Novel HBsAg mutations correlate with hepatocellular carcinoma, hamper HBsAg secretion and promote cell proliferation in vitro

Abstract

Background: An impaired HBsAg-secretion can increase HBV oncogenic-properties. Here, we investigate genetic-determinants in HBsAg correlated with HBV-induced hepatocellular carcinoma (HCC), and their impact on HBsAg-secretion and cell-proliferation.

Methods: This study included 128 chronically HBV-infected patients: 23 with HCC (73.9% D; 26.1% A HBV-genotype), and 105 without cirrhosis/HCC (72.4% D, 27.6% A) as reference-group. The impact of mutations on HBsAg-secretion was assessed by measuring the ratio [secreted/intracellular HBsAg] until day 5 post-transfection. The impact of mutations on cell-cycle advancement was assessed by flow-cytometry.

Results: Two HBsAg mutations significantly correlated with HCC: P203Q (17.4% [4/23] in HCC vs 1.0% [1/105] in non-HCC, P=0.004); S210R (34.8% [8/23] in HCC vs 3.8% [4/105] in non-HCC, P <0.001); P203Q+S210R (17.4% [4/23] in HCC vs 0% [0/110] in non-HCC, P=0.001). Both mutations reside in trans-membrane C-terminal domain critical for HBsAg-secretion. In in-vitro experiments, P203Q, S210R and P203Q+S210R significantly reduced the ratio [secreted/intracellular HBsAg] compared to wt at each time-point analysed (P <0.05), supporting an impaired HBsAg-secretion. Furthermore, P203Q and P203Q+S210R increased the percentage of cells in S-phase compared to wt, indicating cell-cycle progression (P203Q:26±13%; P203Q+S210R:29±14%; wt:18%±9, P <0.01. Additionally, S210R increased the percentage of cells in G2/M-phase (26±8% for wt versus 33±6% for S210R, P <0.001).

Conclusions: Specific mutations in HBsAg C-terminus significantly correlate with HBV-induced HCC. They hamper HBsAg-secretion and are associated with increased cellular proliferation, supporting their involvement in HCC-development. The identification of viral genetic markers associated with HCC is critical to identify patients at higher HCC-risk that may deserve intensive liver monitoring, and/or early anti-HBV therapy.

Keywords: HBsAg mutations; cell proliferation; hepatitis B; hepatitis B surface antigen; hepatocellular carcinoma.

Conflict of interest statement

CONFLICTS OF INTEREST

Salpini R: Speaker fees and consulting fees from BMS, Gilead Sciences and Diasorin.

Andreoni M: Attending symposia, speaking, organizing educational activities, consultancy or advisory board membership, or grant research support from Gilead Sciences, BMS, MSD.

Ceccherini-Silberstein F: Attending symposia, speaking, organizing educational activities, consultancy or advisory board, or grant research support from Abbott Molecular, BMS, Gilead Sciences, Roche Diagnostics.

Locarnini S: Consulting fees and grant research support from Gilead Sciences and Arrowhead Research

Perno CF: Research grants, lecturing fees, advisory boards, scientific consultancies for Gilead Sciences, BMS, MSD, Roche, Abbott Diagnostics.

Valentina Svicher, Research grants, lecturing fees, advisory boards for Gilead Sciences, BMS

Other authors' conflicts of interest: none

Figures

Figure 1. The histogram reports the prevalence of sP203Q, sS210R, and sP203Q+sS210R in 23 HBV-induced HCC patients and in 105 chronically HBV-infected patients (CHB patients) (used as reference group)

Statistically significant differences were assessed by Fisher Exact Test. * P

Figure 2

The graph reports the intra-patient prevalence of sP203Q (white dots), sS210R (grey dots), sP203Q+sS210R (black dots) for each patient in the HCC group (N=13) A. and in the group of chronically-infected patients used as reference (N=27) B. Intra-patient prevalence was expressed as % of reads with the specific mutation. The grey area includes mutations with an intra-patient prevalence <20%, not detected by standard population-based sequencing, and thus defined as minority species.

Figure 3

A. The histogram reports HBsAg secretion factor for the wild-type virus and for mutants carrying P203Q, S210R and P203Q+S210R. Secretion factor is defined as the ratio of extracellular to intracellular HBsAg amount determined by the Alexxis assay. Average of 3 experiments +/− SEM are shown. Statistically significant differences were assessed by 2-tailed unpaired T-test. *P <0.05 **P <0.01, ***P <0.005. B. The histogram reports the percentage of cells in S-phase of cell cycle for wild-type and mutants at 7 days post transfection determined by FACS analysis, selecting GFP+ HepG2 cells. Statistical analysis was assessed by Chi-Square Test based on 2×2 contingency table. *P <0.001