AJCP / Original Article

Clinicopathologic Significance of Combined

Hepatocellular-Cholangiocarcinoma With Stem Cell
Subtype Components With Reference to the Expression
of Putative Stem Cell Markers
Hiroko Ikeda, MD,1 Kenichi Harada, MD,2 Yasunori Sato, MD,2 Motoko Sasaki, MD,2
Norihide Yoneda, MD,3 Seiko Kitamura, MD,1 Yoshiko Sudo, MD,4 Akishi Ooi, MD,5
and Yasuni Nakanuma, MD1,2
From the 1Section of Diagnostic Pathology, Kanazawa University Hospital, Kanazawa, Japan; 2Department of Human Pathology, Kanazawa University Graduate
School of Medicine, Kanazawa, Japan; 3Department of Radiology, Kanazawa University Hospital, Kanazawa, Japan; 4Department of Pathology, Fukui Saiseikai
Hospital, Fukui, Japan; and 5Department of Molecular Cellular Pathology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan.
Key Words: Combined hepatocellular-cholangiocarcinoma; DLK1; Hepatoblast; NCAM/CD56; Stem cells
DOI: 10.1309/AJCP66AVBANVNTQJ

ABSTRACT
Objectives: To examine the clinicopathologic features of
combined hepatocellular-cholangiocarcinoma (HC-CC),
which the World Health Organization (WHO) proposed
classifying into 2 types, and the expression of delta-like 1
homolog (DLK1), as well as putative stem cell markers, such
as NCAM/CD56 and CD133.
Methods: In this study we examined the expression of stem
cell markers using immunohistochemistry.
Results: Thirty-six cases of combined HC-CC were
subclassified into 24 cases, with more than 5% stem cell
features (group B) and 12 cases with less than 5% stem
cell areas (group A). The postoperative overall survival
rate was worse for group B than for group A. DLK1 was
frequently expressed in group B cases compared with
group A, hepatocellular carcinoma, and intrahepatic
cholangiocarcinoma cases.
Conclusions: The 2010 WHO classification seems important
for elucidating the pathogenesis of stem cellrelated liver
cancers.

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Combined hepatocellular-cholangiocarcinoma (HC-CC)

of the liver is a primary liver tumor containing hepatocellular
carcinoma (HCC) and cholangiocarcinoma (CC) elements
that are intimately admixed. In the 2010 World Health Organization (WHO) classification, combined HC-CC is divided
into 2 histologic forms: the classic type and subtypes with
stem cell features.1 The former is the traditional, commonly
used definition of combined HC-CC, in which areas of typical HCC and CC are mixed within the tumor. The category
including subtypes with stem cell features is new and further
subdivided into typical, intermediate-cell, and cholangiocellular subtypes.
Several markers and approaches have been used to detect
and characterize hepatic stem/progenitor cells.2-5 For example, the simultaneous immunohistochemical expression of an
HCC marker (HepPar1 or a-fetoprotein [AFP]) and a biliary
marker (cytokeratin [CK] 19 or carcinoembryonic antigen)
has been recommended for the identification of hepatic stem/
progenitor cells in nonneoplastic and neoplastic livers, and
the expression of nuclear cell adhesion molecule (NCAM/
CD56) and epithelial cell adhesion molecule (EpCAM) has
been reported in small and oval-shaped stem celllike cells.1-5
Delta-like 1 homolog (DLK1) has been reported to be
a biomarker of hepatic stem/progenitor cells in murine liver,
because DLK1 is expressed in fetal liver, but it is restricted to
a subpopulation of oval/hepatic progenitor cells in adult liver.
Moreover, purified DLK1-positive cells can differentiate into
both hepatocyte and biliary epithelial lineages and repopulate
the normal liver in vivo after transplantation.6-8 The expression of DLK1 has been reported in human fetal liver but not in
normal adult liver or diseased livers with viral hepatitis and cirrhosis.9,10 However, upregulation of DLK1 expression recently

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has been reported in hepatoblastomas and some HCCs.10-14 Xu

et al14 suggested that DLK1-positive HCC cells might have
similar characteristics to cancer stem/progenitor cells, because
DLK1-positive HCC cells showed increased chemoresistance,
colony formation, spheroid formation, and in vivo tumorigenicity compared with DLK1-negative cells. To our knowledge,
the expression of DLK1 has not been investigated in combined
HC-CC, and the significance of DLK1 upregulation in liver
cancers needs to be elucidated.
While subtypes with stem cell features in addition to
classic combined HC-CC have been proposed for evaluating combined HC-CC in the 2010 WHO classification, their
clinicopathologic characteristics, pathogenesis, and prognosis
remain unclear. Furthermore, the expression of so-called
hepatic stem/progenitor cell markers in combined HC-CC has
not been reported with respect to the 2010 WHO classification.
In this study, we evaluated the clinicopathologic characteristics of combined HC-CC with or without stem cell
features using a series of primary liver cancer specimens and
attempted to clarify the expression of putative hepatic stem
cell markers, especially DLK1.

Materials and Methods

Selection of Cases and Tissue Preparation
We reevaluated primary liver cancers (4,379 cases) registered in the pathology files of Kanazawa University Hospital
and affiliated hospitals (2001-2011). A total of 36 cases were
diagnosed as combined HC-CC. As a control, 101 cases of
HCC and 23 cases of intrahepatic cholangiocarcinoma (ICC)
were chosen from the pathology files. Six human fetal livers
at the gestational ages of 7 to 10 weeks were also collected
from the files. All HCCs, ICCs, and combined HC-CCs were
surgically resected cases, and fetal livers were collected from
aborted fetuses. The nonneoplastic parts of these primary
liver cancers were available, and 10 cases of liver cirrhosis (5
hepatitis C virusrelated cases and 5 hepatitis B virusrelated
cases) and 5 specimens of normal liver from these cases were
used as nonneoplastic adult liver for immunohistochemistry.
The liver specimens were fixed in formalin and embedded
in paraffin, and more than 20 thin sections (4 mm thick)
were prepared from each paraffin block. Several sections
were subjected to H&E staining, Azan-Mallory staining, and
Gomori reticulin staining for routine histologic observation.
The remaining sections were used for immunohistochemical
staining. This research project was approved by the Kanazawa
University Ethics Committee.
We diagnosed HCC, ICC, and combined HC-CC
according to the 2010 WHO classification of gastrointestinal tumors.1 The HCC components of combined HC-CC
consist of tumor cells that resemble hepatocytes and are
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immunohistochemically positive for HepPar1. The CC components are tubular or papillary or cord-like adenocarcinomas
that are positive for CK7 and CK19. Combined HC-CC was
classified into 2 types in this study: group A and group B.
Group A is the classic form in the 2010 WHO classification
and is composed of typical HCC and CC components, with
less than 5% of the area occupied by subtypes with stem
cell features, and group B is combined HC-CC with more
than 5% stem celllike areas. The components of combined
HC-CC with stem cell features are divided into 3 forms: typical, intermediate, and cholangiocellular. The typical subtype
shows stem celllike cells around the HCC nests facing the
fibrous septa, and these stem celllike cells are small with a
high nucleus to cytoplasm ratio and occasionally positive for
CK19. The intermediate-cell subtype contains carcinoma cells
with features intermediate between those of hepatocytes and
cholangiocytes, which show cytoplasmic immunostaining of
HepPar1 and CK19 simultaneously. The cholangiocellular
subtype has neoplastic components resembling reactive bile
ductules or the canals of Hering, which are arranged in a cordlike and anastomosing pattern, the so-called antler-like pattern
embedded in fibrous stroma, and usually are positive for CK7
and/or CK19. Occasionally, these stem cell features were
focally admixed, and we classified the predominant subtype
when the tumors consisted of 2 or 3 components. In group
B, there was always unequivocal HCC in addition to these
subtypes, with stem cell features within the tumor. It did not
matter whether there were typical CC components.
Immunohistochemistry
Immunostaining was carried out with an autostainer (HX
System BenchMark; Ventana Medical Systems, Tucson, AZ)
according to the manufacturers instructions. Primary antibodies, clones, sources, and dilutions are listed in Table 1.
Appropriate positive and negative controls were included for
each immunostaining.
Table 1
Antibodies Used for Immunohistochemical Staining
in an Autostainer
Antibody Clone

Source

AFP
Polyclonal
DAKO (Glostrup, Denmark)
CD133
AC133
Miltenyi Biotec (Auburn, CA)
CK7
OV-TL 12/30 DAKO
CK19
RCK108 DAKO
DLK1
Polyclonal
Abcam (Tokyo, Japan)
EpCAM HEA125 DAKO
Glypican 3
GC33
Chugai Pharmaceutical
(Tokyo, Japan)
HepPar1 OCH1E5 DAKO
NCAM/CD56 1B6
Novocastra (Newcastle
upon Tyne, England)

Dilution
1:200
1:100
1:75
1:75
1:500
1:100
1:500
1:50
1:75

AFP, a-fetoprotein; CK, cytokeratin; DLK1, delta-like 1 homolog; EpCAM,

epithelial cell adhesion molecule; NCAM, neural cell adhesion molecule.

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Scoring
The immunostaining was semiquantitatively scored as
follows: score 0, no positive cells; score 1+, less than 5% of
tumor cells positive; score 3+, more than 50% positive, and
score 2+, between scores 1+ and 3+. The positive ratio was
the percentage of immunohistochemical positive cells among
morphologic stem celllike cells, not in all tumor cells, in the
stem cell type of combined HC-CC.
Statistical Analysis
The statistical analysis was performed using the MannWhitney test, the Spearman rank correlation coefficient by
rank test, and the Kaplan-Meier method. The differences
between the survival curves were tested using the log rank
test. P < .05 was considered significant.

Results
Clinicopathologic Findings of HC-CC in Comparison
With HCC and ICC
The 36 cases of combined HC-CC were classified into
12 cases in group A and 24 cases in group B. The latter were
subclassified into 6 cases of the typical subtype, 12 cases of the

intermediate-cell subtype, and 6 cases of the cholangiocellular

subtype. The main clinicopathologic features of combined
HC-CC (groups A and B), HCC, and ICC are summarized in
Table 2. The mean age of patients was significantly younger
for HC-CC in group B than for HCC (P = .005) and ICC
(P = .017). There was no significant difference in mean age
between the 3 subtypes of group B (P = .07). There was no statistical difference in sex between the groups. As for the background liver, the ratio of normal liver was higher for ICC than
for HCC, HC-CC (group A), and HC-CC (group B), and the
frequency of cirrhosis was lowest in the ICC group. There was
no significant difference between the 3 subtypes of group B.
The postoperative survival of patients with liver cancers
is shown in Figure 1. The prognosis of patients with HCC
was significantly better than that of patients with ICC (P =
1.0
Overall Survival (%)

Double immunostaining for HepPar1/CK19 (rabbit polyclonal; Abcam, Tokyo, Japan) was carried out according to
the manual supplied. Briefly, deparaffinized sections were
microwaved in citrate buffer and then incubated with each primary antibody overnight. After incubation with Alexa Fluor
594 goat antirabbit immunoglobulin G (IgG) and Alexa
Fluor 488 goat antimouse IgG (Life Technologies, Carlsbad,
CA), nuclei were stained by 4,6-diamino-2-phenylindole
dihydrochloride and examined under a confocal laser microscope (LSM5 7; Carl Zeiss, Oberkochen, Germany).

ICC
HC-CC (group A)
HC-CC (group B)
HCC

0.8
0.6
0.4
0.2
0.0

50

100

150

200

250

Postoperative Period (mo)

Figure 1Overall survival status of patients with liver

cancers. The patients with group B combined hepatocellularcholangiocarcinoma (HC-CC) show a significantly
worse clinical course than those with hepatocellular
carcinoma (HCC) (P = .005). The patients with intrahepatic
cholangiocarcinoma (ICC) show the worst clinical course
(compared with HCC, P = .0004). The prognosis tended to be
worse for group B than for group A (P = .05).

Table 2
Clinicopathologic Findings of Liver Cancers



Characteristic

Combined HC-
CC, Group A,
Combined HC-CC,
Classic Type
Group B, Typical
(n = 12)
Subtype (n = 6)

Combined HC-
CC, Group B,
Intermediate-Cell
Subtype (n = 12)

Combined
HC-CC, Group B,
Cholangiocellular
Subtype (n = 6)

HCC (n = 101)

ICC (n = 23)

Age, mean
64.6 (45-78)
56.8 (41-71)
57.1 (47-71)
61.7 (52-78)
63.6 (38-84)
65.6 (35-84)
(range), y
Sex, M:F
9:3
5:1 10:2 5:1 81:20 16:7
Etiology (No.
HBV (6), HCV (5), HBV (3), HCV (2), HBV (5), HCV (2),
HBV (2), HCV (1), HBV (30), HCV (53),
HBV (2), HCV
of cases) and NL (1) and alcohol (1) unknown (2), NASH (1), Budd- HBV + HCV (1), alcohol (1), unknown
and NL (3) Chiari syndrome (2), NASH (3), unknown (2), PSC (1),
(1), and NL (1) (5), and NL (7) and NL (17)
Cirrhosis, No. (%) 7 (58.3)
4 (66.7)
4 (33.3)
2 (33.3)
49 (48.5)
1 (0.04)
of cases
Previous diagnosis Combined
Combined HC-CC Combined HC-CC (2), Combined HC-CC HCC (101)
ICC (23)
(No. of cases) HC-CC (12) (2) and HCC (4) HCC (7), and ICC (3) (5) and HCC (1)
HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HC-CC, hepatocellular and cholangiocarcinoma; HCV, hepatitis C virus; ICC, intrahepatic cholangiocarcinoma; NASH,
nonalcoholic steatohepatitis; NL, normal liver; PSC, primary sclerosing cholangitis.

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.0004), whereas there were no significant differences between

combined HC-CC (groups A and B) and HCC or ICC. There
was no statistical difference between HCC and HC-CC in
group A, but the prognosis was significantly worse for HCC
in group B (P = .005). There tended to be a poor survival rate
for group B compared with group A (P = .05).
Immunohistochemistry
Expression of DLK1 and Other Stem/Progenitor Cell Markers
Fetal liver and nonneoplastic adult liver. Strong expression of DLK1 in primitive hepatocytes (hepatoblasts) was
seen at 7 gestational weeks, in which liver HepPar-1positive hepatoblasts are arranged in a trabecular pattern and the

biliary system is not established Image 1A and Image 1B.

The staining pattern was mainly cytoplasmic but also seemed
to be membranous, resembling the expression of glypican 3
Image 1C and Image 1D. The expression of DLK1 gradually decreased after 8 weeks, and no staining was found in adult
nonneoplastic livers. Membranous expression of EpCAM was
seen in hepatoblasts at 7 gestational weeks Image 1E, but the
expression shifted to ductal plate cells after 8 weeks. In adult
liver, EpCAM was detected in biliary epithelia of varioussized bile ducts and occasionally in expressed periportal hepatocytes in cases of chronic liver disease. NCAM/CD56 was
expressed focally in small cells around vein-like structures
at 7 gestational weeks Image 1F and located in ductal plate

Image 1 Histology and immunohistochemical findings of fetal liver at 7 gestational weeks. A, Primitive hepatocytes
(hepatoblasts) are arranged around vein-like structures, and no biliary elements are present (H&E). B, HepPar1. Cytoplasmic
staining is seen in hepatoblasts. C, Delta-like 1 homolog. Strong cytoplasmic and also membranous staining is found in
hepatoblasts. D, Glypican 3. Strong cytoplasmic and focal membranous staining is found in hepatoblasts.
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cells after 8 weeks. No staining of CD133 was found in fetal

liver. Basolateral membranous staining of NCAM/CD56 and
luminal staining of CD133 were frequently observed in reactive ductules in cases of liver cirrhosis.
HCC and ICC. DLK1 was expressed in 11 (10.9%) of
101 cases of HCC, all of which contained moderately or
poorly differentiated components. The staining of DLK1 in
HCC cells was cytoplasmic but usually concomitant with
membranous staining with an occasional canalicular pattern.
Diffuse and strong staining of DLK1 (score 3+) was found
in only 5 cases of DLK1-positive HCC. The expression of
glypican 3 was detected in all cases of DLK1-positive HCC,
and AFP expression was also frequently seen. The distribution of DLK1-positive cells in HCC cases was similar to that
of glypican 3 and AFP, and the expression of DLK1 showed
a significant correlation with that of glypican 3 or AFP
Image 2. The cytoplasmic expression of DLK1 in ICC was
seen in 3 cases (13.0%) with papillary components, but few
DLK1-positive cells were detected (score 1+/2+). EpCAM
was expressed in 21.0% of HCC cases. NCAM and CD133
were not detected in HCC. NCAM, EpCAM, and CD133
were expressed in most CC cases (96.0%).
Group A combined HC-CC. All HCC and CC components in group A were moderately or poorly differentiated
HCCs or adenocarcinomas. A score of 1+ for the expression
of DLK1 was seen in just 2 cases, one of which showed
focal positive findings with a canalicular pattern in the HCC
region and the other HC-CC showing focal cytoplasmic
staining in the adenocarcinoma area.
Typical subtype components of group B. Stem/progenitor
celllike small and oval cells were located at the periphery of
differentiated HC nests in all 6 cases; in 1 case there were also
E

CC components. Cytoplasmic expression of DLK1 in stemlike cells was found in 4 (66.7%) cases and focal expression
in the HCC area in 3 (50%) cases. The expression of CK19,
NCAM/CD56, and EpCAM in stem-like cells was rare or focal
and almost never occurred in the same cell. The frequency and
location of DLK1 expression did not correlate to these stem
cellrelated molecules either Image 3.
Intermediate-cell subtype component of group B. All
samples contained morphologic and phenotypical intermediate
tumor cells showing simultaneous expression of HepPar1 and
CK19 Image 4A and Image 4B. The expression of DLK1
was found in 5 (41.7%) cases, and diffuse staining (score 3+)
was seen in 3 (25.0%) cases. The frequency of each marker in
intermediate cells was 75.0% (9 cases) for EpCAM, 50.0% (6
cases) for NCAM/CD56, and 41.7% (5 cases) for CD133. The
positive correlation of immunohistochemical scores between
DLK1 and EpCAM was demonstrated by statistical analysis
(P = .03) Image 4C and Image 4D.
Cholangiocellular subtype of group B. The tumors
showed focal cholangiocellular features, with CK7-positive
small, slender, and arborizing tubules Image 5A and Image
5B. NCAM/CD56 was detected in all cholangiocellular types
to various degrees Image 5D. The expression of DLK1 was
seen in 5 (83.3%) cases, showing a cytoplasmic or canalicular
staining pattern Image 5C. No correlation of immunohistochemical scores between DLK1 and other stem cellrelated
markers was demonstrated by statistical analysis.
Comparison of DLK1 Expression in Liver Cancers
The frequency of DLK1-positive cases (score 1+, 2+,
and 3+) was 10.9% for HCC, 16.7% for combined HC-CC
in group A, 58.3% for HC-CC in group B (66.7% for typical
F

E, Epithelial cell adhesion molecule. Membranous expression is seen in hepatoblasts. F, Neural cell adhesion molecule (NCAM)/
CD56. NCAM/CD56 seems to be expressed in small cells around vein-like structures (A-F, 200).
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subtype, 41.7% for intermediate-cell subtype, and 83.3% for

cholangiocellular subtype), and 13.0% for ICC, as shown in
Figure 2A. The expression of DLK1 tended to be higher in
group B than in HCC, ICC, and group A combined HC-CC.
Notably, the expression rate of DLK1 was significantly higher
in the typical and cholangiocellular subtypes.
The expression of DLK1 in combined HC-CC is shown
in Figure 2B. High scores were obtained for the typical and
cholangiocellular subtypes compared with group A. Diffuse
expression (score 3+) of DLK1 was found in 3 (25.0%) cases
of the intermediate-cell subtype, while the staining of DLK1
was faint (score 1+) in 2 (16.7%) cases in group A (Figure 2B).

DLK1, NCAM/CD56, and CD133 were frequently

expressed in each subtype of group B compared with group
A. The expression of DLK1 and NCAM/CD56 was significantly higher in the typical and cholangiocellular subtypes.
There were no significant differences in EpCAM expression
between any of the groups Figure 2C.

Discussion
We diagnosed primary liver cancers according to the
2010 WHO classification. The mean age of patients was
significantly lower for HC-CC with more than 5% stem

Image 2 Immunohistochemical findings of hepatocellular carcinoma (HCC). A, Moderately differentiated HCC with a
thick trabecular pattern (H&E). B, Delta-like 1 homolog. Diffuse and strong cytoplasmic staining is found, and also a focal
membranous pattern is seen in HCC. C, Glypican 3. Strong cytoplasmic and focal membranous staining is found in HCC.
D, a-Fetoprotein (AFP). Diffuse expression of AFP is seen in HCC (A, 100; B-D, 200).

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Image 3 Immunohistochemical findings of combined hepatocellular-cholangiocarcinoma with stem cell features, typical subtype.
A, Small, oval, and dark-stained cells are distributed between fibrous septa and the periphery of carcinoma cell nests composed
of relatively large cells with an eosinophilic and granular cytoplasm (H&E). B, HepPar1. Almost all carcinoma cells are positive. C,
Delta-like 1 homolog (DLK1). Cytoplasmic staining of DLK1 is found mainly in peripheral cells of hepatocellular carcinoma (HCC)
nests, and DLK1 is focally expressed in stem celllike cells. D, Cytokeratin 19 (CK19). Some expression of CK19 is seen in small
peripheral cells. E, Neural cell adhesion molecule (NCAM)/CD56. Some expression of NCAM/CD56 is seen in peripheral small
cells. F, Epithelial cell adhesion molecule (EpCAM). The peripheral HCC cells tend to show membranous staining of EpCAM,
and stronger staining is observed in stem celllike small cells (A-C, F, 200; D, E, 400).
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celllike areas than that for HCC, ICC, and the classic type
of combined HC-CC. Combined HC-CC with stem cell
features was just as likely to occur in cirrhotic liver as in
HCC. Frequent expression of DLK1 was found in HC-CC
with stem cell features in comparison with HCC, ICC, and
classic HC-CC.
In mice and rats, the expression of DLK1 in hepatoblasts of fetal liver, but not adult liver, has been reported,
and interestingly, a re-upregulation of DLK1 expression in
stem/progenitor (oval) cells has been demonstrated, which
possess the ability to both differentiate and repopulate.6-8

In our study, immunohistochemical staining of DLK1 was

observed in hepatoblasts in the early fetal period, similar
to rodents, but there were no positive findings in normal
or cirrhotic liver after birth. Our results are consistent with
previous studies using human liver.10,12,13 Therefore, there
seem to be differences between humans and rodents in the
role and significance of DLK1 expression, although this
may be related to the immunohistochemical technique used.
The expression of DLK1 in HCC has been reported
in several studies.10-12 DLK1 was detected in 20.5% of
HCCs in a study by Yanai et al10 and in 56.8% of HCCs

Image 4 Immunohistochemical findings of combined hepatocellular-cholangiocarcinoma with stem cell features, intermediatecell subtype. A, Tumor cells have an oval-shaped nucleus and scant cytoplasm (H&E). There is no evident differentiation into
hepatocytes and cholangiocytes. B, Double immunostaining of HepPar1 (green) and cytokeratin 19 (CK19) (red). Tumor cells
showing both membranous red staining and cytoplasmic green/yellow staining are considered positive for both HepPar1 and
CK19. C, Delta-like 1 homolog (DLK1). Cytoplasmic expression of DLK1 is seen in intermediate-cell areas. D, Epithelial cell
adhesion molecule (EpCAM). Membranous staining of EpCAM is found in intermediate-cell areas (A, C, D, 200; B, 400).
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in another study by Huang et al.12 From our data, however, the expression rate was just 10.9%. The degree of
differentiation in the HCC cases examined may explain
the different incidences of DLK1 expression among these
reports because all DLK1-positive HCCs were moderately
or poorly differentiated. However, a positive correlation
between DLK1 and AFP expression is a finding common
to these reports; moreover, DLK1 expression in HCC was
significantly correlated with glypican 3 in our study. DLK1
seemed to act as an oncofetal protein similar to AFP or
glypican 3.

The frequent expression of DLK1 in group B combined

HC-CC was observed in this study. The expression rate
was significantly higher in each subtype of group B than
in HCC and, furthermore, significantly higher in the typical and cholangiocellular subtypes than in HCC, ICC, and
group A combined HC-CC. The expression of DLK1 was
located in stem-like cells in typical HC-CC, in intermediate
cells in intermediate-cell HC-CC, and in cholangiole-like
carcinoma cells in cholangiocellular HC-CC. The staining pattern for DLK1 in group B was different from that
in HCC: cytoplasmic and membranous staining dominated

Image 5 Immunohistochemical findings of combined hepatocellular-cholangiocarcinoma with stem cell features,

cholangiocellular subtype. A, Tumor cells show small, slender, arborizing tubular formation (H&E). B, Cytokeratin 7 (CK7).
Diffuse expression of CK7 is seen. C, Delta-like 1 homolog (DLK1). Weak to faint canalicular staining of DLK1 is observed.
D, Neural cell adhesion molecule (NCAM)/CD56. Membranous staining of NCAM/CD56 is diffusely found (A, B, D, 200;
C, 400).

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A
100
*
80

Frequency (%)

60

40

20

0
HCC

Combined
Group A
(Classical
Type)

Combined
Group B
(Typical
Type)

Combined
Combined
Group B
Group B
(Intermediate (Cholangiocellular
Type)
Type)

ICC

B
100

3+
2+
1+
0

Frequency (%)

80

60

40

20

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Combined
Group A
(Classical
Type)

Combined
Group B
(Typical
Type)

Combined
Group B
(Intermediate
Type)

Combined
Group B
(Cholangiocellular
Type)

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100

DLK1
NCAM/CD56
EpCAM

CD133

80

Frequency (%)

*
60

40

20

Combined
Group A
(Classical
Type)

Combined
Group B
(Typical
Type)

Combined
Group B
(Intermediate
Type)

Combined
Group B
(Cholangiocellular
Type)

Figure 2 The expression rate of delta-like 1 homolog (DLK1) in liver cancers. A, The frequency of DLK1 expression. The rate
of DLK1 is high in each type of combined hepatocellular-cholangiocarcinoma (HC-CC) with stem cell features compared with
hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), and the classic type. *Statistically higher compared with
HCC, ICC, and group A (classic type) of combined HC-CC. Statistically higher compared with HCC. B, The scores for DLK1
expression in HC-CC. There are 2+ or 3+ cases in group B, whereas there are no 2+/3+ cases in group A. C, The frequency
of DLK1, neural cell adhesion molecule (NCAM)/CD56, epithelial cell adhesion molecule (EpCAM), and CD133 in HC-CC. The
frequency of DLK1, NCAM/CD56, and CD133 expression is higher in group B than in group A. The expression of DLK1 and
NCAM/CD56 is significantly more frequent in the typical subtype and cholangiocellular subtype than in group A. *Compared
with group A (classic type).

in HCC, but cytoplasmic staining was predominant in the

stem cell type, and luminal staining was occasionally seen
in the cholangiocellular subtype. Recently, Komuta et
al15 reported that NCAM/CD56 was useful for classifying
mixed ICCs (showing focal hepatocytic differentiation and
ductular areas), which are thought to be almost the cholangiocellular subtype of combined HC-CC. Because NCAM/
CD56 and CD133, considered stem cellrelated markers
in the WHO classification and in several reports,16-18 were
frequently expressed in group B, the expression of DLK1
in stem cell HC-CC seemed to be associated with stem cell
features. These markers may be important to the stem cell
features of liver cancers, but it would be dangerous to make
judgments based on immunohistochemical results because
the expression of these markers is found in HCC, ICC, and
classic-type HC-CC to various degrees. At present, there is
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Ikeda_2012100561.indd 339

a need for both morphologic and phenotypical observations

for the diagnosis of stem cell features.
A progenitor/stem cell origin has been implied for combined HC-CC and also cholangiocellular carcinoma.2,5,19 The
expression of DLK1 in liver neoplasms may be associated
with stem cell features, because DLK1 expression was frequently detected in the stem cell type of combined HC-CC
in this study and also in hepatoblastomas.11,13 However, it
is unclear whether a DLK1-positive result means a progenitor/stem origin, because there was no expression in putative
progenitor cells in nonneoplastic liver. Xu et al14 suggested
that DLK1-positive HCC cells have characteristics similar to
cancer stem/progenitor cells, demonstrating increased chemoresistance, colony formation, spheroid colony formation, and
in vivo tumorigenicity compared with DLK1-negative cells. A
subpopulation of cells called cancer stem cells is now thought

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Ikeda et al / Liver Cancer With Stem Cell Features and DLK1

to be responsible for tumor initiation, growth, metastasis, and

recurrence.20,21 It seems possible that the expression of DLK1
in liver cancers, mainly in group B combined HC-CC, is
related to the existence of cancer stem cells, but more studies
are needed.
We were able to classify primary liver cancers according
to the 2010 WHO classification. New subtypes of combined
HC-CC with stem cell featuresnamely, typical, intermediate-cell, and cholangiocellular subtypeswere identified in
more than 5% of the tumors in 24 of 36 cases in this study.
The frequent expression of DLK1 and NCAM/CD56 was
seen in stem-like cells in the typical subtype and also cholangiocellular components. However, in the intermediate-cell
subtype, the expression rate for DLK1 was higher than in
HCC, but DLK1, NCAM/CD56, and CD133 tended to be
expressed less frequently than in other stem cell subtypes.
The intermediate-cell subtype may reflect transdifferentiation
rather than a stem cell origin.
The distribution of DLK1 expression was not the same in
NCAM/CD56 and/or CD133-positive cells. DLK1 seems to be
a candidate for a stem cell marker, although the significance
of its expression is not yet clear. DLK1 could be a potential
therapeutic target against cancer stem/progenitor cells,12,14 and
the analysis of DLK1-positive cells in liver cancers, mainly in
the stem cell type of combined HC-CC, is urgently needed.
In conclusion, the expression of DLK1 in combined
HC-CC seems to be associated with stem cell features, and
DLK1 is likely to be a candidate stem cell marker in liver cancer. There are differences in etiology, morphology, and stem
cell phenotype for HC-CC with stem cell features. Therefore,
the classification of combined HC-CC in the fourth edition
of the WHO system seems to be significant and important to
elucidation of the pathogenesis of stem cellrelated cancers.
Address reprint requests to Dr Ikeda: Section of Diagnostic
Pathology, Kanazawa University Hospital, Kanazawa 920-8641,
Japan; e-mail: h-ikeda@med.kanazawa-u.ac.jp.

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