Characterization of integrated hepatitis B virus DNA harboring pre‐S mutations in hepatocellular carcinoma patients with ground glass hepatocytes

Abstract Ground glass hepatocytes (GGHs) have been associated with hepatocellular carcinoma (HCC) recurrence and poor prognosis. We previously demonstrated that pre‐S expression in some GGHs is resistant to current hepatitis B virus (HBV) antiviral therapies. This study aimed to investigate whether integrated HBV DNA (iDNA) is the primary HBV DNA species responsible for sustained pre‐S expression in GGH after effective antiviral therapy. We characterized 10 sets of micro‐dissected, formalin‐fixed‐paraffin‐embedded, and frozen GGH, HCC, and adjacent hepatitis B surface antigen‐negative stained tissues for iDNA, pre‐S deletions, and the quantity of covalently closed circular DNA. Eight patients had detectable pre‐S deletions, and nine had detectable iDNA. Interestingly, eight patients had integrations within the TERT and CCNE1 genes, which are known recurrent integration sites associated with HCC. Furthermore, we observed a recurrent integration in the ABCC13 gene. Additionally, we identified variations in the type and quantity of pre‐S deletions within individual sets of tissues by junction‐specific PacBio long‐read sequencing. The data from long‐read sequencing indicate that some pre‐S deletions were acquired following the integration events. Our findings demonstrate that iDNA exists in GGH and can be responsible for sustained pre‐S expression in GGH after effective antiviral therapy.