Genomic And Oncogenic Preference Of HBV Integration In Hepatocellular Carcinoma

Background and Purpose:Hepatocellular carcinoma(HCC) is the third leading cause of global cancer death and also ranks sixth in cancer incidence. In developing countries, hepatocellular carcinoma often comes to medical attention when the tumors are detected at an advanced stage and curative therapies are of limited benefits. HCC incidence and death rates are particularly higher in Asian than whites. Chronic infection with hepatitis B virus(HBV) is one of the major risk factors for the development of HCC, and in China, more than 80% of HCC patients have been associated with HBV.HBV DNA could integrate into host genome and has been reported as one of the major causes of hepatocarcinogenesis. Recently there has been mounting evidence on the role of HBV integration involved in liver oncogenesis. These integrations could induce copy number variations(CNV), chromosome changes, or changes in the expression of host genes. HBV has been reported more inclined to integrate into the host genome in tumors with hotspot targeted genes TERT,FAR2,MLL4. There is also report that HBV tends to integrate into adjacent non-tumor tissues genomes with recurrent integrated genes FN1 and SMAD5.. It is controversial about previous reports’ finding. We don’t yet know the reason of the difference. However,the number of HCC samples of previous studies are still limited, the methods to detect the suspectible loci remain unideal, and the genes related to HBV-induced HCC are far from complete. Therefore, there is a need to take an effort to confirm previous findings and, more importantly, to identify additional susceptibility loci for HBV integration.There was a significant male predominance in incidence of HCC.More recently,a number of studies were focus on the relationship between gender factor and HCC incidence and its clinical character. Huang et al reported the risk of HBV-associated HCC was higher in men than in women and displayed distinct clinical features. Hou et al reported HCC gender disparity also varied in molecular level and leaded to different molecular phenotype. HBV integration into human genome is known to be an early event of the initiation of hepatocarcinogenesis. However, there is no study reported the association of the HBV integration and gender factor.Eight genotypes(A-H) of hepatitis B virus(HBV) have been identified. Several research groups have reported that HBV genotypes have distinct geographic distributions and have been shown to differ with regard to clinical outcome, prognosis, and response to antiviral treatment. The most prevalent HBV genotypes in Asia are genotypes B and C. However, the characteristics of different genotype of HBV to integrate into human genome have never been characterized.The majority of paitients with HCC have concurrent liver cirrhosis, when other HCC patients don’t undergo the long-term cirrhosis stage. Globally, at least one-third of liver cirrhosis was attributable to chronic hepatitis B, and a significant proportion of chronic hepatitis B virus(HBV) infections eventually progress to HCC.A number of studies showed cirrhotic HCC and non-cirrhotic HCC displayed significant clinical and virological characteristics. Unfortunately, most previous studies did not particularly categorise the HCC patients into cirrhotics and non-cirrhotics. Furthermore, HBV integration difference of cirrhotic HCC and non-cirrhotic HCC still remain unknown.Technological limitation of PCR and Southern blot also largely restricts previous studies to completely characterize the HBV integration breakpoints. HBV genome has a large number of mutations,it would lead to PCR failure。Even using WGS, the detection sensitivity of HBV intergrants is still far from ideal.We have developed a novel experimental and computational method, namely HIVID(high-throughput Viral Integration Detection),and it is proven more sensitive to identify the HBV integration sites than whole genome sequencing method(WGS)(8)To address the questions above and understand the effect of HBV integration involved in the mechanism of HCC development, we performed HIVID to survey tumor and paired adjacent non-tumor tissues genomes extracted from 450 clinically and pathologically well-characterized HCC samples from individuals who underwent primary hepatectomy in our hospital, and then randomly select 29 paired samples performing RNA-seq.Our research focus on HBV integrations and its function in HCC progression, provide scientific evidence for transforming the HBV integration-related achievements to clinical application.Methods:We collected tumor and adjacent non-tumor samples from 426 patients with HCC who had distinct clinical phenotypes. We then conducted high-throughput viral integration detection(HIVID), a novel and highly sensitive method for assaying viral insertion, to survey HBV integration in HCC genomes.We validated recurrent integrations sites using PCR and RNA-seq assays and assessed the association with genomic instability and clinical characteristics.Results:A total of 6884 HBV integration events were identified with precise HBV-human DNA junctions. HBV is prone to integrate into fragile sites and functional genomic regions including Cp G islands. A distinct pattern in the preferential sites of HBV integration was observed between tumor and non-tumor tissues.HBV insertional sites were significantly enriched in the proximity of telomere in tumor, underlining HBV-induced genomic instability. Recurrent HBV target genes(tumor, 118; non-tumor, 81) were identified, greatly expanding the list of targeted genes. The overall HBV integration frequency is much higher in tumor genomes of males rather than in females, with a significant enrichment of integration into chromosome 2 and 17. Furthermore, a cirrhosis-dependent HBV integration pattern was observed, affecting distinct targeted genes.Conclusions:The preference of integration into specific genomic loci, and gene-dense regions, in addition to the characteristics of the inserted HBV fragments endow HBV integration a greater potential to drive oncogenic transformation.