Cohen Victor, Agulnik Jason S, Jarry Jonathan, Batist Gerald, Small David, Kreisman Harvey, Tejada Neely Adriana, Miller Wilson H, Chong George
Montreal Center for Experimental Therapeutics in Cancer, Lady Davis Institute for Medical Research, Montreal, Quebec, Canada.
Cancer. 2006 Dec 15;107(12):2858-65. doi: 10.1002/cncr.22331.
Somatic mutations of the epidermal growth factor receptor (EGFR) gene in nonsmall-cell lung cancer (NSCLC) may predict responsiveness to tyrosine kinase inhibitors. These mutations are commonly identified using DNA sequencing methods. Although considered the gold standard, this approach is time-consuming. In addition, this approach requires large diagnostic specimens and a high ratio of tumor-to-normal-tissue DNA for optimal results. The use of denaturing high-performance liquid chromatography (dHPLC) as a method to screen for the 2 predominant EGFR mutations is reported.
Clinical specimens from 104 NSCLC patients were analyzed for EGFR mutations in exons 19 and 21. After DNA extraction and polymerase chain reaction (PCR), both direct sequencing and dHPLC were performed and the results were compared.
Sequencing revealed a total of 7 mutations: 3 deletion mutations in exon 19 and 4 missense mutations in exon 21. dHPLC showed the presence of genomic alterations in 23 samples, including the 7 identified by sequencing plus 16 additional samples (10 in exon 19 and 1 in exon 21). dHPLC fractions were isolated, reamplified, and sequenced to confirm the results. In serial dilution studies, dHPLC was able to detect mutations in samples containing as little as 1.6% to 6.25% mutated DNA, whereas direct sequencing required at least 30%.
dHPLC is an efficient and more sensitive method for screening for genomic alterations in exons 19 and 21 of the EGFR gene compared with direct sequence analysis. These data suggest that dHPLC should be implemented as a screening tool for detection of EGFR mutations.
非小细胞肺癌（NSCLC）中表皮生长因子受体（EGFR）基因的体细胞突变可能预测对酪氨酸激酶抑制剂的反应性。这些突变通常使用DNA测序方法来识别。尽管被认为是金标准，但这种方法耗时。此外，这种方法需要大量的诊断标本以及高比例的肿瘤与正常组织DNA才能获得最佳结果。本文报道了使用变性高效液相色谱（dHPLC）作为筛选两种主要EGFR突变的方法。
对104例NSCLC患者的临床标本进行EGFR基因第19和21外显子突变分析。DNA提取和聚合酶链反应（PCR）后，同时进行直接测序和dHPLC，并比较结果。
测序共发现7个突变：第19外显子有3个缺失突变，第21外显子有4个错义突变。dHPLC显示23个样本存在基因组改变，包括测序鉴定出的7个样本以及另外16个样本（第19外显子10个，第21外显子1个）。分离dHPLC组分，重新扩增并测序以确认结果。在系列稀释研究中，dHPLC能够检测到含有低至1.6%至6.25%突变DNA的样本中的突变，而直接测序至少需要30%。
与直接序列分析相比，dHPLC是一种用于筛选EGFR基因第19和21外显子基因组改变的高效且更灵敏的方法。这些数据表明，dHPLC应作为检测EGFR突变的筛选工具来应用。
