Abstract
Simple SummaryThe quantitative assessment of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is essential to the development of next generation antiviral therapies against hepatitis B. Here, we developed a peptide nucleic acid (PNA)-clamping qPCR method to quantify cccDNA, which was comparable to the recently proposed exonuclease-based cccDNA assays. Using this method, we showed that cccDNA levels in the para-neoplastic liver tissues were independently correlated with overall survival, as well as extrahepatic metastasis in patients with or without virological suppression. These results suggest that in HBV-related hepatocellular carcinoma, patients under antiviral suppression might further benefit from new antivirals, which are designed to reduce cccDNA.New antiviral therapies against hepatitis B virus (HBV) focus on the elimination of covalently closed circular DNA (cccDNA). However, traditional cccDNA-specific quantitative PCR (qPCR) has a narrow effective range, hindering a reliable comparison between the levels of biopsy-derived cccDNAs. Collaterally, the prognostic role of cccDNA in HBV-related hepatocellular carcinoma (HCC) cannot be clearly defined. Here, we developed a peptide nucleic acid (PNA)-clamping qPCR method to provide a wider range of specific cccDNA quantification (up to 5 logs of effective range). Extrachromosomal DNA was extracted from para-neoplastic tissues for cccDNA quantification. In total, 350 HBV-related HCC patients were included for an outcome analysis. Without differential pre-dilution, cccDNA levels in para-neoplastic liver tissues were determined, ranging from < 2 × 103 to 123.0 × 106 copies/gram. The multivariate linear regression analysis showed that cccDNA was independently correlated with the HBV e antigen (p < 0.001) and serum HBV-DNA levels (p = 0.012). The Cox proportional hazard model analysis showed that cccDNA independently predicted overall survival (p = 0.003) and extrahepatic metastasis-free survival (p = 0.001). In virologically suppressed HCC patients, cccDNA still effectively predicted intrahepatic recurrence-free (p = 0.003) and extrahepatic metastasis-free (p = 0.009) survivals. In conclusion, cccDNA independently predicted postoperative extrahepatic metastasis-free survival.
Highlights
Worldwide, hepatitis B virus (HBV) chronically infects an estimate of 240 million patients, resulting in an increased lifetime risk of cirrhosis, liver failure, and hepatocellular carcinoma [1,2]
Clinical data were reviewed, including gender, age, anti-hepatitis C virus antibody, antiviral therapy used after operation, cirrhosis, alcoholism, ascites, hepatitis B e antigen (HBeAg), serum HBV-DNA levels, alpha-fetoprotein (AFP), albumin, bilirubin, prothrombin time, creatinine, aspartate transaminase (AST), alanine transaminases (ALT), Child-Pugh functional class, maximum tumor size, tumor number, microvascular invasion, macrovascular invasion, Edmondson-Steiner histology grade, tumor capsule, Barcelona Clinic Liver Cancer (BCLC) tumor stage, immunohistochemistry (IHC) staining patterns of HBV surface antigen (HBsAg), and IHC staining patterns of hepatitis B core antigen (HBcAg)
Upper panel, it was found that the effective range for the closed circular DNA (cccDNA) specific quantitative PCR (qPCR) in the absence of clamping primers was only 1 to 102 copies
Summary
Hepatitis B virus (HBV) chronically infects an estimate of 240 million patients, resulting in an increased lifetime risk of cirrhosis, liver failure, and hepatocellular carcinoma [1,2]. In Taiwan, individuals who were born before 1986, the carrier rate is around 15% [1]. For those born after 1986, due to the success of the universal vaccination program, the prevalence rate decreases progressively to about 1% [3]. For patients with chronic HBV infection (CHB), two major types of approved therapeutics are available, interferon (IFN)-based and nucleos(t)ide analogs (NAs)-based treatments. These treatments have significantly improved the outcomes of CHB [4,5,6,7]. The current therapeutic goal in clinical practice is set as an optimal and sustained suppression of viral replication, so that hepatic inflammation can be ameliorated and the progression of fibrosis can be curbed
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