The Correlation Between Clinical Pathological Features And EGFR Gene Mutation Status In Primary Lesion Of Newly Diagnosed NSCLC

Lung cancer is the No.1cause of cancer-related morality worldwide and about80%ofthe lung cancer cases were non-small cell lung cancer (NSCLC) whose5-year survival rate isless than15%when diagnosed at advanced stages. Epidermal growth factor receptor (EGFR)is the first discovered driver of NSCLC that plays a central role in proliferation, growth andinvasion of the tumor cells through its tyrosine kinase activity. Accordingly, tyrosine kinaseinhibitors (TKIs) have been proved effective in treatment of NSCLC by multiple phase IIIclinical studies, and have become first-line drugs for NSCLC patients with activationmutation in the19th and21th exons in the kinase domain of EGFR gene. Currently,commercially available TKIs include Icotinib, gefitinib and erlotinib. These TKIs caneffectively inhibit cancer cell growth, angiogenesis, cancer invasion and metastasis, and theinhibition effect is mainly due to its competitive binding to the ATP-binding site of the EGFRtyrosine kinase domain. Direct sequencing of the tumor tissue-derived DNA has become thegold standard for detecting EGFR mutation. To analyze EGFR mutation, high quality tissuesample is required, which is not always possible for all patients due to multiple limitingfactors. Large number of studies have explore the possibility of using peripheral blood inplace of cancer tissue as sample for DNA sequencing to detect EGFR mutation. However,results of these studies varied about the consistency between results obtained from DNA ofdifference sources. Using Chi-square test, this study explores the consistency between plasmaand tissue sample EGFR mutation in NSCLC patients, so as to evaluate the reliability ofplasma EGFR mutation test as an alternative, and to analyze its correlation with clinicalbaseline. The second part of this study explored the status of EGFR mutation in newlydiagnosed NSCLC patients and its correlation with pathological indexes of the patients, in anattempt to use clinical pathological indexes to predict EGFR mutation. For some of thepatients for whom tissue sampling for EGFR mutation test is restricted, we used clinical pathological indexes that predict good efficacy of TKI therapy, providing theoretical basis fortargeting therapy. About25%-54%NSCLC patients showed brain metastasis during treatment,studies have shown that the occurrence of brain metastases of NSCLC patients may beassociated with EGFR mutations, but there is no report of using the occurrence of NSCLCpatients with brain metastasis to predict EGFR mutation in primary lesion of previouslyuntreated NSCLC. Therefore, this study intended to explore the correlation between brainmetastases of NSCLC and EGFR mutation in primary lung cancer by clinical samples andvitro experiments, providing additional reference for NSCLC with brain metastasis failing todetect EGFR mutation in primary lesion to use TKI therapy by using the occurrence of brainmetastases of NSCLC patients to predict EGFR mutation in primary lesion of previouslyuntreated NSCLC.Objective1. To investigate the consistency of EGFR activation mutation determined fromperipheral blood-derived free DNA and paraffin-embedded tissue sample. So as to evaluatethe possibility of using peripheral blood as an alternative to tissue sample to detect EGFRmutation and the correlation between clinical features of NSCLC with brain metastasis andEGFR mutation．to provide additional reference for some of the patients for whom tissuesampling for EGFR gene mutation test is restricted using TKI therapy．2. To investigate the correlation between clinical pathological features of NSCLCpatients and the status of EGFR mutation and use clinical pathological indexes to predictEGFR mutation.so as to provide clinical feature-based reference for application ofindividualized TKI therapy for patients not suitable for sampling.To investigate the correlation between clinical pathological features of NSCLC patientswith brain metastasis and the status of EGFR mutation, so as to provide reference forapplication of TKI therapy for NSCLC patients with brain status and was not suitable fortissue sampling.3. To investigate the correlation between the occurrence of brain metastases of NSCLCpatients and EGFR mutation in primary lung cancer, providing additional reference for brainmetastases of NSCLC patients with failing to detect EGFR mutation in primary lesion to useTKI therapy by using the occurrence of brain metastases of NSCLC patients to predict EGFRmutation in primary lesion of previously untreated NSCLC. Method1. Comparison between EGFR mutation in plasma free DNA and tumor tissue DNAFrom446cases of NSCLC confirmed by pathological analysis that were admitted by theaffiliated Daping hospital of the Third Military Medical University from June2011toDecember2013,40pairs of tissue samples was tested for EGFR mutation by Sanger directsequencing, and corresponding peripheral blood was also tested for EGFR mutation byDHPLC. To test the coherence of EGFR mutation in40plasma sample and in40match intissue sample and relationship between EGFR mutation and clinical features of NSCLCPatients.2. The correlation between clinical pathological features and EGFR gene mutation statusin primary lesion of newly diagnosed NSCLC253cases of previously untreated NSCLC admitted by cancer center of the affiliatedDaping hospital of the Third Military Medical University from July2011to May2013wereselected, and their tissue material were tested by fluorescent PCR Kit to detect EGFRmutation. To investigate the correlation between clinical pathological features of NSCLCpatients and the status of EGFR mutation in tissue sample Using Chi-square test.To investigate the correlation between clinical pathological features of NSCLC patientswith brain metastasis and the status of EGFR mutation in tissue sample using Chi-square test.3. The correlation between the brain metastases of NSCLC patients and EGFR genemutation in primary lung cancerMorphological features of A549human lung adenocarcinoma cell line with wild typeEGFR and HCC827cell line with a deletion mutation in the19thexon of the EGFR gene wereobserved and compared with microscopy; proliferation of the two were compared by MTT;and invasion ability of the two was compared by Transwell in vitro invasion assay.4. The correlation between EGFR gene mutation and clinical baseline was tested by χ2analysis or Fisher exact possibility test and Logistic regression. P<0.05was consideredstatistically significant. And all analyses were performed with SPSS16.0softwarepackage.Comparison of the MTT OD value, number of cells invade through were comparedby one-way analysis of variance.Results1. Comparison between EGFR mutation in plasma free DNA and tumor tissue DNA EGFR mutation in the446plasma sample and in40tumor tissue sample were19.7%and37.5%respectively. Using results obtained from tumor tissue as standard, the negativepredictive value, positive predictive value, sensitivity and specificity of plasma-derived EGFRmutation results were76.7%(23/30),80.0%(8/10),53.3%(8/15), and92.0%(23/25)respectively.The correlation between EGFR mutation in plasma free DNA and clinical baseline:Compared with males, non-adenocarcinomas, the mutation rates of primary lung cancer’sEGFR gene inplasma sample were higher in females, non-smokers, adenocarcinomas, but thedifferences were not statistically significant（(P=0.148, P=0.262).2. The correlation between clinical pathological features and EGFR gene mutation statusin primary lesion of newly diagnosed NSCLCThere are four types mutation in253newly diagnosed Non-small cell lung cancerpatients.they are19Del,21L861Q,21L858R and20ins.There are109mutation cases and144wild cases in253cases,Their rates are43.1%(109/253)，56.9%(144/253) respectively.Of the253cases of previously untreated NSCLC,107had TKI-sensitive mutations inEGFR gene (42.3%,107/253), including L861Q or L858Rmutation in exon21in32cases(39.1%), exon19deletion in75cases (70.1%). While insertion mutation in exon20is TKIresistant, and occurred in2of the253cases (0.8%).TKI-sensitive EGFR mutation casesoccurred in25.8%of the male patients (41/61) and70.2%(66/94) of the female patients; in18.1%(19/105) in patients with smoking history and60.9%(81/133) of non-smokingpatients;8.5%(4/47) of non-adenocarcinoma and50.0%(103/206) of adenocarcinoma cases.The differences were statistically significant(P<0.05).31cases of253cases NSCLC had brain metastasis,12.3%（31/253）. The31cases ofNSCLC brain metastasis,8had EGFR mutation showed EGFR mutation in primary cancerlesion (8/31). The mutation rates of exon20and21of EGFR gene were22.6%(7/31) and3.2%(1/31), respectively. The relationship between mutation status of pulmonary lesion andclinical pathological features were not statistically significant (P>0.05).Clinicalopathologicalfeatures include the number of brain metastases, BMT, primary tumor controlled or notextracranial metastases status.3. The correlation between brain metastasis and EGFR gene mutation in primary cancerlesion. MTT assay showed that A549cell line with wild-type EGFR had significantly fasterproliferation than HCC827cell line with mutant EGFR (P<0.05%); and transwell assayshowed significant lower invasion of A549than HCC827cell line (P<0.05); and of the31cases of NSCLC brain metastasis,8had EGFR mutation showed EGFR mutation in primarycancer lesion (8/31), no significant correlation was detected between the two.Conclusion1. Detection of EGFR mutation using plasma in place of tissue sample is of some value,but the sensitivity is low and there are false negatives.2. TKI-sensitive mutation of EGFR occurs in newly diagnosed NSCLC cases, and it’smore common in female than male, in adenocarcinoma than non-adenocarcinoma, innon-smoker than smokers. These results provide evidence that suggests application of TKItherapy for non-smoking female patients with adenocarcinoma when tissue sampling fordetection of EGFR mutation is not available.3. EGFR mutation can significantly increase invasion of NSCLC cells, brain metastasisof NSCLC patients showed some correlation with EGFR mutation in primary cancer lesion, itprovides theoretical basis to apply TKI therapy for NSCLC patients with brain metastasiswhen tissue sampling for detection of EGFR mutation in primary cancer lesion is not possible,which needs further in-depth investigation.