Detection of hepatitis B virus-host junction sequences in urine of infected patients

Background & Aims: Integrated hepatitis B virus (HBV) DNA, found in >85% of HBV-associated hepatocellular carcinomas (HBV-HCC), can play a significant role in HBV-related liver disease progression. HBV-host junction sequences (HBV-JS9s), created through integration events, have been used to determine HBV-HCC clonality. Here, we investigate the feasibility of analyzing HBV integration in a noninvasive urine liquid biopsy. Approach & Results: Utilizing an HBV-targeted NGS assay, we first identified HBV-JS9s in 8 HBV-HCC tissues and designed short-amplicon junction-specific PCR assays to detect HBV-JSs in matched urine. We detected and validated tissue-derived junctions in 5 of 8 matched urine samples. Next, we screened 32 urine samples collected from 25 HBV-infected patients (5 with hepatitis, 10 with cirrhosis, 4 with HCC, and 6 post-HCC). Encouragingly, all 32 urine samples contained HBV-JS9s detectable by HBV-targeted NGS. Of the 712 total HBV-JS9s detected in urine, 351 were in gene-coding regions, 11 of which, including TERT, had previously been reported as recurrent integration sites in HCC tissue and were found in urine of cirrhosis or HCC patients only. The integration breakpoints of HBV DNA detected in urine were found predominantly (~70%) at a previously identified integration hotspot, HBV DR1-2. Conclusions: HBV viral-host junction DNA can be detected in urine of HBV-infected patients. This study is the first study to demonstrate the potential for a noninvasive urine liquid biopsy of integrated HBV DNA to monitor HBV-infected patients for HBV-associated liver diseases and the efficacy of antiviral therapy.