| Objective The abnormities of four driver genes such as EGFR、KRAS、EML4-ALK and c-Met were detected by multiple methods in NSCLC,thus to analyze the association of driver genes with the clinicopathologic features,sample types and detection methods and the correlation among the four genes,in order to direct the gene detection and targeted therapy in clinical practice.Methods A total of 1144 NSCLC samples including permanent paraffin sections,pleural effusion,seroperitoneum and pericardial effusion were collected for the following experiments.Mutation status of EGFR gene were detected by direct sequencing,ARMS and HRM assays.Mutation status of KRAS gene were detected by direct sequencing.The EML4-ALK fusion gene status were detected by qRT-PCR.The c-Met gene amplification status were detected by FISH.The expressions of c-Met gene on the mRNA levels were detected by qRT-PCR.The associations of driver genes with the clinicopathologic features,sample types and detection methods and the correlation among the driver genes were analyzed.Results 1.The overall mutation rate of EGFR gene of 1144 NSCLC samples was 45.10%.The frequency of the activating(TKI-sensitive)mutations was 40.47% and was more common in non-smoking,female,adenocarcinoma patients.The mutation rate of adenocarcinoma patients was 47.94%.In squamous cell carcinoma,the activating EGFR mutations in female were significantly higher than male(36.84% vs.16.67%,P=0.043).The EGFR mutation rate of surgical resections was significantly higher than bronchoscopic biopsies(47.92% vs.41.46%,P=0.041).The EGFR mutation rates of surgical resections were similar detected by direct sequencing,ARMS and HRM assays.The EGFR mutation rate of bronchoscopic biopsies detected by direct sequencing was significantly higher than by ARMS assay(P=0.037).The EGFR mutation rate of pleural effusion detected by ARMS assay was significantly higher than by direct sequencing(P=0.027).The same cases were detected by different methods.The accordance was 73.68% between direct sequencing and ARMS and was 78.67% between direct sequencing and HRM.2.The KRAS mutation rate of 159 cases from 1144 samples was 6.92%,and 2 cases were analyzed with both KRAS and EGFR mutations.The rate of dual driver genes was 1.26%.3.The positive rate of EML4-ALK fusion gene of 221 cases from 1144 samples was 19.91%,and 14 cases were analyzed with both EML4-ALK fusion and EGFR mutation.The rate of dual driver genes was 6.33%.The EML4-ALK fusion gene was more common in EGFR-widetype patients than in EGFR-mutant patients(21.13% vs.17.72%),especially in female(P=0.035).4.Among 108 cases from NSCLC samples,c-Met gene amplification was observed in two cases(1.85%),which were analyzed with no abnormity in EGFR,KRAS and EML4-ALK genes.The two cases harboring c-Met gene amplification were detected with high mRNA levels of c-Met gene,while others had a low expression.Conclusions 1.The activating EGFR mutation was more common in non-smoking,female,adenocarcinoma patients.The KRAS mutation was more common in young,smoking,male,adenocarcinoma patients.The EML4-ALK fusion gene was more common in young,non-smoking,female,adenocarcinoma patients.2.The non-smoking,female,squamous cell carcinoma patients or biopsy specimens were also used to detecte the EGFR mutation.3.The abnormalities of driver genes can be successfully detected in variety of surgical and biopsy specimens.The EGFR mutation rate of surgical resections was significantly higher than bronchoscopic biopsies.4.There were significantly differences in the EGFR mutation rates among different samples detected by direct sequencing,ARMS and HRM assays.5.The rate of both KRAS and EGFR mutations was 1.26%.The rate of both EML4-ALK fusion and EGFR mutation was 6.33%.6.The consistency rate of the gene amplification of c-Met detected by FISH and c-Met mRNA expression levels analysed by qRT-PCR was 66.67%.7.The amplification of c-Met was not coexisted with the mutations of EGFR,KRAS,and the fusion of EML4-ALK. |
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