The Effect Of EGFR Gene Mutation Status On The Clinical Outcomes Of Patients With Non-small Cell Lung Cancer

Lung cancer is the most commonly occurring cancer in the world in terms of incidence and mortality.Based on histology,lung cancer is divided into two main subtypes:small cell lung cancer and non-small-cell lung cancer(NSCLC),of which NSCLC accounts for approximately 80%-85%of all cases.NSCLC is further classified into subcategories including:adenocarcinoma,squamous-cell carcinoma,and large-cell carcinoma.The most common type of lung cancer is adenocarcinoma,which comprises around 40%of all lung cancer.Compared with non-adenocarcinoma,adenocarcinoma was more frequent in women-who were young and had never smoked.EGFR mutations were also usually detected in female,nonsmoking patients with adenocarcinoma.EGFR is one of four members of the ErbB family and is known to facilitate tumorigenesis and cancer progression.EGFR is structurally comprised of an extracellular ligand binding domain,a transmembrane region,a tyrosine kinase domain.Ligands including the epidermal growth factor,bind to the extracellular domain of EGFR,causing a conformational change that facilitates homo-and heterodimerization with members of the ErbB family.Dimerization induces activation of the tyrosine kinase activity of the receptor,leading to autophosphorylation.Binding of activating ligands leads to EGFR dimerizationand transphosphorylation of the tyrosine residues in the carboxytail,with activation of downstream pathways involved in cell proliferation,survival,invasion and angiogenesis.Somatic mutations in the kinase domain of the EGFR gene are usually detected in exons 18-21 in patients with NSCLC.Common mutations,that is,exon 19 deletions(19-del)and L858R mutation in exon 21,comprise approximately 90%of all the mutations and are associated with sensitivity to EGFR tyrosine kinase inhibitors(EGFR-TKIs).Patients with NSCLC harboring common EGFR mutations benefit remarkably from EGFR-TKIs treatment,demonstrating by less side effect,longer progression-free survival time and overall survival time,when compared with conventional chemotherapy.However,the relationship between EGFR-sensitive mutations and lung cancer metastasis has not been fully elucidated,and the mechanism under which EGFR-sensitive mutations affect lung cancer cell metastasis has not been fully addressed.In the present study,we analyzed the distribution of EGFR-L858R mutation in 2333 patients with ADC undergoing thoracic surgery.The correlation between EGFR-L858R mutation and ADC lymph node metastases(LNM)was evaluated by univariate analysis.A549 and H1299 cell lines were used as in vitro models.Cell proliferation and migration were evaluated with colorimetric assay and wound healing assay,respectively.Cell cycle and cell death were measured by flow cytometry.The ability of cells to form colonies was performed with colony formation assay.The growth and invasion of cell spheroids were determined with 3D-spheroidsprouting assay.The mRNA expressions of CD133,C-kit,SOX2 and OCT4 were quantified by qRT-PCR,and the protein expressions of CD133,C-kit,SOX2 and OCT4 were assessed by Western blot.We observed that EGFR-L858R was inversely correlated with ADC lymph node metastasis(LNM).The overexpression of EGFR-L858R reduced the invasive ability and colony formation capacity of H1299 cells,compared to the wild-type EGFR.In agreement with the inhibitory effect on colony formation,the overexpression of EGFR-L858R led to the decreased expression of C-kit,CD133,SOX2,and OCT4,which are important for maintaining the stemness of cancer cells.Consistently,the reduced levels of these genes were also observed in the ADC lesions with EGFRL858R.In addition to EGFR TKI-sensitive mutations,point mutation(T790M)and insertion mutation(20-ins)in exon 20 of EGFR are resistant to EGFR-TKI.Patients with NSCLC who are harboring those mutations are unlikely to achieve response with EGFR-TKI.Previous studies have shown that the presence of EGFR-T790M mutation may reduce the treatment efficacy of tyrosine kinase inhibitors(TKIs)in EGFR-mutant lung cancer.However,little is known about the clinical features and outcomes of EGFR-T790M mutation in pretreated patients with NSCLC.In the present study,a total of 16,347 patients diagnosed with NSCLC were recruited from the Shanghai Pulmonary Hospital affiliated to Tongji University in China between January 2013 and December 2017.The clinical features of EGFRactivating and T790M mutations were assessed in a large cohort of patients with EGFR-TKI-naive NSCLC(all/EGFR mutations,n=16,347/7,687).The correlation between the pretreatment T790M mutation status and clinical outcomes was evaluated using univariate and multivariate analyses.Pretreatment T790M mutation was reported in 1.39%of patients and coexisted with an EGFR-activating or uncommon mutation.The dual EGFR-T790M and common-activating EGFR mutations were more likely to be detected in lung adenocarcinoma,whereas single T790M mutations were more prevalent in non-adenocarcinomas.The presence of de novo T790M mutation correlated with the reduced RFS in patients with NSCLC(odds ratio[OR]=3.37,95%confidence interval[CI]=1.67-6.79,P=0.001).After molecular stratification,T790M mutation was shown to exert adverse effects on the RFS of EGFR-19-del group(OR=2.89,95%CI=1.10-7.91,P=0.028)and EGFRL858R group(OR=3.43,95%CI=1.33-8.88,P=0.013).Furthermore,pretreatment T790M mutation promoted tumor metastasis to different sites.Our findings suggest that:(1)The decrease of stemness might be responsible for the inverse correlation between EGFR-L858R and LNM..(2)The T790M-positive tumors presented special imageologic features.4.The coexistence of T790M and common EGFR-activating mutations was associated with poor prognosis in patients with NSCLC.