Max Planck Institute for Neurological Research with Klaus-Joachim-Zülch Laboratories of the Max Planck Society and the Medical Faculty of the University of Cologne, Cologne, Germany.
PLoS One. 2011 May 5;6(5):e19601. doi: 10.1371/journal.pone.0019601.
Treatment of EGFR-mutant non-small cell lung cancer patients with the tyrosine kinase inhibitors erlotinib or gefitinib results in high response rates and prolonged progression-free survival. Despite the development of sensitive mutation detection approaches, a thorough validation of these in a clinical setting has so far been lacking. We performed, in a clinical setting, a systematic validation of dideoxy 'Sanger' sequencing and pyrosequencing against massively parallel sequencing as one of the most sensitive mutation detection technologies available. Mutational annotation of clinical lung tumor samples revealed that of all patients with a confirmed response to EGFR inhibition, only massively parallel sequencing detected all relevant mutations. By contrast, dideoxy sequencing missed four responders and pyrosequencing missed two responders, indicating a dramatic lack of sensitivity of dideoxy sequencing, which is widely applied for this purpose. Furthermore, precise quantification of mutant alleles revealed a low correlation (r(2) = 0.27) of histopathological estimates of tumor content and frequency of mutant alleles, thereby questioning the use of histopathology for stratification of specimens for individual analytical procedures. Our results suggest that enhanced analytical sensitivity is critically required to correctly identify patients responding to EGFR inhibition. More broadly, our results emphasize the need for thorough evaluation of all mutation detection approaches against massively parallel sequencing as a prerequisite for any clinical implementation.
表皮生长因子受体（EGFR）-突变型非小细胞肺癌患者接受酪氨酸激酶抑制剂厄洛替尼或吉非替尼治疗可获得高缓解率和延长无进展生存期。尽管已经开发出了敏感突变检测方法，但在临床环境中对这些方法进行全面验证仍缺乏研究。我们在临床环境中，对双脱氧“桑格”测序和焦磷酸测序与作为最敏感的突变检测技术之一的大规模平行测序进行了系统验证。对临床肺肿瘤样本的突变注释表明，在所有对 EGFR 抑制有明确反应的患者中，只有大规模平行测序检测到了所有相关的突变。相比之下，双脱氧测序错过了 4 名应答者，焦磷酸测序错过了 2 名应答者，这表明双脱氧测序的敏感性明显不足，而该方法广泛应用于该目的。此外，对突变等位基因的精确定量揭示了组织病理学估计的肿瘤含量和突变等位基因频率之间的低相关性（r²=0.27），从而对使用组织病理学对用于个体化分析程序的标本进行分层提出了质疑。我们的结果表明，为了正确识别对 EGFR 抑制有反应的患者，需要增强分析敏感性。更广泛地说，我们的结果强调了需要对所有突变检测方法进行大规模平行测序的全面评估，这是任何临床实施的前提。
