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.