Identification of hepatitis B virus integration in hepatitis C virus-infected hepatocellular carcinoma tissues

The integration of HBV DNA is thought to be involved in the initial stage of hepatocarcinogenesis, and it has been reported that transactivating factors encoded by the X and preS2/S genes stimulate transcription of multiple viral and cellular genes. We assessed the possible contributions of hepatitis B virus integration to the occurrence of hepatocellular carcinoma in hepatitis C virus-infected as well as in hepatitis B virus-infected patients by identifying the integrated HBV DNA sequence, and the X and preS2/S regions were further investigated in HBV DNA-integrated cases.

Southern blot hybridization for detecting HBV DNA in tumor tissues from 28 hepatocellular carcinoma patients was carried out with full-length HBV DNA, and then with X and preS2/S regions as probes. We also carried out reverse transcription-polymerase chain reaction for detecting HCV RNA to confirm hepatitis C virus-infection in liver tissues.

Clonally integrated HBV DNA sequences were demonstrated in 16 of 28 patients (57.1%), including five HBsAg seropositive and 11 HBsAg seronegative patients. Of these 11 HBsAg seronegative patients, 10 were also positive for anti-HCV in their sera, and all nine examined cases had HCV RNA in liver. Furthermore, the X region was identified in 14 of 16 HBV DNA integrated cases (87.5%), and the preS2/S region in 6/16 (37.5%).

The present Southern blot analysis demonstrates that clonally integrated HBV DNA sequences were identified even in hepatitis C virus-infected hepatocellular carcinoma patients at a high rate (10/18, 55.6%), and suggests that integrated hepatitis B virus, whose major component is the X gene, may play an important role in hepatocarcinogenesis in hepatitis B virus-integrated cases with and without hepatitis C virus infection.