Victor Ka-Tai Lau,t Ching-Lung Lai,t. Chi-Kin Lo,* and Johnson Yiu-Nam Laut. Fromi thec Depar-tlnents of/Pah(ology* and M1edicinCe,I. lUn'ilvt}' O//loig Kong, ...
Amricau njournal q/ Pathology, 1 a1. 149. \o. 4, October- 1996 (,op}! bght cD Amricrcan Soci t)'yftr ho sti'gative' Pathbology!
Classification of Hepatocellular Carcinoma According to Hepatocellular and Biliary Differentiation Markers Clinical and Biological Implications
Pui-Chee Wu,* Jane Wing-Sang Fang,*t Victor Ka-Tai Lau,t Ching-Lung Lai,t Chi-Kin Lo,* and Johnson Yiu-Nam Laut Fromi thec Depar-tlnents of/Pah(ology* and M1edicinCe,I lUn'ilvt}' O//loig Kong, Hong Kon=g:1 canld the SecCtion 0/ Hcpatobi/ifart Diseases, I)epartmncnt C)!f Mi/dic inC., Ul iv'el/si of Florida. Ga,;iCsvI'il/c. Florida
Hepatocellular carcinoma (HCC) is a heterogeneous disease. HCC derivedfrom different stages of cellular differentiation may have different clinical and pathobiological behavior. To test the hypothesis that HCC can be classified into two types based on its phenotypic markers (hepatocellular and biliary differentiation), liver tissues from 290 Chinese patients with HCC were studied. Expression of hepatocytic differentiation marker (HEP-PAR-reactive antigen), biliary differentiation markers (AEl-AE3, cytokeratin-19), proliferation markers (Ki-67, proliferating cell nuclear antigen), a-fetoprotein, p53, and transforming growth factor- a in the tumor tissue were assessed by immunohistochemistry. Hepatocytic differentiation marker was detected in 99. 7% and biliary differentiation markers were detected in 29.3% of these tumors. Clinically, no patient with HCC with biliary markers survived for more than 27 weeks compared with a 22.6% survival rate in patients with HCC negative for biliary markers. HCCs positive for the biliary differentiation markers showedfeatures of more aggressive disease in terms ofpoorer cellular differentiation (P < 0ao01) and high-level expression of proliferation markers (Ki-67, P < 0.001; proliferating cell nuclear antigen, P = 0.0114) compared with HCCs without biliary markers. HCCs with biliary markers also had a higher level of expression of a-fetoprotein (P < 0.001) and p53
(P = 0.0077). Classification of HCCs based on its phenotypic (differentiation) markers has both clinical and pathobiological implications. (AmJ Pathol 1996, 149:1167-11 75)
Primary liver carcinomas have been classified into hepatocellular carcinoma (HCC) and cholangiocarcinoma with a rare combined type of hepatocellularcholangiocellular carcinoma. HCC has always been considered a single disease based on the assumption that it originates from a single cell type, the hepatocyte or hepatocyte precursor cell. Cholangiocarcinoma is believed to originate from biliary epithelial cells. However, the diverse clinical and pathobiological behavior suggest that HCC is a heterogeneous disease. Epidemiological studies have also shown that HCC from different geographic locations, which have different patterns of etiological associations, had different profiles of oncogene or anti-oncogene involvement.` 1 HCC from regions where aflatoxin Bi contamination of food is high was found to have a higher incidence of mutations in the p53 gene.6 7 It is possible that cells at different stages of differentiation may have a different susceptibility to carcinogens. HCCs transformed from cells at different stages of differentiation may also have different clinical and pathobiological patterns. Advancements in the understanding of malignancies like leukemia and lymphoma were achieved when the concept of cell lineage was established. In Supported by a research grant from the Hong Kong Jockey Club Charities Trust (P-C. Wu) and a grant from the Cancer Center of the University of Florida (J. Y-N. Lau). Accepted for publication June 11, 1996. Address reprint requests to Dr. Johnson Yiu-Nam Lau, Section of Hepatobiliary Diseases, Division of Gastroenterology, Hepatology, and Nutrition, Department of Medicine, University of Florida, PO Box 100214, JHMHC, Gainesville, FL 32610.
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lymphoma, malignant transformation can occur in precursor cells without T or B cell markers, in cells with both T and B cell markers, or in cells already differentiated into T or B cells. When this concept was applied to the study of lymphoma, it was clear that this classification has clinical, pathobiological, and therapeutic implications. The two major cell types in liver, hepatocytes and biliary epithelial cells, are believed to originate from the same progenitor cells.8 Evidence from experimental chemical carcinogenesis models of HCC have suggested that oval cells, which possess both hepatocytes and biliary cell markers, are closely related to pleuripotent stem cells and have dual potential to differentiate into either hepatocytes or biliary epithelial cells.9- If the hypothesis of a single liver progenitor cell is true, HCC may consist of two types, those derived from progenitor cells that have features of both hepatocytic and biliary differentiation and those derived from differentiated hepatocytes that should only be positive for hepatocytic markers.12 The recognition of a rare combined hepatocellular-cholangiocellular type of liver carcinoma,13-16 which shows histological features of both hepatocellular and biliary epithelial carcinoma, supports this hypothesis. By analogy with the pathobiological behavior of other tumors, these two types of HCC, as determined by their phenotypic markers, may have different clinical and pathobiological behavior. The aim of the present study was to test the hypothesis that HCC can be classified into two types based on the presence or absence of biliary markers and that these two types of HCC have different clinical and pathobiological behavior. For the sake of clarity, HCC with biliary differentiation markers are abbreviated as HCC-B+ and those without biliary markers as HCC-B-. The term mixed hepatocellularcholangiocellular carcinoma is avoided here as HCCs included in this study, as defined in Materials and Methods, showed no histological features of
cholangiocarcinoma differentiation.
Materials and Methods Patient Population The diagnostic criteria of HCC for inclusion into this study consisted of compatible clinical and radiological features of HCC, with confirmation by histology. HCCs that were too poorly differentiated for identification of hepatocellular differentiation by the criteria set by the World Health Organization,17 ie, hepatocyte-like malignant cells, trabecular or sinusoidal growth pattern, bile production, or intercellular bile
canaliculi, were excluded. Based on these criteria, formalin-fixed, paraffin-embedded diagnostic HCC tissue blocks from 290 Chinese patients admitted to Queen Mary Hospital, Hong Kong, between 1986 and 1991 were included in this study. Hepatitis B virus surface antigen was positive in 86.1% and histological evidence of cirrhosis was identified in 122 of 151 (80.8%) tissue blocks in which adequate liver tissue was available for assessment. The liver specimens included 201 needle biopsies, 73 surgical resection specimens, and 16 autopsies. In 162 patients who did not receive either medical or surgical treatment and had complete follow-up till death, survival was recorded dating from the time of the confirmatory diagnostic liver biopsy. All cases that underwent any form of treatment for HCC were excluded from the assessment of natural survival.
Histological Assessment For this series, none of the HCCs showed identifiable histological evidence of cholangiocarcinoma differentiation when 5-,um sections stained with hematoxylin and eosin (H&E), Gordon and Sweet silver for reticulin, and mucicarmine for mucin secretion were examined. No fibrolamellar type of HCC was identified. The histological features of HCC including tumor differentiation (well, moderately, and poorly differentiated, scored as 1 to 3),18 architecture (trabecular, solid, and pseudoacinar), and the cytological features (ie, presence or absence of small, medium, or large tumor cells with reference to the size of normal hepatocytes, clear cells, and multinucleated giant cells) were assessed blindly. The degree of cellular and nuclear pleomorphism and mitosis as well as pseudoacini formation was also evaluated and scored 0 to 4 with increasing severity.
Immunohistochemistry Five-micron formalin-fixed paraffin-embedded tissue sections were prepared. The standard indirect streptavidin-biotin horseradish peroxidase immunohistochemical method (Amersham, Chicago, IL) with diaminobenzidine (Sigma Chemical Co., St. Louis, MO) as substrate was employed for the detection of phenotypic markers as well as other related markers. The antigen retrieval system with microwave was used to enhance the detection of Ki-67 antigen before immunohistochemical detection. Hepatocytic differentiation was sought by detection of immunoreactivity to the monoclonal antibody HEP-PAR (kindly provided by Dr. Nalesnik, University of Pittsburgh, Pittsburgh, PA; dilution, 1:4)19 Monoclonal antibod-
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ies directed against two epithelial cell markers, cytokeratin 19 (CK-19; Dako, Carpinteria, CA) at a dilution of 1:100 and AE1-AE3 (ICN, Costa Mesa, CA) at a dilution of 1:200, known to be expressed only in biliary cells and not in hepatocytes, were used as phenotypic markers of biliary differentiation. Two cellular proliferation markers were assessed using monoclonal antibodies against proliferation cell nuclear antigen (PCNA; Dako) at a dilution of 1:100 and Ki-67 (Dako) at a dilution of 1:20. In addition, the expression of a-fetoprotein (AFP) by a rabbit polyclonal antibody (Dako; 1:200 dilution; tested in only 254 of 290 patients), transforming growth factor (TGF)-a (Oncogene Science, Uniondale, NY; 1:800 dilution), and p53 (Dako; 1:50 dilution) were also determined. All antibodies were diluted in Tris-buffered saline (TBS, pH 7.4) with 1% bovine serum albumin (Sigma). Briefly, the tissue sections were deparaffinized with xylene, rehydrated through a graded alcohol series, and blocked with 3% bovine serum albumin and 10% normal rabbit serum or normal goat serum (for AFP) in Tris-buffered saline for 15 minutes to minimize nonspecific absorption. Primary antibodies in appropriate dilutions were applied and incubated for 45 minutes at 37°C. The detection with biotin-peroxidase-conjugated streptavidin as tracer was performed according to the manufacturer's instructions. All sections were counterstained with hematoxylin. Positive controls included sections from nonliver organs that are known to express certain antigens, tumors known to express these antigens, or positive controlled slides purchased from the respective suppliers. Negative controls included using nonimmune mouse and rabbit serum (for AFP) as a substitute for the primary antibodies. The expression of these markers was assessed independently and blindly by two investigators and graded as follows: 0, no positive cells; 1,
