Detection of Covalently Closed Circular DNA and HBV DNA Integration in HCC Patients with Occult Hepatitis B

Abstract

Background: Occult hepatitis B infection (OBI) is a risk factor for hepatocellular carcinoma (HCC) in hepatitis B surface antigen (HBsAg)-negative patients . One mechanism of HBV-induced HCC is through the integration of HBV DNA into human chromosomes. While the current diagnosis of OBI is based on nested PCR reactions to detect HBV DNA, some postulated that it should be based on the detection of the viable form of HBV DNA, the covalently closed circular DNA (cccDNA). This study aimed to detect cccDNA and HBV DNA integration in HCC patients with OBI . Methods: Liver tissues from 110 HBsAg-negative patients (90 with HCC and 20 without HCC) were studied . OBI was detected using 4 independent nested PCR reactions targeting 4 HBV genomic regions (“four-region PCR”) and was defined by positive PCR detection at ≥2 regions (out of 4 regions). cccDNA was detected by “over-gap PCR”, with primers spanning across the nicked region of the HBV genome. HBV DNA integration were detected Alu-PCR . Results: Of the 90 HCC patients, 53 had cryptogenic cause of HCC, and the remaining 37 had identifiable etiologies of HCC including alcoholic liver disease (n = 18), non-alcoholic fatty liver disease/steatohepatitis (NAFLD/NASH) (n = 14), primary biliary cholangitis (PBC) (n = 2), recurrent pyogenic cholangitis (n = 2), and autoimmune hepatitis (AIH) (n = 1) . The 20 non-HCC patients had NAFLD/NASH (n = 7), PBC (n = 7), or AIH (n = 6). OBI was identified, by “four-region PCR”, in 62/90 (69%) HCC patients and 3 (15%) non-HCC patients (P < 0 .0001) . Among the 62 HCC patients with ≥2 positive reactions by “four-region PCR”, cccDNA was detectable in 29 (47%) of them . cccDNA was not detectable in the non-HCC patients . Using Alu-PCR, we detected HBV DNA integration in 43/62 (69%) HCC patients with OBI (i.e. those with ≥2 positive reactions by “four-region PCR”) but in none of the 3 non-HCC patients with OBI . More patients with HBV DNA integration were found in HCC patients with undetectable cccDNA (29/33; 88%) than in patients with detectable cccDNA (14/29; 48%; P = 0 .001) . Some frequently observed HCC-associated HBV integration sites such as TERT and KMT2B were identified. Conclusion: HBsAg-negative HCC patients had higher percentages of OBI and HBV DNA integration than non-HCC patients. More than half of the OBI patients had undetectable cccDNA, but a higher proportion of patients with HBV DNA integration was found in those with undetectable cccDNA. The detectability of HBV DNA integration near HCC-related genes, even when cccDNA was undetectable, suggests that OBI induces HCC via HBV DNA integration in HBsAg-negative patients. [https://doi.org/10.1002/hep.30940 ]