Ishida Yasuaki, Takano Shinichi, Maekawa Shinya, Yamaguchi Tatsuya, Yoshida Takashi, Kobayashi Shoji, Iwamoto Fumihiko, Kuno Toru, Hayakawa Hiroshi, Matsuda Shuya, Fukasawa Mitsuharu, Shindo Hiroko, Inoue Taisuke, Nakayama Yasuhiro, Ichikawa Daisuke, Sato Tadashi, Enomoto Nobuyuki
First Department of Internal Medicine, Faculty of Medicine University of Yamanashi Yamanashi Japan.
First Department of Surgery, Faculty of Medicine University of Yamanashi Yamanashi Japan.
JGH Open. 2020 Jun 27;4(5):978-986. doi: 10.1002/jgh3.12379. eCollection 2020 Oct.
Liquid biopsy is a method that can efficiently detect tumor genetic abnormalities from body fluids such as blood and urine. Detection sensitivity and the available number of mutations in cell-free DNA (cfDNA) are limited. In this study, we develop a highly sensitive and comprehensive method to detect mutations from cfDNA by concentrating tumor fractions of small cfDNA in advanced colorectal cancers.
Biopsied specimens and 37 serum samples were collected from 27 patients with advanced colorectal carcinoma. A serum-extracted cfDNA was divided into enriched fractionated small cfDNA and unfractionated cfDNA. Both cfDNAs were subjected to digital polymerase chain reaction (PCR) to evaluate their , and status. Consequently, their mutant allele frequencies (MAFs) were compared and analyzed by next-generation sequencing (NGS) in conjunction with tissue-derived DNA.
NGS analyses revealed mutations in (63%), (63%), (30%), and (22%). Digital PCR could detect mutations in 25 of 27 samples (93%) of unfractionated cfDNA, a rate that increased to 100% when samples were enriched with fractionated small cfDNA (6.8 10.7%, < 0.001). NGS also showed increased MAFs in fractionated small cfDNA compared to unfractionated cfDNA (16.3 18.8%, = 0.012) and a tendency to detect a greater number of cancer-related genes in fractionated cfDNA.
Fractionated small cfDNA increased MAFs of gene mutations and increases the possibilities to detect cancer-related genes even in advanced cancer patients from whom it is difficult to obtain tissue samples.
液体活检是一种可从血液和尿液等体液中有效检测肿瘤基因异常的方法。游离DNA（cfDNA）的检测灵敏度和可用突变数量有限。在本研究中，我们开发了一种高灵敏度且全面的方法，通过富集晚期结直肠癌中小cfDNA的肿瘤组分来检测cfDNA中的突变。
从27例晚期结直肠癌患者中收集活检标本和37份血清样本。将血清提取的cfDNA分为富集的小片段cfDNA和未分级的cfDNA。对这两种cfDNA都进行数字聚合酶链反应（PCR）以评估其 状态。随后，结合组织来源的DNA，通过下一代测序（NGS）对它们的突变等位基因频率（MAF）进行比较和分析。
NGS分析揭示了 （63%）、 （63%）、 （30%）和 （22%）中的突变。数字PCR可检测27份未分级cfDNA样本中的25份（93%）的突变，当样本用小片段cfDNA富集时，该比率增至100%（6.8 10.7%， < 0.001）。NGS还显示，与未分级的cfDNA相比，小片段cfDNA中的MAF增加（16.3 18.8%， = 0.012），并且在分级cfDNA中检测到更多癌症相关基因的趋势。
小片段cfDNA增加了基因突变的MAF，即使在难以获取组织样本的晚期癌症患者中，也增加了检测癌症相关基因的可能性。
