Hepatocyte clonal expansion and hepatitis B virus DNA integration in patients with long-term nucleos(t)ide analogue therapy

Abstract

Chronic hepatitis B (CHB) infection increases the risk of hepatocellular carcinoma (HCC). The exact mechanisms of how CHB induces HCC remain unclear. One proposed mechanism is via the integration of hepatitis B virus (HBV) DNA into the host genome. Nucleos(t)ide analogues (NUCs), are therapeutic agents against CHB. NUCs effectively suppress HBV replication in CHB patients, but their effects on HBV integration have not been reported. This thesis aimed to investigate the effect of NUCs on HBV integration, expressed in terms of HBV integration frequency and clonal expansion of hepatocytes. The underlying mechanism of NUC-induced HBV integration reduction was also studied. This thesis comprised three parts. The first part was the detection of HBV DNA integration and measurement of hepatocyte clone size by an end-point, serial dilution PCR-based method called inverse PCR. We investigated HBV integration in 28 CHB patients and found that both HBV integration frequency and hepatocyte clone size were significantly reduced after 1 year of treatment and were further reduced after 10 years of treatment. Significant reductions in HBV viral load and serum virologic markers were seen after treatment. However, no significant correlation between the reductions of virological parameters and HBV integration was found, implying that the reductions of HBV viral replication and HBV integration by NUCs may be independent on each other. Next, we performed a more comprehensive detection of HBV integration by next-generation sequencing (NGS). Paired liver tissues (baseline and at year 1 of treatment) from 7 patients were analyzed by NGS, followed by the functional and pathway enrichment analysis. HBV integration was significantly reduced after treatment, and the integration breakpoints were dispersed throughout the HBV genome. Significant clustering of HBV integration in liver cancer-related genes was found especially in the baseline samples but not in the year 1 samples. This further suggests a link between NUCs, HBV integration and HCC. We performed in-vitro experiments to study the underlying mechanism of NUC-induced HBV integration reduction. HepG2 cells with transposon-mediated insertion of HBV genome (named HepG2/SB/HBV) was used to mimic cells with HBV integration and was treated with entecavir. Entecavir selectively inhibited cell proliferation of HepG2/SB/HBV but not of the parental HepG2 cells. Such inhibition was brought by reducing the abundance of phospho- histone H3, a protein that is important in chromosome condensation, the reduction of which resulted in cell cycle arrest and subsequent clone size reduction of HepG2/SB/HBV. In conclusion, to our knowledge, this thesis is the first to demonstrate that NUCs can reduce both HBV DNA integration and hepatocyte clonal expansion in CHB patients. One mechanism may be via the suppression histone H3 phosphorylation, which results in cell cycle arrest. As both HBV integration and hepatocyte clonal expansion are possible mechanisms leading to HCC, this study suggested that NUCs treatment should be given early even to CHB patients

without active viral replication or liver injury in order to reduce HCC risk. This study also provided a theoretical basis on assessing the effects of newer therapeutic agents on the reduction of HBV integration.