Individualized Mutation Detection in Circulating Tumor DNA for Monitoring Colorectal Tumor Burden Using a Cancer-Associated Gene Sequencing Panel.
Individualized Mutation Detection in Circulating Tumor DNA for Monitoring Colorectal Tumor Burden Using a Cancer-Associated Gene Sequencing Panel.
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DOI：
10.1371/journal.pone.0146275
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Nishizuka SS
中科院分区：
文献类型：
--
作者：
Sato KA;Hachiya T;Iwaya T;Kume K;Matsuo T;Kawasaki K;Abiko Y;Akasaka R;Matsumoto T;Otsuka K;Nishizuka SS
循环肿瘤 DNA (ctDNA) 携带肿瘤负荷信息。然而，肿瘤之间的突变谱是不同的。本研究旨在检验 ctDNA 在基于个体突变谱监测肿瘤负荷方面的效用。从总共 176 个样本中提取 DNA，包括术前和术后血浆、原发性肿瘤和外周血单核细胞 (PBMC)，这些样本来自 44 名接受结直肠肿瘤根治性切除的结直肠肿瘤患者以及 9 名健康个体。使用一组 50 个癌症相关基因，通过使用 Ion PGM 测序仪比较来自肿瘤和 PBMC 的单核苷酸变异 (SNV)，鉴定了肿瘤独特的突变。来自单个肿瘤的一组肿瘤独特突变被指定为个体标记突变 (MM)，以使用液滴数字 PCR (ddPCR) 通过 ctDNA 追踪肿瘤负荷。从这些实验中，评估了三个主要目标：（a）肿瘤独特的突变； (b)肿瘤的突变谱； (c) 肿瘤根治性切除后 ctDNA 中 MM 等位基因频率的变化。在 27 个结直肠肿瘤中总共鉴定出 128 个基因点突变。至少 1 个样本中有 26 个基因发生突变，而仅 1 个样本中有 14 个基因发生突变。每个肿瘤平均有 2.7 个基因发生突变。随后，从SNV中选择24个MM进行肿瘤负荷监测。在通过 ddPCR 发现的血浆 DNA 变异等位基因频率 > 0.1% 的 MM 中，100%（8 个中的 8 个）表现出术后 ctDNA 下降，而通过 ddPCR 发现血浆 DNA 变异等位基因频率 < 0.1% 的 16 个 MM 中没有一个显示出术后 ctDNA 下降。这组 50 个癌症相关基因似乎足以识别癌症患者中个体、肿瘤独特的突变 ctDNA 标记。如果血浆 DNA 中 MM 的等位基因频率高于 0.1%，则 MM 在监测治疗性结直肠肿瘤负荷方面显示出临床实用性。
Circulating tumor DNA (ctDNA) carries information on tumor burden. However, the mutation spectrum is different among tumors. This study was designed to examine the utility of ctDNA for monitoring tumor burden based on an individual mutation profile. DNA was extracted from a total of 176 samples, including pre- and post-operational plasma, primary tumors, and peripheral blood mononuclear cells (PBMC), from 44 individuals with colorectal tumor who underwent curative resection of colorectal tumors, as well as nine healthy individuals. Using a panel of 50 cancer-associated genes, tumor-unique mutations were identified by comparing the single nucleotide variants (SNVs) from tumors and PBMCs with an Ion PGM sequencer. A group of the tumor-unique mutations from individual tumors were designated as individual marker mutations (MMs) to trace tumor burden by ctDNA using droplet digital PCR (ddPCR). From these experiments, three major objectives were assessed: (a) Tumor-unique mutations; (b) mutation spectrum of a tumor; and (c) changes in allele frequency of the MMs in ctDNA after curative resection of the tumor. A total of 128 gene point mutations were identified in 27 colorectal tumors. Twenty-six genes were mutated in at least 1 sample, while 14 genes were found to be mutated in only 1 sample, respectively. An average of 2.7 genes were mutated per tumor. Subsequently, 24 MMs were selected from SNVs for tumor burden monitoring. Among the MMs found by ddPCR with > 0.1% variant allele frequency in plasma DNA, 100% (8 out of 8) exhibited a decrease in post-operation ctDNA, whereas none of the 16 MMs found by ddPCR with < 0.1% variant allele frequency in plasma DNA showed a decrease. This panel of 50 cancer-associated genes appeared to be sufficient to identify individual, tumor-unique, mutated ctDNA markers in cancer patients. The MMs showed the clinical utility in monitoring curatively-treated colorectal tumor burden if the allele frequency of MMs in plasma DNA is above 0.1%.