Transforming growth factor-alpha in human hepatocellular carcinoma and coexpression with hepatitis B surface antigen in adjacent liver.

BACKGROUND. Hepatitis B virus (HBV) infection is closely associated with the development of hepatocellular carcinoma (HCC) in many patients, but the mechanisms by which HBV contributes to HCC are not known. Transforming growth factor-alpha (TGF-alpha), a regulator of growth and regeneration in rat liver that can be found in high levels in some human cancers, theoretically could play such an intermediate role in the development of HCC. METHODS. The expression of TGF-alpha and its relation to the HBV antigens were evaluated in human HCC and adjacent nontumorous livers from 33 patients from the United States and China using immunoperoxidase staining of paraffin-embedded sections. RESULTS. TGF-alpha was detected in HCC from 27 of 33 (82%) patients; the frequencies were similar in patients from the United States and China. TGF-alpha was detected in HCC more frequently in patients whose adjacent nontumorous livers had detectable hepatitis B surface antigen (HBsAg) and/or hepatitis B core antigen (HBcAg) than in those whose adjacent livers lacked HBsAg and HBcAg. Detection of TGF-alpha was not affected by tumor size, histologic type, or grade. TGF-alpha was detected in adjacent nontumorous livers from 31 of 33 patients (94%). Coexpression at a high intensity of TGF-alpha and HBsAg in the same hepatocytes could be demonstrated by specific staining of consecutively cut sections for 17 of 33 patients (52%). CONCLUSIONS. TGF-alpha is expressed at a high level in 82% of human HCC. Localization of HBsAg within the same hepatocytes as TGF-alpha suggests a possible interaction between HBV and TGF-alpha during hepatocarcinogenesis in humans. Stimulation of TGF-alpha expression could be part of a chain of events by which HBV contributes to the development of HCC in some patients.     

Detection of hepatitis B surface antigen mutants and their integration in human hepatocellular carcinoma

Vaccine escape hepatitis B virus surface antigen (HBsAg) mutants are capable of independent replication and have been implicated in acute hepatitis. We now report the detection of these mutants with changes at various positions of the antigenic 'a' determinant in human hepatocellular carcinoma (HCC). Southern blot analysis indicated that the HBsAg mutant with the Glycine to Arginine change at position 145 was integrated in HCC, whereas those with a Threonine at position 133 instead of a Methionine were identified in the serum of aggressive HCC. Further studies on HBsAg mutants in HCC should provide new insights on their involvement in the hepatocarcinogenesis.     

Hepatitis B surface antigen in hepatocellular carcinoma

Hepatitis B surface antigen (HBs-Ag) was detected histologically (by victoria blue-nuclear fast red staining) in the liver in 20-40% of liver cirrhosis and/or hepatocellular carcinoma (hepatoma) cases. In hepatoma cases, HBs-Ag was usually found in non-cancerous areas of the liver. However, some HBs-Ag positive cells were also found dispersed in cancerous areas; these were regarded as HBs-Ag-infected non-cancerous hepatic cells remaining undestroyed. There were a few cases where inconsistency was found between the results of the immunofluorescence technique using anti HBs-Ag serum and victoria blue staining in detecting HBs-Ag. This phenomenon was rare in non-cancerous areas, but was relatively frequent in cancerous areas.     

Alteration of gene expression in human hepatocellular carcinoma with integrated hepatitis B virus DNA.

PURPOSE: Integration of hepatitis B virus (HBV) DNA into the human genome is one of the most important steps in HBV-related carcinogenesis. This study attempted to find the link between HBV DNA, the adjoining cellular sequence, and altered gene expression in hepatocellular carcinoma (HCC) with integrated HBV DNA. EXPERIMENTAL DESIGN: We examined 15 cases of HCC infected with HBV by cassette ligation-mediated PCR. The human DNA adjacent to the integrated HBV DNA was sequenced. Protein coding sequences were searched for in the human sequence. In five cases with HBV DNA integration, from which good quality RNA was extracted, gene expression was examined by cDNA microarray analysis. RESULTS: The human DNA sequence successive to integrated HBV DNA was determined in the 15 HCCs. Eight protein-coding regions were involved: ras-responsive element binding protein 1, calmodulin 1, mixed lineage leukemia 2 (MLL2), FLJ333655, LOC220272, LOC255345, LOC220220, and LOC168991. The MLL2 gene was expressed in three cases with HBV DNA integrated into exon 3 of MLL2 and in one case with HBV DNA integrated into intron 3 of MLL2. Gene expression analysis suggested that two HCCs with HBV integrated into MLL2 had similar patterns of gene expression compared with three HCCs with HBV integrated into other loci of human chromosomes. CONCLUSIONS: HBV DNA was integrated at random sites of human DNA, and the MLL2 gene was one of the targets for integration. Our results suggest that HBV DNA might modulate human genes near integration sites, followed by integration site-specific expression of such genes during hepatocarcinogenesis.     

Additive effect modification of hepatitis B surface antigen and e antigen on the development of hepatocellular carcinoma.

To assess the role of hepatitis B e antigen (HBeAg) and its interaction with hepatitis B surface antigen (HBsAg) on the development of hepatocellular carcinoma (HCC), this case-control study included 361 age- and sex-matched pairs of patients with histologically proven HCC and healthy control subjects. HBsAg, HBeAg and antibody to HBeAg (anti-HBe) were detected by radioimmunoassay. Antibodies to hepatitis C virus (anti-HCV) were detected by second-generation enzyme immunoassay. The prevalences of HBeAg (20.2%), HBsAg (80.3%) and anti-HCV (29.5%) in cases were higher than in controls (1.9%, 20.7%, and 2.7% respectively; each P < 0.0001). Using patients negative for HBsAg, HBeAg and anti-HBe as a referent group, univariate analysis indicated that HBsAg alone or HBsAg and HBeAg were risk factors for HCC (P for trend < 0.0001). Calculation of incremental odds ratio indicated that there was additive interaction between HBsAg and HBeAg. Multivariate analysis indicated that HCC development was strongly associated with the presence of HBeAg (odds ratio, 8.1; 95% confidence interval, 2.4-27.1), HBsAg (odds ratio, 68.4; 95% confidence interval, 20.5-227.8) and anti-HCV (odds ratio, 59.3; 95% confidence interval, 13.6-258.4). In conclusion, HBsAg, HBeAg and anti-HCV are independent risk factors for HCC. There is additive and independent effect modification between HBsAg and HBeAg on the development of HCC.     

Liver cell dysplasia and hepatitis B surface antigen in liver cirrhosis and hepatocellular carcinoma

Liver tissues of 223 autopsy cases of cirrhosis and hepatocellular carcinoma were examined for liver cell dysplasia in relation to hepatitis B surface antigen (HBsAg) detected with orcein stain. Liver cell dysplasia was found in 94 cases (42.2%): 37 were from cases of cirrhosis only, and 53 were from cases of cirrhosis with hepatocellular carcinoma. There was a significant difference in the overall incidence of HBsAg in cases with and without dysplasia (70.2%:32.6%). A similar difference was found in all groups, i.e., those with cirrhosis, cirrhosis with hepatocellular carcinoma, and hepatocellular carcinoma only, in which none of 11 cases of HBsAg negative had dysplasia. A good correlation was seen between the semiquantitative grade of dysplasia and the incidence of HBsAg. These findings suggest a close relationship of HBsAg with liver cell dysplasia.     

Liver cell dysplasia in association with hepatocellular carcinoma, cirrhosis and hepatitis B surface antigen in Hong Kong

Liver-cell dysplasia was identified in 60% of 558 cases of cirrhosis with and without hepatocellular carcinoma (HCC) in Chinese coming to necropsy in Hong Kong from 1963 to 1978. A significant correlation was found between liver-cell dysplasia and the identification of hepatitis B surface antigen (HBsAg) in the livers, suggesting that dysplasia may be casually related to hepatitis B virus (HBV). Applying Bayes' theorem to our series of male deaths, we calculated that for Chinese male non-cirrhotics showing liver-cell dysplasia at necropsy, compared with male non-cirrhotics showing no liver-cell dysplasia, the approximate risk factor for HCC is 13:1, whereas for male cirrhotics with liver-cell dysplasia, the risk factor for HCC is approximately 2:1. In the presence of dysplasia, the risk for HCC is increased two-fold in males who are also HBsAg-positive. Thus, the presence of liver-cell dysplasia in cirrhotic or non-cirrhotic livers accompanies a significantly higher risk of developing HCC, especially in the presence of HBsAg.     

Geographic correlation between mortality from primary hepatic carcinoma and prevalence of hepatitis B surface antigen in Greece.

Average annual age-adjusted mortality rates per 100,000 from primary hepatic carcinoma (PHC) among males for 1971-1973 in the urban and rural areas of the 9 geographical regions of Greece were estimated. Hepatitis-B surface antigen (HBsAg) prevalence by region and area was evaluated in a sample of 22,844 Greek Air Force recruits from all parts of the country. Mortality from PHC was found significantly higher in urban areas (28-30 vs. 18-81) whereas prevalence of HBsAg was higher in rural areas (5-3% vs. 3-90%). Nevertheless further statistical analysis showed that there is a strong correlation between HBsAg prevalence and mortality from PHC, which is higher in rural (r = + 0-88) than in urban (+ 0-57) areas. The latter findings indicate that hepatitis B infection and PHC may be causally related.     

Association of human hepatocellular carcinoma and cirrhosis with hepatitis B virus surface and core antigens in the liver.

Paraffin sections of livers obtained at autopsy from 50 cases of hepatocellular carcinoma (HCC), 58 cases of cirrhosis and 54 cases of other miscellaneous liver disorders (controls) were stained for both surface (HBsAg) and core (HBcAg) components of hepatitis B virus (HBV) by immunoperoxidase and immunofluorescence techniques and rigidly controlled for antigen specificity, and in addition stained by orcein for HBsAg. The material was collected from different regions of India and adequate amounts of tissue were examined in most specimens to overcome possible sampling error caused by random distributions of the antigens in liver. HBsAg was detected in 94% of HCC, 71% of cirrhosis and only 2% of control livers, while HBcAg was found in 22%, 31% and none respectively. Antigen positivity seems to be directly related to the amount of tissue examined. Peroxidase staining detected smaller amounts of HBcAg than fluorescence and was also much more convenient for identifying the antigen. Both antigens were present in 9 of 41 HCC cases, 12 of 39 cirrhosis and none of 25 controls. Most of these livers contained 1+ HBsAg and 1+ to 2+ HBcAg, an antigen expression pattern suggestive of a carrier state or, rarely, of mild chronic liver disease. Among all livers tested, HBsAg alone was present in 48, both antigens were found in 21, and HBcAg alone in none. HBsAg was seen inside tumour cells in four cases, but no tumour showed HBcAg. Most HCC was associated with cirrhosis (92%) and antigen-positive cirrhosis had a higher chance of harbouring HCC than antigen-negative disease. HBsAg was detected in all four non-cirrhotic livers associated with HCC, while two of these also had HBcAg. Active cirrhosis was very frequently associated with HBsAg. These results and the overwhelming evidence of sero-logical and epidemiological studies from various parts of the world suggest a strong association of the hepatitis B virus with HCC. The possible ways in which the two could be related are discussed.     

Liver cell dysplasia: association with hepatocellular carcinoma, cirrhosis and hepatitis B antigen carrier status.

Liver cell dysplasia was noted on histological examination of nontumorous liver from 24 of 50 (48%) black southern African males with hepatocellular carcinoma (HCC). Macronodular cirrhosis was present in 40 (80%). There was no statistically significant difference between the frequency of dysplasia in 50% of 40 cirrhotic and 40% of 10 noncirrhotic livers, or in 52.6% of 38 hepatitis B antigen (HBAg) positive and 33.3% of 12 HBAg negative HCC patients. HBAg positivity was present in 80% of 40 cirrhotic and in 60% of 10 noncirrhotic HCC patients. This lack of significant correlation between liver cell dysplasia, and both cirrhosis and HBAg positivity in HCC patients in contrast to findings in Uganda and the United States, suggests a different pathogenetic mechanism for dysplasia in southern Africa. Liver cell dysplasia in man appears to be analogous to preneoplastic experimentally-induced hyperplastic foci or areas.     

Role of hepatitis B surface antigen in the development of hepatocellular carcinoma: regulation of lymphoid enhancer-binding factor 1

Background

There are around 350 million of hepatitis B surface antigen (HBsAg) carriers worldwide, and among them, high risk of developing hepatocellular carcinoma (HCC) has been identified by epidemiological studies. To date, the molecular role of HBsAg in HCC development has not been fully studied. We have previously reported that in cell cultures, HBsAg up-regulated the expression of lymphoid enhancer-binding factor 1 (LEF-1), a key component of the Wnt pathway. In this study we aimed to study this effect of HBsAg on LEF-1 in the development of HCC.

Methods

Expression of HBsAg, LEF-1 and its downstream effector genes were compared among 30 HCCs, their peritumor tissue counterparts and 9 normal control liver tissues by quantitative real-time PCR. In addition, immunohistochemical staining studies on HBsAg and LEF-1 expression were conducted among these samples.

Results

The expression of LEF-1 was compared between 13 HBsAg positive HCC tissues and 17 HBsAg negative HCC tissues. Simultaneous detection of LEF-1 and HBsAg was observed in HBsAg positive HCC tissues and, additionally, the simultaneous detection of HBsAg and LEF-1 was more pronounced in peritumor tissues, compared to that in the tumor tissues. The distribution of cellular LEF-1 in peritumor tissues was predominantly in the cytoplasm; while LEF-1 in the tumor tissues was located either exclusively in the nucleus or both in the nucleus and cytoplasm. By real-time PCR, the expression levels of LEF-1 downstream effector genes cyclin D1 and c-myc were higher in peritumor cells compared to that of the tumor cells. However, a 38 kDa truncated isoform of LEF-1, rather than the 55 kDa wild-type LEF-1, was significantly elevated in the HBsAg positive tumor cells.

Conclusion

Data indicate that deregulation of the Wnt pathway by HBsAg occurred in HBV-associated HCCs, but was more pronounced in the peritumor cells. It is speculated that HBsAg could stimulate proliferation and functional modification of hepatocytes via LEF-1 through the Wnt pathway at the pre-malignant stage.     

Hepatitis B antigen in black patients with hepatocellular carcinoma: correlation between orcein stained liver sections and serology.

Formalin-fixed paraffin-embedded autopsy tissue of liver and tumor from 50 male black mineworkers with hepatocellular carcinoma were examined by orcein stain for the presence of cytoplasmic hepatitis B surface antigen. The results were correlated with the serum hepatitis B antigen (HBAg). In 72% serum HBAg was positive. Orcein staining of nontumor liver cell cytoplasm was present in 18 (36%). Sixteen (89%) of these orcein-positive cases were serum HBAg positive. The two false negative serum HBAg results were obtained by immunodiffusion, immunoelectrophoresis and complement fixation. Serum HBAg, measured by radio-immunoassay and hemagglutination, was positive in 14 orcein-negative cases. Six other negative orcein results appeared to be due to sampling error. Orcein staining was noted in tumor cells of three serum HBAg positive patients. Provided the limitations of the technique are realized, orcein staining of liver tissue from hepatocellular carcinoma patients may prove useful for retrospective screening surveys to assess the prevalence of HBAg positivity in these patients.     

Gene expression-based recurrence prediction of hepatitis B virus-related human hepatocellular carcinoma.

PURPOSE: The poor prognosis of hepatocellular carcinoma (HCC) is, in part, due to the high rate of recurrence even after "curative resection" of tumors. Therefore, it is axiomatic that the development of an effective prognostic prediction model for HCC recurrence after surgery would, at minimum, help to identify in advance those who would most benefit from the treatment, and at best, provide new therapeutic strategies for patients with a high risk of early recurrence. EXPERIMENTAL DESIGN: For the prediction of the recurrence time in patients with HCC, gene expression profiles were generated in 65 HCC patients with hepatitis B infections. RESULT: Recurrence-associated gene expression signatures successfully discriminated between patients at high-risk and low-risk of early recurrence (P=1.9 x 10(-6), log-rank test). To test the consistency and robustness of the recurrence signature, we validated its prognostic power in an independent HCC microarray data set. CD24 was identified as a putative biomarker for the prediction of early recurrence. Genetic network analysis suggested that SP1 and peroxisome proliferator-activated receptor-alpha might have regulatory roles for the early recurrence of HCC. CONCLUSION: We have identified a gene expression signature that effectively predicted early recurrence of HCC independent of microarray platforms and cohorts, and provided novel biological insights into the mechanisms of tumor recurrence.     

Factors determining the development of hepatocellular carcinoma in hepatitis B surface antigen carriers. A comparison between families with clusters and solitary cases

This article documents five families with clusters of hepatocellular carcinoma (HCC), including one in which three successive generations were involved. All the 12 patients in these five families and 96.3% of the patients in 54 families with solitary cases of HCC seen during the same period were hepatitis B surface antigen (HBsAg)-positive. The prevalence of HBsAg in families with clusters and solitary cases of HCC was compared. The clustering of HCC in the five families reported could not be accounted for by a higher HBsAg carrier rate or an earlier age of onset of the hepatitis B virus infection. An attempt was made to identify the factors that determine the development of HCC in HBsAg carriers.     

Decreased expression of Bid in human hepatocellular carcinoma is related to hepatitis B virus X protein

As a mitochondrial membrane death ligand, Bid oligomerises Bak to release cytochrome C and its deficiency renders hepatocytes resistant to apoptosis induced by Fas. The Bid level in hepatocellular carcinoma (HCC) is unknown. In this report, we examined the expression of Bid protein and mRNA in HCC cancerous tissues and their corresponding non-cancerous ones. The effect of the hepatitis B x protein (HBx) on the expression of Bid was also evaluated by transfecting hepatoma cells with the HBx gene. The results showed that the expression of Bid was significantly lower in cancerous tissues than that in their corresponding non-cancerous tissues. Immunohistochemical study revealed that Bid molecule was mainly localised in hepato-cytoplasm. Some nuclei were also positive for Bid antigen though to a lesser degree. In vitro experiments demonstrated that the expression of Bid in cells transfected with HBx was significantly lower than that in the cells without HBx transfection. This finding suggests that HBx may play a causative role in the reduction of Bid expression in HCC. This in vitro result is, to some degree, supported by clinical data that all the HCC examined are positive for hepatitis B virus (HBV). We conclude from this data that the expression of Bid in HCC is significantly decreased and the reduction of Bid may result from a mechanism associated with HBx, a major hepatocarcinogenic product from HBV. The imbalance of increased anti-apoptosis and decreased pro-apoptosis seen in HCC is a critical mechanism leading to the uncontrolled growth of tumour cells. Therefore, this study suggests that a deficiency in the expression of Bid may contribute to the development of such an imbalance in HCC.     

Hepatitis B surface antigen and hepatocellular carcinoma in the People's Republic of China

Eighteen cases of heptocellular carcinoma from the People's Republic of China were investigated for the presence of hepatitis B surface antigen (HBsAg) in the cytoplasm of hepatocytes and tumor cells. The Sternberger-PAP immunoperoxidase technique utilizing monospecific antibody to HBsAg and a modified orcein method demonstrated cytoplasmic HBsAg in hepatocytes of 15 cases (83.3%) and tumor cells of 3 cases (16.7%). Thirteen of these cases were also investigated for HBs antigenemia and of these 11 were positive (84.6%). These hepatomas were often associated with macronodular cirrhosis and/or a persistent inflammatory process in the hepatic parenchyma. The high association of HBsAg and hepatoma indicates that the hepatitis B virus plays an important role in the pathogenesis of this malignancy in China. It is concluded that a major public health effort to eradicate endemic hepatitis B infection is the most reasonable way to decrease the incidence of this cancer, which is common in China.     

Increased expression of angiogenin in hepatocellular carcinoma in correlation with tumor vascularity.

PURPOSE: Neovascularization is known to be one of the major characteristics of human hepatocellular carcinoma (HCC). Angiogenin (ANG), originally discovered in a human colon cancer cell line, is a liver-derived polypeptide that shows strong angiogenic activity in vivo. However, the role of ANG on the development of HCC remains unknown. The present study was designed to examine the implication of ANG in the neovascularization of human HCC. EXPERIMENTAL DESIGN: Forty-one HCC patients who had undergone conventional celiac angiography were used in this study. ANG protein expression and microvessel density (MVD) in HCC specimens obtained by liver biopsy or surgical resection were examined by immunohistochemistry, and the levels were quantified by the KS-400 image analyzing system. ANG mRNA expression in liver tissues was evaluated by in situ hybridization. Serum ANG concentrations were measured by an ELISA. Survival rates were calculated using the Kaplan-Meier method. RESULTS: Immunohistochemistry and in situ hybridization showed greater increments of ANG protein expression and mRNA expression, respectively, in HCC tissues than in the surrounding nontumorous tissues. MVD within tumorous tissues increased according to dedifferentiation of the histological grade of HCC, showing a significant correlation (r = 0.877, P = 0.0009) with ANG expression levels. Mean +/- SD serum ANG levels of healthy subjects and chronic hepatitis (CH) patients were 362.3 +/- 84.1 ng/ml and 331.9 +/- 133.8 ng/ml, respectively, with no significant difference. Serum ANG levels of liver cirrhosis patients (242.4 +/- 126.9 ng/ml) were lower than those of healthy subjects or CH patients and decreased as the fibrosis grade advanced. In HCC patients, despite the cirrhotic background, serum ANG levels increased as the tumor vascularity increased (197.8 +/- 64.9 ng/ml for hypovascular, 326.7 +/- 148.6 ng/ml for hypervascular, and 405.0 +/- 121.3 ng/ml for very hypervascular), in good accordance with histological grading, and significantly decreased (P = 0.015) after successful treatment with transcatheter arterial embolization or percutaneous ethanol injection. HCC patients were conventionally divided into two groups according to the serum level of ANG, those with values higher than the mean level (332.9 +/- 143.8 ng/ml) and those with values lower than the mean,; the 5-year survival rate of the latter group was determined to be significantly higher than that in the former group. CONCLUSIONS: These results suggest that ANG is one of the neovascularization defining factors of HCC. Thus, measuring serum ANG may assist in monitoring the disease, and targeting ANG may provide a new strategy for treating advanced HCC.     

Localization of hepatitis B surface and core antigens in human hepatocellular carcinoma by immunoperoxidase methods. Replication of complete virions of carcinoma cells

The localization of hepatitis B surface antigen (HBsAg) and core antigen (HBcAg) was investigated by an indirect immunoperoxidase method in formalin-fixed, paraffin-embedded liver specimens obtained from 95 Japanese patients with hepatocellular carcinomas. Routine and immune electron microscopic examinations were done in one case. The correlation between expression of hepatitis B virus antigens in the tissue and serum hepatitis B e antigen (HBeAg)/antibody to HBeAg (anti-HBe) status was examined. Hepatitis B surface antigen was detected in the cytoplasm of noncarcinomatous hepatocytes in 28 (29.5%) cases and of carcinoma cells in 11 (11.6%) cases. Hepatitis B core antigen was stained in noncarcinomatous hepatocytes in 13 (13.7%) cases and in carcinoma cells in 4 (4.2%) cases. Hepatitis B core antigen was present mainly in the nuclei, and all HBcAg-positive cases were positive for HBsAg. The routine electron microscopic examination revealed many round particles, 25 to 30 nm in diameter both in the nuclei and in the cytoplasm, and larger particles, 40 to 45 nm in diameter in the cytoplasm of carcinoma cells. Both types of particles had reaction products of HBcAg by immunoelectron microscopic study. Therefore, it was confirmed that the former were cores and the latter were Dane particles. There was a tendency that HBeAg-seropositive cases showed localization of HBcAg in the noncarcinomatous tissue. Among four cases with positive HBcAg in carcinoma cells, two were positive for HBeAg, one was positive for anti-HBe, and the other was negative both for HBeAg and anti-HBe in the sera. The data suggested occasional production of complete hepatitis B viruses of carcinoma cells in anti-HBe-positive as well as in HBeAg-positive hepatocellular carcinomas.