The Value Of Exosomal PD-L1 Expression And MiRNA Profiling In Predicting The Efficacy Of Immunotherapy In Non-small Cell Lung Cancer

Objective : Immune checkpoint inhibitors(ICIs)represented by programmed death-1 / programmed death ligand-1(PD-1/PD-L1)inhibitors have become one of the important treatment options for patients with advanced NSCLC.Since it is not effective for all NSCLC patients,one of the keys to improve efficacy is to screen out the “benefit population”.On one hand,PD-L1 is considered as an important biomarker for predicting the efficacy of immunotherapy.The expression of PD-L1 was assessed using tumor tissue samples.However,tumor tissue sometimes may be unavailable for those patients which are unable to undergo an invasive tumor biopsy.In addition,the efficacy of PD-L1 as a biomarker is often compromised by the spatial and temporal heterogeneity of the tumor biopsy.PD-L1 expression showed inconsistency in 18%-48% of clinical cases when comparing surgically resected specimen with specimen from matched biopsy.On the other hand,exosomes carry a large amount of biological active molecules such as DNA,RNA and proteins,which play an important role in cell-to-cell communication,and can affect the function of the immune system through various mechanisms,thereby affecting the efficacy of immunotherapy.Therefore,the aim of this study was to:(1)investigate the potential of using plasma exosomal PD-L1(e PD-L1)as a biomarker for NSCLC;(2)find out the differential expressed plasma exosomal mi RNAs between NSCLC patients and healthy people,and to explore the potential of plasma exosomal mi RNAs as biomarkers to predict the efficacy of immunotherapy in NSCLC.This will further optimize the screening criteria for benefit population from immunotherapy,so that more NSCLC patients can benefit from immunotherapy.Methods: This study consists of two parts.Part ?: Two NSCLC cell lines,H226(low-expressed PD-L1 cell line)and H1975(high-expressed PD-L1 cell line)and one breast cancer cell line MCF7(PD-L1 negative cell line)were selected for this study.The exosomes secreted by cells into culture medium were separated by high-speed centrifugation,and the cell lysate was also collected.CD9 was used as a marker for exosomes detection.The e PD-L1 expression level and the cell line PD-L1 expression level were detected by western blot.Part ?: This study prospectively collected baseline plasma samples of EGFR/ALK-wild type(confirmed by ARMS and IHC method)advanced NSCLC patients before the administration of PD-1/PD-L1 inhibitors.The efficacy evaluation was conducted every 3 cycles of the immunotherapy and blood samples were collected at the same time until the disease progression.According to the best efficacy evaluation,samples of patients with complete or partial response(CR/PR group)and disease progression(PD group)were selected out.Plasma from 7 healthy individuals was collected as control.Exosomes were extracted by ultracentrifugation and exosome-derived mi RNAs were analyzed by small RNA next-generation sequencing.Results: Part ?: H1975 and MCF7 cell lines showed stronger CD9 expression level compared to H226 cell line.H1975 cell line with stronger PD-L1 expression showed higher e PD-L1 level compared to H226 cell line.PD-L1 negative MCF7 cell line was also found to be e PD-L1 negative.Part ?I: 5 PR patients and 4 PD patients were enrolled in this study.Based on unsupervised hierarchical clustering,exosomal mi RNA expression profile was significantly altered in NSCLC patients compared to normal controls with a total of 155 differentially expressed exosomal mi RNAs between the advanced NSCLCs and healthy individuals(Cutoff: log CPM>4,FDR?0.1,P<0.05 by exact test).Interestingly,three mi RNAs(hsa-mi R-320 d,hsami R-320 c,hsa-mi R-320b),all from the mi R320 family,were identified up-regulated in the PD groups compared to the PR group before the treatment,which might be potential predictors for the response of anti-PD-1/PD-L1 therapy.In addition,we identified that hsa-mi R-125b-5p,a previously reported ?? T cell suppressor,was down-regulated in the post-treatment plasma exosomes compared to pre-treatment samples of the PR patients,which can serve as a biomarker for monitoring the efficacy of immunotherapy.Conclusions: Plasma ePD-L1 expression is regulated by the expression level of PD-L1 in tumor cells and the secretion level of exosomes from tumor cells.When e PD-L1 expression is positive,it can reflect the expression level of PD-L1 in tumor tissues;conversely,in the case of negative e PD-L1,it is recommended that the detection result of tumor tissue PD-L1 shall prevail.NSCLC patients represent unique plasma exosomal mi RNA profiles.hsa-mi R-320 d,hsa-mi R-320 c and hsa-mi R-320 b were identified as potential biomarkers for predicting the efficacy of immunotherapy in advanced NSCLCs.When T cell suppressor hsa-mi R-125b-5p was down-regulated during the treatment,the patients may obtain increase T cell function and response well to immunotherapy.