High Rate of HBV DNA Integration in Hepatocellular Carcinoma Patients with Occult Hepatitis B Infection

Abstract

Background: Hepatitis B surface antigen (HBsAg)-negative patients can have occult hepatitis B infection (OBI), which may cause hepatocellular carcinoma (HCC) (Wong et al. Hepatology 54:829-36). Integration of hepatitis B virus (HBV) DNA into host DNA can also lead to HCC. We aimed to examine the significance of OBI and HBV integration in HBsAg-negative HCC patients. Methods: Of the 90 HBsAg-negative HCC patients recruited, 84 had paired tumor and non-tumor tissues available, 3 had only tumor tissues and 3 had only non-tumor tissues available. OBI was detected in the liver DNA by nested PCR, with primers targeting the 4 HBV genomic regions. OBI was diagnosed when ≥2 positive PCR were detected in either tumor or non-tumor tissues. HBV integration was detected by Alu-PCR sequencing. Results: Of the 90 patients, 18 (21%) had alcoholic liver diseases (ALD), 14 (16%) had nonalcoholic fatty liver disease (NAFLD), 2 had primary biliary cholangitis (PBC), 2 had recurrent pyogenic cholangitis (RPC), 1 had autoimmune hepatitis (AIH), and the remaining 53 patients (60%) had unknown causes of (cryptogenic) HCC. OBI was found in 62/90 (69%) HCC patients, including 9/18 (50%) ALD, 9/14 (64%) NAFLD, 2 (100%) PBC, 2 (100%) RPC, and 40/53 (75%) patients with cryptogenic HCC. Unlike other forms of HCC, the majority (53/62; 85%) of OBI patients did not have liver cirrhosis histologically. Tumor-extracted DNA from 41 OBI patients (6 ALD, 6 NAFLD, 2 PBC, 2 RPC and 25 cryptogenic) were examined by Alu-PCR at the time of writing. Among these 41 OBI patients, 16 yielded amplifiable DNA, 12 (75%) of whom were found to have integrated HBV DNA at chromosomes 3 and 5 (3 cases each), chromosome 11 (2 cases), chromosomes 4, 10, 19 (1 case each), and at repetitive elements at various unidentifiable genomic sites (1 case). Integration was found at the preS/S (9 patients; 75%) and core region (3 patients; 25%). Only 1 patient with HBV integration had cirrhosis. One patient had HBV integration near the telomerase reverse transcriptase (TERT) promoter region, and 1 with integration at the intronic region of cyclin A2. Conclusion: 69% of HBsAg-negative HCC patients had OBI. 85% of HCC patients with OBI had no background of cirrhosis, and the oncogenic process might be explained by the high percentage (75%) of HBV integration detected. This suggests that HBV integration, especially those near apoptosis- or cell cycle-related genes such as TERT or cyclin A2, can be a likely cause of HCC in OBI patients.