Hepatobiliary Pancreat Dis IntVol 11No 4 August 152012 www.hbpdint.

com 349
Author Afliations: Second Department of Internal Medicine, University
of Athens Medical School, Hippokration General Hospital, 114 Vas Soas
Avenue, Athens 11527, Greece (Vasilieva LE and Dourakis SP); Molecular
Cytogenetics Unit, Haematology Laboratory, "G. Genimatas" Regional
General Hospital, Athens, Greece (Papadhimitriou SI)
Corresponding Author: Larisa E Vasilieva, MD, Themistokleous 39, Holargos,
Athens 15562, Greece (Tel: 302106526388; Email: larisatheo@yahoo.gr)
2012, Hepatobiliary Pancreat Dis Int. All rights reserved.
doi: 10.1016/S1499-3872(12)60192-1
BACKGROUND: Cholangiocarcinoma is a very aggressive tumor
with poor survival. Therefore, early diagnosis and surgical
resection are of paramount importance. Its diagnosis is difcult
because access to the tumor is not easy. Biopsy is possible only
for intrahepatic cholangiocarcinoma, which accounts for 10%
of cases. Routine brush cytology from endoscopic retrograde
cholangiopancreatography (ERCP) has a high specicity of 100%
but unfortunately a low sensitivity of 30%. In this review we
briey describe new diagnostic techniques applicable to ERCP
brush cytology specimens and targeting the genetic background
of the disease, in particular uorescence in situ hybridization
(FISH) and digital image analysis (DIA).
DATE SOURCES: The PubMed database up to 2011 was used
for the retrieval of relevant articles. The search terms FISH,
uorescence in situ hybridization, DIA, digital image analysis
and cholangiocarcinoma were used. Both original and review
articles were used.
RESULTS: FISH identies cells with chromosomal abnormalities,
mainly numerical aberrations, using a mixture of uorescence-
labeled probes. FISH offers a higher sensitivity than routine
cytology, retaining a high level of specicity. The DIA criterion
for malignancy is demonstration of aneuploidy. This technique
increases the sensitivity to 40%, but the specicity remains low.
Preliminary data from application to other tumors suggest that
combination of FISH and DIA may be of further benet.
CONCLUSIONS: The new techniques offer a signicantly
enhanced diagnostic efcacy in the evaluation of ERCP brush
specimens. Apart from contributing to a more timely diagnosis,
their wider application to cholangiocarcinoma may also facilitate
the genetic study of the disease and add to our understanding
of oncogenesis at the molecular level, with the prospect of
identifying targets for novel therapeutic interventions.
(Hepatobiliary Pancreat Dis Int 2012;11:349-359)
KEY WORDS: cholangiocarcinoma;
uorescence in situ hybridization;
digital image analysis
Introduction
C
holangiocarcinoma is a malignant neoplasm
originating from biliary epithelial cells.
[1, 2]

It accounts for 3% of the malignant tumors
of the gastrointestinal system and ranks second
in frequency among primary liver tumors, after
hepatocellular carcinoma.
[3]
The incidence and
mortality of the disease are rising worldwide.
[4-8]
About
90% of cholangiocarcinomas are adenocarcinomas
and, with regard to location, may be intrahepatic and
extrahepatic.
[9, 10]
The extrahepatic cholangiocarcinoma
consists of either a liver hilar tumor (also known as
a Klatskin tumor) (60%-70%) or a distal bile duct
tumor (20%-30%).
[11]
The mortality of this tumor is
high because, on diagnosis, it is usually at an advanced
stage.
[12-14]
Survival for extrahepatic cholangiocarcinoma
in patients undergoing palliative resection is 43% (1st
year), 27% (3rd year), and 27% (5th year), while in
those treated with palliative drainage, the respective
gures are 23%, 7%, and 0%. Survival for unoperated
patients is 18% (1st year) and 0% (3rd year).
[15]
Farley et
al
[16]
reported even lower survival rates for patients in
whom the tumor proved unresectable, despite attempted
curative resection: 53% (1st year), 19% (2nd year), 9%
(3rd year), and 4% (5th year).
Risk factors for the development of cholangiocarcinoma
include: i) primary sclerosing cholangitis (PSC) leading
to cholangiocarcinoma at a rate of 8%-40%;
[17-20]
ii)
parasitic biliary infection with Opisthorchis viverrini
[21]

or Clonorchis sinensis;
[22, 23]
iii) bropolycystic malforma-
tions of the liver, Caroli's disease, congenital hepatic
brosis, and choledochal cysts (cystic dilatations of
the bile ducts); a 15% of risk for cholangiocarcinoma
development after the second decade of life;
[11]
iv) hepa-
tolithiasis;
[24-26]
v) exposure to chemical carcinogens such
as dioxins,
[27]
nitrosamines, alcohol, and smoking,
[11, 28]

Modern diagnostic approaches to
cholangiocarcinoma
Larisa E Vasilieva, Stefanos I Papadhimitriou and Spyros P Dourakis
Athens, Greece
Review Article
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330 Hepatobiliary Pancreat Dis IntVol 11No 4 August 152012 www.hbpdint.com
as well as thorotrast, a radiological contrast agent
widely used in the sixties, which is associated with
a 300-fold increase of risk for the development of
cholangiocarcinoma;
[29]
vi) lesions caused by hepatitis C
virus and hepatitis B virus;
[30, 31]
and vii) inammatory
bowel disease.
[32]
Primary symptoms depend on anatomical location.
Intrahepatic cholangiocarcinoma is manifested by
non-specic complaints like abdominal pain, anorexia
and palpable abdominal mass lesions.
[33]
In the case of
extrahepatic cholangiocarcinoma the patient usually
presents with painless jaundice, pruritus, dark urine
and pale stools.
[34]
Current diagnostic methods
The diagnosis of cholangiocarcinoma is based on
imaging techniques, i.e., i) endoscopic retrograde
cholangiopancreatography (ERCP), with or without
routine brush cytology, ii) magnetic resonance
cholangiopancreatography (MRCP), iii) magnetic
resonance imaging, iv) computerized tomography
(CT), v) magnetic resonance angiography, and vi) CT
angiography.
[35-38]
The main features of the imaging
methods are summarized in Table 1.
Histological documentation of cholangiocarcinoma
is not always possible because of the difcult access
to the tumor. In most patients, routine cytology by
brushing during ERCP is the mainstay of diagnosis. This
procedure has a sensitivity of 9%-24% and a specicity
of 61%-100%, respectively.
[1]

Other methods for cholangiocarcinoma diagnosis
are transpapillary cholangioscopy and endoscopic
ultrasound-guided ne needle aspiration (EUS-FNA).
Transpapillary cholangioscopy is used to directly
visualize the bile duct and obtain biopsies. It has a high
sensitivity of 89%-92% and a specicity of 93%-96% for
extrahepatic cholangiocarcinoma, which may further
increase to 71% and 100%, respectively. The advantages
of the single-operator peroral cholangiopancreatoscopy
system, Spyglass, are as follows: i) the examination can
be carried out by one endoscopist because of the good t
of the duodenoscope; ii) the increased maneuverability
of the Spyglass system allows for 4-quadrant biopsies; iii)
it provides better visibility; and iv) it is not fragile.
[39-41]
Another new method allowing access to the tumor
is EUS-FNA.
[42-45]
The application of this method to
patients with suspected hilar cholangiocarcinoma with
negative brush cytology has an accuracy of 91%, a
sensitivity of 89% and a specicity of 100%. Thus, EUS-
FNA is effective and less invasive for the diagnosis of
this subset of patients.
[46]
Serum tumor markers play an auxiliary role in the
diagnostic approach. Such is the case of CA19-9, which
however is not specic.
[47, 48]
For example, when its
value is >100 U/mL in patients with PSC, its sensitivity
is 89% and its specicity 86%.
[1]
In patients without
PSC its sensitivity decreases to 53%.
[49]
In PSC patients,
serum bilirubin may also prove useful in predicting
malignant strictures, although the cutoff levels vary
widely between published studies (75-145 mol/L).
[50-53]

However, CA19-9 increases in patients with bacterial
cholangitis, hepatolithiasis, chronic biliary parasitosis
and other cancers.
[54, 55]
Other nonspecic serum tumor
markers which may be elevated in cholangiocarcinoma
are CA125 and carcinoembryonic antigen.
In summary, the diagnosis of cholangiocarcinoma
is based on clinical and imaging ndings, while
differentiation from benign bile duct strictures is often
problematic. In patients who present with clinical and
radiographic features consistent with hilar lesions and
are operated for suspected cholangiocarcinoma, a benign
tumor (e.g. chronic brosing or erosive inammation,
sclerosing cholangitis, granular cell tumors) is nally
documented in 10%-15% of cases.
[56-58]
A similar
percentage (13%) of benign lesions was found among
patients operated for suspected malignant proximal bile
duct stricture.
[59]
A cytologically documented diagnosis
occurs in only a small minority of patients (30%).
[60, 61]

Table 1. Imaging methods for the diagnosis of cholangiocarcinoma
(modied from references 35-46)
Methods Target
Sampling
method
ERCP Location of tumor, biliary dilatation Routine
cytology
MRCP Location of tumor, hepatic parenchyma,
biliary dilatation
-
MRI Location of tumor, hepatic parenchyma,
biliary dilatation, lymph nodes
-
CT Location of tumor, hepatic parenchyma,
biliary dilatation, lymph nodes
-
MRA Vasculature -
CTA Vasculature -
EUS-FNA Location of tumor, hepatic parenchyma,
biliary dilatation, lymph nodes, acquisi-
tion of material
FNA
Transpapillary
cholangioscopy
Directly visualize bile duct Biopsy
ERCP: endoscopic retrograde cholangiopancreatography; MRCP:
magnetic resonance cholangiopancreatography; MRI: magnetic resonance
imaging; CT: computerized tomography; MRA: magnetic resonance
angiography; CTA: computerized tomography angiography; EUS-FNA:
endoscopic ultrasound-guided ne needle aspiration.
Diagnostic approaches to cholangiocarcinoma
Hepatobiliary Pancreat Dis IntVol 11No 4 August 152012 www.hbpdint.com 331
Biliary brush cytology
Biliary brush cytology is currently the most
appropriate method for the diagnosis of biliary tract
strictures. Although its specicity is high, the sensitivity
for malignancy detection in routine cytology samples
is low. Several studies have shown that the sensitivity
ranges from 30% to 88%, while the specicity is nearly
100%.
[61]
The reasons for this discrepancy include:
i) difculties in sampling, often due to lesion size
and location, extensive brosis or benign epithelium
overlying the tumor; ii) technical errors in handling the
specimen before and after delivery to the laboratory;
iii) errors in interpretation (including subtle changes
in well-differentiated lesions, and unfamiliarity with
the diagnostic criteria for precancerous lesions),
and underestimating the signicance of the smear
background.
[61]
Table 2 shows the diagnostic criteria of biological
evolution from normal bile duct epithelium to dysplasia
and nally to overt malignancy. The categories of
dysplasia were determined in 1990 by Laylied, who
described the cellular characteristics of low and high
grade dysplasia by ERCP brush cytology.
[61]
The
cholangiocarcinoma criteria are clear in contrast to
those of the grey-zone lesions.
[61]

Diagnostic techniques targeting the genetic
background of cholangiocarcinoma
Recent advances in molecular biology have led to
the development of new diagnostic tools and therapeutic
innovations in oncology. Among others, one major
benet is the potential to detect premalignant conditions
and thus elicit diagnostic alertness. Early diagnosis
allows for timely therapeutic intervention, which in
turn results in longer survival and better quality of
life. For example, patients with PSC have a high risk for
development of cholangiocarcinoma and, therefore, it
is advisable for them to be under close supervision for
early indications. When such indications do present,
liver transplantation improves survival and quality of
life of patients. The interpretation of cytological ndings
in patients with PSC is generally difcult and early
diagnosis of cholangiocarcinoma may rely entirely on
the application of newer diagnostic techniques, namely
uorescence in situ hybridization (FISH) and digital
image analysis (DIA)
[1, 13, 62]
Table 2. Diagnostic criteria for biliary tract brush cytology during ERCP (modied from reference 61)
Morphological
criteria
Benign bile duct epithelium
Reactive or inammatory
changes
Cytological grey-zone changes:
(i) atypical, (ii) low-end
high-grade dysplasia,
(iii) suspicious
Malignant
(adenocarcinoma)
Epithelium Cohesive monolayer sheets
of small to medium-sized
epithelial cells
Epithelial layers with slight
nuclear overlap
Epithelial degree 1 to 3 arranged
in sheets and clusters with
nuclear crowding and overlap
Isolated malignant cells
appearing singly
Nuclei Round to oval centrally
located, uniform
Slightly enlarged nuclei with
smooth nuclear membranes
Marked nuclear overlap
and crowding
Nuclear molding of the
nucleus
*
, anisonucleosis,
enlarged nuclei, irregular
nuclear membranes,
disordered cellular sheets,
nuclear grooves, altered
cell polarity
#

Nuclear/cytoplasmic
(N/C) ratio
Low Medium increase (degree 1 to 3) High
*
Chromatin Uniform, nely granular,
evenly distributed
Uniform, nely granular Mild coarsening (degree 1 to 3) Chromatin clumping
*
,
chromatin clearing
#
,
granular, coarse chromatin
Nucleoli Insignicant or completely
absent
Small, discernible Small distinct (degree 1 to 3) Prominent
#
*: Main criteria for differential diagnosis of malignancy; #: secondary criteria for differential diagnosis of malignancy.
Hepatobiliary & Pancreatic Diseases International
332 Hepatobiliary Pancreat Dis IntVol 11No 4 August 152012 www.hbpdint.com
FISH and related techniques
Conventional cytogenetics (chromosomal banding
and karyotype analysis) has proved valuable in the
diagnosis, prognosis and evaluation of treatment
response of hematological neoplasias. Moreover, it
has revealed that practically all solid tumors are
characterized by numerical and structural chromosome
aberrations.
[63]
However, conventional karyotypic
analysis requires the high mitotic activities of target
cells, adequate chromosomal morphology, a long culture
period, and experience.
FISH is a molecular technique developed in the mid-
eighties. It uses uorescence-labeled polynucleotide
probes, complementary to the DNA sequence under
investigation. The hybridization of the probe to its
target allows for its visualization under a uorescence
microscope, either on mitotic preparations or in
the interphase nucleus. The technique has certain
advantages: i) interphase FISH avoids the need for living
cells with mitotic potential, necessary for conventional
studies, and is feasible on practically all kinds of cytologic
or histopathologic specimens; ii) FISH identies small-
scale genetic alterations (down to the level of genes) as
opposed to karyotype analysis, the resolution of which is
limited to the band level; iii) with certain modications,
it allows for the recognition of cellular characteristics
(e.g. size, shape of nucleus or immunophenotype); iv) by
estimating the rate of positive cells, it provides a simple
quantication of malignant spread in the biological
material under investigation.
Although FISH is mainly used in the study of
hematological malignancies and is of clear diagnostic
and prognostic value, it is also used successfully in the
eld of solid tumors. For example, it has been shown
that FISH is more sensitive than routine cytology in
detecting urothelial cancer cells in the urine, retaining a
comparable degree of specicity.
[60]

Comparative genomic hybridization is an important
approach to the genetic analysis of tumors since it is
the rst efcient way to study the entire genome for
variations in DNA copy number. Total genomic DNA
is isolated from test and reference cell populations,
differentially labeled, and hybridized in equal amounts
to reference metaphase chromosomes. The relative
hybridization intensity of the test and reference signals
at a given location is then proportional to the relative
copy number of those sequences in the test and reference
genomes.
Genetic analysis of cholangiocarcinoma is difcult
because access to the tumor is not easy and biopsy is
only possible for intrahepatic tissue, which accounts
for 10% of cases. Therefore genetic data obtained by
conventional karyotypic analysis are limited. In contrast,
several genetic changes have been identied by FISH and
comparative genomic hybridization. The standard FISH
approach relies on the application of a 4-probe mixture
(comprising centromeric probes for chromosomes 3, 7,
and 17 and a probe for the INK4 locus, spanning the p16/
p14 and p15 genes in region 9p21) on brush specimens
obtained during ERCP. A result is considered positive if
at least 5 cells in the specimen show overrepresentation
of the respective chromosomes or deletion of the INK4
locus.
[1, 13, 60, 62]
As shown by several studies, the specicity of FISH
for cholangiocarcinoma is the same as that of routine
cytology, while its sensitivity is signicantly higher
(34% versus 15% with a specicity of 98% and 91%,
respectively).
[62]
The sensitivity of FISH is as high as
67% and its specicity as 75% for cholangiocarcinoma
in patients with PSC and negative cytology.
[1, 13]
The
high sensitivity of FISH relies mainly on its ability
to identify a small number of malignant cells. This
is particularly helpful in patients with PSC, in
whom early identication of the malignancy makes
feasible the application of radical therapies like liver
transplantation.
[1, 13, 60, 62]
To date, the application of
FISH to cholangiocarcinoma diagnosis is based almost
exclusively on the detection of hyperdiploidy, and
has followed the approach for tumors of the urinary
system.
[60]
Nevertheless, although the rst results
were encouraging, the method has certain limitations.
First, there is no proof that all cases are hyperdiploid.
Even if this was true, we know little about the degree
of overrepresentation of individual chromosomes in
hyperdiploid tumors.
[63]
Second, this approach cannot
identify small-scale karyotypic aberrations, such
as amplications or deletions involving oncogenes
or tumor suppressor genes.
[64, 65]
The detection of
such abnormalities would substantially enhance our
understanding of the pathogenesis and, perhaps, clinical
behavior of cholangiocarcinoma.
In this context, an obvious choice would be a study
of the tumor suppressor p53 in the chromosomal region
17p13. Deletion or inactivating mutations of this gene
have been reported in a wide spectrum of hematological
and solid tumors and have been implicated in
oncogenesis and the clinical course of diseases. However,
p53 involvement in cholangiocarcinoma has not been
fully claried.
[66-68]
For instance, beyond losses and
point mutations of 17p13,
[69]
cases of amplications of
the region have also been observed by PCR, and these
seem to confer a favorable prognosis.
[70]
The p16/p14 and p15 genes on 9p21 have been
extensively studied in lymphoid malignancies. In
Diagnostic approaches to cholangiocarcinoma
Hepatobiliary Pancreat Dis IntVol 11No 4 August 152012 www.hbpdint.com 333
particular, for these diseases, loss or inactivation of
p16 is a strong adverse prognostic factor, sometimes
overriding the favorable impact of other aberrations.
[71]

p16 is often deleted or inactivated in solid tumors too,
and cholangiocarcinoma, either sporadic or related to
PSC, is no exception.
[72, 73]
In cholangiocarcinoma, p16
is also deleted or inactivated independently of the p53
status.
[74, 75]
However, the interpretation of available data
is not easy, since the ndings come from a small number
of cases and concern various changes, e.g. whole gene
deletions, point mutations in the coding sequence, or
silencing due to promoter inactivation.
Of course, the genetic prole of cholangiocarcinoma
involves many other genes or chromosomal bands as
candidates for oncogenesis. In a study of 19 intrahepatic
cholangiocarcinoma cases, several genetic changes
were found. Gains were detected in the regions 8q22-
qter (58%), 5p14-pter (32%), 2q33-qter (26%), 7p
(26%), 17q21-q22 (26%), 18q12-q21 (26%), and 19q13.1
(26%). DNA amplication of the 17q21 region was
found in 47% of cases. Losses concerned the Y and X
chromosomes (60% and 32%, respectively), 1p34-pter
(37%), 4q (32%), 18q21-qter (32%), 19p (32%), X (32%),
5q11-q14 (26%), 8p (26%), 9p (26%) and 17p (26%).
[76]
Another study used screening for genetic alterations
and compared the results between 24 hepatocellular
carcinoma (HCC) and 11 intrahepatic cholangiocarci-
noma cases. Characteristic genetic changes for intrahepatic
cholangiocarcinoma were gains of 20q, 5p, 7q, and 13q,
as opposed to gains of 1q and loss of 4q, 10q and 13q for
HCC. Losses of 16q, 17p, and 18q and gain of 8q were
frequently found in both tumors.
[65]

In 14 patients with primary distal bile duct
carcinoma, the most frequently gained regions were
8q and 20q (43%), 12p, 17q and Xp (36%) and 2q, 6p,
7p, 11q, 13q and 19q (29%). The most frequently lost
regions were 18q (57%), 6q and 10p (50%), 8p, 12q and
17p (43%) and 7q, 12p and 22q (29%).
[77]
Genetic changes were studied in 50 biliary tract
carcinomas and their presence was associated with
the development and progression of the tumor. Gains
in part or in whole of 1q, 8q and 20q and losses of 5q,
8q, 9p and 18q were frequently found at the early stage
(T1/T2) (40%), but were also found at the advanced
stage of the disease (T3/T4). In particular, loss of 9p
was the most frequent aberration, both at the early
(78%) and the advanced stage (68%). The frequencies
of gains of 7p12-p14 (P<0.003), 7p21-pter (P<0.007)
and 7q31 (P<0.01) were signicantly different in biliary
tract carcinoma with or without distant metastasis. In
addition, gains of 5p and 19q13 and losses of 6q14-q16
were more frequent in tumors with lymph node
metastasis. Thus, it was assumed that loss of 9p is
critical for the development of biliary tract carcinoma,
while gains of 5p, 7p, 7q and 19q and losses of 6q were
associated with tumor progression and metastatic
potential.
[78]
Although at present FISH is not considered
routine in the diagnostic approach, it could easily
be incorporated into the standard laboratory work-
up of suspected cholangiocarcinoma. A laboratory
experienced in applying interphase FISH to specimens
from FNA or biopsy touch preparations, which today
is common practice in lymphoma investigation,
would work equally well on ERCP brushing material
and provide a reliable result within 2 or 3 days. In
most countries, the cost of the main reagents would
not exceed $300. In this setting, the standard FISH
application with the 4-probe system described above
could be modied to combine diagnostic efcacy with
investigation of the genetic background of the disease.
For example, specic probes could be used for the study
of individual oncogenes
[79]
and the appropriate probe
combination would be useful in the delineation of clonal
evolution or heterogeneity.
[80]
Cholangiocarcinoma diagnosis is applied when the
patient presents jaundice, but unfortunately this is the
case when the biliary duct is obstructed. Thus, before
the onset of jaundice, the patient is asymptomatic and
therefore one does not suspect cholangiocarcinoma.
This is the reason why early diagnosis of premature
cholangiocarcinoma is to date impossible. But when
there is strong suspicion of this tumor in ERCP and
routine cytology is negative or dubious, adoption of
modern techniques could avoid repetitions of ERCP for
obtaining new cytology or worse, the patient could be
taken for surgery without a proper diagnosis.
Flow cytometry and DIA
Flow cytometry is a technique for measuring the
DNA content of a large number of individual cells
as they ow in a liquid stream through a counting
chamber. The presence of cells with an increased
amount of DNA (hyperdiploidy) is interpreted as a
marker of malignancy or a pre-malignant state. In a
study involving routine cytology and ow cytometry,
51 specimens were taken with ERCP from 48 patients
with a stricture of the biliary tree.
[81]
From these, in 38
cases (75%) the stricture was attributed to malignancy:
in 22 it was caused by pancreatic adenocarcinoma, in
10 by biliary malignancy (gallbladder or biliary tree),
and in 6 it was due to malignant metastasis (3 from
colon, 1 from breast, 1 from lung and 1 from ovarian
cancer). Thirteen cases (25%) had their origin in benign
Hepatobiliary & Pancreatic Diseases International
334 Hepatobiliary Pancreat Dis IntVol 11No 4 August 152012 www.hbpdint.com
lesions. Sensitivity for the detection of malignancy was
equal for routine cytology and ow cytometry (42.1%).
The specicity of routine cytology was 92.3% and that
of ow cytometry 77%. If the diagnosis of cancer was
assumed when at least one of the tests was positive,
the sensitivity increased to 63.2% and the specicity
dropped to 69.2%. It was thus concluded that although
ow cytometry had the same sensitivity as routine
cytology, it lacked specicity.
[81]
DIA is a relatively new application which gives a
qualitative account of the cellular constituents, using
spectroscopic data. Unlike traditional spectrophotometers,
a video camera captures the light transmitted through a
cytological specimen slide and converts the absorption
values into pixels of white, gray, or black. Subsequently,
computer analysis of the pixels produces a digital image
of the nucleus and other cellular compartments, thus
quantifying DNA content (diploidy, tetraploidy, and
aneuploidy in general). Thus, chromatin distribution and
nuclear morphology can be digitally determined and may
suggest features of malignancy.
[82]
Although the principle
of DIA is closely related to that of ow cytometry, the
technique is applicable to specimens with a limited
number of cancer cells, in contrast to ow cytometry,
which requires a large population to discriminate between
benign and malignant proliferation.
[82]

Using both DIA and cytology, Baron et al
[83]
studied
100 patients who had undergone brushing of the biliary
tract strictures during ERCP. Of these patients, 56
showed malignant strictures (33 cholangiocarcinoma,
14 pancreatic carcinoma and 9 other cancers). The
diagnosis of malignancy was histologically conrmed
in 39 of the 56 (69.6%) and was established with clinical
follow-up for 7.7 months in the rest 17. Of the remaining
17 patients without a biopsy diagnosis, 16 died of
disease progression (1 with cholangiocarcinoma while
awaiting liver transplantation and 1 with conrmation
of malignancy in tissue obtained at a subsequent ERCP).
Benign stricture diagnosis was established after a follow-
up for 16 months. The criterion for malignancy in DIA
was non-diploidy in general. The results are presented
in Table 3. Combination of DIA with routine cytology
increased the sensitivity from 18% to 42.9%. Therefore,
DIA has a high sensitivity, in particular when combined
with routine cytology. However, specicity is low due to
many false-positives.
[83]

Other investigators have examined specimens
from PSC, choledocholithiasis, chronic pancreatitis,
operative injury, cholangiocarcinoma and pancreatic
carcinoma.
[82]
Designation as a malignant stricture
required conrmation by biopsy. A stricture was
considered as benign if the patient fullled at least one
of the following criteria: cancer-free clinical course for
a minimum of 2 years, surgical exploration with benign
histological ndings, or follow-up ERCP showing
resolution of the stricture. Among 16 patients with
malignant strictures and negative routine cytology, DIA
identied malignancy in 13 of them. In conclusion, the
sensitivity of DIA was 85% and the method proved
useful for malignancy detection in 13 cases where
routine cytology failed.
[82]
In another study,
[84]
the authors proposed a scoring
system based on DIA, using morphometric (nuclear
area), densitometric (nuclear DNA content) and
textural (chromatin distribution) parameters. The score
was based on the index calculated by the formula
=DI+DHT+(2PI), where DI is the DNA index, DHT
is a parameter related to the DNA histogram type and
PI is the proliferation index. A score of 5 was taken
as the criterion of malignancy. This score allowed
discrimination between normal, inammatory and
malignant epithelia of the biliary tract. However, it must
be noted that in this study no cytology results were
presented.
Attempts were also made to establish a correlation
between DNA ploidy and tumor stage. For this
purpose, histological specimens from 6 intrahepatic
cholangiocarcinomas, 6 carcinomas of the gallbladder,
and 5 extrahepatic bile duct carcinoma patients were
analyzed retrospectively. In all cases, the tumor was
in stage 3 or 4, except for a case of carcinoma of the
gallbladder which was in stage 2. When DNA ploidy was
associated with the tumor stage, the 3 well-differentiated
adenocarcinomas (2 intrahepatic cholangiocarcinomas
and 1 extrahepatic bile duct carcinoma) proved
predominantly diploid. Diploid peaks were also found
in moderately-differentiated adenocarcinomas in all
3 sites and of all 3 types and in a poorly differentiated
adenocarcinoma of the extrahepatic bile duct. However,
68% of the cases (15/22) showed only aneuploid
populations. Multiple populations were found in 19/22
cases, a nding which may reect tumor heterogeneity
and is possibly associated with the advanced stage and
aggressive nature of malignancies of the gallbladder
Table 3. Sensitivity and specicity of DIA and RC for malignant
biliary tract strictures with brushing specimens during ERCP
(modied from reference 83)
DIA (%) RC (%)
Sensitivity 39.3 17.9
Specicity 77.3 97.7
False-positives 22.7 2.3
DIA: digital image analysis; RC: routine cytologic examination.
Diagnostic approaches to cholangiocarcinoma
Hepatobiliary Pancreat Dis IntVol 11No 4 August 152012 www.hbpdint.com 333
and biliary system. On the other hand, no correlation
was found between DNA ploidy and tumor stage. This
is probably due to the fact that all cancers were at an
advanced stage.
[85]
DIA and FISH combination
Attempts were made recently by Levy et al
[86]

to combine the novel techniques, DIA and FISH.
Specimens from patients suffering from various
types of cancer (esophageal squamous cell carcinoma,
esophageal adenocarcinoma, gastric adenocarcinoma,
pancreatic mucinous cystic neoplasia, intraductal
papillary mucinous neoplasia, metastatic forearm
sarcoma, small cell and non-small cell lung cancer,
thyroid carcinoma, malignant gastrointestinal stromal
tumor, melanoma and lymphoma) were evaluated by
EUS-guided FNA. These specimens were taken from
lymph glands (lymphadenopathy), pancreatic mass,
esophageal or gastric wall mass, or thyroid mass and
studied with cytology, DIA and FISH. The results are
given in Table 4, from which it was concluded that the
combined DIA/FISH approach provides very satisfactory
results. And although it has not yet been evaluated in
cholangiocarcinoma, there is a good reason to anticipate
that the results will be equally encouraging.
Mechanisms of biliary carcinogenesis and growth
The understanding of the molecular mechanisms
triggering carcinogenesis of the biliary tract plays an
important role in the development of new diagnostic
tools and therapeutic modalities. Recently, Wise et
al
[87]
proposed molecular pathways contributing to
cholangiocarcinogenesis. During the course of chronic
cholestasis and associated liver inammation, factors
such as IL-6, TGF-, IL-8, TNF- and PDGF are released
into the local environment, mobilizing a series of events
that induce genomic damage leading to autonomous
proliferation and escape from apoptosis.
The same authors reported at least two factors
regulating cholangiocarcinoma growth which could
potentially be useful for the development of prevention
and treatment strategies. In particular, inhibition
of the CIX-2 pathway during PSC warrants further
investigation. Also, modulation of cholangiocarcinoma
growth by regulating neural input or the endo-
cannabinoid system might prove fruitful.
In another study, the signicance of cell cycle and
apoptosis-related markers in 112 cholangiocarcinomas
(42 intrahepatic and 70 extrahepatic) and 16 gallbladder
carcinomas combined in a tissue microarray was analyzed
immunohistochemically. Follow-up was available for
44.5% of the patients. The authors concluded that the p53,
Bcl-2, Bax and COX-2 genes play important roles in the
pathogenesis of cholangiocarcinomas. The differential
expression of p16, Bcl-2 and p53 between intrahepatic and
extrahepatic tumors demonstrates that there are location-
related differences in the phenotypes and genetic proles
of these cancers. Moreover, p16 was identied as an
important prognostic marker in cholangiocarcinomas.
[88]

Mechanisms of cholangiocarcinogenesis are presented
diagrammatically in Fig. 1, after modifying the gure of
reference 87 according to the mechanisms proposed in
references 88-93.
In a recent review,
[94]
Gatto and Alvaro pointed out
the crucial role of the insulin-like growth factor 1 (IGF1)
bile biomarker in modulating cholangiocarcinoma
growth and proliferation. This conclusion was mostly
based on a study by Alvaro et al,
[95]
where measurements
of IGF1 in the bile of patients undergoing ERCP for biliary
obstruction showed that biliary IGF1 concentration
Fig. 1. Mechanisms of cholangiocarcinogenesis (modified from
reference 87). IL-6: interleukin-6; IL-8: interleukin-8; TGF-:
transforming growth factor beta; TNF-: tumor necrosis factor
alpha; PDGF: platelet-derived growth factor; NO: nitric oxide;
EGFR: epidermal growth factor receptor; K-ras: K-ras oncogene;
HGF c-met: hepatocyte growth factor; APC: adenomatous
polyposis coli.
Table 4. Summary of the results obtained with the application
of newer techniques on specimens from EUS-guided FNA for
the diagnostic evaluation of gastrointestinal and other tumors
(modied from reference 86)
Technique RC (%) DIA/FISH (%) DIA (%) FISH (%)
Sensitivity 87 97 70 77
Specicity 100 100 100 100
Accuracy 90 98 79 83
RC: routine cytologic examination; DIA: digital image analysis; FISH:
uorescence in situ hybridization.
Hepatobiliary & Pancreatic Diseases International
336 Hepatobiliary Pancreat Dis IntVol 11No 4 August 152012 www.hbpdint.com
was 15-20-fold higher in cholangiocarcinoma than in
pancreatic cancer or benign biliary abnormalities. Apart
from its pathophysiologic signicance, this nding is
of obvious diagnostic value, since it appears that bile
IGF1 levels differentiate cholangiocarcinoma from either
pancreatic cancer or benign biliary stricture as both its
sensitivity and specicity are 100%.
[94]
Conclusions
The current diagnostic algorithm for the investigation
of biliary strictures is presented diagrammatically in
Fig. 2, after modifying gures from references 96 and 97
according to the mechanisms proposed in references 1,
13, 60, and 62. The newer diagnostic techniques described
in this review, targeting the genetic background of
cholangiocarcinoma (mainly hyperdiploidy), increase
considerably the specicity of routine cytology applied
to ERCP brush specimens. The main drawback of ow
cytometry and DIA is a relative lack of specicity, due
to many false-positives. This may be corrected in the
near future through the accumulation of additional
data and a more accurate denition of the cut-off values
for the diagnosis of malignancy. The possibility of a
correlation between tumor stage and the degree of
hyperdiploidy is another promising perspective. More
investigation is also warranted in order to evaluate the
overall diagnostic effectiveness of FISH. This technique
is not only a powerful diagnostic tool but may also
prove very useful for the study of the genetic features
of cholangiocarcinoma. Thus, the availability of a
wide range of probes makes it possible to investigate
the presence of aberrations of a multitude of genes,
beyond whole chromosome polysomies. Conventional
comparative genomic hybridization and its microarrays
can also provide a genome-wide overview of copy-
number abnormalities. A critical improvement will be
the possibility to differentiate between the genomic
aberrations characterizing the premalignant states
and those found in overt cancer. Finally, elucidating
the involvement of individual oncogenes and tumor
suppressor genes in oncogenesis may indicate new lines of
research on the molecular biology of cholangiocarcinoma
and hopefully open the way for targeted therapeutic
intervention for this currently incurable disease.
Contributors: DSP proposed the study. VLE, PSI and DSP collected
and analyzed the data. VLE and PSI wrote the rst draft under
the supervision of DSP. All authors contributed to the design and
interpretation of the study and to further drafts. PSI is the guarantor.
Funding: None.
Ethical approval: Not needed.
Competing interest: No benets in any form have been received
or will be received from a commercial party related directly or
indirectly to the subject of this article.
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Received October 19, 2011
Accepted after revision April 11, 2012