运用TaqMan-MGB探针通过实时荧光定量PCR检测非小细胞肺癌中表皮生长因子受体突变

[Detection of epidermal growth factor receptor mutations in non-small cell lung cancer by real-time PCR using TaqMan-MGB probes].

作者信息

Zhou Cai-Cun, Zhou Song-Wen, Pan Hong, Su Bo, Gao Zhi-Qiang

机构信息

Department of Oncology , Shanghai Pulmonary Hospital, Tongii University, Shanghai 200433, China.

出版信息

Zhonghua Zhong Liu Za Zhi. 2007 Feb;29(2):119-23.

PMID:17645848

Abstract

OBJECTIVE

To investigate mutations of EGFR TK gene in non-small cell lung cancer (NSCLC) and the diagnostic value of the mutations assayed by real-time PCR using TaqMan-MGB probes.

METHODS

Tyrosine kinase genes of EGFR (exons 18, 19 and 21) were amplified by PCR technology, and sequenced and analyzed by Chromas software in 80 NSCLC patients. Based on the results of sequencing, TaqMan-MGB probes were designed and used to detect the mutations of EGFR by real-time PCR. The results of detected mutations were compared between real-time PCR and direct sequencing. The sensitivity and specificity of real-time PCR using TaqMan-MGB probes were analyzed by adding different number of PC-9 cells (exon 19 deleted EGFR) into A549 cells (Wild-type EGFR).

RESULTS

Somatic mutations were identified in the tyrosine kinase domain of the EGFR gene in 21 of 80 NSCLC patients with an incidence rate of 26.3%. In-frame deletions of exon 19 occurred in 13 patients and point mutation occurred in codon 858 (exon 21) in 8 patients. Real-time PCR with the TaqMan MGB probes could detect all the mutations of EGFR found by sequencing. The sensitivity and specificity of the detection of EGFR mutations were both 100%. Real-time PCR with TagMan MGB probes could detect EGFR mutation in as rare as 50 EGFR mutant cells and in a proportion of 10% of mutant cells in a cell population. The mutations were significantly higher in the adenocarcinoma than in non-adenocarcinoma (16/38 vs. 5/42, chi2 = 9.702, P <0.01), in the female patients than in the male patients (14/29 vs. 7/51, chi2 = 11.4, P <0.01) and in non-smokers than in smokers (16/40 vs. 5/40, chi2 = 7.812, P < 0.01). The mutations were not related to patients'age, TNM staging, etc.

CONCLUSION

Somatic mutations of EGFR gene develop in NSCLC and are more common in female, non-smoker and adenocarcinoma patients. Real-time PCR using TaqMan-MGB, which can be used to detect the EGFR gene mutations, is easy to operate and deserves widespread application.

摘要

目的

探讨非小细胞肺癌（NSCLC）中表皮生长因子受体（EGFR）酪氨酸激酶（TK）基因的突变情况，以及应用TaqMan-MGB探针实时荧光定量聚合酶链反应（PCR）检测该突变的诊断价值。

方法

采用PCR技术扩增80例NSCLC患者的EGFR酪氨酸激酶基因（第18、19和21外显子），产物经Chromas软件测序及分析。根据测序结果设计TaqMan-MGB探针，应用实时荧光定量PCR检测EGFR基因的突变情况，并将实时荧光定量PCR与直接测序结果进行比较。通过向A549细胞（野生型EGFR）中加入不同数量的PC-9细胞（第19外显子缺失的EGFR），分析TaqMan-MGB探针实时荧光定量PCR检测EGFR突变的灵敏度和特异性。

结果

80例NSCLC患者中，21例患者的EGFR基因酪氨酸激酶结构域存在体细胞突变，发生率为26.3%。其中，13例患者发生第19外显子框内缺失，8例患者第21外显子858密码子发生点突变。TaqMan-MGB探针实时荧光定量PCR能够检测出测序发现的所有EGFR基因突变，其检测EGFR基因突变的灵敏度和特异性均为100%。TaqMan-MGB探针实时荧光定量PCR能够检测出低至50个EGFR突变细胞，以及细胞群体中比例为10%的突变细胞。腺癌患者的EGFR基因突变率显著高于非腺癌患者（16/38比5/42，χ2 = 9.702，P <0.01），女性患者高于男性患者（14/29比7/51，χ2 = 11.4，P <0.01），非吸烟者高于吸烟者（16/40比5/40，χ2 = 7.812，P <0.01）。EGFR基因突变与患者年龄、TNM分期等无关。