Feasibility and Robustness of Amplification Refractory Mutation System (ARMS)-based KRAS Testing Using Clinically Available Formalin-fixed, Paraffin-embedded Samples of Colorectal Cancers
Feasibility and Robustness of Amplification Refractory Mutation System (ARMS)-based KRAS Testing Using Clinically Available Formalin-fixed, Paraffin-embedded Samples of Colorectal Cancers
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DOI：
10.1093/jjco/hyq151
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发表时间：
2011-01-01
影响因子：
2.4
通讯作者：
Esumi, Hiroyasu
中科院分区：
文献类型：
--
作者：
Ogasawara, Naomi;Bando, Hideaki;Esumi, Hiroyasu
KRAS突变检测被推荐用于鉴别不太可能从抗表皮生长因子受体抗体中获益的转移性结直肠癌患者。最近开发的一种扩增难解突变- scorpion系统正在成为KRAS突变检测的标准方法。该系统的可行性和稳健性使用临床可获得的福尔马林固定、石蜡包埋标本的DNA样本进行了评估。使用商用扩增难解突变- scorpion系统试剂盒，应用宏观解剖的110个标本的基因组DNA进行KRAS突变检测。评估试验的成功率和突变体检出率。检测试剂盒的批内和批间偏差较小，不同实时聚合酶链反应系统之间的一致性较好，表明扩增难解突变- scorpion系统的可靠性。虽然110个样本中有三分之一的样本DNA含量不足以检测1%浓度的突变等位基因，但通过宏观解剖浓缩的肿瘤DNA并没有降低突变的检出率。使用较高数量的模板DNA，可能含有丰富的干扰物质，阻止了外源控制扩增子的检测，导致成功率降低。根据DNA的总浓度调整模板的数量可以降低失败率。采用福尔马林固定、石蜡包埋的标本来源DNA样品的扩增难解突变- scorpion系统显示出可接受的可行性和稳健性，适合常规临床实践。
KRAS mutation testing is recommended for the discernment of metastatic colorectal cancer patients who are unlikely to benefit from anti-epidermal growth factor receptor antibodies. A recently developed amplification refractory mutation-Scorpion system is becoming a standard method for KRAS mutant detection. The feasibility and robustness of this system using DNA samples from clinically available formalin-fixed, paraffin-embedded specimens were evaluated.Genomic DNA from macro-dissected 110 specimens was applied for the KRAS mutant detection using a commercial amplification refractory mutation-Scorpion system kit. Success rate and mutant detection rate of the test were evaluated.Small intra- and inter-lot deviations of the testing kit and a good concordance among different real-time polymerase chain reaction systems suggested the reliability of the amplification refractory mutation-Scorpion system. Though one-third of the 110 samples that were tested did not contain a sufficient amount of DNA to detect a 1% concentration of mutant alleles, the mutant detection rate was not impaired using tumor DNA concentrated by macro-dissection. Using a higher amount of template DNA, which supposedly contained abundant interfering substances, prevented the detection of the exogenous control amplicons, resulting in a reduced success rate. Adjusting the template amount according to the total DNA concentration might reduce the failure rate.The amplification refractory mutation-Scorpion system with formalin-fixed, paraffin-embedded specimen-derived DNA samples exhibited an acceptable feasibility and robustness suitable for routine clinical practice.