Detecting Circulating Tumor DNA in Hepatocellular Carcinoma Patients Using Droplet Digital PCR Is Feasible and Reflects Intratumoral Heterogeneity

Purpose: Circulating tumor DNA (ctDNA) is increasingly recognized as liquid biopsy to profile tumor genome. Droplet digital PCR (ddPCR) is a highly sensitive and easily operable platform for mutant detection. Here, we tried to detect ctDNA in hepatocellular carcinoma (HCC) patients using ddPCR. Methods: Studies sequencing the genome of HCCs and COSMIC (Catalogue of Somatic Mutations in Cancer) database were reviewed to identify hotspot mutations. Circulating cell-free DNAs (cfDNAs) extracted from 1 ml preoperative plasma sample were analyzed to detect circulating mutants using ddPCR. The DNAs from matched tumor and adjacent liver tissues or peripheral blood mononuclear cells (PBMCs) were sequenced to identify the origin of circulating mutants. Results: Forty-eight HCC patients were enrolled and four gene loci, TP53 (c.747G>T), CTNNB1 (c.121A>G, c.133T>C), and TERT (c.1-124C>T) were chosen as targets for ddPCR assay. Serial dilution demonstrated the detection limit of ddPCR to be 0.01%. Twenty-seven patients (56.3%, 27/48) were found to have at least one kind of circulating mutants, with the mutant allele frequency ranging from 0.33% to 23.7%. Six patients (22.2%, 6/27) also had matched mutants in tumor tissues while none of the mutants were detected in adjacent liver tissues or PBMCs in all patients, which excluded the nonneoplastic origin of these circulating mutants and qualified them as ctDNA. Conclusions: ctDNA could be readily detected in HCC patients by targeting hotspot mutations using ddPCR and might reflect intratumoral heterogeneity. ctDNA detecting may serve as a promising liquid biopsy in HCC management.