Deep sequencing analysis of quasispecies in the HBV pre-S region and its association with hepatocellular carcinoma.

Abstract

The association between the evolution of hepatitis B virus (HBV) quasispecies and the development of hepatocellular carcinoma (HCC) is unknown. We used deep sequencing to examine the dynamics of HBV quasispecies and their relationship to HCC development. Thirty-two chronic hepatitis B (CHB) patients with HCC (HCC group) and 32 matched CHB patients without HCC (controls) were recruited. Fourteen patients from each group had serial sera available up to 9years before the time of the present study. Deep sequencing of the HBV pre-S regions was performed. HBV quasispecies complexity, diversity, and intrapatient prevalence of pre-S deletions/mutations were analyzed. Compared with control patients, HCC patients had a significant greater quasispecies complexity (p=0.04 at the nucleotide level), greater diversity (p=0.004 and 0.009 at the nucleotide level and the amino acid level respectively), and a trend of greater complexity at the amino acid level (p=0.065). HCC patients had a higher intrapatient prevalence of pre-S deletions and point mutations (at codons 4, 27, and 167) compared with the control patients (all p<0.05). Longitudinal observation in the sera of 14 HCC patients showed that quasispecies complexity (p=0.027 and 0.024 at the nucleotide level and the amino acid level respectively) and diversity (p=0.035 and 0.031 at the nucleotide level and the amino acid level respectively) increased as the disease progressed to HCC. Increased HBV quasispecies complexity and diversity in the pre-S region, probably reflecting enhanced virus-host interplay, was associated with disease progression from CHB to HCC.