Study Of Prognostic Implication Of Different EGFR Types In Patients With Lung Adenocarcinoma And Related Mechanism

Part Ⅰ Prognostic differences of different EGFR types in stage Ⅰ lung adenocarcinoma patientsObjective:To investigate the difference of postoperative survival in patients with stage Ⅰ lung adenocarcinoma with different status and mutation subtypes of epidermal growth factor receptor（EGFR）.Methods:119 patients of completely resected pathological stage Ⅰ lung adenocarcinoma in Shandong cancer hospital and Tianjin cancer hospital from 2009 to 2014 were retrospectively included.All patients’ fresh tumor tissues or wax embedded tissues underwent EGFR gene mutation detection.According to the detection results,the patients were divided into EGFR wild-type group,EGFR 19del mutation group,EGFR L858R mutation group and other mutation group.The primary endpoint was disease-free survival（DFS）and the secondary endpoint was overall survival（OS）.Results:Among the patients,there were 65 cases of EGFR wild type and 54 cases of EGFR mutation type,including 32 cases of 19del mutation,19 cases of L858R mutation and 3 cases of other mutations（2 cases of G719X and 1 case of L861Q）.EGFR mutations were more common in non-smoking（P=0.000）and female patients（P=0.042）.After a median follow-up of 43.5 months,there was no significant difference in DFS between patients with EGFR mutations and those without（P=0.461）,but DFS and OS in patients with 19del mutation were longer than those with L858R mutation（P=0.028 and P=0.001）.The results were consistent with the above results by using TCGA data.Conclusions:For postoperative stage Ⅰlung adenocarcinoma patients,there is no survival difference between patients with EGFR mutations and those without,but 19del mutation patients have longer DFS and OS than L858R mutation patients.This suggests that EGFR mutation subtype has different potential prognostic value,and there is a difference in the prognosis between 19del mutation and L858R mutation.Part Ⅱ Differences of protein expression in lung adenocarcinoma between 19del and L858R mutation subtypesObjective:E-cadherin,N-cadherin,Vimentin,Chemokine receptor type 4（CXCR4）and Matrix metalloproteinase-9（MMP-9）are all related to the invasion,metastasis and prognosis of lung cancer.The study of part one shows that there are prognosis differences between the two EGFR mutation subtypes.Is there also a difference in the expression of the above protein markers?The protein markers with different expression are screened by immunohistochemical method,which lays a foundation for subsequent cell,animal experiment and study of molecular mechanism.Methods:The expression of five protein markers E-cadherin,N-cadherin,Vimentin,CXCR4 and MMP-9 in lung adenocarcinoma patients with 19del mutation and L858R mutation were detected by immunohistochemical method.The different expression was calculated by Chi-square test.Results:Immunohistochemistry showed that N-cadherin and Vimentin were not positively expressed in the lung adenocarcinoma tissues,E-cadherin was mainly expressed in the cell membrane,CXCR4 and MMP-9 were mainly expressed in the cytoplasm.Chi-square test showed that the expression of CXCR4 in L858R mutation group was higher（P=0.003）,and the expression of E-cadherin in 19del mutation group was higher（P=0.004）,but there was no significant difference in the expression of MMP-9 between two mutation subtypes（P=0.180）.Conclusions:There are differences in the expression of CXCR4 and E-cadherin between different EGFR mutant subtypes of lung adenocarcinoma patients.The expression of CXCR4 in L858R mutation patients is higher,and the expression of E-cadherin in 19del mutation patients is higher.Part Ⅲ L858R mutation upregulates the expression of CXCR4 to enhance cell malignant behavior through MEK-ERK pathwayObjectie:Based on the results of the previous two parts,we further explored the differences of migration,proliferation,invasion and metastasis in cells with different EGFR subtypes,and explored the relevant pathways and possible molecular mechanisms of these differences around CXCR4 and E-cadherin.Methods:EGFR wild-type A549 cells were selected to obtain exon 19 deletion mutation cells and exon 21 L858R point mutation cells by CRISPR/Cas9 technology,and control cells were obtained by blank plasmid.After stable screening,three kinds of cells were obtained,named EGFR-19del cells,EGFR-21mut cells and CON cells.The three kinds of cells were subjected to scratch test,MTT test,western blot test and transwell test respectively.The three kinds of cells were injected subcutaneously and through tail vein into nude mice in order to explore the differences in migration,proliferation,invasion and metastasis among the three cells.At the same time,the relevant molecular pathways causing the above differences were explored by pathway blockers and molecular antagonists.Results:Scratch test showed that the migration rate of EGFR-21mut cells was faster than that of EGFR-19del cells.MTT test and subcutaneous tumorigenesis test in nude mice showed that the proliferation activity of EGFR-21mut cells was stronger than that of EGFR-19del cells.Western blot test,transwell test and lung metastasis model test showed that the invasion and metastasis ability of EGFR-21mut cells was stronger than that of EGFR-19del cells.Blocking MEK-ERK pathway with U0126 could downregulate the expression of CXCR4 and reduce the above ability of EGFR-21mut cells.Conclusions:L858R mutation cells are stronger than 19del mutation cells in terms of migration rate,proliferation activity,invasion and metastasis ability,which may be related to L858R mutation upregulating the expression of CXCR4 through MEK-ERK pathway downstream of EGFR.CXCR4 may be a potential target for the combined treatment of lung adenocarcinoma patients with L858R mutation.