Evolutionary changes of hepatitis B virus pre-S mutations prior to development of hepatocellular carcinoma

Abstract

BACKGROUND AND AIMS: Hepatitis B virus (HBV) pre-S deletions/mutations have been associated with hepatocellular carcinoma (HCC). However, the evolutionary changes of pre-S mutations prior to HCC diagnosis remain unexplored. METHODS: HBV pre-S sequences were determined in 74 chronic hepatitis B (CHB) patients with HCC and 146 CHB patients who have been followed up for >3 years with no HCC (HCC-free). Samples collected at the following time points were studied: 1–3 years, 4–6 years, 7–9 years and ≥10 years prior to HCC development and at the same time points for the HCC-free group from last follow-up. HBV pre-S mutations were assessed by comparison with 54 wild-type reference sequences retrieved from the NCBI Genbank. RESULTS: Pre-S deletions were detected in 31.1%, 25%, 48.5%, and 42.8% of HCC patients at 1–3 years, 4–6 years, 7–9 years, and ≥10 years prior to HCC development, respectively. In the HCC-free group, the frequencies of pre-S deletions at the 4 corresponding time points were lower (9.6%, 7.9%, 8.8%, and 17.6%, respectively; with p values of <0.001, 0.036, <0.001, and 0.096, respectively). At 1–3 years prior to HCC development, higher frequencies of pre-S point mutations at 11 codons (codons 4, 27, 51, 54, 60, 62, 100, 125, 137, 166 and 167) were observed in the HCC patients (range: 14.9–75.7%) than in the HCC-free patients (range: 5.5–26%; all p < 0.05). Ten out of these 11 pre-S mutations (except codon 4, which emerged at 4–6 years before HCC) existed ≥10 years before HCC. Among these 11 codons, the number of mutated codons with differential distribution between HCC and HCC-free patients increased with time prior to HCC: a significantly higher frequency of mutations was detected in the HCC patients in 2/11 codons at ≥10 years, in 9/11 codons at 7–9 years, and in 10/11 codons at 4–6 years prior to HCC. The total number of HCC-associated pre-S point mutations increased with time prior to HCC and correlated with the time to HCC development (r = 0.151, p = 0.042), while no correlation was observed in the HCC-free group (r = 0.073, p = 0.398). Multivariable logistic regression analysis showed that pre-S deletions and point mutations at codons 27, 51 and 167 were independent factors associated with increased HCC risk (all p < 0.05). CONCLUSIONS: High prevalence and cumulative evolution of pre-S mutations preceding HCC development confirmed the carcinogenic role of pre-S mutations. The effect of individual pre-S mutations on HCC development warrants further studies.