San Raffaele Scientific Institute, Genomic Unit for the Diagnosis of Human Pathologies, Center for Translational Genomics and Bioinformatics, Milan, Italy.
PLoS One. 2013;8(3):e59939. doi: 10.1371/journal.pone.0059939. Epub 2013 Mar 25.
Molecular diagnostics of human cancers may increase accuracy in prognosis, facilitate the selection of the optimal therapeutic regimen, improve patient outcome, reduce costs of treatment and favour development of personalized approaches to patient care. Moreover sensitivity and specificity are fundamental characteristics of any diagnostic method. We developed a highly sensitive microarray for the detection of common KRAS and BRAF oncogenic mutations. In colorectal cancer, KRAS and BRAF mutations have been shown to identify a cluster of patients that does not respond to anti-EGFR therapies; the identification of these mutations is therefore clinically extremely important. To verify the technical characteristics of the microarray system for the correct identification of the KRAS mutational status at the two hotspot codons 12 and 13 and of the BRAF(V600E) mutation in colorectal tumor, we selected 75 samples previously characterized by conventional and CO-amplification at Lower Denaturation temperature-PCR (COLD-PCR) followed by High Resolution Melting analysis and direct sequencing. Among these samples, 60 were collected during surgery and immediately steeped in RNAlater while the 15 remainders were formalin-fixed and paraffin-embedded (FFPE) tissues. The detection limit of the proposed method was different for the 7 KRAS mutations tested and for the V600E BRAF mutation. In particular, the microarray system has been able to detect a minimum of about 0.01% of mutated alleles in a background of wild-type DNA. A blind validation displayed complete concordance of results. The excellent agreement of the results showed that the new microarray substrate is highly specific in assigning the correct genotype without any enrichment strategy.
人类癌症的分子诊断可以提高预后的准确性，有助于选择最佳的治疗方案，改善患者的治疗效果，降低治疗成本，并促进针对患者的个性化治疗方法的发展。此外，敏感性和特异性是任何诊断方法的基本特征。我们开发了一种高度敏感的微阵列，用于检测常见的 KRAS 和 BRAF 致癌突变。在结直肠癌中，KRAS 和 BRAF 突变已被证明可以识别一组对抗 EGFR 治疗无反应的患者；因此，这些突变的鉴定在临床上非常重要。为了验证微阵列系统用于正确识别 KRAS 突变状态的技术特征，我们选择了 75 个样本，这些样本之前已经通过传统方法和 CO-扩增在低变性温度-PCR（COLD-PCR）下进行了特征分析，随后进行了高分辨率熔解分析和直接测序。在这些样本中，60 个是在手术中采集的，并立即浸入 RNAlater 中，而其余 15 个是福尔马林固定和石蜡包埋（FFPE）组织。所提出的方法的检测限因所测试的 7 种 KRAS 突变和 V600E BRAF 突变而有所不同。特别是，微阵列系统能够在野生型 DNA 背景下检测到约 0.01%的突变等位基因。盲法验证显示结果完全一致。结果的高度一致性表明，新的微阵列基质在没有任何富集策略的情况下，高度特异性地分配正确的基因型。
