Project Details
Abstract
Hepatitis B virus (HBV) chronically infects an estimate of 240 million patients worldwide. In Taiwan, individuals who were born before 1986 (> 31 years of age), the carrier rate remains around 15%. For those born after 1986, because of universal vaccination, the prevalence rate decreases progressively to < 1%. Current anti-HBV treatments are capable of continuously suppressing HBV replication to an undetectable level. Next generation anti-HBV drugs are targeted at complete eradication of HBV, that is, elimination of tissue cccDNA. Current methods to quantify cccDNA remain clumsy. The “traditional” qPCR method for cccDNA relies on differential PCR efficiencies, where the cccDNA “specific” PCR primers were across the genomic gap of HBV rcDNA. However, because of the capability of nucleotide sequence jumping during PCR, when the concentration of HBV rcDNA is high, rcDNA-related PCR product can still be produced. As such, it has been estimated that effective differentiation between cccDNA and rcDNA can only be achieved between 0 to 10(3) copies or less per reaction mixture. When total HBV DNA concentration is high, extensive dilutions have to be made so that the sample’s HBV DNA levels can fall into the effective range. However, after extensive dilution, low levels of cccDNA can no longer be detected. As a result, fair comparison of cccDNA levels among different samples with a wide range of intrahepatic HBV DNA levels becomes impossible. To achieve fair comparison, a qPCR method with wider effective range to quantify cccDNA is urgently needed, especially when developing next generation anti-HBV drugs. In this proposal, we devised a new method using peptide nucleic acid (PNA)-clamping based qPCR. The PNA clamping is used to inhibit amplification of rcDNA, while cccDNA can be amplified because it cannot be “clamped”. We examined the differential efficiency for amplification of rcDNA and cccDNA. Our preliminary data showed that an effective range of 0 to 10(5) can be achieved without sample pre-dilution.
The aims of this proposal are:
Year-1. Determination of sensitivity and specificity of the PNA-clamping qPCR method for detection of tissue cccDNA. Preliminary data showed that we could already widen the specific range to 0 – 10(5) copies. Further optimization will be attempted in this proposal.
Year-2. Quantification of intrahepatic cccDNA using paired HCC tissues (both cancerous and noncancerous parts) and correlation of the cccDNA levels with other clincopathological factors as well as postoperative outcomes. Our preliminary data showed that the cccDNA levels (noncancerous parts) correlated well with postoperative survival as well as distant metastasis.
Year-3. We have collected 4 hepatocellular carcinoma (HCC) tissues (paired) from patients who had been treated with entecavir and achieved complete virological suppression. Yet, HCC still developed. Our preliminary data showed that in one patient, cccDNA remained in the noncancerous tissues, and next generation sequencing showed differential distributions of oncogenic S truncation mutations in cccDNA and mRNA. We will complete next generation sequencing for all 4 pairs of HCC tissues.
Project IDs
Project ID:PC10901-0310
External Project ID:MOST107-2314-B182-004-MY3
External Project ID:MOST107-2314-B182-004-MY3
Status | Finished |
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Effective start/end date | 01/08/20 → 31/07/21 |
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