishchandran 1. Introduction 1.1. Advantaes of DNA Vaccines DNA or enetic
vaccines are currently
ein evaluated for safety and efficacy in human clinica
l trials in the areas of infectious disease and cancer. Since DNA vaccines induc
e anti
odies and cytotoxic T lymphocytes (CTLs), they are currently
ein evalua
ted in humans for
oth prevention and therapy of HSV-2, HIV-1, and HBV infection
s, for prevention of influenza and malaria, and therapy of cutaneous T-cell lymp
homa (CTCL) and colorectal cancer. DNA vaccines offer advantaes over su
unit va
ccines, peptide vaccines and whole-killed vaccines in their a
ility to enerate

oth humoral and cellular immune responses (1). DNA vaccines are also potentiall
y safer than live-attenuated vaccine approaches, since they are non-replicatin
and present no daner of reversion to a pathoenic form. DNA vaccines also offer
manufacturin advantaes over other vaccine technoloies, since they are manufa
ctured in lare quantities as plasmids in Escherichia coli, and different vaccin
e vectors can
e made
y the same process. Furthermore, since purified DNA is re
latively sta
le in solution, and since it can
e lyophilized, DNA vaccines offer
sta
ility advantaes over live-virus vaccines. The enhanced sta
ility of DNA va
ccines may
e particularly important for their distri
ution in developin countr
ies.
From: Methods in Molecular Medicine, vol. 29, DNA Vaccines: Methods and Protocol
s Edited
y: D. B. Lowrie and R. G. Whalen Humana Press Inc., Totowa, NJ
473
474
Ciccarelli et al.
Fi. 1. Immunoloical consequences followin introduction of DNA vaccines into m
uscle. Followin administration, plasmid DNA is taken up
y muscle cells and oth
er cell types in the animal. The transfected cells serve as factories that produ
ce encoded ene products. The immune response is initiated
y the professional a
ntien-presentin cells (APCs) either throuh transfection
y the administered D
NA, or throuh the acquisition of the protein product. These APCs interact with
CD4+ T cells in proximal lymphoid tissues, stimulatin a cascade of events leadi
n to an expansion of antien specific CD4+ or CD8+ T cells and B cells (plasma
cells) that serve as effectors of cellular or humoral immune responses.
1.2. Overview of DNA Vaccine Technoloy
A DNA vaccine is typically a dou
le stranded supercoiled plasmid DNA produced in
E. coli that is enineered for mammalian expression of enes from pathoens or
tumors. The purified supercoiled DNA is formulated in isotonic saline, or with t
ransfection facilitatin components such as
upivacaine and cationic lipids. The
DNA vaccine formulation stimulates cellular and humoral immune responses to the
expressed proteins antiens (1). DNA vaccines have stimulated immune responses
usin several parenteral and mucosal routes of administration, with the intramus
cular route
ein the most widely practiced. When plasmid DNA is delivered to mu
scle, transfected myofi
ers express the ene for plasmid-encoded antien(s) tran
siently, with expression usually peakin at 7 to 14 d post-transfection as measu
red
y reporter enes such as luciferase (2). Uptake of the plasmid
y cells in
muscle as measured
y expression and the immune response to the encoded antien
can
e enhanced
y preDistri
ution of Plasmid DNA in Animal Models
475
treatment with the local anesthetic
upivacaine, or alternatively
y directly fo
rmulatin the DNA with
upivacaine (3,4). DNA vaccines introduced into muscle, s

timulate
oth humoral and cellular components of the host immune response, as sh
own in Fi. 1. Myofi
er expression of antiens is not required to enerate immun
e responses, since other routes of delivery have also produced
oth anti
odies a
nd CTLs (5). This is primarily due to the role of professional antien presentin
 cells (APCs) present in muscle and other tissues. In muscle, transfected cells
produce plasmid encoded proteins and display their processed peptide framents o
n the MHC-I complex. Althouh specific CD8+ T-cells are capa
le of reconizin p
eptide-
ound MHC-I complexes, muscle cells lack specific co-stimulatory molecule
s such as B7.1/B7.2 needed to enerate a lon-lastin memory CTL response. There
fore, antien presentin cells (APCs) such as macrophaes and dendritic cells tr
affickin in muscle are presumed to pick up framents of the antienic proteins,
and process them
y the MHC class II pathway. These APCs may then travel to dra
inin lymph nodes. It is also likely that such APCs enulf and express plasmid D
NA, and the expressed antiens are also processed throuh the MHC class I pathwa
y in these cells. In addition, cytokines are released from T-helper cells which
stimulate
oth T- and B-cell directed responses. In this manner
oth antien spe
cific anti
odies and CTLs are produced (1,6). Immune responses enerated
y this
approach have proven to
e protective aainst live challene
y infectious aen
ts in a num
er of mammals, includin chimpanzees (3,7,8). The a
ility of DNA vac
cines to induce CTL responses raises the possi
ility of their use in therapy as
well as in protection. The utility of DNA vaccines for therapy was recently demo
nstrated in HIV-1 positive chimpanzees, which cleared HIV-1 virus after vaccinat
ion with a plasmid expressin p160 (9). Althouh DNA vaccines have
een well to
lerated and have produced ro
ust immune responses in animal models, their safety
, tolera
ility and efficacy in humans have only
eun to
e evaluated. In the fi
rst human trial of a DNA vaccine, in an HIV-1 seropositive patient population, a

upivacaine-formulated plasmid expressin the HIV-1 env and rev enes was well
tolerated and increases in immune responses to p 120 were o
served (10). Recent
ly, DNA vaccines for HIV-1, HSV-2, HBV, influenza, malaria, and cancer have
een
advanced to clinical trials. Advancement of DNA vaccines to the clinic requires
extensive preclinical research and development aimed at demonstratin
oth safe
ty and immune responses in animal models. This article descri
es preclinical dev
elopment studies necessary for advancement of these vaccines into humans, and pr
esents a detailed method for one of these studies, the analysis of plasmid DNA d
istri
ution in animal tissues.
476 1.3. Preclinical Safety Studies for DNA Vaccines 1.3.1. General Consideratio
ns
Ciccarelli et al.
Preclinical safety studies for DNA vaccines should provide enouh information in
the area of their intended clinical use to allow initiation of a phase I clinic
al study (11). Preclinical studies are carried out with the clinical formulation
in accordance with ood la
oratory practices (GLP). Desin of the preclinical s
afety assessment plan should include the standard animal studies that are requir
ed of other types of vaccines, as well as studies for specific safety issues ass
ociated with DNA vaccines (12). The specific safety studies should address devel
opment of anti-DNA anti
odies, rescue of latent viruses, recom
ination with rela
ted sequences, oncoenesis resultin from expression of the antien, and plasmid
interation. Furthermore, DNA vaccines could potentially
e used as therapeutic
s, either alone or in com
ination with other dru therapies. Preclinical support
for the use of therapeutic DNA vaccines miht require their assessment of safet
y and immune responses in a relevant animal model for the specific disease. Stan
dard safety studies to assess local site reactoenicity as well as systemic toxi
city are carried out with DNA vaccine formulations in a similar manner to other
types of vaccines. Local reactoenicity studies in well esta
lished models such
as ra

it or pi should address
oth the method (e.., needle and syrine) and t
he route (e.., intramuscular) of vaccine administration. The DNA vaccine formul
ation should
e administered at or a
ove the intended clinical dosae. Tissues a

t or near the injection site(s) should
e evaluated histoloically. This evaluat

ion should include determination of the local effects due to administration of D
NA formulation (e.., local irritation), as well as effects of forein antien e
xpression and presentation to the immune system. Systemic toxicity studies shoul
d also
e assessed in a well characterized animal model, such as ra

it. The int
ended clinical dose rane and protocol should
e addressed. The animals eneral h
ealth and clinical patholoy are evaluated, alon with toxicity to tissues and t
aret orans as determined
y ross patholoy and histopatholoy. Specific toxic
oloical assessment of cells and orans of the circulatory and immune systems sh
ould also
e evaluated. In addition to standard toxicoloical studies, specific
safety issues for DNA vaccines should
e addressed. Such issues include: plasmid
DNA tissue distri
ution and persistence, includin the persistence of low level
s of antien expression leadin to immune tolerance, or in the case of human pro
teins, adverse autoimmune responses; the development of anti-DNA anti
odies, and
immune responses to DNA or chromatin; and insertion of plasmid DNA sequences in
to enomic DNA (interation), leadin to tumorienesis (11,12).
Distri
ution of Plasmid DNA in Animal Models
477
The potential development of anti-DNA anti
odies and the immuno-stimulatory effe
cts of
acterial DNA in humans has
een the su
ject of a recent review (20). Alt
houh DNA is a su
stance of relatively low immunoenicity, preclinical safety st
udies in animals with specific plasmids should include an assessment of anti-DNA
anti
odies followin multiple immunizations, in particular with the specific va
ccine formulation to
e used in humans. Impure DNA preparations or DNA associate
d with proteins miht
e more immunoenic than hihly purified plasmid DNA. In o
ne study, mice immunized with an SV40 T-antien expressin plasmid developed ant
i
odies aainst DNA and DNA-histone complexes. The anti
odies presuma
ly develop
ed due to the DNA-
ound T-antien, since mutation of the DNA
indin domain of t
he SV40 T-antien a
olished the anti-DNA anti
ody response (20). Another safety
issue associated with DNA vaccines is DNA interation. The pro
a
ility of a DNA
interation event
ein oncoenic has
een estimated to
e exceedinly low (15).
DNA interation
y a DNA vaccine is defined as random insertion (non-homoloous
recom
ination) of plasmid DNA sequences into the enomic DNA of host tissues. I
n theory, plasmid DNA could cause an insertional mutation leadin to activation
of an oncoene or disruption of a tumor suppressor ene. Even if the insertion e
vent occurred in a cell type capa
le of propaatin the mutation, this still wou
ld only
e one step in the multistep process of tumorienesis. In addition, the
pro
a
ility of a DNA insertion is decreased throuh the use of non-replicatin p
lasmids and DNA transfection in terminally differentiated, non-replicatin cells
. Althouh the pro
a
ility of a DNA insertion event leadin to tumorienesis has

een predicted to
e
elow the
ackround mutation frequency (14), it is pruden
t to evaluate the possi
ility of plasmid interation in preclinical animal model
s.
1.3.2. Persistence of DNA, Antiens, and Anti
odies
In rodents, anti
ody responses have remained elevated for periods of at least on
e yr followin intramuscular (i.m.) DNA injection. However, this has enerally n
ot
een the case in primates (13). Lonevity of the anti
ody response miht
e r
elated to persistent expression of the DNA, which requires that the DNA itself r
emain in an intact and expressi
le state in tissues. Plasmid DNA persistin in t
issues is likely to
e only in an extrachromosomal (noninterated) form. Persist
ent expression of plasmid encoded enes miht also
e the result of interation
into the cellular enome (discussed in Su
headin 1.3.1.). However, studies with
a site-specific interatin adenovirus (Ad8) showed silencin of adenoviral en
es followin chromosomal insertion (21,22). In theory, cells expressin forein
antien should
e eventually elimi-

478
Ciccarelli et al.
nated
y a host immune response, so it is possi
le that persistence of antien e
xpression miht also
e due to delivery of the plasmid DNA to cells that are poo
rly reconized
y the host immune system. Early studies with reporter enes have
indicated that ene expression could occur at low levels for periods of at leas
t 18 mo after a sinle intramuscular injection in mice (16). In a recent study w
ith a DNA vaccine vector expressin a secreted viral antien (HBsA) in mice, ex
pression was maximum at 7 to 10 d, decreased throuh 30 d and was undetecta
le a
t 50 d. However, the presence of anti-HBsA anti
odies which developed in the se
rum of these animals complicates the analysis of expression in this study (17).
Lon term expression of antiens is a concern since it could possi
ly lead to an
immunoloically unresponsive state (tolerance), althouh this has yet to
e o
s
erved experimentally (18). To address the issue of plasmid DNA persistence, it i
s important to determine
oth the distri
ution and the kinetics of distri
ution
followin administration of the plasmid. It is also important to reconize that
the persistence and distri
ution of the plasmid DNA is hihly dependent on the D
NA formulation and route of delivery. DNA plasmids formulated with
upivacaine a
nd delivered intramuscularly have
een cleared rapidly from the site of injectio
n (19). Startin with initial doses of plasmid correspondin to 1013 molecules,
plasmid was not detected at a sensitivity of 104 molecules (9 lo
elow the init
ial dose) in 23 d in muscle and skin at the site of injection (19). Althouh plas
mid DNA was rapidly cleared from the site of injection, it was useful to analyze
for a low num
er of copies of plasmid in a wide variety of tissues at later tim
e points (mo). To achieve this oal, a hihly sensitive, PCR
ased method was dev
eloped and is descri
ed in the followin sections. Usin this method, enomic DN
A from ra

it tissues was analyzed for the presence of DNA plasmid sequences thi
rty days followin intramuscular administration of 0.4 m of a DNA vaccine formu
lation desined to express the HIV-1 apol enes (Fi. 2). Total DNA was isolat
ed from a wide variety of tissues collected from six injected ra

its, and was a
nalyzed for the presence of the plasmid sequences. All the animals analyzed had
PCR-positive plasmid-sinals in DNA isolated from muscle and skin at the site of
injection, spleen, thymus and drainin lymph nodes (inuinal). Liver and
one m
arrow in some animals also was positive for plasmid sequences. However, other ti
ssues were neative, includin ovary, testes, semen, PBL, and kidney. In other e
xperiments, the PBL fraction has
een positive in some animals, which is consist
ent with the presence of plasmid sinals in the drainin nodes. This experiment
indicates that tissues
eyond the site of injection, and in particular, tissues
associated with the mammalian immune system, have plasmid associated with them a
t least 30 d post-injection, which miht
e related to the mechanism of lon-ter
m immune responses enerated in animals
y DNA
Distri
ution of Plasmid DNA in Animal Models
479
Fi. 2. Distri
ution of plasmid DNA in animal models. Tissue DNA was isolated an
d analyzed as descri
ed. Sensitivity of the PCR-
ased detection as determined
y
spike recovery was 102 copies of plasmid DNA in over 108 enome equivalents. Th
e fiure summarizes the studies with four separate study plasmids, with 6 ra

it
s/study (3 males and 3 females), 30 d followin intramuscular administration of

upivacaine formulated plasmid SNA. Plasmid DNA was not detected in
rain and lu
n tissues.
vaccines. However, this experiment cannot determine whether these plasmidspecifi
c PCR sinals indicate the presence of intact, expressi
le plasmid in these tiss
ues.

480
Ciccarelli et al.
2. Materials 2.1. Isolation of Total DNA from Tissues 2.1.1. Isolation of Total
DNA from Tissues Usin DNAzol 2.1.1.1. SPECIAL EQUIPMENT
1. 2. 3. 4. 5. 6. 7. Mini food processor. Liht Phase Lock Gels (availa
le from 5
3, Boulder, CO). Fine wire mesh strainers; Teflon microcentrifue sample pestle
. 65C and 37C water
aths. General centrifue with swinin
ucket and fixed rotor
s. Vacuum centrifue; microcentrifue. Laminar flow hood.
2.1.1.2. ENZYMES 1. Proteinase K: Prepare a stock solution of 20 m/ml in steril
e distilled water. Aliquot and store at 20C. 2. RNase A: Prepare a stock solution
of 50 m/mL in sterile distilled water. Store in aliquots at 20C. 2.1.1.3. REAGENT
S 1. Buffer saturated phenol:chloroform (1:1): store in the dark at 4C. 2. Sodium
acetate, 3M, pH 5.2/acetate. 3. Ethanol and 70% ethanol in water (v/v). 4. SDS
(10%) 5. CIP-200: Manufactured
y Calon-Vestal Corp. (St. Louis, MO). Prepare a
1% solution (v/v) usin distilled water. 6. DNAZOL: manufactured
y Life Technol
oies (Grand Island, NY).
2.1.2. Isolation of Total DNA from Muscle and Skin
1. All equipment and reaents descri
ed in Su
headin 2.1.1. except DNAzol. 2. Di
estion
uffer: 25 mM EDTA; pH 8.0/NaOH, 10 mM Tris; pH 8.0/HCl, 100 mM NaCl, 0.
5% SDS. Sterilize
y filtration throuh a 0.45 m filter. Store at room temperatur
e.
2.2. Chromatoraphic Separation of Plasmid Sequences from Genomic DNA 2.2.1. Res
triction Diestion
1. 2. 3. 4. Restriction enzymes, selected as descri
ed in Su
headin 3.2.1. Prot
einase K. SDS (10%) Buffer saturated phenol/chloroform (1:1): store in the dark
at 4C.
2.2.2. Column Chromatoraphy
1. Special equipment: FPLC; Sephacryl S-1000 column (75 mL
ed volume). Bed volu
mes of 40150 mL can
e used. Choice of the
ed volume is dependent on
Distri
ution of Plasmid DNA in Animal Models
481
2. 3. 4. 5.
the amount and volume of the sample to
e applied on the matrix. Optimal resolut
ion, and minimal loss of an added marker DNA was o
tained when 0.5 m DNA in 0.5
mL was applied to a 40-mL
ed. Dul
eccos Phosphate Buffered Saline (DPBS)(pH 7.5
): 0.2 /L KCl, 0.2 /L, KH2PO4, 8/L NaCl, 1.15 /L Na2HPO4. Column
uffer: 1 m
M EDTA, pH 8.0, 0.1% SDS, 5% ethanol in DPBS. Sodium acetate, 3.0M, pH 5.2/aceta
te. Ethanol and 70% ethanol in water (v/v).
2.2.3. Reeneration of Column
1. Freshly prepared 1.0M sodium hydroxide 2. Column
uffer.
2.3. Screenin of Column Fractions
y PCR 2.3.1. Preparation of Column Fractions
for PCR
Ethanol and 70% ethanol in water (v/v).
2.3.2. PCR of Column Fractions
1. Special equipment: Thermal cycler; aarose el electrophoresis equipment; sui
ta
le power supply; materials for the preparation and ethidium
romide stainin
of DNA aarose els. 2. Olionucleotide primers: selection of appropriate primer

s sets is descri
ed in Su
headin 3.3. Prepare 100 m stock solutions in sterile d

istilled water. Store at 20C. 3. 10 PCR
uffer: 500 mM KCl, 100 mM Tris, 15 mM MCl
2, pH 8.3 / HCl , 0.01% (w/v) elatin. 4. Deoxyri
onucleotide triphosphate (dNTP
) mixture: Prepare a stock solution mixture of dATP, dGTP, and dTTP, at 2.5 mM e
ach. The mixture is stored at 20C. 5. Dithiothreitol (DTT): Prepare a 100-mM stock
solution in sterile distilled water and filter sterilize usin a 0.22-m filter.
Aliquot and store at 20C. 6. Taq polymerase is availa
le from Perkin-Elmer/Cetus (
Norwalk, CT).
3. Methods 3.1. Isolation of Total DNA from Tissues Hih sensitivity of the assa
y requires requires decontamination of all work areas and equipment prior to tis
sue processin (see Note 1). All tissue manipulations are performed in a laminar
flow hood to ensure elimination of air
orne plasmid contamination. The method d
escri
ed is the preferred method for isolatin DNA from all tissues types. Howev
er, DNA is isolated in low yield from certain tissues such as muscle and skin
y
this method and may contain inhi
itors that affect latter steps of the assay. T
herefore, we have modified the method to yield pure DNA from muscle and skin, an
d the modification is also descri
ed.
482
Ciccarelli et al.
For
oth methods, frozen tissue must
e fully thawed on ice, and its weiht dete
rmined prior to processin. Purity of isolated DNA is essential to ensure comple
te diestion
y restriction enzymes, while havin the isolated DNA as a hih-mol
ecular-weiht species (>20 k
p) ensures efficient separation of chromosomal and
plasmid sequences durin column chromatoraphy. Optimal performance of
oth thes
e steps is required to achieve the sensitivity that this method offers.
3.1.1. Isolation of Total DNA from Tissues Usin DNAzol
1. Place the whole tissue in a mini food processor, and mince at maximum settin
until the tissue is completelyfully homoenized (see Note 2). 2. Transfer the h
omoenate into an appropriate size container (see Note 3), and add 25 mL of DNAz
ol per ram of tissue. Resuspend the tissue homoenate (see Note 4) and incu
ate
it at room temperature for 45 min. Centrifue to remove any undissolved tissue i
n a centrifue at 5000 rpm. 3. Recover the aqueous DNAzol phase
y pourin it int
o a new container throuh a fine wire mesh (see Note 5). 4. Add one-half volume
of ice-cold ethanol to the recovered DNAzol phase to precipitate the DNA. Mix en
tly
y inversion (see Note 6). 5. Pellet the precipitated DNA
y centrifuation
at 5000 rpm for 10 min and ently pour off the supernatant. Wash the pellet twic
e with 70% ethanol. 6. Dry the DNA pelletsample in a vacuum centrifue, and diss
olve in a minimal volume of sterile distilled water (see Note 7). 7. Add 50  RNa
se A per milliliter of DNA sample, mix
y inversion. Incu
ate at 37C for 1 h. 8.
Add 100  proteinase K stock per milliliter of DNA sample, and SDS to 0.5%. Incu

ate at 65C for 30 min. 9. Transfer the solution to a Phase-Lock tu
e (see Note 8),
and add an equal volume of
uffer-saturated phenol:choroform solution. Mix ent
ly
y inversion and centrifue at 3500 rpm for 10 min to separate the phases. 10
. Transfer the aqueous phase to a new container, and precipitate the DNA
y addi
n one-tenth volume of 3.0 M sodium acetate and two volumes of ice-cold ethanol.
Gently mix
y inversion. 11. Pellet the DNA
y centrifuation at 5000 rpm for 1
0 min. Pour off the supernatant, and wash the pellet twice with 70% ethanol. 12.
Dry the DNA pellet in a vacuum centrifue, and dissolve in a minimal volume of
sterile distilled water (see Note 7). 13. Repeat steps 913 as needed (see Note 9)
.
3.1.2. Isolation of Total DNA from Muscle and Skin Samples
1. Place the whole muscle in a mini food processor and mince it until the muscle
is fully homoenized (see Note 2). 2. Transfer homoenized muscle to an appropr
iate container (see Note 3), and suspend the muscle in preheated (65oC) diestio

n
uffer (2.0 mL/ tissue) (see Note 4).

Distri
ution of Plasmid DNA in Animal Models
483
3. Add SDS (0.5%), and 100  of proteinase K stock per milliliter of homoenate.
Incu
ate in a 65C water
ath for 1 h. Repeat proteinase K addition twice for a to
tal incu
ation time of 3 h. 4. Recover the aqueous solution
y pourin it into a
n appropriate container throuh a fine wire mesh strainer. Transfer the aqueous
phase into a PhaseLock tu
e, and add an equal volume of
uffer-saturated phenol:c
hloroform (see Note 8). Gently mix
y inversion. Centrifue at 3500 rpm for 10 m
in. to separate the phases. 5. Transfer the aqueous phase to an appropriate cont
ainer, and add one-tenth volume of 3.0M sodium acetate and two volumes of ice-co
ld ethanol to precipitate the DNA. Gently mix
y inversion. 6. All further proce
ssin of the DNA is identical to that descri
ed for steps 613 in Section 3.1.1.
3.2. Chromatoraphic Separation of Plasmid Sequences Form Genomic DNA 3.2.1. Res
triction Enzyme Diestion of Total Isolated Tissue DNA
Choice of the restriction enzyme is
ased on its a
ility to enerate plasmid fra
ments of no more than 2500
ase pairs in lenth while eneratin enomic DNA fr
aments that are larer than 10 k
p. Enzymes that are employed for pulse-field 
el electrophoresis (PFGE) separation of hih molecular weiht DNA (e.., NotI, N
ruI, RsrII) are suita
le. If necessary, multiple restriction enzymes may
e used
in conjunction to enerate plasmid framents of less than 2500
p in size (see
Note 10). The appropriate restriction enzyme reaction conditions should
e deter
mined prior to sample DNA processin (see Note 11).
1. Diest the DNA sample isolated from tissue with an appropriate amount of rest
riction enzyme for the pre-determined time and temperature conditions. 2. Follow
in diestion, add proteinase K to 100 /mL and SDS to 0.5%. Incu
ate at 65C for 3
0 min. 3. Apply the sample to the column. The sample may
e stored at 20C. A porti
on of the sample is analyzed
y Southern
lot to determine the extent of diesti
on
y restriction enzymes.
3.2.2. Gel Filtration Column Chromatoraphy
1. Equili
rate the Sephacryl S-1000 column with at least two volumes of column

uffer. 2. Apply 1 mL of the restriction enzyme diested DNA to the column. Elute
the DNA usin the column
uffer at a flow rate of 1 mL/min. 3. Collect 40 2.0-m
L fractions.
3.2.3. Reeneration of the Column Matrix
1. Remove residual DNA not eluted from the matrix
y washin the column with one
column volume of a 1.0M sodium hydroxide.
484
Ciccarelli et al.
2. Wash the column with three volumes of sterile water and two volumes of column

uffer. The column may
e indefinitely stored in column
uffer.
3.3. PCR Screenin of Column Samples Olionucleotides primer pairs for amplifica
tion of plasmid DNA sequences should
e desined to amplify each frament of the
plasmid enerated
y cleavae with the restriction enzyme chosen in Su
headin
3.2. Use of multiple sets of PCR primers increases the pro
a
ility of detection
of plasmid framents, and is therefore recommended (see Note 12). 3.3.1. Prepara
tion of Column Fractions for PCR
1. Precipitate column fractions
y addin two volumes of ethanol. Mix well. 2. C
entrifue at 10,000 rpm for 10 min to pellet the DNA. Gently pour off the supern
atant, and wash the pellet twice with 1 mL of 70% ethanol. 3. Dry the DNA sample

s under vacuum in a vacuum centrifue, and dissolve them in 100 mL of sterile wa

ter. Samples may
e stored at 20C until they are analyzed
y PCR.
3.3.2. PCR of Column Fractions
1. Prepare a master mixture containin all necessary components for the reaction
. The followin recipe is for 20 PCR reactions; sterile distilled water (474 L),
10X PCR
uffer (100 L), mixture of 2.5 mM each deoxyri
onucleotide triphosphate (
80 L), 100 M olio primer 1 (8 L), 100 M olio primer 2 (8 L), 100 mM DTT (10 L), and
Taq polymerase (20 L). 2. Add 35 L of master PCR mixture to 15 L of each sample, al
on with appropriate control reactions (see Note 13). 3. Carry out 25 cycles of
amplification reaction in a thermal cycler as follows: 30 s at 94C, 30 s at 55C, 3
0 s at 72C (see Note 14). 4. Analyze 20 L of each reaction
y aarose el electrop
horesis. Visualize the amplified DNA sequences
y ethidium
romide (0.5 /mL) sta
inin.
4. Notes 4.1. Isolation of Total DNA from Tissues
1. Decontamination of all equipment and work surfaces is accomplished throuh th
e use of a phosphoric acid deterent solution such as CIP-200 (Calon-Vestal Cor
p.). A 10-min treatment with a 1% CIP-200 solution is sufficient to remove conta
minatin DNA from surfaces. However, routinely the surfaces should
e soaked in
the acid deterent for 1 h. It is recommended that filter pipette tips
e utiliz
ed for pipettin manipulations to further decrease the potential of sample conta
mination, with plasmid DNA. Aerosol contaminated pipette
arrels disseminate pla
smid molecules, and these contaminate PCR samples. 2. Maximal recovery of DNA ca
n
e achieved
y complete homoenization of tissues. The homoenization in a min
i food processor, such as a Black & Decker Handy Chopper (Shelton, CT), should
e
performed at a continuous settin for the
Distri
ution of Plasmid DNA in Animal Models
485
3. 4.
5.
6.
7.
8.
9.
required time (usually 12 min). Small tissues, such as lymph nodes, cannot
e hom
oenized efficiently in a food processor. These tissues are homoenized in a mic
rocentrifue tu
e usin a Teflon pestle. DNA can
e isolated from skin
y usin
a decontaminated razor
lade, or a dissection knife,
y separatin skin layers a
nd
y cuttin them into small pieces, prior to the addition of SDS and proteinas
e K. The size of the tissue, and the amount of solution required, should
e take
n into account when selectin an appropriate container. Complete suspension of t
he homoenate is required for maximal recovery of DNA. It is recommended that on
ly a portion of the required re-suspension solution
e used initially,
y pipett
in the homoenate up and down. Remainin tissue chunks in the homoenate should

e small enouh to
e drawn throuh a wide aperture tip. Rapid re-suspension of
the tissues in
uffer minimizes nuclease action. To avoid coaulation pro
lems,
resuspend tissues such as liver and kidney immediately. Recoverin the aqueous
phase throuh a wire mesh strainer effectively removes any tissue framents from
the sample. For small tissue samples, the aqueous phase may
e recovered
y pip
ettin. Mixin
y inversion is required for most steps to prevent shearin of e

nomic DNA. DNAzol and ethanol phases
lend poorly, and multiple inversions are re
quired for complete mixin. Dissolution of the precipitated DNA sample is perfor
med
y pipettin with a wide
ore pipette tip. Smaller samples can
e dissolved
in 500 L of sterile water, while larer tissue samples such as liver may require
up to 25 mL of sterile water. DNA pellets may also
e dissolved at 4C overniht.
Avoid usin harsh methods (e.., vortexin) that could shear the enomic DNA. Ov
erdryin the DNA pellet could make dissolution difficult. For sample volumes up
to 500 L, microcentrifue Phase-Lock tu
es may
e used. Up to 25 mL of sample my

e used in 50 mL Phase-Lock tu
es for phenol: chloroform extractions. Proteinase K
/SDS treatment should
e performed until the DNA is pure. Usually two to three t
reatments are sufficient for smaller tissue samples. However larer tissues may
require multiple rounds of diestion and extraction with phenol:chloroform. Puri
ty of the DNA preparation is characterized
y the ratio of its a
sor
ance at 260
and 280 nm. Determination of the rate or the extent of diestion of the spiked
plasmid are also used to characterize the purity of the DNA sample. Due to possi

le contamination of DNA preparations
y nucleases, it is recommended that the D
NA
e stored as an ethanol precipitate at 20C. Storae of enomic DNA solutions at
20C for extended periods of time (>2 wk) results in the appearance of sinificant
amounts of deraded DNA. Quality of the enomic DNA preparation is also assesse
d
y electrophoresis on aarose els and visualized
y ethidium
romide stainin
.
4.2. Chromatoraphic Separation of Plasmid Sequences from Genomic DNA
10. Pre-testin of the column fractionation process is performed to determine th
e effectiveness of the restriction enzyme(s) chosen to separate plasmid sequence
s
486
Ciccarelli et al.
from the
ulk of enomic DNA. Genomic DNA isolated from control tissue spiked with
a known amount of plasmid may
e employed as a test sample. Most of enomic DNA
should elute in early fractions, while plasmid sequences should elute in a few
fractions toward the end of the column profile. Plasmid DNA is detected
y PCR a
nalysis of column fractions. Generation of a lare amount of smaller molecular w
eiht enomic framents will shift the elution profile of enomic DNA to overlap
that of plasmid DNA framents. Also the use of multiple restriction enzymes for
sample processin, the use of certain com
inations of restriction enzymes, or n
uclease contamination of the sample increases the possi
ility of an elution prof
ile overlap. Co-elution of enomic and plasmid DNA decreases sensitivity of the
PCR assay. An overlap of the elution profiles of enomic and plasmid sequences d
ue to incomplete diestion
y restriction enzymes, contaminatin proteins or ove
rloadin the column could result in the detection of plasmid sequences in the en
tire
ed volume of the column profile. 11. Diestion conditions must
e optimize
d for the enzyme(s) chosen. Enzyme concentrations, time and temperature of incu

ation, enzyme sta
ility durin the diestion process, and the concentration of D
NA are pre-tested to ensure complete diestion of plasmid in the presence of en
omic DNA. Genomic DNA isolated from control tissue spiked with a known amount of
plasmid is employed as a test sample. Completeness of plasmid diestion is
est
visualized
y Southern
lot analysis. Incomplete plasmid diestion will result
in overlap of enomic and plasmid DNA elution profiles durin column fractionati
on as descri
ed in Note 10. Poor restriction enzyme diestion of spiked plasmid
DNA is indicative of exchanea
le inhi
itors in the enomic DNA preparation. The
se samples may
e further treated with proteinase K/SDS, extracted with phenol:c
hloroform, and precipitated
efore use.
4.3. PCR Screenin of Column Samples
12. The sensitivity of olionucleotide primer sets should
e determined
y PCR a
nalysis. Primer sets capa
le of detectin a minimum of 1500 copies of plasmid or

less in the presence and a
sence of 1  enomic DNA usin the PCR conditions des
cri
ed in Section 3.2.2. are suita
le for analysis of fractions. An ideal primer
set is characterized
y its specificity in a enomic
ackround, and its a
ilit
y to produce visi
le quantities of product
y stainin of aarose els with ethi
dium
romide. One olio set for each plasmid frament enerated
y restriction c
leavae was chosen to analyze column fractions. 13. Control PCR reactions (no DN
A control, 1 n plasmid DNA control) should also
e carried out for each primer
set analyzed. 14. The PCR cyclin conditions descri
ed are suita
le for use with
a Perkin Elmer (Branch
ur, NJ) GeneAmp 9600 PCR System employin thin-walled PC
R tu
es. For other thermocycler systems, cyclin conditions of 1 min at 94C, 2 mi
n at 55C, and 3 min at 72C are recommended.
Distri
ution of Plasmid DNA in Animal Models
487
Acknowledments The authors wish to acknowlede the followin individuals for ad
ditional intellectual and la
oratory contri
utions to this work: Richard Carrano
, Leslie Coney, Kathleen Herold, Terry Hiins, Larry Smith, Khushroo Shroff, He
nry Klepser, Gary Gates, Maria Bam
erer, Anthony Gayle, Edward Watson, Meiqin
Lu, Donna Sosnoski, Dan McCallus, Malcolm Montomery, James Semler, Lisa Bores,
Linda Snyder, Ro
ert Troutman, David Weiner, Dan Zurawski, and Vincent Zurawski
. References
1. McDonnell, W. M. and Askari, F. K. (1996) DNA Vaccines. New Enl. J. of Med.
334, 4225. 2. Manthorpe, M., Cornefert-Jensen, F., Hartikka, J., Felner, J., Run
dell, A., Maralith, M., and Dwarki, V. (1993) Gene therapy
y intramuscular inj
ection of plasmid DNA: studies on firefly luciferase ene expression in mice. Hu
m. Gene Ther. 4, 419431. 3. Wan, B., Uen, K. E., Srikantan, V., Aadjanyan, M.
G., Dan, K., Refaeli, Y., Sato, AI., Boyer, J., Williams, W. V., and Weiner, D.
B. (1993) Gene inoculation enerates immune responses aainst human immunodefic
iency virus type I. Proc. Natl. Acad. Sci. USA 90, 41564160. 4. Davis, H. L., Bra
zolot Millan, C. L., Mancini, M., McCluskie, M. J., Hadchouels, M., Comanita, L.
, Tiollais, P., Whalen, R. G., and Michel, M.-L. (1997) DNA
ased immunization a
ainst hepatitis B surface antien (HBsA) in normal and HBsA-transenic mice. V
accine 15, 849852. 5. Fynan, E. F., Ro
inson, H. L., and We
ster, R. G. (1993) Us
e of DNA encodin influenza hemalutinin as an avian influenza vaccine. DNA Cel
l Biol. 12, 791797. 6. Ro
inson, H. L. (1997) Nucleic acid vaccines: an overview.
Vaccine 15, 785787. 7. Ro
inson, H. L., Hunt, L. A., and We
ster, R. G. (1993) P
rotection aainst a lethal influenza virus challene
y immunization with a haem
alutininexpressin plasmid DNA. Vaccine 11, 957960. 8. Boyer, J. D., Uen, K. E
., Wan, B., Aadjanyan, M., Gil
ert, L., Baarazzi, M. L., Chatteroon, M., Fro
st, P., Javadian, A., Williams, W. V., Refaeli, Y., Ciccarelli, R. B., McCallus,
D., Coney, L., and Weiner, D. B. (1997) Protection of chimpanzees from hih-dos
e heteroloous HIV-1 challene
y DNA vaccination. Nature Med. 3, 526532. 9. Boye
r, J. D., Uen, K., Chatteroon, M., Wan, B., Shah, A., Baarazzi, M. L., Javad
ian, A., Carrano, R., Coney, L., Williams, W. V., and Weiner, D. B. (1997) DNA v
accination as anti-HIV immunotherapy in infected chimpanzees. J. Infect. Dis. 17
6, 15011509. 10. MacGreor, R. R., Gluckman, S., Lacy, K., Boyer, J., Baarazzi,
M., Weiner, D., Francher, D., Gins
er, R., and Hiins, T. (1996) First Human T
rial of a Facilitated DNA Plasmid Vaccine for HIV-1: Safety and Host Response. E
leventh International Conference on AIDS, Vancouver, BC, Canada.
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11. (1997) Center for Bioloics Evaluation and Research (HFM-630), Food and Dru
Administration, Rockville, MD. 12. Smith, H. (1994) Reulatory considerations f
or nucleic acid vaccines. Vaccine 12, 15151519. 13. Donnelly, J. J., Ulmer, J. B.
, Shriver, J. W., and Liu, M. A. (1997) DNA vaccines. Ann. Rev. Immunol. 15, 6176

48. 14. Nichols, W. W., Ledwith, B. J., Manam, S. V., and Troilo, P. J. (1996) P
otential DNA vaccine interation into host cell enome. Ann. N.Y. Acad. Sci. 772
, 3039. 15. Kurth, R. (1996) Risk potential of the chromosomal insertion of forei
n DNA. Ann. N.Y. Acad. Sci. 772, 140151. 16. Wolff, J. A., Ludtke, J. J., Acsadi
, G., Williams, P., and Jani, A. (1992) Lonterm persistence of plasmid DNA and
forein ene expression in mouse muscle. Hum. Mol. Gen. 1, 363369. 17. Geissler,
M., Tokushie, K., Chante, C. C., Zurawski, V,R., and Wands, J. R. (1997) Cellul
ar and humoral immune response to hepatitis B virus structural proteins in mice
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ased immunization. Gastroenteroloy 112, 13071320. 18. Xian, Z. Q., S
pitalnik, S. L., Chen, J., Erikson, J., Wojczyk, B., and Ertl H. C. (1995) Immu
ne responses to nucleic acid vaccines to ra
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Winear, R. A., Monforte, J. A., Suin, K. D., OLouhlin, K. G., Rudd, C. J., and
Macreor, J. T. (1996) Determination of tissue distri
ution of an intramuscula
r plasmid vaccine usin PCR and in situ hy
ridization. Hum. Gene Ther. 7, 2185219
4. 20. Moens, U., Seternes, O. M., Hey, A. W., Sisland, Y., Traavik, T., Johasen
, B., and Rekvi, O. P. (1995) In vivo expression of a sinle viral DNA-
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protein enerates systemic lupus erythematosus-related autoimmunity to dou
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randed DNA and histones. Proc. Natl. Acad. Sci. USA 92, 12,39312,397. 21. Doerfle
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ortive infection and malinant transformation
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teration of viral DNA and control of viral ene expression
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tion of forein DNA into mammalian enomes and its consequences: a concept in on
coenesis. Adv. Cancer Res. 66, 313344.
40
DNA Vaccination
Tolerance and Autoimmunity
Gil Mor and Mariel Eliza 1. DNA Vaccines: An Overview Infectious diseases result
in sinificant mor
idity and mortality worldwide. Preventin these infections i
s
oth a pu
lic health priority and the primary oal of vaccine research. The di
scovery that cell-mediated and/or humoral immune responses aainst viruses, para
sites,
acteria and tumor antiens can
e induced
y antien-encodin DNA plasmi
ds is revolutionizin the vaccine development field (13). DNA vaccines (also know
n as plasmid DNA or nucleic acid vaccines) have
een proven to successfully prev
ent infection in a variety of animal models, and are currently underoin clinic
al trials in humans for the prevention of a variety of infections, includin HIV
(1,4,5). DNA vaccines consist of a plasmid DNA
ack
one containin an antienen
codin ene and a stron mammalian promoter that controls its expression. When i
njected intramuscularly or intradermally (6,7) the antien is transcri
ed, trans
lated and presented to the immune system in the context of a self major histocom
pati
ility complex (MHC) (8,9). More specifically, circulatin lymphocytes encou
nter plasmid-encoded antien in the muscle
ed at the site of injection, initiat
e a humoral response in the drainin lymph nodes and then seed distal lymphoid o
rans (9,10). Althouh the a
ility of DNA vaccines to elicit stron and specific
immune responses is well esta
lished, concerns have
een raised reardin their
safety. More specifically, their potential to induce deleterious immune respons
es, such as autoimmunity and the development of tolerance in response to the per
sistent expression of a forein antien.
From: Methods in Molecular Medicine, vol. 29, DNA Vaccines: Methods and Protocol
s Edited
y: D. B. Lowrie and R. G. Whalen Humana Press Inc., Totowa, NJ
489
490
Mor and Eliza
1.1. Autoimmunity The potential of DNA vaccines to result in the formation of an
ti-DNA anti
odies is of special concern. This is important not only in healthy i

ndividuals,
ut also in those with autoimmune diseases, such as systemic lupus e
rythematosus (SLE) where anti
odies aainst sinle- and dou
le-stranded DNA, dou

le-stranded RNA, polyri
onucleotides, ri
onucleoprotein, and ri
osomes are prod
uced. The
acterial oriin and intrinsic immunostimulatory activity of the plasm
id
ack
one of DNA vaccines hihliht concerns reardin the potential of DNA va
ccines to induce deleterious immune responses. Specifically, DNA motifs composed
of an unmethylated CpG dinucleotide flanked
y two 5 purines (optimally a GpA)
and two 3 pyrimidines (optimally a TpC or TpT) activate the innate immune syst
em to produce a series of immunomodulatory cytokines such as interleukin-6, inte
rferon- (IFN-), IL-12, and tumor necrosis factoralpha (TNF-), s well s immunoglo

ulin M (IgM) nti
odies (11). Hex mers
e ring this sequence motif re 20 times
more common in micro
i l th n m mm li n DNA due to differences in the frequency
of utiliz tion nd methyl tion p ttern of CpG dinucleotides in prok ryotes versu
s euk ryotes. These immunomodul tory properties of
cteri l DNA c n result in i
mmune ctiv tion, the production of IgG nti-DNA uto nti
odies nd the developm
ent of glomerulonephritis in mice. B cteri l DNA lso h s
een shown to cceler
te uto nti
ody production in lupus-prone (NZB X NZW)F1 mice (12). An ddition l
s fety concern ssoci ted with the use of DNA v ccines is th t myocytes th t m
y t ke up the injected pl smid nd express the encoded ntigen could potenti lly

ecome t rgets for ntigen-specific T-cells or the developments of utoimmune m
yositis. In series of studies in mice, DNA v ccin tion h s
een shown to trigg
er low level IgG nti-DNA,
ut not ntimuscle cell uto nti
ody production. In
ddition v ccin tion did not induce or cceler te the development of systemic or
muscle cell-specific utoimmune dise se (13). 1.2. Toler nce
DNA v ccines ( dministered intr muscul rly or intr derm lly) induce strong prote
ctive immune responses in dult nim ls. Yet, most v ccines intended for hum n u
se re dministered to inf nts nd children. Due to the imm turity of their immu
ne system, new
orns exposed to foreign ntigens re t risk for developing toler
nce r ther th n immunity. A num
er of f ctors influence the development of neon
t l toler nce, including the n ture, concentr tion nd mode of ntigen present
tion to the immune system, nd the ge of the host. Experiment l evidence sugges
ts th t recognition of foreign determin nts is cquired t distinct ges of m tu
r tion,
Toler nce nd Autoimmunity
491
r nging from e rly gest tion until d ys or weeks fter
irth (14). Since the pro
tein encoded
y DNA v ccine is produced endogenously nd is expressed in the c
ontext of self MHC, the potenti l exists for the neon t l immune system to recog
nize it s self, nd therefore for the development of toler nce. This is confirm
ed
y studies demonstr ting th t pl smid v ccine encoding the circumsporozoite
protein of the m l ri p r site Pl smodium yoelii induces toler nce r ther th n
immunity when dministered to 25-d-old mice. Neon t lly tolerized nim ls were u
n
le to mount nti
ody, cytokine, or cytotoxic responses when rech llenged with
the DNA v ccine in vivo or in vitro. This toler nce, however, ppe rs to
e spe
cific for immunogenic epitopes expressed
y the v ccine-encoded, endogenously pr
oduced ntigen. Mice ch llenged with exogenous circumsporozoite protein produced
nti
odies g inst different set of epitopes, nd were not tolerized (15). Th
e n ture nd loc liz tion of the immune response elicited
y DNA v ccin tion c n

e determined
y sensitive nd specific ss ys such s the enzymelinked immunos
or
ent ss y (ELISA) nd enzyme-linked immunospot ss y (ELISPOT). We here
y des
cri
e the pplic tion of these ss ys to the study of the following immune respo
nse p r meters: (i) ntigen-specific nti
ody production
y B-cells, nd (ii) cy
tokine production
y lymphoid cells. 2. M teri ls Use the following m teri ls fo
r st nd rd ELISA nd ELISPOT ss ys:
1. 2. 3. 4. 5. 6. 7. Polystyrene microtiter pl tes. Inverted microscope. ELISA r
e der. Incu
tor. ELISA pl te w sher (option l). Multich nnel pipette with dispo
s
le tips for 50200 L. Micropipette with dispos
le tips for 2001000 L nd 20200 L.

2.1. Re gents
1. 2-Amino-2-methyl-1-prop nol lk line
uffer solution: Sigm , St. Louis, MO: 1
00 mL. 2. 5-Bromo-4-chloro-3-indolyl phosph te (BCIP). Sigm , 500 mg. 3. Se Pl q
ue g rose. FMC Corp. (Fockl nd, ME): 25 g. 4. Strept vidin lk line phosph t se
(AP). J ckson ImmunoRese rch L
or tories (West Grove, PA). 5. Phosph t se su
s
tr te kit. Kirkeg rd-Perry L
or tories (G ithers
urg, MD). 6. Anti
odies.
492 2.2. Buffers
Mor nd Eliz
1. Culture medium: RPMI 1640 cont ining 10% (v/v) fet l c lf serum (FCS), 20 mM
HEPES, 10 mM sodium pyruv te, 1.5 mM L-glut mine nd supplemented with 100 U/mL
penicillin /streptomycin. 2. PBS/BSA Stock solution: Add 50 g BSA to 500 mL 10X
PBS. Dilute 1:10
efore use. 3. Blocking
uffer: dd 25 g (5% (w/v)) BSA nd 0.1
25 mL (0.025% (v/v)) of Tween-80 to 500 mL of 1X PBS. Sterilize
y filtr tion. 4
. PBS/Tween: Stock solution: 10X PBS nd 0.25 % (v/v) Tween 80. Dilute 1:10
efo
re use. 5. DDW-Tween: Dou
le-distilled w ter (DDW) nd 0.025% (v/v) Tween-80 (DD
W-Tween). 6. Buffer for second ry nti
ody nd lk line phosph t se vidin D (SA
B): 1X PBS, 5% (v/v) FCS nd 0.05% (v/v) Tween. 7. BCIP Ag rose mixture: Add 1 m
L of w ter nd 0.03 g of g rose to 4 mL of BCIP. Mix gently nd melt the g ros
e
y
oiling in microw ve oven for 13 min depending on the volume. 8. Phosph t
se su
str te solution: Add 2 mL of dieth nol mine
uffer (5X) nd two p-nitrophe
nyl t
lets to 10 mL of w ter.
3. Methods 3.1. New
orn Immuniz tion
1. Remove the mother mouse from the c ge cont ining the pups nd pl ce her on
sep r te c ge t the opposite side of the room. 2. Pl ce the c ge under l mp t
o keep the pups w rm. 3. Use 32-g uge needle with yellow tip on it to inject
the pups (see Note 1). Le ve ~2 mm of free needle t the end of the pipet tip t
o limit the depth of injection. 4. Inject 10 L of pl smid on e ch of the pups thig
hs while holding the pup
etween the index nd middle fingers. 5. Cle n ll
loo
d on the pup with w rm w ter
efore returning the pup to the c ge. 6. Hold the m
other on your h nd for t le st 23 min
efore returning her to the c ge with the
pups.
3.2. Enzyme-Linked Immunosor
ent Ass y (see Note 2) 3.2.1. Antigen-Specific ELIS
A Ass y
Use the ntigen-specific ELISA ss y for epitope m pping of specific nti
odies
in the serum of nim ls injected with pl smid DNA. Microtiter pl tes re co ted
with either synthetic peptides or the recom
in nt protein.
3.2.1.1. COATING PLATES
1. Co t fl t-
ottom 96-well Immulon I (Dyn tech L
s, Inc., Alex ndri , VA) micr
otiter pl tes with purified protein or synthetic peptide (120 g of ntigen/ mL) in
0.1M c r
on te
uffer (pH 9.6) (see Note 3). Cover with pl stic wr p. 2. Incu

te for 6 h t room temper ture or overnight t 4C.
Toler nce nd Autoimmunity
493
3. Remove the wr p nd disc rd the excess co ting ntigen. 4. Block the pl tes w
ith
locking
uffer for 2 h t room temper ture. 5. Disc rd the
locking
uffer
nd fill the wells with the w shing solution PBSTween. Dip the pl te in cont i
ner filled with w shing solution to fill ll the wells nd let it sit for
out
5 min. W sh pl tes the s me w y t le st three times with PBS-Tween followed
y
3 ddition l w shings with DDW. Store the pl tes in the refriger tor for l ter u
se. 6. Add s mple to the wells, diluting it in PBS s desired. 7. Incu
te for 2
h t room temper ture. 8. W sh pl tes s descri
ed in step 5 using DDW-Tween in

ste d of PBS-Tween. 9. Add 1/1000 dilution of phosph t se conjug ted nti-mouse

IgG, IgM, IgG1 or IgG2 nti
odies in PBS-BSA nd incu
te for 2 h t room tempe
r ture.
3.2.1.2. DETECTION
1. W sh the pl tes s in step 5, Su
he ding 3.2.1.1. 2. Add 100-L of fresh phos
ph t se su
str te per well, cover the pl tes, nd keep them in d rk pl ce unti
l you re re dy to run the ss y. 3. Develop the color for 1530 min nd re d the
optic l densities t 405 nm (see Note 4). 4. The concentr tion of serum nti
ody

ound to the pl tes is determined from the colorimetric re ction. Use st nd r
d curve gener ted with known dilution of high-titer nti-serum.
3.3. Enzyme-Linked Immunospot Ass ys (ELIspot)
ELISPOT ss y is the most ppropri te technique for the detection nd qu ntific
tion of individu l nti
ody or immunoglo
ulin-secreting cells in vitro. B sed on
the s me principle s the enzyme-linked immunosor
ent ss y, the ELISPOT ss y
c n
e used to study ntigen specific responses g inst
oth solu
le (protein n
d polys cch ride) ntigens nd p rticul te ntigens or peptides. In ddition, th
is ss y c n
e used not only for the detection of nti
ody secreting cells,
ut
of ny cell which secretes specific product. In this c p city, the ELISPOT s
s y h s proved to
e powerful technique for the study of cytokine-producing ce
lls (16).
3.3.1. Cell Prep r tion
Prep re single cell suspension from spleen or lymph nodes in RPMI-1640 supplem
ented with 10% (v/v) he t-in ctiv ted FCS, 1.5 mM L-glut mine, nd 100U/mL penic
illin/streptomycin.
494 3.3.2. Antigen-Specific ELIspot Ass y
3.3.2.1. COATING PLATES
Mor nd Eliz
1. Co t fl t-
ottom Immulon II microtiter pl tes with 50 L of the ntigen (protei
n or peptide) in co ting
uffer. PBS (pH 7.4) nd 0.1M c r
on te
uffer (pH 9.6)
re the most frequently used co ting
uffers (see Note 5). 2. If the co ting w
s c rried out t 4oC , le ve the pl te t room temper ture for 15 min to llow t
he temper ture to equili
r te. Disc rd the excess co ting
uffer from the wells
nd dd 200 L
locking
uffer for 1 h t room temper ture. 3. Disc rd the
lockin
g
uffer nd w sh the pl tes with PBS-Tween 3 5 min nd 5X with DDW s well to r
emove the Tween. 4. Add the spleen or lymph node cell suspension (1 106/well) to
the ntigen-co ted pl tes in seri l dilutions nd incu
te for 5 h t 37 oC in
humidified 5% (v/v) CO2 environment. Do not move or sh ke the pl tes during th
is time. 5. W sh the pl tes s descri
ed in step 3. 6. Add 50 L of the isotype-sp
ecific lk line phosph t se-l
eled second ry nti
ody in SAB
uffer nd incu
t
e for 2 h t room temper ture
3.3.2.2. DETECTION
1. Prep re the strept vidin AP in SAB
uffer 30 min
efore dding it to the pl t
es. 2. W sh the pl tes with DDW-Tween, 3 5 min. e ch. 3. Add 50 L of the strept v
idin AP per well (1:1000) nd incu
te the pl tes for 1 h t room temper ture. 4
. W sh the pl tes s in step 2, Su
he ding 3.3.2.2. 5. Add 50 L of BCIP/0.6% (w/v
) g rose mixture. Do not move the pl tes until the g rose solidifies.
3.3.2.3. READING PLATES
A colored precipit te will form to indic te the presence of ntigen. After 24 h,
count the spots using dissecting microscope.
3.3.3. Cytokine-Specific ELISPOT

3.3.3.1. COATING PLATES

1. Co t 96-well nitrocellulose-
cked microtiter pl tes with 50 L/well of 110 g/mL
of the desired nti-cytokine nti
ody in 0.1M c r
on te
uffer (pH 9.6). Cover t
he pl tes with pl stic lid/cover nd incu
te for 4 h. t room temper ture. 2.
Block the pl tes
y dding 200 L/well of
locking
uffer for 2 h t room temper
ture (see step 3, Su
he ding 2.2.). 3. W sh 3 5 min with PBS-Tween nd 3 with DDW
. 4. Add seri l two-fold dilutions of the single-cell suspension to the co ted p
l tes, st rting with n initi l concentr tion of 106 cells/well nd incu
te for
5h. t
Toler nce nd Autoimmunity
495
37C in humidified 5% (v/v) CO2 environment. 5. W sh pl tes 3 5 min with DDW-Twe
en nd 3 with DDW. 6. Add 50 L of the
iotinyl ted nti-cytokine nti
ody (1 g/mL)
in SAB nd incu
te overnight t 4oC. 7. W sh the pl tes s in step 5. 8. Using
the s me
uffer s for the second nti
ody, tre t the w shed pl tes with 1/200
0 dilution of vidin-conjug ted lk line phosph t se for 1 h t room temper ture
. Longer incu
tions m y
e c rried out t 4C. 9. W sh the pl tes s in step 5.
3.3.3.2. DETECTION
1. Add 50 L of BCIP/NBT per well nd incu
te for 1020 min t room temper ture. 2.
Disc rd nd w sh 3 5 min with DDW.
3.3.3.3. READING PLATES
1. A colored precipit te, or ELISPOT, will form t sites were individu l cells s
ecreted the cytokine(s) of interest. 2. Select cell dilution resulting in ppr
oxim tely 50 spots/well to c lcul te the tot l num
er of cytokine-secreting cell
s/s mple. 3. C lcul te the tot l num
er of cytokine-secreting cells per experime
nt l nim l s follows: (num
er of cytokine secreting cells/106 in e ch org n) (n
um
er of cells/org n)/106.
3.4. Assessing Ren l P thology nd Proteinuri in Experiment l Su
jects
Auto nti
ody production m y result in the deposition of nti
ody complexes in th
e glomeruli nd therefore in the development of ren l p thology. Anti
ody induce
d ren l dise se is m nifested t the microscopic level
y the visu liz tion of
nti
ody complexes deposited t the glomeruli nd t the m croscopic level
y cli
nic lly evident proteinuri .
3.4.1. Tissue Preserv tion nd St ining
1. Remove org ns t the time of s crifice. 2. Fix one kidney in 10% (w/v) form l
in, em
ed in p r ffin nd section for st ining with hem toxylin nd eosin. 3. Fl
sh freeze the second kidney in liquid nitrogen, fix nd section for st ining wi
th fluorescein-conjug ted go t nti-mouse IgG.
3.4.2. Ren l P thology nd Proteinuri
1. Ex mine sections microscopic lly for evidence of immune complex deposition (s
ee Note 6). 2. Monitor urine s mples using Al
ustix (B yer, Elkh rt, IN) to est

lish the pres496
ence nd monitor the progression of proteinuri .
Mor nd Eliz
4. Notes
1. Speci l c re must
e given to the h ndling of the pups when immunizing new
or
n mice to ensure th t the mother will not reject the pups. Alw ys we r gloves n
d m sk when h ndling new
orn mice. 2. ELISA provides s fe nd simple method

of me suring ntigen specific nd tot l immunoglo
ulins. Engv ll nd Perlm nn (1

971) (17) nd Schuurs nd Weemen (1977) (18) first descri
ed this method. V riou
s types of ELISA h ve
een developed nd descri
ed in the liter ture since then.
3. The protein should
e titr ted to determine the optim l co ting concentr tio
n. 4. The degree of development tt ined correl tes in line r f shion with the
time to re ch the re ction pl te u, therefore we k sign ls c n
e improved
y l
onger developing times. In contr st, dding 50 L/well of 0.2 M N OH c n stop f
st re ction. If n ss y is overdeveloped, it c n
e re-re d fter w shing with
DDWTween, nd DDW nd then developing it g in s in steps 2 nd 3, Su
he ding 3
.2.1.2. 5. It is import nt to determine which one is the optim l co ting
uffer
for your experiment. Co ting m y
e c rried out for 6 h t room temper ture or o
vernight t 4C. Alw ys cover the pl tes with pl stic wr p to prevent ev por tion
of the ntigen/
uffer co ting. 6. Qu ntit tion m y
e possi
le on the
sis of f
luorescence intensity.
References
1. Donnelly, J., Ulmer, J. nd Liu, M. (1997) DNA V ccines. Life Scien. 60, 16317
2. 2. Donnelly, J. J., Ulmer, J. B., nd Liu, M. A. (1994) Immuniz tion with DNA
. J. Immunol. Methods 176, 145152. 3. Liu, M. A. (1995) Overview of DNA v ccines.
Ann. N.Y. Ac d. Sci. 772, 1520. 4. Must f , F., Johnson, E., S ntoro, J. C., Wis
sink, J., Mullins, J. I., H ynes, J. R., Letvin, N. L., Wy nd, M., nd Ro
inson,
H. L. (1996) Simi n Immunodeficiency Virus DNA V ccine:Tri l in M c ques. J. Vi
rol. 70, 39783991. 5. Y sutomi, Y., Ro
inson, H. L., Lu, S., Must f , F., Lekutis
, C., Arthos, J., Mullins, J. I., Voss, G., M nson, K., Wy nd, M., nd Letvin, N
. L. (1996) Simi n immunodeficiency virus-specific cytotoxic T-lymphocyte induct
ion through DNA v ccin tion of rhesus monkeys. J. Virol. 70, 678681. 6. Wolff, J.
A., Ludtke, J. J., Acs di, G., Willi ms, P., nd J ni, A. (1992) Long-term pers
istence of pl smid DNA nd foreign gene expression in mouse muscle. Hum. Mol. Ge
net. 1, 363369. 7. Wolff, J. A., Dowty, M. E., Ji o, S., Repetto, G., Berg, R. K.
, Ludtke, J. J., Willi ms, P., nd Sl utter
ck, D. B. (1992) Expression of n ke
d pl smids
y cultured myotu
es nd entry of pl smids into T tu
ules nd c veol
e of m mm li n skelet l muscle. J. Cell Sci. 103, 12491259. 8. Ulmer, J. B., Deck
, R. R., De Witt, C. M., Friedm n, A., Donnelly, J. J. nd Liu,
Toler nce nd Autoimmunity
497
9.
10.
11.
12.
13. 14.
15.
16.
17. 18.
M. A. (1994) Protective immunity
y intr muscul r injection of low doses of infl
uenz virus DNA v ccines. V ccine 12, 15411544. Ulmer, J. B., Donnelly, J. J., P
rker, S. E., Rhodes, G. H., Felgner, P. L., Dw rki, V. J., Gromkowski, S. H., De
ck, R. R., De Witt, C. M., Friedm n, A., H we, L., Le nder, K., M rtinez, D., Pe
rry, H., Shiver, J., Montgomery, D., nd Liu, M. (1993) Heterologous protection
g inst influenz
y injection of DNA encoding vir l protein. Science 259, 174

51749. Mor, G., Klinm n, D., Sh piro, S., H giw r , E., Sedeg h, M., Norm n, J.,
Hoffm n, S., nd Stein
erg, A. (1995) Complexity of the cytokine nd nti
ody re
sponse elicited
y immunizing mice with Pl smodium yoelii circumsporozoite prote
in pl smid DNA. J. Immunol. 155, 20392046. Klinm n, D. M., Yi, A.-K., Be uc ge, S
. L., Conover, J., nd Krieg, A. M. (1996) CpG motifs present in
cteri l DNA r
pidly induce lymphocytes to secrete interleukin 6, interleukin 12, nd interfer
on g mm . Proc. N tl. Ac d. Sci. USA 93, 28792883. Gilkeson, G. S., Pippen, A. M.
, nd Pisetsky, D. S. (1995) Induction of crossre ctive nti-dsDNA nti
odies in
pre utoimmune NZB/NZW mice
y immuniz tion with
cteri l DNA. J. Clin. Invest.
95, 13981402. Mor, G., Singl , M., Stein
erg, A., Hoffm n, S., Okud , K., nd Kl
inm n, D. (1997) Do DNA v ccines induce utoimmune dise se? Hum. Gene Ther. 8, 2
93300. M rodon, G. nd Roch , B. (1995) Activ tion nd deletion of self-re ctive m
ture nd imm ture T cells during ontogen of Mls-1 : implic tions for neon t l to
ler nce induction. Int. Immunol. 6, 18991904. Mor, G., Y mshchikov, G., Sedeg h,
M., T keno, M., W ng, R., Houghten, R., Hoffm n, S., nd Klinm n, D. (1996) Indu
ction of neon t l toler nce
y pl smid DNA v ccin tion of mice. J. Clin. Invest.
98, 27002705. H giw r , E., A

si, F., Mor, G., Ishig tsu
o, Y., nd Klinm n, D
. (1995) Phenotype nd frequency of cells secreting IL-2, IL-4, IL-6, IL-10, IFN
nd TNF lph in hum n peripher l
lood. Cytokine 7, 815822. Engv ll, E. nd Perl
m nn, P. (1971) Enzyme-linked immunosor
ent ss y (ELISA) qu ntit tive ss y of
immunoglo
ulin G. Immunochem. 8, 871874. Schuurs, V. nd V n Weemen, B. (1977) En
zyme immuno ss y. Clin. Chim. Act 81, 140.

41
Assuring the Qu lity, S fety, nd Effic cy of DNA V ccines
J mes S. Ro
ertson nd Elwyn Griffiths 1. Introduction Scientists in c demi wh
ose rese rch is imed t the development of novel v ccine or ppro ch to v cci
n tion m y not lw ys
e fully w re of the regul tory process
y which c ndid
te v ccine
ecomes licensed product. This ch pter will provide n overview of
the regul tory process nd will discuss in more det il the qu lity nd pre-clin
ic l s fety issues of pl smid DNA v ccines intended for hum n use. It is useful
for rese rch scientists to
e w re of these processes s the development of n
ovel v ccine could
e pro
lem tic due to the st rting m teri l often
eing devel
oped in rese rch l
or tory under illdefined conditions. 2. Development of N
ovel V ccine The initi l nucleic cid v ccines produced for m rketing re likely
to
e pl smid DNAs derived from
cteri l cells. Future v ccines m y consist of
RNA inste d of DNA or m y
e nucleic cid molecules complexed with other entiti
es. In ny c se, the development of novel v ccine from l
or tory to licensed
product is likely to t ke consider
le num
er of ye rs nd s the process dv
nces, there will
e gre ter inter ction
etween the v ccine m nuf cturer nd t
he ppropri te regul tory gencies. M jor milestones in the development of nov
el v ccine include:
l
or tory demonstr tion of proof of concept design nd est
lishment of m nuf ct
ring demonstr tion of qu lity nd pre-clinic l s fety pprov l for nd conduct o
f clinic l tri ls pplic tion for nd tt ining product license
From: Methods in Molecul r Medicine, vol. 29, DNA V ccines: Methods nd Protocol
s Edited
y: D. B. Lowrie nd R. G. Wh len Hum n Press Inc., Totow , NJ
499
500
Ro
ertson nd Griffiths
3. The Regul tory Process Although the first offici l inter ction
etween v cc
ine m nuf cturer nd regul tory uthority is likely to
e when permission is s
ought to proceed to clinic l tri ls, it is import nt nd useful for industry nd
regul tors to work together closely in the development of ny novel v ccine. In
form l cont ct during ll st ges of development is to
e gre tly encour ged. In
order to proceed to clinic l tri ls, the following inform tion will
e required,

 Sufficient l
or tory-derived scientific d t th t indic tes the potenti l of th

e c ndid te v ccine. Inform tion on the qu lity of the c ndid te v ccine. Inform
tion on the pre-clinic l s fety of the c ndid te v ccine.
3.1. Qu lity
There re two m jor ppro ches to ssuring the qu lity of DNA v ccine:
1. The pplic tion of v riety of l
or tory tests on the fin l purified v ccin
e,
efore nd/or fter formul tion. 2. The pplic tion of in-process control.
In-process control is n ppro ch th t h s proven very useful in the qu lity con
trol of v ccines in gener l for m ny dec des. It involves documenting the l
or
tory development of the v ccine, ensuring the qu lity of the st rting m teri ls,
provision of full description of the m nuf cturing process nd the perform nc
e of ppropri te tests t v rious st ges of m nuf cture. Qu lity spects re dd
ressed in more det il in Su
he ding 4.
3.2. Pre-clinic l S fety
Concerns
out the s fety of DNA v ccines re gener lly hypothetic l in n ture
nd due to limited underst nding of the complex
iologic l systems involved. Th
ere re two princip l concerns: (i) th t pl smid molecule m y integr te into t
he host chromosome nd disrupt the control of cell division, nd (ii) th t n un
expected nd untow rd immunologic l re ction m y result from the use of DNA v
ccine. Pre-clinic l s fety concerns re de lt with in more det il l ter.
3.3. Clinic l Tri ls
Permission to proceed with clinic l tri l must
e sought from n ppropri te r
egul tory uthority. Within the Europe n Union, clinic l tri ls re regul ted
y
the licensing uthorities within individu l mem
er st tes; in the USA, n IND (
Investig tion l New Drug) is su
mitted to the U.S. Food nd Drug
Assuring Qu lity, S fety, nd Effic cy
501
Administr tion (FDA). After document tion of the qu lity nd pre-clinic l s fety
of new v ccine, clinic l tri ls proceed through three progressive ph ses. Typ
ic lly, in ph se 1 tri l, which involves sm ll num
er of volunteers, short-t
erm clinic l toler nce nd gross ssessment of immunogenicity of the v ccine
re ssessed. The ph se 2 tri l, involving l rger num
er of volunteers, is to i
nvestig te dos ge nd v ccin tion schedules. Ph se 2 tri ls will lso provide fu
rther inform tion on s fety nd immunogenicity nd will
e pivot l in determinin
g whether or not to proceed to l rge-sc le ph se 3 tri l in which the protecti
ve effic cy of the v ccine will
e ssessed with gre ter precision. A ph se 3 tr
i l will typic lly involve thous nds of v ccinees nd will lso provide further
d t on s fety. Clinic l tri ls of new v ccine gener lly t ke sever l ye rs to
complete.
3.4. Product License
Upon successful completion of clinic l tri ls, the dossier su
mitted to the regu
l tory uthorities for m rketing uthoris tion should provide evidence of the fo
llowing spects of the c ndid te v ccine:
Effic cy S fety Qu lity Consistency
The effic cy of the v ccine will
e ssessed from d t gener ted during the ph s
e 3 clinic l tri l. Evidence for s fety will ccrue from the clinic l tri ls, su
ch s the frequency nd n ture of ny dverse re ctions, nd from pre-clinic l s
fety (l
or tory) tests gener lly performed prior to initi tion of the clinic l
tri ls. Much of the inform tion on the qu lity of the v ccine will h ve
een v
il
le t the time of initi tion of the clinic l tri ls. However, during the cl
inic l tri l period, it is likely th t further development of the m nuf cturing

process will t ke pl ce nd this in turn will result in further refinements to t

he qu lity control of the v ccine. By the time of su
mission for m rketing utho
ris tion, the fin l m nuf cturing procedure t the proposed production sc le mus
t
e est
lished nd d t supporting the consistency of the process will
e n i
mport nt p rt of the su
mission. St
ility d t on the v ccine should lso now

e v il
le long with full toxicology d t . In ddition, it will
e necess ry f
or the m nuf cturing pl nt to
e inspected to ensure th t the v ccine is
eing p
rep red under good m nuf cturing pr ctice, which involves m nuf cturing under hi
ghly defined, c refully controlled nd reproduci
le conditions.
502
Ro
ertson nd Griffiths
3.5. Post-licensing In the c se of v ccines, fter product license h s
een o

t ined, the regul tory work continues with ph se 4, during which ny dverse re
ctions continue to
e reported, with the incre sing num
ers of recipients provid
ing more ccur te estim tion of the frequency nd n ture of dverse re ctions,
especi lly r re events. Thus, the full long-term effic cy nd s fety of v cci
ne will only
e properly est
lished fter m ny ye rs of use nd ssessment of i
ts perform nce. Further improvements or ch nges to the m nuf cturing procedure,
such s production t l rger sc le, or lter tions to the