Analysis of hepatitis B virus genetic heterogeneity in chronic hepatitis B patients with hepatocellular carcinoma

Abstract

The involvement of hepatitis B virus (HBV) genetic heterogeneity in liver disease progression to hepatocellular carcinoma (HCC) is not fully understood. We aimed to analyze HBV genetic heterogeneity in chronic hepatitis B (CHB) patients with HCC.

Full-length HBV genome sequences were characterized in both tumor and non-tumor tissues from 14 HCC patients by direct sequencing. HBV DNA sequences from HCC tumor tissues displayed different mutation patterns from those in non-tumor tissues. Hotspot mutations at HBx aa130-131 and preS deletions were detected in 13 (93%) and 6 (43%) patients, respectively. Deletions in the X/preC/C gene were more frequently detected in the tumor tissues than non-tumor tissues (p=0.031).

HBV quasispecies characteristics within the preS region were further investigated in the HCC tumor and non-tumor tissues using clonal sequencing. Compared with the nontumor tissues, tumor tissues had a lower quasispecies complexity and lower diversity at both the nucleotide and amino acid levels (all p<0.05). Phylogenetic analysis showed that HBV sequences derived from tumor and non-tumor tissues were separately clustered, suggesting the occurrence of compartmentalization, which was confirmed by correlation coefficient testing (all p<0.02).

The prevalence and evolutionary changes of HBV preS mutations in up to 10 years prior to the development of HCC were studied in 74 HCC and 148 age- and sex-matched HCC-free CHB patients. Higher frequencies of preS deletions and point-mutations at 11 codon positions were observed in the HCC patients than in the HCC-free patients (all p<0.05). Multiple logistic regression analysis showed that preS deletions, and pointmutations at codons 51 and 167 were independent risk factors for HCC development. Longitudinal observation showed that preS deletions and most of the 11 point-mutations existed at least 10 years before HCC development, and were more prevalent in HCC patients than in HCC-free patients. The number of HCC-associated preS point-mutations increased over time prior to HCC development, and correlated positively with the time to HCC diagnosis (r=0.194, p=0.006).

Deep sequencing analysis of HBV quasispecies characteristics in preS region prior to HCC was performed in the sera of 32 HCC and 32 matched HCC-free CHB patients. Compared with the HCC-free patients, patients who eventually developed HCC had a higher quasispecies complexity (p=0.001 and 0.002, at the nucleotide and amino acid levels, respectively) and greater diversity (p=0.005 and 0.007, at the nucleotide and amino acid levels, respectively). HCC patients also had a higher intrapatient prevalence of preS deletions and point-mutations compared to HCC-free patients (all p<0.05). Longitudinal observation in the sera of 14 HCC patients and 14 matched HCC-free patients showed that quasispecies complexity and diversity increased as the disease progressed to HCC development.

In summary, this study identified a dynamic change of viral heterogeneity (pointmutations, deletions and quasispecies characteristics) during the development of HCC. PreS mutations and increased quasispecies complexity and diversity were associated with HCC development. Following HCC development, selection of certain HBV mutations was found in the tumor tissues, inducing a decreased quasispecies complexity and diversity in tumor compartment.