Lee Kyung Ha, Lee Tae Hee, Choi Min Kyung, Kwon In Sun, Bae Go Eun, Yeo Min-Kyung
Department of Surgery, Chungnam National University Hospital, Daejeon 282, Korea.
The Biobank of Chungnam National University Hospital, Daejeon 282, Korea.
J Clin Med. 2020 Jul 18;9(7):2283. doi: 10.3390/jcm9072283.
KRAS (Kirsten rat sarcoma 2 viral oncogene homolog) is a major predictive marker for anti-epidermal growth factor receptor treatment, and determination of KRAS mutational status is crucial for successful management of colorectal adenocarcinoma. More standardized and accurate methods for testing KRAS mutation, which is vital for therapeutic decision-making, are required. Digital droplet polymerase chain reaction (ddPCR) is an advanced digital PCR technology developed to provide absolute quantitation of target DNA. In this study, we validated the clinical performance of ddPCR in determination of KRAS mutational status, and compared ddPCR results with those obtained by Sanger sequencing and peptide nucleic acid-clamping. Of 81 colorectal adenocarcinoma tissue samples, three repeated sets of KRAS mutation were measured by ddPCR, yielding high consistency (ICC = 0.956). Receiver operating characteristic (ROC) curves were constructed to determine KRAS mutational status based on mutant allele frequency generated by ddPCR. Using the best threshold cutoff (mutant allele frequency of 7.9%), ddPCR had superior diagnostic sensitivity (100%) and specificity (100%) relative to the two other techniques. Thus, ddPCR is effective for detecting the KRAS mutation in colorectal adenocarcinoma tissue samples. By allowing definition of the optimal cutoff, ddPCR represents a potentially useful diagnostic tool that could improve diagnostic sensitivity and specificity.
KRAS（ Kirsten大鼠肉瘤2病毒癌基因同源物）是抗表皮生长因子受体治疗的主要预测标志物，KRAS突变状态的测定对于成功管理结肠腺癌至关重要。需要更标准化、准确的KRAS突变检测方法，这对治疗决策至关重要。数字液滴聚合酶链反应（ddPCR）是一种先进的数字PCR技术，旨在对目标DNA进行绝对定量。在本研究中，我们验证了ddPCR在测定KRAS突变状态方面的临床性能，并将ddPCR结果与通过桑格测序和肽核酸钳夹获得的结果进行了比较。在81份结肠腺癌组织样本中，通过ddPCR对三组重复的KRAS突变进行了检测，结果具有高度一致性（组内相关系数=0.956）。构建了受试者工作特征（ROC）曲线，以根据ddPCR产生的突变等位基因频率确定KRAS突变状态。使用最佳临界值（突变等位基因频率为7.9%），相对于其他两种技术，ddPCR具有更高的诊断敏感性（100%）和特异性（100%）。因此，ddPCR对于检测结肠腺癌组织样本中的KRAS突变是有效的。通过确定最佳临界值，ddPCR是一种潜在有用的诊断工具，可以提高诊断敏感性和特异性。
