Detection Of EGFR Mutations In NSCLC Tumor Specimens From Various Ways By DHPLC And Comparison Of EGFR And K-ras Genotypes Between Primary Tumors And Corresponding Lymph Node Metastases In NSCLC

Lung cancer is a major cause of cancer related mortality worldwide. The clinical manifestations and Early Detection of lung cancer are lack, only 16% lung cancer patients are diagosed in early stage, most of these cases are non-small-cell lung cancer (NSCLC). The main therapeutic methods include surgery, radiotherapy, chemotherapy and targeted therapy, among which operation is the only radical method, but the therapeutic effect is disappointed.Epidermal growth factor receptor(EGFR) tyrosine kinase domain has become the fastest developing Therapeutic target now.Gefitinib and erlotinib are the most common tyrosine kinase inhibitors, and have been widely used in the treatment of patients with advanced NSCLC. The efficacy of TKIs is associated with EGFR mutation. K-ras is one of the most important members of RAS family. K-ras mutation may leading to the primary resistance of EGFR TKIs and poor prognosis of NSCLC patients. It is very necessary to comform the EGFR gene type before the treatment of patients suffering from advanced NSCLC. Currently, direct sequencing is the most common method of detection EGFR mutation, and is also as the "gold standard". But direct sequencing is complex, time-and cost-intensive, less sensitive. As a result, direct sequencing isn't able to fulfill the requirement. The Denaturing High-Performance Liquid Chromatography(DHPLC) is a new technology of high-throughput screening of gene variations.wild type and mutant DNA were detected and separated in partial denaturing condition by ion-pair-reverse-phase liquid chromatography.DHPLC is a rapid automated sensitive and specific method in mutant gene detection. TKIs had be successful in the treatment of advanced NSCLC.The neoadjuvant TKIs in operable NSCLC patients would be a new treatment model. But the heterogeneity of malignant tumors is significant. Many studies showed discrepancy in EGFR mutation between primary tumors and corresponding metastasis. We evaluated that DHPLC as a rapid detection method for EGFR mutation in NSCLC tumor specimens and compared the EGFR and K-ras genotypes between primary tumors and corresponding metastatic lymph node in NSCLC.This research consists two partsPart oneDetection of EGFR mutations in NSCLC tumor specimens from various ways by DHPLC and comparison of EGFRObjectiveEvaluation DHPLC as a rapid detection method for EGFR mutations of NSCLC tumor specimens from CT guided transthoracic needle biopsy of lung, lymph node biopsy and surgical resection. Correlation analysis of EGFR mutations and clinical characteristic of NSCLC patients.Method1.83 NSCLC patients hospitalized in guangdong general hospital from January 2008 to June 2009 were included. There were 37 tumor specimens from CT guided transthoracic needle biopsy of lung,15 from Lymph Node Biopsy, and 31 from surgical resection. The tumor specimens were divided into two, a section fixed by 10% neutral formalin and paraffin embedded was used for pathological diagnosis. The other section was put into liquid nitrogen and then transferred to -80℃low temperature refrigerator. All the patients were naive chemotherapy or targeting therapy, the specimens included in the study have be pathologically confirmed.2. DNA extraction:The genomic DNA was extracted from lung cancer tissues.The DNA purity and content were detected by the Eppendorf nucleic acid protein cryoscope.3. TA cloning method was used to constructed EGFR gene exon 19, exon 21 wild-type and mutant (del746-750, L858R) plasma. The 1:1,1:5,1:10,1:20,1:50,1:100 dilutions of mutant and wild type EGFR plasma DNA were using in evaluation the sensitivity of DHPLC and direct sequencing.4. The EGFR exon 19,21 were amplified by PCR. PCR products were used for direct sequencing and DHPLC detection after being evaluated by 2% agarose gel electrophoresis.5. The PCR products were purified.Direct sequencing of the PCR products was performed using ABI PRISM 3100 DNA Analyzer. Chromas software was used to analyze the sequencing results.6. PCR products were detected by DHPLC without any purification. We compared the sensitivity of non-denatured condition and the partial denatured condition in the detection of the EGFR exon 19. Exon 21 was evaluated by partial denatured condition. The exon 19,21 were detected by partial denatured condition in tumor specimens.7. Statistics analysis:All data were analyzed with SPSS 13.0 software. Chi-square test was used in examination of the relationship between EGFR mutations and gemder, smoking status, pathological type. P< 0.05 was considered statistically significant for all analyses.Result1. evaluation of DHPLC sensitivity:Mutant plasma DNA can be detected in the serial dilution of 1:100 by DHPLC and 1:10 by direct sequencing. The sensitivity of detecting EGFR exon 19 is similar in the condition of non-denaturing and partial denaturing.2. DHPLC showed 22 EGFR mutations in 83 NSCLC patients,19 samples had been conformed by direct sequencing except 3 samples, the other 61 samples are wild type by DHPLC and direct sequencing. Compared with direct sequencing, the sensitivity of DHPLC was 100%, and the specificity was 95.31%.The sensitivity of detection tumor specimens from CT-guided transthoracic needle lung biopsy, lymph node Biopsy and surgical resection were 100%,and the specificity of detection tumor specimens from CT-guided transthoracic needle lung biopsy, lymph node Biopsy and surgical resection were 96.55%,100.00% and 92.00% respectively.3. Compared with DHPLC the sensitivity of direct sequencing was 86.36%.4. EGFR mutations were present in 37.93% female and 14.81% male(x2=5.712, P=0.017);in 32.14% adenocarcinoma and 3.70% non-adenocarcinoma(x2=8.347, P=0.004), There was no statistical difference. EGFR mutations were present in 40.74%.Feminine, non-smoking, the adenocarcinoma NSCLC patients. But the EGFR genotype was not associated with smoking status (P>0.05).ConclusionDHPLC is a sensitive method for screening EGFR mutations, partial denatured condition is applicable to detecting EGFR exon 19, and can detect unknown mutation. DHPLC is in accordance with the "gold standard" and is a precise rapid preliminary screening method for detection of NSCLC EGFR genotype. The tumor specimens were from CT-guided transthoracic needle lung biopsy, lymph node Biopsy and surgical resection which are the most commonly ways, the results of DHPLC is in accordance with direct sequencing. EGFR mutations were present in 22.89% of 83 NSCLC patients.Female and adenocarcinoma patients showed higher mutation frequency. Smoking status and age were no significant correlation with EGFR mutations. In conclusion DHPLC is a precise rapid preliminary screening method for detection of NSCLC EGFR genotype.Part two Comparison of EGFR and K-ras genotypes between primary tumors and corresponding lymph node Metastases in NSCLCObjectiveCorrelation analysis of EGFR and K-ras genotypes between primary tumors and corresponding metastatic lymph node in NSCLC.Method1. Macthed primary tumors and metastatic lymph nodes in 21 NSCLC patients hospitalized in guangdong general hospital from may 2008 to February 2010 were included. There were 14 primary tumor specimens from surgical resection and the other 7 from CT guided transthoracic needle biopsy of lung. There were 7 metastatic lymph nodes from surgical resection,11 from Mediastinoscopy and the other 3 from infraclavicula lymph node biopsy. The tumor specimens were divided into two, a section fixed by 10% neutral formalin and paraffin embedded was used for pathological diagnosis. The other section was put into liquid nitrogen and then transferred to-80℃low temperature refrigerator.2. DNA extraction:The genomic DNA was extracted from lung cancer tissues.The DNA purity and content were detected by the Eppendorf nucleic acid protein cryoscope.3. The EGFR exon 18-21 and K-ras codon 12,13,59 were amplified by PCR. PCR products were used for direct sequencing and DHPLC detection after being evaluated by 2% agarose gel electrophoresis.4. The PCR products were purified.Direct sequencing of the PCR products was performed using ABI PRISM 3100 DNA Analyzer. Chromas software was used to analyze the sequencing results.5. Statistics analysis:All data were analyzed with SPSS 13.0 software. Kappa test was used in examination of the homogeneity in EGFR and K-ras genotypes between primary tumors and corresponding metastatic lymph node in NSCLC. P< 0.05 was considered statistically significant for homogeneity. Examination of the homogeneity combined the Kappa value.ResultA K-ras mutation was present in 4.76% of 21 patients, and was concordant in primary tumors and corresponding metastatic lymph node. We found concordant EGFR mutations in 6 patients.4 cases harbored exon 21 L858R mutation,1 case harbored exon 21 L858R and exon 20 T790M mutations,1 case harbored exon 19 delection. Discordant EGFR mutation was present in only one case, primary tumor harbored EGFR wild type, but metastatic lymph node harbored EGFR exon 19 delection. Concordant EGFR and K-ras wild type were found in 13 patients. EGFR and K-ras genotypes of primary tumors and corresponding metastatic lymph node in , NSCLC were concordant(Kappa value of EGFR and K-ras is 0.889 and 1.000, P<0.001).ConclusionEGFR and K-ras genotypes of primary tumors and corresponding metastatic lymph node in NSCLC were concordant by detecting fresh tissues, but we need enlarge the sample size for further research.