Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing.
Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing.
复制标题
DOI：
10.1371/journal.pone.0121891
复制
发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Nishio K
中科院分区：
文献类型：
--
作者：
Sakai K;Tsurutani J;Yamanaka T;Yoneshige A;Ito A;Togashi Y;De Velasco MA;Terashima M;Fujita Y;Tomida S;Tamura T;Nakagawa K;Nishio K
KRAS、NRAS 和 BRAF 基因的体细胞突变与结直肠癌中抗 EGFR 抗体的耐药性有关。我们已经使用下一代测序仪建立了扩展的 RAS 和 BRAF 突变测定法来分析这些突变。进行多重深度测序以检测 KRAS、NRAS 和 BRAF 内的体细胞突变，包括轻微突变成分。我们首先使用 10 个正常 DNA 和 20 个福尔马林固定石蜡包埋 (FFPE) 肿瘤样本验证了多重深度测序的技术性能。为了证明我们的检测的潜在临床实用性，我们分析了从结直肠癌患者获得的 100 个 FFPE 肿瘤样本和 15 个血浆样本。我们使用基于泊松分布的变体调用方法。假设突变阳性群体的分布遵循泊松分布，突变阳性状态被定义为大于错误率显着性水平的值 (α = 2 x 10-5)。截止值被确定为平均错误率加上 7 个标准差。对100例临床FFPE肿瘤标本进行突变分析，无无效病例。检测到突变的频率为 59% (59/100)。该检测与 Scorpion-ARMS 之间的 KRAS 突变一致性为 92% (92/100)。还分析了从 15 个血浆样本中获得的 DNA。在 6 名患者的血浆和组织样本中均发现了 KRAS 和 BRAF 突变。使用下一代测序仪进行的基因筛查测定经过验证，可用于使用 FFPE 和液体样本检测临床相关的 RAS 和 BRAF 突变。
Somatic mutations in KRAS, NRAS, and BRAF genes are related to resistance to anti-EGFR antibodies in colorectal cancer. We have established an extended RAS and BRAF mutation assay using a next-generation sequencer to analyze these mutations. Multiplexed deep sequencing was performed to detect somatic mutations within KRAS, NRAS, and BRAF, including minor mutated components. We first validated the technical performance of the multiplexed deep sequencing using 10 normal DNA and 20 formalin-fixed, paraffin-embedded (FFPE) tumor samples. To demonstrate the potential clinical utility of our assay, we profiled 100 FFPE tumor samples and 15 plasma samples obtained from colorectal cancer patients. We used a variant calling approach based on a Poisson distribution. The distribution of the mutation-positive population was hypothesized to follow a Poisson distribution, and a mutation-positive status was defined as a value greater than the significance level of the error rate (α = 2 x 10-5). The cut-off value was determined to be the average error rate plus 7 standard deviations. Mutation analysis of 100 clinical FFPE tumor specimens was performed without any invalid cases. Mutations were detected at a frequency of 59% (59/100). KRAS mutation concordance between this assay and Scorpion-ARMS was 92% (92/100). DNA obtained from 15 plasma samples was also analyzed. KRAS and BRAF mutations were identified in both the plasma and tissue samples of 6 patients. The genetic screening assay using next-generation sequencer was validated for the detection of clinically relevant RAS and BRAF mutations using FFPE and liquid samples.
登录
查看更多内容
评估血清 DNA 中表皮生长因子受体突变状态作为吉非替尼 (IRESSA) 反应的预测因子。
影响因子：
8.8
作者：
Kimura, H.;Suminoe, M.;Kasahara, K.;Sone, T.;Araya, T.;Tamori, S.;Koizumi, F.;Nishio, K.;Miyamoto, K.;Fujimura, M.;Nakao, S.
通讯作者：
Nakao, S.
影响因子：
8.4
作者：
Bokemeyer, Carsten;Van Cutsem, Eric;Koehne, Claus-Henning
通讯作者：
Koehne, Claus-Henning
影响因子：
50.5
作者：
Bokemeyer, C.;Bondarenko, I.;Koralewski, P.
通讯作者：
Koralewski, P.
影响因子：
45.3
作者：
Giantonio, Bruce J.;Catalano, Paul J.;Benson, Al B., III
通讯作者：
Benson, Al B., III
影响因子：
158.5
作者：
Jonker, Derek J.;O'Callaghan, Chris J.;Moore, Malcolm J.
通讯作者：
Moore, Malcolm J.