Clinical History
This 42 year old woman underwent liver transplantation for primary sclerosing cholangitis.
She had a long history of ulcerative colitis and was diagnosed with PSC seven years after colectomy for
UC. Cholangiogram showed beading of large bile ducts, with a dominant stricture near the hepatic hilum.
The patient underwent orthotopic liver transplantation. This section is from near the hilum of her
native liver.
Morphologic Findings
Most of the tissue on the slide provided is connective tissue and large nerve
bundles from the hepatic hilum; a small portion of hepatic parenchyma is present. A large bile duct with
a fibrotic and necrotic wall is embedded in the hilar tissue. The epithelial lining of this duct is
flattened and sloughed; the residual cells have a high nuclear to cytoplasmic ratio. More diagnostic are
the distorted glands with poorly formed lumens and single atypical cells infiltrating the wall of the
duct. These rudimentary glandular structures are composed of cells with pleomorphic, vesicular,
sometimes angular nuclei. The tumor infiltrates the dense fibrosis of the hilum and perineural invasion
is present. The adjacent liver is also involved by tumor and in addition shows concentric periductal
fibrosis typical of primary sclerosing cholangitis. In other sections of liver taken near the hilum,
dysplastic papillary mucosal lesions were present in large bile ducts.
Click on each slide thumbnail image for an enlarged view:
Slide 23
Perihilar Cholangiocarcinoma Arising in Primary Sclerosing Cholangitis
The typical cholangiocarcinoma is well to moderately differentiated, and the tumor cells closely resemble biliary epithelium. A dense desmoplastic stroma usually accompanies the tumor, even in cases not arising in primary sclerosing cholangitis.
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Slide 24
Perineural Invasion in Cholangiocarcinoma
Peri- and intraneural spread is common in cholangiocarcinomas and may serve as a helpful clue to diagnosis of malignancy in small specimens.
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Diagnosis: Hilar Cholangiocarcinoma Arising in Primary Sclerosing Cholangitis
Clinical Course
Tumor recurrence at the hepatic hilum of the transplanted liver occurred within one
year of transplantation. The patient died of complications of the tumor nine months later.
Cholangiocarcinoma, the second most frequent primary hepatic malignancy, makes up from 5 to 30% of
malignant hepatic tumors. Although several classification schemes for these malignant bile duct tumors
have been proposed, the most widely accepted divides these lesions into two broad categories,
intrahepatic (peripheral), the most common type worldwide ;1 and hilar (central). This division is
supported by the different clinical presentations and surgical strategies associated with these
locations. The term "cholangiolocarcinoma" is reserved by some investigators for intrahepatic tumors
confined to the liver and not involving the extrahepatic biliary tree. Hilar tumors, the majority of
surgically treated cholangiocarcinomas in most series from the United States ,2 are further subdivided
based on the duct involved, or the position of the neoplasm along the common bile duct. An alternative
proposed classification based on anatomy and preferred surgical treatment divides cholangiocarcinomas
into intrahepatic, perihilar, and distal tumors .2 In this classification, perihilar tumors involve
the hepatic duct bifurcation. Distal tumors involve the distal extrahepatic or intrapancreatic portion
of the common bile duct.
Cholangiocarcinoma and PSC
The development of cholangiocarcinoma is a major complication of PSC,
seen in up to 16% of PSC patients. Risk factors for development of cholangiocarcinoma in PSC are not
well defined: age, gender, smoking history, indices of disease severity, and the presence or duration of
inflammatory bowel disease have not been shown to have predictive value .3 In one report, alcohol
consumption by PSC patients appeared to be associated with development of cholangiocarcinoma .3
Accurate diagnosis of cholangiocarcinoma remains a problem in many cases, as tumor may be
indistinguishable from stricture on cholangiogram and accurate cytologic diagnosis from bile duct
brushings may be exceedingly difficult. Elevated CA19-9 levels, if greatly elevated, may be of utility,
although considerable overlap with PSC without cancer is seen .4
Molecular Mechanisms of Oncogenesis in Cholangiocarcinoma
Molecular mechanisms involving both cell proliferation and apoptosis appear to be involved in the
pathogenesis of cholangiocarcinoma. Common features of factors predisposing to cholangiocarcinoma
include chronic inflammation and/or cholestasis, with production of cytokines during inflammation
resulting in production of nitric oxide by macrophages. NO and its derivatives can induce DNA damage by
deaminating DNA-nucleobases and causing DNA strand breaks. In addition, downregulation of the GSH and
UGT pathways of cellular detoxification has been demonstrated in cholangiocarcinomas. These pathways
remove damaging cellular carcinogens, and their down-regulation predisposes to genetic alterations .5
Comparative genomic hybridization studies show distinctive features of genetic alterations in
intrahepatic cholangiocarcinoma compared to hepatocellular carcinoma, with intrahepatic
cholangiocarcinoma showing changes similar to pancreatic and colorectal carcinomas, in the form of gains
of 5p, 7p, 13q, and 20q .6 K-ras and p53 mutations are found both in sporadic cholangiocarcinomas and
those arising in primary sclerosing cholangitis .7 K ras mutations detected in bile of PSC patients
may represent an early event in carcinogenesis but cannot be used for diagnosis, as most patients with
positive findings in one study remained tumor free on follow up .8 Aberrant CpG island methylation
contributes to carcinogenesis in ~80% of intrahepatic cholangiocarcinomas .9
| Gene | Anormalities in cholangiocarcinoma |
| c-myc | Expressed in up to 95% |
| Ki-ras | Mutated/activated in 80-90% |
| C-erb-B-2 | Expressed in 75% |
| APC | Allelic loss in 30% |
| bcl-2 | Expressed in 60-70% |
| p53 | Allelic loss or mutation in 40% |
Utility of Serum CA 19-9 in Diagnosis of Cholangiocarcinoma
Serum CA 19-9 levels are in general higher in patients with cholangiocarcinoma, with or without
primary sclerosing cholangitis, than in those with benign biliary strictures or non-malignant liver
disease .3,10 However, there is considerable overlap in serum levels for patients with PSC with and
without cholangiocarcinoma, and a negative test is not helpful in excluding malignancy. Patients without
PSC who have unresectable cholangiocarcinoma typically have much higher CA 19-9 levels (mean ~15,000
U/ml) than those with lower stage disease (344 U/ml) .10 CA 19-9 levels may be in the thousands in
patients with bacterial cholangitis; however, the serum level will decrease rapidly with resolution of
the infection. Ca 19-9 has not be shown to be useful in establishing malignancy in patients undergoing
pre-transplant evaluation .11 Serum CEA is elevated in about 40% of PSC patients with
cholangiocarcinoma but is less sensitive and specific than CA 19-9 .12
Central/hilar (perihilar) Cholangiocarcinoma
These tumors share many etiologic associations, such as primary sclerosing cholangitis and ulcerative
colitis, fibropolycystic liver diseases, and parasite infestation, with intrahepatic cholangiocarcinoma.
The incidence of cholangiocarcinoma in patients with primary sclerosing cholangitis is estimated at 8% to
16%. In contrast to most patients with intrahepatic cholangiocarcinoma, patients with perihilar tumors
usually present with jaundice and other evidence of large bile duct obstruction.
Gross and Microscopic Features
The typical gross appearance of perihilar cholangiocarcinomas is
dense white scar infiltrating the hepatic hilum and extending into the adjacent parenchyma. In cases of
sclerosing cholangitis, the presence of tumor on gross examination may be obscured by dense fibrosis.
The bile duct may be encircled and thickened by dense desmoplastic tumor. In some cases, the tumor is
papillary and protrudes into the lumen of the bile duct. In general the microscopic appearance is
similar to that of intrahepatic cholangiocarcinoma, with most of the tumors composed of small well-formed
ducts. Desmoplasia is a prominent feature in many perihilar cholangiocarcinomas, and perineural invasion
is commonly found. The differential diagnosis included benign reactive changes and bile ductular
proliferation; in patients with biliary stents, diagnosis may be particularly difficult because of the
significant degree of cellular atypia associated with reactive change in bile duct epithelium.
Prognostic Factors
Incomplete resection and positive regional lymph nodes appear to be the two most
important factors predictive of shortened survival .13-15 Although univariant analysis has shown
various factors such as tumor grade and size to be significant prognostic factors in hilar
cholangiocarcinoma, multivariant analysis in several studies showed only residual tumor stage after
surgery and the presence of lymph node metastases to be of independent statistical significance (1 4,1
5). Hepatic resection may improve survival .16 Other investigators report that histologic grade
influences survival ,13 ,16 with patients with well differentiated carcinomas having a median
survival of 58 months, compared to 9 months for patients with poorly differentiated tumors .13
Perineural invasion, present in 36 of 43 cases, was not shown to be an independent prognostic factor
(10), probably because of its high prevalence in these tumors. Two studies have shown that high total
bilirubin concentration preoperatively is a poor prognostic indicator .13,17 In a series of 207
patients with cholangiocarcinoma who underwent liver transplantation, 5-year survival estimate was 23%.
Survival after recurrence was typically less than 1 year. Incidentally found cholangiocarcinomas carried
the same poor prognosis as those with known malignancies prior to transplantation .18
Stage
Perihilar cholangiocarcinoma is staged using a tumor/node/metastasis (TNM) classification
scheme devised by the American Joint Commission on Cancer for staging extrahepatic bile duct carcinomas
(1 9). Stage I tumors are confined to the bile duct, while Stage II tumors have spread to periductal
tissues or have regional lymph node metastases. Stage III tumors invade large regional vessels such as
the portal vein or its main branches bilaterally , the common hepaticartery, or other adjacent structures such as colon , stomach, and duodenum. Stage IV tumors have
evidence of distant metastases.
Staging of Perihilar Cholangiocarcinoma 19
| TNM Definitions |
| Primary Tumor |
| T1 | Tumor confined to the bile duct histologically |
| T2 | Tumor invades beyond the wall of the bile duct |
| T3 | Tumor invades the liver, gallbladder, pancreas, and/or unilateral branches of the portal vein or hepatic artery |
| T4 | Tumor invades any of the following: main portal vein or its branches bilaterall, dommon hepatic artery, or other adjacent structures such as colon, stomach, duodenum, or abdominal wall |
| Regional Lymph Nodes |
| N0 | No regional lymph node metastasis |
| N1 | Metastasis in cystic duct, pericholedocal, and/or perihilar lymph nodes |
| N2 | Metastasis in regional lymph nodes near duodenum or head of pancreas |
| Metastasis |
| M0 | No distant metastasis |
| M1 | Distant metastasis |
| Stage Grouping |
| Stage IA | T1, N0, M0 |
| Stage IB | T2, N0, M0 |
| Stage IIA | T3, N0, M0 |
| Stage IIB | T1, T2, or T3, N1, M0 |
| Stage III | T4, any N, M0 |
| Stage IV | Any T, any N, M1 |
Carcinoma of the Extrahepatic Bile Duct
is a relatively uncommon malignancy, and much less common
than carcinoma of the gallbladder. This tumor has a male preponderance and is more common in the
elderly. While a palpable mass may be evident at surgery, in many cases only diffuse thickening of the
bile duct wall is appreciated. Lesions of the confluence of the hepatic bile duct and upper common
hepatic duct account for over half of cases of extrahepatic biliary cancer. Lesions involving the middle
third of the common bile duct account for approximately 20%, as do cases involving the lower third of the
common bile duct. Over 95% of these tumors are adenocarcinomas, and most have an associated desmoplastic
stroma; when these tumors are well differentiated, frozen section diagnosis may be particularly
difficult, especially in the setting of stent placement and inflammation.
Diagnosis of Hilar Cholangiocarcinoma and Bile Duct Carcinoma by Endobiliary Brush Cytology
As
endoscopic cholangiogram techniques become more ever more sophisticated and widely used, cytologic
examination is used more and more in the evaluation of biliary strictures. Such specimens often pose
diagnostic challenges for even the experienced pathologist, much less those of us who rarely see these
difficult specimens. Key cytologic criteria for malignancy that have been identified by multiple
investigators include a background of tissue damage, nuclear overlap and crowding, irregular nuclear
membranes, nuclear molding, coarse chromatin pattern, and increased nuclear to cytoplasmic ratio .20,21
Small three- dimensional epithelial clusters with marked atypia are considered by some to be highly
suspicious for malignancy in brush smears .22 Rare small epithelial clusters with some overlapping at
the periphery but without marked cytologic atypia could be found in brushings from benign strictures in
this study .19 In general, sensitivity (37% to 85%) is lower than specificity (93% to 100%) .23,24
Some investigators report that sensitivity of bile duct brushings is higher for cholangiocarcinoma
(80%)than for pancreatic carcinoma (35.5%) .25 p53 immunostaining of bile duct brushings has been
advocated by some as a means of improving sensitivity .26 While false positive diagnoses are rare, a
negative result does not reliably exclude malignancy.
Peripheral or Intrahepatic Cholangiocarcinomas
The Liver Cancer Study Group of Japan has defined
peripheral cholangiocarcinoma as cholangiocarcinoma arising in a segmental duct or a more peripheral duct
.27
Etiology
The etiology of intrahepatic cholangiocarcinoma is usually unknown. However, these tumors
are associated with all forms of fibropolycystic liver disease, including the presence of multiple
biliary microhamartomas .1 Chronic inflammatory lesions of the bile ducts and conditions associated
with bile stasis also predispose to the development of intrahepatic cholangiocarcinoma; these conditions
include primary sclerosing cholangitis, parasitic infections with liver flukes such as Clonorchis and
Opisthorchis, and recurrent bacterial cholangitis with hepatolithiasis. Intrahepatic cholangiocarcinomas
have also been reported in association with exposure to Thorotrast 28 and have been associated with
anabolic steroid use. In contrast to hepatocellular carcinoma, most cases of intrahepatic
cholangiocarcinoma arise in a non-cirrhotic liver and are not associated with hepatitis B infection. In
one series of 85 intrahepatic cholangiocarcinomas, less than 5% were associated with non-biliary
cirrhosis. The cholangiocarcinomas in this series did not differ in morphologic features from
cholangiocarcinomas arising in non-cirrhotic livers, and displayed similar immunohistochemical staining
patterns with respect to carcinoembryonic antigen, CA19-9, DU-PAN-2, and biliary-type cytokeratins .29
Clinical Associations
The global incidence of intrahepatic cholangiocarcinoma, but not extrahepatic
biliary malignancies, appears to be increasing .30 Intrahepatic cholangiocarcinoma generally occurs
in older adults, with most patients between 50 and 70 years of age. The tumor is often clinically silent
until late in the course; patients typically complain of fever, weight loss, anorexia, and vague
abdominal pain. In contrast to hilar cholangiocarcinoma, patients with intrahepatic cholangiocarcinoma
rarely present with jaundice.
Prognostic Factors and Staging
Intrahepatic cholangiocarcinoma is staged using the same TNM
classification and stage grouping as hepatocellular carcinoma .19 Complete resection of the tumor
appears to be an important factor in prognosis in intrahepatic cholangiocarcinoma. Median survival for
resectable intrahepatic cholangiocarcinoma is as high as 30 months in some series, and the five year
survival ranges between 35% and 45% .2,31 Median survival for unresectable intrahepatic tumors is
only 6 to 7 months, even with adjuvant therapy. Tumor grade is significantly associated with outcome in
some series 32 but not others .31 In one series of 19 patients with intrahepatic
cholangiocarcinoma who underwent surgical resection, positive hilar lymph nodes were a poor prognostic
sign; most of these patients died within 9 months of surgery, in contrast to node-negative patients, who
had a median survival of over 36 months. Tumor grade and size in this small series had no effect on
survival .31
Gross and Microscopic Features
On gross examination, intrahepatic cholangiocarcinomas are
generally gray-white to tan masses; larger lesions may contain areas of central necrosis or, less
commonly, hemorrhage. Most tumors are firm because of the prominent desmoplastic stroma, which may be
gritty because of dystrophic calcifications. In general the intrahepatic cholangiocarcinoma consists of
a single non-encapsulated mass in a non-cirrhotic liver, although satellite lesions may be present. The
margins may be deceptively well circumscribed on gross examination, but microscopic examination shows
infiltrative borders. Rarely, involvement of portal or hepatic veins may be seen, and occasionally
intraductal growth occurs. Some investigators have subdivided intrahepatic cholangiocarcinomas based on
the pattern of growth, and report that tumors without biliary strictures behave more like hepatocellular
carcinomas, in that they are more likely to occur in a diseased liver and have frequent intrahepatic
spread without lymph node metastases .27
Most cholangiocarcinomas are adenocarcinomas; rarely, areas of squamous differentiation may be seen,
and sarcomatoid variants have been reported .33 Other variants include papillary adenocarcinoma,
found generally within larger ducts, and signet ring cell carcinoma. The most common microscopic pattern
is a well to moderately differentiated adenocarcinoma forming small tubular glands and duct-like
structures. The tumor cells are low cuboidal to columnar, with clear to eosinophilic cytoplasm and round
to oval nuclei. Intracellular mucin production may be scant, but is usually demonstrable with special
stains for mucin; typically a mixture of neutral and acidic mucins is found. A desmoplastic stroma is
generally prominent, but is not always present. Perineural and lymphovascular invasion is common, and
cholangiocarcinomas often involve portal tracts, either by spread within portal vein radicals or by
spread within the intrahepatic biliary tree. Bile ducts in adjacent portal tracts may demonstrate
varying degrees of epithelial dysplasia; however, it is usually not possible to identify a specific bile
duct of origin.
Differential Diagnosis
The primary challenge for the pathologist in diagnosing most intrahepatic
cholangiocarcinomas is distinction from metastatic adenocarcinoma. Primary sites producing tumors with
similar histology include pancreas, extra-hepatic biliary tree, breast, and occasionally lung.
Immunohistochemical stains are of limited use in distinguishing cholangiocarcinoma from other primaries,
and mucin stains are helpful only in distinguishing cholangiocarcinoma from hepatocellular carcinoma.
The distinction between cholangiocarcinoma and metastatic adenocarcinoma therefore depends heavily on the
exclusion of a primary site elsewhere. Comparative immunohistochemical studies suggest that cytokeratins
7 and 20 may be useful in some cases in distinguishing peripheral cholangiocarcinomas, which are
generally CK 7+/CK 20-, from colorectal metastases, which are usually CK 7-/CK 20+ .34,35 The
distinction between hepatocellular carcinoma and cholangiocarcinoma is usually more straightforward,
although there is some overlap in morphology and combined tumors do exist. Hepatocellular carcinomas
display a trabecular architecture with scant fibrous stroma, a distinctly different morphology from the
usual cholangiocarcinoma. In problematic cases, a panel of immunohistochemical stains can be employed to
distinguish between the two. Polyclonal or cross-reactive CEA will positivity in cholangiocarcinoma will
usually show a cytoplasmic staining pattern, without the "chicken wire" pattern of cross-reactivity to
biliary glycoprotein seen in hepatocellular carcinoma. Immunostain for a -fetoprotein is negative in
cholangiocarcinoma. Ultrastructural examination is seldom indicated, but electron microscopy of
cholangiocarcinoma cells shows typical features of adenocarcinoma, such as microvilli and true lumen
formation.
Differential Diagnosis of Cholangiocarcinoma 36
| Diagnosis | Distinguishing Features |
| Non-neoplastic reactive change in periductal glands | Cribriform glands, mitoses, isolated tumor cells in stroma, perineural invasion, nuclear atypia in cholangiocarcinoma |
Bile duct adenoma
Bile duct hamartoma | Small lesions, no mitoses, no nuclear atypia |
| Metastatic adenocarcinoma | CK7/CK20 panel has been suggested (29,30,34) |
| Hepatocellular carcinoma | HCC has trabecular architecture, minimal fibrous stroma; cross-reactive CEA; -fetoprotein; HepPar 1, albumin mRNA |
Intrabiliary growth of tumors metastatic to liver or large bile ducts may mimic cholangiocarcinoma.
In particular, metastasis from colorectal carcinoma may involve the large bile ducts, leading to
obstructive changes in the liver .38,39 Colorectal carcinoma has a propensity for growth along
mucosal surface, leading to the erroneous interpretation of origin of the tumor in dysplasia of primary
biliary neoplasia. Hepatocellular carcinoma may also present as an intraluminal mass involving a large
bile duct, at times posing diagnostic difficulties .40,41
Staging of Intrahepatic Cholangiocarcinoma 19
| TNM Definitions |
| Primary Tumor |
| T1 | Solitary tumor, without vascular invasion |
| T2 | Solitary tumor with vascular invasion or multiple tumors, none > 5 cm |
| T3 | Multiple tumors more than 5 cm, or tumor involving a major branch of the portal or hepatic vein(s) |
| T4 | Tumor with direct invasion of adjacent organs other than the gallbladder, or with perforation of the visceral peritoneum |
| Regional lymph nodes |
| N0 | No regional lymph node metastases |
| N1 | Regional lymph node metastases |
| Distant Metastases |
| M0 | No distant metastases |
| M1 | Distant metastases |
| Stage Grouping |
| Stage I | T1, N0, M0 |
| Stage II | T2, N0, M0 |
Stage IIIA
IIIB
IIIC | T3, N0, M0
T4, N0, M0
Any T, N1, M0 |
| Stage IV | Any T, any N, M1 |
Mixed Hepatocellular/cholangiocarcinoma
Occasional primary epithelial malignancies in the liver
will show divergent differentiation, with features of both cholangiocarcinoma and hepatocellular
carcinoma. Such tumors may be especially common in genetic hemochromatosis .42 These mixed tumors
assume one of two patterns, termed "collision tumors" and "transition tumors" by Goodman in one of the
earlier studies of this relatively rare entity .43 In the "collision tumor", different areas of the
neoplasm or separate tumor masses in the liver show different patterns of differentiation, with separate
areas of hepatocellular carcinoma and cholangiocarcinoma. The "transition tumors" show more intermixed
patterns. Combined hepatocellular/cholangiocarcinomas have been reported to share the same associations
with cirrhosis, hepatitis B, hepatitis C, and elevated a-fetoprotein levels as hepatocellular carcinomas.
However, a recent study using in situ hybridization for mRNA, a sensitive and specific marker for
hepatocellular differentiation , has shown that many tumors what would be classified by usual means as
cholangiocarcinomas are positive for albumin mRNA. The tumors in this series were not associated with
cirrhosis, hepatitis B, or hepatitis C 44 . Combined tumors have a poor prognosis and disseminate
widely, spreading to regional lymph nodes and distant organs. Metastases maintain the mixed pattern or
exhibit hepatocellular differentiation .45
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