Study of HBV DNA integration in patients with HBV-related HCC by a high-throughput viral integration detection method

The aim of the study was to detect HBV integration sites across the genome and decipher viral-host interaction, by applying a new a high-throughput targeted sequencing of enriched HBV integrants, in tumor (T) and non-tumor (NT) liver tissues from HBsAg-positive patients with HCC, on PLC/PRF/5 cell line as positive control, and on 3 different tissue samples from HBV-negative patients as negative control. Methods: DNA was fragmented by sonication, blunted, adenosine-tailed, and ligated to annealed adaptors. HBV integrations were recovered by PCR using specific HBV primers covering the different HBV regions and primer-adapters. Results: A total of 5,539 HBV integration breakpoints (covered by a total of 44,141 chimeric reads) were detected in tumor liver tissues from 7 HBsAg-positive HCC patients, in paired non-tumor tissues, availabe from 6 of them, and in PLC/PRF/5 human hepatoma cells (4,369 in T,1,139 in NT, and 31 in cells). Among the 5,539 HBV integration sites, 3,211 were located in repeated DNA sequences. Most of the HBV integrations were found in SINEs (23% in T,10% in NT, and 22,6% in cells), in simple repeats (12,5% in T, 28% in NT, and 0% in cells), in LINE (10,3% in T, 11,5% in NT and 9,7% in cells), and in LTR (8% in T, 4% in NT and 6% in cells). Among the remaining 2,328 viral integration sites, 289 occurred within exons [242 in T, 47 in NT (P=0.02), and 0 in cells] and 772 within introns (604 in T,164 in NT, and 4 in cells). Interestingly, 215 breakpoints were found in lncRNA [175 in T, 36 in NT (P=0.03), and 4 in cells]. A total of 1,052 integrations were found in intergenic regions and most of them were located within 100kbp from genes. An enrichment of microhomology (MH) sequences between host DNA and integrated HBV DNA was found at the site of integration in a high percentage of chimeras from each sample. PreS-S genomic sequences and sequences including ENHI/X promoter/3’ deleted-X gene were the most frequent HBV integrants detected. In PLC/PRF/5 cells, 11 distinct HBV integration breakpoints were recognized. All HBsAg-negative control patients showed no HBV integration event. The developed high-throughput HBV integration sequencing enabled us to perform a large scale and unbiased analysis of HBV DNA integration sites in liver cancer. This new approach allowed the definition of preference sites of integration occurring within regions of the genome prone to DNA mutations or rearrangements and novel genomic elements recurrently affected by HBV integration.