Integrated state of subgenomic fragments of hepatitis B virus DNA in hepatocellular carcinoma from mainland China

Abstract

Hepatocellular carcinoma (HCC) samples from mainland China were examined for the presence and state of hepatitis B virus (HBV) DNA sequences. HBV DNA was detected by dot-blot hybridization in 13 of 17 cases of HCC from the Shanghai area and in three of six samples from Hangzhou. The HCC cases from Shanghai were then analyzed in more detail. Fifteen of the 17 patients had serologic evidence of past or present infection with HBV (with inadequate information available for the other two), and the 13 HCC samples positive for HBV DNA all came from serologically positive patients. Southern blot analysis showed that the HBV DNA sequences were always integrated in the HCC high-molecular-weight DNA; only one or two viral copies were present per tumor cell, and no common integration site was evident. Hybridization analyses using subgenomic probes of HBV DNA revealed that the tumors seldom retained an entire HBV genome. HBV S-region sequences were always present, X-region sequences were usually represented, and C-region sequences were rarely detectable in virus-positive tumors. A fragment within the HBV DNA X-region, between nucleotides 1441 and 1526, was found to hybridize nonspecifically with cellular DNA; reported sequence data indicated that this fragment would contain approximately 70% guanine + cytosine. Histologic sections were prepared from some of the frozen tissue specimens and stained by an indirect immunoperoxidase technique for hepatitis B surface antigen (HBsAg). Only 1 of 10 HBV DNA-positive samples contained HBsAg in the cytoplasm of tumor cells, although abundant HBsAg was present in adjacent normal cells in all 10 cases. There were no significant differences in histology between HCC that contained HBV DNA sequences and those that were virus negative. These data support the premise that HBV represents a major etiologic factor in the development of HCC in the Shanghai area of China, although the molecular basis of viral involvement remains obscure.