R a diof r equ e nc y a bl at ion

of li v er t u mor s
An experimental and clinical study

Lars Frich
The Department of Surgery
and
The Interventional Centre
Rikshospitalet University Hospital

Thesis submitted for the degree of Dr. med.

October, 2006
Faculty of Medicine
University of Oslo

 Lars Frich, 2007

Series of dissertations submitted to the

Faculty of Medicine, University of Oslo
No. 476
ISBN 978-82-8072-693-3
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urgery is a conservative art. It takes to

novel methods reluctantly as an old dog
to new tricks. It was slow to adopt the
ligature; slow to adopt the principles of
antisepsis; slow to adopt the fastidious
technique and painstaking haemostasis
that have largely put a stop to operating by the clock.
It has been equally slow to adopt the principles of
electrosurgery which, from a technical standpoint,
are likely to be no less revolutionizing.
Harvey Cushing,

III

Ac k now l e d g e m e n ts

The present work was carried out at the Department of Surgery and the Interventional Centre,
Rikshospitalet University Hospital during the years 2001-2006. The work was financially
supported by the Norwegian Cancer Society, the University of Oslo, Rikshospitalet University
Hospital, Skaugens foundation (Bergliot og Sigurd Skaugens fond til bekjempelse av kreft) and
Henrik Homans foundation (Legatet til Henrik Homans minde).
I would like to express my gratitude to my primary supervisor Ivar Gladhaug, whose unique
combination of clinical proficiency, scientific judgment, openness to pursue new concepts and
gentle personality is highly appreciated. Co-supervisor Professor Atle Bjrnerud provided invaluable guidance in the exciting world of magnetic resonance imaging.
My sincere thanks to Professor Erik Fosse at the Interventional Centre who provided excellent
facilities for conducting experimental research in an inspiring multidisciplinary environment.
I am in debt to former head of the Department of Surgery Anstein Bergan, present head of the
Department of Surgery Karl-Erik Giercksky, and head of the Section for Gastrointestinal Surgery
ystein Mathisen for supporting the clinical study and for providing good working facilities.
As my predecessor research fellow, Tom Mala introduced me to the field of thermal ablation of
liver tumors. His research and experience provided me with a foundation for which I am most
grateful. I am also grateful to Bjrn Edwin whose early enthusiasm for thermal ablation and
incorporation of these techniques into clinical practice was an important inspiration for conducting experimental research. I would like to thank the staff at the Interventional Centre whose
professionalism and positive spirit have been a most valuable resource. Especially, I would like to
thank Sumit Roy and Per Kristian Hol for inspiring scientific discussions and Terje Tillung for assistance in magnetic resonance imaging. It has been a pleasure to cooperate with Knut Brabrand,
Gaute Hagen and Trond Mogens Aalkken at the Department of Radiology in the treatment and
follow-up of patients.

IV

I have much appreciated discussions of electrosurgical principles with Professor Sverre Grimnes at
the Department of Biomedical and Clinical Engineering. Sigrid Fossheim kindly provided liposomes. Laura Killingbergtr and the staff at Section for Gastrointestinal Surgery provided unique
care to our patients. Kristin Bjrnland and Solveig Pettersen provided expertise in zymography.
I am grateful to Professor Ole Petter F. Clausen and the staff at the Department and Institute
of Pathology for preparation and examination of histopathologic specimens. Dag Srensen and
the staff at the Center for Comparative Medicine provided excellent care of animals. My sincere
thanks also goes to the patients who participated in the clinical study. Hopefully, you have benefited from our common research effort.
Finally, I would like to thank my family and my family-in-law. The ones not here are deeply missed.
Special thanks to my children Ingeborg and Jens Andreas who have been a highly loved source of
joy. I am immensely grateful for the support my wife Ellen provided during these years. This thesis
would not have been possible without her encouragement, love and patience.

Con t e n ts

List of papers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII

Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
RadiofreQuency ablation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Aims of the study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Summary of papers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Appendix I - The bioheat eQUAtion . . . . . . . . . . . . . . . . . . . . . . . 45
Appendix II - A note on terminology . . . . . . . . . . . . . . . . . . . . . 47
Appendix III - A note on animal models . . . . . . . . . . . . . . . . . . . . 49
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Errata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

VI

L i s t of pa pe r s

Frich L, Mala T, Gladhaug IP.

Hepatic radiofrequency ablation using perfusion electrodes in a pig model:
Effect of the Pringle manoeuvre.
Eur J Surg Oncol. 2006;32(5):527-32.

II

Frich L, Bjrnerud A, Fossheim S, Tillung T, Gladhaug I.

Experimental application of thermosensitive paramagnetic liposomes for monitoring
magnetic resonance imaging guided thermal ablation.
Magn Reson Med. 2004;52(6):1302-9.

III

Frich L, Hol PK, Roy S, Mala T, Edwin B, Clausen OPF, Gladhaug IP.
Experimental hepatic radiofrequency ablation using wet electrodes:
electrode-to-vessel distance is a significant predictor for delayed portal vein
thrombosis.
Eur Radiol. 2006;16(9):1990-9.

IV

Frich L, Bjrnland K, Pettersen S, Clausen OPF, Gladhaug IP.

Increased activity of matrix metalloproteinase 2 and 9 after hepatic
radiofrequency ablation.
J Surg Res. 2006;135(2):297-304.

Frich L, Hagen G, Brabrand K, Edwin B, Mathisen , Aalkken TM, Gladhaug IP.

Radiofrequency ablation of colorectal liver metastases: evaluation of local tumor
recurrence by 3D volumetric analysis.
Submitted.

VII

A bbr e v i at ions

2D
3D
CEA
CRC
CT
ECM
HCC
MDCT
MMP
MR
MRI
PET
RF
ROI
RPV
SD
SI
T1
T2
VOI

2-dimensional
3-dimensional
carcinoembryonal antigen
colorectal cancer
computed tomography
extracellular matrix
hepatocellular carcinoma
multi-detector computed tomography
matrix metalloproteinase
magnetic resonance
magnetic resonance imaging
positron emission tomography
radiofrequency
region of interest
right portal vein
standard deviation
signal intensity
longitudinal relaxation
transversal relaxation
volume of interest

VIII

I n t roduc t ion

Colorectal cancer is the second most common malignancy in Norway, following cancer of the prostate in men and the breast in
women. Average age-adjusted incidence rates
in the period 2000-2004 for cancer of the
colon were 27.0 per 100 000 person-years in
men and 23.9 in women, with corresponding
rates for cancer of the rectum 15.9 in men
and 10.5 in women. Approximately 3 400 patients are diagnosed with colorectal cancer in
Norway per year, and more than 1 600 deaths
per year are attributed to this entity. The 5year survival for localized colorectal cancer is
80-90%. The corresponding 5-year survival in
patients with metastatic disease is below 10%
[1]. Synchronous metastases, i.e. those recognized at the time of diagnosis of the primary
tumor, are found in approximately 15-25% of
patients with colorectal cancer. Metachronous
metastases, those detected after diagnosis of
the primary tumor, are found in additional
25-50% [2-5].
Metastasis (from Greek methistanai, change
of place) is the result of a series of interrelated steps in which cancer cells leave the
original tumor and migrate to distant organs
via the blood or the lymphatic system [6, 7].

Different primary malignant tumors have a

propensity to spread to specific organs [7].
The patophysiology of organ specificity in
tumor metastasis can be considered in terms
of two mutually not exclusive hypotheses: the
seed-and-soil hypothesis and the mechanical hypothesis. The seed-and-soil hypothesis was proposed by Paget in 1889 and focuses
on the interaction between the cancer cells
and the specific organ microenvironment [8].
The mechanical hypothesis was proposed by
Ewing in 1928, and suggests that the greater
the number of tumor cells delivered to a specific site, the more likely it is that a metastasis
will develop at that site. The mechanical
hypothesis is therefore mainly concerned
with the anatomy of possible dissemination
routes from the organ containing the primary
malignancy [7, 9, 10]. Primary tumors in organs drained by the portal circulation have a
higher probability of metastasis in the liver
without metastasis elsewhere than primary
tumors in organs drained by the systemic
venous circulation [11, 12]. In line with the
mechanical hypothesis, this may be related
to the fact that the liver is the first visceral organ that malignant cells encounter following
release into the portal circulation [13]. These


data suggest that survival may be improved in

selected patients if metastatic disease in the
liver can be controlled.
The first documented liver resection was
performed on a trauma patient in 1716 [14].
Successful elective liver resection for a mass
lesion was performed in 1888 [15]. A total
of 59 cases of partial hepatectomy had been
described worldwide by 1899 [16]. Resection
for metastatic tumors was first described in
1940. Cancer metastastic to the liver was
for a long time considered incurable, and
systematic attempts to treat liver metastasis
by surgical resection were not made until the
1960s. In a retrospective study from 1963,
an average survival of 35 months was found
in 25 patients with liver metastasis from different primaries treated by surgical resection
[17]. A 1978 literature review of more than
400 patients treated with liver resection for
metastastic cancer from various primary tumors concluded that cure could be achieved,
especially in patients with primary colorectal
tumors [18]. Liver resection was associated
with a mortality of nearly 30% in early series
[14]. Several technical advances have gradually transformed liver resection into its present
state as a safe routine surgical procedure [19].
Due to improvements in surgical technique
and intra- and postoperative patient support,
the current operative mortality rates are less
than 5% with a 5-year survival of 30-50% [2022]. Recent studies have shown improved
survival compared with historical series,
with 5-year survival of 58% [23-25]. With
the reduced perioperative mortality, a more


aggressive approach has emerged towards resection of colorectal liver metastases. Repeat
liver resections in patients with isolated liver
recurrence after the first hepatectomy have
shown survival rates comparable to those
achieved by first hepatectomies [26-28].
Curative hepatectomy has been performed
in selected patients with initially unresectable
colorectal liver metastases after downsizing by
chemotherapy [29]. However, at present only
10-15% of patients with colorectal liver metastases are candidates for potential curative
hepatic resection [5, 29].
Evaluating the possible benefit of a treatment
requires knowledge of the natural course
of the disease. Patients with colorectal liver
metastases accepted for surgical treatment
represent a subset with limited disease and
favorable prognosis. Patient selection bias
may therefore present problems in interpreting the results of interventions in patients
with limited hepatic colorectal metastases.
Most studies of the natural history of malignant liver metastases are retrospective studies
conducted in the 1960s and 1970s, without
the advantage of modern imaging modalities.
Median survival for patients with untreated
liver metastases from colorectal cancer rarely
exceeds 9 months [30, 31]. In a retrospective
study of 252 patients with biopsy proven hepatic metastases that were not resected, 5-year
survival was less than 3% [32]. A prospective
study of 484 patients conducted between
1980-90 found a median 3-year survival of
0.9% [33]. Improved short-term survival, but
no 5-year survival was found in 62 patients

with potentially resectable metastases that

were not resected [34, 35]. Although the efficacy of liver resection versus no treatment has
not been assessed in prospective randomized
studies, it is generally accepted that resection
of colorectal liver metastases improves longterm survival [21, 36, 37].
Surgical treatment is by its nature directed
towards localized disease. Precise knowledge
of the extent and anatomical location of malignant disease constitute a fundamental prerequisite for rational surgical management.
Accurate preoperative staging is dependent
on the sensitivity of the imaging modality
used. Although the sensitivity of sonography,
computed tomography (CT) and magnetic
resonance imaging (MRI) is high for hepatic
metastases above 1-2 cm diameter, hepatic
metastases below 1 cm diameter can only occasionally be detected by these imaging modalities. Thus, colorectal cancer patients may
have more widespread disease than anticipated from preoperative staging [38]. Several
classification systems are currently used for
staging of colorectal cancer. Dukes classification for rectal cancer, proposed in 1932 and
modified in 1958 [39, 40], is widely used as a
valuable prognostic indicator in patients with
colorectal cancer. According to this classification, colorectal cancer is staged into A, B or C
based on the tumor involvement of the wall
of the colon and rectum. Dukes classification
has later been expanded to include stage D for
patients with distant metastasis. The TNM
classification of malignant tumors was introduced by the International Union Against

Cancer in 1958 [41]. The TNM system provide anatomic information on primary tumor,
lymph node involvement and the presence or
absence of distant metastasis. These staging
systems do not separate between patients
with resectable or non-resectable hepatic
metastases. It has therefore been argued that
new staging systems should be developed for
patients with advanced disease [42].
Treatment of cancer can be provided as palliative or curative therapy. Palliative therapy
is provided in the context that symptoms
are present that can possibly be ameliorated
by the treatment, but that eradication of the
cancer is not expected. Curative therapy implies that a chance, albeit sometimes small,
exists for complete eradication of the cancer.
It is generally accepted that surgical resection for malignant liver disease should only
be performed with a curative intent [31].
Criteria used for evaluating if a patient with
malignant disease confined to the liver is
eligible for hepatectomy can be categorized
as technical criteria, such as tumor proximity to major intrahepatic vessels precluding
safe resection, biological criteria assumed to
be associated with inferior prognosis such as
the number, size or distribution of tumors, or
general criteria related to the physical condition of the patient. Disease in patients selected to hepatectomy was previously limited to
one to three unilobar metastases, preferably
presenting metachroneously and resectable
with a margin of 1 cm [42]. The definition of
resectability has evolved, and the current definition of resectability with curative intent is


based on the ability of the surgeon to achieve

clearance of all measurable disease from the
liver, while leaving a healthy future remnant
liver of at least 20% of the total liver volume
[42, 43]. Extrahepatic malignant disease has
been considered an absolute contraindication
to hepatectomy for colorectal liver metastases
[21, 44], but this dogma has recently been
challenged [45, 46].

laser ablation [50] and radiofrequency ablation [51] are currently in clinical use for
treatment for hepatic tumors. High-intensity
focused ultrasound offers a non-invasive approach to tissue destruction, but has not yet
come into clinical use for treatment of malignant liver tumors [52]. Several recent reviews
present current methods for local ablation of
hepatic tumors [53-57].

Methods for local ablationi of malignant tumors were developed in order to achieve local
tumor control and possibly increased survival
in patients that could not be offered curative
hepatic resection. Ablation techniques can be
categorized into chemical or thermal methods according to the physical principle used
for induction of tissue damage. Early attempts
in using cold to treat liver tumors were crude
and involved direct application of liquid nitrogen to tumors or to metallic instruments in
contact with tumors [47]. Modern cryoablation devices use nitrogen or argon gas under
high pressure, which is led into a cryoprobe
whose tip is cooled down to subzero temperatures. However, the inability to visualize deep
liver tumors limited the use of local ablation
until intraoperative sonography became available in the late 1980s [48]. Several minimallyinvasive methods producing localized heat
such as microwave coagulation therapy [49],

The therapeutic approach to colorectal hepatic metastases has changed dramatically over
the last three decades, from an evaluation of
whether surgical resection was indicated or
not, to a complex multidisciplinary field involving surgeons, radiologists and oncologists
offering an increasing number of treatment
modalities [54, 58, 59]. Current treatment of
colorectal hepatic metastases includes strategies for achieving hepatic resection in previously non-resectable patients such as neoadjuvantii chemotherapy, preoperative portal vein
embolization to increase the future remnant
liver [60], 2-stage resection approaches [61]
and ex situ resection with liver autotransplantation [62]. Non-resection treatments
currently used are tumor ablation [63, 64],
locoregional or systemic adjuvant chemotherapy [65], local radiation by microspheres
[66], external beam radiotheraphy [67], immunotherapy [68] and gene therapy [69].

From Latin ablatio, removal or destruction. In the context of this thesis used to describe methods used for local
tissue destruction by heat, cold or chemical substances.

ii

From Latin adiuvans, to help. Adjuvant chemotherapy: chemotherapy given after tumor surgery to prevent cancer
recurrence. Neoadjuvant chemotherapy: chemotherapy given prior to surgical removal of a tumor to shrink the
tumor, with the intent of making the patient operable or the surgical procedure less extensive.

Radiofrequency ablationiii is an electrosurgical

method utilizing the flow of electrical current
through tissue to dissipate heat. An electrode
is placed inside the tumor, and tissue in the
vicinity of the electrode is heated, producing
localized tissue destruction [70]. This modality has recently been introduced in the treatment of patients with non-resectable hepatic
metastases [51, 71].
This study was carried out to investigate various experimental aspects of hepatic radiofrequency ablation, as well as the clinical application of this technique in a well-defined patient
population with colorectal liver metastases.

iii The term radiofrequency refers to the frequency of the alternating current used in these systems (300 - 500 kHz),
which is within the radiofrequency spectrum.

Ra diof r equ e nc y a bl at ion

Electrosurgical principles
Electrosurgery can be defined as the therapeutical use of heat dissipated by high-frequency
electrical current passing through biological
tissue. Electrosurgery can be performed with
either monopolar or bipolar techniques;
monopolar techniques are more commonly
used for tumor ablation. For monopolar electrosurgery an asymmetrical electrode system
is utilized where a small active electrode is
placed in the tissue to be treated and a large
dispersive electrode is placed on the skin of
the patient. With the bipolar technique, two
identical electrodes are placed in the tissue
close to each other, and the current passes
through tissue locally between the electrodes.
Fundamental quantities when describing an
electrical circuit is the electrical potential
(volt, V), the flow of electric charge per time
unit (ampere, A), and the resistance through
which the current passes (ohm, ). Electrical
current may be unidirectional (direct current,
DC) or may change direction (alternating current, AC). Alternating current can be characterized by the frequency of cycles per second
(hertz, Hz). The opposition to alternating

current is denominated impedance, and is

determined not only by the resistance, but
also by the reactance, which is the capacitive
and inductive components of the electrical
circuit. The reactance and hence the impedance is dependent on the current frequency.
The impedance is represented by the symbol
Z, and is measured in ohm ().
The relationship between the electrical potential, current and resistance in a resistive
direct current circuit is expressed by Ohms
law, where V is the potential difference, I is
the current, and R is the resistance:
(1)

V = R I

Power in a purely resistive circuit is represented by the symbol P (watt, W), and is determined by the potential and current:
(2)

P = IV

By combining Ohms law (1) with equation

(2), the power can also be expressed as:
(3)

P = I2 R

The relationship between energy, current, resistance and time can be determined by Joules
law where Q is energy (joule, J), I is current
(A), R is electrical resistance () and t is the
length of time the current is permitted to flow
(sec):
(4)

Q = I2 R t

The central concept in electrosurgery is that

current passing through tissue will dissipate
electrical energy as heat. Equation (4) shows
that energy generated in an electrical circuit is
a function of the current to the second power.
The current passing through the active electrode, the tissue and the dispersive electrode is
the same per time unit. Because the resistance
of the metal electrodes is negligible, virtually
no heat will be dissipated in the electrodes.
Both the active and the dispersive electrode
are therefore cold during electrosurgery.
Biological tissue consists of cells containing
ionic electrolytes, surrounded by cell membranes with capacitive properties. The cells are
surrounded by extracellular electrolytes and
organized into organs enclosed by membranes
with different electrical properties than the intracellular and extracellular electrolytes. The
impedance of tissues is much higher than the
impedance of electrodes [72]. Hence, current
passing through tissue will dissipate heat.

tion of the cable is constant and the material

of the cable is homogeneous. When currentcarrying electrodes are brought into contact
with tissue in a closed circuit, current spreads
out in the tissue volume. An idealized model
of a spherical electrode positioned in a homogeneous medium illustrate that the current
spread out from the electrode in the shape of
a sphere:

(5)
J=
r a
2
4

r

where J is the current density, I is the current
passing through the electrode, r is the distance from the electrode, a is the radius of the
electrode.
In a monopolar system, the active electrode
is small, leading to a high current density. In
contrast, the dispersive electrode is large in
order to permit the limited heat produced
by the low current density to be dissipated by
conduction [73]. Assuming adiabatic conditions, i.e. that no heat is transferred to or from
the tissue (for instance by blood flow), and no
phase difference between the current density
and the electric field, the temperature rise in a
tissue volume is a function of the current density to the second power:
(6)

T =

J2 t
c

where T is the temperature rise, J is the curThe current density ( J), defined as the electric
current per cross-sectional area (A/m2), is
constant throughout a cable if the cross sec-

rent density, t is the time, is the tissue conductivity, c is specific heat capacity and is
the density.

The power density Wv (watt/cubic metres)

is an expression of the electrical energy per
time unit that tissue is exposed to under ideal
conditions [74]. The power density falls off
extremely rapidly with distance from the electrode, as it is inversely proportional to the distance from the electrode to the fourth power:
(7)

a2
W v = V 4
r
2

where is the tissue electrical conductivity, V

the voltage, a the radius of the electrode, and r
the distance from the electrode.

to a high power density which may cause a

rapid temperature rise. Consequently, tissue
in proximity to the active electrode is susceptible to carbonization. Tissue carbonization
leads to increased tissue impedance and an
abrupt fall in the flow of electrical current,
which limits tissue heating and thereby the
extent of the coagulation [75]. The balance
between obtaining a sufficient power density
at a distance from the electrode while avoiding tissue carbonization at the electrode-totissue interface is a fundamental biophysical
limitation of electrosurgery which was noted
as early as 1928 [73].

Although the relationship between the temperature rise in a tissue volume and the current
density described by equation (6) is valid for
an adiabatic model system, additional factors
such as heat loss and metabolic tissue heating
influence tissue temperature in vivo. The bioheat equation (Appendix I) is a mathematical
model that incorporates these factors [76].
The general concept of the bioheat equation
can be simplified to [55]:

extent of coagulation = energy deposited

tissue interactions heat loss

This model can be used to predict the extent

of tissue coagulation produced by thermal
energy under a variety of circumstances [77].
During thermal ablation in a given tissue volume in vivo, increase of coagulation volume
can be achieved by increasing energy deposition or by reducing heat loss. During monopolar electrosurgery, the electrode-to-tissue
interface at the active electrode is exposed


Tissue response to heat

The tissue response to heat is a function of
temperature and exposure time. Tumors are
believed to be more thermosensitive than
normal tissue, possibly due to inability to
dissipate heat effectively [78-80]. Cellular
homeostasis is maintained with a mild elevation of temperature to approximately 40C.
When temperatures are increased to 42-45C
cells become more susceptible to damage by
other agents such as chemotherapy and radiationiv [81]. Below a tissue temperature of
45C thermal changes are largely reversible
[82]. With a temperature of 50-52C, cellular death occurs within 4-6 minutes [70, 80,
83]. At temperatures above 60C near instantaneous protein coagulation and irreversible
cellular damage occurs [55]. The liquid in the
tissue evaporates at temperatures above 90C,
resulting in desiccation if the tissue is heated
slowly, or vaporization if the heat is delivered
rapidly [84, 85]. Carbonization of the tissue
occurs at temperatures above 105C.
The aim of radiofrequency ablation is to
achieve and maintain a temperature of 50
100C throughout the entire target volume.
Thermally treated tissue is associated with
different findings on postprocedural imaging
(Appendix II). The term coagulation is used
in this thesis to describe the localized tissue
alterations caused by thermal ablation.

Hepatic radiofreQuency
ablation: a conceptual
history
Development of electrosurgery
The term electricity was introduced in 1600
by the natural philosopher and physician
William Gilbert to describe the phenomenon
that rubbing furs against amber (Greek elektron) would cause an attraction between the
two materials. Gilbert found that a number
of materials had similar abilities, but erroneously believed that the attraction was caused
by a subtle effluvium that was released after
rubbing had pushed away the air between the
two materials [86]. The first device generating static electricity was invented in 1663 by
Otto von Guericke by using a rotating sphere
of sulphur on a shaft. By 1740 electrostatic
machines were in widespread use in Europe.
In 1745 the Leyden jar was invented, which
made it possible to store and discharge static
electricity [87].
In the early eighteenth century, dissected
frogs were regularly used to explore the effects of static electricity. While investigating these effects the Italian physicist Luigi
Galvani accidentally discovered in 1780 that
muscle spasms were induced in frog legs after
the sciatic nerve was brought into contact
with two dissimilar metals. Galvani believed
that the muscle contractions were caused by

iv The use of loco-regional or whole body hyperthermia with a temperature of 42-44C to sensitize tumor cells to
the cytotoxic effects of ionizing radiation and chemotherapy should not be confused with thermal ablation, which
require temperatures > 50C.

10

a disturbance of the equilibrium of electrical

fluid carried to the muscles by the nerves. He
coined the term animal electricity to describe
what was believed to be a new form of electricity [85, 86]. This discovery and subsequent
experiments led to the concept of electrical
current and the birth of bioelectricity. In 1799
Allesandro Volta constructed the first device
that produced continuous electrical current
by a chemical reaction, thereby showing that
this assumed new form of electricity could be
generated independently of animals. The device produced by Volta, known as the voltaic
pile, is the progenitor of modern batteries. The
current produced by this device was known
as Galvanic current, corresponding to the
modern term direct current. Michael Faraday
discovered electromagnetic induction in
1831 and thereby the principles necessary for
construction of the dynamo, which was able
to deliver alternating current.

Nagelschmidt developed the theory that

creation of heat observed during treatment
was caused by molecular agitation induced by
high frequency electrical currents, and introduced the concept diathermy in 1897 (from
Greek dia, through; therma, heat) [86, 90].
The first surgical use of electrical current is
believed to be the treatment of a carcinomatous ulcer of the hand by the French physician
Joseph Rivre in 1900 [91]. During the next
decade, the use of electricity in treating lesions
of the skin, oral cavity as well as coagulation
of vascular tumors and hemorrhoids became
widespread [85]. Pozzi introduced the term
fulguration (from Latin fulgur, lightning) referring to the superficial carbonization resulting when an electrical spark was used to treat
skin [92].

Exposing living organisms to direct current

or low-frequency alternating current is associated with neuromuscular stimulation
and an involuntary tetanic response. In 1891
Jacques-Arsne dArsonval discovered that by
using frequencies above 10 kHzv, electrical
current could be passed through tissue without producing other physiological effect than
that of heat. Based on these findings, an apparatus was built that produced a spark across
a gap, which could be used to generate super-

In 1909 Eugne-Louis Doyen introduced the

term electrocoagulation (from Latin coagulare,
to curdle) to describe a method where the tissue was touched directly with the treatment
electrode and a neutral electrode was attached
to the patient. This arrangement produced
deep tissue coagulation rather than carbonization on the tissue surface [93, 94]. In 1911
William Lawrence Clark used a high voltage
spark gap oscillator to remove skin tumors. By
microscopy of the treated tissue he observed
that it shrunk from dehydration, and used
the term desiccation (from Latin desiccare, to
dry out) to describe this process [95]. In 1923

ficial tissue destruction [88-90]. Carl Franz

George A. Wyeth used electrosurgery for cut-

It has later been shown that nerve and muscle exitation can be elicited at frequencies well above 10 kHz, but that
increasingly higher current density is needed to elicit such a response with increasing frequency (LaCourse, J.R., et
al., Effect of high-frequency current on nerve and muscle tissue. IEEE Trans Biomed Eng, 1985. 32(1): p. 82-6.)

11

ting tissues, utilizing an apparatus he termed

an endotherm knife (from Greek endo, within;
therm, heat) [96]. In 1925 Ward discovered
that a continuous sine wave from a vacuum
tube oscillator was effective for cutting tissue, whereas coagulation was achieved by a
damped sinusoid from a spark gap oscillator
[97].
These discoveries inspired the physicist
William T. Bovie to develop a device that
could be used both for cutting and for obtaining hemostasis by tissue coagulation (Fig
1). In 1926 the device, known as the Bovie
unit, was used by the neurosurgeon Harvey
Cushing for surgical treatment of patients
with intracranial tumors that had previously
been considered inoperable [73, 94]. The
Bovie unit consisted of an alternating current

Figure 1. The Bovie electrosurgical unit, manufactured

from 1926 by the Liebel-Flarsheim Co, Cincinnati,
OH.

12

generator, interchangeable small active electrodes mounted on an insulated pistol-grip

of bakelite, and a large dispersive electrode
placed on the skin of the patient. By varying
the modulation of the current used, the unit
was capable of superficial dehydration, cutting or coagulation of a limited tissue volume
[73]. Modern electrosurgical units are based
on the electrophysiological principles of the
Bovie unit.
Development of hepatic radiofrequency
ablation
Radiofrequency ablation was suggested for
treatment of hepatic malignancies in 1990
[98, 99]. The same year an experimental study
using a needle electrode with 10 mm noninsulated tip showed that it was possible to
produce an ellipsoid coagulation in porcine
liver with maximal diameter perpendicular
to the electrode axis of 1.4 cm [99]. In 1992
percutanous ultrasound guided radiofrequency ablation was performed in 10 pigs,
with typical coagulation diameters of 1-2 cm
[100]. The first publication presenting hepatic malignancies treated with radiofrequency
ablation appeared in 1993, and presented 13
patients with hepatocellular carcinoma. The
unit which was used achieved a coagulation
volume of 1.8 mL [101], corresponding to
a coagulation diameter of 1.5 cm. In a 1995
study using needle electrodes connected to
a generator commonly used for cardiac ablations, various combinations of electrode tip
exposure, gauge, duration of treatment and
temperature were examined to assess the op-

timal settings for generating the largest possible tissue coagulation volume. The authors
concluded that coagulations larger than 1.6
cm in diameter could not be produced by any
combination of factors with a needle electrode [102]. Numerous patient series were
published in the second half of the 1990s
[103-110]. The populations treated were often heterogeneous with both primary hepatic
tumors and metastases from various primary
tumors. The majority of early reports are observational studies with short follow-up and
missing data on long-term patient survival.
Established oncological criteria suggest that
hepatic malignancies should be eradicated
radically including a 1-cm margin of apparently healthy tissue to eliminate microscopic

Figure 2. The figure shows an electrode placed in a

tumor. In order to obtain a 1-cm tumor-free margin,
the tumor with a diameter of 2 cm requires a tissue
coagulation with a diameter of at least 4 cm.
Figure modified after Rhim, H., et al., Essential
techniques for successful radio-frequency thermal ablation
of malignant hepatic tumors. Radiographics, 2001. 21
Spec No: p. S17-35.

foci of malignant cells, and to compensate

for the uncertainty in determining the exact
tumor margin [111, 112]. In order to adhere
to the principle of a 1-cm tumor-free margin,
a tumor must be completely enveloped by a
continuous coagulation that encompasses
the tumor as well as a 1-cm margin on all
sides. The diameter of a spherical coagulation would therefore need to be at least 2 cm
larger than the largest diameter of the tumor
(Fig 2) [113]. This implies that a coagulation
volume several times the tumor volume must
be created (Fig 3). The needle electrode was
not capable of generating adequate coagulation volumes with one electrode insertion
[55, 102]. Furthermore, the use of overlapping coagulations is not an effective method
to increase coagulation volume (Fig 4) [113].

Figure 3. The figure shows the tumor volume (filled)

and the volume of the corresponding 1-cm margin. The
volume of a 2-cm spherical tumor is 4.2 mL, the volume
of the surrounding 1-cm tumor free margin (excluding
the tumor) is 29.3 mL. The required coagulation
volume to treat a 2-cm spherical tumor is therefore 33.5
mL, approximately 8-fold the tumor volume. Present
radiofrequency ablation systems produce coagulation
volumes of approximately 40 mL.

13

Devices producing larger coagulations with

one electrode insertion would therefore have
to be developed to make radiofrequency ablation useful for treatment of most clinically
relevant hepatic tumors (i.e. those measuring
> 1-2 cm in diameter). Several strategies were
proposed in the last half of the 1990s in order to increase the coagulation volume [114].
Although generators capable of adjusting
power output according to tissue impedance
and temperature were introduced, it was principally the rapid development in electrode
design that enabled substantially larger tissue
coagulations to be obtained.

2.0 cm

2.8 cm

4 cm
Figure 4. The figure illustrates the result of overlapping
coagulations for a device generating a spherical tissue
coagulation of 2 cm (thick line). The coagulation
diameter can be increased by positioning four
coagulations in the x-y plane (thin lines) and two
additional coagulations along the z-axis (not shown).
The positioning of six coagulations will produce a
spherical coagulation with a diameter of 2.8 cm (dotted
line). A coagulation with a diameter of 2 cm correspond
to a volume of 4.2 mL, whereas a coagulation with
a diameter of 2.8 cm correspond to a volume of 11.5
mL. Hence, increasing the number of coagulations by
a factor of 6 only increase the spherical coagulation
volume by a factor of 2.7.
Figure modified after Rhim, H. and G.D. Dodd, 3rd,
Radiofrequency thermal ablation of liver tumors. J Clin
Ultrasound, 1999. 27(5): p. 221-9.

14

Loop electrode. The loop electrode

consisted of a super-elastic metal with a distal
loop with a radius of 1 cm, which was introduced into the tissue by a cannula. After the
electrode had regained its loop shape, the
electrode was rotated simultaneously with
activation of the electrosurgical unit in cut
mode. This produced a spherical coagulated
rim of 1 mm which physically separated a
non-coagulated spherical tissue volume with
a diameter of 2 cm from the surrounding vital
parenchyma [115, 116].
Bipolar electrode.The use of bipolar electrodes, i.e. insertion of two identical
electrodes in the target tissue, generates high
current density at both electrodes and in tissue between the electrodes [117]. A bipolar
system was found to produce larger coagulations than the needle electrode, but also more
irregular coagulation geometry [118].
Cluster electrode.The use of cluster
electrodes, i.e. insertion of three or more electrodes that are activated simultaneously, was
examined in 1995. Simultaneous application
of energy to three electrodes placed 1.5 cm
apart would lead to a coagulation diameter of
3 cm [119].
Internally cooled electrode.The internally cooled electrode was described in
1996 [120, 121]. This design consisted of a
hollow electrode with two internal lumens
extending to the electrode tip. One lumen
delivered cooled saline to the electrode tip,
and the other lumen returned the saline to a

collection unit outside the body. The perfusate did not come into contact with patient
tissues. A thermocouple was embedded at
the tip of the electrode to allow for continuous temperature measurement. By cooling the
electrode tip, the risk of excessive heating and
carbonization was reduced, enabling a higher
current density and higher deposition of electrical energy in the tissue than was tolerated
with conventional electrodes. Using this principle coagulation with a diameter of 2.5 cm
could be made in porcine liver in vivo [120].
Perfusion electrode. It was noted in
1990 that adding saline to the electrode-totissue interface would increase coagulation
volume [98]. The explanation proposed for
this finding was that installation of saline
produced a more electrically uniform tissue, thereby limiting tissue carbonization. It
was further hypothesized that a high local
ion concentration at the electrode-to-tissue
interface would increase the effective surface
of the electrode and thereby the coagulation

volume. In 1997 the effect of continuous saline infusion during ablation was evaluated
[105]. This study showed that radiofrequency
ablation with a needle electrode produced coagulations with a diameter of less than 1 cm in
vivo, whereas continuous intraparenchymal
infusion of 0.9% saline at 1 mL/min during
ablation increased maximum coagulation diameter to 4 cm.
Multitined expandable electrode.
A device utilizing four expandable electrodes
was presented in 1998 [122, 123]. The device
consisted of an insulated shaft containing
four retractable electrodes which could be deployed to a maximum diameter of 3 cm. Each
electrode tip contained a thermocouple for
temperature monitoring. Coagulations with
diameters of 3.1 cm were created in an experimental study [122].
Commercially available hepatic radiofrequency ablation systems have been developed
based on the principle of the cluster electrode,

Table 1. The four most commonly used systems for hepatic radiofrequency ablation.

Generator name

3

4

1
2

Frequency (kHz)

Power output (W)

Electrode type

HiTT 1061

375

60

Cool-tip RF2

480

200

Internally cooled

RF 30003

480

200

Multitined expandable

1500X4

460

250

Multitined expandable

Perfusion

Integra LifeSciences, Tuttlingen, Germany, formerly Berchtold Tuttlingen, Germany

Valleylab, Boulder, CO, formerly Radionics, Burlington, MA
Boston Scientific Corp., Boston, MA, formerly RadioTherapeutics Corp., Sunnyvale, CA
RITA Medical Systems, Mountain View, CA

15

the internally cooled electrode, the perfusion

electrode, the multitined expandable electrode
as well as combinations of these principles.
Technical details of the four most commonly
used systems for hepatic radiofrequency ablation are summarized in Table 1.

An impedance-controlled radiofrequency
ablation system with perfusion electrodes
(Elektrotom HiTT 106, Berchtold GmbH &
Co, Germany) based on the principle of continuous intraparenchymal infusion of saline
during ablation was used in this study (Fig 5).

Figure 5. The figure shows the perfusion electrodes used in this study.
a. The diameter of the electrodes are 1.7 mm with a insulated shaft with a length of 15 or 20 cm incorporating a lumen
for saline perfusion.
b. The terminal non-insulated 1.5 cm segment of the electrode has three pairs of two side holes, placed at an angle of
120 from each other.
c. Saline is distributed to the tissue around the electrode through the side holes.

16

Hepatic radiofrequency
ablation: clinical
challenges
Patient survival
Following the first report in 1996 of 6 patients with colorectal liver metastases treated
with radiofrequency ablation [104], larger
series were published in 1999 and 2000 [110,
124, 125]. Follow-up was short and survival
at standardized time intervals (i.e. 1, 3 and 5
years) was not presented. More recent patient
series indicate that radiofrequency ablation of
non-resectable colorectal liver metastases is
associated with a 5-year survival of approximately 30% [126-129].
Facilitated by downsizing as a result of neoadjuvant chemotherapy, and the ability of local
ablation techniques to treat non-resectable
tumors, patients with previously non-resectable tumors are offered therapy with curative
intent. The natural course of the disease in
this novel and highly selected patient category is not known. Treatment is offered within
a complex multimodal framework consisting
of repeat surgical interventions, local ablation procedures and different adjuvant and
neoadjuvant chemotherapy regimens. The
interventions may be implemented in varying
order and at non-standardized time points.
No randomized studies comparing radiofrequency ablation with hepatic resection or
with chemotherapy in patients with colorec-

tal hepatic metastases have been performed to

date. Although preliminary data indicate that
improved survival can be achieved in selected
patients with non-resectable colorectal hepatic metastases treated with radiofrequency
ablation, formal scientific proof is lacking.
Local tumor control
Complete eradication of malignant growth
is considered mandatory to achieve increased
survival in patients with colorectal liver metastases [35]. This assumption constitutes
the oncological rationale for local ablation of
non-resectable hepatic metastases. However,
local tumor control, i.e. lack of tumor recurrence at the site of treated tumors, is not a
sufficient condition for increased patient survival, as intrahepatic recurrence not related to
the treatment site and extrahepatic recurrence
is common in patients treated with R0-resectionvi [130] and radiofrequency ablation
[24, 131] of colorectal liver metastases. Local
tumor control should therefore be considered
a surrogate endpoint, but is nevertheless a
valuable indicator of the technical ability to
eradicate a tumor.
Local tumor recurrence rates after radiofrequency ablation are higher than after hepatic
resection, with reported rates varying from
less than 5% to above 50% [110, 125, 132134]. A recent meta-analysis of 5227 liver
tumors treated with radiofrequency ablation
found that tumor diameter > 3 cm, perivas-

vi R0-resection implies that no microscopic residual tumor is present in the resections margin(s), and no residual
macroscopic malignant disease is detected.

17

cular tumor localization and multiple tumors

were independent predictors for local tumor
recurrence. Thus, the ability of radiofrequency ablation to eradicate liver tumors is
limited by a number of tumor-related factors.
Furthermore, higher local recurrence rates
were found for procedures using a percutaneous approach [135].
In order to attain reliable tumor destruction
by radiofrequency ablation, knowledge of the
expected coagulation geometry and volume
as well as the relationship of the coagulated
volume to the position of the electrode is
crucial. A systematic review of the literature
from 1990 to 2003 found that adequate experimental data were missing for most of
the commercially available electrodes [136].
Reproducibility of coagulation geometry and
volume is low under standardized treatment
protocols in experimental studies [137, 138]
and in clinical series [139]. Reliable intraprocedural monitoring of the extent of tissue
coagulation is therefore essential to ensure adequate treatment of a tumor and to avoid damage to abutting structures. Transabdominal
sonography provide accurate guiding of the
ablation electrode, but is unreliable in assessment of the extent of tissue coagulation [100,
140]. The use of CT [141], MR [142, 143]
and contrast enhanced sonography [144]
have been evaluated for intraprocedural monitoring of radiofrequency ablation. Real-time
monitoring of the temperature distribution
during the procedure would allow the operator to optimize the treatment and possibly
avoid damaging normal structures adjacent to
18

the treatment site. Temperature sensors are

attached to or embedded in the ablation electrodes of most commercial radiofrequency
ablation electrodes. However, thermal information is restricted to a predefined number
of measuring points, and provide no information on tissue temperatures at a distance away
from the electrode. Non-invasive temperature
measurement is an attractive concept with the
potential to visualize the three-dimensional
temperature distribution during treatment
[145].
Cooling caused by blood flow in large vessels
passing through or near the treatment site is
an important limitation of the extent of the
coagulation in vivo [122, 146]. Several studies
have examined the effect of temporary reduction of intrahepatic perfusion by pharmacological modulation [147, 148] or temporary
mechanical occlusion of hepatic vessels [146,
149-152]. Interruption of hepatic inflow during ablation increases efficiency [153], and
enables coagulations that are larger, with a
more spherical geometry [149, 151], which
would be beneficial in order to achieve complete treatment.
Assessment of local tumor recurrence
Malignant tissue treated by radiofrequency
ablation is left in situ, necessitating repeated
imaging follow-up to determine if the tumor
has been eradicated. Early detection of local
tumor recurrence may facilitate early reintervention with potential benefits for patient
survival. The use of fine needle aspiration cy-

tology is controversial due to the risk of needle

tract seeding [154, 155]. Consequently, noninvasive methods should preferably be used
for detection of persistent or residual malignant tissue at the coagulated site. However,
it can be difficult to distinguish benign tissue
alterations caused by radiofrequency ablation
from residual tumor on post-ablation followup imaging [156-158]. Non-invasive methods for assessment of local tumor recurrence
should therefore be refined.
Complications
Two large retrospective studies have assessed
complications associated with hepatic radiofrequency ablation [159, 160]. The reported
mortality in both studies was below 1%, with
complications occurring in less than 10% of
patients. Complications directly related to
the application of thermal energy such as
damage to bile ducts or intrahepatic vessels
occurred in less than 1% of patients. In one of
these studies of 3670 patients, 3 of 20 deaths
was attributed to portal vein thrombosis and
1 death to bile duct stricture [159]. The risk
of damage to intrahepatic vessels and bile
ducts [150, 161-163], as well as the feasibility of protecting bile ducts by intraductal
cooling have been examined in recent studies
[164-166].

induced in the treated tumor and surrounding hepatic parenchyma have not been well
characterized due to the fact that the treated
tumors and surrounding parenchyma are left
in situ. It has been hypothesized that radiofrequency ablation generates an antigen source
that may induce antitumor immunity [167],
which may be a beneficial effect. However, of
special interest are recent experimental findings that radiofrequency ablation strongly
promotes proliferation of residual neoplastic
cells [168]. Examination of biological effects
induced by radiofrequency ablation may be
of importance for understanding mechanisms
for post-ablation tumor recurrence, and
should be further characterized.

Biological alterations at the coagulated site

Although the gross and microscopic findings after hepatic radiofrequency ablation are
well characterized [140], biological effects
19

20

A i m s of t h e s t u dy

This thesis examines the use of hepatic radiofrequency ablation in four experimental studies in
non-tumor animal models and in one clinical study in patients with colorectal liver metastases.
An impedance-controlled perfusion electrode radiofrequency ablation system was used in all
studies. Specifically, the aims were:
1.

Characterize coagulation geometry and coagulation volume generated in porcine liver

during maintained and interrupted hepatic inflow.

2.

Investigate the feasibility of a thermosensitive liposomal agent for thermal monitoring of

MRI-guided hepatic radiofrequency ablation in rabbit liver.

3.

Determine if electrode-to-vessel distance or the use of hepatic inflow occlusion are associated with acute or delayed portal vein thrombosis in porcine liver.

4.

Examine if hepatic radiofrequency ablation increases the expression of matrix metalloproteinase-2 and matrix metalloproteinase-9 in the transition zone separating coagulated
tissue from normal porcine hepatic tissue.

5.

Determine if alterations in coagulation volume on post-ablation CT images acquired

every third month facillitate early detection of local tumor recurrence in patients with
colorectal hepatic metastases treated with radiofrequency ablation.

21

22

Su m m a ry of pa pe r s

Frich L, Mala T, Gladhaug IP.

Hepatic radiofrequency ablation using perfusion electrodes in a pig model:
Effect of the Pringle manoeuvre.
Eur J Surg Oncol. 2006;32:527-32.

Coagulation geometry, coagulation volume and delivered energy per coagulated tissue
volume was assessed in a non-tumor porcine model. Mechanical occlusion of the portal
vein and the hepatic artery (Pringle maneuver) during ablation was performed in 6 of 12
animals. One coagulation was made in each animal close to the portal vein. All animals
were sacrificed 4 days after ablation. Use of hepatic inflow occlusion increased the effective coagulation diameter by 1 cm, but was associated with elongated coagulations
extending outside the expected treatment site. Displacement of the geometrical centre of
the coagulation relative to the position of the electrode was observed. The energy delivered during ablation could not be used to predict coagulation volume.

23

II.

Frich L, Bjrnerud A, Fossheim S, Tillung T, Gladhaug I.

Experimental application of thermosensitive paramagnetic liposomes for monitoring
magnetic resonance imaging guided thermal ablation.
Magn Reson Med. 2004;52:1302-9.

The utility of a thermosensitive liposomal agent to assess the extent of tissue coagulation
during MRI-guided laser ablation and radiofrequency ablation in non-tumor rabbit liver
was examined. One coagulation was made in each animal prior to administration of the
agent, and two additional coagulations were made after administration of the agent.
Tissue temperature during treatment was measured by a MRI-compatible temperature
fiber. T1-weighted MR images were acquired prior to heating, during heating and after
heating for each coagulation. Signal intensity (SI) of the coagulations generated prior
to injection of the agent decreased 7% below baseline during heating, but increased 5%
above baseline at normalization of tissue temperature. Both coagulations generated after
administration of the agent showed increased SI during heating (5% vs. 19%), followed
by a further increase after tissue temperature had normalized (14% vs. 26%). Increased
SI was found for coagulations made 50-55 min after injection of the agent compared to
those made 12-14 min after injection of the agent. Persistent signal enhancement on T1weighted MR images was found in areas assumed to be exposed to a temperature above
the phase transition temperature of 57C, indicating that thermosensitive liposomes
may be used for monitoring of radiofrequency ablation.

24

III

Frich L, Hol PK, Roy S, Mala T, Edwin B, Clausen OPF, Gladhaug IP.
Experimental hepatic radiofrequency ablation using wet electrodes: electrode-to-vessel
distance is a significant predictor for delayed portal vein thrombosis.
Eur Radiol. 2006;16:1990-9.

Possible explanatory variables associated with acute and delayed portal vein thrombosis
after hepatic radiofrequency ablation close to portal vessels were assessed in an experimental study. Coagulations were created within 1.5 cm of the right portal vein branch
in 12 pigs with (n=6) or without (n=6) hepatic vascular occlusion (Pringle maneuver).
Sham operations with Pringle maneuver were performed in four animals. Vessel patency
was assessed by rotational portal venography prior to ablation, 10 min after ablation and
4 days after ablation. Distance between the ablation electrode and the right portal vein
branch was measured from 3-dimensional reconstructions of the portal venograms. No
animals developed acute portal vein thrombosis. Delayed portal vein thrombosis occurred in 2 of 6 animals in the Pringle group, and in 3 of 6 animals in the group where
coagulations were made during physiological hepatic circulation. All five occurrences of
delayed portal vein thrombosis occurred in animals with a distance between the ablation
electrode and the right portal vein branch of 5 mm or less. Delayed portal vein thrombosis may occur after hepatic radiofrequency ablation during physiologic hepatic circulation. An electrode-to-vessel distance of < 5 mm was a significant predictor for delayed
portal vein thrombosis.

25

IV

Frich L, Bjrnland K, Pettersen S, Clausen OPF, Gladhaug IP.

Increased activity of matrix metalloproteinase 2 and 9 after hepatic radiofrequency
ablation.
J Surg Res. 2006;135(2):297-304.

Activity of MMP-2 and MMP-9 in plasma and in tissue lysates from the transition zone
surrounding coagulated hepatic tissue was examined in a non-tumor porcine model.
Hepatic vascular occlusion was performed in 6 of 12 animals. MMP activity was quantified by gelatin zymography. Cellular localization of MMPs was determined by immunohistochemistry. MMP-2 and MMP-9 activity in tissue lysates from the transition zone
was significantly increased compared to normal hepatic parenchyma with ratios of 3.0
and 2.6, respectively. MMP-2 and MMP-9 activity in plasma 1 h after radiofrequency
ablation was significantly increased compared to baseline levels, with ratios of 1.2 and
1.5, respectively, but had normalized 4 days after radiofrequency ablation. Hepatic
inflow occlusion during ablation did not inflence MMP activity. Expression of MMP-2
and MMP-9 was localized to macrophages in the transition zone.

26

Frich L, Hagen G, Brabrand K, Edwin B, Mathisen , Aalkken TM, Gladhaug IP.

Radiofrequency ablation of colorectal liver metastases: evaluation of local tumor recurrence by 3D volumetric analysis.
Submitted.

In this clinical study, 11 patients with colorectal liver metastases were treated with radiofrequency ablation. Multi-detector computed tomography (MDCT) was performed at
1 and 3 months after radiofrequency ablation, and then at intervals of 3 months until 24
months, and each sixth month thereafter. A total of 81 post-ablation scans were analyzed,
with a median follow-up of 27 months (17-43 months). The theoretical treatment margin
1 month after radiofrequency ablation was assessed retrospectively. Post-ablation coagulation volume was retrospectively determined using a semi-automatic three-dimensional
(3D) method. Local tumor recurrence was found in 7 patients, and was detected median 9
months (6-21 months) after radiofrequency ablation. A positive margin was not achieved
in 3 patients. In patients without local tumor recurrence, coagulation volume was less than
30% of baseline value at 24 months. In contrast, coagulation volume increased markedly
in 6 of 7 patients with local tumor recurrence. Local tumor recurrence was identified
simultaneously by conventional morphological criteria and semi-automatic 3D volumetric
analysis. Semi-automatic 3D volumetric analysis provides added diagnostic information
compared with conventional morphological evaluation, and may increase specificity of
non-invasive diagnosis of local tumor recurrence after radiofrequency ablation of colorectal
cancer liver metastases.

27

28

Di sc us sion

Experimental studies
Study I
Hepatic radiofrequency ablation systems were
not compared in experimental studies using
standardized treatment protocols until 2003
[137, 138]. All evaluated systems showed
substantial variability in coagulation volume
and geometry. Furthermore, coagulation volumes were higher in ex vivo experiments than
in vivo, leading to the conclusion that ex vivo
findings did not predict the in vivo efficacy
of these systems [137]. Although the systems
were able to produce coagulations with transverse diameters in the range 2.5 to 4 cm, the
coagulations were smaller than expected and
deviated from the assumed spherical geometry. The perfusion electrode system used in
the present study was the most efficient [153]
(i.e. requiring the least amount of electrical energy per coagulated tissue volume), produced
among the largest volumes of coagulated tissue [138], but with larger variations in coagulation geometry and coagulation volume than
systems based on other principles [137, 138].

Active heating of tissue during radiofrequency ablation is counteracted by heat loss caused
by circulating blood, as shown by the bioheat
equation (Appendix I). Temporary interruption of hepatic blood flow during ablation
would therefore be expected to influence coagulation geometry and coagulation volume.
Interruption of hepatic inflow during ablation is associated with two different effects.
The term heat-sink effect refers to cooling by
adjacent blood vessels with diameter > 3 mm,
which may lead to irregular coagulation geometry in proximity to the vessels [122, 169].
Additionally, parenchymal perfusion on the
capillary level is reduced. In line with the bioheat equation, reduction of heat loss caused
by parenchymal perfusion would be expected
to lead to a general increase in the coagulation
volume [146]. Interruption of hepatic inflow
during ablation with multitined expandable
electrodes and internally cooled electrodes
produced coagulations that were larger, less elliptic and less distorted in experimental studies [149, 151, 170] and clinical series [152,
171]. In contrast, the impact of interruption
of hepatic inflow on coagulation geometry

29

and coagulation volume made with the perfusion electrode system had not been investigated. In study I we examined coagulation
geometry and coagulation volume produced
by the perfusion electrode system in a porcine
model with and without hepatic inflow occlusion (Pringle maneuver). The portal vein and
the hepatic artery was mechanically occluded
during ablation in 6 of 12 animals, based on
the assumption that reduction of heat loss
would lead to a coagulation geometry more
in line with an ideal sphere as suggested by
equation (7).
Our results confirmed that the use of a standardized ablation protocol during physiological hepatic circulation did not produce predictable coagulation geometry or predictable
coagulation volumes. As expected, reduction
of heat loss by Pringle maneuver was associated with increased coagulation volume,
which may be considered a beneficial effect.
However, the coagulation geometry was characterized by elongated coagulations extending outside the expected treatment site, and
only a fraction of the increased coagulation
volume contributed to a spherical shape. The
explanation for the elongated coagulations
observed in our study may be related to the
effect of saline infusion during ablation. With
the settings used in this experimental study,
saline was infused at a rate of 105 mL/hour,
corresponding to 16 mL during a 9 min coagulation, which was larger than the mean
coagulation volume of 10 mL. A recent clinical study confirmed that saline was distrib-

30

uted outside the planned treatment volume

with the perfusion electrodes used in this
study [172]. Thermal damage was observed
to abutting organs, which may be caused by
direct thermal damage from heated saline.
Additionally, uneven intraparenchymal distribution of saline may alter both the local
current density and tissue conductivity, which
would influence the tissue temperature, as
seen from equation (6). After determination
of the largest circle that could be inscribed in
each slice from a coagulation, the term effective coagulation diameter was assigned to the
diameter of the largest of these circles. This
entity was used to calculate the effective coagulation volume, which is the volume of the
largest sphere that could be inscribed into the
coagulation volume. Knowledge of the largest
spherical coagulation volume produced is of
clinical interest as it would allow the operator
to adequately coagulate a tumor without considering variation in coagulation geometry
relative to the axis of the electrode. In this
study, determination of geometrical properties were based on photographs of tissue slices
from the coagulation which had been cut in
parallel sections. A limitation of this method
is that it is dependent on the plane of cutting,
and that the accuracy decreases with increasing slice thickness. Nevertheless, the clinically
important geometrical concepts introduced
in this study are robust, easily comprehensible
and may be applied for evaluation of geometrical properties of future ablation systems.
The terminology to describe coagulation geometry is still under development. Of interest,

standardization of the methods and terminology for describing coagulation geometry has
recently been proposed [173].
Our findings indicated displacement of the
geometrical centre of the effective coagulation volume relative to the position of the
electrode. Inability to predict the position of
the coagulated area relative to the electrode
constitutes a fundamental limitation in the
use of local ablation techniques (Fig 6). The
ablation electrode was positioned close to
the right portal vein branch in all animals.
It is reasonable to assume that displacement
of the geometrical center of the coagulation
relative to the electrode may have been caused
by the heat-sink effect [146, 174]. If this is
the case one would expect displacement to
be less pronounced in the animals where hepatic inflow occlusion was performed during
ablation. Displacement of the geometrical

Figure 6. The figure shows an electrode placed in a

tumor and the position of the anticipated coagulation
diameter (dotted line). The actual position of the
coagulation is indicated by a circle. Due to displacement
of the coagulation, a part of the tumor is incompletely
treated.

centre of the coagulation relative to the electrode could not be quantified reliably by the
methods used in our study because the exact
position of the ablation electrode was not
known at reoperation 4 days after the ablation
procedure. Lastly, due to the dependence on
electrically conducting saline in generating a
large coagulation volume, displacement of the
coagulation volume may also be a limitation
of the perfusion electrode design.
Study II
As shown in study I, coagulation geometry
and volume is not predictable under standardized experimental ablation protocols.
Furthermore, coagulations are smaller and
more irregular than expected in clinical series
of patients treated with hepatic radiofrequency ablation [139, 175]. Intraoperative visualization of the thermally induced necrosis may
be beneficial to achieve complete treatment
of the index tumor and to avoid damage to
organs adjacent to the planned treatment site.
Assessment of the extent of tissue coagulation by sonography, CT or MRI during and
immediately after ablation is unreliable [55,
140, 141, 175]. Non-invasive thermometry
is an attractive concept with the potential to
visualize the three-dimensional temperature
distribution created during ablation. Several
methods have the potential to measure temperature non-invasively, including MRI-based
thermometry, ultrasound thermometry, microwave-based thermometry and electrical
impedance tomography [176, 177]. MRI-

31

based methods can provide non-invasive thermal mapping of relative temperature changes
with acceptable accuracy, spatial resolution
and temporal resolution [145]. However,
radiofrequency ablation generates significant
electromagnetic interference in the frequencies used for transmission and reception in
MRI, precluding the use of several MRI-based
methods [178]. Temperature-sensitive MRI
contrast agents represent a novel approach
for thermal imaging by indicating an absolute
temperature threshold.
In study II we examined the feasibility of
a thermosensitive liposomal agent with a
membrane phase transition temperature
of 57C and a size of 110 nm to assess the
extent of tissue coagulation during intraprocedural MRI-guided laser ablation and
radiofrequency ablation. The liposomal membrane can be tailormade to a predetermined
transition temperature by modulating the
composition of membrane phospholipids.
The liposomes used in our study contained
gadodiamide (GdDTPA-BMA), which is a
MRI contrast agent that acts by reducing the
longitudinal relaxation time (T1) of tissue water protons. At physiologic temperatures the
paramagnetic compound is retained within
the liposome and does not influence the T1relaxation of the tissue water protons due to
slow transmembrane water exchange conditions. At temperatures above the transition
temperature the T1-relaxation efficacy of the
liposomal agent increases markedly either
due to fast transmembrane water exchange

32

conditions or leakage of gadodiamide into

the tissue. T1-shortening of the tissue water
protons will be shown as high signal intensity
(SI) on standard T1-weighted MR images. A
transition temperature of 57C of the liposomal membrane was chosen because protein
denaturation and irreversible cellular damage
would be expected to have occurred above
this temperature threshold [55]. Increased SI
on T1-weighted MR images would therefore
indicate the extent of irreversible tissue damage. In study II coagulations made after injection of the agent showed slightly increased SI
during heating, followed by a nonreversible
increase in SI after the tissue had regained
physiological temperature. We speculated
that this may have been caused by leakage of
GdDTPA-BMA from the liposome interior
and subsequent entrapment due to coagulation of vessels. This mechanism has also been
suggested by a recent study in which the feasibility of using these liposomes as indicators
for local drug delivery during hyperthermia
has been examined [179]. The diameter of the
coagulations as measured from photographs
of macroscopic specimens and as determined
from MR images were inconsistent. This may
possibly be explained by the fact that irreversible thermal damage may have occurred
at a temperature below the phase transition
temperature of the agent [80]. Nevertheless,
a consistent pattern in the diameters was not
observed. This finding might have been caused
by the introduction of saline into the tissue
during treatment, which would be expected
to decrease SI on T1-weighted images and lead

to local inhomogeneities in the concentration

of the liposomal agent or of GdDTPA-BMA
released in the tissue. Although the mechanisms responsible for our observed results are
not completely understood, consistent results
were found that confirm the ability of the
liposomal agent to differentiate between tissue exposed to thermal treatment and normal
liver tissue. The persistent signal enhancement
in areas exposed to a temperature above the
threshold temperature may be of clinical value
during MRI-guided radiofrequency ablation.
Study III
The presence of large vessels in the proximity of tumors treated with radiofrequency
ablation is associated with a high rate of incomplete tumor destruction and local tumor
recurrence, assumed to be caused by cooling
effects [135, 169, 180]. Temporary reduction
of flow in the portal vein and hepatic artery
during radiofrequency ablation may enhance
coagulation necrosis close to hepatic vessels [169]. However, as maintained flow in
large vessels serve to protect the vessels from
thermal damage [181], vessels may be more
vulnerable to thermal injury and subsequent
thrombosis if radiofrequency ablation is performed during interruption of hepatic vascular inflow. Although segmental portal vein
thrombosis may be asymptomatic, patients
with a deficit in functional hepatic reserve
may be at risk of postoperative hepatic failure after portal vein thrombosis. The safety
of performing radiofrequency ablation in

proximity to large intrahepatic vessels has not

been established. It has been suggested that
radiofrequency ablation should only be performed for tumors at least 2 cm from major
intrahepatic vessels [105]. By adhering to this
principle, potentially operable patients could
be excluded from treatment. We therefore
considered it important to examine factors
associated with risk of damage to intrahepatic
vessels during radiofrequency ablation in an
experimental model.
In study III we examined risk factors associated with portal vein thrombosis after hepatic
radiofrequency ablation, with special attention to the effect of temporary hepatic inflow
occlusion and the electrode-to-vessel distance.
The presence or absence of portal vein thrombosis was examined by performing rotational
portal venography prior to radiofrequency
ablation, 10 min after radiofrequency ablation and 4 days after radiofrequency ablation.
Consequently, we could determine by an
objective method in the living animal when
portal vein thrombosis occurred and the anatomical location of a portal vein thrombosis.
Acute portal vein thrombosis was defined as
thrombosis on portal venograms acquired 10
min after completion of the ablation, whereas
delayed portal vein thrombosis was defined
as thrombosis on portal venograms acquired
4 days after ablation. Delayed portal vein
thrombosis occurred in 2 of 6 animals in the
group in which hepatic inflow occlusion was
performed, and in 3 of 6 animals in the group
where coagulations were made during physi-

33

ological hepatic circulation. Hence, the use of

temporary hepatic inflow occlusion was not
associated with portal vein thrombosis. We
found that an electrode-to-vessel distance of
less than 5 mm was a significant predictor for
delayed portal vein thrombosis. Our findings
conflict with those of a study which reported
that portal vein thrombosis invariably occurred when hepatic radiofrequency ablation
was performed during vascular inflow occlusion, and that no portal vein thrombosis
was found during physiological hepatic circulation [163]. These apparently diverging
results may be caused by differences in study
design and the method used for detection
of portal vein thrombosis. The electrodeto-vessel distance, which was quantified by
3D portal venography in our study, proved
to be of significance for the occurrence of
portal vein thrombosis. Due to variations in
electrical power and electrode designs used
in different ablation systems, we consider our
findings to be valid only for the specific radiofrequency ablation system used in this study.
Even though the electrode-to-vessel distance
may be of importance in regard to portal vein
thrombosis in humans, the pig is considered
to be more susceptible to activation of the coagulation system following endothelial damage than humans [182]. Consequently, the
electrode-to-vessel distance needed to induce
post-ablation thrombosis in humans may be
shorter than the 5 mm distance found to be
of significance in our experimental study. Our
results indicate that portal vein thrombosis
may occur without temporary occlusion of

34

hepatic vessels. Another finding of clinical

importance is that normal portal venography
10 min after radiofrequency ablation does not
exclude later occurrence of portal vein thrombosis. This finding may be of importance for
postoperative evaluation of patients with assumed high risk of post-ablation portal vein
thrombosis.
Study IV
Several established risk factors for local tumor recurrence after radiofrequency ablation
of hepatic metastases exist, such as tumor diameter > 3 cm and tumor proximity to major
vessels [135]. Local tumor recurrence after
radiofrequency ablation can be attributed to
incomplete treatment of the index tumor or
growth of micrometastatic disease around
the tumor [181]. Micrometastatic disease
in the vicinity of the macroscopic tumor
has been detected in 2% to 70% of patients
who underwent liver resection for colorectal
liver metastases [183, 184]. These contradictory results possibly illustrate methodological
limitations. Therefore, the true rate of micrometastatic disease in this patient category remains uncertain. Patients treated with hepatic
radiofrequency ablation have higher local, intrahepatic and distant recurrence rates, as well
as lower overall survival than comparable resected patients [24]. A reasonable explanation
for these findings is negative patient selection
bias, with micrometastatic disease being present in a larger proportion of patients treated
with radiofrequency ablation. An alternative

explanation is that thermal ablation may induce biological alteration in the tumor tissue
or peritumoral hepatic parenchyma that facilitate spread and invasion of malignant cells.
However, biological effects induced by radiofrequency ablation in the treated tumor and
surrounding hepatic parenchyma are not well
characterized due to the fact that the treated
tumors and surrounding parenchyma are left
in situ. In a recent experimental tumor model
in mice, hepatic radiofrequency ablation promoted intrahepatic growth of residual neoplastic cells [168]. The mechanisms explaining these findings is not well understood,
although increased expression of growth factors have been found after thermal ablation in
experimental tumor models [185].
Matrix metalloproteinases (MMPs) are a
family of matrix-degrading endopeptidases
involved in a variety of physiological and
pathological events [186-188]. The gelatinases
MMP-2 and MMP-9 are involved in colorectal cancer tumor cell invasion, metastasis and
angiogenesis [188]. In study IV we examined
the activity of MMP-2 and MMP-9 in the
transition zone separating the coagulated tissue and normal hepatic parenchyma after hepatic radiofrequency ablation in a non-tumor
porcine model. MMP-2 and MMP-9 activity
in tissue lysates from the transition zone 4
days after radiofrequency ablation was threefold increased compared to normal hepatic
parenchyma. Increased activity of MMPs
probably represents an inflammatory response
caused by radiofrequency ablation. However,

up-regulation of MMP activity may provide a

microenvironment that facilitates the risk of
local tumor recurrence and distant metastasis
by vascular invasion in the presence of viable
malignant cells. This mechanism would be of
particular importance if a tumor is incompletely treated by radiofrequency ablation,
leaving viable malignant cells, or if micrometastases are present in or near the transition
zone. Furthermore, hepatic radiofrequency
ablation is increasingly combined with hepatic resection, which induces hematogenous
dissemination of colorectal cancer cells [189,
190].
The data generated in this non-tumor preliminary animal study does not allow us to
draw conclusions concerning the possible role
of MMPs in local tumor recurrence, invasion
or metastasis after radiofrequency ablation of
colorectal liver metastases. However, based
on the assumption that increased expression
of MMPs in peritumoral tissue is associated
with increased risk of growth and metastasis
of malignant cells, our results indicate that hepatic radiofrequency ablation should only be
performed when complete eradication of the
target tumor including an adequate margin is
possible.

35

Clinical study
Study V
Hepatic radiofrequency ablation has been
used for treatment of patients with non-resectable liver metastases from colorectal cancer at our institution since 2003. Prior to introduction of hepatic radiofrequency ablation
in clinical practice, a non-randomized phase
II study was designed and approved by the
Regional committee for medical research ethics. Written informed consent was obtained
from all patients prior to radiofrequency ablation. All patients in the study were systematically followed by multi-detector computed
tomography (MDCT) of the liver at 1 and
3 months after radiofrequency ablation, and
then at intervals of 3 months until 24 months,
and each sixth month thereafter.
Non-invasive diagnosis of local tumor recurrence is desirable due to the risk of needle
tract seeding and negative impact on patient
survival if biopsy is performed in operable patients [191, 192]. However, it can be difficult
to distinguish between local tumor recurrence and benign tissue alterations on postablation follow-up imaging [156-158]. In
study V our hypotheses were that inadequate
treatment margins as determined from scans
1 month after radiofrequency ablation may
identify patients who would develop local
tumor recurrence, and that increase in coagulation volume during follow-up may be used
to detect local tumor recurrence earlier than
conventional morphological criteria. The
36

geometrical concepts of effective coagulation

diameter and effective coagulation volume developed in study I were applied in the analysis
of the treatment results.
Based on previous published results from
other groups, occurrences of local tumor recurrence at the treated site were expected to
be diagnosed during follow-up within the
first 6 months. However, local recurrence had
also been reported to appear up to 23 months
after the procedure [125, 193, 194]. In order
to compare groups with and without local tumor recurrence, we retrospectively identified
11 patients who had either developed local
tumor recurrence or who had been followed
for at least 24 months without local tumor
recurrence, assuming that local tumor recurrence would not develop in this last category.
Local tumor recurrence was found in 7 of
11 patients. Intrahepatic recurrence not related to the radiofrequency ablation site was
found in 6 patients, and extrahepatic tumor
growth in 6 patients. Of the 7 patients with
local tumor recurrence, 4 patients presented
with inoperable intrahepatic or extrahepatic
tumors at the time of diagnosis of local tumor
recurrence. Fine needle aspiration cytology of
the area treated with radiofrequency ablation
was performed in 4 patients during follow-up.
In 3 patients malignancy was verified, whereas
no malignant cells were found in 1 patient
who did not develop local tumor recurrence.
Malignant growth is common in this patient
category despite successful treatment of the
index tumor, with inoperable intrahepatic

or extrahepatic malignant growth occurring

in 3 of 4 patients where the index tumor had
been successfully treated with radiofrequency
ablation. These findings are comparable to a
previous study [24]. The largest coagulation
diameter 1 month post-ablation was larger
than the pre-ablation tumor diameter in all
patients. However, when considering the effective coagulation diameter 1 month post
ablation, a positive treatment margin had
only been achieved in 8 patients. The 3 patients with an effective coagulation diameter
smaller or equal to the largest tumor diameter
developed local tumor recurrence. A negative treatment margin may be of clinical use
to identify patients who later develop local
tumor recurrence. However, the prognostic
value of this parameter should be examined in
prospective studies.
In the patients who did not develop local tumor recurrence, coagulation volume decreased
during follow-up, with all values below 30%
of baseline values at 24 months. In contrast,
coagulation volume increased significantly in
the patients who developed local tumor recurrence. Our findings suggest that the combination of semi-automatic 3D volumetric analysis
and conventional morphological evaluation
may increase the specificity of non-invasive
diagnosis of local tumor recurrence after radiofrequency ablation of colorectal cancer
liver metastases, thereby possibly avoiding unnecessary biopsies. Furthermore, systematic
post-ablation follow-up is associated with detection of treatable malignant disease, with 3
of 11 patients re-treated with curative intent.

We found a high rate of local recurrence in

this study, which can partly be explained by
the fact that the selection criteria used in this
study leads to an overrepresentation of patients with local tumor progression. Hepatic
radiofrequency ablation using expandable
and internally cooled electrodes increase
parenchymal pressure [195], and it has been
speculated that this may be a mechanism for
spread of malignant cells to surrounding tissue. One would expect that intraparenchymal
infusion of saline by the ablation system used
in this study would lead to further increase in
parenchymal pressure. Furthermore, study I
confirmed that the perfusion electrodes produced irregular and non-predictable coagulations, which may be unfavorable in regard to
achieving adequate treatment margins. This
was in line with our clinical experience, and is
one of the reasons we no longer use the perfusion electrode system in clinical treatment at
our institution. Finally, a learning curve exists
for new procedures. Therefore, as this patient
series represents our initial experience with
radiofrequency ablation, a lower rate of local
tumor recurrence would be expected with increasing experience.
Study V is a small clinical study. Some authors
regard small clinical studies as unscientific
and unethical [196]. On the other hand, one
could argue that in rapidly lethal diseases, one
should look for large effects in small trials
rather than small effects in large trials [197].
This non-randomized study with a median
follow-up of 27 months does not allow us
to determine long-term survival effects of
37

radiofrequency ablation of colorectal liver

metastases. However, of the 11 patients included in the study, 9 patients are alive with
a median follow-up of 28 months (20-43
months). This compares favorably to recent
studies of patients with metastastic colorectal
cancer treated with chemotherapy, in which
a median survival of 20-22 months has been
achieved [198, 199]. However, the patients
in our study is a highly selected group, with
liver-only involvement which may have better prognosis than patients included in chemotherapy studies. Patient selection bias can
therefore not be excluded as an explanation
for the apparent short-term benefit of radiofrequency ablation. In 7 of the 11 patients in
study V, liver resection had been performed
previously. Therefore, a time-effect is also
present, with these 7 patients having a median
survival of 12 months (2-77 months) after
development of metastatic disease to the liver
prior to entering our study.

38

F u t u r e pe r spec t i v e s

New technology that offers the promise of

improved patient care is attractive, and may
be adopted despite lack of evidence of either
efficacy or superiority over existing procedures [200]. When hepatic radiofrequency
ablation was introduced in the early 1990s, it
was viewed as a palliative minimally-invasive
experimental method [135]. Within a decade
after its introduction, the use of hepatic radiofrequency ablation is increasing despite lack
of formal scientific evidence.
In order to investigate the therapeutic potential of the concept of local ablation of liver
metastases, local tumor recurrence at the site
treated with radiofrequency ablation should
be minimized. This involves development of
radiofrequency ablation systems capable of
generating predictable tissue coagulations,
and improved intra-procedural monitoring in
order to secure adequate treatment margins.
The ability of radiofrequency ablation to
eradicate liver tumors is limited by a number
of tumor-related factors [135] which should
be taken into consideration when determining which patients are eligible for this treatment modality. Novel diagnostic modalities

such as positron emission tomography (PET)

may improve staging of malignant disease,
which would facilitate selection of patients
with localized non-resectable disease who
may benefit from local tumor ablation [201].
It is important to assess whether high local
control rates are of clinical significance in regard to long-term survival, since intrahepatic
tumor recurrence not related to the area treated with radiofrequency ablation or extrahepatic tumor recurrence significantly limits the
value of local tumor control. Furthermore, an
unresolved apparent inconsistency exists in
post-treatment recurrence rates in patients
with colorectal liver metastases treated with
hepatic resection or radiofrequency ablation,
with significantly higher recurrence rates observed in patients treated with radiofrequency ablation [24]. If these differences cannot
be attributed to biological differences in the
treated tumors, differences in recurrence rates
must be attributed to the treatment, and not
the disease [202]. Further exploration of the
biological effects induced by radiofrequency
ablation may provide insights in the underlying mechanisms of these findings.

39

Long-term patient survival is the primary

clinical endpoint in evaluation of potential
curative interventions in cancer patients. Even
though recent non-randomized series indicate
that radiofrequency ablation of non-resectable colorectal liver metastses is associated
with a 5-year survival of approximately 30%
[126, 129], these patients belong to a highly
selected group treated within a multimodal
framework. Patient survival in non-randomized series cannot reliably be attributed to
any single intervention, as the outcome may
reflect patient selection bias and the cumulated effect of a multimodal treatment regime
[51]. Randomized studies should therefore be
performed to determine the possible impact
of radiofrequency ablation on long-term survival. However, randomization of resectable
patients to radiofrequency ablation is considered unethical, as superior long-term survival
in well-defined patient populations is established for surgical resection [134, 202, 203].
Randomization of non-resectable patients
to chemotherapy or radiofrequency ablation
may be ethically acceptable. In 2002 a multicenter randomized phase III study examining
the possible benefit of hepatic radiofrequency
ablation was initiated by the European organization for research and treatment of cancer
(EORTC). The objective of the study was to
examine the effect of performing radiofrequency ablation in addition to chemotherapy
by randomizing 390 patients with non-resectable colorectal liver metastases to chemotherapy vs. chemotherapy and radiofrequency ablation. However, in this multicenter study with

40

nearly 50 participating centers, recruitment

of patients was insufficient, and the study was
redesigned to a randomized phase II study,
with planned inclusion of 152 patients [204].
The lack of sufficient recruitment may reflect
ethical or practical concerns of the individual
physicians in charge of patient inclusion, or
that patients refuse to volunteer for randomization. Nevertheless, this study will probably be able to determine if radiofrequency
ablation provide an added survival benefit to
chemotherapy.
Despite expanding indications for resection of colorectal liver metastases, several
recent studies have shown improved survival
compared with historical series, with 5-year
survival of 58% [23-25]. These studies may
establish a new gold standard for long-term
survival after resection of colorectal hepatic
metastases against which other potentially
curative treatments must be compared. Longterm results after radiofrequency ablation
are promising, but do not currently match
those achieved by resection. Radiofrequency
ablation has been promoted by some as an
inexpensive minimal-invasive alternative to
resection [205]. However, the short-term
benefits of less invasiveness should not be
considered a valid argument for performing
a treatment which may be less effective than
established treatment. Therefore, based on
currently available studies, only non-resectable tumors should be treated with radiofrequency ablation [202, 206]. Considering
the complexity of the multi-modal treatment

available to patients with colorectal liver metastases, radiofrequency ablation should only
be performed in patients in which resection is
not considered possible after evaluation by a
multidisciplinary team including oncologists,
radiologists and surgeons experienced in hepatic surgery [202].

41

42

Conc lusions

1.

The perfusion electrode system produces irregular coagulations during physiological

hepatic perfusion. Mechanical occlusion of the portal vein and the hepatic artery during
ablation increases the effective coagulation volume, but is associated with elongated
coagulations extending outside the expected treatment site.

2.

A liposomal thermosensitive agent showed persistent signal enhancement on T1-weighted MR images of tissue exposed to a temperature above the phase transition temperature
of 57C, and may be of use during MRI-guided radiofrequency ablation.

3.

Delayed portal vein thrombosis may occur after radiofrequency ablation during physiological hepatic flow. An electrode-to-vessel distance of < 5 mm was a significant predictor for delayed portal vein thrombosis.

4.

Radiofrequency ablation of normal porcine liver is associated with significantly

increased activity of matrix metalloproteinase-2 and -9 in the transition zone separating
tissue coagulation from normal hepatic parenchyma. The expression of matrix metalloproteinases was localized to macrophages in the transition zone.

5.

Semi-automatic 3D volumetric analysis of post-ablation CT images acquired every third

month does not facilitate earlier detection of local tumor recurrence than conventional
morphological evaluation, but may increase specificity of non-invasive diagnosis of local
tumor recurrence after radiofrequency ablation of colorectal cancer liver metastases.

6.

Local tumor progression was found in a high proportion of patients with colorectal liver
metastases treated with radiofrequency ablation.

43

7.

Intrahepatic and extrahepatic malignant growth is commonly found during follow-up in

patients treated with radiofrequency ablation despite successful treatment of the index
tumor.

8.

Systematic post-ablation follow-up may identify patients with malignant disease that can
be offered re-resection or re-ablation in curative intent.

44

A ppe n di x I - T h e bioh e at equat ion

In 1948 Pennes developed a mathematical model for heat transfer in perfused tissue based on
temperature measurements in the human forearm [76]. Pennes suggested that the rate of heat
transfer between blood and tissue is proportional to the product of the volumetric perfusion
rate and the difference between the arterial blood temperature and the local tissue temperature.
The equation proposed by Pennes is known as the bioheat equation [55]:

t c tT(r,t) /t = (k t T) c b b m t (T Tb ) + Qp (r,t) + Qm (r,t)

where

c
k
m
Qp
Qm

density of tissue, blood (kg/m3)

specific heat of tissue, blood (W sec/kg C)
thermal conductivity
perfusion (blood flow rate/unit mass tissue) (m3/kg sec)
power absorbed per unit volume tissue
metabolic heating per unit volume of tissue.

For the sake of this thesis, the most important contribution of the bioheat equation is to offer
a biophysiological explanation of the effects of reduction of intrahepatic blood flow during
radiofrequency ablation.

45

46

A ppe n di x II - A not e on t e r m i nolog y

The terminology used for description of radiofrequency ablation systems, electrodes, and
postoperative image findings is inconsistent. Incompatible sets of terminology have been
proposed by different groups [181, 207]. The terminology used in this thesis adheres to the
proposal of the International working group on image-guided tumor ablation [181]. Due to the
general evolvement of the terminology and the fact that authors are encouraged to implement
the terminology used by a specific scientific journal, the terminology used in the papers in this
thesis is inconsistent.
1.

Radiofrequency ablation system and electrodes

A single radiofrequency ablation system was used for all studies in this thesis.
The current generator is denominated impedance-controlled, whereas the system
including the electrodes is denominated as saline-enhanced radiofrequency ablation
system. The preferred term for the electrodes used in this study is perfusion electrodes,
which correspond to the term saline-cooled used in paper II and wet electrode used
in paper III. In the literature, the terms open perfused system and Berchtold system
are occasionally used to describe this system.

2.

Thermally treated tissue

Postprocedural imaging may identify zones with altered signal intensity (at magnetic
resonance imaging), echogenicity (at sonography), or attenuation (at computed tomography) that can be attributed to the thermal treatment. When describing findings
on postprocedural images, the zone in which the induced treatment effect is present is
called coagulation or ablation zone. This corresponds to the terms lesion or ablation used in paper II.

47

3.

Failure of local therapy

A distinction is made between tumor growth within or at the site treated with radiofrequency ablation, intrahepatic foci of malignancy not related to the site treated with
radiofrequency ablation, and extrahepatic tumor growth. The preferred terms for tumor
growth within or at the site previously treated with radiofrequency ablation are local
tumor progression and local tumor recurrence.

48

A ppe n di x III - A not e on a n i m a l mode l s

All animals were supplied by the Department of Comparative Medicine, Rikshospitalet

University Hospital. Two different non-tumor animal models were used in this study.
In paper I, III and IV we used a pig model. The porcine liver is not morphologically similar to
the human liver. Nevertheless, the pig is widely used as a model for hepatic thermal ablation.
Furthermore, this model has proved to be valuable to our group in previous experimental research. A total of 26 pigs were operated. Eight pigs were included in a pilot study to establish the
model. Of the consecutive 13 pigs who were randomized to hepatic vascular occlusion, one pig
was euthanized within 2 hours of the primary operation due to respiratory distress attributed
to complicated tracheal intubation. Consequently, 12 animals were successfully randomized,
operated and reoperated 4 days later. Data from these 12 animals are included in paper I, III and
IV. Additionally, 5 animals constituted a control group where only hepatic vascular occlusion
but no radiofrequency ablation was performed. One of the animals in the control group was
euthanized 2 days postoperatively due to symptoms of paralytic ileus. Data from the remaining
4 animals in the control group were used in paper III.
In paper II we studied a liposomal paramagnetic agent which was only available in limited
quantities. In order to obtain adequate plasma levels, the experiments were performed in a smallanimal model. The rabbit was chosen because it is a well-established liver model in magnetic
resonance imaging research. A total of 14 rabbits were operated. Five rabbits were included in
a pilot study to establish the model. Of the remaining 9 animals, one animal with preoperative
weight loss was euthanized during the ablation procedure due to signs of distress. In the remaining 8 animals the procedure was successfully completed.

49

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65

66

E r r ata

Paper III, page 1995

The correct heading of the second column of Table 2 is:
Control group (n=6)

67

EJSO 32 (2006) 527532

www.ejso.com

Hepatic radiofrequency ablation using perfusion electrodes in a pig model:

Effect of the Pringle manoeuvre
L. Fricha,b,*, T. Malab, I.P. Gladhaugb,c
a

The Interventional Centre, Rikshospitalet University Hospital, N-0027 Oslo, Norway

b
Department of Surgery, Rikshospitalet University Hospital, N-0027 Oslo, Norway
Department of Surgery, Faculty Division Rikshospitalet, University of Oslo, N-0027 Oslo, Norway

Accepted 27 February 2006

Available online 3 April 2006

Abstract
Aim: To assess the inuence of the Pringle manoeuvre on volume and geometry of coagulations close to the portal vein using an impedancecontrolled radiofrequency ablation system with perfusion electrodes.
Methods: Twelve pigs were randomly assigned to a control group (nZ6) and a group where the Pringle manoeuvre was applied during
ablation (nZ6). One coagulation was made in each animal close to the portal vein. All animals were sacriced 4 days after ablation, and the
livers were removed for gross and histopathologic analysis.
Results: Effective coagulation volume in the Pringle group (10.8G5.0 cm3) was signicantly increased (pZ0.03) compared to the control
group (4.1G4.1 cm3). The efcacy ratio, dened as the effective coagulation volume divided by the coagulation volume, was not
signicantly different in the Pringle group (0.47G0.27) compared to the control group (0.33G0.22). The geometrical centre of the effective
coagulation volume did not correspond to the position of the ablation electrode. Thermal damage of the gallbladder was found in three
animals, all belonging to the Pringle group.
Conclusions: The Pringle manoeuvre was associated with increased effective coagulation volume, but did not signicantly inuence the
predictability of coagulation volume or geometry.
q 2006 Elsevier Ltd. All rights reserved.
Keywords: Radiofrequency; Liver; Neoplasm; Animal experimentation

Introduction
Radiofrequency ablation (RF ablation) is used for in situ
destruction of malignant liver tumours in patients who are
not candidates for hepatic resection.1 RF ablation should be
subject to established oncological criteria for surgical
treatment of malignant liver tumours including complete
tumour clearance and treatment margins of 0.51 cm.2,3
Coagulation geometry is as important as coagulation
volume in order to achieve adequate treatment margins.35
Interruption of hepatic inow during ablation enable RF
coagulations that are larger, less elliptic and less distorted in
experimental studies using multitined expandable electrodes.4,6 In contrast, effects of the Pringle manoeuvre on
* Corresponding author. Address: Department of Surgery, Rikshospitalet
University Hospital, N-0027 Oslo, Norway. Tel.: C47 23071825; fax: C47
23072267.
E-mail address: lars.frich@labmed.uio.no (L. Frich).

0748-7983/$ - see front matter q 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ejso.2006.02.021

coagulation volume and geometry made with an impedancecontrolled RF ablation system using perfusion electrodes
have not been investigated.
Perfusion mediated tissue cooling is believed to be
particularly prominent in the perivascular regions.7 It is
therefore of special interest to characterize volume and
geometry of coagulations made in proximity to large
intrahepatic vessels. The aim of this study was to assess
the impact of the Pringle manoeuvre on the volume and
geometry of coagulations made with an impedancecontrolled perfusion electrode system close to the right
portal vein branch (RPV) in normal porcine liver.
Materials and methods
Study design
Twelve domestic pigs with a mean weight of 30.3 kg
(SDZ3.4 kg) were randomly assigned to a control group

528

L. Frich et al. / EJSO 32 (2006) 527532

(nZ6) and a group where the Pringle manoeuvre was

applied during ablation (nZ6). One coagulation was made
in each animal. Coagulation volume and geometry was
assessed from specimens retrieved 4 days after ablation.
RF ablation protocol
An impedance-controlled RF ablation system (Elektrotom 106 HiTT, Berchtold GmbH and Co., Germany) was
used. The system incorporates a generator that delivers
alternating current at 375 kHz. The cumulative energy
output is displayed in real-time. The 1.7 mm diameter
perfusion electrode (EZ 707-15-04, Berchtold, Germany)
has a 15 cm long shaft incorporating a lumen for saline
perfusion, with a terminal non-insulated segment of 15 mm.
RF energy was applied for 1 min with an effect of 30 W,
immediately followed by 8 min with 50 W.
Operative procedure
Surgery was performed during general anaesthesia
induced by intravenous pentobarbital 150400 mg and
morphine 1030 mg and maintained by inhaled isourane
11.2% in oxygen. The analgesic effect was supplemented
with morphine infusion at 10 mg/h. A 134 cm2 dispersive
plate (EZ 344-02, Berchtold, Germany) was placed over the
right hip. Laparatomy was performed and a silicone vessel
loop (Sterion, MN, USA) was passed twice loosely around
the hepatoduodenal ligament.
The ablation electrode was positioned 2 cm into the liver
parenchyma in the anteriorposterior plane within 1.5 cm of
RPV. In the Pringle group, the vessel loop was tightened
around the hepatoduodenal ligament 1 min prior to application of RF energy and released immediately after ablation
had been completed. The needle track was coagulated by
applying 25 W during withdrawal of the electrode. The
animals were euthanised 4 days after ablation and the livers
were removed for gross and histopathological analysis.
Assessment of coagulation volume and geometry
All coagulations were excised with wide margins and cut at
46 mm intervals transverse to RPV. All fresh tissue slices
were photographed with a L-shaped ABFO no. 2 photomacrographic scale (Lightning Powder Company, Jacksonville, FL,
USA) (Fig. 1(a)). The images were imported into the scientic
image analysis program ImageJ version 1.33 (National
institute of mental health, Bethesda, MA, USA). The border
of normal liver tissue vs tissue with macroscopically visible
changes was traced on both sides of each slice, and the
perimeter and area of the coagulation was measured.
Maximum coagulation diameter was determined by measuring the Ferets diameter, which is the longest distance between
any two points along the perimeter of the coagulation.
Effective coagulation diameter was dened as the diameter
of the largest circle that could be t into the coagulation

Figure 1. (a) Macrophotograph of tissue coagulation made during Pringle

manoeuvre. The right portal vein branch is seen in the periphery of the
coagulated area. The assumed position of the ablation electrode is indicated
by an arrowhead. (b) Geometrical evaluation of the coagulation in (a). The
thick line denotes the border of normal liver tissue vs tissue with
macroscopically changes consistent with coagulation. Dmax denotes
maximum coagulation diameter and Deff effective coagulation diameter.
The geometrical centre of the inscribed circle does not correspond to the
assumed position of the ablation electrode (black dot).

(Fig. 1(b)). The coagulation volume of each slice was

calculated by multiplying the slice thickness with the mean
of the coagulation area from both sides of the slice.
Coagulation volume was determined by addition of the
corresponding slice coagulation volumes. Delivered energy
per coagulation volume was calculated for all coagulations.
Effective coagulation volume was dened as the volume of the
largest sphere that could be inscribed into the coagulation
volume. The efcacy ratio was dened as the effective
coagulation volume divided by the total coagulation volume.
An image navigation and display software system8 was used to
create 3D surface renderings of the coagulation volume and
the inscribed effective coagulation volume on the basis of the
electronically traced perimeters of the coagulation slices.
Histopathological examination
Tissue slices were xed in 4% formaldehyde for at least
10 days, then dehydrated and embedded in parafn.

L. Frich et al. / EJSO 32 (2006) 527532

529

Table 1
Summary of coagulation characteristics and delivered energy per coagulation volume in the two experimental groups

Maximal coagulation diameter (cm)

Effective coagulation diameter (cm)
Coagulation volume (cm3)
Effective coagulation volume (cm3)
Efcacy ratio
Delivered energy per coagulation volume
(J/cm3)
a

Control group (nZ6),

meanGSD (range)

Pringle group (nZ6),

meanGSD (range)

pa

4.1G1.5 (1.66.3)
1.7G0.8 (0.62.7)
9.9G7.6 (0.921.9)
4.1G4.1 (0.110.6)
0.33G0.22 (0.10.7)
7450G11,004 (114329,623)

7.6G4.2 (3.413.3)
2.7G0.4 (2.13.2)
31.2G25.5 (11.677.2)
10.8G5.0 (4.917.9)
0.47G0.27 (0.20.9)
1224G768 (3152135)

0.10
0.03
0.08
0.03
0.36
0.20

Independent samples t-test with unequal variances assumed.

A 45 mm thick section was cut from each slice, stained

with haematoxylin and eosin and examined by light
microscopy by an experienced pathologist.
Statistical analysis
The animals were allocated to the two groups by block
randomization, using a block size of two. SPSS for Mac
OSX version 11.0.2 (SPSS, Chicago, IL, USA) was used for
statistical analyses. Data are presented as meanGSD unless
otherwise indicated. Independent samples t-test was used
for comparison of the two groups. A p-value !0.05 was
considered statistical signicant.

(Fig. 2(b)). Delivered energy per coagulation volume

was six-fold lower, although not signicantly, in the
Pringle group.
Coagulation geometry
Maximal coagulation diameter was increased, although
not signicantly, in the Pringle group. Effective coagulation
diameter was signicantly increased in the Pringle group.
The efcacy ratio was not signicantly different in the two
groups. The geometrical centre of the effective coagulation
volume did not correspond to the position of the ablation
electrode (Fig. 1(b)).

Results
Complications
Coagulation characteristics and delivered energy per
coagulation volume in the two groups are summarized in
Table 1.
Coagulation volume
Coagulation volume was increased three-fold, although
not signicantly, in the Pringle group compared to the
control group (Fig. 2(a)). Effective coagulation volume
was signicantly increased in the Pringle group

Thermal damage to abutting organs was present on

re-operation in three animals, all belonging to the Pringle
group. In two animals, gallbladder perforation was caused
by severely deformed coagulations with maximum lengths
of 11.3 and 13.7 cm extending to the gallbladder (Fig. 3).
Additionally, perforation of the gastric wall was found in
one of these animals. Perforation of the gallbladder was also
found in a third animal where the coagulation had not
extended to the gallbladder.

Figure 2. Coagulation volume (a) and effective coagulation volume (b) in the control group (nZ6) and the Pringle group (nZ6). Boxes represent the
interquartile range, horizontal bars within the boxes indicate median values. Mean values are indicated by a plus sign (C).

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L. Frich et al. / EJSO 32 (2006) 527532

preserved in parts of the coagulated area. Cytoplasm of

coagulated hepatocytes was more eosinophilic, and showed
pycnotic nuclei compared to viable hepatocytes. In other
coagulated areas, the hepatocytes had undergone karyolysis
and were denucleated.
Discussion

Figure 3. Thermal injury to abutting organ. The coagulation has extended to

the gallbladder (G) and caused a full wall damage. The main portal vein
trunk (P) has been incised. The arrowhead indicates the assumed position of
the ablation electrode.

Histopathology
A sharp demarcation between coagulated tissue and
normal hepatic parenchyma was found at gross examination, corresponding to the 12 mm border between
coagulated and non-coagulated tissue at histopathologic
examination (Fig. 4). The hepatic microstructure was

Figure 4. Haematoxylin and eosin stained section. A sharp demarcation

(arrowheads) is seen between the coagulation (R) and normal hepatic
parenchyma (N).

Coagulation volumes in our control group were smaller

than in previous experimental studies using perfusion
electrodes,5,9 but in conformity with clinical results.10
These diverging ndings can be attributed to the use of
different ablation protocols and methods used for volume
estimation. Furthermore, as the coagulations in our study
were made close to a portal vein, perfusion mediated tissue
cooling possibly limited the coagulation volume. These
studies nevertheless support our ndings of unpredictable
ablation volumes when no hepatic vascular occlusion is
used. Use of the Pringle manoeuvre was associated with
three-fold increase in coagulation volume, which is
comparable to previous studies with multitined expandable
electrodes.4 The effective coagulation volume was nearly
three-fold increased in the Pringle group, demonstrating a
benecial effect of the Pringle manoeuvre. The
maximal coagulation diameter is of importance in order to
avoid thermal damage to abutting organs, but is oncologically irrelevant.3 In contrast, knowledge of the effective
coagulation diameter is important to avoid
incomplete coagulation of the index tumour.11 Although
the effective coagulation diameter was increased by 1 cm
in the Pringle group, the Pringle manoeuvre was associated
with elongated coagulations extending outside the expected
treatment site.
The isoperimetric ratio has been used as an indicator of
coagulation circularity, but is not appropriate for characterization of irregular non-spherical volumes as different ratios
can be acquired depending on the orientation of the
measurement planes. In this study, we, therefore, introduced
the concept of the efcacy ratio which directly relates to the
ability to produce a spherical coagulation volume (Fig. 5).
A low efcacy ratio indicates that a large proportion of the
coagulation occurs in locations not relevant for the
oncological goal of the treatment. The efcacy ratio was
below 0.5 in both groups, but tended to increase in the
Pringle group. Our observation of a displacement of the
geometrical centre of the effective coagulation volume
relative to the position of the electrode may be caused by the
proximity of the electrode to large portal vessels. This
nding may be a of clinical importance and should be
further assessed.
Direct heat transfer from the treatment site can cause
burns of adjacent viscera, which is a well known
complication of hepatic RF ablation regardless of ablation
system used.12,13 However, when using perfusion electrodes, diffusion of heated saline may cause thermal damage to
structures not in direct contact with the treatment site.10,14 In

L. Frich et al. / EJSO 32 (2006) 527532

531

microperfusion due to hepatic inow occlusion may

increase the risk of low resistance electrical paths or heated
saline to cause thermal damage.
Our ndings are valid only for the specic RF ablation
system and electrodes used in the study. The lack of
statistical signicance for several of the examined variables
may be explained by the limited statistical power of the
study. In contrast to the human liver, the deep ssures and
the thin lobes in the porcine liver may inuence the volume
and geometry of the coagulations. Extrapolation of our
ndings to clinical settings should, therefore, be done with
caution.
In conclusion, the reproducibility of the coagulation
volumes and geometry including the efciency ratio was
low in the control group. The Pringle manoeuvre was
associated with increased effective coagulation volume, but
did not signicantly increase the predictability of coagulation volume, coagulation geometry or the efcacy ratio.
Furthermore, the Pringle manoeuvre was associated with
coagulations extending outside the expected treatment site
causing thermal damage to adjacent organs.
Acknowledgements
Figure 5. 3D surface renderings of coagulation volume (blue) and the
corresponding effective coagulation volume (red). Pringle manoeuvre was
used for both coagulations. The volumes are shown with correct
proportions relative to each other. (a) Coagulation volume is 42.9 cm3,
effective coagulation volume is 15.6 cm3. The efcacy ratio is 0.36. (b)
Coagulation volume is 12.8 cm3, effective coagulation volume is 11.5 cm3.
The efcacy ratio is 0.90.

the present study, thermal damage to abutting structures was

observed in three animals. Gallbladder perforation
was observed in one of these animals even though
the coagulation did not involve the gallbladder, suggesting
that this perforation may be caused by diffusion of heated
saline.
The effect of saline infusion on coagulation volume and
geometry has not been completely elucidated, but is
believed to be three-fold; a liquid electrode effect where
the effective surface of the ablation electrode is increased by
the electrical conductive qualities of saline, reduction of
charring in the proximity of the electrode due to cooling
effects, and extended thermal damage by diffusion of heated
saline into the tissue.15 A marked divergence from a
spherical geometry for coagulations produced with perfusion electrodes during physiological liver perfusion has
been reported previously,5,9 and may be caused by diffusion
of saline along vessels, tissue planes or surfaces, generating
low resistance electrical paths that could lead to heating
outside the expected target area. In our study, use of the
Pringle manoeuvre resulted in unpredictable coagulation
volumes and geometry as well as thermal damage to the
gallbladder and the gastric wall. A possible explanation for
this nding is that reduced tissue capillary level

We thank the staff at the Interventional Centre at

Rikshospitalet for providing excellent assistance. We also
thank the Department of Comparative Medicine at
Rikshospitalet for support in animal care, and Ole Petter
F. Clausen, Department of Pathology, Rikshospitalet
University Hospital for examination of the histopatological
specimens. This work was supported by the Norwegian
Cancer Society, grant no. D02042/001.
References
1. Ni Y, Mulier S, Miao Y, Michel L, Marchal G. A review of the general
aspects of radiofrequency ablation. Abdom Imaging 2005;30(4):
381400.
2. Blumgart LH. Liver resection for benign disease and for liver and
biliary tumors. In: Blumgart LH, Fong Y, editors. Surgery of the liver
and biliary tract. 3rd ed. London: W.B. Saunders; 2000. p. 1639713.
3. Mulier S, Ni Y, Miao Y, Rosiere A, Khoury A, Marchal G, et al. Size
and geometry of hepatic radiofrequency lesions. Eur J Surg Oncol
2003;29(10):86778.
4. Chinn SB, Lee Jr FT, Kennedy GD, Chinn C, Johnson CD, Winter
III TC, et al. Effect of vascular occlusion on radiofrequency ablation of
the liver: results in a porcine model. AJR Am J Roentgenol 2001;
176(3):78995.
5. Pereira PL, Trubenbach J, Schenk M, Subke J, Kroeber S,
Schaefer I, et al. Radiofrequency ablation: in vivo comparison of
four commercially available devices in pig livers. Radiology 2004;
232(2):48290.
6. Chang CK, Hendy MP, Smith JM, Recht MH, Welling RE. Radiofrequency ablation of the porcine liver with complete hepatic vascular
occlusion. Ann Surg Oncol 2002;9(6):5948.
7. Lu DS, Raman SS, Limanond P, Aziz D, Economou J, Busuttil R,
et al. Inuence of large peritumoral vessels on outcome of
radiofrequency ablation of liver tumors. J Vasc Interv Radiol
2003;14(10):126774.

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8. Rosset A, Spadola L, Ratib O. OsiriX: an open-source software for

navigating in multidimensional DICOM images. J Digit Imaging 2004;
17(3):20516.
9. Denys AL, Baere TD, Kuoch V, Dupas B, Chevallier P, Madoff DC,
et al. Radio-frequency tissue ablation of the liver: in vivo and ex vivo
experiments with four different systems. Eur Radiol 2003;13(10):
234652.
10. Kettenbach J, Kostler W, Rucklinger E, Gustorff B, Hup M, Wolf F,
et al. Percutaneous saline-enhanced radiofrequency ablation of
unresectable hepatic tumors: initial experience in 26 patients.
AJR Am J Roentgenol 2003;180(6):153745.
11. Patterson EJ, Scudamore CH, Owen DA, Nagy AG, Buczkowski AK.
Radiofrequency ablation of porcine liver in vivo: effects of blood ow
and treatment time on lesion size. Ann Surg 1998;227(4):55965.

12. Mulier S, Mulier P, Ni Y, Miao Y, Dupas B, Marchal G, et al.

Complications of radiofrequency coagulation of liver tumours. Br
J Surg 2002;89(10):120622.
13. Livraghi T, Solbiati L, Meloni MF, Gazelle GS, Halpern EF,
Goldberg SN. Treatment of focal liver tumors with percutaneous
radio-frequency ablation: complications encountered in a multicenter
study. Radiology 2003;226(2):44151.
14. Gillams AR, Lees WR. CT mapping of the distribution of saline during
radiofrequency ablation with perfusion electrodes. Cardiovasc Intervent Radiol 2005;28(4):47680.
15. Livraghi T, Goldberg SN, Monti F, Bizzini A, Lazzaroni S,
Meloni F, et al. Saline-enhanced radio-frequency tissue ablation
in the treatment of liver metastases. Radiology 1997;202(1):
20510.

II

Magnetic Resonance in Medicine 52:13021309 (2004)

Experimental Application of Thermosensitive

Paramagnetic Liposomes for Monitoring Magnetic
Resonance Imaging Guided Thermal Ablation
Lars Frich,1,2* Atle Bjrnerud,3 Sigrid Fossheim,4 Terje Tillung,1 and Ivar Gladhaug2
The use of a liposomal paramagnetic agent with a T1-relaxivity
that increases markedly at temperatures above the phase transition temperature (Tm) of the liposomal membrane was evaluated
during magnetic resonance imaging (MRI) guided hyperthermia
ablation. A neodymium-yttrium aluminum garnet (Nd-YAG) laser
unit and a radiofrequency ablation system were used for tissue
ablation in eight rabbit livers in vivo. One ablation was made in
each animal prior to administration of the liposomal agent. Liposomes with a Tm of 57C containing gadodiamide (GdDTPA-BMA)
were injected iv, and two additional ablations were performed.
T1-weighted scans were performed in heated tissue, after tissue
temperature had normalized, and 1520 min after normalization of
tissue temperature. Increase in signal intensity (SI) for ablations
prior to injection of the agent was 13.0% (SD 5.7) for the laser
group and 9.1% (SD 7.9) for the radiofrequency group. Signal
intensity after administration of the agent unrelated to heating was
not statistically signicant (SI 1.4%, P 0.35). For ablations
made after injection of the agent, a signicant increase was found
in the laser (SI 34.5%, SD 11.9) and radiofrequency group
(SI 21.6%, SD 22.7). The persistent signal enhancement
found in areas exposed to a temperature above the threshold
temperature above Tm allows thermal monitoring of MRI guided
thermal ablation. Magn Reson Med 52:13021309, 2004. 2004
Wiley-Liss, Inc.
Key words: thermometry; contrast agent; liver; radiofrequency
ablation

Hyperthermia ablation is used for treatment of benign and

malignant tumors (1 4). The tissue response to heating is
related to the absolute temperature induced in the tissue
and exposure time. Irreversible tissue destruction occurs
when tissue is exposed to temperatures in excess of 50
55C (5,6). Hyperthermia ablation has been widely used
for treating malignant liver tumors not amenable to conventional surgical resection. Heating modalities in clinical
use include laser, microwave, radiofrequency, and focused
ultrasound (1,79). The exact size and shape of the resulting ablation is difcult to predict because of heterogeneous
distribution of the thermal energy due to local disparity in
tissue composition, tissue anatomy, and vascularization

The Interventional Centre, Rikshospitalet University Hospital, 0027 Oslo,

Norway.
2
Department of Surgery, Rikshospitalet University Hospital, 0027 Oslo, Norway.
3
Department of Radiology, Rikshospitalet University Hospital, 0027 Oslo,
Norway.
4
Amersham Health AS, Part of GE Healthcare, Postbox 4220 Nydalen, 0401
Oslo, Norway.
Grant sponsor: Norwegian Cancer Society; Grant number: D02042/001.
*Correspondence to: Lars Frich, Department of Surgery, Rikshospitalet University Hospital, N-0027 Oslo, Norway. E-mail: lars.frich@labmed.uio.no
Received 2 February 2004; revised 28 July 2004; accepted 28 July 2004.
DOI 10.1002/mrm.20289
Published online in Wiley InterScience (www.interscience.wiley.com).
2004 Wiley-Liss, Inc.

(10,11). In clinical series, local tumor progression after

ablation therapy has been reported to be as high as 30
60%, indicating the presence of viable malignant cells at
the treated site (8,12,13). Improved intraprocedural monitoring may reduce the local tumor progression rates associated with thermal ablation and prove benecial in regard
to long-term patient survival.
Multiplanar imaging capabilities and excellent soft tissue contrast make magnetic resonance imaging (MRI) ideally suited for peroperative visualization, positioning of
energy applicators, and monitoring of thermal ablation
procedures. Additionally, noninvasive, three-dimensional
mapping of relative temperature changes is feasible with
MRI, based on the longitudinal relaxation time (T1), the
diffusion coefcient (D), or proton resonance frequency
(PRF) of tissue water. These methods rely on image subtraction and are sensitive to tissue motion (14). Thermal
imaging of the liver therefore presents certain challenges
as the liver is displaced during respiration due to movement of the diaphragm. A substantial body of research
exists on MRI-based methods for temperature mapping
during laser ablation (1517). These methods can be applied to thermal monitoring of other MRI-compatible local
thermal ablation therapies such as focused ultrasound
(18). Thermal monitoring of radiofrequency ablation poses
certain challenges. MRI-based methods for thermal monitoring cannot be used during radiofrequency ablation, as
the application of radiofrequency energy leads to electromagnetic noise that severely deteriorates image quality.
Simple switching circuits (19) and ltering of the RF signal
by modication of the hardware (20) have been developed
that allow image acquisition in near real-time or during RF
ablation. At present such solutions are not incorporated in
commercially available systems used for RF ablation. Consequently, reliable MRI-based thermometry of radiofrequency ablation requires development of new methods for
thermal imaging.
Thermosensitive MRI contrast agents represent a novel
approach for thermal imaging that offers a robust, motioninsensitive method for indication of an absolute temperature threshold on standard imaging hardware using conventional MRI sequences. Mechanisms of action include
temperature-induced transition from a diamagnetic to a
paramagnetic state using bistable molecular complexes
(21) and temperature-dependent relaxivity by incorporation of conventional MRI contrast agents within thermosensitive liposomes (22,23). In the present study we used
liposomes with a phase transition temperature (Tm) of
57C containing gadodiamide (GdDTPA-BMA) during
thermal ablation in vivo. The agent is designed to be MR
dormant at physiologic temperatures and MR activated

1302

Thermosensitive Paramagnetic Liposomes

1303

supplied by Amersham Health AS. Figure 1 shows the in

vitro temperature dependence of the T1-relaxivity for liposomal gadodiamide (GdDTPA-BMA) used in this study. For the
relaxometric measurements, the liposomal dispersion was
diluted with pH-adjusted isosmotic glucose solution to an
effective Gd concentration of 1 mM and dispended in NMR
tubes. Each tube was heated to a predetermined temperature
within a temperature range of 3757C in a thermostated
heating block at a heating rate of 10C/min. When the desired
temperature was reached, each sample was inserted into a
0.47-T relaxometer (Minispec PC-120b, Bruker GmbH, Germany) for immediate relaxometric measurements.
FIG. 1. In vitro temperature dependence of the T1-relaxivity for
liposomal and nonliposomal gadodiamide (GdDTPA-BMA). The vertical dashed line indicates the phase transition temperature of the
liposomal membrane at 57C.

when reaching the predened Tm of the liposome membrane. At physiologic temperatures, the T1-relaxation enhancement of the liposomal agent is low due to limited
water exchange. As the temperature approaches Tm, the
transmembrane water exchange kinetics is faster and the
T1-relaxivity increases rapidly and markedly before leveling off as Tm is exceeded (Fig. 1). Tissue with a temperature above Tm would therefore be expected to appear with
increased signal intensity (SI) on T1-w images. An important secondary mechanism in vivo is leakage of GdDTPABMA from the liposomes into the surrounding tissue at
temperatures above Tm. Following thermal coagulation of
parenchyma and vessels, it is assumed that the agent cannot migrate and therefore will interact with the tissue
where it was released. Consequently, a persistent signal
intensity enhancement on T1-w images may verify that a
temperature of Tm or above was achieved.
The purpose of this study was to quantify relative signal
intensity alterations associated with the use of paramagnetic thermosensitive liposomes during and after laser and
radiofrequency ablation of rabbit liver in an interventional
MRI system.

Laser and Radiofrequency Equipment

The laser apparatus consisted of a neodymium-yttrium
aluminum garnet (Nd-YAG) laser unit with a wavelength
of 1064 nm (Dornier mediLas Fibertom 4100, Dornier MedTech GmbH, Germany). The laser light was distributed
through 1.9-mm-diameter exible MRI-compatible light
guides designed for interstitial tissue ablation (Dornier
MedTech GmbH, Germany) with a 20-mm diffuser at the
tip of the ber.
A saline-enhanced, impedance-controlled radiofrequency system (Elektrotom HiTT 106, Berchtold GmbH &
Co, Germany) was used for radiofrequency ablation. The
375-kHz radiofrequency generator delivered a nonmodulated high-frequency alternating current to a maximum of
1.2 amp. Maximum power output of the generator could be
set between 5 and 60 W. The actual power output was
adjusted automatically in accordance with the impedance
of the patient circuit. A syringe pump (Pilot C, Fresenius
Vial, France) was connected by a RS-232 connection to the
RF generator. Output of a saline solution of 154 mmol
(0.9%) NaCl per liter water was controlled by an algorithm
built into the RF generator. The saline-cooled MRI-compatible RF electrodes had a diameter of 1.7 mm and a
15-cm shaft. The saline solution was distributed to the
tissue in the immediate vicinity of the tip of the electrode
through three groups of two side holes each in the 15-mm
exposed tip, placed at 120 angles from each other.
MRI-Compatible Fiber-Optic Thermometry System

MATERIALS AND METHODS

Liposomes
Liposomal gadodiamide (GdDTPA-BMA), whose membrane
consisted of 90% (w/w) distearoylphosphatidylcholine
(DSPC) and 10% distearoylphosphatidylglycerol (DSPG),
was prepared by the thin lm hydration method (24). The
liposomes were subsequently sized down by membrane extrusion and untrapped GdDTPA-BMA was removed by dialysis. Key physicochemical liposomal properties, as measured by standard methods described elsewhere (22,24),
were effective Gd concentration 27 mM, extraliposomal pH
7.2, and osmolality 355 mosmol/kg. Intensity weighted liposome size was 110 nm with a polydispersity index of 0.16,
indicating a highly monomodal size distribution. The transition temperature (Tm) of the liposomal membrane was 57C
with a SD of the Tm measurements of 1%. The liposomal
agent was injected iv at a dose of 1.1 mL/kg, corresponding to
30 mol Gd/kg. The thermosensitive liposomal agent was

Tissue temperature during the thermal treatment was measured by a MRI-compatible ber-optic thermometry system consisting of a exible ber-optic probe and an optoelectronic signal-processing unit (Multitemp 1601, Optomed, Norway). The outer diameter of the temperature
probe was 1.5 mm. The probe had 16 measuring points
with a sensor interval of 3 mm. All 16 sensors could be
read out simultaneously in real-time with a temporal resolution of 1 sec and an accuracy of 0.1C. The system
was calibrated to a temperature range of 0 150C. The
temperature monitoring system was connected by a RS232 interface to a laptop with specially designed software
(Multitemp). Temperature readings for all 16 sensors were
sampled each second.
Animals and Operative Procedure
The experimental protocol was approved by the Department of Comparative Medicine at Rikshospitalet Univer-

1304

sity Hospital under the surveillance of the Norwegian Animal Research Authority. Eight chinchilla-bastard rabbits
with a mean weight of 3.2 kg (2.7 4.0 kg) were treated in
accordance with current legislation governing animal care.
After premedication with a subcutaneous injection of 0.1
mL/kg fentanyl 0.315 mg/mL uanisone 10 mg/mL
(Hypnorm, Janssen), an iv cannula (Neoon, BectonDickinson) was inserted in the dorsal ear vein and 0.1 mL/kg
fentanyl 0.315 mg/mL uanisone 10 mg/mL (Hypnorm,
Janssen) was administered iv. All rabbits were shaved in
the upper abdominal midline using a hair clipper (Oster
Golden A5). The four rabbits to undergo radiofrequency
ablation were shaved in an area of 10 20 cm on the lower
back, and a 134-cm2 dispersive plate (EZ 344 02, Berchtold, Germany) was applied. The rabbits were intubated
with a 3- or 3.5-mm tracheal tube. All animals were anesthetized by Isourane 11.2%, 50% oxygen, using a
minute volume of 1.2 liter and respiratory frequency of
30/min. A midline incision was made and the rabbits were
placed inside the MRI head coil. To eliminate motion
artifacts during scanning, the right medial, left medial, and
left lateral liver lobes were exteriorized and placed on a
horizontal 5-mm polycarbonate sheet xated to the head
coil. Care was taken not to compromise the circulation of
the liver.
Four animals were treated with laser ablation and four
animals with radiofrequency ablation. The laser ber or
the radiofrequency electrode was inserted into one of the
liver lobes axially. The temperature ber was inserted
parallel in close proximity to the laser ber or the RF
electrode, with the tip of the temperature sensor extending
15 mm in front of the energy applicator. Each treatment
session consisted of 9 min active heating. For the animals
treated with laser ablation, a maximum power setting of 20
W and duration of 3 min was applied three times successively for each ablation. Automated stepwise reduction of
the laser effect (20/15/10 W each for 30 sec and 7 W for 90
sec) was used. For the radiofrequency system a power
setting of 20 W and a continuous treatment time of 9 min
was used. One ablation was created in each of the three
exteriorized liver lobes of each animal. The rst ablation in
each animal, denoted lesion A, was made prior to injection
of the agent. Tissue temperature during active heating and
passive cooling was monitored by real-time readout from
the temperature probe in the liver. When all sensors on the
temperature probe were below 37C, the laser ber/RF
electrode and the temperature probe were removed and
inserted into one of the two remaining liver lobes. The
liposomal agent was injected in a dorsal ear vein with an
infusion speed of 1 mL/min, and the vein was ushed with
10 mL saline solution of 154 mmol NaCl. The second
ablation, denoted lesion B, was made 1214 min after
administration of the agent, using the same energy modality and protocol as the rst ablation. The tissue temperature was allowed to normalize, and the third ablation,
denoted lesion C, was made accordingly in the remaining
lobe 50 55 min after injection of the agent. The A, B, and
C lesions were randomly assigned to each of the three
exteriorized liver lobes. After the MRI scans had been
completed, the animals were killed by injecting100 mg/kg
pentobarbital iv.

Frich et al.

MRI Protocol
All procedures were performed on a 0.5-T vertically open
whole-body MRI system (Signa SP2, GE Medical Systems)
using a transmit and receive head coil. An axial T1weighted fast spin echo (FSE) sequence was used for imaging. The imaging parameters were repetition time (TR)
300 msec, echo time (TE) 19 msec, ip angle (FA) 90,
slice thickness 4 mm, spacing 4.5 mm, 256 256 acquisition matrix, eld of view 180 180 mm, and four signal
averages. Total duration of the sequence was 2 min, 16 sec.
A scan was performed after each repositioning of the laser
ber or RF electrode, but before energy was applied. For
the four animals operated with laser, one scan was performed every 3 min during heating for each of the ablations. The mean signal intensity of the three scans during
laser heating was used for further analysis. Due to the
electromagnetic noise generated by the radiofrequency circuit while operating, scanning in heated tissue during
treatment was not possible for the four animals treated
with radiofrequency ablation. A scan was therefore performed within 10 sec after termination of the radiofrequency treatment. The liver tissue was allowed to reach
baseline temperature and a postablation scan at baseline
temperature was performed for all animals. This scan protocol was repeated for each of the three ablations. Scans
performed prior to heating, in heated tissue, and after
cooling of the tissue were denoted t0, t1, and t2, respectively. The scan at t0 for B and C lesions was made
1520 min after cooling of the tissue of the previous ablations. Signal intensity of A and B lesions 1520 min after
cooling of the tissue, denoted t3, could therefore be measured from the t0 scans of B and C lesions, respectively. No
scan was done 1520 min after normalization of the tissue
temperature for C lesions. Hence, signal intensity at t3 is
therefore not available for C lesions, and statistical analysis is performed only for the values measured at t1 and t2
for all lesion types. To evaluate signal intensity changes
due to the agent unrelated to heating, signal intensity of
the reference regions of interest (ROI)s was measured previous to administration of the agent and 3 min after injection of the agent, but prior to the generation of the B
lesions.
Analysis of MR Images
The signal intensity of the ablated area versus the untreated tissue was measured from uncompressed DICOM
images by image analysis software designed for quantitative image analysis (DIMview, Rikshospitalet University
Hospital, Oslo, Norway) running on a Windows XP computer system. Ablations as seen on T1-w images consisted
of a dark central area at the needle track with diameters of
15 mm surrounded by a brighter rim. Each ablation was
segmented manually using a polygon tool, avoiding the
central darker part. The mean value of the signal intensity
of this ROI (SIlesion) was used for further analysis. To
compensate for a potential general variation of signal intensity in the liver during the procedure, each signal intensity measurement was normalized using two independent regions of interest (SIref1 and SIref2) from liver tissue
not exposed to the local thermotherapy on the same image

Thermosensitive Paramagnetic Liposomes

1305

as the ablations. The subtraction of the mean signal intensity of these two reference ROIs from the signal intensity of
the ablation was used as an expression of the relative
alteration of signal intensity between the ablation and the
surrounding liver tissue.

SI % SI lesion

SI ref1 SI ref2
100
2

The largest diameter of each ablation was measured

from the MR images by an investigator blinded to the
results of the corresponding measurements on the macroscopic specimens.
Analysis of Liver Tissue
The livers were harvested immediately after the animals
were killed. To retain the orientation of the ablated area, a
marker was inserted into the tracks from the laser ber or
RF electrode, and each ablation was excised with wide
margins. Using a custom-built slicer, the ablated tissue
was cut in 4-mm slices axial to the inserted marker. All
slices were photographed with a 2.1-megapixel digital
camera (Canon, Tokyo, Japan) under standardized lightning conditions with a L-shape ABFO No. 2 photomacrographic scale (Lightning Powder Company, Jacksonville,
FL). The images were imported into the scientic image
analysis program ImageJ version 1.3 (National Institute of
Mental Health, Bethesda, MD) running on an Apple
Macintosh OSX computer system. Each image was calibrated by measuring the number of pixels per unit length
of the photomacrographic scale. Image resolution was typically 10 pixel/mm. Slices from each ablation were manually segmented using the border of normal liver tissue
versus tissue with macroscopically visible changes, and
the largest diameter of the ablation was determined.

FIG. 2. a: Temperature distribution in rabbit liver during and after

laser ablation. Laser energy was applied in three repeated cycles,
each of 3 min. The distance between each temperature sensor was
3 mm. The horizontal dashed line indicates the transition temperature of 57C for the MRI agent used. Temperature has reached 37C
in all sensors 15 min after the ablation. b: Temperature distribution
in rabbit liver during and after radiofrequency ablation. The distance
between each temperature sensor was 3 mm. The horizontal
dashed line indicates the transition temperature of 57C for the MRI
agent used. Temperature reached 37C in all sensors 15 min after
the ablation.

Statistical Analysis
SPSS for Mac OSX version 11.0.2 (SPSS, Chicago, IL) was
used for statistical analysis. Analysis of the normalized
signal intensity of the ablations was performed based on a
variable component model, with animal as random component and lesion type and relative time as xed components. This statistical model accounts for dependency in
observations from the same animal. Post hoc tests were
based on Scheffes method. Paired samples t test was used
for analysis of signicance of the observed signal intensity
alterations of the agent unrelated to heating.
RESULTS
In one of the rabbits in the laser treatment group only 2
ablations were made due to the anatomy of the liver. In the
remaining seven animals 1 ablation was made in each of
the three exteriorized liver lobes. Hence, 11 laser ablations
were made in four animals and 12 radiofrequency ablations were made in four animals. At the time of removal of
the liver, macroscopic lesions were identiable for all
attempted ablations. Energy deposited during each radiofrequency ablation was typically 10,500 J. Examples of
temperature distribution during laser and radiofrequency

ablation are shown in Fig. 2a and b. During laser ablation,

temperatures in close proximity to the laser applicator tip
exceeded the 150C calibration limit of the temperature
sensor for several of the ablations. With the radiofrequency
system, the highest registered temperature was 104C. A
temperature above 57C was registered by at least one of
the temperature sensors in the rst 3 min of passive cooling following the treatment. During scanning in this thermal imaging window, increased signal intensity of the
areas with a temperature above the transition temperature
of the agent could be expected. Tissue temperature returned to baseline within 10 min following termination of
the energy application.
Signal Intensity prior to Administration of the Agent
For the animals in the laser group, decreased SI was seen
for the A lesions for scans at t1 during heating (SI
3.5%, SD 6.4) followed by a signal increase (SI
12.2%, SD 2.3) at t2 when the tissue had returned to
baseline temperature. On scan t3 1520 min after the tissue had reached baseline temperature, no further discernable increase in SI (SI 13.0%, SD 5.7) was seen (Fig.

1306

Frich et al.

3a). A similar pattern was seen for the A lesions in the

animals in the radiofrequency group, where scans at t1
showed decreased SI (SI -6.9%, SD 8), followed by a
signal increase at t2 (SI 4.7%, SD 10.5) and t3 (SI
9.1%, SD 7.9) (Fig. 3b).
Signal Intensity after Administration of the Agent

FIG. 3. a: Mean relative signal intensity on T1-weighted images

during and after laser ablation of four rabbit livers. A lesions were
made prior to injection of the agent. B lesions were made 1214 min
after injection of the agent and C lesions 50 55 min after injection of
the agent. Relative time is indicated on the horizontal axis; during
heating (t1), after the tissue temperature is normalized (t2), and
1520 min after normalization of tissue temperature (t3). The value
of t1 is the average of three scans made during application of laser
energy. Intensity measurement at t3 is not available for C lesions. b:
Mean relative signal intensity on T1-weighted images during and
after radiofrequency ablation of four rabbit livers. A lesions were
made prior to injection of the agent. B lesions were made 1214 min
after injection of the agent and C lesions 50 55 min after injection of
the agent. Relative time is indicated on the horizontal axis; during
heating (t1), after the tissue temperature is normalized (t2), and
1520 min after normalization of tissue temperature (t3). Intensity
measurement at t3 is not available for C lesions.

The change in signal intensity prior to and 3 min after iv

injection of the contrast agent unrelated to heating was not
statistically signicant (SI 1.4%, SD 4.5, P 0.35).
For animals in the laser group a slight increase in signal
intensity was measured for B lesions at t1 (SI 2.3%,
SD 6.1), followed by an increase at t2 (SI 18.8%,
SD 20.0) and a further increase at t3 (SI 34.5%, SD
11.9). For C lesions an increase during ablation at t1 (SI
16.4%, SD 7.1) followed by a further increase at t2 (SI
31.5%, SD 8.9) was found (Fig. 3a). Test for comparisons of SI measurements found signicance at the 0.05
level between A lesions and C lesions with a P value less
than 0.05 and a condence interval (CI) of 6.9522.05. The
difference between B lesions and C lesions was also signicant (P 0.049, CI 0.0315.13). Difference between
A lesions and B lesions did not reach signicance when
performing an analysis including only SI measurements at
t1 and t2 (P 0.053, CI -0.0713.91). The results for the
animals in the radiofrequency group (Fig. 3b) were comparable to those in the laser treatment group. The B lesions
showed a minor signal increase at t1 (SI 5.4%, SD
9.5) followed by a further increase at t2 (SI 13.7%, SD
9.7) and at t3 (SI 21.6%, SD 22.7). For C lesions an
increase at t1 (SI 19.3%, SD 12.7) was found, followed by a further increase at t2 (SI 26.3%, SD 10.9).
In the radiofrequency group, test for comparisons of means
of SI found a signicance between A lesions and C lesions
with a P value less than 0.05 and a CI of 7.6727.66. The
difference between B lesions and C lesions was also signicant (P 0.048, CI 0.09 20.08). The difference between A lesions and B lesions did not reach signicance
when performing an analysis including only SI measurements at t1 and t2 (P 0.165, CI -2.4117.58). Typical
MR images of an ablation made after administration of the
agent are shown in Fig. 4. Figure 5 shows the corresponding macroscopic specimen.

FIG. 4. T1-weighted images of radiofrequency ablation (lesion B) in rabbit liver after injection of liposomal contrast. Prior to heating (t0),
during heating (t1), after normalization of tissue temperature (t2), and 1520 min after normalization of tissue temperature (t3). Note the
increasing signal intensity in the periphery of the thermal lesion in the right liver lobe (white arrows). The radiofrequency electrode has been
repositioned in the left liver lobe at t3 (white arrowhead).

Thermosensitive Paramagnetic Liposomes

FIG. 5. Macrophotograph of the ablated liver tissue depicted in

Fig. 4.

Diameter of Ablations on MRI and Macroscopic

Specimens
The largest diameter of the ablations perpendicular to the
probe track was measured from the photographs of the
macroscopic specimens by one of the investigators (LF).
The mean diameter of the 11 laser ablations was 16.3 mm
(SD 1.2; 14 18 mm). The corresponding gure for the 12
radiofrequency ablations was 24.3 mm (SD 3.8; 18
32 mm). Another investigator (AB) blinded for the results
of the analysis of the macroscopic specimens measured the
largest diameter for all ablations from the MR images at t2.
For the 11 laser ablations the mean and SD of the difference between the diameter as measured from the photographs and the MR images were 1.7 1.3 mm. For 3 of the
12 radiofrequency ablations, a reliable measurement of the
largest diameter could not be established from the MR
images. For the 9 radiofrequency ablations where a diameter could be determined, the corresponding gures were
0.3 3.4 mm. Figure 6 shows a scatterplot of the diameter of the ablations as measured from the MR images and
the photographs of the specimens.

1307

mal GdDTPA-BMA contributed to the alterations in signal

intensity on T1-w images observed in the present study.
Additionally, a direct thermal T1-effect was present for
scans in heated tissue. The correlation between tissue
T1-relaxation time and temperature is complex and nonlinear if one considers the entire temperature range investigated in the present study (37 to 150C) (26,29). In the
temperature range of 37 45C, a linear increase in T1 with
temperature is expected (14,30). At higher temperatures, coagulation and tissue denaturation occur, leading to nonlinear
and irreversible T1 changes, and the T1-temperature correlation becomes complex and tissue dependent (29,31).
Alterations of T1 related to the ablation and a direct
thermal T1-effect contributed to the observed signal intensity for A lesions made prior to injection of the liposomal
agent. A lesions therefore provided a reference that facilitated quantication of T1-changes that could be attributed
to the T1-effect of the liposomal agent for subsequent B and
C lesions. Signal intensity of A lesions decreased to 4 7%
below baseline in both the laser and the radiofrequency
groups on scans at t1 performed in heated tissue. On scans
at t2 after normalization of the tissue temperature, SI
increased 512% above baseline. These ndings are in
accordance with previous results from laser ablation of
rabbit liver (32). No further increase in SI was seen for A
lesions at t3, 1520 min after normalization of the tissue
temperature. The slope between t1 and t2 can possibly be
explained by the direct thermal T1-effect causing a net
increase in T1 for scans acquired in heated tissue at t1. In
scans at normalized temperature at t2, the direct thermal
T1-effect is no longer present and the observed 512%
increase in signal intensity at t2 can be attributed to structural and biologic tissue changes related to the ablation.
Intravenous injection of the liposomal agent in tissue with
normalized temperature unrelated to heating did not cause
a general increase in signal intensity, conrming that the
T1-relaxation enhancement of the agent was negligible at a
temperature well below the phase transition temperature
of the liposomal membrane (Fig. 1). For B lesions a mean
increase in SI of 25% compared to baseline was found at

DISCUSSION
Several active and passive methods for noninvasive thermometry within the human body exist, utilizing transmission or reection of microwaves, ultrasound, or MRI (25).
In the present study, we examined the feasibility of the
liposome encapsulated paramagnetic agent gadodiamide
(GdDTPA-BMA) as a thermosensitive probe during MRIguided laser ablation and radiofrequency ablation of rabbit
liver in vivo.
Local application of heat in tissues leads to alternations
in metabolic, macromolecular, extracellular, and vascular
parameters (26). Irreversible cellular damage is a function
of temperature and exposure time and is dependent on the
type of tissue. Denaturation of proteins occurs after exposure for temperature above 50 55C and leads to macroscopically visible tissue changes in liver tissue. Tissue
becomes desiccated when reaching 90 100C and carbonized when temperatures exceed 150C (5,27,28). Reversible and irreversible structural and biologic tissue changes
related to ablation (26) as well as the inuence of liposo-

FIG. 6. Largest diameter of 11 laser ablations and 9 radiofrequency

ablations measured from macroscopic specimens and T1-weighted
MR images.

1308

t1. The signal intensity for B lesions increased to 14 19%

at t2 and further to 2235% at t3. The nonreversible contrast enhancement after cooling could be explained by a
leakage of GdDTPA-BMA from the liposome interior and
subsequent entrapment due to coagulation of vessels. The
increased signal intensity stabilized and was present on
scans performed more than 60 min after termination of the
thermal treatment. Signal intensity for C lesions was signicantly increased to 16 19% at t1 with a further increase to 26 32% at t2. The magnitude of the normalized
signal intensity enhancements for B and C lesions was comparable to that found in a recent study of focused ultrasound
ablation of rabbit liver using the same liposomal agent (23).
The slope between t1 and t2 observed for B and C lesions was
comparable to the slope between t1 and t2 observed for A
lesions, suggesting that the considerably lower SI seen during
scanning in heated tissue at t1 compared to t2 for B and C
lesions was primarily caused by two counteracting processes
taking place during heating. The alterations of tissue parameters induced by the thermal treatment and the direct thermal T1-effect caused a net increase of tissue T1 as seen on t1
scans of A lesions, whereas the activated liposomal agent
decreased T1 of the tissue.
Increased signal intensity by a constant offset was observed in C lesions compared to B lesions and in B lesions
compared to A lesions in both the laser group and the radiofrequency group. Conventional liposomes are rapidly opsonized in blood after iv injection and taken up by the mononuclear phagocyte system (MPS) with the liver as a major
organ. For a liposome size of 110 nm as used in this study,
Kuppfer cells are responsible for the liposome uptake in
liver, but a minor uptake by the hepatocytes is also to be
expected. This intracellular uptake increases with time
postinjection and reaches a plateau, with the latter dependent on factors such as liposome size and coating (33). Biodistribution studies of the current liposome formulation in
rabbits using the same dosage as used in the present study
show a rapid blood clearance with only 35% of the injected
dose remaining in the blood 5 min postinjection. The liver
uptake at 3 h postinjection was about 40%, a typical gure
for the liposome size and membrane investigated (unpublished data). The offset between B lesions made 1214 min
after administration of the agent and C lesions made 50
55 min after administration of the agent may therefore be
explained by a time-dependent hepatic intracellular uptake
of the liposomal agent, where the intracellular concentration
of GdDTPA-BMA was higher at the time of creation of C
lesions compared to B lesions.
An increase in signal intensity was observed for B lesions
at t3 compared to t2 in both in the laser and the radiofrequency group (Fig. 3a and b). A corresponding slope between
t2 and t3 was not present for A lesions. The reason for this
delayed enhancement is not established. However, delayed
focal signal increase can be seen on T1-w images in infarcted
myocardial tissue following injection of extracellular gadolinium agents (34). This nding is believed to be caused by
loss of integrity of the cell membranes in the infarcted tissue,
with a subsequent local increase of the distribution volume
of the agent. A hypothesis could be made that a similar local
increase in the distribution volume occurs in liver tissue
exposed to thermal ablation, giving rise to a time-dependent
accumulation of GdDTPA-BMA released from the liposome

Frich et al.

interior into the surrounding tissue at tissue temperatures

above Tm.
The correlation between the largest diameters of the ablations as measured from the MR images and the macroscopic
specimens was not comparable to that found in a study
examining the correlation between GdDTPA-enhanced MRI
and histomorphologic ndings of laser ablation in rabbit liver
(35). Several factors might contribute to this inconsistency.
The contrast agent used in this study enhances the ablation
site, and not the surrounding normal liver tissue, as is the
cause for conventional MRI liver contrast agents. Irreversible
tissue damage is dependent on both the temperature and the
exposure time. The temperature needed for irreversible tissue damage with an exposure time of 9 minutes as used in
this study might be lower than the 57C temperature threshold of the liposomal agent (5). Hence, irreversible tissue
damage could occur in areas exposed to a temperature below
the transition temperature of the liposomal agent. Interestingly, the diameters measured from the laser specimens were
larger than those measured from the MR images for all ablations except for one, suggesting that enhancement is only
present in limited areas within the thermal lesion. For the
radiofrequency ablations, which were larger and more irregular than the laser ablations, the correlation was poorer.
During radiofrequency ablation, saline was introduced at the
ablation site. Saline is associated with increasing T1-relaxation times and could cause a decrease in signal intensity on
T1-w images. Additionally, the presence of saline might lead
to local inhomogeneities in the concentration of the liposomal agent. The lack of correlation between images and specimens could also partly be explained by methodological limitations. For example, even if care was taken to retain the
orientation of the ablated area, slicing of the liver specimens
were not necessarily identical to the plane of the axial T1-w
images.
This experimental study was performed in a small animal model subject to several limitations. Findings in rabbit
liver tissue are not necessarily transferable to those in a
human liver. Furthermore, all treatments were conducted
in normal liver parenchyma. Vascular divergence between
intratumoral and peritumoral blood ow and surrounding
normal liver (36) could lead to signicant inhomogeneous
distribution of the contrast agent of possible importance.
In our study, the temperature-sensitive liposomal agent
was not compared to other established MRI contrast-enhanced methods. Compared to our A lesions, conspicuity
can probably be improved by conventional MRI contrast
agents (37) or by using other imaging modalities such as
contrast-enhanced ultrasound (38). However, consistent
results were found that conrm the ability of the liposomal
agent to differentiate between tissue exposed to the thermal treatment and normal liver tissue.
In conclusion, a signicant nonreversible increase in
signal intensity was found for B and C lesions made after
injection of the liposomal agent compared to control A
lesions in both the laser ablation group and the radiofrequency group. The persistent signal enhancement in areas
exposed to a temperature above the threshold temperature
may be of clinical value during MRI-guided thermal ablation, especially for local heating modalities, such as radiofrequency ablation, where other MRI-based thermometry
methods are not feasible.

Thermosensitive Paramagnetic Liposomes

ACKNOWLEDGMENTS
The authors thank Unni Nordby Wiggen and Astrid Rogstad, Amersham Health AS, for the preparation and characterization of the liposomes. Thore Egeland, Rikshospitalet University Hospital, provided statistical assistance. We
thank the staff at the Interventional Centre at Rikshospitalet for providing excellent assistance. We also thank the
Department of Comparative Medicine at Rikshospitalet for
support in animal care.
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III

Eur Radiol (2006) 16: 19901999

DOI 10.1007/s00330-006-0177-6

Lars Frich
Per Kristian Hol
Sumit Roy
Tom Mala
Bjrn Edwin
Ole Petter F. Clausen
Ivar P. Gladhaug

Received: 8 December 2005

Revised: 19 January 2006
Accepted: 20 January 2006
Published online: 16 March 2006
# Springer-Verlag 2006
L. Frich . P. K. Hol . S. Roy . B. Edwin
The Interventional Centre,
Rikshospitalet University Hospital,
0027 Oslo, Norway
L. Frich (*) . T. Mala .
B. Edwin . I. P. Gladhaug
Department of Surgery,
Rikshospitalet University Hospital,
0027 Oslo, Norway
e-mail: lars.frich@labmed.uio.no
Tel.: +47-230-71825
Fax: +47-230-72267
O. P. F. Clausen
Department and Institute of Pathology,
Rikshospitalet University Hospital,
0027 Oslo, Norway
I. P. Gladhaug
Department of Surgery,
Faculty Division Rikshospitalet,
University of Oslo,
0027 Oslo, Norway

EXPERIME NTAL

Experimental hepatic radiofrequency ablation

using wet electrodes: electrode-to-vessel
distance is a significant predictor for delayed
portal vein thrombosis

Abstract The aim of this study was to

examine possible explanatory
variables associated with acute and
delayed portal vein thrombosis after
hepatic radiofrequency (RF) ablation
using wet electrodes. Coagulations
were created within 1.5 cm of the right
portal vein (RPV) branch in 12 pigs
with (n=6) or without (n=6) Pringle
manoeuvre. Sham operations with
Pringle manoeuvre were performed in
four animals. Rotational portal
venography was performed prior to
ablation, 10 min after ablation and 4
days after ablation. Vessel diameters
and vessel patency was determined
from the portal venograms. Distance
between the ablation electrode and
RPV was measured from 3-dimensional reconstructions of the portal
venograms. The portal veins were
examined by microscopy. Delayed
portal vein thrombosis was found in
two of six animals in the Pringle group
and three of six animals in the control

Introduction
Hepatic resection may be contraindicated in patients with
tumours located close to central hepatic vessels, if resection
of these vessels is associated with unacceptable risk of
hepatic insufficiency due to extensive loss of functional liver
parenchyma. For cirrhotic patients with a deficit in functional
hepatic reserve this risk is greatly increased [1]. Radiofrequency (RF) ablation minimises loss of functional liver
parenchyma and is a treatment option for patients with nonresectable malignant liver tumours [25]. Treatment of perivascular tumours not amenable to resection is a potentially
important clinical application for hepatic RF ablation.

group 4 days after ablation (P=1.0,

Fishers exact test). All five occurrences of delayed portal vein thrombosis were found in the six animals
with a distance between the ablation
electrode and RPV of 5 mm or less
(P=0.030), indicating that the
electrode-to-vessel distance may be an
independent explanatory factor for
delayed portal vein thrombosis after
RF ablation with wet electrodes.
Keywords Radiofrequency (RF)
ablation . Portal vein thrombosis .
Complication . Experimental
animal study

Large vessels in the proximity of tumours treated with

RF ablation are a predictor of incomplete tumour destruction and, consequently, local tumour progression [68].
Ablation of perivascular tumours, i.e. tumours contiguous
with a vessel with a diameter of 3 mm or more, is restricted
by the heat-sink effect, where the region to be coagulated is
cooled by blood flow in large vessels [9, 10]. Occlusion of
hepatic vascular inflow, by clamping of the portal vein and
the hepatic artery (Pringle manoeuvre), can be expected to
decrease the heat-sink effect and reduce perfusion of the
hepatic parenchyma. Use of the Pringle manoeuvre is
associated with increased energy efficiency [11] and
increased ablation volume [9, 12]. Additionally, hepatic

1991

vascular occlusion enhances coagulation necrosis close to

hepatic vessels [6] and may be advantageous for ablation of
perivascular tumours [8]. However, vascular inflow occlusion may make hepatic vessels more vulnerable to thermal
injury and subsequent thrombosis [13]. Portal vein thrombosis related to RF ablation may lead to greater loss of
functional liver parenchyma than anticipated, increasing
the risk of postoperative hepatic insufficiency.
Risk factors associated with occurrence of portal vein
thrombosis following RF ablation have not been fully
established. The aim of this experimental study was to
examine possible explanatory variables associated with
acute and delayed portal vein thrombosis after RF ablation
in proximity to the right portal vein (RPV) branch in
porcine liver. Specifically, the impact of performing hepatic
vascular occlusion, and the distance between the ablation
electrode and RPV was investigated.

Materials and methods

Study design
The protocol was approved by the institutional Department
of Comparative Medicine. Sixteen domestic pigs with a
mean weight of 30.7 kg (SD=3.1) were included in the
study. Twelve animals were randomly allocated to two
experimental groups. In one group Pringle manoeuvre was
applied during the ablation (n=6). In the control group, RF
ablation was performed without Pringle manoeuvre (n=6).
Four animals were assigned to a sham group where Pringle
manoeuvre, but no RF ablation, was performed. Patency of
portal vessels in all groups was determined by rotational
portal venography prior to ablation, 10 min after ablation
and 4 days after ablation. The portal veins were examined
by microscopy after euthanasia.
Radiofrequency equipment
An impedance-controlled RF ablation system (Elektrotom
HiTT 106, Berchtold GmbH & Co, Germany) was used for
creating coagulations. The system incorporated a generator
that delivered alternating current at 375 kHz to a maximum
of 1.2 A. Power output of the generator could be set
between 5 W and 60 W. The actual power output was
adjusted by the generator in accordance with the impedance of the patient circuit. Tissue impedance and cumulative deployed energy were displayed in real-time. The
active radio-opaque electrode was 1.7 mm in diameter,
with a 15 cm long shaft incorporating a lumen for perfusion
of saline solution. The terminal non-insulated segment of
the electrode was 15 mm in length. A syringe pump (Pilot
C, Fresenius Vial, France) was used for infusion of isotonic
saline solution (0.9% NaCl) through the electrode. The
saline solution was distributed to the tissue via three pairs

of two side holes in the electrode tip, placed at a 120 angle

from each other. The pump was connected by a RS-232
serial port to the RF generator. The infusion rate of saline
solution was determined by the pre-selected power output
of the generator and the circuit impedance. Under a power
setting of 50 W, saline solution was infused at a rate of
105 ml/h, which corresponded to a volume of 16 ml during
9 min ablation. If an upper impedance threshold of 900
was reached, five-times the value of the nominal flow was
infused for 1.2 s. Radiofrequency energy was applied for
1 min with an effect of 30 W, immediately followed by
8 min with 50 W. Maximum cumulated electrical energy
generated during one treatment cycle with this protocol
was 25,800 J. A new electrode was used for each animal.
Angiography system
A floor mounted C-arm based angiography system
(Angiostar O.R., Siemens, Erlangen, Germany) was used
for acquisition of rotational venographic images of the
portal veins. During each portal venography, 60 ml
iodixanol 320 mg I/ml (Visipaque, Amersham Health,
Oslo) was injected into the splenic vein by an injector at
5 ml/s. Starting 3 s after the injection commenced, a series
of 160 portal venograms was obtained while the C-arm
rotated through 198 over 8 s. The set of image data was
transferred to a post-processing workstation (Leonardo,
Siemens), and a 3-dimensional (3D) portal venogram was
created using a volume-rendering algorithm.
Operating procedure
After the animals had undergone premedication with
intramuscular injection of ketamine 1520 mg/kg (Ketalar,
Pfizer), atropine sulphate 1 mg (Atropin, Nycomed
Pharma) and azaperone 24 mg/kg (Stresnil, JanssenCilag), an intravenous cannula was placed in an ear vein,
and the animal was transported to a combined angiography
and operating theatre. Ampicillin 250 mg was administered
intramuscularly prior to surgery and after the surgical
procedure had been completed. General anaesthesia was
induced with intravenous administration of pentobarbital
150400 mg and morphine 1030 mg. After endotracheal
intubation, anaesthesia was maintained with isoflurane 1
1.2% in oxygen. The analgesic effect was supplemented
with morphine infusion at 10 mg/h. A thermodilution
SwanGanz catheter 7.5 F (Edwards Lifesciences, USA)
was introduced via the right external jugular vein and
advanced to the pulmonary artery for monitoring of vital
signs. A 134 cm2 dispersive plate (EZ 344-02, Berchtold,
Germany) was placed over the right hip. A midline incision
with a transverse right subcostal extension was made. The
hepatoduodenal ligament, containing the hepatic artery, the
portal vein and the bile duct, was identified, and a silicone

1992

vessel loop (Sterion, MN) was passed twice around all

structures. To minimise the risk of unintended damage to
portal vessels, we did not use surgical diathermy when
exposing the hepatoduodenal ligament. The spleen was
mobilised, and a 5 F 100 mm long introducer (Radifocus,
Terumo, Leuven, Belgium) was introduced into the distal
24 cm of the splenic vein and advanced 5 cm in an
antegrade direction for administration of intraportal contrast medium. A dilute heparin solution 2.5 IU/ml heparin
(Hepaflex, Baxter, Lessines, Belgium) was infused through
the introducer at 60 ml/h to maintain catheter patency.
The porcine intrahepatic portal vein shows a consistent
pattern of ramification into four segments with the RPV
coming off at an almost right angle to the portal arch [14].
The extrahepatic portal vein with the bifurcation to the
RPV was identified in all animals. The ablation electrode
was positioned in the anteriorposterior plane under direct
visual guidance based on the point of entry of the RPV into
the hepatic parenchyma. The electrode was inserted 2 cm
into the liver parenchyma between the portal arch and
within 1.5 cm of RPV, perpendicular to the vessel (Fig. 1).
The distance between the active tip of the ablation
electrode and RPV was determined by portal venography.
The electrode was repositioned if the active tip was not
within 1.5 cm of the RPV. When the position of the
electrode was found to be acceptable, rotational portal
venography was performed. In animals randomly allocated
to hepatic inflow occlusion, the silicone vessel loop was
tightened around the hepatoduodenal ligament 1 min prior
to application of RF energy and released immediately after
ablation had been completed for a total duration of 10 min.
In the sham group hepatic inflow was occluded for 10 min
in all animals, without insertion of an ablation electrode
into the liver. Portal flow was measured in the sham group
by a transmit time flowmeter (VeriQ, Medi-Stim ASA,
Norway) using a 12 mm flow probe (Cardiac Output,
Medi-Stim ASA).
The acute effect of the ablation on the portal veins was
assessed by portal venography 10 min after completion of
the ablation, while the RF electrode was still positioned in
the hepatic parenchyma. The electrode was removed after
completion of the portal venography. To prevent bleeding
from the needle track, we activated the electrode at 25 W
during its withdrawal. The introducer in the splenic vein
was removed, and the distal 24 cm of the splenic vein was
ligated. The SwanGanz catheter was removed, and the
right external jugular vein was ligated. Postoperatively, the
animals were kept under observation until full recovery
from anaesthesia and were allowed free access to food and
water. Postoperative pain was managed with intramuscular
injection of buprenorphine hydrochloride 0.01 mg/kg
(Temgesic, Schering-Plough, N.J., USA) twice daily for 3
days. The animals underwent repeat laparotomy 4 days
after ablation, and the delayed effect of the ablation was
evaluated by rotational portal venography as described
previously. The animals were subsequently euthanised with

Fig. 1 Anteroposterior projection of the porcine extrahepatic and

intrahepatic portal vein and the splenic vein. The radiofrequency
ablation electrode (a) was positioned 2 cm into the liver parenchyma
between the portal arch (b) and within 1.5 cm of the right portal vein
branch (c). Vessel diameter was measured along a perpendicular
from the electrode tip to the vessel wall (dashed lines). Contrast
agent was distributed to the portal vein (d) by injection through an
introducer placed in the splenic vein (e)

intravenous injection of pentobarbital 500 mg and potassium chloride 23 mmol/kg. The liver was excised
immediately after asystole had occurred.
Interpretation of portal venograms
Diameter of the portal arch and RPV was measured at the
perpendicular from the tip the ablation electrode to the
vessel on native portal venograms by image acquisition
software (Polytron T.O.P., Siemens). The 3D reconstructions of the portal venograms allowed multidirectional view
of the volume data set consisting of the radio-opaque
electrode and contrast medium-filled portal veins. The distance between the non-insulated 15 mm tip of the ablation
electrode and the portal arch and RPV was determined by
rotation of the 3D reconstruction to the projection where the
largest perpendicular distance between the tip of the electrode and the corresponding portal vein segment could be
measured. If areas relevant to the examination were hidden
by other structures, the volume data could be trimmed and a
volume of interest generated by post-editing of the volume
data set (Fig. 2). To correct for possible displacement of the
electrode relative to the vessels during ablation, we measured the electrode-to-vessel distance on portal venograms
obtained both prior to and 10 min after ablation. The mean
of these measurements was considered to be the electrodeto-vessel distance, and was used in the statistical analysis.
Measured vessel diameters and electrode-to-vessel distances
were rounded off to the nearest millimetre.

1993

Fig. 2 Three-dimensional
reconstructions of the porcine
extrahepatic and intrahepatic
portal vein before and after
radiofrequency ablation. Orientation of the volume is indicated
by the square on the lower right
of each image. a After positioning of the radiofrequency
electrode but before ablation.
The electrode (white arrow) is
positioned in proximity of the
right portal vein branch.
b Reoperation 4 days after
ablation. Note lack of contrast in
the right portal vein branch
(white arrowhead). c, d Volume
data have been trimmed to
elucidate the spatial relationship
between the electrode, the right
portal vein branch and the portal
arch

Occurrence of portal vein thrombosis was determined by

independent examination of the native 2-dimensional (2D)
portal venograms and the 3D reconstructions by a surgeon
and two experienced radiologists unaware of the group to
which the particular animal had been randomly allocated.
Agreement between the examiners was reached by
consensus. Owing to the pseudostenosis phenomenon
observed at volume-rendered 3D angiography [15], native
2D images were used to confirm abnormal findings on the
reconstructed 3D images. The splenic vein, the extrahepatic portal vein, the portal arch and the RPV were
classified as normal or abnormal. Abnormal vessels were
categorised as either having a filling defect or as being
occluded. An occluded portal vessel with lack of peripheral
filling on post-ablation portal venograms was considered to
be a portal vein thrombosis. Acute portal vein thrombosis
was defined as thrombosis on portal venograms acquired
10 min after completion of the ablation, whereas delayed
portal vein thrombosis was defined as thrombosis on portal
venograms acquired 4 days after ablation.

Histopathological examination
The portal arch was incised longitudinally, and the
presence of intraluminal thrombi and macroscopic endothelial damage was noted. The opening of the RPV was
identified. All coagulations were excised with wide
margins and cut in 46 mm slices transverse to the RPV,
fixed in 4% formaldehyde for at least 10 days, dehydrated,
and embedded in paraffin. One 45 m-thick section was
cut from each paraffin block and stained with haematoxylin
and eosin for microscopic examination by an experienced
pathologist blind to the findings on the portal venograms.
The presence of viable hepatocytes between the coagulated
area and the portal vessel wall, the degree and extent of
damage to the portal vessel wall itself, and the presence of
thrombotic material adherent to the intimal surface of the
vessel was noted.
Statistical analysis
SPSS for Mac OSX version 11.0.4 (SPSS, Chicago, Ill.,
USA) was used for statistical analyses. A P value less than
0.05 was considered statistically significant. Data are

1994

presented as mean SD unless otherwise indicated. A

sample size of six animals in each experimental group was
needed to demonstrate a significant difference between the
two groups with proportions of portal thrombosis of 0.05
and 0.95 using a two-group continuity corrected 2 test
with 80% power at the 0.05 two-sided significance level.
The animals were allocated to the two experimental groups
by block randomisation, using a block size of two.
Occurrence of delayed portal vein thrombosis in the two
experimental groups was analysed by Fishers exact test.
Independent samples t-test assuming unequal variances
was used for comparison of the two experimental groups
with respect to energy delivered during the ablation,
diameter of the portal arch, diameter of RPV, distance
between the electrode and the portal arch and distance
between the electrode and RPV. Subsequently, the animals
were regrouped on the basis of presence of delayed portal
vein thrombosis on post-ablation portal venograms, and the
same variables were compared using independent samples
t-tests. The distance between the RPV and the ablation
electrode was recoded into a binary variable with a cut-off
value of 5 mm. Fishers exact test with Bonferroni
correction for multiple tests was used for examination of
the relationship between Pringle manoeuvre and the
recoded electrode-to-vessel distance as explanatory variables, and delayed portal vein thrombosis as outcome
variable.

Results
One ablation was completed in each animal in the two
experimental groups. Reversible systemic hypotension
with a reduction of mean arterial pressure of 10 mmHg
or more during the ablation was observed in five animals in
the Pringle group and two in the control group. Increase in
tissue impedance was frequently observed during ablation
in the animals in the Pringle group.
Portal venograms acquired prior to RF ablation were
considered normal in all animals. A filling defect in the
vicinity of the electrode tip was seen 10 min after RF
ablation in one animal in each experimental group. Neither
of these two animals was considered to have an acute portal
vein thrombosis. The remaining ten portal venograms
acquired 10 min after ablation were normal. Delayed portal
vein thrombosis was found 4 days after ablation in two of
six animals in the Pringle group and three of six animals in
the control group (Table 1). Delayed portal vein thrombosis
was present in the two animals with abnormal portal
venograms 10 min after ablation, and in three animals with
normal portal venograms 10 min after ablation. Four of the
occurrences of portal vein thrombosis were located in the
RPV (Fig. 2). A fifth portal vein thrombosis was found in
one animal in the Pringle group, where the portal arch with
a diameter of 13 mm was found to be occluded. Gross
examination of the liver of this animal revealed thermal

Table 1 Acute and delayed portal vein thrombosis in the two

experimental groups
Parameter

Acute portal vein thrombosisa

Delayed portal vein thrombosisb

Pringle
group
(n=6)
0/6
2/6

Control
group
(n=6)
0/6
3/6

P*

1.0

*Fishers exact test

Ten minutes after ablation
Four days after ablation

damage to the luminal surface at the opening of the RPV,

with a thrombus extending into the portal arch. The
endothelium of the portal arch was macroscopically
normal. Diameters of the occluded RPV branches ranged
from 49 mm (5.61.9 mm). The splenic vein and the
extrahepatic portal vein were normal in all animals in the
two experimental groups.
No occurrences of thrombosis or filling defects were
found in the splenic veins, extrahepatic portal veins or
intrahepatic portal veins in the sham group 10 min after
Pringle or at follow-up 4 days later. Portal flow measurements in the sham-group animals during Pringle manoeuvre confirmed cessation of portal flow. Ligation of the
distal 24 cm of the splenic vein after removal of the
introducer used for administration of intraportal contrast
agent did not significantly decrease portal flow compared
to baseline values (88460 ml/min vs 934146 ml/min,
P=0.455).
Histopathology
A sharp demarcation between the coagulated area and
normal hepatic parenchyma was found at gross examination, corresponding to the border between viable and nonviable tissue found at histopathological examination
(Fig. 3a,b). The hepatic microstructure was preserved in
parts of the coagulated area. The cytoplasm of coagulated
hepatocytes was more eosinophilic and showed pyknotic
nuclei compared to viable hepatocytes. In other coagulated
areas, the hepatocytes had undergone karyolysis and were
denucleated. Fibroblast proliferation with a variable degree
of inflammation was prominent between the coagulation
and the portal vein in all animals. The portal vein could not
be identified in one animal in the Pringle group with
delayed portal vein thrombosis due to extensive tissue
necrosis. In the remaining four animals with delayed portal
vein thrombosis, thrombosis was confirmed by microscopy. In addition, subtotal thrombotic occlusion of the RPV
was found in one animal in the Pringle group. The
corresponding portal venogram was interpreted as normal,
and this animal was therefore not considered to have a
portal vein thrombosis in the statistical analysis.

1995

Fig. 3 Haematoxylin and eosin stained sections of hepatic radiofrequency ablation. A sharp demarcation between the coagulated
areas (RF) and normal hepatic parenchyma is present (arrowheads).
Note thrombus in portal veins in both specimens (arrows) a Pringle
manoeuvre during the ablation. The coagulation merges with the
adjacent vessel. Necrosis of the vessel wall was present in 2030%

of the circumference. b No hepatic vascular occlusion during

ablation. The coagulated area has fragmented. Note that the contour
of the coagulation has been altered by the adjacent portal vein,
illustrating the heat sink effect. Extensive inflammation of the vessel
wall was present

Viable hepatocytes were present between the vessel wall

and the coagulated area in one of five animals in the Pringle
group and in four of six in the control group. Damage to the
endothelial lining was occasionally present without thermal
damage to the adjacent hepatocytes. Necrosis of all layers
of the portal vein wall covering 20100% of the circumference was present in all five pigs in the Pringle group in
whom the portal vein could be assessed. In the control
group, necrosis of the vessel wall was seen in four of six
animals, covering 2080% of the circumference. In one
animal in the control group with portal vein thrombosis,
extensive inflammation of the portal vein was found. In the
remaining three animals where the portal vein could be
assessed, necrosis of all layers of the portal vein was
present.

Factors associated with portal vein thrombosis

Mean energy delivered during ablation was lower in the
Pringle group than in the control group (22,5602,520 J vs
25,670360 J, P=0.046). No significant differences
regarding preoperative vessel diameter or electrode-tovessel distances were found in the two experimental groups
(Table 2). Hepatic vascular occlusion was not associated
with occurrence of portal vein thrombosis (P=1.0, Fishers
exact test). The largest diameter of a thrombosed RPV with
Pringle manoeuvre was 9 mm, whereas the corresponding
vessel diameter in the control group was 5 mm. The longest
distance between the ablation electrode and a thrombosed

Table 2 Deployed energy, preoperative vessel diameters

and electrode-to-vessel distances in the two experimental groups
Parameter

Pringle
group
(n=6)
Mean SD

Control
group
occlusion(n=6)
Mean SD

Pa

Energy
22,560 2,520 J 25,270 361 J
0.046
Diameter of portal
11.3 1.5 mm
11.2 1.6 mm 0.856
arch
Diameter of right
5.8 1.6 mm
5.7 1.8 mm 0.867
portal vein branch
Distance of electrode
10.0 4.2 mm
17.2 7.4 mm 0.074
to portal arch
Distance of electrode
6.0 2.2 mm
6.8 5.6 mm 0.745
to right portal
vein branch
a

Independent samples t-test with unequal variances assumed

Fig. 4 Relationship between use of Pringle manoeuvre, applied

electrical energy, distance between the ablation electrode and right
portal vein branch and delayed portal vein thrombosis in the two
experimental groups (n=12). Occurrence of delayed portal vein
thrombosis is indicated by a filled symbol. PVT delayed portal vein
thrombosis, NOR no portal vein thrombosis, P+ Pringle manoeuvre,
P no hepatic vascular occlusion

1996

Table 3 Deployed energy, preoperative vessel diameters and

electrode-to-vessel distances, using delayed portal vein thrombosis
as the categorising variable
Parameter

Delayed portal
vein thrombosis
(n=5)
Mean SD

No delayed portal Pa
vein thrombosis
(n=7)
Mean SD

Energy
24,313 1,384 J 23,630 2751 J
Diameter of portal
11.6 1.8 mm
11.0 1.3 mm
arch
Diameter of right
5.6 1.9 mm
5.9 1.5 mm
portal vein branch
Distance of electrode
10.2 6.7 mm
16.0 6.3 mm
to portal arch
Distance of electrode
3.6 1.5 mm
8.4 4.2 mm
to right portal vein
branch
a

0.586
0.547
0.810
0.167
0.023

Independent samples t-test with unequal variances assumed

RPV with Pringle manoeuvre was 5 mm. The corresponding distance in the control group was 4 mm. The shortest
distance between the electrode and the RPV without
occurrence of portal vein thrombosis was 3 mm and was
found in an animal with a RPV diameter of 5 mm in the
Pringle group. This animal received lowest cumulated
energy of all animals (Fig. 4).
The mean distance between the ablation electrode and
the RPV was significantly shorter in the five animals that
developed delayed portal thrombosis (3.61.5 mm) than in
the seven animals with normal portal venograms (8.4
4.2 mm), with a P value of 0.023. No other significant
differences were found in these two groups (Table 3).
Delayed portal vein thrombosis occurred in five of six
animals, with a distance between the electrode and the RPV
of 5 mm or less, while no occurrences of portal vein
thrombosis were found in the six animals where this
distance was 6 mm or more (Fig. 4). A distance between
the electrode and the RPV of 5 mm or less was a significant
predictor for delayed portal vein thrombosis (P=0.030,
Fishers exact test).

Discussion
Although a non-cirrhotic liver may tolerate a resection of
up to 80%, reduction of liver parenchyma by even 50%
may be associated with postoperative hepatic insufficiency
[1]. The hepatic artery normally supplies 25% of the total
blood flow to the liver but may provide up to 3050% of
the normal oxygen requirement of the hepatic parenchyma,
because of the higher oxygen content in the arterial blood
than in the partly deoxygenated portal blood. Increased
oxygen extraction from the hepatic arterial blood and

increased arterial hepatic blood flow can minimise the

effect of a significant reduction in portal flow [16]. Owing
to these compensatory mechanisms the clinical implication
of segmental portal vein thrombosis after RF ablation could
be questioned. Although segmental portal vein thrombosis
may be asymptomatic [13], transient liver failure has been
reported after portal vein embolisation in patients with
reduced hepatic reserve [17]. Liver failure caused by portal
vein thrombosis was the most frequent cause of death in a
recent study of 312 patients with hepatic tumours treated by
radiofrequency ablation [18]. Patients with a deficit in
functional hepatic reserve may be at risk of postoperative
hepatic failure after portal vein thrombosis. Of special
clinical concern are patients in whom an extensive hepatic
resection has been performed and concomitant RF ablation
in the remnant liver is planned [18]. Determination of risk
factors associated with occurrence of segmental portal vein
thrombosis related to RF ablation may, therefore, be of
value for selecting patients suitable for RF ablation.
Use of the Pringle manoeuvre was not associated with
increased risk of delayed portal vein thrombosis in this
experimental randomised study. Histopathological examination confirmed necrosis of the portal vein wall in all pigs
in the Pringle group where the integrity of the portal vein
wall could be assessed. Viable hepatocytes between the
vessel wall and the coagulated area were present in only
one of five animals in the Pringle group, compared with
four of six in the control group. These findings suggest that
hepatic vascular occlusion during RF ablation enhances
cellular devitalisation close to portal vessels and confirm
the increased risk of thermal damage to vessels during
hepatic inflow occlusion found in previous experimental
studies [19, 20]. Nevertheless, this did not translate into a
higher incidence of acute or delayed portal vein thrombosis
in this study. Even though the distribution of all variables
under consideration, except energy, was similar in the two
experimental groups, as expected from randomisation
(Table 2), these figures are derived from a small data set
of five occurrences of delayed portal vein thrombosis in 12
animals and should be interpreted with caution. Hence, our
finding that the Pringle manoeuvre was not associated with
acute or delayed portal vein thrombosis may be explained
by the limited sample size.
Less energy was delivered during ablation in the Pringle
group than in the control group. This finding can be
explained by the fact that the impedance-controlled
radiofrequency system employed in this study reduced
power output if tissue impedance increased during ablation. The reduced or absent hepatic blood flow caused by
the Pringle manoeuvre can be expected to limit the
perfusion-mediated cooling of the electrode-to-tissue interface, leading to rapid desiccation and subsequent
impedance rise. As the ablation protocol used in this
study consisted of a predefined time interval of 9 min and a
fixed power setting of 50 W, reduced power output resulted
in lower accumulated energy output. The importance of the

1997

lower mean energy deposited in the Pringle group is

unclear, as no statistical significant difference was found
when we compared deployed cumulated energy in the
animals with and without delayed portal vein thrombosis
(Table 3).
A recent study by Ng et. al. reported that use of the
Pringle manoeuvre during RF ablation invariably resulted
in delayed portal vein thrombosis, whereas no such risk
was present for ablation without hepatic vascular occlusion
[20]. In our study, delayed portal vein thrombosis occurred
only in two of six animals in the Pringle group. Perhaps of
more importance was that three of six animals in the control
group developed delayed portal vein thrombosis. These
apparently diverging results may have several explanations. In the study by Ng et. al., the distance between the
vessel and the electrode was stated to be 5 mm in all
animals. Our results suggest that the distance between
electrode and vessel may be of significant importance for
development of delayed portal thrombosis. The RF ablation systems used in the two studies were different. In the
study by Ng et. al., an internally cooled electrode with a
non-insulated tip of 30 mm was used, whereas we used a
wet ablation electrode design with a non-insulated tip of
15 mm. The generator and generator setting used by Ng et.
al. was not specified, but the electrodes used in the study
are usually connected to a generator that can deliver up to
200 W, compared to the 50 W used in our study. The
duration of ablation (12 min vs 9 min) also varied. In the
study by Ng et. al. the presence of delayed portal vein
thrombosis was investigated 1 week after ablation. Histopathological examination revealed complete necrosis of the
portal vein wall in the majority of the animals 4 days after
the ablation in our study. We cannot exclude that the
proportion of portal vein thrombosis could have been
higher if our animals had been examined at 7 days after
ablation compared to 4 days after ablation. Other dissimilar
aspects are the methods used for assessment of portal vein
thrombosis. The study by Ng et al. measured portal flow by
means of intraoperative Doppler ultrasonography, while
repeat portal venography was used for determination of
portal vein thrombosis in our study. The use of portal
venography was based on the assumption that venography
would facilitate objective detection of segmental portal
vein thrombosis that might go unnoticed when alterations
of flow velocity at the portal trunk were measured.
The approach used in our study involved exposing portal
endothelium injured by ablation to a non-ionic contrast
agent and ligation of the distal 24 cm of the splenic vein.
Both the use of intraportal contrast medium and ligation of
the distal splenic vein could be potential influential factors
for portal vein thrombosis, especially in the group without
Pringle manoeuvre. Even though the non-ionic contrast
agent used in this study is associated with a low incidence
of thrombus-related events [21], the safety profile of the

agent has not been evaluated for porcine portal endothelium damaged by thermal ablation. We cannot, therefore,
completely rule out the possibility that use of this contrast
agent in the presence of portal endothelium damaged by
thermal ablation could lead to formation of thrombi that
may have influenced the results. Significantly reduced
portal vein flow caused by decreased inflow from the
splenic vein could be a contributing factor for intrahepatic
portal thrombosis. However, flow measurements in the
sham group showed that ligation of the distal splenic vein
was associated with a statistically not significant reduction
of portal flow of 5%. It is, therefore, not likely that reduced
portal flow due to distal splenic vein ligation was a
confounding factor for the development of portal thrombosis. The distal splenic vein is a possible source of emboli
to intrahepatic portal veins. However, filling defects were
not observed in the splenic veins or extrahepatic portal
veins on any portal venogram. Furthermore, thrombosis
developed in the RPV, which comes off at almost a right
angle to the portal arch and is not a probable site of
thrombus formation caused by distant emboli.
The absence of acute portal vein thrombosis following
RF ablation in proximity to portal veins has been
established by previous studies [20, 22]. Only two of the
five animals with delayed portal vein thrombosis 4 days
after ablation had abnormal findings on the portal
venograms 10 min after ablation. Hence, a normal portal
venography obtained 10 min after ablation did not exclude
later occurrence of thrombosis, whereas an abnormal portal
venography 10 min after ablation was associated with
delayed portal vein thrombosis. Thrombus formation
related to RF ablation is believed to be initiated by
endothelial cell injury with interruption of endothelial
continuity, leading to platelet adhesion and activation as
well as thrombin production [23]. In the present study,
histopathology confirmed that portal vein thrombosis was
invariably accompanied by transmural necrosis of the
portal wall or extensive inflammation of the portal
endothelium. This suggests that acute endothelial damage
caused by the ablation represents a risk for subsequent
thrombosis of the vessel.
Our finding of an electrode-to-vessel distance of 5 mm
or less as a significant predictor of portal thrombosis is
biologically plausible. The relationship between factors
that influence tissue temperature after deposition of energy
is described by the bioheat equation [24]. According to
this, an increase in tissue temperature is dependent on the
energy deposited in the tissue, modified by local tissue
interactions, minus the heat loss [25]. In accordance with
the bioheat equation, our findings suggest that the critical
distance from the electrode at which portal vessels are at
risk of thrombosis may be increased if perfusion-mediated
heat loss is minimised due to hepatic vascular occlusion.
The tissue power density Wv (watts/cubic metres) is an

1998

expression of the electrical energy per time unit that tissue

is exposed to under ideal conditions:
Wv V 2

a2
r4

where is the tissue conductivity, V the voltage, a the

radius of the electrode, and r the distance from the
electrode. Thus, the power density can be expected to fall
off extremely rapidly with increasing distance from the
electrode, as it is inversely proportional to the distance
from the electrode to the fourth power [26]. The RF
ablation system used in this study infuses saline solution to
the tissue through the active electrode during ablation.
Although the effect of saline infusion has not been
completely elucidated, the conductive properties of saline
solution are believed to increase the effective radius of the
electrode [27] and thereby increase power density at a
given distance from the electrode. The magnitude of this
effect has not been quantified for the RF ablation system
used in this study.
From an electrical point of view, the liver is inhomogeneous, with large variations in tissue conductivity. Consequently, the flow pattern of the electrical current around the
electrode is determined by the tissue morphology and
composition. The temperature distribution is also related to
non-electrical tissue factors, such as the amount of heat loss
by conduction. The complexity of the association between
these factors and the resulting thermal tissue damage is
demonstrated by the fact that size and shape of the
coagulated volume cannot be predicted, even under
standardised ablation protocols [28]. Although the relationship between these factors and the resulting tissue temperature is not a trivial one, it seems reasonable to assume that,
when the electrode-to-vessel distance is below a critical
limit, the vessel wall will be exposed to temperatures high
enough to cause endothelial damage. Factors related to the
specific RF ablation equipment used, such as the generator
output, size and shape of the active electrode and the
presence of internal or external cooling of the electrode,
influence the energy efficacy [11] and the extent of the
coagulation [29]. The RF ablation system used in this study
has been shown to produce irregular coagulations when
compared with other RF ablation systems [29, 30].
Therefore, our finding of an electrode-to-vessel distance
of 5 mm or less as being associated with delayed portal
vein thrombosis is only valid for the specific RF ablation
system used in this study.
Several limitations of this study must be addressed. First,
findings after experimental RF ablation in normal porcine

liver are not necessarily applicable to clinical settings. The

pig has a coagulation pathway similar to that of humans,
but it is considered to be more susceptible to activation of
the coagulation system following endothelial damage [31].
Hence, thermal ablation and endothelial damage causing
thrombosis in pigs would not necessarily lead to portal
thrombosis in humans. Secondly, for the prevention of
clotting in the introducer in the splenic vein, heparin at
150 IU/h was administered through the introducer between
the contrast examinations during the 12 h procedure. The
accumulated heparin dose used in our study was less than
5% of the 100150 IU/kg per hour considered a normal
dose for surgical procedures in the pig [31]. We consider it
unlikely that administration of heparin at 150 IU/h into the
splenic vein would have influenced the occurrence of
portal vein thrombosis or its natural course. Reversible
systemic hypotension with a reduction of mean arterial
pressure of 10 mmHg or more during ablation was
observed in both experimental groups during ablation.
Consequently, reduced hepatic blood flow may have
occurred even in the control group. This study was
designed to detect thrombosis in the portal veins. Concurrent thrombosis of segmental hepatic arteries would be
expected to increase the loss of hepatic function but was
not assessed in this study. Finally, the experiments were
carried out in an animal model with an adequate hepatic
reserve, which is unsuitable for demonstration of altered
hepatic function after segmental portal vein thrombosis.
In conclusion, delayed portal vein thrombosis associated
with hepatic RF ablation close to portal veins occurred in
both the Pringle and the control group. Use of the Pringle
manoeuvre during RF ablation was not associated with a
higher risk of delayed portal thrombosis. On the other
hand, our findings indicate that the Pringle manoeuvre
rendered portal veins more susceptible to thermal damage.
A distance between the ablation electrode and the RPV of
5 mm or less was significantly associated with development of delayed portal vein thrombosis after RF ablation
with wet electrodes. Further research is warranted to
examine the role of the electrode-to-vessel distance in the
development of acute or delayed portal vein thrombosis for
other hepatic RF ablation systems, and to assess alterations
of hepatic function associated with segmental portal vein
thrombosis.
Acknowledgements We thank the staff at the Interventional Centre
for providing excellent assistance, and the Department of Comparative Medicine for support in animal care. Marijke Veenstra and Marte
Olstad at the Institute of Biostatistics provided statistical assistance.
This work was supported by the Norwegian Cancer Society, grant no.
D02042/001.

1999

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IV

Journal of Surgical Research 135, 297304 (2006)

doi:10.1016/j.jss.2006.05.010

Increased Activity of Matrix Metalloproteinase 2 and 9 After Hepatic

Radiofrequency Ablation
Lars Frich, M.D.,*,,1 Kristin Bjrnland, M.D., Ph.D.,,, Solveig Pettersen, B.S.,
Ole Petter F. Clausen, M.D., Ph.D., and Ivar P. Gladhaug, M.D., Ph.D.,
*The Interventional Centre, Department of Surgery, Institute for Surgical Research, Department and Institute of Pathology,
Rikshospitalet University Hospital; and Department of Surgery, Faculty Division Rikshospitalet, University of Oslo, Oslo, Norway
Submitted for publication December 22, 2005

Background. Radiofrequency (RF) ablation of hepatic metastases from colorectal cancer (CRC) is associated with a high rate of local and intrahepatic tumor
recurrence. Matrix metalloproteinases (MMPs) play
an important role in inammation, tissue repair and
tumor cell invasion and metastasis. MMP-2 and MMP-9
are associated with increased risk of recurrence and
decreased survival in patients with colorectal cancer.
The primary aim of the study was to determine if hepatic RF ablation increased MMP-2 and MMP-9 activity in the transition zone surrounding the coagulated
hepatic tissue.
Materials and methods. Twelve pigs were randomized to hepatic RF ablation with (n 6) or without
(n 6) hepatic vascular occlusion (Pringle maneuver).
Four days after ablation tissue specimens were collected from the transition zone surrounding coagulated hepatic tissue, and from normal hepatic parenchyma. MMP activity was quantied by gelatin
zymography. Cellular localization of MMPs was determined by immunohistochemistry using antibodies
against MMP-2, MMP-9, and the macrophage marker
CD68.
Results. MMP-2 and MMP-9 activity was increased in
the transition zone compared to normal hepatic parenchyma, with ratios of 3.0 (P 0.005) and 2.6 (P
0.001), respectively. Pringle maneuver did not inuence MMP activity. MMP-2 and MMP-9 expression was
localized to macrophages in the transition zone.
Conclusions. Hepatic RF ablation is associated with
increased expression of MMP-2 and MMP-9 in macrophages in the transition zone surrounding the coagulated hepatic parenchyma. These ndings may contrib1
To whom correspondence and reprint requests should be addressed at Department of Surgery, Rikshospitalet University Hospital, N-0027 Oslo, Norway. E-mail: lars.frich@labmed.uio.no.

ute to the understanding of possible mechanisms for the

high recurrence rates observed in patients after RF ablation of CRC hepatic metastases. 2006 Elsevier Inc. All rights
reserved.

Key Words: matrix metalloproteinases; radiofrequency

ablation; liver; neoplasm; animal experimentation.
INTRODUCTION

Hepatic resection is the gold standard for treatment

of liver metastases from colorectal cancer (CRC), with
5-year survival rates of approximately 30% in selected patients [1, 2]. The majority of patients with
hepatic metastases from CRC are not candidates for
potential curative hepatic resection because of bilobar
tumor location, underlying hepatic disease or extrahepatic tumor growth. Radiofrequency (RF) ablation is
increasingly used for in situ tumor destruction in patients with non-resectable metastases limited to the
liver [3]. Focal tissue necrosis is achieved by applying
alternating electrical current within a frequency range
of 400 to 500 kHz to an active electrode positioned in
the target tumor. At temperatures above 55C, protein
denaturation and cell death occur. Heating of tissue is
associated with increased tissue impedance and charring which limit the coagulation necrosis to 3 to 5 cm
diameter [4]. Furthermore, in vivo the extent of tissue
coagulation is restricted by the heat-sink effect, where
the region to be coagulated is cooled by blood ow in
large vessels [5, 6]. Increased coagulation volume can
be achieved by reduction of hepatic perfusion during
ablation by temporary mechanical occlusion of the portal vein and the hepatic artery (Pringle maneuver)
[79].
Patients with CRC liver metastases treated with
hepatic RF ablation have higher local, intrahepatic,

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2006 Elsevier Inc. All rights reserved.

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JOURNAL OF SURGICAL RESEARCH: VOL. 135, NO. 2, OCTOBER 2006

and distant recurrence rates, as well as lower overall

survival than comparable resected patients [10]. A
plausible explanation for these ndings is negative
patient selection bias, with micrometastatic disease
being present in a larger proportion of patients treated
with RF ablation [11]. An alternative explanation that
warrants further investigation is that thermal ablation
of malignant tumors may induce biological alterations
in the tumor tissue or peritumoral hepatic parenchyma
that facilitate spread and invasion of malignant cells
[12, 13].
Matrix metalloproteinases (MMPs) are a family of
zinc-dependent matrix-degrading endopeptidases involved in a variety of physiological and pathological
events [14 16]. MMPs are associated with inammation and tissue repair and play an important role in the
normal physiological turnover of the extracellular matrix (ECM). Increased expression of MMPs is necessary
for tumor cell invasion, metastasis and angiogenesis.
The gelatinases MMP-2 and MMP-9 degrade type IV
collagen, which is a major component of the basement
membrane. Disruption of the basement membrane allows tumors to spread locally and distally. MMP-2 and
MMP-9 contribute to CRC progression in experimental
models [14], are overexpressed in patients with CRC
hepatic metastasis [1719], and are associated with
increased risk of tumor recurrence and decreased survival in patients with CRC [16]. Increased MMP activity may provide a physiological mechanism of tissue
remodeling that facilitates growth and spread of malignant cells.
Our study hypothesis was that hepatic RF ablation
increases expression of MMPs and cytokines in the
transition zone separating coagulated tissue from normal hepatic parenchyma. The primary aim of the study
was to determine activity of MMP-2 and MMP-9, and
expression of the cytokines tumor necrosis factor-
(TNF-), interleukin-10 (IL-10) and prostaglandin E 2
(PGE 2) in hepatic tissue lysates from the transition
zone. Additionally, we wanted to examine if use of the
Pringle maneuver during RF ablation was associated
with alterations in MMP activity or cytokine levels.
Tissue was harvested 4 days after ablation to allow
examination of MMP activity and cytokine levels during an established inammatory process with a well
demarcated transition zone.
MATERIALS AND METHODS
Experimental Protocol
The protocol was approved by the institutional Department of
Comparative Medicine. Twelve domestic pigs with a mean weight of
30.3 kg (SD 3.4 kg) were randomized to two experimental groups.
In the rst group RF ablation was performed without occlusion of
hepatic vessels (n 6). In the second group Pringle maneuver was
performed during RF ablation (n 6).
Premedication consisted of intramuscular injection of ketamine

15 to 20 mg/kg (Ketalar; Pzer, Cambridge, MA), atropine sulfate 1

mg (Atropin; Nycomed Pharma, Rockslide, Sweden) and azaperone 2
to 4 mg/kg (Stresnil, Janssen-Cilag). Ampicillin 250 mg was administered intramuscularly before and after the surgical procedure. Surgery was performed during general anesthesia induced by intravenous pentobarbital 150 to 400 mg and morphine 10 to 30 mg and
maintained by inhaled isourane 1 to 1.2% in oxygen. The analgesic
effect was supplemented with morphine infusion at 10 mg/h. A 7.5 F
pulmonary artery catheter (CCOmbo 774HF75; Edwards Lifesciences, Irvine, CA) was introduced via the right external jugular
vein and advanced to the pulmonary artery for monitoring of vital
signs. A 134 cm2 dispersive plate (EZ 344-02; Berchtold GmbH & Co.,
Tuttlingen, Germany) was placed over the right hip. Laparatomy
was performed and a silicone vessel loop (Sterion, Minneapolis, MN)
was passed twice loosely around the hepatoduodenal ligament containing the hepatic artery, the portal vein and the bile duct.
An impedance-controlled RF ablation system (Elektrotom 106
HiTT, Berchtold GmbH & Co., Germany) was used for ablation. The
system incorporates a generator that delivers alternating current at
375 kHz to a maximum of 1.2 ampere. The active electrode is 1.7 mm
in diameter, with a 15 cm long shaft incorporating a lumen for saline
perfusion, and a terminal non-insulated segment of 15 mm. A syringe pump (Pilot C; Fresenius Vial, Grenoble, France) was used for
infusion of saline through the active electrode. The saline was distributed to tissue in the vicinity of the electrode tip through three
pairs of two side holes. The electrode was positioned 2 cm into the
liver parenchyma under visual guidance. RF energy was applied for
1 min with an effect of 30 Watts, immediately followed by 8 min with
50 Watts. In animals randomized to the Pringle group, the silicone
vessel loop was tightened around the hepatoduodenal ligament 1
min before application of RF energy and released immediately after
ablation had been completed. After ablation had been completed the
pulmonary artery catheter was removed, and the right external
jugular vein was ligated. Postoperatively, the animals were kept
under surveillance until full recovery from anesthesia and were
allowed free access to food and water. Pain was managed with
intramuscular buprenorphine hydrochloride 0.01 mg/kg (Temgesic;
Schering-Plough, Berkeley Heights, NJ) twice daily for 3 days. Four
days after ablation, the animals were sacriced by intravenous injection of pentobarbital 500 mg and potassium chloride 2 to 3 mmol/
kg, and the livers were removed.

Blood Samples
Systemic MMP activity and cytokine expression was determined
in blood samples collected at three time points as follows: baseline
(i.e., 1 h after laparotomy, but before RF ablation), 1 h after RF
ablation and 4 days after RF ablation. Blood samples were drawn
into 5 mL citrate tubes, stored on ice during the operative procedure,
and centrifuged at 1000 g for 10 min. The plasma fraction was
separated and stored at 70C until analyzed. Protein content of
each sample was measured with a Bio-Rad BCA assay.

Tissue Samples
Immediately after removal of the liver, fresh tissue cubes of 4
4 4 mm were excised from the transition zone, incorporating the
edge of the coagulated area and the surrounding hepatic parenchyma. Corresponding samples were excised from normal hepatic
parenchyma at least 5 cm from macroscopic signs of tissue coagulation. The samples were immediately frozen in liquid nitrogen. Proteins were extracted using 10 L lysis buffer per 1 mg tissue and
stored at 70C until analyzed. Protein content was measured with
a Bio-Rad BCA assay. As RF ablation denaturizes protein, measured
protein content of the tissue lysates would not necessarily reect the
protein content before ablation. The amount of tissue lysates used in
the gelatin zymography was therefore determined by dry weight of
each tissue sample before homogenization.

FRICH ET AL.: INCREASED MMP-2 AND MMP-9 AFTER RADIOFREQUENCY ABLATION

299

Gelatin Zymography

Statistical Analysis

MMP-2 and MMP-9 enzyme activity was analyzed using gelatin

zymography. Equal amounts of plasma or liver tissue lysates were
applied to a non-reducing 10% sodium dodecyl sulfate (SDS)polyacrylamide gel containing 0.1% gelatin (Bloom 300; SigmaAldrich, St. Louis, MO), and run as previously described with minor
modications [20]. The gelatin zymograms were calibrated with human MMP-2 and MMP-9 standard (CC073; Chemicon, Temecula,
CA) and activated with 0.2% Brij 35 (Sigma-Aldrich). Gels were
stained with Coomassie brilliant blue R-250 (Bio-Rad, Hercules, CA)
and destained before being scanned. MMP enzyme activity was detected as clear bands against a staining background of undegraded
substrate. The gels were photographed under standardized lightning
conditions, and clear bands representing gelatinase activity was
quantied by densitometry using TotalLab software (Nonlinear Dynamics, Newcastle upon Tyne, UK). Latent and active forms of
MMP-2 and MMP-9 were pooled in the statistical analysis.

SPSS for Mac OSX version 11.0.4 (SPSS, Chicago, IL) was used for
the statistical analyses. A P value of less than 0.05 was considered
statistical signicant. Data are presented as mean SD unless
otherwise indicated. Plasma values of MMPs and cytokines 1 h after
laparotomy, but before RF ablation were normalized to a value of 1
in each animal, and relative alterations in the corresponding plasma
samples collected 1 h after RF ablation were analyzed by a one
sample t test, using a test value of 1.0. MMP activity and cytokine
expression in lysates from normal hepatic parenchyma were normalized to a value of 1 in each animal, and relative alterations in the
corresponding values from the transition zone were examined by a
one sample t test, using a test value of 1.0. Data from all animals
were pooled in the statistical analysis. Differences in the two groups
were analyzed by a two-group t test. Sample size of the study was
estimated by nQuery Advisor 4 (Statistical Solutions, Saugus, MA).
With a sample size of 6 in each experimental group, a single group t
test with a two-sided signicance level of 0.05 would have 80% power
to detect a difference between a null hypothesis mean of 1 and an
alternative mean of 1.75, assuming a standard deviation of 0.5.
Concerning differences between the two experimental groups, a sample size of 6 in each group would have 80% power to detect a
difference in means of 0.9 between the two groups assuming a common standard deviation of 0.5 when using a two group t test with a
0.05 two-sided signicance level. The animals were allocated to the
two experimental groups by block randomization, using a block size
of two.

Enzyme-Linked Immunosorbent Assays

Cytokine expression in plasma and tissue lysates was determined
by commercially available enzyme-linked immunosorbent assays
(ELISAs) performed according to the instructions from the manufacturer (R&D Systems, Minneapolis, MN) for the following markers:
TNF- (PTA00), IL-10 (P1000) and PGE 2 (DE0100). Values below
the detection limit of an assay were treated as missing in the statistical analysis.

Immunohistochemical Analysis
Primary antibodies used in the study were a mouse anti-humanMMP-2 monoclonal antibody (MAB3308; Chemicon, Temecula, CA)
and a rabbit anti-human MMP-9 polyclonal antibody (AB13458,
Chemicon), both diluted at 1/150. Western immunoblotting using a
7.5% polyacrydamide gel with a dual color protein standard (Precision Plus Dual Color, Bio-Rad) and antibodies against MMP-2 and
MMP-9 was performed to verify that the MMP antibodies used in the
study recognized porcine MMP-2 and MMP-9, with protein bands
staining at approximately 72 kDa and 92 kDa, respectively. A monoclonal mouse anti-human CD68 antibody (M0876; DakoCytomation,
Glostrup, Denmark) diluted at 1/100 was used as a macrophage
marker.
Coagulations were excised from fresh liver tissue and cut in 4 to
6 mm thick slices. Each slice contained the central coagulated area
and the surrounding transition zone between coagulated and normal
hepatic parenchyma. The slices were xed in 4% formaldehyde for at
least 10 days, then dehydrated and embedded in parafn. One 3 to
5 m thick section was cut from each parafn block and stained with
hematoxylin and eosin. Additionally, 3 to 5 m thick sections were
mounted on SuperFrost Plus adhesion slides (Menzel-Glser, Braunschweig, Germany). The sections were dried for 50 min at 56C
followed by 37C overnight and deparafnized, hydrated through
alcohols, rinsed in water and pre-treated with dual endogenous enzyme block for 10 min (EnVision Dual Link System-HRP (DAB);
Dako, Glostrup, Denmark). Thereafter the sections were rinsed in
water and incubated in a wash solution (APK Wash Solution; Ventana Medical Systems, Tucson, AZ) and stained using an automated
immunohistochemical stainer (NexES, Ventana Medical Systems)
using iView DAB Detection Kit (Ventana Medical Systems) according to the manufacturers protocol. The sections were rinsed in running tap water for 10 min and counterstained with Lillies hematoxylin for 7 s, rinsed in lukewarm water for 15 min, dehydrated
through alcohols and xylene and mounted in Eukitt Mounting Medium (Electron Microscopy Sciences, Hateld, PA). Sections were
examined by light microscopy by an experienced pathologist.

RESULTS

Blood samples from one animal in the non-Pringle

groups were missing. Hence, plasma was available for
analysis in ve of the six animals in the non-Pringle
group and six of the six animals in the Pringle group.
MMP Activity in Liver Tissue Lysates and Plasma

Five separate bands of gelatinolytic activity were

detected on the zymograms. A band with an approximate molecular weight of 135 kDa represented the
heterodimer of proMMP-9 and neutrophil gelatinaseassociated lipocalin [21]. The next four bands represented proMMP-9 (92kDa), active MMP-9 (82 kDa),
proMMP-2 (72 kDa), and active MMP-2 (62 kDa)
(Fig. 1). The active form of MMP-2 was observed in the
normal hepatic parenchyma and the transition zone in
10 animals, whereas the active form of MMP-9 was
observed in 6 animals, and only in specimens from the
transition zone.
MMP-2 and MMP-9 activity in tissue lysates from
the transition zone was signicantly increased compared to normal hepatic parenchyma with ratios of 3.0
and 2.6, respectively (Table 1). MMP-2 and MMP-9
activity in plasma 1 h after RF ablation was signicantly increased compared with baseline levels with
ratios of 1.2 and 1.5, respectively (Table 2). MMP-2 and
MMP-9 activity in plasma was normalized 4 days after
RF ablation.

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JOURNAL OF SURGICAL RESEARCH: VOL. 135, NO. 2, OCTOBER 2006

FIG. 1. Gelatin zymography showing MMP activity in three representative animals. Hepatic tissue was obtained 4 days after radiofrequency ablation. Normal porcine hepatic parenchyma (N), transition zone separating coagulated and normal hepatic parenchyma (T). The
positions of a 135-kDa complex of ProMMP-9 and neutrophil gelatinase-associated lipocalin, proMMP-9, active MMP-9, proMMP-2, and active
MMP-2 are indicated.

Cytokine Expression in Liver Tissue Lysates and Plasma

PGE 2 levels in tissue lysates from the transition zone

was signicantly increased compared to normal hepatic parenchyma with a ratio of 1.9. Corresponding
tissue levels of TNF- and IL-10 were not increased,
with ratios of 1.1 and 1.4, respectively (Table 1). Levels
of TNF- and PGE 2 were not increased in plasma 1 h
after RF ablation compared with baseline levels, with
ratios of 1.0 and 0.8, respectively (Table 2). PGE 2 was
only detected in three of 11 available plasma samples.
IL-10 levels were below the detection limit in all
plasma samples. Levels of TNF- and PGE 2 in plasma
were normalized 4 days after RF ablation.
Immunohistochemical Analysis

A sharp demarcation between coagulated tissue and

the surrounding hepatic parenchyma was present at
TABLE 1
Lysates from Tissue Samples Obtained 4 Days After
Radiofrequency Ablation. Activity of Matrix Metalloproteinase 2 and 9, and Levels of Tumor Necrosis
Factor-, Interleukin-10 and Prostaglandin E 2 in Lysates from the Transition Zone Relative to Values in
Normal Hepatic Parenchyma

MMP-2
MMP-9
TNF-
PGE 2
IL-10

Ratio

12
12
8*
12
12

3.0 2.0 (0.968.57)

2.6 1.1 (1.385.52)
1.1 0.3 (0.811.70)
1.9 1.2 (0.323.81)
1.4 1.0 (0.273.91)

0.005
0.001
0.234
0.025
0.230

Note. MMP matrix metalloproteinase; TNF- tumor necrosis

factor-; IL-10 interleukin-10; PGE 2 prostaglandin E 2. Values
are mean standard deviation (min, max).
* TNF- was below the detection limit in 4 animals.

gross examination of the fresh tissue specimens in both

experimental groups. This demarcation corresponded
to a 1 to 3 mm transition zone between the coagulated
tissue and surrounding hepatic parenchyma seen on
hematoxylin and eosin stained sections (Fig. 2). The
transition zone consists of an inner necrotic zone
with dead hepatocytes, a rim of hemorrhagic material and thrombosed vessels, and an outer rim with
hemorrhage and viable hepatocytes, as described
previously [22]. Immunohistochemical analysis using antibodies directed against MMP-2 and MMP-9
in sections from the transition zone showed cells
strongly positive of MMP-2 and MMP-9 surrounded
by proliferating broblasts and inammatory cells.
Positive staining for MMP-2 or MMP-9 antibodies was
not detected in normal hepatic parenchyma (Fig. 3).
TABLE 2
Plasma Samples Obtained 1 Hour After Radiofrequency Ablation. Activity of Matrix Metalloproteinase
2 and 9, and Levels of Tumor Necrosis Factor-,
Interleukin-10 and Prostaglandin E 2 Relative to Baseline Values 1 hour After Laparotomy

MMP-2
MMP-9
TNF-
PGE 2
IL-10

n2

Ratio

11
11
11
3

1.2 0.2 (0.841.67)

1.5 0.6 (0.842.45)
1.0 0.2 (0.681.18)
0.8 0.2 (0.660.93)

0.036
0.013
0.830
0.107

Note. MMP matrix metalloproteinase; TNF- tumor necrosis

factor-; IL-10 interleukin-10; PGE 2 prostaglandin E 2. Values
are mean standard deviation (min, max).
PGE 2 was not detected in 8 animals.
IL-10 was not detected in any animal.
* Plasma was not available for analysis for one of the animals in
the non-Pringle group.

FRICH ET AL.: INCREASED MMP-2 AND MMP-9 AFTER RADIOFREQUENCY ABLATION

301

TNF- (P 0.951), IL-10 (P 0.751), and PGE 2 (P

0.282). No differences were found in tissue lysates from
the transition zone compared to normal hepatic parenchyma: MMP-2 (P 0.531), MMP-9 (P 0.775), TNF-
(P 0.107), PGE 2 (P 0.612), and IL-10 (P 0.787).
Thus, the Pringle maneuver did not affect the absolute
values measured in normal hepatic tissue lysates or
the ratio in tissue lysates from the transition zone
compared to normal parenchyma. No qualitative differences between the two experimental groups were
seen on immunohistochemical analysis of sections from
the transition zone. No signicant differences were
found in plasma values for MMPs or cytokines in the
non-Pringle group and the Pringle group (data not
shown).
FIG. 2. Hematoxylin and eosin stained section from porcine liver
4 days after radiofrequency ablation. Normal hepatic parenchyma
(N) and coagulated parenchyma (C) is separated by a sharply demarcated transition zone (T).

Sections from the transition zone contained cells staining positive for the macrophage marker CD68, whereas
cells in the normal hepatic parenchyma did not stain
positive for the macrophage marker (Fig. 3).
Effect of Pringle Maneuver

Absolute MMP activity and cytokine values measured in lysates from normal hepatic tissue were not
signicantly different in the non-Pringle group and the
Pringle group: MMP-2 (P 0.542), MMP-9 (P 0.592),

DISCUSSION

RF ablation of malignant liver tumors is associated

with local recurrence rates ranging from 2% to 60%
[23]. Tumor size larger than 3 to 4 cm, perivascular
tumor localization and multiple tumors are established
risk factors for local recurrence after hepatic RF ablation [23, 24]. Although local tumor recurrence rates comparable to those found after hepatic anatomical and
wedge resection has been achieved [25], patients treated
with RF ablation have higher intrahepatic recurrence
rates and inferior survival rate compared with resected
patients. This difference cannot fully be explained by
selection bias or local tumor recurrence at the site of the

FIG. 3. Immunohistochemical analysis of sections from hepatic parenchyma harvested 4 days after radiofrequency ablation. Original
magnication 200 or 400 as indicated far left. Sections were incubated with antibodies directed against MMP-2 (not shown), MMP-9 and
the macrophage marker CD68. Large cells strongly positive for MMP-9 (arrow) are seen in the transition zone separating coagulated and
normal hepatic parenchyma (rst column). Normal hepatic parenchyma showing the portal triad with hepatocytes and some inammatory
cells. No cells stained positive for MMP-9 (second column). Macrophages strongly positive for CD68 (arrow) are seen in the transition zone
(third column). Normal hepatic parenchyma showing the portal triad with hepatocytes and some inammatory cells. No cells stained positive
for CD68 (fourth column). f, broblast proliferation and inammatory cells; h, hepatocytes; p, portal triad; m, macrophages.

302

JOURNAL OF SURGICAL RESEARCH: VOL. 135, NO. 2, OCTOBER 2006

coagulation [10]. The biological effects induced by RF

ablation in the index tumor and surrounding hepatic
parenchyma are not well characterized because of the
fact that the treated tumors and surrounding parenchyma are left in situ. Although cellular necrosis occurs when tissue is exposed to temperatures above 50
to 55C [26], cells in the periphery of a coagulated area
are exposed to sub-lethal temperatures associated with
a wide range of cellular responses including protein
denaturation, inactivation of protein synthesis, cell cycle progression, and DNA repair processes [27]. Experimental animal data suggest that hepatic RF ablation
promotes intrahepatic growth of residual neoplastic
cells [12]. Although the biological mechanisms leading
to induction of neoplastic growth after thermal RF
ablation remain unresolved, experimental studies have
shown that hepatic thermal ablation increase the expression of heat shock proteins [28] and growth factors
[13] adjacent to the treated site.
In the present study MMP-2 and MMP-9 activity
was signicantly increased in tissue lysates from the
transition zone 4 days after RF ablation. Increased
MMP-2 and MMP-9 activity at the tumor-stroma interface in human tumor tissue sections are predominantly expressed by reactive stromal cells and represent a tumor-induced host response that can be
adopted by malignant cells to promote tumor growth
and invasion [29, 30]. The cellular origin of the increased MMP activity found in tissue lysates by gelatin
zymography was examined in tissue sections from the
transition zone by immunohistochemical staining with
human MMP-2 and MMP-9 antibodies as well as the
macrophage marker CD68. The results of the CD68
staining and the typical macrophage morphology
clearly show that the cells staining for MMP antibodies
in the transition zone were macrophages. Even though
MMP-2 and MMP-9 activity was detected in normal
hepatic tissue by zymography, no MMP-2 or MMP-9
staining was found in normal hepatic tissue by immunohistochemical analysis of tissue sections. Western
blotting veried that the anti-human MMP-2 and
MMP-9 antibodies used in the study recognized porcine
MMP-2 and MMP-9. Therefore, we believe that the
lack of staining for MMP-2 and MMP-9 in normal
hepatic tissue was because of the very low MMP levels
in normal tissue, which most probably were below the
detection limit for immunohistochemistry.
Increased plasma MMP levels were found 1 h after
RF ablation, possibly indicating that RF ablation induced acute MMP release from cells other than tissue
macrophages at the treatment site. However, our approach of serial blood samples followed by analysis of
hepatic tissue lysates after euthanasia 4 days after RF
ablation does not allow us to draw conclusions regarding the temporal relationship between systemic and
local tissue MMP activity.

Pringle maneuver during RF ablation is generally

associated with increased coagulation volume [8],
which is considered benecial in treatment of large
tumors to avoid viable residual tumor cells. To our
knowledge, no randomized studies have evaluated the
impact of hepatic vascular occlusion during RF ablation on local tumor recurrence. In a recent metaanalysis of local recurrence after hepatic RF ablation of
liver tumors, univariate analysis found slightly lower
local recurrence rates in patients in which hepatic vascular occlusion was used (9.3% versus 12.8%, P
0.038). In a multivariate analysis of the same data,
hepatic vascular occlusion was not signicant [23]. Although RF ablation with Pringle maneuver has not
been shown to have higher recurrence rates compared
to RF ablation without Pringle, experimental data suggest that hepatic ischemia and reperfusion (I/R) in the
presence of hepatic micrometastases is a strong stimulus of tumor growth [31]. This is of importance as
hepatic RF ablation is increasingly combined with hepatic resection, which has been shown to induce hematogenous and intrahepatic dissemination of CRC cells
[32]. Increased activity of MMP-9 in serum 1 week
after I/R has been reported [33]. We therefore considered it of interest to examine if Pringle inuenced the
tumor microenvironment by altering the expression of
MMPs and cytokines. Our data suggest that Pringle
maneuver for 10 min does not have an impact on absolute values of MMP-2 or MMP-9 activity or cytokine
expression in normal hepatic parenchyma. These apparently diverging ndings may be explained by the
short duration of hepatic vascular occlusion used in our
study.
The activity of MMPs is regulated at several levels
including gene transcription of a number of growth
factors, tumor promoters, oncogenes, and hormones
[34]. Additionally, MMPs are regulated by proenzyme
activation and reversible inhibition of activated enzymes by tissue inhibitors of MMP [15, 35]. Increased
MMP expression can be detected in repair or remodeling processes as well as in diseased or inamed tissues
and may act on pro-inammatory cytokines, chemokines and other proteins to regulate inammation [15].
TNF- mediates many aspects of the acute inammatory process, and is one of the earliest indicators of
subsequent host responses, with peak plasma values
1 h after intravenous injection of endotoxin [36]. In our
study, plasma levels of the pro-inammatory mediator
TNF- or the anti-inammatory mediators IL-10 and
PGE 2 was not present in plasma 1 h after hepatic RF
ablation, conrming ndings in previous experimental
[37] and clinical studies [38, 39]. Cytokine levels were
only examined in blood samples drawn 1 h after ablation and 4 days postoperatively. We cannot exclude the
existence of a cytokine response in between these time
points. In fact, a recent experimental study examining

FRICH ET AL.: INCREASED MMP-2 AND MMP-9 AFTER RADIOFREQUENCY ABLATION

the systemic inammatory response after hepatic RF

ablation, cryotherapy or resection in porcine liver reported increased TNF- levels 6 h after RF ablation
[39]. These diverging ndings may therefore be inuenced by the time of blood sample collection relative to
the procedure, but could also be related to different
extent of the coagulation induced in these studies. In
our study increased MMP activity in tissue lysates was
found without a concurrent increase in tissue TNF-
levels. Increased levels of PGE 2 were found in tissue
lysates from the transition zone, in accordance with the
roles of prostaglandins in inammatory reactions after
tissue damage [40].
MMP activity is up-regulated in many forms of cancer,
including CRC [16, 30]. In a recent clinical study of 32
patients with CRC liver metastasis treated by hepatic
resection, both active and latent forms of MMP-2 and
MMP-9 were increased in tissues containing metastatic
tumor compared to normal liver tissue, with median ratios of 17.6 for proMMP-2 and 2.9 for proMMP-9. Of
importance, MMP expression was higher in the metastatic tumor itself than in the transitional tissue containing the invasive edge and immediate surrounding tumor
and normal tissue [18]. Our results show that RF ablation of normal hepatic parenchyma is associated with a
three-fold increased activity of MMP-2 and MMP-9.
Therefore, if tumor tissue is incompletely coagulated,
viable cancer cells are exposed to a microenvironment
that may increase the risk of tumor progression and
distant metastasis. On the other hand, one could hypothesize that in situ tumor destruction might provide
a benecial antigen source for induction of antitumor
immunity [41]. However, based on the assumption that
increased expression of MMPs in peritumoral tissue is
associated with increased risk of tumor cell growth, our
results indicate that RF ablation should only be attempted when complete eradication of the target tumor
including a safety margin is possible.
This study was performed in an animal model subject to several limitations. All coagulations were performed in normal hepatic porcine parenchyma. MMP
activity in tissue was assessed at one time point 4 days
after ablation. Our study design does not allow us to
draw conclusions regarding the prognostic role of the
increased activity of MMP-2 and MMP-9 activity on
clinical outcome data such as local tumor recurrence or
patient survival. Furthermore, all coagulations were
made with a RF ablation system using perfusion of
saline into the tissue during ablation that may affect
our results. Therefore, further research should be conducted to assess the expression of MMPs after hepatic
RF ablation using other RF ablation systems.
In conclusion, hepatic RF ablation was associated with
a three-fold increased activity of MMP-2 and MMP-9 in
the transition zone separating coagulated tissue from
normal hepatic parenchyma. The use of Pringle ma-

303

neuver for 10 min during ablation did not inuence

MMP activity. The clinical signicance of the combination of residual tumor cells and induction of a microenvironment that facilitates growth and of tumors is
not fully explored and should be assessed in animal
tumor models with extended observation time.
ACKNOWLEDGMENTS
We thank the staff at the Interventional Centre for providing
excellent assistance, the Department of Comparative Medicine for
support in animal care and Aasa R. Schjlberg at the Institute of
Pathology for assistance with the immunohistochemical analysis.
LF was supported by the Norwegian Cancer Society, grant no.
D02042/001. KB was supported by a postdoctoral fellowship from
University of Oslo, Norway.

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Radiofrequency ablation of colorectal liver metastases:

evaluation of local tumor recurrence by 3D volumetric analysis

Lars Frich1, 2
Gaute Hagen 3
Knut Brabrand 3
Bjrn Edwin 1, 2
ystein Mathisen 1
Trond Mogens Aalkken 3
Ivar P. Gladhaug 1, 4
1.
2.
3.
4.

Department of Surgery, Rikshospitalet University Hospital, 0027 Oslo, Norway

The Interventional Centre, Rikshospitalet University Hospital, 0027 Oslo, Norway
Department of Radiology, Rikshospitalet University Hospital, 0027 Oslo, Norway
Department of Surgery, Faculty Division Rikshospitalet, University of Oslo,
0027 Oslo, Norway

Word count

Abstract
193 words
Manuscript including references 4548 words

Running title

Volumetric evaluation of local tumor recurrence

Subject category

Radiology, gastrointestinal, oncology

Corresponding author

Lars Frich
Department of Surgery
Rikshospitalet University Hospital
N-0027 Oslo
NORWAY
Tel.
+ 47 23071825
Fax.
+ 47 23072267
E-mail:
lars.frich@labmed.uio.no

This work was supported by the Norwegian Cancer Society, grant no. D02042/001.

Abstract
Background: The purpose of this study was to characterize the relationship between local tumor
recurrence after radiofrequency (RF) ablation of colorectal liver metastases and post-ablation
alterations in coagulation volume.
Materials and methods: 11 patients with hepatic metastases from colorectal cancer were treated
with RF ablation. All patients were followed for at least 24 months or until local tumor recurrence.
Multi-detector computed tomography (MDCT) was performed at 1 and 3 months after RF ablation,
and then at intervals of 3 months until 24 months and each sixth month thereafter. Median followup was 27 months (17-43 months). Local tumor recurrence was found in 7 patients. Coagulation
volume was measured from 81 follow-up MDCT scans using a semi-automatic three-dimensional
(3D) method.
Results: Patients with local tumor recurrence had a median increase in coagulation volume of 50%
(range 0-378%) compared to images acquired 3 months previously. Local tumor recurrence was
LGHQWLHGVLPXOWDQHRXVO\E\VHPLDXWRPDWLF'YROXPHWULFDQDO\VLVDQGFRQYHQWLRQDOPRUSKRORJLF
criteria.
Conclusions: Semi-automatic 3D volumetric analysis provides added diagnostic information
FRPSDUHGZLWKFRQYHQWLRQDOPRUSKRORJLFDOHYDOXDWLRQDQGPD\LQFUHDVHVSHFLFLW\RIQRQLQYDVLYH
diagnosis of local tumor recurrence after RF ablation of colorectal liver metastases.
Key words: Radiofrequency ablation, liver, computed tomography

Introduction
Metastatic disease to the liver is a frequent event in patients with colorectal cancer [1]. Surgical
resection is a potentially curative treatment of liver metastases from colorectal cancer, with 5year survival rates of 30-50 per cent in selected patients [2-4]. The majority of patients with
colorectal liver metastases are not candidates for hepatic resection. Radiofrequency (RF) ablation is
increasingly used for in-situ tumor destruction in patients with non-resectable metastases limited to
WKHOLYHU>@,QDUHFHQWPHWDDQDO\VLVRIQLQHW\YHVHULHVDQGOLYHUWXPRUVWUHDWHGZLWK5)
ablation, the reported rate of local tumor recurrence varied between 2% and 60%. Follow-up was
short in several studies, probably contributing to an underestimation of the true rate of local tumor
UHFXUUHQFH>@/RFDOWXPRUUHFXUUHQFHDWWKHWUHDWHGVLWHXVXDOO\RFFXUVZLWKLQWKHUVWPRQWKV
but have been reported to appear up to 23 months after the procedure [7-9].
(DUO\GHWHFWLRQRIORFDOWXPRUUHFXUUHQFHPD\IDFLOLWDWHHDUO\UHLQWHUYHQWLRQZLWKSRWHQWLDOEHQHWV
for patient survival. Malignant tissue treated by RF ablation is left in situ, necessitating repeated
follow-up imaging or biopsy to determine if the tumor has been eradicated. Furthermore, biopsy
of potentially operable colorectal liver metastases is controversial due to the risk of needle tract
seeding and negative impact on patient survival [10, 11]. Consequently, non-invasive methods
should preferably be used for detection of persistent or residual malignant tissue at the site of
5)DEODWLRQ,WFDQEHGLIFXOWWRGLVWLQJXLVKEHWZHHQORFDOWXPRUUHFXUUHQFHDQGEHQLJQWLVVXH
alterations on follow-up imaging [12-14]. Analysis of coagulation geometry and coagulation
volume on early post-ablation scans may identify patients with inadequate treatment margins
relative to the index tumor, which would be expected to have a high risk of local tumor recurrence.
Various morphologic patterns are associated with local tumor recurrence on post-ablation computed
tomography (CT) [13]. An enhancing peripheral tissue rim surrounding the non-enhancing
coagulation may represent local tumor recurrence, but may also represent benign periablational
HQKDQFHPHQWZKLFKLVFRQVLGHUHGWREHWKHUHVXOWRIDEHQLJQSK\VLRORJLFLQDPPDWRU\UHDFWLRQ
to thermally damaged cells [15, 16]. Additionally, local tumor recurrence may be diagnosed based
RQDQLQFUHDVHLQWKHRYHUDOOFRDJXODWLRQVL]HFRPSDUHGZLWKSUHYLRXVVFDQV>@,WLVGLIFXOW

to quantify small alterations in coagulation diameter reliably due to inter-scan displacement and
deformation of the liver. Increase in coagulation volume may be a more sensitive indicator of
growth than increase in coagulation diameter; for a spherical volume, an increase in diameter of
20% corresponds to a volume increase of 73%. However, the relationship between local tumor
recurrence after RF ablation of hepatic colorectal metastases and alterations in coagulation volume
is not well characterized.
In this clinical pilot study, patients with colorectal liver metastases treated with RF ablation were
followed for at least 24 months or until local tumor recurrence. To assess theoretical treatment
margins, we determined the largest tumor diameter on pre-ablation images, and the effective
coagulation diameter 1 month after RF ablation. Post-ablation coagulation volumes were measured
on repeat follow-up scans using a semi-automatic three-dimensional (3D) technique. Our study
hypotheses were: (1) inadequate treatment margins as determined from scans 1 month after
radiofrequency ablation may identify patients who would develop local tumor recurrence, and (2)
increase in coagulation volume during follow-up may be used to detect local tumor recurrence
earlier than conventional morphological criteria.

Materials and methods

Study population
This study was approved by the Regional committee for medical research ethics. From 2003 to
2006 a total of 23 patients underwent RF ablation for non-resectable malignant liver tumors at
our institution. Written informed consent was obtained from all patients prior to the procedure.
$OOSDWLHQWVZHUHHQWHUHGLQWRDSURVSHFWLYHGDWDEDVH:HLGHQWLHGSDWLHQWVZKRVDWLVHGWKH
following criteria: (a) non-resectable liver metastasis from colorectal cancer; (b) complete clearance
of all liver tumors possible either by RF ablation alone or combined with surgical resection; (c)
diameter of tumor treated with radiofrequency ablation less than 4 cm; (d) no extrahepatic disease;
(e) follow-up > 24 months, or to local tumor recurrence. The reasons for exclusion were noncolorectal cancer liver metastasis (n=1), tumor diameter > 4 cm (n=2), extrahepatic tumor growth
present at time of treatment (n=1), and follow-up less than 24 months without occurrence of local
tumor recurrence (n=8).
All patients were treated with a curative intent. Patient age was 60.3 8.0 yr (49-80 yr). The
primary tumor location was the colon in 8 patients and the rectum in 3 patients. All patients had
undergone curative resection of the primary colorectal tumor. Liver resection of colorectal liver
metastases had previously been performed in 7 of 11 patients. The median interval between
operation of the primary tumor (n=11) and RF ablation was 17 months (3-101 months). The median
interval between liver resection (n=7) and RF ablation was 12 months (2-77 months). The patients
included in this study had 1-4 liver tumors, with a median of 1 tumor. One tumor was treated by RF
DEODWLRQLQHDFKRIWKHSDWLHQWV6HYHQRIWKHVHWXPRUVZHUHFODVVLHGDVSHULYDVFXODU
Operative procedure
Patients were treated with a RF ablation system with perfusion electrodes (Elektrotom HiTT 106,
Berchtold GmbH & Co, Germany). This system incorporates a generator that delivers alternating
current at 375 kHz to a maximum of 1.2 ampere, and a syringe pump (Pilot C, Fresenius Vial,
France) for infusion of isotonic saline through the electrode tip during ablation. All procedures

were performed under general anesthesia. The approach used for RF ablation was percutaneous
(n=4), laparoscopic (n=4) and by laparotomy (n=3). Intraprocedural image guidance was contrastenhanced ultrasonography (n=6), intraoperative ultrasonography (n=4) and magnetic resonance
imaging (MRI) (n=1). RF ablation and hepatic resection was performed during the same procedure
in 4 patients, one of these resections was performed as a laparoscopic procedure. The median
number of electrode activations in each procedure was 2 (2-4). Hepatic vascular occlusion was not
used during RF ablation in any patients. CT or contrast-enhanced ultrasonography was performed in
all patients prior to discharge to assess the completeness of the treatment.
Follow-up
All patients were followed with a standardized imaging protocol. Sixteen or 64-channel multidetector computed tomography (MDCT) (GE LightSpeed Pro 16 or GE LightSpeed VCT; GE
Healthcare, Milwaukee, WI, USA), was performed using 150-200 mL iodixanol 320 mgI/mL
(Visipaque, GE Healthcare, USA). Images were acquired in portal venous phase. Scans were
performed at 1 and 3 months after RF ablation, and then every 3 months until 24 months, and every
sixth month thereafter. A total of 81 post-ablation scans from 1 month after ablation and onwards
were reviewed, with a follow-up of 27 months (17-43 months). CT of the chest was performed
every sixth month. Carcinoembryonic antigen (CEA) with a cut-off level of 5 g/L was used a
marker of malignancy, and was measured at all time points. All patients were followed for at least
24 months or until local tumor recurrence.
Assessment of local tumor recurrence
3DWLHQWVZHUHFRQVLGHUHGWRKDYHORFDOWXPRUUHFXUUHQFHEDVHGRQDWOHDVWRQHRIWKUHHSUHGHQHG
morphological criteria on follow-up CT examinations; new lesion(s) in the periphery of the
coagulated area; at least 20% growth of the largest diameter of the coagulated area; persistent
attenuation in the periphery of the coagulated area different from the coagulated area and from
normal hepatic parenchyma. During follow-up a strict distinction was kept between local tumor
recurrence, i.e. tumor growth within or at the ablation margin, intrahepatic tumors not related to the
areas treated with RF ablation, and extrahepatic tumor growth.

Treatment margin
The largest coagulation diameter and the effective coagulation diameter were retrospectively
TXDQWLHGIURP&7VFDQVDFTXLUHGPRQWKDIWHU5)DEODWLRQXVLQJWKHVFLHQWLFLPDJHDQDO\VLV
program ImageJ version 1.37 (National institute of mental health, Bethesda, MA, USA). A region
RILQWHUHVW52,ZDVGHQHGE\WUDFLQJWKHERUGHUEHWZHHQQRUPDOOLYHUWLVVXHDQGDOWHUDWLRQV
attributed to RF ablation on all slices where tissue coagulation was present. Measurement
of geometric properties of each ROI was performed using automatic algorithms. The largest
FRDJXODWLRQGLDPHWHUZDVGHQHGDVWKHORQJHVWGLVWDQFHEHWZHHQDQ\WZRSRLQWVDORQJWKH
SHULPHWHURIWKH52,VIRUDVLQJOHFRDJXODWLRQ(IIHFWLYHFRDJXODWLRQGLDPHWHUZDVGHQHGDVWKH
GLDPHWHURIWKHODUJHVWFLUFOHWKDWFRXOGEHWLQWRWKH52,VGHQLQJDVLQJOHFRDJXODWLRQ)LJ
The largest pre-ablation tumor diameter was subtracted from the effective coagulation diameter, and
this difference was divided by 2 to achieve the theoretical treatment margin surrounding the tumor
on all sides.
Coagulation volume
All images were retrospectively reviewed by an experienced radiologist unaware of the course of
the disease in each patient, who had not been involved in the RF ablation procedures or prospective
interpretation of follow-up scans. Images of each patient were reviewed in chronological order.
Tumors on preopoerative images contiguous to a vessel with diameter of 3 mm or more were
FODVVLHGDVSHULYDVFXODU9ROXPHWULFDQDO\VHVZHUHSHUIRUPHGRQDZRUNVWDWLRQ*($GYDQWDJH
Workstation 4.2; GE Healthcare, USA). The coagulation volumes were calculated by the software
package Volume Viewer (GE Healthcare, USA). Using a seeding based semi-automatic technique,
DYROXPHRILQWHUHVW92,ZDVGHQHGXVLQJWKHERUGHUEHWZHHQWKHK\SRDWWHQXDWLQJYROXPH
representing tissue coagulation and normal hepatic parenchyma. The extent of the VOI was
controlled on coronal and axial source images. The volume of the selected VOI was calculated by
the software package.
Statistical analysis
SPSS for Mac OSX version 11.0.4 (SPSS, Chicago, Illinois, USA) was used for the statistical
DQDO\VHV$SYDOXHRIOHVVWKDQZDVFRQVLGHUHGVWDWLVWLFDOVLJQLFDQW'DWDDUHSUHVHQWHGDV
7

mean SD (range), or median (range). All post-ablation coagulation volumes were normalized to
the 1-month baseline value for each patient. Independent samples t-test was used for comparison
between groups. Wilcoxon signed rank test was used for repeated measurements within each group.

Results
Local, intrahepatic and extrahepatic tumor recurrence
Diameter of the tumors treated with RF ablation was 2.2 0.8 cm (1.0-3.5 cm), with a tumor
volume of 7.1 6.6 mL (0.5-22.5 mL) (Table 1). Treatment of the index tumor was initially
considered complete in all patients. Local tumor recurrence was found during follow-up in 7
patients, and was detected median 9 months (6-21 months) after RF ablation. Tumor diameter
in the patients who developed local tumor recurrence was 2.2 0.3 cm (1.0-3.5 cm) compared
with 2.1 0.4 cm (1.2-3.1 cm) in the patients without local tumor recurrence. This difference was
QRWVWDWLVWLFDOO\VLJQLFDQW)LQHQHHGOHDVSLUDWLRQF\WRORJ\RIWKHDUHDWUHDWHGZLWK5)DEODWLRQ
ZDVSHUIRUPHGLQSDWLHQWVGXULQJIROORZXS,QSDWLHQWVPDOLJQDQF\ZDVYHULHGZKHUHDVQR
malignant cells were found in 1 patient who did not develop local tumor recurrence. Local tumor
recurrence was found in 5 of 7 perivascular tumors. Of the 7 patients with local tumor recurrence, 4
patients presented with inoperable intrahepatic (n=1) or extrahepatic (n=3) malignant growth at the
time of diagnosis of local tumor recurrence. The 3 patients with local tumor recurrence only were
re-treated with resection (n=1) or repeat RF ablation (n=2). Considering all 11 patients, intrahepatic
recurrence not related to the RF ablation site was found in 6 patients with a median of 14.5 months
(4-24 months) after RF ablation, and extrahepatic tumor growth was found in 6 patients with a
median of 11.5 months (4-22 months) after RF ablation, with the following distribution; lungs
(n=4), lymph nodes (n=1), bone (n=1). Two patients who had developed extrahepatic disease died
17 and 20 months after RF ablation. The remaining 9 patients, including the 3 patients who were retreated, were alive with a median follow-up of 28 months (20-43 months).
Treatment margin
Largest coagulation diameter 1 month after RF ablation was 4.8 1.4 cm (2.9-7.5 cm), and was
larger than the corresponding pre-ablation tumor diameter in all patients. Effective coagulation
diameter was 2.9 0.8 cm (1.6-4.2 cm). The effective coagulation diameter was larger than the
corresponding tumor diameter in 8 patients (Table 1), with a positive margin of 0.6 0.3 cm (0.31.0 cm). The 3 patients in which the effective coagulation diameter was smaller or equal to the
tumor diameter developed local tumor recurrence. In the 7 patients with local tumor recurrence
9

the margin was 0.3 0.6 cm (-0.4-1.0 cm), compared to 0.6 0.3 cm (0.3-0.9 cm) in the 4 patients
ZLWKRXWORFDOWXPRUUHFXUUHQFH7KLVGLIIHUHQFHZDVQRWVWDWLVWLFDOO\VLJQLFDQWS 
Coagulation volume
Baseline coagulation volume 1 month after RF ablation was 29.7 16.0 mL (13-70 mL), and was
larger than the corresponding tumor volume in all patients. At 3 months, coagulation volume had
decreased in all patients, and was 54.1 16.9% (38-88%) of the baseline value. In the patients
without local tumor recurrence (n=4), subsequent measurements showed generally decreasing
coagulation volumes, with all values below 50% of baseline values at 12 months and below 30%
of baseline values at 24 months. One patient had a volume increase of 3% between two successive
measurements, but decreasing volume on the following measurement (Figure 2a). Patients who
GHYHORSHGORFDOWXPRUUHFXUUHQFHQ VKRZHGDVLJQLFDQWLQFUHDVHLQFRDJXODWLRQYROXPHRI
85 131% (0-378%) from the examination 3 months prior to diagnosis of local tumor recurrence
until diagnosis of local tumor recurrence (p=0.028). The median volume increase in these patients
was 50% (Figure 2b). One patient had local tumor recurrence without an associated alteration in
coagulation volume (Figure 3). Coagulation volume measurements 3 months after diagnosis of local
tumor recurrence were available in 5 patients, showing further increase in coagulation volume in 4
patients (Table 2). Of importance, 4 of the 7 patients with local tumor recurrence had a coagulation
volume less than the baseline volume at time of diagnosis.

10

Discussion
Several methods have been proposed for early detection of local tumor recurrence after RF ablation
RQSRVWDEODWLRQ&7VFDQVVXFKDVDOWHUDWLRQVLQ+RXQVHOGXQLWV>@RUDQDO\VLVRIFRDJXODWLRQ
geometry [17]. In the present study we retrospectively examined if inadequate treatment margins
assessed 1 month post-ablation could predict local tumor recurrence, and if increase in coagulation
volume on post-ablation MDCT images could be used to detect local tumor recurrence earlier than
conventional morphological criteria.
Established oncological criteria suggest that hepatic malignancies should be eradicated radically
including a 1-cm margin of apparently healthy tissue to eliminate microscopic foci of malignant
cells, and to compensate for the uncertainty in determining the exact tumor margin [18, 19].
Considering the effective coagulation diameters produced in this study, we did not coagulate
DVSKHULFDOWLVVXHYROXPHVXIFLHQWWRDFKLHYHDFPPDUJLQRQDOOVLGHVRIWKHWXPRULH
a coagulation diameter at least 2 cm larger than the largest tumor diameter) in any patients.
Theoretically, a margin of at least 0.5 cm could have been achieved in 5 patients, whereas 3
patients had a positive margin less than 0.5 cm. An effective coagulation diameter smaller or equal
to the pre-ablation tumor diameter was found in 3 patients, and was invariably associated with
ORFDOWXPRUUHFXUUHQFH7DEOH+RZHYHUSURJQRVWLFVLJQLFDQFHRIDQHJDWLYHPDUJLQUHPDLQV
to be examined in larger prospective studies. Even though one would expect a large margin to
EHEHQHFLDOWRDYRLGORFDOWXPRUUHFXUUHQFHWKHSDWLHQWZLWKWKHODUJHVWPDUJLQLQWKLVVWXG\
developed local tumor recurrence (Table 1). The calculated margins surrounding the tumors may
have been overestimated if the tumor diameter was larger than anticipated from pre-ablation images.
Furthermore, even if an adequate coagulation volume was achieved, suboptimal positioning of the
ablation electrode or displacement of the geometrical center of the coagulated volume relative to the
ablation electrode may result in inadequate treatment [20].
7KHFRDJXODWLRQYROXPHDVGHQHGLQWKLVVWXG\PLJKWLQFRUSRUDWHERWKYLDEOHDQGQRQYLDEOH
malignant and non-malignant tissue. Coagulation volume decreased from baseline at 1 month to

11

3 months in all patients. Consequently, decreasing coagulation volume from 1 to 3 months after
RF ablation cannot be used as a predictor for the absence of later development of local tumor
recurrence. A marked increase in coagulation volume when local tumor recurrence was diagnosed
was seen in 6 of 7 patients. The lack of increase in coagulation volume in one patient was probably
due to the occurrence of nodules at a small distance from the coagulated area which were not
LGHQWLHGE\WKHVHPLDXWRPDWLFPHWKRGXVHGIRUYROXPHHVWLPDWLRQLQWKLVVWXG\)LJXUH
This indicates that semi-automatic volumetric analysis should only be used as a supplement to
conventional morphological criteria. With a follow-up interval of 3 months, increase in coagulation
volume was not present until local tumor recurrence was detected based on conventional criteria
(Figure 2b).
The natural course of adequately treated colorectal metastases is a decrease in coagulation volume,
as illustrated by the patients who did not develop local tumor recurrence. In the 6 patients where
coagulation volume was increased at the time of local tumor recurrence, the increase in volume
was at least 19% compared to the previous examination 3 months earlier, with a median increase
RI:HDWWULEXWHWKHREVHUYHGYROXPHLQFUHDVHWRDJURZWKRIPDOLJQDQWWLVVXH,WLVGLIFXOWWR
determine with certainty whether local tumor recurrence was caused by incomplete coagulation of
a tumor that continued to grow, or if a new tumor grew at or near the site of the adequately treated
tumor [16]. Untreated colorectal liver metastases have a tumor volume doubling time of 3-5 months
[21]. Viable malignant tissue after RF ablation would in most cases be expected to constitute a small
fraction of the coagulated volume. It is therefore somewhat surprising that a median coagulation
volume increase of 50% in 3 months was observed in our patients with local tumor recurrence.
However, recent experimental research indicate that RF ablation induces biological alterations in
the tissue of possible importance for tumor invasion and metastasis [22], and that thermal ablation
promotes growth of residual neoplastic cells, possibly through stimulation by growth factors [23,
24]. Therefore, one could speculate that the growth rate of viable residual malignant cells exposed
for thermal ablation may be higher than for untreated malignant tissue.
Three patients with local tumor recurrence were biopsied. In the remaining 4 patients with local
tumor recurrence, biopsy was not obtained due to simultaneous intrahepatic or extraheptic tumor
12

growth, which precluded potential curative surgical resection or local ablation. In these 4 patients
WKHUHVXOWRIDELRSV\ZRXOGQRWLQXHQFHWKHSODQQHGWUHDWPHQWRIWKHSDWLHQWDQGZDVZDLYHG,Q
retrospect, operable patients with suspected local tumor recurrence by morphological criteria and
a concurrent marked increase in coagulation volume could be considered for repeated treatment
without performing biopsy, which delays potentially curative treatment and may lead to tumor
VHHGLQJ>@2XUQGLQJVVXJJHVWWKDWWKHFRPELQDWLRQRIVHPLDXWRPDWLF'YROXPHWULF
DQDO\VLVDQGFRQYHQWLRQDOPRUSKRORJLFDOHYDOXDWLRQPD\LQFUHDVHWKHVSHFLFLW\RIQRQLQYDVLYH
diagnosis of local tumor recurrence after RF ablation of colorectal cancer liver metastases, and
thereby possibly avoid unnecessary biopsies.
In this study we found local tumor recurrence in 7 of 11 patients, corresponding to a rate of 64%.
Reported local tumor recurrence rates varies from less than 20% [25], to recently reported rates
of 47% and 58% [26, 27]. Tumor proximity to major vessels is a well-established risk factor of
local tumor recurrence [6]. A high proportion of the tumors treated in this study were perivascular,
SRVVLEO\LQXHQFLQJWKHUDWHRIORFDOWXPRUUHFXUUHQFH7KH5)DEODWLRQV\VWHPXVHGLQRXUVWXG\
produces irregular coagulations [20, 28, 29], which may contribute to the high rate of local tumor
recurrence. Furthermore, a learning curve exists for new procedures, including RF ablation [6, 30].
Therefore, as this patient series represents our initial experience with RF ablation, a lower rate of
local tumor recurrence would be expected with increasing experience.
This retrospective pilot study has some important limitations. It presents data from only 11 patients.
This sample size is too small to allow conclusions regarding the prognostic value of the treatment
PDUJLQ4XDQWLFDWLRQRIODUJHVWSUHDEODWLRQWXPRUGLDPHWHUODUJHVWFRDJXODWLRQGLDPHWHUDQG
effective coagulation diameter were made from 2D axial images. Local tumor recurrence was
YHULHGE\ELRSV\LQSDWLHQWV$OWKRXJKZHEHOLHYHWKDWWKHFULWHULDXVHGIRULGHQWLI\LQJSDWLHQWV
ZLWKUHVLGXDOWXPRUKDYHDKLJKVSHFLFLW\DQGWKHIROORZXSSHULRGRIDWOHDVWPRQWKVZDV
adequate to detect local tumor recurrence, the number of patients with malignant residual tumor
PD\EHXQGHUHVWLPDWHG7KHSDWWHUQRISRVWDEODWLRQYROXPHWULFDOWHUDWLRQVPD\EHVSHFLFWR
the perfusion electrode RF ablation system used in this study. Dual-modality positron emission
tomography (PET)/CT may have higher sensitivity to detect viable malignant tumor after RF
13

ablation compared to CT [31, 32], however this image modality was not evaluated in this study.
In conclusion, by acquiring follow-up MDCT images each third month after hepatic RF ablation,
ORFDOWXPRUUHFXUUHQFHZDVLGHQWLHGVLPXOWDQHRXVO\E\WKHXVHRIFRQYHQWLRQDOPRUSKRORJLFDO
criteria and semi-automatic 3D volumetric analysis. In patients with local tumor recurrence an
increase in median coagulation volume by 50% was seen, whereas in patients without local tumor
occurrence decreasing coagulation volume during the follow-up period was observed, with all
coagulation volumes less than 30% of baseline at 24 months. Semi-automatic 3D volumetric
analysis provides added diagnostic information compared with conventional morphological
HYDOXDWLRQDQGPD\LQFUHDVHVSHFLFLW\RIQRQLQYDVLYHGLDJQRVLVRIORFDOWXPRUUHFXUUHQFHDIWHU
RF ablation of colorectal cancer liver metastases.

14

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17

32.

Veit, P., G. Antoch, H. Stergar, A. Bockisch, M. Forsting, and H. Kuehl (2006) Detection of
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18

/LVWRIJXUHVDQGJXUHFDSWLRQV
Figure 1
a. 55-year old man with liver metastases from colorectal cancer. Portal venous phase multi-detector
computed tomography acquired 1 month after resection of segment II/III and radiofrequency
DEODWLRQRIDWXPRUZLWKDGLDPHWHURIFPLQVHJPHQW9VKRZVDZHOOGHQHGORZDWWHQXDWLRQ
coagulation.
b. Geometric analysis of the coagulation in (a). The thick line denotes the border of normal liver
tissue vs. alterations attributed to coagulation. Dmax denotes maximum axial coagulation diameter
and Deff effective coagulation diameter. On the shown slice, Dmax is 4.6 cm, whereas Deff is 3.4 cm.
Figure 2
Coagulation volumes 1-24 months after radiofrequency ablation of colorectal liver metastases.
Coagulation volumes for each patient have been normalized to the corresponding coagulation
volume 1 month after radiofrequency ablation.
a. Patients without local tumor progression (n=4). All values were below 50% of baseline values at
12 months and below 30% of baseline values at 24 months.
b. Patients with local tumor progression (n=7). The time of diagnosis of local tumor progression is
marked by a circle. The patient marked by as asterisk had a volume increase to above 400% when
local tumor progression was diagnosed at 6 months.
Figure 3
55-year-old man with liver metastases from colorectal cancer in which local tumor progression
was not detected by semi-automated 3D volumetric analysis. Portal venous phase multi-detector
computed tomography. The preoperative CEA was 64 g/L. Resection of segment II and III, local
resection in segment VI and radiofrequency ablation of a tumor in segment VIII was performed
in the same procedure. 1 month after radiofrequency ablation coagulation volume was 26 mL,
and CEA was 3 g/L. a. 9 months after radiofrequency ablation. The coagulation volume was
19

FDOFXODWHGWRP/&($ZDVJ/$ZHOOGHQHGORZDWWHQXDWLRQFRDJXODWLRQZLWKRXWSHULSKHUDO
hyperemia is seen.
b. 12 months after radiofrequency ablation. The coagulation volume was calculated to 7 mL. CEA
was 16 g/L. A heterogeneous low-attenuation nodule representing local tumor progression is
VHHQDWWKHPDUJLQRIWKHFRDJXODWHGDUHDORFDWHGGRUVDODQGPHGLDOWRWKHZHOOGHQHGFRDJXODWLRQ
(arrow).

20

Table 1
Patient characteristics.
Table 2
Post-ablation coagulation volumes in patients with local tumor progression (n=7).

21

Figure 1
a. 55-year old man with liver metastases from
colorectal cancer. Portal venous phase multidetector computed tomography acquired 1
month after resection of segment II/III and
radiofrequency ablation of a tumor with a
diameter of 3.1 cm in segment V shows a wellGHQHGORZDWWHQXDWLRQFRDJXODWLRQ

b. Geometric analysis of the coagulation in (a).

The thick line denotes the border of normal
liver tissue vs. alterations attributed to coagulation. Dmax denotes maximum axial coagulation diameter and Deff effective coagulation
diameter. On the shown slice, Dmax is 4.6 cm,
whereas Deff is 3.4 cm.

22

Figure 2
Coagulation volumes 1-24 months after radiofrequency ablation of colorectal liver metastases. Coagulation volumes for each patient have been normalized to the corresponding coagulation volume
1 month after radiofrequency ablation.

a. Patients without local tumor progression

(n=4). All values were below 50% of baseline values at 12 months and below 30% of
baseline values at 24 months.

b. Patients with local tumor progression

(n=7). The time of diagnosis of local tumor
progression is marked by a circle. The patient
marked by as asterisk had a volume increase
to above 400% when local tumor progression
was diagnosed at 6 months.

23

Figure 3
55-year-old man with liver metastases from colorectal cancer in which local tumor progression was
not detected by semi-automated 3D volumetric analysis. Portal venous phase multi-detector computed tomography. The preoperative CEA was 64 g/L. Resection of segment II and III, local resection in segment VI and radiofrequency ablation of a tumor in segment VIII was performed in the
same procedure. 1 month after radiofrequency ablation coagulation volume was 26 mL, and CEA
was 3 g/L.

a. 9 months after radiofrequency ablation. The

coagulation volume was calculated to 7 mL.
&($ZDVJ/$ZHOOGHQHGORZDWWHQXation coagulation without peripheral hyperemia is seen.

b. 12 months after radiofrequency ablation.

The coagulation volume was calculated to
7 mL. CEA was 16 g/L. A heterogeneous
low-attenuation nodule representing local
tumor progression is seen at the margin of the
coagulated area located dorsal and medial to
WKHZHOOGHQHGFRDJXODWLRQDUURZ

24

Table 1
Patient characteristics
Patient
no.

1
2
3
4
5
6
7
8
9
10
11

Tumor
diameter
(cm)
2.3
2.5
1.2
1.6
1.0
1.8
3.5
2.5
3.1
2.3
2.1

Largest
coagulation
diameter
(cm)
6.7
4.8
2.9
3.9
4.2
3.9
4.3
7.5
4.6
5.9
4.2

Effective
coagulation
diameter
(cm)
4.2
3.0
2.1
2.7
2.8
2.5
2.7
4.2
3.6
1.6
2.1

Theoretical Local
treatment
tumor
margin
recurrence
(cm)
1.0
0.3
0.5
0.6
0.9
0.4
-0.4
0.9
0.3
-0.3
0.0

Yes
Yes
No
No
Yes
Yes
Yes
No
No
Yes
Yes

Time
to local
tumor
recurrence
(months)
21
6
12
6
12
9
9

25

Table 2
Post-ablation coagulation volumes in patients with local tumor progression (n=7).
Post-ablation coagulation volume
(mL)
Patient no.
1
2
5
6
7
10
11

Baseline
volume at
1 month
43
34
26
22
22
13
22

3 months
prior to
diagnosis
19
30
7
10
11
2
9

At
diagnosis
27
47
7
93
26
10
14

3 months
after
diagnosis
80
54
6
41
17

26