Study On The Role Of EML4-ALK Regulating PD-L1 Expression In Non-small Cell Lung Cancer

Background and Purpose:Studies have indicated that PD-L1 expression was associated with EGFR driver mutation in non-small cell lung cancer and regulated by it.EML4-ALK fusion gene is the second important driver genes besides EGFR driver mutation in NSCLC,how the PD-L1 expression was related to EML4-ALK fusion gene and what the regulation mechanism was to be remain mostly unknown.The purpose of this study is to investigate the association between the PD-L1 expression and EML4-ALK fusion gene and its clinical significance in NSCLC,and further explore the potential regulatory mechanism of PD-L1 by EML4-ALK.Materials and Methods:1.One hundred and forty one patients with lung adenocarcinoma stages I-III who underwent complete surgical resection at Tianjin Medical University Cancer Institute&Hospital between January 2011 and December 2014 were studied.All patients were performed ALK D5F3 Ventana IHC detection and defined as the wild-type of EGFR by the department of pathology.We performed the IHC analysis of PD-L1 in 141 paraffin-embedded primary lung adenocarcinoma tissues.Association between the PD-L1 expression and EML4-ALK rearrangement was analyzed and prognostic factors were identified.2.Western blot,RT-PCR and cell immunofluorescence were performed to detect of PD-L1 expression in immortalized human lung bronchial epithelial cell line(Beas-2B),human NSCLC cell lines(A549,GLC-82)and EML4-ALK positive cell lines(H2228,H3122).Select Beas-2B and H3122 cell lines for subsequent tests.3.H3122 cell line was transfected with 3 different ALK siRNAs,and ALK inhibitor(TAE684)was selected to downregulate the ALK expression;EML4-ALK(V1)over-expression plasmid was transfected into Beas-2B cell line to overexpression the ALK fusion gene,and the negative control group was set at the same circumstance;Western blot,RT-PCR were performed to detect of PD-L1expression after transfection and further detect the expression of JAK3,STAT3,AKT and ERK1/2 to explore the potential molecular regulatory mechanism of PD-L1 by EML4-ALK in NSCLC.Results:1.In the present study,the IHC test of PD-L1 expression was performed to the141 patients.The prevalence rate of PD-L1 positivity was 22.0%(31/141),42.6%(60/141)and 58.9%(83/141)(50%cut-off value,5%cut-off value,and 1%cut-off value,respectively).PD-L1 positivity was significantly associated with the smaller tumor size(52.1%vs 32.4%,P=0.018,5%cut-off value;67.1%vs 50.0%,P=0.039,1%cut-off value),invasive adenocarcinoma(45.3%vs 18.2%,P=0.038,5%cut-off value),ALK rearrangement(38.1%vs 15.2%,P=0.003,50%cut-off value;69.0%vs 31.3%,P<0.001,5%cut-off value;73.8%vs 52.5%,P=0.019,1%cut-off value).Kaplan-Meier survival analysis showed that patients with PD-L1 positivity have a shorter DFS(log-rankc~2=7.087,P=0.008)and OS(log-rankc~2=4.705,P=0.030)than PD-L1 negative patients at the 50%cut-off value.Univariate analysis indicated that the DFS was significantly associated with the pathological stage(HR,5.31;95%CI 2.81-10.03;P<0.001)and PD-L1 overexpression(HR,2.07;95%CI 1.16-3.70;P=0.014);the OS was significantly associated with pathological stage(HR,9.99;95%CI 3.36-29.70;P<0.001),histopathologic subtypes(HR,3.76;95%CI 1.39-10.14;P=0.009),adjuvant therapy(HR,0.18;95%CI 0.07-0.49;P<0.001)and PD-L1overexpression(HR,2.63;95%CI 1.05-6.60;P=0.039).Multivariate analysis using a Cox proportional hazards model compared the DFS and OS of all patients.After adjusting for pathologic nodal staging,tumor staging,tumor size,and adenocarcinoma subtypes,the variables that remained significantly associated with DFS were histopathologic subtypes(HR,3.95;95%CI 2.26-6.93;P<0.001)and PD-L1 overexpression(HR,1.95;95%CI 1.14-3.33;P=0.015);the OS was significantly associated with pathological stage(HR,8.15;95%CI 3.01-22.07;P<0.001),histopathologic subtypes(HR,3.75;95%CI 1.44-9.79;P=0.007)、adjuvant therapy(HR,0.23;95%CI 0.09-0.57;P=0.002).PD-L1 overexpression is an adverse prognostic factor for a shorter DFS and OS,PD-L1 overexpression is not an independent prognostic factor in NSCLC.2.Western blot showed that the expression of PD-L1 protein in ALK-positive cell lines(H2228 and H3122)was higher than that of ALK-negative cell lines(A549,GLC-82 and Beas-2B).RT-PCR showed that the expression of PD-L1 mRNA was also significantly higher in ALK-positive cell lines.Cell immunofluorescence indicated that PD-L1 was mainly concentrated in the cytomembrane and the fluorescence intensity of H3122 cell line was significantly higher than that of A549cells.3.Western blot and RT-PCR showed that TAE684,ALK siRNA-1 and ALK siRNA-1 significantly knocked down the ALK expression,followed by the down-regulation of PD-L1 in H3122 cell line;EML4-ALK was significantly overexpressed in the Beas-2B cell line after transfection of EML4-ALK(V1)over-expression plasmid,followed by the up-regulation of PD-L1 in Beas-2B cell line.When applied TAE684 on H3122 cell,the activity of JAK-STAT signaling pathway,PI3K-AKT signaling pathway and EKR1/2 signaling pathway was restrained,and Beas-2B cell was transfected by the exogenous EML4-ALK fusion gene,followed by the up-regulation of JAK-STAT3 pathway protein,PI3K-AKT pathway protein and EKR1/2 pathway protein.It shows that those signaling pathways may be involved in the process of EML4-ALK regulating the PD-L1 expression.Conclusion:PD-L1 was highly expressed in NSCLC with ALK rearrangement,and PD-L1was an adverse prognostic biomarker in lung adenocarcinoma.Inhibiting EML4-ALK expression downregulated PD-L1 expression and over-expression exogenous EML4-ALK upregulated PD-L1 expression.JAK-STAT signaling pathway,PI3K-AKT signaling pathway and EKR1/2 signaling pathway may be involved in the process of EML4-ALK regulating the PD-L1 expression.Our findings provide the theoretical evidence of targeted therapy combined with immunotherapy for advanced NSCLC.