Complete genome sequencing and clinical analysis of intrahepatic hepatitis B virus cccDNA from HCC

Abstract

More than half of hepatocellular carcinomas (HCCs) are etiologically attributed to hepatitis B virus (HBV) infection, but it remains unclear whether HBV mutations are virological factors that contribute to formation of HCC or instead reflect accumulation during the progression of HBV-related disease. Rolling-cycle amplification and PCR sequencing were used to characterize covalently closed circular DNA (cccDNA) mutations in tumor tissues. Paired non-tumor tissues were used as controls. High frequencies of C1653T, T1753V, and A1762T/G1764A cccDNA mutations were observed in both tumor and non-tumor tissues. T1719G, C1329A, and T3098C mutations were related to the overall survival of HCC patients. Patients with G1719 tended to be in the high Barcelona Clinic Liver Cancer stage and had lower levels of total DNA and cccDNA per cell than patients with T1719. Additionally, invitro analysis revealed that T1719G mutation reduced viral replication efficacy. Finally, significantly higher levels of preoperative alpha-fetoprotein were observed in patients harboring the G1078T, C1653T, G1727A, C1913A, T1978C, or C3116T mutations at the cccDNA level. We speculated that HBV cccDNA mutations accumulated over the course of HBV-related disease development, and that some key mutations had prognostic value for patients with HBV-related HCC.