Challenges in Diagnosing

and Reporting
C h o l a n g i o c a rc i n o m a
Tony El Jabbour, MDa, Attila Molnar, MDb,
Stephen M. Lagana, MDc,*

KEYWORDS
 Cholangiocarcinoma  Intrahepatic  iCCA  Small duct type iCCA  Large duct type iCCA
 Bile duct hamartoma  Biliary duct adenoma  cHCC-CCA

Key points
 Benign/pre-neoplastic lesions and bland looking subtypes/forms of cholangiocarcinomas share multi-
ple morphologic features. Usage of well-established microscopic criteria and ancillary tests along with
clinical and radiological data is crucial to avoid diagnostic pitfalls.
 Every option should be used when classifying poorly differentiated malignancies of the liver, as some
entities may have a specific therapeutic strategy or/and targeted therapy.
 Historically, combined hepatocellular-cholangiocarcinoma were difficult to define and to diagnose.
The last WHO classification of digestive tumors has simplified this concept rendering the diagnosis
and subsequent staging of combined hepatocellular-cholangiocarcinoma more feasible.

ABSTRACT neoplasms arising from the bile ducts and

I
showing biliary differentiation.1 Based on their
ntrahepatic cholangiocarcinoma is a challenge anatomical origin they can be classified as intra-
to the practicing surgical pathologist for several hepatic cholangiocarcinoma (iCCA), perihilar
reasons. It is rare in many parts of the world, cholangiocarcinoma (pCCA) or extrahepatic chol-
and thus practical exposure may be limited. angiocarcinoma (eCCA).1 This article is devoted
Related to the fact of its rarity is the fact that to iCCA and its mimics with a special emphasis
more common tumors which frequently metasta- on the challenges they can possibly bring to sur-
size to the liver can be morphologically indistin- gical pathologists.
guishable (eg, pancreatic ductal adenocarcinoma).
Immunohistochemical testing is generally non-
contributory in this context. Other difficulties arise HISTOLOGIC FEATURES OF INTRAHEPATIC
from the protean morphologic manifestations of CHOLANGIOCARCINOMAS
cholangiocarcinoma (ie, small duct vs. large
duct) and the existence of combined cholangio- iCCA is a type of invasive adenocarcinoma which
carcinoma and hepatocellular carcinoma. These, generally displays variable sized tubular, acinar,
and other issues of concern to the practicing diag- and or micropapillary structures.2–4 The malignant
nostic pathologist are discussed herein. cells are usually small to medium sized, cuboidal
more than columnar, and demonstrate clear or
eosinophilic cytoplasm, closely resembling biliary
OVERVIEW epithelial cells.2–4 The nuclei are frequently small,
and the nucleoli are not especially prominent. The tu-
Cholangiocarcinoma (CCA) is a heterogeneous mors may produce mucin, and do not produce bile.
surgpath.theclinics.com

group of tumors composed of malignant epithelial These characteristics are usually the opposite in
a
West Virginia University; b Mount Sinai Morningside and Mount Sinai West, Department of Pathology, 1000
Tenth Avenue, First floor, Room G183, New York, NY 10019, USA; c New York-Presbyterian /Columbia Univer-
sity, Irving Medical Center, 622 W168th St, Vc14-209, New York, NY 10032, USA
* Corresponding author.
E-mail address: [email protected]

Surgical Pathology 16 (2023) 599–608

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600 El Jabbour et al

hepatocellular carcinoma (HCC).5 Moreover, distinc- Assuming a malignant primary tumor of liver
tive features of invasion such as desmoplasia, peri- with biliary phenotype has been established, the
neural invasion and proximity of glands to larger 5th edition of WHO, has further categorized
arteries are common findings supporting the diag- iCCAs into small or large duct types.20 While the
nosis of malignancy.5,6 On a scant biopsy material etiology in many cases of iCCAs is unknown,
however, a few glands may only be available for eval- some risk factors seem to contribute more to
uation. In such cases, the main differential is the the formation of one type over to the other.
normal biliary glands, and the main goal is to estab- Furthermore, some of these risk factors
lish a diagnosis of malignancy. Multiple studies contribute to both subtypes or are shared with
attempted to provide guidance for such scenarios, HCCs or eCCAs, see Table 1.21–25 The character-
specifically when the above listed invasive features, istics and mimickers of each iCCA subtype will be
are absent.7–9 Immunohistochemistry is not typically detailed in the following sections.
useful in this distinction, although studies on the
topic are available. For example, the Ki 67 prolifera-
tion index may be considered, given benign glands INTRAHEPATIC CHOLANGIOCARCINOMA,
often have a Ki67 index less than 10% while in LARGE DUCT TYPE
some cases of iCCA this index is higher.10 Aberrant
(mutational) p53 immunoreactivity (strong diffuse Large duct type iCCA recapitulates the large intra-
staining or complete absence of staining) certainly hepatic ducts and the peribiliary glands.2 Histolog-
increases the likelihood that a proliferation is malig- ically, the tumor is formed by ductal or tubular
nant.11,12 However many iCCAs exhibit a normal mucin secreting glands appearing as large
(wild type) p53 staining pattern (scattered nuclear cancerous bile ducts with columnar to cuboidal
reactivity), and so p53 is meaningful only when it is epithelial lining and surrounding desmoplastic
clearly abnormal.13 Cytokeratins (CK), such as pan- stroma.26,27 Intracellular or luminal mucin secretion
CK cocktails, CK7, and CK19 strongly mark benign is significantly more frequent in large duct type
and malignant biliary proliferations (typically), but iCCA.28 One particular location is critical for these
they may be useful to highlight single cells within tumors: the intrahepatic portions of the right and
fibrotic stroma, or to better illustrate an invasive left hepatic ducts. Due to their proximity to the hilar
pattern. Identification of a rare single cell does not region, tumors arising in the intrahepatic portions of
entirely establish the diagnosis of malignancy either, the right and left hepatic ducts may present as hilar
as the possibility of benign entrapped cells and a masses, confounding a practicing pathologist to
glancing cut of the “start” of a duct profile need to incorrectly classify them as pCCA. However, the
be excluded.14–17 Loss of nuclear BAP1 occurs in correct classification of such tumors is iCCA large
16-32% of iCCA and virtually never in benign biliary duct type. The term perihilar cholangiocarcinoma
glands.18,19 Thus, sensitivity is poor, but specificity is reserved for the tumors arising from the extrahe-
is high (nearly 100%). Other malignancies may lose patic portion of the right or left hepatic ducts or the
BAP1, in particular mesothelioma, so the tumor common hepatic duct.4 The difference between
must be determined to be of biliary phenotype before these two anatomic locations is illustrated in
interpreting BAP1 loss by immunohistochemistry. Fig. 1. This differentiation is crucial since iCCAs

Table 1
Examples of subtype-specific risk factors for iCCA

Both Small and Large Duct Type

Small Duct Type iCCAa Large Duct Type iCCAb iCCA
HBV Caroli disease Asbestos
HCV Bile duct cyst Nitrosamine
Alcoholic liver disease Congenital hepatic fibrosis Dioxins
Obesity/Metabolic syndrome/ Opisthorchis viverrini/Clonorchis Vinyl chlorides
Diabetes sinensis
Hemochromatosis PSC Thorotrast
Wilson‘s disease Choledocholithiasis/Cholangitis
Hepatolithiasis

Abbreviations: HBV, hepatitis B virus; HCV, hepatitis B virus; PSC, primary sclerosing cholangitis.
a
Shared risk factors with HCC.
b
Shared risk factors with eCCA.

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 Challenges in Diagnosing Cholangiocarcinoma 601

be the hepatic progenitor cells and stem cells of

the canals of Herring or the stem cells of bile duct-
ules.2 As the name implies, small duct type iCCA
with DPM pattern are markedly different and
resemble ductal plate malformation.26,35 Fig. 2
summarizes the three patterns of small duct
iCCA in comparison to large duct iCCA. Isocytrate
dehydrogenase 1 (IDH1) mutation is displayed by
many carcinomas including iCCA.36 Multiple
studies show that small duct type iCCA more
frequently harbors IDH1 mutation than large duct
type iCCA, raising the possibility of using it as a
potential biomarker in the diagnostic pro-
cess.2,4,36–38 The gain of function mutation of
IDH1 results in the accumulation of D-2-hydroxy
glutarate (D-2HG).36,39 The accumulation of this
Fig. 1. Short arrow: Tumor of the right hepatic duct metabolite appears to inhibit hepatocellular differ-
localized intrahepatically: this tumor can present as entiation and leads to the uncontrolled prolifera-
a perihilar mass and should be classified as intrahe- tion of liver progenitor cells.40 Based on the
patic cholangiocarcinoma. Long arrow: Tumor of the
above findings it is suggested that IDH1 mutation
left hepatic duct localized extrahepatically: this tumor
will present as a perihilar mass and should be classi-
can be an initial step in the carcinogenesis of
fied as perihilar cholangiocarcinoma. iCCA, whether by itself or in combination with
other genetic changes remains unclear.41

and pCCAs are established as two separate en- MIMICKERS OF CHOLANGIOLOCARCINOMAS:

tities.29 They are also different at the molecular level BILE DUCT HAMARTOMA, BILIARY DUCT
and are staged differently according to the AJCC ADENOMA AND BILIARY ADENOFIBROMA
cancer manual (8th edition).29,30 KRAS mutation
seems to be an important driver of oncogenesis in As previously described, cholangiolocarcinoma is
iCCA especially in large duct types.31 When a small duct type iCCA where the tumor displays
KRAS is mutated, it means constant activation of homogenous growth with a small tubular or acinar
MAPK and AKT pathways, leading to signal inde- pattern and well to moderately differentiated cells,
pendent cell proliferation and mitosis as well as resembling proliferating bile ductules.26 Conse-
increased metastatic activity.32,33 Moreover, muta- quently, the main mimickers of this lesion are
tions in KRAS, TP53 and CDKN2A were shown to benign and pre-neoplastic lesions that are formed
predict worse overall survival in patients with by bland looking ducts or ductules such as: bile
iCCA, regardless of stage and treatment status.34 duct hamartoma (BDH) also known as Von Meyen-
burg complex (VMC), bile duct adenoma (BDA)
and biliary adenofibroma (BAF).
INTRAHEPATIC CHOLANGIOCARCINOMA,
The characteristics of these entities in compari-
SMALL DUCT TYPE son to cholangiolocarcinoma are summarized in
Table 2 and demonstrated in Fig. 3. This diagnosis
Small duct iCCAs arise in the peripheral parts of the is very difficult to establish on biopsy, as tissue in-
liver recapitulating septal and interlobular bile vasion is the feature most likely to allow for
ducts.2 Histologically they display small tubules diagnosis.
lined by cuboidal to low columnar cells.26 While
not exactly stated in the last edition of the WHO, it
INTRAHEPATIC CHOLANGIOCARCINOMA
seems that the vast majority of small iCCA do not
need to be further subtyped and are referred to as VERSUS METASTATIC PANCREATOBILIARY
iCCA, small duct type (conventional).20 That said, ADENOCARCINOMA TO THE LIVER: AN
iCCA small duct type can be further classified as ONGOING DIAGNOSTIC DILEMMA
cholangiolocarcinoma or small duct type iCCA
with ductal plate malformation (DPM) pattern.2,20,35 Adenocarcinomas of the biliary tree and the
Small duct type iCCA, cholangiolocarcinoma pancreatic duct (including perihilar cholangiocar-
subtype (or simply cholangiolocarcinoma) has fea- cinoma, extrahepatic/distal cholangiocarcinoma,
tures resembling reactive bile ductules.26 The pancreatic ductal adenocarcinoma (PDAC) and
possible origin of these tumors is postulated to ampullary/periampullary adenocarcinoma with

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602 El Jabbour et al

Fig. 2. (A). Large duct type iCCA (20x) showing cancerous large caliber bile ducts with irregular shapes. The lumen
is lined by multilayered tumor cells displaying relatively small and pale nuclei with occasional, small nucleoli as
shown in the insert (60x). (B). Small duct type iCCA (20x) displays small tubular and focally anastomosing ducts,
embedded into a desmoplastic stroma. In some areas, the malignant cells are organized into cords (black arrow)
or single cells (blue arrow). The ducts or cords are formed by small cuboidal cells as shown in the insert (60x). (C).
Cholangiolocarcinoma (20x) displays small malignant ducts, resembling ductular reaction. The malignant ducts
are lined by small cuboidal cells as shown in the insert (60x). (D). iCCA with ductal plate malformation like fea-
tures (20x) shows irregular, focally dilated malignant ducts surrounded by fibrous stroma. The malignant cells lin-
ing these irregular ducts show nuclear pleomorphism as shown in the insert (60x).

pancreatobiliary differentiation) share the albumin ISH.57 It is also important to note that al-
morphology and non-specific (CK7 positive/collo- bumin immunostain is very difficult to interpret due
quially”CK7-oma”) immunophenotype with to the background staining of normal cells which
iCCAs.1,15,16 Such tumors frequently metastasize could be minimized using the ISH technique
to the liver (mostly pancreatic ductal adenocarci- instead.58 Unfortunately, a common phenomenon
noma, due to incidence) making the distinction be- is when more tumors are subjected to a new test,
tween an iCCA and a metastasis to the liver always there is some loss of specificity and sensitivity.
challenging. Immunohistochemistry cannot typi- This was seen with respect to the albumin ISH,
cally resolve the differential, although the loss of which shows fairly frequent positivity in gall-
SMAD4 expression is more in favor of a metastasis bladder adenocarcinoma and rare positivity in
from a pancreatic adenocarcinoma. Nonetheless, many other tumors.59
loss of SMAD4 can be seen in tumors other than
PDAC, including iCCA, and some PDAC retains
POORLY DIFFERENTIATED INTRAHEPATIC
SMAD4, so this is not a deterministic test.14,54,55
Albumin in-situ hybridization (albumin ISH) has CHOLANGIOCARCINOMA VERSUS POORLY
been established as a promising biomarker of DIFFERENTIATED HEPATOCELLULAR
the hepatic lineage/origin.56 One study highlighted CARCINOMA
that a small duct like appearance of an invasive tu-
mor coupled with a positivity for albumin ISH is Both iCCA and HCC can present as a poorly differ-
diagnostic for iCCA over a metastatic adenocarci- entiated carcinoma and the distinction between
noma.57 According to the same study the utiliza- these two entities has significant challenges. How-
tion of albumin ISH presents with few pitfalls ever, this distinction is still crucial due to the differ-
worth mentioning. These include the positivity of ence in the subsequent therapeutic approach.60
pancreatic acinar cell carcinoma and the tendency Since “poorly differentiated” tumors demonstrate
of large duct type iCCA to show negativity for little resemblance to their benign counterparts,

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 Challenges in Diagnosing Cholangiocarcinoma 603

Table 2
Gross, histologic, and immunophenotypic comparison of cholangiolocarcinomas to its mimickers: bile
duct hamartoma, bile duct adenoma, and biliary adenofibroma

Gross Histology Notes References

Bile Duct  Multiple  Interanastomosing  CK7 1 20,42,43
Hamartoma nodules dilated biliary ducts,  CK19 1
 < 0.5 cm lined by cuboidal or
flat epithelium
 May contain inspis-
sated bile or eosino-
philic debris
 Surrounded by myxo-
matous or fibrotic
stroma
Bile Duct Adenoma  Solitary  Uniform benign ap-  CK7 1 20,44–47
nodule pearing small caliber  CK19 1
 Subcapsular bile ducts within a  BRAF V600 E muta-
 < 1 cm fibrous stroma tions (50%)
 No cytological atypia  Ki67 < 10%,
or pushing borders  p53 wild type
 p16 1
 EZH2 -
Biliary  Solitary  Acinar, microcystic and  CK7 1 20,48–51
Adenofibroma nodule tubuloglandular ele-  CK19 1
 Microcystic, ments lined by
 Size is up to cuboidal flat or low
16 cm columnar epithelium
 Cells with round nuclei
without obvious
nucleoli
 Fibrous stroma
 Possible papillary in-
traluminal projections
with fibrovascular core
 Possible nuclear atypia
with elongated, peni-
cillate hyperchromatic
nuclei, resembling
dysplasia
Cholangiolo-  Variable sizes  Innocent-looking tu-  CK7 1 2,20,26,52,53
carcinoma mor resembling reac-  CK19 1
tive bile ductules  Ki67 > 10%
 Variable degrees of  p53 mutated
cytologic atypia, des-  p16 variable
moplasia, and single  EZH21
cells infiltration  CD561

morphologic examination is often inconclusive. general, a poorly differentiated iCCA should still
Small biopsies in which a precursor lesion, or a express CK7 and/or CK1914–17,61 and will not ex-
more differentiated component is absent, am- press any of the hepatocytic lineage markers
plifies the problem. That said, morphology should such as HepPar1 and Arginase.54,61–65 On the
not be discounted, since a small focus of bile pro- other hand, poorly differentiated HCC rarely ex-
duction by tumor cells confirms the diagnosis of press CK7 or CK19 and may continue to express
HCC, whereas contrariwise, mucin production is hepatocytic markers such as Arginase,54,62
seen only in adenocarcinoma, such as iCCA. although HepPar1 expression diminishes as differ-
Immunohistochemistry can be a helpful addition entiation gets worse.54,60 Importantly, the pres-
to the diagnostic armamentarium with some prac- ence of aberrant CK19 expression in HCC has
tical considerations worth keeping in mind. In been demonstrated to carry a poorer

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604 El Jabbour et al

Fig. 3. (A). Cholangiolocarcinoma (20x) displays small malignant ducts, resembling ductular reaction. The malig-
nant ducts are lined by small cuboidal cells as shown in the insert (60x). (B). Von Meyenburg complex (20x) is
comprised by variable sized irregularly shaped bile ducts separated by collagenous stroma. These dilated bile
ducts are lined by benign, bland cuboidal to flat cells as seen in insert (60x). (C). Bile duct adenoma (20x) displays
branching, irregular, non-dilated, and predominantly tubular shaped ducts embedded to a fibrous stroma. The
cells lining the irregular ducts are small, cuboidal without cytological atypia as seen in insert (60x). (D). Biliary
adenofibroma (20x) shows cystic, dilated ducts within a moderately fibrous stroma. The dilated biliary ducts
are lined by low columnar to cuboidal epithelium with mildly hyperchromatic nuclei and eosinophilic cytoplasm
as seen in the insert (60x).

Fig. 4. (A). cHCC-CCA (10X) displays blending tumor cells of HCC on the left side and iCCA on the right side of the
image. The area of tumor blending is marked by black discontinuous line. (B). HepPar-1 immunostain (10X) shows
marked reactivity in the HCC area and no reaction in the iCCA cells. (C). CK7 (10x) immunostain shows positive
staining pattern in the iCCA cells and no reactivity in the HCC area. (D). Glypican-3 (10X) is strongly positive in
the HCC area and patchy non-reactive, nonspecific (negative) staining is seen in the iCCA area.

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 Challenges in Diagnosing Cholangiocarcinoma 605

prognosis.66,67 CD10, bile salt export pump This term (cHCC-CCA, intermediate cell carci-
(BSEP), and polyclonal CEA are occasionally use- noma subtype or simply intermediate cell carci-
ful when attempting to classify a poorly differenti- noma) is reserved for tumors that are
ated liver tumor as they stain hepatocellular morphologically and immunohistochemically uni-
carcinoma with a distinctive “canalicular” form: all the cells are of intermediate morphology
pattern.14,54,63,68,69 Although, the recognition of between a hepatocyte and a cholangiocyte and
this canalicular pattern carries significant diffi- all the cells express both hepatocytic and cholan-
culties, especially in poorly differentiated tumors giocytic marker.20
and may require consultation from an experienced
liver pathologist.14,54 Since BSEP is expressed
(practically) only by hepatocytes, it does not CLINICS CARE POINTS
require the identification of a canalicular pattern
(though this pattern is the most common). The
problem with these 3 markers is that very poorly  Differentiating benign peribiliary glands
differentiated HCCs do not commonly recapitulate from cholangiocarcinoma on a scant biopsy
the bile canaliculus. Therefore, the best marker to may be challenging. A panel of immunostains
distinguish poorly differentiated HCC from poorly comprising Ki-67, p53, CK7, CK19 and BAP1
may be helpful.
differentiated iCCA is often glypican 3, which is
often positive in poorly differentiated HCC, but  Tumors arising in the intrahepatic portions of
only rarely positive in iCCA.70 Albumin ISH is the right and left hepatic ducts may present
non-contributory in the scenario since it is a as hilar masses but should be classified as in-
marker of liver primaries and is commonly positive trahepatic cholangiocarcinomas. The term
in HCCs and iCCAs alike.56–58,69 perihilar cholangiocarcinoma is reserved for
the tumors arising from the extrahepatic
portion of the right or left hepatic ducts or
the common hepatic duct.
COMBINED HEPATOCELLULAR-
CHOLANGIOCARCINOMA  Cholangiolocarcinoma, a bland subtype of in-
trahepatic cholangiocarcinoma can be
Another entity to consider in the differential of pri- mimicked by 3 benign/preneoplastic lesions:
bile duct hamartoma, bile duct adenoma
mary liver carcinoma is combined hepatocellular-
and biliary adenofibroma.
cholangiocarcinoma (cHCC-CCA). The pathologic
definition of cHCC-CCA has evolved dramatically  Albumin-ISH, an established marker of the
over the years. However, the last edition of the hepatic lineage/origin may be rarely positive
WHO classification of gastrointestinal and hepato- in many other tumors and consequently a
biliary tumors has simplified this concept. Accord- diagnostic pitfall.
ing to the new criteria, cHCC-CCA is a primary
liver carcinoma with both hepatocytic and cholan-
giocytic differentiation in the same tumor. This DISCLOSURE
diagnosis is made regardless of the percentage
of each component within the tumor. Immunohis- None.
tochemistry is confirmatory; however routine H&E
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