应用蝎形探针扩增阻滞突变系统检测非小细胞肺癌表皮生长因子受体基因突变与病理改变的关系

目的 探讨非小细胞肺癌(NSCLC)肿瘤组织中表皮生长因子受体(EGFR)基因突变的检出率,比较蝎形探针扩增阻滞突变系统(scorpions amplifieation refractory mutation system,以下简称Scorpions ARMS)即时荧光PCR和直接测序检测EGFR基因突变的敏感性.方法 应用显微切割术、Scorpions ARMS及直接测序检测82例NSCLC石蜡包埋肿瘤组织以及癌旁正常肺组织中EGFR基因的第18、19、20和2l外显子的突变.结果 Scorpions ARMS检测82例NSCLC中42例的瘤组织中存在EGFR突变,突变检出率为51.2%,其中20例为EGFR第19外显子框架内多核苷酸的缺失,18例为EGFR第21外显子L858R点突变,1例为EGFR第21外显子L861Q点突变,2例为EGFR第20外显子插入突变,另外1例为EGFR第20外显子$7681点突变.而PCR反应后行直接测序,82例标本中仅58例得到可分析的测序结果,其中25例瘤组织中出现EGFR突变,因此82例的突变检出率为30.5%,25例中13例为EGFR第19外显子框架内多核苷酸的缺失,10例为EGFR第21外显子L858R点突变,1例为EGFR第21外显子L861Q点突变,另外1例为EGFR第20外显子$768I点突变.直接测序检测出25例突变模式与Scorpions ARMS检测结果一致.结果 显示Scorpions ARMS具有很高的敏感度,部分直接测序阴性的病例经Scorpions ARMS检测出EGFR突变.结论 EGFR基因的点突变或缺失在中国NSCLC的患者中的检出率明显高于其他国家,以女性、腺癌和细支气管肺泡以及不吸烟患者突变率较高.Scorpions ARMS技术能快速、敏感、准确检测EGFR突变,能为临床冶疗及预后提供重要信息.

Detection of epidermal growth factor receptor gene mutations in non-small cell lung cancers by real-time polymerase chain reaction using scorpion amplification refractory mutation system

Objective To investigate mutations of EGFR gene in non-small cell lung cancers(NSCLC) using scorpions amplification refractory mutation system (Scorpions ARMS)is in comparing the detection sensitivity with that by PCR. direct sequencing method, and in addition to study the correlation between the mutations and the clinicopathological characteristics of the patients. Methods Tumot cells were collected by microdissection from paraffin embedded tumor specimens and adjacent normal lung tissues 0f 82 NSCLC patients. The genomic DNA was extracted. Mutations of EGFR gene(exons 18, 19, 20 and 21)were detected bv PCR-direct sequencing and Scorpions ARMS methads respectively. Results Somatic mutations were identified involving the tyrosine kinase domain of the EGFR gene in 42 of 82 cases with a mutation detection rate of 51. 2%by Scorpions ARMS assay. In-frame deletions of exon 19 occurred in 20 patients and point mutation occurred at codon 858, 861(exon 21) in 18 and 1 patients respectively. Two patients had insertions mutations and 1 patient had point mutation occurring at codon 768(exon 20). Among the 58 informative cases analyzed by PCR-direct sequencing, 25 mutations (detection rate of 30. 5%)were identified. In-frame deletions of exon 19 occurred in 13 patients and point mutation occurred at codon 858. 861(exon 21)in 10 and 1 patients respectively. In addition, 1 patient had point mutation at codon 768 (exon 20). Overall, Scorpions ARMS assay was more sensitive in detecting mutations of EGFR than PCRdirect sequencing. Conclusions A higher incidence of somatic mutations of EGFR gene was detected in NSCLC of Chinese patients. Mutations were more common in female, non-smoking patients with adenocarcinoma and bronchioloalveolar carcinoma histology. Scorpions ARMS method is quicker, more sensitive and accurate in detecting the EGFR gene mutations and should provide important therapeutic and prognostic information to the clinicians.