Background: Epigenetic alteration through methylation is one of the most important steps in carcinogenesis. However, the relation between hepatitis virus infection and epigenetic alterations is poorly understood. Methods: Sixteen patients without hepatitis B virus (HBV) and hepatitis C virus (HCV) and 35 patients with HBV or HCV who underwent liver resection for hepatocellular carcinoma (HCC) were studied. Mutation of p53 was detected by direct sequencing. Methylation status of p16 was evaluated in tumor and noncancerous liver tissues by methylation-specific polymerase chain reaction. Results: In HCC without HBV and HCV, p53 mutations were detected in 5 (31%) of 16 HCCs. Methylation of p16 promoter was detected in 2 (25%) of 8 moderately differentiated HCCs, 6 (75%) of 8 poorly differentiated HCCs, and none of 16 noncancerous tissue specimens. In HCC with HBV or HCV, p53 mutations were detected in 8 (23%) of 35 HCCs. Methylation of p16 promoter was detected in 2 (100%) of 2 well-differentiated HCCs, 13 (76%) of 17 moderately differentiated HCCs, 12 (75%) of 16 poorly differentiated HCCs, and 9 (26%) of 35 noncancerous liver tissue specimens. Conclusions: Our results suggest that hepatitis viruses might induce methylation of p16 promoter in liver with chronic inflammation, before appearance of HCC.

Few studies have examined the etiology of hepatocellular carcinoma (HCC) in patients without hepatitis virus infection. We evaluated the role of occult hepatitis B virus (HBV) infection in the development of HCC in Japanese patients without hepatitis B surface antigen (HBsAg) and antibodies to hepatitis C antigen (anti-HCV). Twenty-one HBsAg negative and anti-HCV negative (non-B, non-C) patients with HCC were studied. HBV DNA in serum and HBV transcripts in liver were examined by polymerase chain reaction (PCR) or reverse transcription and PCR. HBV DNA integration was examined by Southern blot analysis or cassette-ligation-mediated PCR as described previously. p53 mutations were examined by direct sequencing. HBV DNA was not detected in serum from any patients. HBV-related transcripts were detected in 5 of 7 HCCs from patients with antibodies to hepatitis core antigen (anti-HBc) and in 3 of 14 HCCs from patients without anti-HBc (P = 0.0261). HBV DNA was integrated into human genome in two non-B, non-C HCCs. Of the 14 patients without anti-HBc, 5 had a history of excessive alcohol intake. In exons 5 through 8 of the p53 gene, mutations were detected in 2 of 8 HCCs with HBV-transcripts and in 5 of 13 HCCs without such transcripts. p53 mutation at codon 159 was found in 2 of 6 patients with excessive alcohol intake without HBV-transcripts. These results suggested that occult HBV infection might play an important role in hepatocarcinogenesis in non-B, non-C patients with anti-HBc and that excessive alcohol intake might be related to HCC in non-B, non-C patients in Japan.

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