Hepatitis B virus DNA detection by in situ hybridization in human hepatocellular carcinoma

Abstract

The distribution of hepatitis B virus (HBV) DNA in tumor tissue sections from six Korean patients with HBsAg positive hepatocellular carcinoma (HCC) was examined by in situ hybridization using a biotin-labeled recombinant, cloned HBV DNA probe. All patients tested were positive for both HBeAg and anti-HBc in their sera. HBV DNA was distributed abundantly in the cytoplasm and rarely in the nuclei of tumor cells. The validity of the in situ hybridization assay was confirmed by the dot blotting technique using a ³²P-labeled HBV DNA probe obtained by nick translation. In conclusion, it is speculated that integration of HBV DNA into host DNA as well as persistant amplified replication of the HBV DNA within the hepatocytes is linked etiologically to the development of human hepatocellular carcinoma.

Fig. 1.

In situ hybridization of HBV DNA in hepatocellular carcinoma. The cytoplasm of HCC show abundant HBV DNA granules. The nuclei rarely reveal DNA grains. (Methyl green counterstain, × 400)

Fig. 2.

In situ hybridization of HBV DNA in the cirrhotic areas around HCC. The cytoplasm of the cirrhotic nodule shows plentiful HBV DNA grains. (×200)

Fig. 3.

High power details of figure 2. The nuclei reveal no discernible grains of HBV DNA. (Methyl green counter stain, × 400)

Fig. 4.

Autoradiography of the dot blot hybridization of sera of patients with HCC utilizing a³²P-labeled HBV DNA probe (specific activity, 10⁶ cpm/μg DNA). To determine the concentration, the indicated amounts of unlabeled recombinant lambda-HBV DNA with serial dilutions were directly spotted on the nitrocellulose paper in 50μl volumes, followed by hybridization with labeled DNA, and then autoradiographed for signal detection.