Detecting Circulating Tumor DNA in Hepatocellular Carcinoma Patients Using Droplet Digital PCR Is Feasible and Reflects Intratumoral Heterogeneity.
Detecting Circulating Tumor DNA in Hepatocellular Carcinoma Patients Using Droplet Digital PCR Is Feasible and Reflects Intratumoral Heterogeneity.
复制标题
使用微滴式数字 PCR 检测肝细胞癌患者的循环肿瘤 DNA 是可行的，并反映了瘤内异质性
DOI：
10.7150/jca.15823
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发表时间：
2016
影响因子：
3.9
通讯作者：
Zhou J
中科院分区：
文献类型：
--
作者：
Huang A;Zhang X;Zhou SL;Cao Y;Huang XW;Fan J;Yang XR;Zhou J
目的：循环肿瘤DNA（ctDNA）作为一种液体活组织检查，越来越被人们所认识。液滴数字PCR（ddPCR）是一种高灵敏度、易操作的突变检测平台。在这里，我们尝试使用ddPCR检测肝细胞癌（HCC）患者的ctDNA。研究方法：回顾了肝癌基因组测序和COSMIC（癌症体细胞突变目录）数据库的研究，以确定热点突变。使用ddPCR分析从Iml术前血浆样品提取的循环无细胞DNA（cfDNA）以检测循环突变体。从匹配的肿瘤和邻近肝组织或外周血单个核细胞（PBMC）的DNA测序，以确定循环突变体的起源。结果如下：选取48例HCC患者，选取TP 53（c.747G>T）、CTNNB 1（c.121A>G、c.133T>C）和TERT（c.1- 124 C>T）4个基因位点作为ddPCR检测的靶基因。连续稀释证明ddPCR的检测限为0.01%。27例（56.3%，27/48）患者至少存在一种循环突变，突变等位基因频率为0.33%~ 23.7%。6例患者（22.2%，6/27）在肿瘤组织中也有匹配的突变体，而在所有患者的邻近肝组织或PBMC中均未检测到突变体，这排除了这些循环突变体的非肿瘤来源，并将其鉴定为ctDNA。结论：ctDNA可以很容易地检测到肝癌患者的靶向热点突变，使用ddPCR，并可能反映肿瘤内的异质性。ctDNA检测有可能成为HCC治疗中一种有前途的液体活检方法。
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.