Abstract 876: Comprehensive analysis of the complexity of HBV DNA integration sites in the circulation of patients with HBV-related liver disease
Authors: Lin Selena , Jain Surbhi , Boldbaatar Batbold , Block Timothy , Song Wei , Su Ying-Hsiu 
Published: May 2015

HBV related liver disease ranges from hepatitis, cirrhosis, and hepatocellular carcinoma (HCC), which has a 5-year survival rate of 14% because it is difficult to diagnose at its curative stages. Chronic hepatitis B virus (HBV) infections is the major etiology of HCC, associated with over 50% of HCC cases worldwide and up to 70-80% in endemic areas. 85-90% of HBV related HCCs (HBV-HCC) contain integrated HBV DNA. During chronic HBV infection, HBV DNA has been shown to integrate at random sites in the host chromosome creating a unique genetic signature in each HBV infected hepatocytes. This makes the pool of HBV DNA integration sites highly complex in the liver. This genetic signature in HBV-infected liver along with the fundamental mechanism of carcinogenesis, which is clonal expansion, would result in HCC tissue containing only one to few clonally expanded integration sites, resulting in reduced complexity. Thus, we hypothesize the detection of reduced complexity of HBV DNA integration sites in the circulation can be a potential biomarker for HBV-HCC.

In order to study the complexity of cell free circulating HBV DNA integration sites in the circulation, a targeted next generation sequencing (NGS) assay for urine DNA was developed. We chose to use urine in order to avoid detecting of HBV DNA from infectious viruses. Targeted enrichment was performed using an in-solution hybridization platform with biotinylated RNA baits covering the entire HBV genome. The assay was tested using library constructed DNA controls: Hep3B, which contains integrated DNA, and HepG2, which contains no HBV DNA to confirm targeted capture of HBV DNA. Next, matching tissue and urine DNA and DNA from urine collected after surgical removal of HCC tumor from seven HBV-HCC patients were collected and underwent library construction. The library DNA samples were subsequently enriched with HBV and HCC-specific RNA baits. Enriched library DNA was sequenced on Illumina's MiSeq platform. Although the analysis is still ongoing, we have successfully analyzed the complexity of cell-free circulating HBV DNA integration sites in urine. We detected 46 integration sites in the tissue and urine where the location of integrated HBV DNA mapped primarily (89%) to a known HBV integration breakpoint region, HBV DR1-2. Furthermore in 3 HBV-HCC patients, a pattern of reduced complexity of cell-free circulating HBV DNA integration sites was found in urine before the removal of HCC, as compared to urine collected after surgery suggesting reduced complexity of HBV DNA integration sites in HBV-HCC patients. A similar NGS analysis of the urine of hepatitis and cirrhosis patients is in progress. Overall, if successful this study will demonstrate the potential of detecting reduced complexity of HBV DNA integration sites as a biomarker for HBV-HCC screening and provide insight to the involvement of HBV DNA integration sites in HBV-HCC carcinogenesis.

Citation Format: Selena Lin, Surbhi Jain, Batbold Boldbaatar, Timothy Block, Wei Song, Ying-Hsiu Su. Comprehensive analysis of the complexity of HBV DNA integration sites in the circulation of patients with HBV-related liver disease. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 876. doi:10.1158/1538-7445.AM2014-876