Detection EMl4-ALK Fusion Genes And Analysis Its Clinical Features In Nsclc Patients Possesing Clinical Characteristics Related To Egfr Mutations

AIM:To detect the frequency of EML4-ALK fusion gene in NSCLC patients who have the clinical characteristics related to EGFR mutation and analyze the relationship between EML4-ALK fusion gene and clinical features as well as provide a reference for the individual treatment of NSCLC.MATERIAL AND METHODS:1.102fresh tumor tissue specimens of NSCLC were screened from the specimen bank of Affiliated Tumor Hospital of Guangxi Medical University and all the patients have accepted the surgical treatment from January2006to August2011.102Chinese patients with NSCLC were enrolled on the basis of one or more of the following characteristics:female gender, never/light smoking history, and adenocarcinoma histology.2. we selected there pairs of primers for PCR amplification of EML4-ALK fusion gene by references.First, We extracted the total RNA of102fresh tumor tissue specimens by using the RNA extraction kit, and RNA was reverse transcribed into cDNA by reverse transcription kit, then cDNA was amplified by using a single-tube multiplex RT-PCR.Second, PCR products were identified by agarosegel electrophoresis in order to detect the EML4-ALK fusion gene mutation, then PCR products that emergenced the positive bands in the electrophoretic were directly sequenced. Ultimately, the measurement sequence was matched with known sequence of EML4-ALK and determine whether there is the EML4-ALK fusion gene mutation and which types they belong to.3. As to EML4-ALK-positive samples, we extracted the DNA by using a DNA extraction kit. Exon18to21EGFR mutations and exon1and2KRAS mutations were detected by DNA directly sequencing after PCR amplification.4.The comparison of the EML4-ALK-positive and-negative patients were made in age, gender, smoking history, pathologic type and clinical stage.5.Statistical analysis were performed by SPSS13.0statistical software. Comparison their clinical features between EML4-ALK positive and negative patients by Continuity Correction X2Test and Fisher’s exact test. All p values were based on a two-sided hypothesis,P<0.05was defined as statistical difference.Results1. The average age of102NSCLC patients was58.79±9.93years old and the range is30years old to80years old.45patients were younger than average age, accounting for53.9%,57patients were older than average age, accounting for56.1%.54patients were male, accounting for52.9%,48patients were female, accounting for47.1%. In the smoking history,73patients were non-smoker, accounting71.6%,29patients were smoker, accounting28.4%. In the pathological type,73patients were adenocarcinoma, accounting71.6%,14 patients were squamous cell carcinoma, accounting13.7%,14patients were adenosquamous carcinoma, accounting13.7%,1patient was lymphoepithelial-like carcinoma, accounting1%. In the clinical stage,34patients were stage I, accounting33.3%,17patients were stage II, accounting16.7%,40patients were stage III, accounting39.2%,11patients were stage IV, accounting10.8%.2. Eight cases(7.8%) among102tumor tissue samples had EML4-ALK fusion gene mutation.7cases were variant1(V1),1case was variant2(V2).3. Both EML4-ALK positive and negative patients have not statistical difference in age (p=0.47), gender (p=0.20), smoking history (p=0.53), pathologic type (p=0.64) and clinical stage (P=0.58)4. According to the results of constitute of the clinical features of8EML4-ALK positive patients, positive patients are more common in younger(62.5%), non-smoking(87.5%), female(75%), adenocarcinoma(62.5%).5.The8EML4-ALK positive patients were the wild type on the exon18-21of EGFR and exon land2of KRAS.Conclusions:1. EML4-ALK fusion gene mutation represents a new molecular subtype of NSCLC, the frequency of EML4-ALK fusion gene is slightly higher in NSCLC patients who have the clinical characteristics related to EGFR mutation than that the unselected NSCLC patients, and it is consistent with the previous reported in the literature.2. EML4-ALK fusion gene-positive patients are more common in the NSCLC patients who are younger, no smokers, women and adenocarcinoma histology. It will provide some reference for screening potential clinical advantages population of EML4-ALK mutations in the future. 3.The gene mutations of EML4-ALK, EGFR and KRAS are not co-exist simultaneously. It will provide a theoretical basis and important referencement for the standardization of NSCLC genetic screening and the individual treatment of NSCLC base on genotyping.