rs10204525 binding to miR-4717-3p modulates PD-1 expression and predicts the development of immune-related adverse events in advanced cancer patients treated with anti-PD-1/PD-L1 therapy

Background Predictive biomarkers for anti-programmed cell death-1 (PD-1) and -programmed cell death ligand 1 (PD-L1) therapy are needed. Here, we validated the role of PD-1 single nucleotide polymorphisms (SNP) in predicting the development of immune-related adverse events (irAEs) in advanced cancer patients treated with anti-PD-1/PD-L1-based immunotherapy and define the molecular mechanisms underlying the role of identified SNP candidate. Methods Blood samples, clinical-pathological characteristics, survival outcomes and irAEs were collected from two cohorts of advanced cancer patients: i) advanced cancer patients treated with anti-PD-1/PD-L1 alone and ii) advanced non-small cell lung cancer (NSCLC) patients treated with anti-PD-1 in combination with platinum-based chemotherapy with or without anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4) therapy. PD-1 SNPs including rs2227981, rs7421861, rs11568821, rs36084323, rs2227982 and rs10204525 were genotyped and correlated with clinical-pathological characteristics and irAEs. Putative miRNAs binding to PD-1 SNP candidate were identified by in silico analysis. Validation of miRNA binding to PD-1 SNP allele specificity as well as evaluation of the induced PD-1 modulation was performed in vitro utilizing patient derived peripheral blood mononuclear cells (PBMCs). Susceptibility of non-cancer cells to immune cells incubated with anti-PD-1 based on PD-1 SNP allele specificity and miRNA modulation was performed by co-culturing non-cancer human epidermal keratinocyte (HaCaT) cells and bronchial epithelial BEAS-2B cells with human leucocyte antigen (HLA)-matched PBMCs, obtained from cancer patients treated with anti-PD-1/PD-L1-based immunotherapy therapy and carrying a different PD-1 SNP. Results Most of the analyzed PD-1 SNPs were not associated with the development of irAEs. In contrast, rs10204525 exhibited a significant association with the occurrence of both grade 1-2 and 3-4 irAEs in both cohorts of patients. Specifically, patients carrying C/C had higher rate of irAEs as compared to those carrying C/T. rs10204525 mapped on 3’-UTR region of PD-1. miR-4717-3p bound to rs10204525 based on its allele-specificity. Modulation of miR-4717-3p expression as well as of miR-4717-3p binding to rs10204525 differentially regulated PD-1 expression and induction in PMBCs harboring C/C or C/T genotypes as well as their ability to recognize and destroy HLA-matched HaCaT cells, even more in presence of anti-PD-1 therapy. Specifically, PBMCs carrying a C/T genotype displayed a significant lower ability to recognize and destroy non-cancercells as compared to those carrying C/C. These results were further validated by co-culturing of both BEAS-2B and HaCaT non-cancer cells with PBMCs carrying differential rs10204525 genotypes, isolated from additional cancer patients, incubated with anti-PD-1 or anti-PD-1 in combination with anti-CTLA-4 therapy. Conclusions These findings have high clinical relevance since define rs10204525 binding to miR-74 4717-3p-mediated PD-1 expression and induction as a mechanism modulating the reactivity of immune cells to non-cancer cells as well as a novel biomarker for predicting irAEs in advanced cancer patients treated with anti-PD-1/PD-L1-based immunotherapy.