QUANTITATION OF INTRAHEPATIC cccDNA USING DROPLET DIGITAL PCR IN PATIENTS WITH HBV-RELATED HCC AND OBI-RELATED HCC

Abstract

Chronic infection with Hepatitis B virus (HBV) is a common health issue worldwide and cannot be completely cured. Patients are required to continuously take medication due to the high mutational rate of HBV, with the persistence of closed circular DNA (cccDNA) in the nucleus of hepatocyte. The cccDNA was difficult to eliminate and undetectable in the serum, poses a significant challenge. The standard approach to cccDNA examination requires the liver tissues and highly sensitive methods. Therefore, droplet digital PCR (dd-PCR) was performed to quantify the intrahepatic cccDNA level in liver tissues from 27 patients with HBV-related hepatocellular carcinoma (HBV-related HCC) and 8 patients with occult HBV-related HCC (OBI-related HCC). To access the performance of intrahepatic cccDNA detection from different DNA extraction methods, digested with Plasmid-safe ATP-dependent of DNase (PSAD) assay and without digestion by PSAD techniques were compared. The results showed that there was no statistically significant difference in intrahepatic cccDNA detection between the PSAD digestion after DNA extraction and without PSAD digestion (without PSAD; 6.53 copies/ug vs. PSAD; 15.50 copies/ug, P = 0.249). Then, we selected the PSAD digestion method in detecting cccDNA in liver tissues from patients. The result demonstrated that the difference of intrahepatic cccDNA level between patients with OBI-related HCC and HBV-related HCC was not statistically significant (8.5 copies/ug vs. 5.2 copies/ug, P=0.232). Digestion of DNA with PSAD before measuring cccDNA expression may be a suitable method for cccDNA quantitation. However, this study still requires more sample size to validate by this technique in further study.