Clinically Experimental Investigation Of Biomarkers Of Peripheral Blood As Potential Predictors Of The Efficacy Of Immune Checkpoint Blockade In Patients With Solid Tumors

Background:Tumor immunotherapy represented by immune checkpoint inhibitors targeting cytotoxic T lymphocyte-associated antigen-4(CTLA-4),programmed cell death-1(PD-1)and programmed death-ligand 1(PD-L1)revolutionized the tumor treatment paradigm,providing significant survival benefits.ICB has been approved by the Food and Drug Administration(FDA)or the China Food and Drug Administration(CFDA)for a variety of solid and hematologic cancer treatments,and its application in lung cancer is particularly significant.However,although the efficacy of ICB immunotherapy has been verified by many parties,considering the low response rate of treatment,the specific immunotoxicity and side effects of treatment,and the high cost of treatment,finding reliable biological or clinical biomarkers to screen out the clinical patients most likely to benefit from ICB treatment is still a critical issue that is urgently needed in clinical practice.Biomarkers in peripheral blood include T cell receptor(TCR)immune repertoire(IR),soluble cytokine receptors(sCKR)and soluble immune checkpoint proteins levels are closely related to immune function.Aim:The purpose of this study was to investigate the correlation between the dynamic changes in peripheral blood biomarkers(T cell receptor immune repertoire,soluble cytokine receptors and soluble immune checkpoint proteins)and the efficacy of tumor patients treated with PD-1/PD-L1 checkpoint inhibitors.Methods:We collected samples from 25 patients with solid tumors(16 cases of lung cancer,5 cases of urothelial carcinoma,2 cases of gastric cancer,1 case of liver cancer and 1 case of cholangiocarcinoma)treated with PD-1/PD-L1 checkpoint inhibitors at baseline,6 weeks and 12 weeks after treatment for anticoagulation peripheral blood,and isolated peripheral blood mononuclear cells(PBMC)and plasma;we conducted high-throughput sequencing(HTS)of T cell TCRβ chain genes contained in PBMCs by multiplex PCR,and bioinformatics analysis of the changes of diversity and clonality of TCRβ chains complementarity-determing region 3(CDR3)sequences;Plasma soluble cytokine receptors and soluble immune checkpoint proteins levels were detected using a multiplex cytokine assay kit.According to the therapeutic effect of PD-1/PD-L1 checkpoint inhibitors,the above 25 patients were divided into clinical benefit(CB)group and no clinical benefit(NCB)group or partial response(PR),stable disease(SD)and progressive disease(PD)groups.The expression levels of TCR immune repertoire,soluble cytokine receptors and soluble immune checkpoint proteins were analyzed and compared between 2 groups or among 3 groups.Results:The results of TCR cloning frequency analysis showed that patients in the clinical benefit group had a greater difference in the frequency of use of the V J gene in the CDR3 region of the TCR than in the no clinical benefit group(P<0.05).The analysis result of Chao index showed that the TCR-CDR3 diversity in the PR group was significantly increased after treatment than SD or PD group(P=0.001),and the number of CDR3 AA clonotypes also increased significantly(P=0.012).A total of 61 VP gene fragments and 15 Jβ gene fragments were uniquely identified during immunotherapy.There was a significant difference in the number of VP gene fragments between baseline samples and after treatment in PR group(P=0.042).The expression profile of soluble cytokine receptors and soluble immune checkpoint proteins showed that CD27 had opposite regulation in the PR and SD groups.In the condition of clinical benefit group,sRAGE and CD27 had significant changes in the fold change before and after immunotherapy.Further in-depth analysis revealed that the dynamic changes in expression of soluble cytokine receptors and soluble immune checkpoint proteins with similar functions showed a holistic trend of significant Signature characteristics.Signature-1 changes in immune checkpoint proteins with significant differences(P=0.044).NCB group patient Signature-1 was significantly higher than CB group,and most patients with no signature-1 characteristics in baseline samples benefited clinically from ICB treatment.Conclusions:This study revealed the dynamic changes of TCR immune repertoire before and after treatment in patients with solid tumors treated with ICB which were associated with the efficacy of ICB treatment.The TCR-CDR3 diversity and the number of amino acid clonotypes in responding patients were significantly increased,suggesting that T cell expansion induced by ICB treatment is associated with therapeutic response.In view of the soluble cytokine receptors and soluble immune checkpoint proteins in peripheral blood,signature is a holistic indicator that integrates multiple molecules with similar functionalities.Compared with single molecules,signature has a better predictive value for ICB efficacy.In addition,there is a correlation among the indicators of TCR immune repertoire(richness and evenness),soluble cytokine receptors and soluble immune checkpoint proteins,which may be directly or indirectly related to the immune changes induced by ICB treatment.In the future,further integration of the TCR immune repertoire,soluble cytokine receptors and soluble immune checkpoint proteins in peripheral blood(especially Signature-1)will enhance our ability to predict the efficacy of ICB.