T. Saito1, R. Suzuki2,3, A. Rahman4, S.A. Rajib4, K. Mori2, N. Kobayashi1,2, T. Orimo1, L. Liang2, S. Suzuki2,3, T. Tamura2,3,5, Y. Satou4, A. Taketomi1, F. Takasuke2,3,5,6 1Department Of Gastroenterological Surgery I, Graduate School Of Medicine, Hokkaido University, Sapporo, HOKKAIDO, Japan 2Department of Microbiology and immunology, Faculty Of Medicine, Hokkaido University, Sapporo, HOKKAIDO, Japan 3Institute for Vaccine Research and Development (IVReD), Hokkaido University, Sapporo, HOKKAIDO, Japan 4Joint Research Center for Human Retrovirus Infection, Kumamoto University, Kumamoto, KUMAMOTO, Japan 5One Health Research Center, Hokkaido University, Sapporo, HOKKAIDO, Japan 6Laboratory of Virus Control, Research Institute for Microbial Diseases, Osaka University, Suita, OSAKA, Japan
Introduction: In Japan, the incidence of non-B non-C hepatocellular carcinoma (NBNC-HCC), which is negative for hepatitis B surface antigen (HBsAg) and hepatitis C virus antibody, is increasing. Recently, it is reported that a significant number of patients with NBNC-HCC have positive HBc antibody which means they had HBV infection and cured.. This suggests that pre-existing HBV infection may contribute to the development of NBNC-HCC, although the details remain unclear. This study aimed to investigate whether HBV genomes are integrated into the cancerous tissues of HBc antibody-positive NBNC-HCC cases and whether such integrations are involved in hepatocarcinogenesis and cancer progression.
Methods: HBc antibody-positive NBNC-HCC resection samples were collected in Japan. DNA was extracted from the cancerous tissues, and PCR was performed using five primer sets targeting comprehensive HBV sequences (HBV detecting PCR). Additionally, DNA samples from specimens positive in HBV detecting PCR were analyzed for integration sites using next-generation sequencing (NGS) based on the Viral DNA Capture Approach (VCDA method, Katsuya H et al., 2019, Cell Reports). To confirm the correlation between tissue and blood results, Hepatitis B Core-related Antigen (HBcrAg) was measured in blood samples.
Results: Among 90 HBc antibody-positive NBNC-HCC cases, 18 cases showed amplification with HBV-specific primers. In the NGS analysis of these 18 positive cases, HBV reads were found in 17 cases, and HBV genome was integrated in 10 cases. 7 out of the 10 cases had HBV genome insertion near the TERT (telomerase reverse transcriptase) gene. In these cases, TERT mRNA levels were significantly increased compared to cases which did not have HBV genome integration near the TERT. This suggests that a certain number of NBNC-HCC cases may develop due to HBV genome integration into the TERT gene. Additionally, there was a positive correlation between PCR results and HBcrAg levels.
Conclusion: In some cases of HBc antibody-positive NBNC-HCC, HBV genomes were integrated into the cancerous tissues, and a subset of these cases had HBV integration in the TERT oncogenes. Since TERT is a cancer-promoting gene, it is possible that HBV genome integration increased the expression of the TERT gene, contributing to hepatocarcinogenesis. In NBNC-HCC, HBcrAg might potentially predict the presence of HBV genome integration into the host genome without the need for invasive tests such as liver biopsy.