J Cancer 2016; 7(13):1907-1914. doi:10.7150/jca.15823

Research Paper

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

Ao Huang1*, Xin Zhang1*, Shao-Lai Zhou1, Ya Cao2, Xiao-Wu Huang1, Jia Fan1,3, Xin-Rong Yang1, Jian Zhou1,4✉

1. Liver Surgery Department, Liver Cancer Institute, Zhongshan Hospital, Fudan University
Key Laboratory of Carcinogenesis and Cancer Invasion (Fudan University), Ministry of Education, No.180, Fenglin Rd, Shanghai, 200032, China.
2. Cancer Research Institute, Central South University
Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, No.110, Xiangya Rd, Changsha, 410078, China.
3. Institute of Biomedical Sciences, Fudan University, Shanghai, No.130, Dong'an Rd, 200032, China.
4. State Key Laboratory of Genetic Engineering Fudan University, No.220, Handan Rd, Shanghai, 200433, China.
* co-first author

Abstract

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.

Keywords: circulating tumor DNA, hepatocellular carcinoma, liquid biopsy, droplet digital PCR, intratumoral heterogeneity.

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How to cite this article:
Huang A, Zhang X, Zhou SL, Cao Y, Huang XW, Fan J, Yang XR, Zhou J. Detecting Circulating Tumor DNA in Hepatocellular Carcinoma Patients Using Droplet Digital PCR Is Feasible and Reflects Intratumoral Heterogeneity. J Cancer 2016; 7(13):1907-1914. doi:10.7150/jca.15823. Available from http://www.jcancer.org/v07p1907.htm