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1. A Clinicopathologic Study of Intrahepatic
Cholangiocarcinoma Containing a Component
of Squamous Cell Carcinoma
Tohru Nakajima, MD, and Yoichiro Kondo, MD
Eleven cases of cholangiocarcinoma containing a component of squamous cell
carcinoma (CC-SCC) were reviewed clinicopathologically in comparison with 82
cases of common cholangiocarcinoma presenting as a pure form of adenocarcinoma
(CC-AC). The former type was prone to develop in a rather advanced stage of the
disease, as indicated by short survival time, large tumor size, aggressive modes of
intrahepatic spreading, and frequent metastasis. Histologically, the area of
squamous cell carcinoma ranged from focal to overwhelming. The occurrence of
the squamous element could be interpreted as the result of the metaplastic
transformation of adenocarcinoma cells because of the intimate coexistence of the
two types in the primary and metastatic lesions. It also was suggested that
anaplastic carcinoma cells could sometimes differentiate into squamous cell
carcinoma. In no instances was there any squamous metaplasia of the bile ducts
demonstrated therefore, there was no supporting evidence for the possibility of
squamous metaplasia of the bile duct epithelium and subsequent malignant
transformation. Cancer 65:1401-1404,
1990.
RIMARY squamous or adenosquamous carcinomas
Pare common in the gallbladder,' whereas such car-
cinomas are rare in the liver; only seven squamous2-8and
six adenosquamous have been reported in
the English literature. Although the individual clinicaland
pathologic features and the histogenesis of these unique
variants of cholangiocarcinoma have been described in
these previous reports, a comprehensive study based on
a certain number of cases is still lacking.
The current study was undertaken to clarify some of
the clinicopathologic characteristics of intrahepatic cho-
langiocarcinoma containing a component of squamous
cell carcinoma (CC-SCC) in comparison with common
intrahepatic cholangiocarcinoma composed of pure ad-
enocarcinoma (CC-AC).
Materials and Methods
From 1975 through 1988, 117 histologically defined
cases of intrahepatic cholangiocarcinoma were surveyed
in our laboratory and in affiliated hospitals. Of these, 1 1
From the Department of Pathology, School of Medicine, Chiba Uni-
versity, Chiba, Japan.
Addressfor reprints: Tohru Nakajima, MD, Department of Pathology,
Schoolof Medicine,Chiba University, Inohana 1-8-1, Chiba 280,Japan.
Accepted for publication September 18, 1989.
cases showed a feature of CC-SCC and consisted of one
surgical and ten autopsy cases. Eighty-two autopsy cases
of conventional CC-AC with relevant clinicopathologic
data were available as controls.
Formalin-fixed tissueblocks were obtained from at least
two different areas of each primary lesion, and also were
obtained from various metastatic sites. Four-pm paraffin
sections were stained with hematoxylin and eosin (H &
E). Periodic acid-Schiff (PAS)and alcian blue stainswere
applied to confirm the adenocarcinomatous nature, i.e.,
the presence of mucin in the glandular lumina or cyto-
plasm. Identification of the squamous cell carcinoma
component was based on the presence of solid nests of
polygonal cells showing either keratinization or intercel-
lular bridge formation. In addition, an immunohisto-
chemical examination (peroxidase-antiperoxidase
method [PAP]I4)was performed using rabbit antibody
for involucrin (BiomedicalTechnologiesInc., Cambridge).
Involucrin is a precursor of the keratinocyte cross-linked
envelope protein or marginal band present in the human
stratum corneum and can be used for the assessment of
squamous cell differentiation.15,16 The specificity of the
staining was confirmed by substituting normal rabbit
serum for the primary antibody, with normal human skin
being used as a positive control and heart muscle as a
negative control.
1401

2. 1402 CANCER
March 15 1990 Vol. 65
All cases were studied with regard to age, sex, initial
symptoms, frequency of -associatedgallstones, liver cir-
rhosis, survivaltime, site of tumor in the liver,tumor size,
gross type, histologic features, modes of intrahepatic
spreading,and incidence of metastasis. The survivaltime
was calculated in months from the date of the onset of
initial symptoms. The gross type and modes of intrahe-
patic spreading were classified according to our previous
report.l7
Results
The clinicopathologic features of CC-SCC and CC-AC
are summarized in Table 1. Although these two groups
shared many common features, the mean survival rate of
the patients with CC-SCC was shown to be less than that
of the selectedautopsy casesof CC-AC (4.0 f 1.2 months
versus 6.9 f 1.2 months). The association of gallstones
was more frequent in CC-SCC than CC-AC. Liver cir-
rhosis was observed in CC-AC (6.1%), but not in CC-
SCC. The most common gross appearance in CC-SCC
was the nodular type, in contrast to the massive type in
CC-AC. The mean size of the tumor at its greatest di-
mension of CC-SCC (10.2 f 2.2 cm) was slightly larger
than that of CC-AC (8.3 f 1.0cm).
Table 2 represents the proportions of adenocarcinoma
and squamous cell carcinoma in the liver. There was no
instanceof a pure form of squamouscellcarcinoma.Three
cases were composed mostly of squamous cell carcinoma
(Fig. l), containing focal areas of glandular formation or
TABLE1. Clinicopathologic Analysis of CC-SCC and CC-AC
Variable cc-SCC CC-AC
No. of cases
Mean age (yr)
Sex ratio (M:F)
Frequent initial symptoms (%)
Abdominal pain
Jaundice
Fever
Anorexia
Others
Survivaltime (mo)
Associated gall stones (%)
Associated liver cirrhosis (%)
Site of tumor in the liver (9%)
Peripheral
Hilar
Mean size (cm)
Gross type (%)
Massive
Nodular
Diffuse
Sclerosing
Intraluminal
11
60.5 ? 6.8
7:4
6 (54.5)
3 (27.3)
l(9.1)
1 (9.1)
0 (0)
4.0 k 1.2
3 (27.0)
0 (0)
7 (63.6)
4 (36.4)
10.2 ? 2.2
3 (27.3)
6 (54.5)
1 (9.1)
1 (9.1)
0 (0)
82
62.6 f2.1
52:30
24 (29.3)
23 (28.0)
10 (12.2)
8 (9.8)
17 (20.7)
6.9 f 1.2
16 (19.5)
5 (6.1)
59 (72.0)
23 (28.0)
8.3 k 1.0
44 (53.7)
24 (29.3)
4 (4.9)
8 (9.8)
2 (2.4)
CC-SCC cholangiocarcinoma containing squamous cell carcinoma;
CC-AC cholangiocarcinoma containing pure adenocarcinoma.
TABLE
2. Components of SCC in 11 Cholangiocarcinomas
Patterns No. of cases
~~
SCC with focal AC 3
Adenosquamous carcinoma 3
AC with focal SCC 3
Anaplastic carcinoma with AC and SCC 1
Anaplastic carcinoma with SCC 1
SCC squamous cell carcinoma; AC: adenocarcinoma.
clusters of vacuolated cells with intraluminal or intracel-
lular mucin as demonstrated by PAS and alcian blue
stains. Three casesthat contained equal amounts of squa-
mous and glandular elements were designated as adeno-
squamous carcinoma. Admixed within the predominant
area of adenocarcinoma were tiny foci of squamous cell
carcinoma in three other cases. In these cases, more than
ten specimens were examined to show the presence of
squamous cell carcinoma. The squamous cell carcinoma
component was scattered in each specimen. The remain-
ing two cases were anaplastic carcinoma with partial
squamous cell differentiation (Fig. 2), one of which also
presented scattered, definite glandular structures within
undifferentiated tumor cell clusters. Keratinization was
present in eight cases,whereasseven exhibited intercellular
bridges. In ten cases, positive staining for involucrin oc-
curred in the cytoplasm of somewhat well-differentiated
portions of squamous cell carcinoma (Fig. 3). PAS and
alcian blue stains showed the presence of mucinous sub-
stances, mostly in conjunction with glandular formation.
The stainings were completely negative in one case of
anaplasticcarcinoma that showedno glandularformation.
In general, a similar histologic pattern (ie.,the con-
current presence of glandular and squamous components)
also could be observed at various metastatic sites.
RG. 1. A portion of well-differentiated squamous cell carcinoma
showing keratinization in the center of a nest (H & E, original magni-
fication X 115).

3. No. 6 INTRAHEPATIC
CHOLANGIOCARCINOMA
- Nakajima and Kondo 1403
FIG. 2. Anaplastic carcinoma showingfocal squamous differentiation
(arrow) (H & E, original magnification X230).
Gallstones were present in three cases. Their histologic
patterns were not different from those of the other cases
with no gallstones.
As for the histogenesisof CC-SCC, eosinophilic plump
cells were often observed within a gland (Fig. 4). These
plump cells gradually became prevalent by replacing the
preexisting tumor cells and showed formation of inter-
cellular bridges or keratinization as demonstrated by im-
munostaining for involucrin. In the two casesof anaplastic
carcinoma, one showed direct differentiation of anaplastic
tumor cells into squamous cell carcinoma and the other
displayed dual differentiation into adenocarcinoma and
squamous cell carcinoma. No squamous metaplasia was
encountered in the intrahepatic bile duct.
Regarding intrahepatic tumor spreading, CC-SCC
showed a higher incidence in sinusoidal invasion, vascular
involvement, permeation in the portal connective tissue,
perineural or intraneural invasion, and lymphatic in-
FIG.4. A transitional area from adenocarcinoma to squamous cell
carcinoma. Clusters of plump cells are seen in the glandular portion
(arrow). On the right, a large nest of squamous cell carcinoma can be
seen (H & E, original magnification X 115).
volvement as compared with CC-AC (Table 3).Replacing
growth along the bile duct epithelium was observed in 13
cases (1 5.8%)of CC-AC, but not in cases of CC-SCC.
The CC-SCC cases had 11 intrahepatic (loo%), 8 re-
mote organ (72.8%), and 9 lymph node metastases
(81.8%), whereas these numbers were 55 (67.1%), 57
(69.5%),and 56 (68.3%),respectively,in the CC-AC cases
(Table 3).
Discussion
Although several reports of CC-SCC in the liver have
been published, the clinical behavior of this diseaseis still
poorly understood. It is speculated that its clinical course
and prognosis do not vary significantly from other types
of cholangiocarcinoma.'' In previous reports, distant or-
gan or lymph node metastases were recorded in eight of
13 ~ a s e s . ~ - l ~
TABLE
3. Spreading Patterns and Incidence
of Metastasis of CC-SCC and CC-AC
No. of cases ('70)
Variable cc-SCC CC-AC
FIG.3. Squamous cell carcinoma showingpositive staining for invol-
ucnn corresponding to keratinized cells (counterstained with methyl
green, original magnification X230).
Intrahepatic spreading
Sinusoidal invasion 11 (100) 66 (80.5)
Vascular involvement 8 (72.9) 39 (47.6)
Permeation in portal connective tissue 6 (54.5) 14 (17.1)
Neural invasion 5 (45.5) 14 (17.1)
Lymphatic involvement 4 (36.4) 14 (17.1)
0 (0) 13 (15.8)
Replacing growth
Intrahepatic 11 (100) 55 (67.1)
Remote organ 8 (72.8) 57 (69.5)
Lymph node 9 (81.8) 56 (68.3)
CC-SCC: cholangiocarcinoma containing squamous cell carcinoma;
Metastasis
CC-AC: cholangiocarcinoma containing pure adenocarcinoma.

4. 1404 CANCER
March 15 1990 Vol. 65
Although the number of CC-SCC cases was not sub-
stantial enough to draw a meaningful statistical conclu-
sion, the results of the current study suggested that this
rare variant tended to present rather aggressive clinico-
pathologic features when compared with common cho-
langiocarcinomas.Despite the close similarityin age and
sex ratios between the two groups, the patients with CC-
SCC seemed to have a poor prognosis. In addition, CC-
SCC were prone to spread into the hepatic tissue by var-
ious ways. These findings suggested that squamous dif-
ferentiation may be partly responsible for promoting the
stageof cholangiocarcinoma.A similartrend alsohasbeen
reported in squamous and adenosquamous carcinoma of
the gallbladder.I These considerations may be of some
use for determining the treatment and prognosis of
CC-SCC.
The histogenesis of tumors containing a squamous
component in the liver has been debated, and there was
a defined case consistent with malignant transformation
of a hepatic teratoma.* Some examplesof squamous cell
carcinoma arising in the wall of a hepatic cyst were con-
sidered to occur through the squamous metaplasia of the
lining epithelium before malignant tran~formation.~-~
Such a metaplastic process also was assumed in the in-
trahepatic biliary epithelium with concomitant hepato-
lithia~is.~
On the contrary, Arase et al. stressed that the
incidenceof squamousmetaplasiain the intrahepatic bile
duct was too rare to support the hypothesis mentioned
above.' The origin of mucoepidermoid carcinoma was
attributed to the terminal ramifications of the bile cana-
liculi that had undergone squamous metaplasia.' Moore
et al. described a case of biliary cystadenocarcinoma
showing metaplastic adenosquamous alteration.l 3
In our study, cysticremnants, componentsof teratoma,
or squamousmetaplasiain the bile duct epithelium could
not be found in the liver. If heterotopic or metaplastic
squamous epithelium does undergo malignant transfor-
mation, the tumor tissue may be composedof pure squa-
mous elements. Such a process is not applicable for in-
terpreting the pathogenesis of our cases that showed the
coexistence of adenocarcinoma and squamous cell car-
cinoma.
Generally,the component of squamous cell carcinoma
was intimately mixed with adenocarcinoma. In fact, the
two components were often observed in the same tumor
cell nest. Therefore, it seems reasonable to assume that
CC-SCCdevelopson the basis of the squamous transfor-
mation of preexisting adenocarcinoma. This concept is
in agreementwith that of Barr and Hancock" and Willis.''
However, one case of anaplastic carcinoma showed di-
rect squamous differentiation, bypassing the process of
glandular formation. Another case of anaplastic carci-
noma exhibited concurrent squamous and glandular dif-
ferentiation of the tumor cells. These observations sug-
gestedthat anaplasticcarcinoma retained the propertyfor
differentiating into adenocarcinoma or squamous cell
carcinoma.
Whatever the events, the fact that under some condi-
tions the growth rate of adenocarcinoma can be exceeded
by that of squamous cell carcinoma as evaluated by the
analysis of the doubling time" would, at least in part,
accountfor heterogeneitiesin the histologicmanifestations
of CC-SCC. However, the pathogenesis that induces the
evolution of squamous cell carcinoma in cholangiocar-
cinoma remains unknown.
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