| Part one Clinical investigation on EGFR-TKIs resistance in patients of lung adenocarcinomaObjective: Retrospective study was used to investigate the clinical data and gene detection results of patients with advanced lung adenocarcinoma(LUAD)before and after epidermal growth factor receptor-tyrosine kinase inhibitors(EGFR-TKIs)therapy and to analyzed the clinical characteristics of patients with EGFR mutation.Median progression-free survival(PFS)and drug resistance mechanism were analyzed in advanced lung adenocarcinoma patients treated with EGFR-TKIs in first-line therapy.Methods:1.LUAD patients with stage ⅢB/Ⅳ were selected to detect EGFR gene expression in the Department of Tumor Immuno-therapy and Respiratory of the Fourth Hospital of Hebei Medical University from January 2017 to December 2017.According to the results of EGFR gene detection,patients were divided into EGFR mutant type(EGFR-MUT)group and EGFR wild type(EGFR-WT)group.The expression of EGFR mutation and its relationship with sex,age,smoking history and other clinical characteristics were analyzed.2.71 LUAD patients with EGFR sensitive mutation(19del or L858R)were treated with EGFR-TKIs in the first-line therapy and followed up regularly,according to Response Evaluation Criteriain Solid Tumors(RECIST)version 1.1.The median PFS treated with first line therapy of EGFR-TKIs was analyzed.3.59 LUAD patients underwent secondary gene detection after EGFRTKIs resistance in the first-line therapy.Drug resistance pattern was analyzed according to the results of gene detection.Results:1.There were 87(50.9%)EGFR mutational patients in all LUAD patients,including 42(48.3%)exon19 deletion patients,35(40.2%)exon21 L858 R mutation patients and 10(11.5%)patients for others mutation.In all LUAD patients with EGFR mutation,there were 54 female patients and 33 male patients(62.1% vs.37.9%,P < 0.001),20 patients with previous smoking history and 67 non-smoking patients(23.0% vs.77.0%,P = 0.002),34 patients > 65 years old and 53 patients ≤ 65 years old(39.1% vs.60.9%,P = 0.434),14 patients with stage ⅢB/C and 73 patients with stage Ⅳ(16.1% vs.83.9%,P = 0.200),respectively.These results suggest that EGFR mutation is associated with sex and smoking history,but not with age and TNM staging.2.71 LUAD patients with EGFR sensitive mutation(19del or L858R)were treated with EGFR-TKIs in the first-line therapy.Among them,there were 37(52.9%)patients treated with icotinib,23(32.2%)patients treated with gefitinib and 11(14.9%)patients treated with erlotinib.The median PFS of these LUAD patients treated with EGFR-TKIs in first-line therapy was 10.6 months(95% CI 9.8-11.4).3.There were 68 LUAD patients with drug resistance to progress,among the patients with EGFR sensitive mutation.59(86.7%)patients underwent secondary hematological or cytological gene detection.There were 32(54.8%)patients with EGFR T790 M mutation,3(5.1%)patients with MET amplification,1(1.7%)patients with HER2 amplification and 23(38.9%)patients with unknown mutation.T790 M mutation was the main mechanism of EGFR-TKIs resistance in LUAD patients treated with EGFR-TKIs in first-line therapy.Conclusions:1.In LUAD patients,EGFR mutation is associated with sex and smoking history,but not with age and TNM staging.Non-smokers and women had the more rate of EGFR mutation.2.The median PFS of LUAD patients treated with EGFR-TKIs in first-line therapy was 10.6 months.T790 M mutation was the main mechanism of drug resistance,followed by MET and HER2 amplification.However,the mechanisms of EGFR-TKIs resistance in 38.9% of the patients were still unknown.Part two Expression and significance of BIN1 in lung adenocarcinoma cells with EGFR-TKIs resistanceObjective: To detect the expression levels of BIN1 in EGFR 19 del LUAD cell line PC-9 and gefitinib resistant LUAD cell line PC-9/GR,and construct PC-9/GR cells which high expression of BIN1 by gene transfection,then investigate the effect of BIN1 overexpression on gefitinib resistance cells.Methods:1.Inverted microscope was used to observe the morphology changes of PC-9 and PC-9/GR cells.2.Quantificational real-time polymerase chain reaction(qRT-PCR)and Western blotting assays were used to evaluate BIN1 expression levels in PC-9 and PC-9/GR cells.3.BIN1 were transfected into PC-9/GR cells with BIN1 eukaryotic expression recombinant vector(BIN1 group),compared with empty vector plasmid group(NC group).PC-9/GR cells was performed as the control group(Con group),additional.qRT-PCR and Western blotting assays were used to detect the effect of BIN1 transfection.4.MTT assays were used to detect the proliferation ability of PC-9/GR cells which overexpressed BIN1.5.Clone formation was used to detect the colony ability of PC-9/GR cells which overexpressed BIN1.6.Flow cytometry assay was performed to evaluate the effect of BIN1 on cell apoptosis level in PC-9/GR cells.Results:1.Compared with PC-9 cells,the morphology of gefitinib resistant PC-9/GR cells was fusiform and bigger.The transmittance of PC-9/GR cells was decreased and there were fused giant cells and vacuoles in the PC-9/GR cells.2.BIN1 overexpressed PC-9/GR cells was constructed by transfected eukaryotic recombinant plasmid.The expression of BIN1 in PC-9/GR cells confirmed by qRT-PCR and Western blot were lower compared to LUAD cell line PC-9(P < 0.05).3.Compared with NC and Con groups,the expressions of BIN1 mRNA and protein in BIN1 PC-9/GR cells were increased significantly(P < 0.05),which confirmed that BIN1 transfected into PC-9/GR cells successfully.4.Compared with NC and Con groups,the relative viability of BIN1 group at 0.1、0.2、0.5、1、5、10、20 gefitinib concentration(μmol/L)were repressed,respectively(P < 0.05).There were no significant difference of relative viability between NC and Con groups.These results demonstrated that BIN1 could inhibit the abilities of proliferation in PC-9/GR cells.5.Colony formation assay suggested that the colony ability of BIN1 cells was decreased compared to NC and Con cells(P < 0.05).These results demonstrated that BIN1 could partly restored the sensitivity to gefitinib in PC-9/GR cells.6.Compared with the NC and control group,the percentage of cells apoptosis was significantly increased in BIN1 group[(3.79±0.51)%,(3.06 ± 0.22)% vs.(10.47 ± 1.23)%,P < 0.05].The result demonstrated that overexpressing BIN1 in PC-9/GR could promote the apoptosis treated with gefitinib.Conclusions:1.The expression of BIN1 in gefitinib resistant PC-9/GR cells were lower than that of EGFR sensitive mutation PC-9 cells.2.Overexpression of BIN1 in gefitinib resistant PC-9/GR cells could partly restore the sensitivity to gefitinib and gefitinib plays an antitumor role by inducing cells apoptosis. |
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