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

Abstract

Background: The association between the evolution of hepatitis B virus (HBV) quasispecies and the development of hepatocellular carcinoma (HCC) is unknown.

Methods: 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 9 years 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.

Results: 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.

Conclusions: 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.

Keywords: Deep sequencing; Hepatitis B virus; Hepatocellular carcinoma; Pre-S deletions; Quasispecies.