基于高分辨率熔解分析的扩展RAS突变高通量筛选以预测结直肠癌抗表皮生长因子受体治疗疗效
High-throughput screening of extended RAS mutations based on high-resolution melting analysis for prediction of anti-EGFR treatment efficacy in colorectal carcinoma.
作者信息
Ishige Takayuki, Itoga Sakae, Sato Kenichi, Kitamura Kouichi, Nishimura Motoi, Sawai Setsu, Matsushita Kazuyuki, Suzuki Kazufumi, Ota Satoshi, Miyauchi Hideaki, Matsubara Hisahiro, Nakatani Yukio, Nomura Fumio
机构信息
Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba, Japan; Division of Laboratory Medicine and Clinical Genetics, Chiba University Hospital, Chiba, Japan.
Division of Laboratory Medicine and Clinical Genetics, Chiba University Hospital, Chiba, Japan.
出版信息
Clin Biochem. 2014 Dec;47(18):340-3. doi: 10.1016/j.clinbiochem.2014.09.013. Epub 2014 Sep 28.
OBJECTIVES
Recent studies have demonstrated that, in advanced colorectal carcinoma (CRC) patients, extended RAS (in KRAS exons 2-4 and NRAS exons 2-4) and BRAF mutations are negative predictors for anti-EGFR treatment efficacy and negative prognostic factor, respectively. Thus, high-throughput and cost-effective methods for identification of the mutation status are required.
DESIGN AND METHODS
We developed a PCR-high-resolution melting (HRM)-based method for screening extended RAS and BRAF mutations, and relative frequency of mutations in formalin-fixed paraffin-embedded samples of CRC was analyzed.
RESULTS
Among 93 CRC samples, 29 harbored mutations in KRAS exon 2, and 9 harbored mutations in BRAF exon 15. Analysis of 55 KRAS exon 2 and BRAF exon 15 wild-type CRC samples identified the following mutations: 1/55 in exon 3 and 2/55 in exon 4 of KRAS; 1/55 in exon 2, 3/55 in exon 3, and 0/55 in exon 4 of NRAS.
CONCLUSIONS
Our PCR-HRM method will enable rapid determination of the extended RAS and BRAF mutation status prior to anti-EGFR treatment in the clinical setting.
目的
近期研究表明，在晚期结直肠癌（CRC）患者中，扩展型RAS（KRAS外显子2-4和NRAS外显子2-4）和BRAF突变分别是抗表皮生长因子受体（EGFR）治疗疗效的阴性预测指标和不良预后因素。因此，需要高通量且经济高效的方法来鉴定突变状态。
设计与方法
我们开发了一种基于聚合酶链反应-高分辨率熔解曲线分析（PCR-HRM）的方法来筛查扩展型RAS和BRAF突变，并分析了CRC福尔马林固定石蜡包埋样本中的相对突变频率。
结果
在93份CRC样本中，29份在KRAS外显子2存在突变，9份在BRAF外显子15存在突变。对55份KRAS外显子2和BRAF外显子15野生型CRC样本的分析确定了以下突变：KRAS外显子3中有1/55、外显子4中有2/55；NRAS外显子2中有1/55、外显子3中有3/55、外显子4中有0/55。
结论
我们的PCR-HRM方法能够在临床环境中抗EGFR治疗前快速确定扩展型RAS和BRAF的突变状态。
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