Mutations within the hepatitis B virus genome among chronic hepatitis B patients with hepatocellular carcinoma

Chronic hepatitis B infection is a major cause of hepatocellular cancer (HCC). The pathogenesis of the carcinogenesis is not fully understood. Viral proteins such as the X protein and the truncated middle S protein have been implicated to be transactivators. In order to investigate whether any mutations within relevant parts of the hepatitis B virus (HBV) genome could be associated with the development of HCC, the genomes of 16 HBV strains from chronic HBV carriers with HCC were studied. Serum samples were subjected to PCR and the HBV DNA sequenced subsequently. Genotypes A-D were represented. The sequence analysis showed that an especially high proportion, 50% (CI 95%, 25-75%), of the patients with HCC carried HBV mutants with deletions or insertions in the N-terminal half of the pre-S2 region or had a point mutation in the start codon of pre-S2 compared with controls with chronic HBV infection, 21% (CI 95%, 3-39%). A high proportion (69%) also had mutations at position 1762 (A --> T) and/or 1764 (G --> A) in the core promoter region, but the proportion of core promoter mutations was no different from what was found in a control group of HBV carriers without HCC (68%). The pre-S2 variants, which involve deletions of immunogenic regions, may have a survival advantage as they are mostly found in long-standing HBV infection. There were no other mutations found frequently within the region coding for the X protein.     

Frequent integration of hepatitis B virus DNA in noncancerous liver tissue from hepatocellular carcinoma patients

To investigate the relationship between hepatocarcinogenesis and integration of hepatitis B virus (HBV) DNA in the cellular DNA of the liver, we studied the integration of HBV DNA in various noncancerous regions of the liver from 31 patients using Southern blot analysis. Of 13 patients without hepatocellular carcinoma (HCC), 4 had heterogeneously integrated HBV DNA. Of the latter four patients, two had chronic liver disease, and two had nonspecific histological changes. In contrast, integration of HBV DNA was found in noncancerous tissue from 11 of 18 patients with HCC. In eight patients, homogeneous integration was found in noncancerous tissue, and restriction fragments of integrated HBV DNA were different from those found in cancerous tissue. Moreover, integration of HBV DNA was found in all portions examined from the same liver, and homogeneously integrated HBV DNA showed different restriction patterns in different areas. These results suggest that integration of HBV DNA may occur in heterogeneous sites of cellular DNA before hepatocarcinogenesis. Subsequently, multi-focal clonal populations develop from these hepatocytes, especially frequently in the case of HCC. Integrated HBV DNA may play an important role in the clonal growth of hepatocytes, although the development of HCC requires additional factors.     

Detection of hepatitis B virus DNA in hepatocellular carcinoma.

DNA-DNA hybridization was used to examine tissue from 31 patients with hepatocellular carcinoma (HCC) in Taiwan for the presence of the hepatitis B virus genome. Twenty-four of the DNA samples had discrete high molecular weight bands when cut with HindIII and hybridized with a radiolabelled HBV DNA probe, and similar results were obtained with 25 of the samples when cut with EcoRI. Thus integrated HBV DNA was present in almost 80% of the specimens. None of the samples had a similar pattern and most contained HBV DNA integrated at multiple sites. Seventeen out of 19 HBsAg carriers and one out of two patients with anti-HBs were found to have integrated HBV DNA in the tumour tissue, providing further evidence of the strong association between HBV infection and development of hepatocellular carcinoma.     

Association of hepatitis B virus with chronic liver diseases and hepatocellular carcinoma

The idea of hepatitis-cirrhosis-hepatocellular carcinoma sequence in liver was proposed by the workers in tropical Africa, the homeland of hepatocellular carcinoma. The discovery of Australia antigen by Blumberg et al provided the missing link and it was observed by several workers as well as the present group that Hepatitis B surface antigen (HBSAg) and that to Hepatitis B Virus (HBV) is someway related with the incidence of chronic liver disease and hepatocellular carcinoma (HCC).     

Hepatitis B virus DNA in primary hepatocellular carcinoma tissue.

Tumour, cirrhotic, and metastatic tissues from four patients with primary hepatocellular carcinoma have been investigated for the presence of hepatitis B viral DNA by nucleic acid hybridization. Tumours from two of three patients with a current HBV infection contained 1--2 genomes per cell of unintegrated viral DNA, while tumours from the third HBs antigen-positive patient contained less than one genome equivalent per ten cells. A tumour from one patient with anti-HBs contained no detectable HBV DNA. A variety of models involving HBV as an etiologic agent may be advanced to explain the statistical correlation of HBV infection with primary hepatocellular carcinoma (PHC). The data presented here argue against the model that HBV DNA integrated into every cell is required to maintain the oncogenic transformation of hepatocytes, but they do not rule out other models.     

Detection of hepatitis B virus DNA in hepatocellular carcinoma

DNA-DNA hybridization was used to examine tissue from 31 patients with hepatocellular carcinoma (HCC) in Taiwan for the presence of the hepatitis B virus genome. Twenty-four of the DNA samples had discrete high molecular weight bands when cut with HindIII and hybridized with a radiolabelled HBV DNA probe, and similar results were obtained with 25 of the samples when cut with EcoRI. Thus integrated HBV DNA was present in almost 80% of the specimens. None of the samples had a similar pattern and most contained HBV DNA integrated at multiple sites. Seventeen out of 19 HBsAg carriers and one out of two patients with anti-HBs were found to have integrated HBV DNA in the tumour tissue, providing further evidence of the strong association between HBV infection and development of hepatocellular carcinoma.     

Epidemiology of chronic hepatitis B virus infection,hepatocellular carcinoma,and hepatitis B virus-induced hepatocellular carcinoma

Approximately 360 million people worldwide are chronically infected with hepatitis B virus (HBV) and are at high risk of developing hepatocellular carcinoma (HCC). Chronic HBV infection is the most prevalent cause of this tumour, accounting for 55% of global cases, and 89% of those in endemic regions for HBV infection. Relative risks for developing HCC in the presence of chronic HBV infection may be as high as 49 in case-control studies, and 98 in cohort studies. HCC is the sixth most common cancer in the world today, with approximately 630,000 new cases occurring each year. It ranks third in annual cancer mortality rates. Approximately 80% of HCCs occur in developing countries where HBV infection is endemic, with the highest incidences being in the Asia-Pacific region, and sub-Saharan Africa. In the chronic carriers of the virus who are at greatest risk of developing HCC, the infection is acquired at birth or in the early months or years of life, either perinatally or horizontally, and frequently becomes chronic. The risks are greater in males, and older individuals, and are increased by co-exposure to aflatoxin B₁, the presence of cirrhosis, obesity, or diabetes mellitus, and possibly co-infection with hepatitis C virus. Viral factors that influence the risk of HCC are high viral load, the presence of certain mutations, and genotypes. Although the incidence of chronic HBV infection is beginning to decrease as a result of the universal infant immunization programme, HBV-induced HCC incidence is projected to increase for at least another two decades.     

Interrupted replication of hepatitis B virus in liver tissue of HBsAg carriers with hepatocellular carcinoma

To search for events underlying reduction of peripheral viremia and integration of hepatitis B virus (HBV) DNA into the liver cell genome in long-term virus carriers with hepatocellular carcinoma, paired samples of liver and tumor tissue were analyzed by molecular hybridization and immunological methods. Most tumor tissues contained integrated viral DNA; in none was extrachromosomal HBV DNA detected. Integrated HBV DNS was also found in peritumor liver tissue in the majority of patients. However, liver of patients either with or without peripheral viremia also contained free HBV DNA and replicative intermediates. In three nonviremic patients with replicative HBV DNA in liver, viral core antigen expression was markedly reduced or absent, whereas viral envelope protein (surface antigen) expression was normal. In one case, replicative intermediates in liver were sensitive to DNase I digestion, indicating that viral DNA was not encapsidated in normal viral core particles. These results suggest that decreased or defective core antigen production can lead to reduced viremia associated with blocked virus assembly/secretion and accumulation of unencapsidated HBV DNA replicative intermediates in the liver cell. Accumulation of such HBV DNA molecular forms in the liver may lead to an increased propensity for HBV DNA to integrate into the host genome, which has been found with high frequency in hepatic neoplasms from patients infected with hepatitis B virus.     

Detection of hepatitis B virus genome in hepatocellular carcinoma tissues with PCR-in situ hybridization.

The detection is described of hepatitis B virus (HBV)-DNA in preserved hepatocellular carcinoma tissues, which were derived from 14 HBV-seropositive patients. Detection was by polymerase chain reaction (PCR) amplification of the target sequence, followed by specific localization of the PCR product with in situ hybridization. PISH (PCR-in situ hybridization) yielded strong positive signals in most of the tumor tissues despite very low copy numbers of chromosome-integrated HBV genome, whereas no signal was detected in control samples, indicating that the signals were specific for HBV. Positive signals were sometimes detected in cirrhotic nodules surrounding the tumor regions, indicating that HBV had infected non-transformed liver cells. HBV-DNA was detected in both nucleus and cytoplasm in some specimens, possibly representing HBV at different stages of the life cycle. In one case, a gradient of viral DNA was revealed, with the highest DNA signal centered at the site of viral antigen expression. Taken together, PISH is shown to be a highly sensitive molecular detection method that is capable of detecting the presence of a low copy number viral genome in situ.     

Occult hepatitis B virus infection in patients with chronic hepatitis C liver disease.

BACKGROUND: Hepatitis B virus (HBV) infections in patients who lack detectable hepatitis B surface antigen (HBsAg) are called occult infections. Although such infections have been identified in patients with chronic hepatitis C liver disease, their prevalence and clinical significance are not known. METHODS: With the polymerase chain reaction, we searched for HBV DNA in liver and serum samples from 200 HBsAg-negative patients with hepatitis C virus (HCV)-related liver disease (147 with chronic hepatitis, 48 with cirrhosis, and 5 with minimal histologic changes). One hundred of the patients had detectable antibodies to the HBV core antigen (anti-HBc); 100 were negative for all HBV markers. Eighty-three were treated with interferon alfa. We also studied 50 patients with liver disease who were negative both for HBsAg and for HCV markers. In six patients found to have occult HBV infection, we evaluated possible genomic rearrangements through cloning or direct sequencing procedures. RESULTS: Sixty-six of the 200 patients with chronic hepatitis C liver disease (33 percent) had HBV sequences, as did 7 of the 50 patients with liver disease unrelated to hepatitis C (14 percent, P=0.01). Among the 66 patients, 46 were anti-HBc-positive and 20 were negative for all HBV markers (P<0.001). Twenty-two of these 66 patients (33 percent) had cirrhosis, as compared with 26 of the 134 patients with hepatitis C infection but no HBV sequences (19 percent, P=0.04). HBV sequences were detected in 26 of the 55 patients in whom interferon therapy was ineffective and 7 of the 28 patients in whom interferon therapy was effective (P=0.06). None of the sequenced HBV genomes had changes known to interfere with viral activity and gene expression. CONCLUSIONS: Occult hepatitis B infection occurs frequently in patients with chronic hepatitis C liver disease and may have clinical significance.     

Prevalence of hepatitis B and hepatitis C virus coinfection in chronic liver disease

Coinfection with HBV and HCV may lead to serious consequences. The present study was done to find out the prevalence of coinfection in patients with chronic liver disease. From patients with hepatitis and chronic liver disease 1673 samples were received and analysed for HBsAg by ELISA. 1342 samples were analysed for anti HCV by third generation ELISA. 493 samples positive for HBsAg were also analysed for Anti HCV to see the prevalence of coinfection. 15(3.0%) were found positive for both HBsAg and anti HCV. Out of 15 patients with coinfection 4 (26.6%) had HCC. Prevalence of HCC in patients with coinfection was higher than either infection alone i.e. HBV-9.1% and HCV-16.5%.     

Hepatitis B virus DNA patterns in the liver of children with chronic hepatitis B

The pattern of hepatitis B virus DNA (HBV-DNA) expression were studied in 2 sequential liver biopsies from 26 children (18 treated with interferon and 8 controls) with chronic hepatitis B. In the basal biopsy replicative forms of HBV-DNA were detected in all of the samples and integrated viral DNA was present in 1 case. At the end of the study, 8 children had lost serum HBV-DNA although 2 of the children were still HBeAg positive. (Six had been treated with interferon.) In all of the cases, HBV-DNA was not detectable in the final biopsy. For the rest of the patients, HBV-DNA was positive in serum and all of them had replicative forms of HBV-DNA in the second liver sample. None of the patients lost hepatitis B surface antigen (HBsAg). Peripheral blood mononuclear cells (PBMC) from these patients were studied. HBV-DNA was not found in the PBMC of the 8 children without serum HBV-DNA, and HBV-DNA was detected in the PBMC of 5/12 patients with serum HBV-DNA. In conclusion, HBV-DNA disappeared from the biopsies of children who lost circulating HBV-DNA, although some of the patients were still HBeAg positive. This result implies that the detection of HBV-DNA in liver is important in order to assess the efficacy of the antiviral therapy. On the other hand, HBsAG remained positive in all children at the end of the study although HBV-DNA was not detected in serum, liver, and PBMC by the conventional hybridization techniques.     

False-positive results with hepatitis B virus DNA dot-hybridization in hepatitis B surface antigen-negative specimens.

Three serum samples derived from healthy hepatitis B surface antigen-negative subjects were found to be reactive for hepatitis B virus (HBV) DNA sequences when assayed by DNA dot-hybridization. All three results were shown to be due to the presence of sequences which reacted with residual bacterial plasmid vector sequences in the DNA probe, and no evidence of HBV markers was demonstrated in the sera. This is the first report of a false-positive result with the HBV DNA dot-hybridization assay.     

Clinical and molecular correlation between chronic hepatitis and hepatocellular carcinoma induced by hepatitis B virus

The role of X-gene of hepatitis B virus (HBV), telomerase gene, structural HBV proteins is considered. Both factors of the virus and those of the host are involved in hepatocarcinogenesis. The duration of chronic B hepatitis may influence mitogenic and mutagenic conditions for accumulation of occasional genetic and chromosomal damage and result in hepatocarcinoma development.     

Electron microscopy of hepatitis B virus components in chronic active liver disease.

Eighteen liver biopsy specimens from patients with hepatitis B surface antigen (HBsAg) positive chronic aggressive hepatitis were studied by electron microscopy. All cases were selected on the basis of positive liver cell membrane fluorescence for HBsAg on immunohistochemical investigation. Striking changes in the morphology of the liver cell membrane were observed in nearly all cases. Furthermore, a dual aspect of hepatitis B core antigen (HBcAg) is described. HBcAg particles may occur as either 'naked' or 'cloudy' particles surrounded by semi electron dense material. The nature of the 'cloud' remains to be identified.     

Hepatitis B virus DNA detected in formalin-fixed liver specimens and its relation to serologic markers and histopathologic features in chronic liver disease.

To examine the relationship of hepatitis B virus (HBV) DNA sequences in the liver with histopathologic features and antigenic markers, the authors determined the hepatocytic status of viral DNA by in situ hybridization in formalin-fixed liver sections using a biotinylated probe in 45 patients with various chronic liver diseases. The results were compared retrospectively with the HBV serologic markers and histopathologic features including the presence of ground-glass cells or Shikata staining positivity. The specificity of this in situ detection of HBV DNA has been proven excellent in a double-blind control study in 18 patients in whom liver HBV DNA was also determined by DNA extraction, gel electrophoresis, and the Southern blotting technique. In 41 patients, the findings of HBV DNA and serologic markers were concordant (17 positive and 24 negative). Twelve of the 20 HBV-DNA-positive patients were HBsAg-positive (6 with chronic hepatitis, 3 with cirrhosis, and 3 with hepatocellular carcinoma). Ground-glass cells or Shikata positivity were found in 10 of these 12 patients. HBV DNA sequences were found in the liver of all patients with chronic liver disease and serologic positivity for HBV infection. In liver with normal histologic features, HBV DNA was not demonstrable, despite the positive anti-HBc and anti-HBs. However, a positive HBV DNA was found in 3 serologically negative patients. In another patient the interpretation of findings was impossible because of severe hemosiderosis. From this study, it is concluded that in situ detection of HBV DNA in formalin-fixed liver sections has a clinical value and is suitable for routine use.     

Hepatitis B virus DNA in patients with chronic liver disease and negative tests for hepatitis B surface antigen

We assessed the presence of hepatitis B virus (HBV) DNA in liver or serum samples from 134 patients with hepatitis B surface antigen (HBsAg)-negative chronic liver disease, including 20 with hepatocellular carcinoma. HBV DNA sequences were detected in 52 of the 88 liver samples (59 per cent), including 17 of the 20 samples from patients with hepatocellular carcinoma. Presumably "replicative forms" of HBV DNA were detected in only 5 of the 88 liver samples, 3 of which were from patients with no serologic marker for HBV. In most of the liver samples the DNA patterns were consistent with the presence of HBV or a closely related virus. Of the 105 serum samples tested, HBV DNA sequences were identified in 10 (9.5 per cent), 6 of which had no HBV serologic marker. Moreover, HBsAg-associated determinants were detected in 5 of 17 patients who were positive for HBV DNA and in none of 14 patients who were negative. This study demonstrates the high frequency of HBsAg-negative HBV DNA-positive viral infection of the liver and suggests that multiplication of HBV may occur in the absence of any conventional serologic marker for HBV.     

Varying nuclear staining intensity of hepatitis B virus DNA in human hepatocellular carcinoma

An in situ hybridization technique with biotinylated hepatitis B virus (HBV)-DNA probes was used to localize HBV-related DNA sequences in cells of human hepatocellular carcinoma (HCC). Formalin fixed, paraffin-embedded liver biopsies of 11 patients with HCC studied; nuclear HBV-DNA hybridization was observed in 7 of the patients. Sensitivity and specificity of the method were determined by examining appropriate controls. The number of cells exhibiting nuclear fluorescence and intensity of fluorescence varied from tumor to tumor. In two instances liver tissue adjacent to HCC exhibited nuclear staining. HBV-DNA nuclear staining did not correlate with tumor localization of HBsAg or HBcAg, nor with type or with differentiation of the tumor. The use of biotinylated HBV-DNA probes offers a powerful and reproducible technique to localize HBV-related DNA sequences even in formalin-fixed, paraffin-embedded tumor tissue, and also to compare the presence of HBV-DNA with that of viral antigens stained in parallel sections. The frequent localization of HBV-DNA in nuclei of HCC cells fortifies the important epidemiologic association between infection and HCC. The random cellular localization of HBV-DNA sequences in HCC suggests that HBV-DNA may be incorporated, or perhaps replicated, unequally in tumor cells.     

Detection of hepatitis C virus RNA in the liver biopsy specimens from patients with chronic hepatitis C and hepatocellular carcinoma by means of in situ RT-PCR

The in situ RT-CPR technique has been first adapted in Russia to detect hepatitis C virus (HCV) RNA in the samples from patients with chronic hepatitis C (CHC) and hepatocellular carcinoma (HC). A total of 18 patients with CHC and HC were examined. Fourteen (78%) samples were ascertained to be positive. A positive reaction was found in 2 of 3 patients with HC. A label was revealed in individual hepatocytes without any regularity in the distribution along the tissue section. A reaction was negative in all samples from control groups. The label was observed in both the cytoplasm and the hepatocytic nuclei. There was no correlation between the degree of hepatic lesion, estimated after Knodelle, the results of the classical RT-CPR and the in situ RT-CPR techniques.