Sakai Kazuko, Tsurutani Junji, Yamanaka Takeharu, Yoneshige Azusa, Ito Akihiko, Togashi Yosuke, De Velasco Marco A, Terashima Masato, Fujita Yoshihiko, Tomida Shuta, Tamura Takao, Nakagawa Kazuhiko, Nishio Kazuto
Department of Genome Biology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, Japan.
Department of Medical Oncology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka, Japan.
PLoS One. 2015 May 8;10(5):e0121891. doi: 10.1371/journal.pone.0121891. eCollection 2015.
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.
KRAS、NRAS和BRAF基因的体细胞突变与结直肠癌中抗表皮生长因子受体(EGFR)抗体的耐药性相关。我们使用新一代测序仪建立了一种扩展的RAS和BRAF突变检测方法来分析这些突变。进行多重深度测序以检测KRAS、NRAS和BRAF内的体细胞突变,包括少量突变成分。我们首先使用10份正常DNA和20份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。为了证明我们检测方法的潜在临床实用性,我们分析了从结直肠癌患者获得的100份FFPE肿瘤样本和15份血浆样本。我们使用了基于泊松分布的变异检测方法。假设突变阳性群体的分布遵循泊松分布,并且将突变阳性状态定义为大于错误率显著性水平的值(α = 2×10⁻⁵)。截止值确定为平均错误率加7个标准差。对100份临床FFPE肿瘤标本进行突变分析,无任何无效病例。突变检测频率为59%(59/100)。该检测方法与蝎形扩增阻滞突变系统(Scorpion-ARMS)之间的KRAS突变一致性为92%(92/100)。还对从15份血浆样本中获得的DNA进行了分析。在6例患者的血浆和组织样本中均鉴定出KRAS和BRAF突变。使用新一代测序仪的基因筛查检测方法在使用FFPE和液体样本检测临床相关RAS和BRAF突变方面得到了验证。