改良的内切酶突变体富集法检测肺癌标本中EGFR基因突变

Modified Restriction Enzyme-based Detection of EGFR Mutations in Lung Cancer

背景与目的表皮生长因子受体(epidermal growth factor receptor,EGFR)基因突变是肺癌靶向药物疗效的可靠预测指标,因此基因突变的检测具有非常重要的临床意义。本研究建立使用常规实验仪器、高灵敏度、简便的检测表皮生长因子受体突变的方法,以利于临床中快速的检测EGFR基因突变。方法采用改良的内切酶法富集法检测251例肺腺癌DNA标本中EGFR基因外显子19缺失突变和21(L858R)点突变,并与直接测序进行比较。利用混合突变/野生型EGFR基因的细胞系测定改良方法的灵敏度。结果在251例腺癌标本DNA中使用测序法检测出EGFR外显子19突变46例、外显子21突变26例。采用改良的突变体富集法检另外测出外显子19突变78例、外显子21突变57例,总突变率53.8%。灵敏度检测显示对于外显子19和21,新方法的检测灵敏度达0.5%。结论本方法具有简便、经济、灵敏度高等特点,便于临床快速筛查非小细胞肺癌病理组织中的EGFR基因突变。

Background and objective Epidermal growth factor receptor（EGFR） gene mutation in lung cancer is a reliable predictor of the efficacy of tyrosine kinase inhibitors,so detection of gene mutation has very important clinical significance.The aim of this study is to optimize conditions for restriction enzyme-based mutant enriched detection of EGFR mutation in lung cancer specimens.Methods EGFR somatic mutations identified in DNA specimens from 251 lung adenocarcinomas with modified mutant enriched method and direct sequencing were compared.The sensitivity of the modified method was determined by serial dilution of mutant-carrying lung cancer cells in wild-type cells.Results 46 deletion mutations in exon 19 and 26 point mutations in exon 21 were found by sequencing,while modified method identified another 78 deletions in exon 19 and 57 substitutions in exon 21 with a mutation rate of 53.8%.Sensitivity of this modified method was 0.5%（mutant/wild type） in serial diluted cells.Conclusion The modified restriction enzyme-based method is convenient for clinical EGFR mutation screening in non-small cell lung cancer for its simpleness,cost-saving and high sensitivity.