Sato Kei A, Hachiya Tsuyoshi, Iwaya Takeshi, Kume Kohei, Matsuo Teppei, Kawasaki Keisuke, Abiko Yukito, Akasaka Risaburo, Matsumoto Takayuki, Otsuka Koki, Nishizuka Satoshi S
Molecular Therapeutics Laboratory, Department of Surgery, Iwate Medical University School of Medicine, Morioka, Japan.
Department of Surgery, Iwate medical University School of Medicine, Morioka, Japan.
PLoS One. 2016 Jan 4;11(1):e0146275. doi: 10.1371/journal.pone.0146275. eCollection 2016.
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%.
循环肿瘤DNA（ctDNA）携带着肿瘤负荷的信息。然而，不同肿瘤的突变谱有所不同。本研究旨在基于个体突变谱检测ctDNA在监测肿瘤负荷方面的效用。
从44例行结直肠癌根治性切除术的结直肠癌患者以及9名健康个体中总共提取了176份样本的DNA，包括术前和术后血浆、原发性肿瘤以及外周血单个核细胞（PBMC）。使用包含50个癌症相关基因的基因panel，通过Ion PGM测序仪比较肿瘤和PBMC中的单核苷酸变异（SNV）来鉴定肿瘤特异性突变。将来自个体肿瘤的一组肿瘤特异性突变指定为个体标记突变（MM），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）肿瘤特异性突变；（b）肿瘤的突变谱；（c）肿瘤根治性切除后ctDNA中MM的等位基因频率变化。
在27例结直肠癌中总共鉴定出128个基因点突变。26个基因在至少1个样本中发生突变，而分别有14个基因仅在1个样本中发生突变。每个肿瘤平均有2.7个基因发生突变。随后，从SNV中选择了24个MM用于肿瘤负荷监测。在血浆DNA中通过ddPCR发现的变异等位基因频率>0.1%的MM中，100%（8/8）在术后ctDNA中出现下降，而在血浆DNA中通过ddPCR发现的变异等位基因频率<0.1%的16个MM中，没有一个显示下降。
这组50个癌症相关基因似乎足以在癌症患者中鉴定个体、肿瘤特异性的突变ctDNA标记。如果血浆DNA中MM的等位基因频率高于0.1%，MM在监测根治性治疗的结直肠癌肿瘤负荷方面显示出临床效用。
