Comparative immunohistochemical profile of hepatocellular carcinoma,cholangiocarcinoma, and metastatic adenocarcinoma

Distinguishing hepatocellular carcinoma (HCC) from cholangiocarcinoma (CC) and metastatic adenocarcinoma (MA) involving the liver can be problematic, often requiring the use of immunohistochemistry to facilitate diagnosis. Hep Par 1, a monoclonal antibody with expression confined primarily to benign and malignant hepatocytes, has recently become commercially available. We evaluated Hep Par 1 along with other immunohistochemical markers used to differentiate HCC, CC, and MA, including AE1/AE3, CAM 5.2, B72.3, monoclonal carcinoembryonic antigen (mCEA), polyclonal CEA (pCEA), alpha-fetoprotein (AFP), factor XIIIa, inhibin, CD10, villin, MOC-31, cytokeratin (CK) 7, CK 19, and CK 20, to determine the markers most useful in differentiating these entities. Forty-two cases of HCC, 9 cases of CC, and 56 cases of MA (24 colon, 15 pancreas, 8 ovary, 5 breast, and 4 stomach) were studied. Hep Par 1 was sensitive and specific for HCC, with 38 of 42 (90%) cases staining positively, whereas reactivity was observed in only 8 of 56 (14%) MAs and 0 of 9 CCs. Though limited somewhat by poor sensitivity, a bile canalicular pattern of staining with pCEA, CD10, and villin was specific for HCC and was not observed in the other tumors. Lack of mCEA and MOC-31 immunoreactivity was also characteristic of HCCs. CK 19 positivity favored CC over HCC, but was not useful in differentiating CC from MA. Expression of AFP, although observed in only about one third of the cases, favored HCC over CC and MA. CK 7 and CK 20 were also useful in this differential diagnosis, particularly when dealing with MA of colonic origin. AE1/AE3, CAM 5.2, B72.3, inhibin, and factor XIIIa were noncontributory in differentiating these entities.     

An immunohistochemical study of hepatic atypical adenomatous hyperplasia, hepatocellular carcinoma, and cholangiocarcinoma with α-fetoprotein, carcinoembryonic antigen, CA19-9, epithelial membrane antigen, and cytokeratins 18 and 19

Eight hepatic atypical adenomatous hyperplasias (AH), 30 hepatocellular carcinomas (HCC) consisting of 11 well-, 13 moderately and six poorly differentiated HCC, and 10 intrahepatic cholangiocarcinomas (CC) were investigated immunohistochemically with anti-alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA19-9, epithelial membrane antigen (EMA), and cytokeratins (CK) 18 and 19 antibodies. Immunostaining was regarded as positive when more than 5% of cells were stained. Alpha-fetoprotein was positive, although focally, in five (17%) of 30 HCC but negative in all AH and CC. Carcinoembryonic antigen (polyclonal antibody) did not stain the cytoplasm of all AH and HCC, but stained two (25%) of eight AH and 10 (33%) of 30 HCC in a bile canalicular staining manner. Carcinoembryonic antigen showed intracytoplasmic or luminal border staining in six (60%) of 10 CC. CA19-9 was negative in all AH and HCC, while six (60%) of 10 CC were positive for CA19-9. Epithelial membrane antigen was positive in one (13%) of eight AH, seven (23%) of 30 HCC and in all 10 cases of CC. Cytokeratin 18 was positive in all AH, HCC and CC. Cytokeratin 19 was negative in both AH and HCC, whereas it stained the cytoplasm of tumor cells in all CC diffusely and intensely. These results suggest that immunostaining of AFP, CEA, CA19-9, EMA, CK18 and CK19 are not useful in the differential diagnosis between AH and well-differentiated HCC, and that CK19 is the most suitable reagent for the differential diagnosis between HCC and CC.     

Copy number aberrations in combined hepatocellular carcinoma and cholangiocarcinoma

Combined hepatocellular carcinoma and cholangiocarcinoma (CHC) is a rare liver cancer which shares unequivocal features of both hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). A greater awareness of genetic relationship between HCC and CC components is limited. To help characterize this rare liver neoplasm, we described clinicopathologic features and evaluated copy number (CN) changes in this study. A total of 13 cases of CHC were collected. Four paired HCC and CC components from four cases were first subject to genome-wide analysis. Nine target genes were subsequently selected for further analysis using quantitative polymerase chain reaction. The paired HCC and CC components in each case had a concordant trend of CN gain or loss in these nine genes. However, the magnitude of concordant CN gain or loss was different. There were significant differences of CN copies between HCC and CC in each case. We demonstrate genetic divergence between HCC and CC components in CHC.     

Combined hepatocellular carcinoma and cholangiocarcinoma with components of mucinous carcinoma arising in a cirrhotic liver

A rare autopsy case of combined hepatocellular and cholangiocarcinoma, occurring in a 54-year-old man with liver cirrhosis, is presented. Initial laboratory data included CEA 52.1 ng/mL, DUPAN-2 1600 U/mL, AFP 2 ng/mL, and negativity for hepatitis B surface antigen, hepatitis B early antigen and hepatitis B core antibody. Ultrasonography and CT scan showed a large tumor node in the liver with ringed enhancement, swelling of several para-aortic lymph nodes, and ascites. Clinically, it was not possible to determine whether the hepatic tumor was an intrahepatic cholangiocarcinoma or a metastatic carcinoma. Histologically, the primary lesion was composed solely of hepatocellular carcinoma (HCC) with a trabecular pattern, and the intrahepatic metastases consisted of a variable admixture of HCC and cholangiocarcinoma (CC) with excessive mucin production. Interestingly, the tumor cell cluster showing a trabecular growth pattern produced mucin and had immunohistochemical expression of hepatocyte, cytokeratins 7 and 8. It is concluded that these hepatic tumor cells had both HCC and CC characters.     

Comparison of alpha-fetoprotein with some other tumour markers in Malaysians with hepatocellular carcinoma

Although alpha-fetoprotein (AFP) is regarded as the reference marker for hepatocellular carcinoma (HCC), it sometimes produces false results. The objective of this study was to see if some of the readily available laboratory markers could complement AFP to improve the laboratory diagnosis of HCC. The markers tested and their sensitivities were: CA 125, 92%; ferritin, 71.3%; CA 19-9, 69.8%; beta-2-microglobulin (B2M), 53.3%; CA 72-4, 13.6%; and carcinoembryonic antigen (CEA), 10.6%. In comparison, AFP had a sensitivity of 58.8%. CA 72-4 and CEA (at the "tumour" cut-off level of 20 ng/ml) had specificities of 100%, and AFP, 97.4%. The specificities of the other markers were less impressive: CEA, 77.8% (at the cut-off level of 5 ng/ml); ferritin, 48.6%; CA 125, 48.5%; B2M, 39.6%; and CA 19-9, 37.3%. The efficiencies of the markers for HCC, which are based on the consideration of sensitivity and specificity together, were as follows: AFP, 77.6%; CA 125, 71.3%; ferritin, 60.5%; CA 19-9, 55.3; B2M, 46.9%; CEA, 40.8%; and CA 72-4, 34.5%. The receiver-operating characteristic plots confirmed AFP to be the most efficient marker for HCC. Nevertheless, it is proposed that CA 125 be combined with AFP for HCC screening because of their excellent sensitivity and specificity, respectively: a negative result for both, or even just CA 125 alone, would indicate that the disease is unlikely while a positive AFP (which would likely occur with a positive CA 125) would make its presence highly probable. A positive CA 125 and negative AFP would be equivocal for HCC. Other markers in combination with AFP are less useful.     

Utilization of spectrins βI and βIII in diagnosis of hepatocellular carcinoma

Spectrins are a group of cytoskeletal proteins which participate in many important cellular functions. It has been suggested that loss of spectrin isoforms may be associated with tumorigenesis of lymphoma, leukemia, gastric cancer and hepatocellular carcinoma (HCC). We recently reported that βI spectrin expression was present in normal hepatocytes but lost in HCC cells, which suggested that spectrins may be helpful markers in diagnosis of HCC. In this study, using immunohistochemical staining, we further investigated the expression pattern of four spectrin isoforms (αII, βI-III) on different benign and malignant liver tumors including focal nodular hyperplasia (FNH), hepatic adenoma (HA), HCC, and cholangiocarcinoma (CC). The results revealed that βI spectrin was moderately to strongly positive in FNH and HA tissues, but was only weakly positive or lost in HCC cases and was weakly positive in all CC cases. In addition, the βIII spectrin, majority of which was moderately positive in both FNH and HA tissues, was mostly lost in poorly differentiated HCC but remained at least moderately positive in most CC cases. These results suggest that spectrins βI and βIII may be used to differentiate well differentiated HCC from FNH or HA, and poorly differentiated HCC from CC, respectively.     

Combined fibrolamellar carcinoma and cholangiocarcinoma exhibiting biphenotypic antigen expression: a case report

Fibrolamellar carcinoma (FLC), a variant of hepatocellular carcinoma (HCC), very rarely occurs in association with cholangiocarcinoma (CC). This report describes the first case of FLC coexisting with CC (FLC-CC) from Japan. Although the major part of the tumour located in the right lobe of the liver showed the typical features of FLC, CC was admixed with the FLC, not only in the primary hepatic tumour, but also in the lymph node metastases. Immunohistochemical analysis revealed that, although carcinoembryonic antigen (CEA), which can be detected with monoclonal antibodies in the cytoplasm and the cell surface of CC cells but not HCC cells, was expressed in only the CC cells in the primary tumour, it was expressed extensively in the cytoplasm of both CC and FLC cells in the metastatic and recurrent tumours. Furthermore, Hep Par 1, a hepatocyte specific antigen, was also expressed in both the FLC and CC cells. These findings suggest that, in this case, both FLC and CC were possibly derived from the same cancer stem cell with the capacity to differentiate into both hepatocytes and bile duct epithelium, and that both the cellular components, therefore, exhibited biphenotypic antigen expression.     

磷脂酰肌醇蛋白聚糖3等抗体在高分化肝细胞肝癌中的表达及其鉴别诊断意义

目的 探讨磷脂酰肌醇蛋白聚糖3(GPC3)、CD10、CD34及甲胎蛋白(AFP)在高分化肝细胞肝癌(HCC)、高级别异型增生性结节(H-DN)、低级别异型增生性结节(L-DN)、肝硬化结节、局灶性结节状增生(FNH)和肝腺瘤中的表达及临床应用价值.方法 应用免疫组织化学(EliVision法)对80例HCC(30例高分化HCC、50例进展期HCC)、30例DN(18例H-DN、12例L-DN)、36例肝硬化结节、20例FNH及20例肝腺瘤分别进行GPC3、CD10、CD34及AFP抗体标记,分析这些抗体在肝结节性病变中的鉴别诊断价值.结果 (1)GPC3的阳性表达率在进展期HCC中为92%(46/50),在高分化HCC中为66.7%(20/30),在H-DN中为2/18,在L-DN、肝硬化结节、FNH和肝腺瘤中无表达.GPC3在高分化HCC中的阳性表达率低于进展期HCC,而高于H-DN、L-DN、肝硬化结节、FNH、肝腺瘤,表达差异有统计学意义(P＜0.05).(2)CD10在进展期HCC中的失表达率为78%(39/50),阳性细胞比率＞50%的仅为2%(1/50);在高分化HCC中失表达率为43.3%(13/30),阳性细胞比率＞50%的为16.7%(5/30),而在H-DN、L-DN、肝硬化结节中失表达率分别为0、0、2.8%(1/36),阳性细胞比率＞50%分别为15/18、11/12、80.6%(29/36);在FNH和肝腺瘤中失表达率均为20%(4/20),阳性细胞比率＞50%均为60%(12/20).CD10在高分化HCC中的阳性细胞比率高于进展期HCC,而低于在H-DN、L-DN、肝硬化结节、FNH和肝腺瘤中的表达,表达差异有统计学意义(P＜0.05).(3)GD34在进展期HCC、高分化HCC中的表达范围绝大部分在25%～100%,阳性细胞数＞50%的为76.0%(38/50)和70.0%(21/30);而在H-DN和L-DN中的表达范围多集中在5%～25%,阳性细胞数＜25%的分别为16/18、10/12;在肝硬化结节表达范围在0～5%,阳性细胞数＜25%的为27.8%(10/36).在FNH与肝腺瘤中的表达范围在25%～50%.CD34在高分化HCC中的阳性细胞数与进展期HCC中无明显差异(P＞0.05),但高于H-DN、L-DN、肝硬化结节、FNH和肝腺瘤中的阳性细胞数,差异有统计学意义(P＜0.05).(4)AFP在高分化HCC中阳性表达率为20%(6/30),在进展期HGG中阳性表达率为44%(22/50),在H-DN、L-DN、肝硬化结节、FNH和肝腺瘤中均未见表达.AFP在高分化HCC中的阳性表达率低于进展期HCC,而高于肝硬化结节、FNH和肝腺瘤,差异具有统计学意义(P＜0.05).结论 GPG3、CD10、CD34及AFP在高分化HCC的诊断与鉴别诊断中,GPC3是一个较敏感及特异的标记物,其与CD34、CD10及AFP联合使用对诊断高分化HCC以及鉴别其与DN、FNH、肝腺瘤、肝硬化结节具有较高的应用价值.

Abstract：

Objective To study the expression and significance of GPC3, CD10 and CD34 in hepatocellular carcinoma (HCC), dysplastic nodules ( DN), cirrhotic regenerative nodules (CRN), focal nodular hyperplasia (FNH) and hepatocellular adenoma (HA). Methods Immunohistocheicical study for GPC3, CD10,CD34 and AFP was performed on 80 cases of HCC (30 cases of well-differentiated HCC and 50 cases of advanced HCC), 30 cases of DN (18 cases of high-grade DN and 12 cases of low-grade DN),36 cases of CRN, 20 cases of FNH and 20 cases of HA. Results (1)The positive expression rate of GPC3was 92% (46/50) in advanced HCC, 66. 7% (20/30) in well-differentiated HCC, 2/18 in high-grade DN, and 0 in low-grade DN, CRN, FNH and HA. The expression rate of GPC3 in well-differentiated HCC was lower than that in advanced HCC and higher than that in high-grade DN (P＜0.05). (2) The negative expression rate of CD10 was 78%(39/50) in advanced HCC, 43.3% (13/30) in well-differentiated HCC,20% (4/20 and 4/20) in both FNH and HA, 2.8% (1/36) in CRN and 0 in both high-grade DN and low-grade DN. The occurrence of CD10-strongly positive cells was 2%(1/50) in advanced HCC, 16.7%(5/30) in well-differentiated HCC, 15/18 in high-grade DN, 11/12 in low-grade DN, 80.6% (29/36) in CRN and 60%(12/20 and 12/20) in both FNH and HA. The positive expression rate of CD10 in well-differentiated HCC was higher than that in advanced HCC and lower than that in high-grade DN,low-grade DN, CRN, FNH and HA(P＜0.05).(3) The positive expression rates of CD34 in advanced HCC and well-differentiated HCC ranged from 25% to 100% [and strongly positive in 76% (38/50) and 70%(21/30), respectively]. The rates in high-grade DN and low-grade DN ranged from 5% to 25% (and weakly positive in 16/18 and 10/12, respectively). In CRN, the rate ranged from 0 to 5% [and weakly positive in 27.8%(10/36)]. In FNH and HA, the positive rates ranged from 25% to 50%. The positive expression rate of CD34 in well-differentiated HCC was significantly higher than that in high-grade DN,low-grade DN, CRN, FNH and HA (P＜0.05). (4) The positive expression rate of AFP was 44%(22/50) in advanced HCC, 20% (6/30) in well-differentiated HCC, no expression in DN, LCN, LCN,FNH and HA. The positive expression rate of AFP in well-differentiated HCC was lower than that in advanced HCC and higher than that in LCN, FNH and HA. The different expression had statistical significance (P＜0.05). Conclusions GPC3 is a relatively sensitive and specific marker in pathologic diagnosis of HCC. When coupled with immunohistochemical results of CD34, CD10 and AFP, GPC3 is useful in differentiating HCC from DN, LCN, FNH and HA.     

Concurrent evaluation of p53, beta-catenin, and alpha-fetoprotein expression in human hepatocellular carcinoma

Recent models suggest that hepatocellular carcinoma (HCC) develops through several independent pathways marked by key mutations in the beta-catenin or p53 gene. An additional pathway potentially is marked by aberrant expression of a-fetoprotein (AFP). To see whether these potential markers are expressed independently, we immunostained sequential sections from 55 HCCs. Of the cases, 30 (55%) were positive for 1 or more proteins: AFP, 19 cases (35%); p53, 12 cases (22%); and beta-catenin, 9 cases (16%). Seven tumors (13%) were positive for more than 1 protein, with 4 of 7 positive in the same area of tumor and 3 of 7 positive in different areas of the carcinomas. By statistical analysis, expression of the markers was independent of one another and of tumor size. Concurrent evaluation of p53, beta-catenin, and AFP protein expression showed no associations, supporting models in which these proteins might serve as markers of independent pathways in the development of HCC.     

Immunocytochemical staining of fine-needle aspiration biopsies of the liver as a diagnostic tool for hepatocellular carcinoma.

Fine-needle aspiration biopsy (FNAB) under ultrasonographic or computerized tomographic guidance is a useful diagnostic procedure for hepatic neoplasms. However, cytologic criteria alone may not allow for the distinction of hepatocellular carcinomas (HCC) from cholangiocarcinomas (CC) and metastatic adenocarcinomas (MA). In an effort to refine the FNAB diagnosis of hepatic malignancies, a panel of immunocytochemical stains was applied to aspiration specimens from primary and metastatic carcinomas in the liver. Anticytokeratin antibodies with different specificities (Cam 5.2 and AE1) were used in conjunction with antibodies to carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), and alpha-1-antitrypsin (AAT). All HCC, CC, and MA were immunoreactive with the antikeratin antibody Cam 5.2. However, only three (15%) HCC were positive with AE1, in contrast to 100% of CC and MA. Antibodies to CEA and AFP were also helpful diagnostic aids, especially for the three HCC that were immunoreactive with AE1. Canalicular staining for CEA was present in 47% of HCC, but in none of the CC or MA. AFP positivity occurred in 45% of HCC, but only one CC and none of the MA. AAT was not a useful marker for HCC due to low sensitivity and specificity. Immunocytochemistry is an effective adjunct to the cytodiagnosis of malignant liver tumors sampled by FNAB.     

The clinicopathological and prognostic relevance of cytokeratin 7 and 19 expression in hepatocellular carcinoma. A possible progenitor cell origin

AIMS: Cytokeratin (CK) 7 and CK19 expression, present in hepatic progenitor cells (HPCs) and in cholangiocytes but not in normal hepatocytes, has been reported in some hepatocellular carcinomas (HCCs); however, the incidence and relevance of this expression in HCC in Caucasians is not known. Therefore, our aim was to study the occurrence and clinicopathological characteristics of HCC expressing CK7 and/or CK19 in 109 Caucasian patients. METHODS AND RESULTS: The expression of hepatocellular differentiation markers (Hepar, canalicular polyclonal carcinoembryonic antigen), biliary/progenitor cell markers (CK7, CK19), alpha-fetoprotein (AFP), p53 and beta-catenin in HCC was semiquantitatively assessed by immunohistochemistry. Of 109 HCCs, 78 were CK7-/CK19- (72%), 13 CK7+/CK19- (12%), seven CK7-/CK19+ (6%), 11 CK7+/CK19+ (10%). CK19 expression was significantly associated with elevated serum AFP (400 ng/ml) (P = 0.023), tumour AFP expression (P < 0.0001), presence in serum of anti-hepatitis B core (P = 0.016), less fibrosis in non-neoplastic parenchyma (P = 0.009) and less nuclear beta-catenin expression (P = 0.021). CK7 expression was significantly associated with elevated serum bilirubin (> 2 mg/dl) (P = 0.0005) and less nuclear beta-catenin expression (P = 0.003). HCC expressing CK19 had a higher rate of recurrence (P = 0.009, hazard ratio 12.5, n = 31) after liver transplantation compared with CK19- tumours. CONCLUSIONS: In our series, 28% of HCCs contained cells expressing CK7 and/or CK19. They potentially derive from HPCs. The higher recurrence rate of CK19+ HCC after transplantation suggests a worse prognosis for these HCCs compared with CK19- HCC.     

Utility of pancreatic digestive enzyme immunohistochemistry in the differential diagnosis of hepatocellular carcinoma, cholangiocarcinoma and metastatic adenocarcinoma of the liver

To test the diagnostic utility of pancreatic digestive enzyme immunohistochemistry in liver cancers, the expression of three pancreatic digestive enzymes (trypsinogen, chymotrypsinogen and pancreatic lipase) was investigated in cholangiocarcinoma (CC) (n = 42), hepatocellular carcinoma (HCC) (n = 35), combined HCC-CC (n = 11) and metastatic adenocarcinoma (MA) of the liver (n = 34; 4 gastric cancer, 5 pancreatic cancer and 25 colon cancer). In CC, 15 (36%) expressed one or more of these enzymes, while the remaining 27 (64%) did not express any enzymes. In MA, 13 (38%) expressed one or more of these enzymes, while the remaining 21 (62%) did not express any enzymes. Expression of trypsinogen, chymotrypsinogen and lipase was noted in 15 CC (36%), 11 CC (25%) and 15 CC (36%), respectively, and in 9 MA (26%), 6 MA (18%) and 13 MA (38%), respectively. There was no significant difference in the positive ratio of each enzyme between CC and MA. In positive cases, the enzymes were expressed with a cytoplasmic granular pattern. In MA, there was no significant difference in the positive ratio of the enzymes among the primary sites. In contrast to CC and MA, these enzymes were not expressed in any cases of HCC and combined HCC-CC. These data suggest that pancreatic digestive enzyme immunohistochemistry may be useful for differential diagnosis between HCC and CC or MA as well as between combined HCC-CC and CC or MA, but it is not useful for differential diagnosis between CC and MA. A positive reaction for these enzymes is indicative of CC or MA and is against the diagnosis of HCC or combined HCC-CC, and a negative reaction is noncontributory to the differential diagnosis.     

Intrahepatic cholangiocarcinoma arising in chronic advanced liver disease and the cholangiocarcinomatous component of hepatocellular cholangiocarcinoma share common phenotypes and cholangiocarcinogenesis

Xu J, Sasaki M, Harada K, Sato Y, Ikeda H, Kim J‐H, Yu E & Nakanuma Y
(2011) Histopathology  59 , 1090–1099
Intrahepatic cholangiocarcinoma arising in chronic advanced liver disease and the cholangiocarcinomatous component of hepatocellular cholangiocarcinoma share common phenotypes and cholangiocarcinogenesis Aims: Intrahepatic cholangiocarcinomas (ICCs) are known to arise in cases of non‐biliary, chronic advanced liver disease (CALD), but their clinicopathological features remain unexplored. The aim of this study was to compare the histological and immunohistochemical ICCs arising inCALD with those arising in livers with non‐specific reactive (NSR) changes. Methods and results: Seventy‐one cases of ICC arising in CALD were compared with ICCs arising in livers with NSR changes, including normal livers (72 cases) and the cholangiocarcinomatous (CC) component of hepatocellular cholangioncarcinomas (HC‐CCs) (30 cases). The expression of mucin was higher in ICC with NSR changes, whereas it was relatively low in ICC with CALD and the CC component of HC‐CC. The expression of biliary markers [cytokeratin (CK)7, CK19, epithelial membrane antigen, and epithelial cell adhesion molecule (EpCAM)] was lower in CC with CALD and in the CC component of HC‐CC than in CC with NSR changes. The expression of hepatic progenitor cell markers [neural cell adhesion molecule (NCAM) and c‐kit] was higher in ICC with CALD and the CC component of HC‐CC than in ICC with NSR changes. EpCAM and CK19 were constantly expressed in cultured CC cells, whereas NCAM was infrequently expressed in cultured CC cells. Conclusions: The carcinogenesis of ICC arising in CALD and the ICC component of HC‐CC, each showing similar features, may involve hepatic progenitor cells.     

Expression of biliary antigen and its clinical significance in hepatocellular carcinoma

In order to classify the hepatocellular carcinomas (HCCs) which had diverse clinicopathologic characteristics, we divided HCCs into two groups according to the expression of biliary antigen on the basis of the hypothesis that the hepatocyte and biliary epithelial cell originate from the same precursor cell, and then we investigated the clinical and pathologic characteristics in the two groups. Forty HCC cases with no preoperative treatment and at least two-year follow-up data were selected among 202 cases of HCC files from 1991 to 1995. Expression of biliary antigen (AE1, cytokeratin 19), p53, AFP, and Ki-67 in the tumor tissue were assessed by immunohistochemistry. Positive cytokeratin 19 was noted in one case (2.5%); AE1 was detected in 40% of patients; p53 was overexpressed in 20% of patients; and AFP was detected in 45% of patients. No statistical difference between the biliary antigen positive group (16 cases) and the negative group (24 cases) were noted in terms of mean age, sex, presurgical serum AFP level, Child class, and tumor size. HBsAg positive rate was 66.7% for the biliary antigen (-) group and 93.8% for the biliary antigen (+) group with a statistically significant difference (p = 0.048). The number of cases for Edmonson-Steiner grade I/II and III/IV were 15 and 9 in the biliary antigen (-) group, and 4 and 12 in the biliary antigen (+) group, respectively, with a statistically significant difference (p = 0.024). The 1, 3 and 5-year disease-free survival rates were 69.7, 40.9 and 40.9% for the biliary antigen (-) group and 73.7, 39.1, 39.1% for the biliary antigen (+) group with no statistically significant difference. The 1, 3 and 5-year overall survival rates were 91.7, 73.8, 66.4% for the biliary antigen (-) group and 68.8, 34.4, 34.4% for the biliary antigen (+) group, with a significantly greater overall survival rate for the biliary antigen negative group (p = 0.045). Poor histopathological differentiation, a high HBsAg positive rate and poor overall survival rate were noted in the biliary antigen positive group and the differences were statistically significant. In conclusion, HCCs with positive biliary antigen, which originates from more primitive cells, is suggested to be more aggressive than HCCs with negative biliary antigen.     

Classification of hepatocellular carcinoma according to hepatocellular and biliary differentiation markers. Clinical and biological implications.

Hepatocellular carcinoma (HCC) is a heterogeneous disease. HCC derived from 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 (AE1-AE3, cytokeratin-19), proliferation markers (Ki-67, proliferating cell nuclear antigen), alpha-fetoprotein, p53, and transforming growth factor-alpha 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 showed features of more aggressive disease in terms of poorer cellular differentiation (P < 0.001) 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 alpha-fetoprotein (P < 0.001) and p53 (P = 0.0077). Classification of HCCs based on its phenotypic (differentiation) markers has both clinical and pathobiological implications.     

Liver cell dysplasia and hepatocellular carcinoma: a histoiogical and immunohistochemical study

Liver cell dysplasia (LCD) was found in 28 (60%) of 47 patients with hepatocellular carcinoma (HCC); 22 (79%) of them had associated liver cirrhosis. LCD was more frequently observed in posthepatitic cirrhosis (82%) than in the other forms. Carcinoembryonic antigen (CEA), alpha-1-antitrypsin (AAT) and alpha-fetoprotein (AFP), as demonstrated by the peroxidase-antiperoxidase method, were similarly expressed both in normal and in dysplastic cells. Hepatitis B surface antigen was found in eight cases (17%), six of which were associated with LCD. HBsAg was rarely found in dysplastic cells and frequently displayed a peculiar perinuclear pattern. The possible preneoplastic role of LCD is stressed.     

Immunohistochemical analysis of hepatocellular and adenocarcinoma in the liver: MOC31 compares favorably with other putative markers.

Distinguishing hepatocellular carcinoma (HCC) from metastatic adenocarcinoma (MA) and cholangiocarcinoma (CC) can, at times, be difficult and sometimes requires immunohistochemical analysis. Recently, MOC31, an antibody directed against a cell surface glycoprotein, has been shown to be useful in separating HCC from both MA and CC; however, no study has compared MOC31 and other frequently used immunostains. We compare MOC31 with other commonly used immunostains for HCC, MA, and CC. Formalin-fixed, paraffin-embedded tissue sections from 57 previously characterized hepatic neoplasms (13 HCC, 14 CC, 3 combined HCC-CC, and 27 MA) were immunostained with antibodies directed against MOC31, cytokeratin (CK) 7, CK20, alpha-fetoprotein (AFP), polyclonal carcinoembryonic antigen, Ber-EP4, and Factor XIII-A. Two pathologists reviewed slides, and positivity was defined as more than 1% of cells staining with the appropriate pattern. Positive MOC31 immunostaining was seen in 0 of 13 HCC, 13 of 14 CC, 3 of 3 HCC-CC, and 27 of 27 MA; the staining was strong and diffuse. CK20 reactivity was observed in 0 of 13 HCC, 2 of 14 CC, 0 of 3 HCC-CC, and 12 of 27 MA; CK7 immunostained 4 of 13 HCC, 13 of 14 CC, 3 of 3 HCC-CC, and 15 of 27 MA; AFP was detected in 4 of 13 HCC and 2 of 3 HCC-CC, whereas all CC and MA were negative; polyclonal carcinoembryonic antigen showed immunoreactivity in 12 of 13 HCC and 3 of 3 HCC-CC in a canalicular pattern, whereas diffuse positivity was identified in 13 of 14 CC and 26 of 27 MA; Ber-EP4 immunostained 1 of 13 HCC, 14 of 14 CC, 2 of 3 HCC-CC, and 26 of 27 MA; and Factor XIII-A was negative in all HCC, CC, and MA. MOC31 expression distinguished HCC from adenocarcinoma in 56 of 57 cases. AFP was specific for HCC but was not sensitive. CK7 and CK20 have limited utility in distinguishing HCC from CC or MA, and Factor XIII-A is not useful. Ber-EP4 staining was similar to MOC31, but one HCC did stain with Ber-EP4. Polyclonal CEA yields similar numerical results as MOC31, but the focal nature of the staining and occasional difficulty in evaluating the pattern can make interpretation problematic. We conclude that MOC31 should be a component of the immunohistochemical panel to distinguish HCC from CC and MA.     

Diagnostic utility of immunohistochemistry in hepatocellular carcinoma, its variants and their mimics.

Hepatocellular carcinoma (HCC) is known for its histomorphologic heterogeneity. Immunohistochemistry (IHC) can help in the comparative morphologic evaluation of HCC, its variants and their mimics. Some of these diagnostic challenges can be attributed to (i) the variety of neoplasms that can arise from the hepatic stem cell lineage; (ii) the spectrum of well-differentiated hepatocellular nodular lesions; (iii) the liver being a target for metastases with some of these histologic entities mimicking variants of HCC or actually arising in the liver; and (iv) the limitations of serum alpha-fetoprotein (AFP). The role of IHC is in the distinction of benign hepatocellular nodules from reactive hepatocytes; WD-HCC from benign hepatocellular nodules; poorly differentiated HCC from cholangiocarcinoma and metastases; and determination of histogenesis of malignant tumor; and of primary site of origin of malignant tumor. A panel of antibodies has more discriminant value. AFP expression usually indicates malignancy in a hepatocellular nodule and hepatocytic histogenesis of a malignancy. Polyclonal carcinoembryonic antigen (pCEA) and CD10 stain bile canaliculi in better-differentiated HCC. HepPar1 is generally accepted as a hepatocytic marker. However, not all HCC stain uniformly and not all HepPar1-positive tumors are of hepatocytic origin or arise in the liver. Mature hepatocytes and hepatocellular nodules stain with CAM 5.2, CK 8, and 18 but not with CK 7, 19, 20, or AE1/AE3. Biliary epithelium expresses CK 7 and 19. CD 34 highlights sinusoidal capillarization. AFP, pCEA/CD10, and CD34 are useful for ascertainment of malignancy in hepatocellular nodules; HepPar1 and cytokeratins to be included if histogenesis is the issue. IHC results should be interpreted in the larger context of the case.     

Distribution of albumin- and/or alpha-fetoprotein-positive cells in hepatocellular carcinoma

Albumin (ALB)-positive cells were identified by an immunoperoxidase technique in 52 of 53 autopsy cases and in all of 13 surgical cases of hepatocellular carcinoma (HCC). The distribution pattern of ALB-positive cells could be classified into three groups: diffuse, localized, and sparse. The diffuse type was the most common pattern and was usually seen in well or moderately differentiated HCC, showing a trabecular growth pattern. The localized or sparse patterns were more frequent in poorly differentiated HCC showing a compact growth pattern. alpha-Fetoprotein (AFP)-positive cells were detected in 37 of the 53 autopsy cases of HCC and 11 of the 13 surgical cases. The number of AFP-positive cells and the intensity of the immunoperoxidase reaction were roughly proportional to serum AFP levels in most cases. In most regions of HCC, there seemed to be an inverse relationship between the number of ALB- and AFP-positive cells, suggesting tht most HCC cells synthesized only one of the two antigens studied. ALB-positive hepatocytes were found in all of the normal or cirrhotic livers examined and in the tumor-free regions of the HCC-containing livers. In contrast, AFP was not detected in nonneoplastic hepatocytes.