The Role of Immunocytochemical Markers to Differentiate Primary from Secondary Neoplastic Hepatic Masses: A Diagnostic Challenge on Cytology
Jenna B BHATTACHARYA1, Shyam Lata JAIN2, Subbarayan DEVI3
1Department of Pathology, Lady Hardinge Medical College, NEW DELHI, INDIA
2Maulana Azad Medical College & Hospital, NEW DELHI, INDIA
3Chettinad Hospital and Research Institute, CHENNAI, INDIA
Keywords: Hepatic neoplasm, Liver metastases, Immunocytochemistry, Cytology, HepPar1
It is challenging and difficult to differentiate primary from metastatic hepatic masses solely on cytology. The present study aimed to
correlate cytomorphological spectrum of hepatic masses with immunocytochemical markers to differentiate primary from metastases in liver.
Material and Method: The present study comprised of 30 clinico-radiologically suspicious cases of neoplastic hepatic masses. Ultrasound-guided
fine needle aspiration smears and cell blocks were prepared as per standard technique; two of the smears were air-dried and Giemsa stained to
study cytomorphological features. A panel of markers (HepPar-1, CD 10, CK7, CK19, CD34, and MOC-31) were studied both in smears and
Results: Cytomorphological features on smears were evaluated and correlated with immunocytochemistry in all cases; the final diagnosis was:
Hepatocellular carcinoma (n=7), cholangiocarcinoma (n=2), hepatoblastoma (n=1) and metastatic carcinoma (n=20). HepPar-1, CD10 and
CD34 demonstrated 86%, 72%, 86% sensitivity and 100% specificity respectively for hepatocellular carcinoma; CK7&CK19 showed 100%
sensitivity for cholangiocarcinoma, MOC 31 showed 90% sensitivity and 100% specificity for metastatic carcinoma.
Conclusion: The present study recommends a panel of minimum three markers (HepPar-1, CD10, and MOC-31) which were helpful to
differentiate hepatocellular carcinoma from metastatic carcinoma that was a major diagnostic challenge solely on cytomorphology. Correlating
cytomorphology with these three markers, 100% of the cases could be diagnosed as primary malignancy and distinguished accurately from
The distinction between primary and metastases in liver on
cytomorphology alone has been challenging and a matter
of much debate. Though some cytomorphological features
help to differentiate hepatocellular carcinoma (HCC) from
metastatic carcinoma (MC), the distinction between the
two still remains a challenge in case of poorly differentiated
tumors. A cytopathologist also faces diagnostic challenges
to differentiate benign/reactive hepatocytes from welldifferentiated
HCC (WDHCC). Hence, a panel of
immunocytochemical markers (ICC) is advised for the
final diagnosis in challenging cases. Various studies were
conducted to assess the role of ICC in hepatic masses, using
a single marker or panel of markers when a diagnosis on
cytomorphology alone was difficult 1-13
. Also, since
the prognosis and treatment of both HCC and MC are
significantly different, ancillary techniques for correct
diagnosis and further follow up are mandatory. Human
hepatocyte antibody-1 (HepPar-1) has been reported as a highly specific marker for hepatocytic differentiation with
high sensitivity (90%). 4,5,11
Despite the availability of many markers, no single marker
is 100% specific and sensitive for either HCC or MC so far
5-16. Hence, the present study aims to evaluate a panel
of markers (HepPar-1, CD10, CK7, CK19, CD34, MOC31)
along with cytomorphology to differentiate HCC from MC.
This prospective study was conducted in the departments
of pathology and gastroenterology surgery after obtaining
ethical clearance and written informed consent from
the patients. 30 clinico-radiologically suspected cases of
neoplastic hepatic mass lesions were aspirated. Patients
with abnormal coagulation profile, vascular lesions and
infective cysts were excluded. Relevant clinic-radiological
and serological findings were recorded. The study was
approved by the Institutional Ethics Committee (Date:
04.08.2011, Ref. No: 11/1EC/MAMC/2011/119)
USG/CT-guided FNAC was performed as per standard
technique using a 23/24 gauge needle. Multiple areas
were aspirated for an adequate and representative sample.
Two smears were air-dried and Giemsa stained to study
morphological features and the rest was preserved at 0°C
for ICC. The remaining aspirate was processed for the cell
block (CB). To prepare CBs, the hemorrhagic aspirate was
allowed to clot for 1-3 hrs, fixed in 10% buffered formalin,
and processed further as routine Hematoxylin & Eosin
(H&E) stained histological sections for cyto-histological
correlation. ICC was performed on fresh smears (n=16)
and on both smears and CB (n=14). For ICC, the smears
were fixed in a mixture of cold acetone and methanol (1:1)
for 5 minutes; and for CBs, 3-5μ thick sections were heated
to deparaffinize followed by changes in xylene, graded
alcohol and hydration in water. All steps were carried out in
a moist and humid chamber throughout the procedure. The
primary antibodies (Ab) used were as follows: HepPar-1
(monoclonal mouse Ab, DAKO, clone OCHIE5), CD10
(monoclonal mouse Ab, DAKO), MOC31 (anti-mouse Ab,
BioSB, clone BerEp4), CK7 (monoclonal mouse Ab, DAKO,
Flex, clone OV-TL) and CK19 (monoclonal mouse Ab, Flex
DAKO, clone RCK 108), CD34 (Skytek, clone 4C4.9). The
Biogenex Super Sensitive Polymer-HRP Detection System
was used in conjunction with rabbit/mouse IgG Primary
Abs. DAB (diaminobenzidine) substrate for peroxidase
was used as an enzyme label forming a stable brown end
product at the site of the antigen (Ag). A fresh citrate buffer
was prepared with every set of staining and used for Ag
retrieval wherever required. Hematoxylin was used as a
counter stain. Positive and negative controls were run with
Two authors independently evaluated every case. Granular
cytoplasmic positivity for HepPar-1; canalicular, cytoplasmic
and membranous stain for CD10; endothelial cell stain for
CD34; membranous/membranous and cytoplasmic stain
for MOC31; and cytoplasmic stain pattern for CK7& CK19
were considered significant and positive. Statistical analysis
was done using the Chi square test and Fisher’s exact test
using SPSS software.
Of the 30 patients included in the study, 17 were male (56.6%)
and 13 female (43.3%), with a male to female ratio of 1.3:1.
The patients ranged in age from <1 to >80 years (mean age-
48.03 ±1.73 years). The most common radiological finding
was a single space-occupying lesion (SOL) in cases of HCC
and multiple SOLs in cases of metastases (Figure 1A, B
Click Here to Zoom
|Figure 1: A) CT image
showing a single spaceoccupying
lesion. B) CT
image showing multiple
C) FNA smears showing
atypical cells with
scattered naked nuclei
HCC showing trabecular
x400). Figure insets:
HepPar-1 showing strong
positivity (ICC; x400).
CD10 showing strong
positivity (IHC; x400).
CD34 showing strong
grade 3 positivity (ICC;
The cytomorphological features studied encompassed
cellularity, pattern of cell arrangement, cytoplasmic and nuclear details and the background. Based on these features
and correlating with ICC, the final diagnosis was HCC
(n=7), CC (n=2), HB (n=1) and MC (n=20) from various
sites (Table I).
The following cytological features were useful in the
diagnosis of HCC: clusters of tumor cells surrounded
by capillaries (peri-sinusoidal pattern) and clusters with
transgressing capillaries (transgressing/centri-sinusoidal
pattern), bile pigment in tumor cells, intranuclear
inclusions (INI), prominent macronucleoli, and atypical
stripped nuclei in the background (Figure 1C, D). Smears
of CC showed a typical smear pattern of adenocarcinomas
(AC) with clusters of columnar epithelial cells in an acinar
pattern (Figure 2A, B). Initially these cases were suggestive
of a metastatic AC; however, only after the panel of ICC
could they be correctly diagnosed as CC since both CK7
and CK19 were positive (Figure 2C, D) and the rest of the
markers were negative ruling out primary HCC/metastases
even though the clinical diagnosis was suggestive of MC
Click Here to Zoom
|Figure 2: A-B) Cholangiocarcinoma
showing atypical columnar
cells in an acinar pattern with
a fibrotic background (Giemsa;
x400). C) CK7 showing strong
cytoplasmic positivity in tumor
cells (ICC; x400). D) CK19
showing strong cytoplasmic
positivity in tumor cells (ICC;
x400). (Inset: Tumor cells
negative for Hep Par 1).
The MC group showed atypical cells with attempted
acinar/papillary pattern, necrosis, and a mucinous and/
inflammatory background. The important feature was the
presence of benign hepatocytes as separate clusters (Figure
3A, B); however, these features were not clear in poorly
differentiated tumors (PD). Based on cytomorphology
alone, 6/7 cases could be diagnosed provisionally as HCC
and confirmed later on ICC; the remaining case was
provisionally diagnosed as PDHCC or PD metastatic AC, as
it did not have the classical cytological features and was later
confirmed on ICC. Cases of HB (n=1) with a provisional
diagnosis of small round cell tumor (SRCT) and CC (n=2)
were confirmed by ICC and histopathology. Out of 20 cases
of MC, one case showed atypical squamous cells admixed
with benign hepatocytes. On further evaluation, the patient
was found to have a primary in the lung. One case had
atypical small cells with nuclear overlapping, smudged
chromatin, and a necrotic background, which on ICC
and histology proved to be a case of metastatic small cell
carcinoma (SmCC) lung. Among the 18 AC cases (NOS),
8 cases could be correctly diagnosed on cytology alone; in
the remaining 10 cases a possibility of metastatic PDAC
was suggested. All these cases of MC were positive for
MOC31 and thus confirmed as MC. In a single case of AC,
focal neuroendocrine (NE) morphology was also noticed
together with an acinar pattern and was later confirmed by
NE markers, i.e. Chromogranin (CG) and Synaptophysin
(Syn) in addition to MOC31 on ICC. Hence the final
diagnosis was AC with NE differentiation. (Table III)
Click Here to Zoom
|Table III: Cases showing staining pattern and grading on immunocytochemistry
HepPar-1: HepPar-1 showed distinct granular cytoplasmic
positivity in 6/7(86%) HCC cases (Figure 1C, inset) and HB
(n=1); while a single case of PDHCC and all cases of MC
(n=20) were negative for HepPar-1.
CD10: 5/7 (71.2%) cases of HCC showed positivity for
CD10 (Figure 1D, inset) including the PDHCC which was
negative for HepPar-1; the staining pattern was canalicular
with long branching and zig-zag linear pattern (n=3) and cytoplasmic stain with membranous accentuation (n=2).
None of the CC and MC showed positive staining for CD10.
CD34: CD34 positive stain was considered when any cell
that stained brown with a dot like, linear, semi-circular/
circular pattern, and was clearly separate from an adjacent
cell. The counting was carried out in 10 high power fields
(HPF) and an average was taken which was further graded
as 0-4: 0 (no stain), 1 (< 25% cells stain positive), 2 (25-50%
cells stain positive), 3 (50-75% cells stain positive), 4 (>75%
cells stain positive). In this study, a positive result was found in 6/7 (86%) cases of HCC; grade-1 in one case (3.37%),
grade-2 in 3 cases (10%), grade-3 in 2 cases (6.67%), and
no staining in one case (3.37%). None of the CC and MCs
showed positive stain. There was no correlation between
the grade of tumor and positive ICC.
CK7: Total of 4 (16.67%) cases were positive for CK7; 2
were CC that showed strong cytoplasmic reactivity and
2 were MC ovary which also showed strong cytoplasmic
immunoreactivity (Figure 2C). None of the cases of HCC
CK19: A positive result was considered when the tumor
cells showed either cytoplasmic staining with membrane
staining or paranuclear dot like accentuation. A total of
seven cases were positive for CK19; 2 were CC (100%)
and 5 (25%) were MC (colonic AC-3, strong cytoplasmic
positivity, gastric AC-2, focal cytoplasmic positivity)
(Figure 2D). None of the HCC’s was positive for CK 19.
MOC31: MOC31 was expressed in 18/20 (90%) cases of
MC; eight showed cytoplasmic positivity with membranous
accentuation (40%), eight showed membranous positivity
(40%), and two showed cytoplasmic positivity (10%).
CC (n=2), in addition to CK 7 and CK19 positivity, also
expressed cytoplasmic positivity for MOC31 (Figure 3B).
None of the cases of HCC were positive.
A few additional markers apart from the primary panel
were used (i.e. CG, Syn, S100) to confirm the metastatic
origin of certain cancers like SmCC lung and AC with NE
Click Here to Zoom
|Figure 3: Metastatic
adenocarcinoma. A) Atypical cells
with intracytoplasmic mucin in
a necrotic background (Giemsa;
x400). B) MOC31 showing strong
membranous accentuation in
tumor cells (ICC; x400).
Distinguishing HCC from CC and MC on FNAC of
neoplastic hepatic masses has been a diagnostic challenge
and it is paramount to differentiate these lesions as the
prognosis and treatment approaches are different for all three
conditions. The areas of challenge include differentiation
of benign reactive hepatocytes from WD HCC, PDHCC
from MCs and, CC from HCC or metastases. This difficulty
is compounded by limited and or non-representative
aspirate, and a hemorrhagic and or necrotic background.
In recent years, various ancillary techniques such as ICC
have contributed greatly to distinguish these lesions and to
overcome the diagnostic pitfalls 1-13
. The present study
highlights certain cytomorphological features helpful in the
diagnosis of HCC, especially in WD and MD HCC such as
round to polygonal cells, peri and/centri-sinusoidal pattern
of tumour cells with small transgressing capillaries, bile
pigment, INI, macronucleoli, variable pleomorphism and atypical stripped nuclei in the background. Features of MC
included atypical cells in acinar and or papillary pattern,
scattered cells, mucinous background and signet ring
cells in some; and most importantly benign hepatocytes
in separate clusters. However, in PD tumors (HCC vs
MC), these features are not clearly identifiable leading to
a diagnostic challenge and pitfall on smears. In such cases,
ICC is essential to solve this dispute. Various markers have
been studied in the past two decades to evaluate the most
specific and sensitive markers to differentiate the primary
versus metastatic lesions. The present study evaluated
HepPar-1, CD 10, CK7, CK19, CD34, and MOC 31 to
differentiate HCC from MC.
HepPar-1 (human hepatocyte Ab) recognizes hepatocyte
mitochondrial epitope with granular cytoplasmic staining
on ICC. Earlier studies showed high sensitivity of HepPar-1
(90%) in detecting HCC 1,4,9,10. However, despite the
high sensitivity, it is not entirely specific for hepatocytes,
nor could it discriminate between benign and malignant
hepatocytes. Also, some studies have reported negative
HepPar-1 in PDHCC, and interestingly positive reactivity
in other malignancies with hepatoid differentiation 16. In
the present study, sensitivity (86%) and specificity (100%)
in detecting HCC with HepPar-1 were observed. Negative
staining was noted in PDHCC (n=1) and all cases of MC.
The above case of PDHCC posed a diagnostic challenge
as initially the provisional diagnosis was PDHCC/PDMC
with history and cytomorphology. The dilemma continued
as HepPar-1 was negative; however, CD10 was the only
marker amongst the panel that was positive and confirmed
the diagnosis of PDHCC, and hence proved that a single
marker i.e. Hep Par1 does not help the cytopathologist in
challenging cases. A case of HB provisionally diagnosed as
pediatric SRCT was later confirmed as HB with HepPar-1
positivity that ruled out other SRCTs.
CD10 is a commonly used marker for hematolymphoid
neoplasms. The positive stain includes a cytoplasmic,
membranous/canalicular pattern. The canalicular pattern
has been considered specific for hepatocytic differentiation
on tissue sections. Saad et al. used CD10 on FNA smears
with satisfactory SN (77%) and SP (100%) 4. The present
study also showed comparable results for SN (72%) and
SP (100%) in detecting HCCs. It was the only positive
marker that was expressed in a case of HCC that was
negative for HepPar1 and CD34. Recently, Singha et al.
showed canalicular, membranous, and membranous and
cytoplasmic staining patterns for CD10 5. The present
study also showed these characteristic staining patterns.
Cui et al. first reported strong expression of CD 34 in
sinusoidal vessels in all cases of advanced HCC with a high sensitivity (100%) in detecting HCC 6. The present study
showed SN of 86% and SP of 100%. Kakar et al. also showed
CD 34 expression in WDHCC in a few cases of adenomatous
hyperplasia, but none in cirrhosis; hence, supporting the
fact that CD 34 positive sinusoids in HCC may suggest
microvessel angiogenesis and tumor cell proliferation due
to hepato-carcinogenesis 15. This finding correlates in
the present study also, since none of the control cases or
MC was positive. Although the assessment of angiogenesis
does not provide prognostic information, it might help as a
diagnostic marker to differentiate HCC from MC.
MOC31 has been proven a sensitive and specific marker of
AC differentiation and thus is used to distinguish AC from
mesothelioma both in tissue sections as well as in body
fluids. Proca and Porcell et al. showed high sensitivity and
specificity (100%) in detecting MC and distinguishing it
from HCC 2,7. Luoquan et al. studied MOC31 expression
in alcohol-fixed, paraffin-embedded liver FNA-CBs and
found a high sensitivity (97%) and specificity (94%) 10.
In the present study, MOC 31 helped in detecting MACs
to liver with a sensitivity of 90% and a specificity of 100%
and ruled out primary malignancy; however, it did not help
to differentiate MC vs. CC indicating the primary site of
Expressions of various cytokeratins (CK) has recently gained
popularity to distinguish primary sites of liver metastases
8,11 because malignant cells usually maintain the CK
profile of their “cells of origin”. Normally, adult hepatocytes
express CK 8 and 18 whereas bile duct epithelium expresses
CK 7, 19, 8 and 18. CK19 expression is normally found
in hepatic progenitor cells and cholangiocytes but not
in adult hepatocytes. CK 19 expression appears relatively
specific for CC, though HCC may sometimes exhibit CK19
positivity. The present study included two cases of CC, both
expressing CK7 & CK19; while none of the cases of HCC
were positive for CK 7 and CK19. Few MC expressed either
of these markers but none showed positivity for both.
In conclusion, based on the present study, it is observed
that a panel of three markers (HepPar, CD10 and MOC31)
would help to differentiate HCC from metastatic AC in
conjunction with cytomorphological correlation. Both
FNA smears and CB can be used for ICC with similar
intensity; however, FNAC may have limitations because of
a non-representative sample and or a hemorrhagic necrotic
CONFLICT of INTEREST
The authors declare no conflict of interest.
Concept: JBB, SLJ, Design: JBB, SLJ, Data collection or
processing: JBB, Analysis or Interpretation: JBB, SLJ,
Literature search: JBB, SD, Writing: JBB, SD, Approval:
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