Clonal expansion of hepatocytes and state of hepatitis B virus DNA integration in patients with nucleos(t)ide analogue therapy

Abstract

Background and Aims: HBV DNA integration and hepatocyte clonal expansion are possible mechanisms of hepatocarcinogenesis. While it has been suggested that nucleos(t)ide analogues (NUCs) treatment can reduce the risk of hepatocellular carcinoma, the effect of NUC on HBV DNA integration has not been examined. This study aimed to investigate the effect of 1 year of NUCs treatment on HBV DNA integration and hepatocyte clonal expansion. Method: This study included 10 chronic hepatitis B patients with paired liver biopsies collected before treatment and after 1 year of NUCs treatment. HBV DNA integration was detected by inverse PCR. Hepatocyte clone size, which reflects the extent of HBV DNA integration, was measured using a semi-quantitative end-point dilution method. Results: All 10 patients (7 males and 3 females; mean age 38 ± 10 years) received entecavir treatment. Six were hepatitis B e antigen (HBeAg)-positive and 4 were HBeAg-negative at baseline. HBV DNA integration was detectable in all 10 patients at baseline. The viral junctions of HBV DNA integration clustered at the HBV direct repeat regions (nucleotides 1698 to 1986), while integration junctions at human chromosomes 1, 7, 8, 11, 16, 20 and 21 were identified. HBV DNA integration was also detected after 1 year of treatment, and the site of HBV DNA integration remained unchanged in each of the patients at year 1. There was no significant association between hepatocyte clone size and patients’ age at baseline. After 1 year of treatment, a significant reduction in hepatocyte clone size was seen (median hepatocyte clone size at baseline vs. at year 1: 1.17 × 106 vs 1.48 × 105; P = 0.005), with a median reduction of 78% (range 21%–95%) in clone size. At year 1, serum HBV DNA, intrahepatic total HBV DNA and covalently closed circular DNA were reduced by 5.2 log, 1.5 log and 0.5 log respectively, but their reductions did not significantly correlate with the magnitude of hepatocyte clone size reduction. Conclusion: HBV DNA integration was detected in patients both before and after 1 year of entecavir therapy. However, hepatocyte clone size, which reflects the degree of HBV DNA integration, was significantly reduced after 1 year of treatment. This suggested that, in addition to reducing serum and intrahepatic HBV DNA, NUCs treatment by reducing the extent of HBV DNA integration, may have an additional anti-oncogenic mechanism.