Nishizuka Satoshi S, Sato Kei A, Hachiya Tsuyoshi
Division of Biomedical Research and Development, Institute of Biomedical Sciences, Iwate Medical University, Morioka, Iwate, Japan.
Department of Surgery, Iwate Medical University School of Medicine, Morioka, Iwate, Japan.
Methods Mol Biol. 2019;1908:229-241. doi: 10.1007/978-1-4939-9004-7_16.
Circulating tumor DNA (ctDNA) is emerging as a promising biomarker for cancer diagnosis. However, the system to detect gene mutations with very low frequencies from plasma remains to be established in terms of technical aspects of sequencing technologies and cost for universal use. One strategy is to employ a cancer sequencing panel to detect mutations in a primary tumor in a time- and cost-effective manner, and subsequently assess these mutations with a digital PCR technology from plasma ctDNA. This strategy enables the accurate detection of low frequency mutations (i.e., less than 1% allele frequency) from ctDNA, since both comprehensive coverage of genes and quantitative mutation detection with very low frequencies are required for cancer diagnosis from plasma samples. Here, we present a pipeline can be used to detect mutations from plasma ctDNA with very low allele frequencies using a next-generation sequencing technology for comprehensive coverage of primary tumors and droplet digital PCR for sensitive detection from plasma ctDNA.
循环肿瘤DNA（ctDNA）正成为一种很有前景的癌症诊断生物标志物。然而，从技术层面的测序技术以及普遍使用的成本来看，用于检测血浆中极低频率基因突变的系统仍有待建立。一种策略是采用癌症测序panel，以具有时间和成本效益的方式检测原发性肿瘤中的突变，随后利用数字PCR技术从血浆ctDNA中评估这些突变。这种策略能够准确检测ctDNA中的低频突变（即等位基因频率小于1%），因为从血浆样本进行癌症诊断需要对基因进行全面覆盖以及对极低频率的突变进行定量检测。在此，我们展示了一种流程，该流程可用于使用下一代测序技术全面覆盖原发性肿瘤，并利用液滴数字PCR从血浆ctDNA中进行灵敏检测，从而检测极低等位基因频率的血浆ctDNA中的突变。
