Serum and bile biomarkers for cholangiocarcinoma

Domenico Alvaro
Division of Gastroenterology, Department of Clinical Purpose of review
Medicine, Polo Pontino, University of Rome
‘La.Sapienza’, Rome, Italy
To discuss recent studies proposing new markers in serum or bile for the diagnosis and
prognosis of cholangiocarcinoma (CCA), which could help in the differential diagnosis
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Correspondence to Domenico Alvaro, MD, via R.

between malignant and benign biliary disorders or for the surveillance of disorders at
o4XMi0hCywCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC4/OAVpDDa8KKGKV0Ymy+78= on 05/28/2023

Rossellini 51, Division of Gastroenterology,

Department of Clinical Medicine, University of Rome
‘La Sapienza’, 00137 Rome, Italy
Tel: +39 06 49972023; fax: +39 06 4453319;
e-mail: domenico.alvaro@uniroma1.it
Current Opinion in Gastroenterology 2009,
25:279–284
risk, including primitive sclerosing cholangitis.
Recent findings
In the last few years, efforts have been made to identify biomarkers with adequate
diagnostic accuracy for CCA in serum or biological fluid. Studies have been focused on
cytokines, growth factors or enzymes produced and secreted by CCA cells as well as on
the proteomic analysis of serum and bile.
Summary
The serum levels of interleukin 6, trypsinogen, mucin-5AC, soluble fragment of
cytokeratin 19 and the platelet–lymphocyte ratio have been recently shown to help in
the diagnosis of CCA with, in some cases, a prognostic value. As far as bile is
concerned, the ratio of pancreatic elastase/amylase, mucin-4, minichromosome
maintenance replication protein and insulin-like growth factor 1 have been explored,
with the insulin-like growth factor 1 biliary concentration capable of completely
discriminating CCA from benign biliary disorders and pancreatic cancer. We have also
discussed advances in the proteomic of serum and bile, which seem promising in
identifying new markers for CCA.

Keywords
biliary biomarkers, cholangiocarcinoma, primitive sclerosing cholangitis, proteomic,
serum biomarkers

Curr Opin Gastroenterol 25:279–284

ß 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins
0267-1379

to aid CCA diagnosis but, unfortunately, none of these

Introduction
Cholangiocarcinoma (CCA) is a devastating cancer
with an incidence and mortality progressively increasing
worldwide. Radical surgery and liver transplantation are
applicable and effective only when CCA is diagnosed at
an early stage but, unfortunately, this occurs in less than
50% of the cases. For many years, efforts have been
performed to identify, in serum or biological fluid, bio-
markers with adequate diagnostic accuracy for CCA,
which could also be useful for population screening or
for the surveillance of disorders at risk, including primary
sclerosing cholangitis (PSC). Unfortunately, these efforts
are complicated by the relative rarity of this neoplasm and
the frequent occurrence, especially in the extrahepatic
form, of cholestasis and cholangitis, which represents
confounding variables in identifying biomarkers specific
for CCA.
Serum tumour markers
Serum tumour markers are attractive because of the ease
of obtaining samples and their relative low cost. There-
fore, they have been the object of extensive investigation
0267-1379 ß 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins
markers has reached adequate specificity for CCA
[1

,2–4,5

,6
,7].
Carcinoembryonic antigen (CEA), which is mainly used
for colorectal cancers, is of scarce utility being increased
only in approximately 30% of patients with CCA [1

,2–
4,5

,6
,7]. The carbohydrate antigen 19-9 is still the most
widely used serum marker for CCA that, however, is
useless in 7% of the population who are Lewisa-antigen
negative. Carbohydrate antigen 19-9 is elevated in pan-
creatic cancer, gastric cancer and primary biliary cirrhosis
and, in addition, it is increased in smokers and during
cholangitis or cholestasis associated with benign biliary
disorders. Recently, Ong et al. [8] investigated the fre-
quency of a false-positive increase in carbohydrate anti-
gen 19.9 and the correlation between carbohydrate anti-
gen 19.9 and serum bilirubin concentration in patients
investigated for supposed biliary malignancies. In a retro-
spective review, 83 consecutive patients presenting
with an abnormal carbohydrate antigen 19.9 (range 38–
10 000 kU/l) and radiological or clinical features sugges-
tive of biliary malignancy, but subsequently shown to
have benign disease, were investigated. These cases
DOI:10.1097/MOG.0b013e328325a894

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 280 Biliary tract
represented 10% of 819 patients, referred to the inves-
tigating centre with an abnormal carbohydrate antigen
19.9 level and radiological or clinical features suggestive
of malignancy. In a multivariate regression analysis, bili-
rubin was identified as an independent variable predict-
ing the carbohydrate antigen 19.9 level. Very importantly,
there was a 44% reduction in the median carbohydrate
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antigen 19.9 level and median bilirubin following inter-
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vention for bile drainage. This study adds further con-
troversy to the discussion about the diagnostic accuracy of
carbohydrate antigen 19-9 for CCA. In general, as exten-
sively discussed in recent reviews [1

,2–4,5

,6
,7], a
large range of sensitivity and specificity have been
reported for carbohydrate antigen 19-9 in patients with
CCA depending on the study population and cutoff
values and this is also true for CCA complicating PSC.
Charatcharoenwitthaya et al. [9
] retrospectively evalu-
ated the diagnostic performance of serum carbohydrate
antigen 19-9 in 230 PSC patients screened for develop-
ment of CCA and followed for 7 years (23 CCA detected).
At the optimal cutoff value (20 U/ml), a sensitivity of
78%, specificity of 67%, positive predictive value (PPV)
of 23% and negative predictive value (NPV) of 96% were
found. Higher cutoff values markedly decreased sensi-
tivity and, importantly, allowed CCA detection only at
advanced stages. When the combination of serum carbo-
hydrate antigen 19-9 with imaging techniques was eval-
uated, either computed tomography, MRI, magnetic
resonance cholangiopancreatography (MRCP) or endo-
scopic retrograde cholangiopancreatography (ERCP) was
found to show the best sensitivity (100%) but with a low
specificity (40%), whereas the combination of serum
carbohydrate antigen 19-9 and ultrasound had intermedi-
ate specificity (62%) with a good sensitivity (91%) for
detecting cancer. On the basis of test properties, cost and
availability, combination of serum carbohydrate antigen
19-9 (cutoff value of 20 U/ml) and abdominal ultrasound
at 12-month intervals was proposed as useful strategy for
screening/surveillance of CCA in PSC. Unfortunately,
this proposal was based on 23 cancers, of which nine were
found on initial screening and only 14 detected during
surveillance. Therefore, prospective trials to validate
these results are necessary.
Current efforts aim to identify novel serum markers for
CCA, which can substitute carbohydrate antigen 19-9 or
can improve (when measured together) the diagnostic
accuracy of carbohydrate antigen 19-9. Previous studies
[1

,2–4,5

,6
,7,10,11
,12] investigated several bio-
chemical markers harbouring determinants different
from the Lewisa antigen, including carbohydrate antigen
50, carbohydrate antigen 242, carbohydrate antigen 195
and detection of pancreatic cancer associated antigen 2
(DU-PAN-2) as well as des-g-carboxy prothrombin, pan-
creatic polypeptide, biliary fibronectin and lactate in
serum or bile but none of these markers have been
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
demonstrated to be satisfactory in terms of diagnostic
accuracy for CCA.
Serum interleukin (IL)-6 level has already been proposed
as a biomarker for CCA since 1998 and has been sub-
sequently suggested, when measured simultaneously
with carbohydrate antigen 19-9, to significantly improve
the diagnostic accuracy of carbohydrate antigen 19-9
alone if high cutoff points had been used. Recently,
Cheon et al. [13
] prospectively measured serum IL-6
levels in 26 patients with CCA, 26 patients with hepa-
tocellular carcinoma and 23 healthy adults. Using a
25.8 pg/ml cutoff, serum IL-6 provided a diagnostic
sensitivity of 73% and a specificity of 92%; PPV and
NPV being 83 and 87%, respectively. Serum levels of
IL-6 were correlated with tumour burden in CCA
patients and, interestingly, 1 month after treatment with
photodynamic therapy, the mean IL-6 level decreased
significantly. Although these findings are encouraging, it
should be considered that serum IL-6 is also elevated in
many patients with hepatocellular carcinoma, benign
biliary disease and metastatic lesions, therefore limiting
its specificity.
Recently, Smith et al. [14] suggested that a high pre-
operative platelet–lymphocyte ratio (PLR), a proposed
index of systemic inflammation, represented a prognostic
marker for resected ampullary adenocarcinoma, being
survival shorter than in patients with low PRL (10.1
vs. >60 months). The same authors, Smith et al. [15]
also provided evidence that the PLR improves the pre-
dictive value of serum carbohydrate antigen 19-9 levels in
selecting patients with ampullary adenocarcinoma for
staging laparoscopy before surgery and, with this method,
21% of laparoscopies were avoidable. Although further
studies are required to validate these findings and to
demonstrate whether PLR is influenced by inflammatory
conditions such as cholangitis or cholestasis, the simple
evaluation of PLR could be taken into consideration as a
prognostic index of periampullary adenocarcinoma.
Tissue expression of tumour-associated trypsinogen has
been observed in several cancers, for example, ovarian,
pancreatic, gastric and colorectal cancer in which tryp-
sinogen-1 and 2 are encoded by the same genes as
the pancreatic trypsinogens, but with posttranslational
modifications. Lempinen et al. [16
] evaluated the diag-
nostic accuracy for CCA of serum trypsinogen-2,
measured by time-resolved immunofluorometric assay,
in comparison with carbohydrate antigen 19-9. Eighty-
four patients with either PSC, CCA or both, referred for
liver transplantation or other liver surgery, were inves-
tigated. Forty-six PSC patients were transplanted, and in
three patients, CCA was found incidentally, whereas
29 out of 38 patients with CCA were candidates for
surgery with eight of these patients having both PSC

and CCA. The area under the ROC (receiver-operating
characteristics) curve (AUC) for serum trypsinogen-2
was 0.804 in comparison with 0.613 for carbohydrate
antigen 19-9. Serum trypsinogen-2 also showed the high-
est accuracy for differentiation between PSC with or
without CCA with an AUC value of 0.759. Serum tryp-
sinogen-2 levels were unaffected by concomitant inflam-
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matory bowel diseases, and no correlation was found
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between serum bilirubin levels and serum trypsino-
gen-2, indicating that this marker was unaffected by
the severity of cholestasis as, in contrast, occurred in
this and other studies [8] for carbohydrate antigen 19-9.
In recent years, measurements of the serum levels of
different mucins have been investigated as biomarkers
for various cancers. As discussed in a recent review [17],
mucins are heavily O-glycosylated proteins mainly
expressed by ductal and glandular epithelial tissues as
transmembrane or secreted gel-forming mucins, with
mucin genes expressed in a cell and tissue-specific man-
ner. As many CCA are mucin-producing adenocarcino-
mas, the aberrant expression of mucin5AC (MUC5AC) in
CCA tissues was, as expected, repeatedly demonstrated
and linked with a bad prognosis. More importantly,
mucin 1 (MUC1) and MUC5AC were not expressed
by hepatocellular carcinoma, suggesting a role in the
differential diagnosis. With these premises, serum
MUC5AC was demonstrated to be increased in CCA
when tested with agarose gel electrophoresis and immu-
noblotting. Recently, the same authors, Bamrungphon
et al. [18
], to detect serum MUC5AC in an easier and
cheaper way, developed a sandwich enzyme-linked
immunosorbent assay (ELISA). Only 0.1% (12 of 120)
of the controls, in contrast with 71% of 169 CCA patients,
had high level of serum MUC5AC and the mean value of
this marker was significantly elevated in CCA as com-
pared with gastrointestinal cancer patients, benign hepa-
tobiliary group and healthy controls. By using a cutoff
value of more than 0.074, the authors correctly identified
120 out of 169 CCA patients (71.01% sensitivity) and
excluded 108 out of 120 controls (90.0% specificity), with
90.01 and 68.79% PPV and NPV, respectively. Median
survival was worst in CCA patients with high serum
MUC5AC (158 days) compared with patients who had
a low level of serum MUC5AC (297 days). Therefore,
sandwich ELISA of serum MUC5AC seems to be prom-
ising and provides clinical value for diagnosis and prog-
nosis of CCA. MUC4 and MUC5AC were also investi-
gated by Matull et al. [19
] using real-time reverse
transcriptase-PCR (qPCR) and Western blot in serum
and bile samples prospectively collected from 72 patients
with biliary obstruction of different causes, including 39
CCA and seven PSC. Serum MUC5AC protein was
detected in 17 out of 39 (44%) CCA but not in the other
malignancies. When bile samples, aspirated proximally to
the stricture via ERCP, were analysed, 9 out of 34 (27%)
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Serum and bile biomarkers for cholangiocarcinoma Alvaro 281
CCA but none of other malignancies was positive for
MUC4 protein. In patients with either biliary MUC4-
positive or serum MUC5AC-positive Western blot
results, the median survival was 6.8 months compared
with 17.6 months in CCA patients with negative results.
In summary, this study indicates that biliary MUC4 and
serum MUC5AC are highly specific CCA-associated
mucins with prognostic values, although the sensitivity
is not currently satisfactory.
Several investigators reported high serum levels of
CYFRA21-1 (a soluble fragment of cytokeratin 19) in
patients with nonsmall-cell lung cancer or other cancers
(cervical, oesophageal, breast and gastric) in which this
biomarker also has a prognostic value. In 2003, Uenishi
et al. [20] showed that serum CYFRA 21-1 can adequately
differentiate intrahepatic CCA with respect to benign
liver disease or hepatocellular carcinoma. More recently,
the same authors, Uenishi et al. [20] measured CYFRA
21-1 in the sera of patients with intrahepatic CCA and
evaluated its prognostic significance. Seventy-one
patients who underwent laparotomy for intrahepatic
CCA were compared with 90 patients with nonmalignant
liver diseases. After excluding operative mortality, 55
CCA patients were followed up from surgery until death.
The AUC (CCA vs. benign conditions) was higher for
CYFRA 21-1 than CEA or carbohydrate antigen 19-9
(0.901, 0.779 and 0.794, respectively). At the optimal
cutoff value of 2.7 ng/ml, CYFRA 21-1 had a sensitivity
of 74.7% and a specificity of 92.2% for CCA. The 3-year
recurrence-free survival rates for patients with high and
low concentrations of CYFRA21-1 were 25.0 and 76.2%,
respectively. On multivariate analysis, a high concen-
tration of CYFRA21-1 was independently associated with
both recurrence and death after surgery. Thus, also in
intrahepatic CCA, as in other cancers, CYFRA 21-1 seems
to be a good prognostic serum marker for patients with
intrahepatic CCA.
Research on cancer biomarkers is currently an important
field in serum proteomic. By using surface-enhanced
laser desorption/ionization (SELDI) technology, an
increasing number of cancer-related biomarkers for diag-
nosis, progression and prognosis are currently under
investigation [21

]. Identification of these candidates
also facilitates the development of traditional multipro-
tein antibody array for early detection of cancer. Liu et al.
[22] used SELDI technology to analyse serum samples
from 56 CCA, 49 hepatobiliary diseases, 269 other cancers
and 53 healthy individuals. The candidates were isolated
and identified by SDS-polyacrylamide gel electrophor-
esis (PAGE), electrospray ionization tandem mass spec-
trometry (ESI/MS–MS), Western blot and immunopre-
cipitation. Three peaks (13.76, 13.88 and 14.04 k m/z) in
sera were significantly decreased in CCA compared with
the control groups, and these peaks were identified as
 282 Biliary tract
native transthyretin (TTR) and its two variants. TTR is a
55-kDa homotetrameric protein involved in the transport
of thyroid hormones in the blood synthesized partly from
the liver and partly from extrahepatic tissues. ELISA
assay indicated that TTR levels were consistent with
SELDI analysis and were significantly decreased in CCA
compared with healthy controls and benign hepatobiliary
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diseases. By combining TTR with carbohydrate antigen
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19-9 to differentiate CCA from benign hepatobiliary
diseases, a sensitivity and specificity of 98.2 and 100%
were reached suggesting levels of TTR as complemen-
tary markers of carbohydrate antigen 19-9 in diagnosing
CCA. We should, however, consider that the levels of
TTR could be decreased in cases of severe liver disease,
malnutrition and acute inflammation and, in addition,
TTR was found to decline in the sera of patients with
ovarian, cervical and endometrial carcinomas.
The proteomic profile of serum samples from CCA
patients was also analysed by Scarlett et al. [23] on
hydrophobic protein chips using SELDI time-of-flight
mass spectrometry (SELDI–TOF-MS). Serum from
patients with CCA (n ¼ 20) was compared with patients
with benign disease (n ¼ 20) and healthy volunteers
(n ¼ 25). Univariate analysis revealed 12 individual
peaks differentially expressed between CCA and sera
of healthy volunteers. The 4462 mass-to-charge serum
peak had superior discriminatory ability with respect to
carbohydrate antigen 19.9 and CEA with an AUC of
0.76, 0.73 and 0.70, respectively. Serum results were
further improved with the inclusion of carbohydrate
antigen 19.9 and CEA (AUC ¼ 0.86 and 0.99 for CCA
vs. benign and healthy serum, respectively). In con-
clusion, proteomic of biomarker panels in serum seems
to have important diagnostic implications for unknown
bile duct stricture.
Bile markers
Given the challenges associated with serum markers, bile
was investigated as a logical alternative fluid for identify-
ing CCA-specific biomarkers. Bile is, in fact, more prox-
imal than serum to the tumour and its flow through the
biliary tree should favour the enrichment of CCA-derived
products. Unfortunately, collection of bile samples could
only be performed by using invasive technique and
therefore the use for screening purposes or surveillance
of population at risk is limited.
Different studies [24–27] have recently shown that
human CCA tissues express insulin-like growth factor
1 (IGF1) and vascular endothelial growth factor (VEGF),
two growth factors involved in cancerogenesis and the
growth and spreading of different cancers. The neoplastic
cells secrete IGF1 and VEGF, and high serum levels of
IGF1 are associated with an increased risk for prostate,
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
breast, pancreatic, lung and colorectal cancer, with prog-
nostic significance in some cases. With this in mind, we
[28

] recently measured IGF1 and VEGF in bile of
patients undergoing ERCP for biliary obstruction and
evaluated the performance of these markers in differen-
tiating extrahepatic CCA from pancreatic cancer or
benign biliary abnormalities. The biliary IGF1 concen-
tration was 15–20-fold higher in extrahepatic CCA
(n ¼ 29) than in pancreatic cancer (n ¼ 19) or benign
biliary disorders (n ¼ 25) and the AUC was 1 (no data
overlap). In contrast, biliary VEGF concentration was
similar in the three groups. This study indicated a marker
(bile IGF1), which could be used, with absolute diag-
nostic accuracy, in patients undergoing ERCP for biliary
obstruction, to differentiate extrahepatic CCA from
either pancreatic cancer or benign biliary disorders.
Recent findings have shown a marked upregulation of
IGF-binding protein 5 (IGFBP5) gene in CCA adding
new support to the role of the IGF1 system in the biology
of CCA [29]. Unfortunately, applications of biliary IGF1
in the screening of CCA or in the surveillance of popu-
lation at risk cannot be proposed given that ERCP is an
invasive manoeuvre. However, if IGF1 could be
measured with the same accuracy in bile-rich duodenal
fluid collected during common upper endoscopy or by
using enteric capsule, this could lead to this test being a
proposal on a large scale.
Pancreatic elastase-3B is a 29-kDa serine protease that
hydrolyzes many extracellular matrix proteins, has a
digestive function in the intestine and is basically iden-
tical to pancreatic elastase 1 with cholesterol-binding
properties. High concentrations of serum elastase 1 were
found in pancreatic carcinoma and combined measure-
ment with carbohydrate antigen 19-9 or CEA was
suggested for the early detection of this cancer. Chen
et al. [30
] analysed fasting bile samples obtained from
patients with bile duct obstruction caused by either CCA
or gallstone and collected by endoscopic or percutaneous
transhepatic catheter drainage. Bile proteins were separ-
ated by two-dimensional electrophoresis and identified
by mass spectrometry. Biliary amylase activity was used
to correct for pancreaticobiliary reflux. The protein
expression, enzyme activity of pancreatic elastase and
pancreatic elastase/amylase ratio were increased in the
bile of CCA patients. The AUC was 0.877 and, using
0.065 as a cutoff value, the ratio distinguished malignant
from benign causes of biliary obstruction with a sensi-
tivity of 82% and a specificity of 89%. The authors [30
]
also identified mRNA for pancreatic elastase 3B (PE3B)
in CCA tissues demonstrating its origin from neoplastic
cells and, therefore, PE3B in bile was suggested as a
biomarker for CCA.
Minichromosome maintenance (MCM) replication
proteins are markers of dysplasia and have been utilized
 Serum and bile biomarkers for cholangiocarcinoma Alvaro 283

Table 1 Serum and bile biomarkers recently proposed for the diagnosis of cholangiocarcinoma
Biomarker Sensitivity (%) Specificity (%) Reference

Serum
Carbohydrate antigen 19-9 53–92a 50–98a [1

,2–4,5

,6
,7,8,9
]
CEA 33–68a 79–100a [1

,2–4,5

,6
,7,8,9
]
IL-6 73 92 [13
]
Trypsinogen-2b AUC ¼ 0.804 [16
]
MUC5AC 71.01 90 [18
]
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CYFRA 21-1 74.7 92.2 [20]

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TTR þ CA19-9 98.2 100 [22]

Bile
Carbohydrate antigen 19-9 46–61a 60–70a [1

,2–4,5

,6
,7,8,9
]
CEA 67–84a 33–80a [1

,2–4,5

,6
,7,8,9
]
IGF1 100 100 [28

]
Pancreatic elastase/amylase ratio 82 89 [30
]
Mcm5 62 92 [31]
Mac-2BPc 69 67 [21

]

Comparison with carbohydrate antigen 19-9 and CEA. AUC, area under the ROC curve; CCA, cholangiocarcinoma; CEA, carcinoembryonic antigen;
CYFRA 21-1, soluble fragment of cytokeratin 19; IGF1, insulin-like growth factor-1; IL-6, interleukin-6; Mac-2BP, tumour antigen 90K-binding protein;
Mcm5, minichromosome maintenance replication protein; MUC5AC, mucin5AC; PSC, primitive sclerosing cholangitis; ROC, receiver operating
characteristic; TTR, transthyretin.
a
The diagnostic performance of carbohydrate antigen 19-9 and CEA in serum and bile has been extensively discussed in different review articles
[1

,2–4,5

,6
,7,8,9
] referenced in the manuscript.
b
The authors reported an AUC of 0.804 for CCA vs. PSC and of 0.759 for CCA þ PSC vs. PSC.
c
Discussed in [21

].

for the diagnosis of cervical and bladder cancer. Ayaru
et al. [31], apart from tissue immunohistochemistry for
Mcm2 and 5, investigated Mcm5 levels by automated
immunofluorometric assay in bile samples prospectively
collected at ERCP from 102 consecutive patients with
biliary strictures of established (n ¼ 42) or indeterminate
causes (n ¼ 60). The authors [31] demonstrated that
Mcm5 levels in bile were more sensitive than brush
cytology (66 vs. 20%) for the detection of biliary malig-
nancy, but with a comparable PPV (97 vs. 100%). There-
fore, this study indicates that biliary Mcm5 is a more
sensitive indicator of biliary malignancy than routine
brush cytology (Table 1).
Proteomic studies in bile are challenging because this
fluid contains a number of contaminants and, therefore, a
variety of sample preparations, including delipidation,
desalination and nucleic acid removal, should be adopted
to remove interfering substances [21

]. Apart from deli-
pidation and desalination, Chen et al. [32] added soni-
cation to decrease the sample viscosity and background
smears caused by nucleic acids and, thereafter, performed
2D electrophoresis (2DE) for proteomic analysis of bile
from one CCA patient and a cholelithiasis control. A large
number of protein spots were separated in 2D maps from
the CCA and control bile, and approximately 20 spots
were differentially expressed in CCA vs. control bile. The
relevance of this study is to have validated a reliable
sample preparation process suitable for 2DE of bile fluid,
and to have indicated the remarkably different proteomic
presentation between the benign and malignant bile.
Other methods of bile sample preparation for proteomic
analysis include prefractionation strategies to induce
enrichment for glycoproteins and concomitant removal
of albumin. By using this method, the biliary membrane
protein carcinoembryonic antigen-related cell adhesion
molecule 1 (CEACAM1), cancer-associated proteins such
as carbohydrate antigen 125, MUC2, Mac-2BP, lipocalin
2 and deleted in malignant brain tumour 1 have been
identified, as discussed in a recent review [21

]. Most
importantly, these methods allow the identification of
the potentially useful biliary Mac-2BP, which has been
previously proposed as a useful biomarker for CCA
especially if combined with bile carbohydrate antigen
19-9 and this has discussed in a recent review [21

].
Recently, Albiin et al. [33] evaluated (1)H magnetic
resonance spectroscopy [(1)H-MRS] of bile from 45
patients (16 CCA, 18 PSC and 11 benign biliary disorders)
and demonstrated that CCA differed from the benign
group in the levels of phosphatidylcholine, bile acids,
lipid and cholesterol. With this method, it was possible to
distinguish CCA from benign conditions with sensitivity,
specificity and accuracy of 88.9, 87.1 and 87.8%, respect-
ively, although how much of these changes depends
on the cancer rather than on coincident variables (i.e.
cholestasis, liver damage, etc.) remains to be clarified.
Conclusion
Although a number of new serum and biliary biomarkers
have been recently proposed for CCA, further confir-
mation is need to support their applicability in a clinical
setting. Most importantly, we are still searching for a
specific and sensitive serum marker to be used for popu-
lation screening. As far as bile is concerned, some bio-
markers seem to be very promising, including IGF1,
elastase and some mucins, but these biomarkers are
useful to discriminate between benign and malignant
biliary conditions, only in patients submitted to bile
drainage. Thus, a significant progress could be the

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
 284 Biliary tract
validation of these biomarkers in bile samples collected
by less-invasive procedures, including bile-rich fluid col-
lected during upper endoscopy or with the aid of an
enterocapsule. Only this progress may lead to measure-
ments of biliary markers for surveillance of CCA.
Acknowledgements
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D. Alvaro is supported by MIUR grant numbers, PRIN #2007 and prot.
2007HPT7BA_003.
We thank Tracie Dornbusch for English editing.
There are no conflicts of interest.
References and recommended reading
Papers of particular interest, published within the annual period of review, have
been highlighted as:

of special interest


of outstanding interest
Additional references related to this topic can also be found in the Current
World Literature section in this issue (pp. 297–298).
1 Blechacz B, Gores GJ. Cholangiocarcinoma: advances in pathogenesis,


diagnosis, and treatment. Hepatology 2008; 48:308–321.
An excellent and extensive review on the modern aspects of the pathogenetic and
clinical aspects of CCA.
2 Blechacz BR, Gores GJ. Cholangiocarcinoma. Clin Liver Dis 2008; 12:131–
150.
3 Khan SA, Miras A, Pelling M, Taylor-Robinson SD. Cholangiocarcinoma and
its management. Gut 2007; 56:1755–1756.
4 Patel T, Singh P. Cholangiocarcinoma: emerging approaches to a challenging
cancer. Curr Opin Gastroenterol 2007; 23:317–323.
5 Yachimski P, Pratt DS. Cholangiocarcinoma: natural history, treatment, and


strategies for surveillance in high-risk patients. J Clin Gastroenterol 2008;
42:178–190.
An excellent review on the natural history, treatment and strategies of CCA in high-
risk patients.
6 Morris-Stiff G, Bhati C, Olliff S, et al. Cholangiocarcinoma complicating

primary sclerosing cholangitis: a 24-year experience. Dig Surg 2008;
25:126–132.
This study describes the prevalence and outcome of CCA arising in patients with
PSC.
7 Schulick RD. Primary sclerosing cholangitis: detection of cancer in strictures.
J Gastrointest Surg 2008; 12:420–422.
8 Ong SL, Sachdeva A, Garcea G, et al. Elevation of carbohydrate antigen 19.9
in benign hepatobiliary conditions and its correlation with serum bilirubin
concentration. Dig Dis Sci 2008; 53:3213–3217.
9 Charatcharoenwitthaya P, Enders FB, Halling KC, Lindor KD. Utility of serum

tumor markers, imaging, and biliary cytology for detecting cholangiocarcinoma
in primary sclerosing cholangitis. Hepatology 2008; 48:1106–1117.
The authors proposed the combination of serum carbohydrate antigen 19-9 (cutoff
value of 20 U/ml) and abdominal ultrasound at 12-month intervals as useful
strategy for screening/surveillance of CCA in PSC.
10 Yubin L, Chihua F, Zhixiang J, et al. Surgical management and prognostic
factors of hilar cholangiocarcinoma: experience with 115 cases in China. Ann
Surg Oncol 2008; 15:2113–2119.
11 Kitiyakara T, Chapman RW. Chemoprevention and screening in primary

sclerosing cholangitis. Postgrad Med J 2008; 84:228–237.
This review summarizes the incidence of malignancies in patients with PSC and
discusses potential strategies for its early detection.
12 Tischendorf JJ, Geier A, Trautwein C. Current diagnosis and management of
primary sclerosing cholangitis. Liver Transpl 2008; 14:735–746.
13 Cheon YK, Cho YD, Moon JH, et al. Diagnostic utility of interleukin-6 (IL-6)

for primary bile duct cancer and changes in serum IL-6 levels following
photodynamic therapy. Am J Gastroenterol 2007; 102:2164–2170.
This study describes that the elevation of IL-6 serum has a good sensitivity and
specificity for CCA and that IL-6 decreased after photodynamic therapy. This
observation may have implications in terms of diagnosis as well in the evaluation of
response to treatment.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
14 Smith RA, Ghaneh P, Sutton R, et al. Prognosis of resected ampullary
adenocarcinoma by preoperative serum CA19-9 levels and platelet-lympho-
cyte ratio. J Gastrointest Surg 2008; 12:1422–1428.
15 Smith RA, Bosonnet L, Ghaneh P, et al. The platelet-lymphocyte ratio
improves the predictive value of serum CA19-9 levels in determining patient
selection for staging laparoscopy in suspected periampullary cancer. Surgery
2008; 143:658–666.
16 Lempinen M, Isoniemi H, Mäkisalo H, et al. Enhanced detection of cholan-

giocarcinoma with serum trypsinogen-2 in patients with severe bile duct
strictures. J Hepatol 2007; 47:677–683.
In this study, serum trypsinogen-2 discriminates between PSC with or without
CCA and was unaffected by the severity of cholestasis suggesting that this marker
should be tested for the surveillance of patients with PSC.
17 Duraisamy S, Ramasamy S, Kharbanda S, et al. Distinct evolution of the
human carcinoma-associated transmembrane mucins, MUC1, MUC4 AND
MUC16. Gene 2006; 373:28–34.
18 Bamrungphon W, Prempracha N, Bunchu N, et al. A new mucin antibody/

enzyme-linked lectin-sandwich assay of serum MUC5AC mucin for the
diagnosis of cholangiocarcinoma. Cancer Lett 2007; 247:301–308.
The authors developed a sandwich ELISA of serum MUC5AC, which seems
promising for the diagnosis and monitoring of CCA patients.
19 Matull WR, Andreola F, Loh A, et al. MUC4 and MUC5AC are highly specific

tumour-associated mucins in biliary tract cancer. Br J Cancer 2008; 98:
1675–1681.
This study indicates that biliary MUC4 and serum MUC5AC are highly specific
CCA-associated mucins with prognostic values, although the sensitivity is not
currently satisfactory.
20 Uenishi T, Yamazaki O, Tanaka H, et al. Serum cytokeratin 19 fragment
(CYFRA21-1) as a prognostic factor in intrahepatic cholangiocarcinoma. Ann
Surg Oncol 2008; 15:583–589.
21 Bonney GK, Craven RA, Prasad R, et al. Circulating markers of biliary


malignancy: opportunities in proteomics? Lancet Oncol 2008; 9:149–158.
An excellent review focusing on the recent advances on proteomic techniques
applied to identify CCA biomarkers.
22 Liu L, Wang J, Liu B, Dai S, et al. Serum levels of variants of transthyretin down-
regulation in cholangiocarcinoma. J Cell Biochem 2008; 104:745–755.
23 Scarlett CJ, Saxby AJ, Nielsen A, et al. Proteomic profiling of cholangiocarci-
noma: diagnostic potential of SELDI-TOF MS in malignant bile duct stricture.
Hepatology 2006; 44:658–666.
24 Alvaro D, Mancino MG. New insights on the molecular and cell biology of
human cholangiopathies. Mol Aspects Med 2008; 29:50–57.
25 Mancino A, Mancino MG, Glaser SS, et al. Estrogens stimulate the prolifera-
tion of human cholangiocarcinoma by inducing the expression and secretion
of vascular endothelial growth factor. Dig Liver Dis 2009; 41:156–163.
26 Alvaro D, Mancino MG, Glaser S, et al. Proliferating cholangiocytes: a
neuroendocrine compartment in the diseased liver. Gastroenterology
2007; 132:415–431.
27 Alvaro D, Barbaro B, Franchitto A, et al. Estrogens and insulin-like growth
factor 1 modulate neoplastic cell growth in human cholangiocarcinoma. Am J
Pathol 2006; 169:877–888.
28 Alvaro D, Macarri G, Mancino MG, et al. Serum and biliary insulin-like growth


factor I and vascular endothelial growth factor in determining the cause of
obstructive cholestasis. Ann Intern Med 2007; 2:451–459.
This study demonstrated that bile IGF1 levels is absolutely sensitive and specific
for extrahepatic CCA suggesting a role of this biomarkers in the differential
diagnosis of patients submitted to ERCP for obstructive cholestasis.
29 Nishino R, Honda M, Yamashita T, et al. Identification of novel candidate
tumour marker genes for intrahepatic cholangiocarcinoma. J Hepatol 2008;
49:207–216.
30 Chen CY, Tsai WL, Wu HC, et al. Diagnostic role of biliary pancreatic elastase

for cholangiocarcinoma in patients with cholestasis. Clin Chim Acta 2008;
390:82–89.
The study indicates that ratio of pancreatic elastase/amylase in bile distinguish
malignant from benign causes of biliary obstruction with a good sensitivity and
specificity.
31 Ayaru L, Stoeber K, Webster GJ, et al. Diagnosis of pancreaticobiliary
malignancy by detection of minichromosome maintenance protein 5 in bile
aspirates. Br J Cancer 2008; 6:1548–1554.
32 Chen B, Dong JQ, Chen YJ, et al. Two-dimensional electrophoresis for
comparative proteomic analysis of human bile. Hepatobiliary Pancreat Dis
Int 2007; 6:402–406.
33 Albiin N, Smith IC, Arnelo U, et al. Detection of cholangiocarcinoma with
magnetic resonance spectroscopy of bile in patients with and without primary
sclerosing cholangitis. Acta Radiol 2008; 18:1–8.