Liver cancer (LC) is presumed to be the sixth most common and newly diagnosed cancer and the fifth leading causing of cancer death around the world in 2018. Hepatocellular carcinoma (HCC) is the most frequent liver cancer and the chronic infection with hepatitis B/C virus is the major risk factor of HCC. Unlike tissue biopsy, liquid biopsy is based on collection of a sample in non-invasive and convenient way at multiple time points over the course of disease. For circulating cell free DNA, it can be used for Next Generation Sequencing (NGS) to detect genetic mutations.
Whole Blood of patients are collected and plasma was separated. The circulating tumor DNA (ctDNA) is extracted from the plasma samples and quantified using Qubit High Sensitive assay and Agilent D1000 assay. The extracted ctDNA is used for molecular barcoded library construction and captured with probe for next generation sequencing. All samples were then analyzed to identify genetic mutations.
Currently, 20 HCC patients were collected and samples were extracted, sequenced and analyzed. Two thirds of the patients were hepatitis B-viral infected and the one third of the patients were Non-B, Non-C HCC. Most patients were staged 3 others were staged 1 or 2 in HCC. The total ctDNA in 2ml plasma of each patietns were ranged from 2.0 ng to 200ng. As total amount of ctDNA were varied, input ctDNA to construct library was differed ranging from 2ng to 50ng. After molecular tagged libraries were constructed, were pooled and captured with probe for next generation sequencing on Nextseq 500/550 300cycles. Data analysis was performed: TP53 mutations were most frequent and the frequency of mutations are ranged from 3% to 40%.
To fully utilize advantages of the liquid biopsy and circulating tumor DNA, detection of low frequency mutations in the cancer is essential. Detection of low frequency mutation in circulating tumor DNA of hepatocellular carcinoma via optimization of circulating tumor DNA Isolation in plasma samples, optimization of library preparation and target capture condition by using molecular barcoding and customized somatic mutation probe, may improve prognosis, diagnosis, prediction and monitoring of therapeutic response and detection of residual tumor burden.
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