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

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.