Persistence of intrahepatic hepatitis B virus DNA integration in patients developing hepatocellular carcinoma after hepatitis B surface antigen seroclearance

Abstract

Background/Aims: The role of hepatitis B virus (HBV) integration into the host genome in hepatocarcinogenesis following hepatitis B surface antigen (HBsAg) seroclearance remains unknown. Our study aimed to investigate and characterize HBV integration events in chronic hepatitis B (CHB) patients who developed hepatocellular carcinoma (HCC) after HBsAg seroclearance.<br/>Methods: Using probe-based HBV capturing followed by next-generation sequencing technology, HBV integration was examined in 10 samples (seven tumors and three non-tumor tissues) from seven chronic carriers who developed HCC after HBsAg loss. Genomic locations and patterns of HBV integration were investigated.<br/>Results: HBV integration was observed in six patients (85.7%) and eight (80.0%) of 10 tested samples. HBV integration breakpoints were detected in all of the non-tumor (3/3, 100%) and five of the seven (71.4%) tumor samples, with an average number of breakpoints of 4.00 and 2.43, respectively. Despite the lower total number of tumoral integration breakpoints, HBV integration sites in the tumors were more enriched within the genic area. In contrast, non-tumor tissues more often showed intergenic integration. Regarding functions of the affected genes, tumoral genes with HBV integration were mostly associated with carcinogenesis. At enrollment, patients who did not remain under regular HCC surveillance after HBsAg seroclearance had a large HCC, while those on regular surveillance had a small HCC.<br/>Conclusions: The biological functions of HBV integration are almost comparable between HBsAg-positive and HBsAgserocleared HCCs, with continuing pro-oncogenic effects of HBV integration. Thus, ongoing HCC surveillance and clinical management should continue even after HBsAg seroclearance in patients with CHB.

Distinct Patterns of HBV Integration and TERT Alterations between in Tumor and Non-Tumor Tissue in Patients with Hepatocellular Carcinoma

“…Probe-based HBV capture followed by NGS technology was performed to survey HBV integration in HCC (Supplementary Figure S3). The probes for HBV hybridization were designed to tile based on the eight Korean full HBV genome sequences (GenBank Accession numbers AY641559.1, DQ683578.1, GQ872211.1, GQ872210.1, JN315779.1, KR184660.1, AB014381.1, AB014395.1, and D23680.1 Hepatitis B virus complete genome sequence (https://www.ncbi.nlm.nih.gov/nuccore, accessed on 2 March 2018), as described elsewhere [23]. We designed 215 probes to span the entire HBV genome.…”

“…The Illumina NGS workflow we used to capture the integrated HBV sequences was described elsewhere [23]. Briefly, 1 ug of genomic DNA was fragmented by adaptive focused acoustic technology (AFA; Covaris) and then repaired; an 'A' was ligated to the 3' end, Agilent adaptors were ligated to the fragments, and the adaptor-ligated product was PCR-amplified.…”

“…The identification of the HBV-human chimeric reads was described previously [23]. Briefly, a modified reference was generated by merging the human (UCSC assembly hg19, original GRCh37 from National Center for Biotechnology Information (NCBI), February 2009) and HBV (DQ683578.1) genome.…”

KASL clinical practice guidelines for management of chronic hepatitis B

“…HBV can integrate in human DNA and promote hepatocarcinogenesis by insertional mutagenesis, increased genomic instability, or expression of viral oncoproteins such as the protein HBx [ 130 , 131 ]. The application of next-generation sequencing led to characterization of HBV integration events.…”

Viral Biomarkers for Hepatitis B Virus-Related Hepatocellular Carcinoma Occurrence and Recurrence

“…Many studies indicate that HBV integration causes genetic damage and chromosomal instability, which has the potential to promote carcinogenic transformation ( Scotto et al, 1983 ; Zhao L.H. et al, 2016 ; Chen et al, 2019 ; Jang et al, 2020 ), or drive downstream host protein expression. HBV DNA can integrate into fragile sites, CpG islands and near telomerase reverse transcriptase, lysine methyltransferase 2B, as well as cyclin A2 ( Wong et al, 2020 ), potentially inducing cancer-initiating genomic instability ( Zhao L.H.…”