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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 &lt; 2 × 10 3 to 123.0 × 10 6 copies/gram. The multivariate linear regression analysis showed that cccDNA was independently correlated with the HBV e antigen ( p &lt; 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.

Hepatitis B Virus Covalently Closed Circular DNA Predicts Postoperative Liver Cancer Metastasis Independent of Virological Suppression