Circulating CD137⁺Treg cells and LOX-1⁺PMN-MDSCs as biomarkers of immunotherapy resistance in (R/M) HNSCC patients

Background: Recurrent/metastatic head and neck squamous cell carcinoma ((R/M) HNSCC) represents one of the most aggressive and immunosuppressive cancers. Despite the introduction of immunotherapy, only a limited number of patients obtain long-term benefits. In (R/M) HNSCC patients, the antitumor immune response is defective, conferring resistance and promoting tumor progression. Therefore, identifying novel biomarkers for better clinical outcomes and easily accessible in standard clinical settings is still an unmet clinical need. Aim: In this study we evaluated, at baseline and during therapy, the levels of circulating immunosuppressive regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs), to investigate their impact as potential biomarkers of immunotherapy resistance. Methods: The levels of Tregs and MDSCs, obtained from blood samples of 80 (R/M) HNSCC patients (40 discovery cohort, 40 validation cohort) undergoing pembrolizumab therapy, were analyzed by flow cytometry, at baseline and during therapy. Correlation between these immunosuppressive cell subsets and clinical parameters were performed along with immunosuppressive assays to evaluate functional mechanism of isolated LOX-1⁺PMN-MDSCs. Results: Univariate analysis showed that higher circulating levels of both CD137⁺Tregs and LOX-1⁺PMN-MDSCs, at baseline, identified patients with significantly worse survival. CD137⁺Tregs resulted also positively correlated with Performance status (p=0.0004), while high levels of LOX-1⁺PMN-MDSCs negatively affected response to pembrolizumab (p=0.021), showing a significant increase in non-responsive patients during therapy (p=0.047). Moreover, functional characterization of LOX-1⁺PMN-MDSC isolated from (R/M) HNSCC patients revealed a significantly stronger immunosuppressive capacity, and an increased production of Arginase-1 and ROS than LOX-1⁻PMN-MDSC. The circulating LOX-1⁺PMN-MDSC subset resulted an independent prognostic factor for survival by multivariate analysis, as confirmed in an independent validation cohort (PFS: p=0.007; OS: p=0.001). Conclusions: The levels of circulating CD137⁺Tregs and LOX-1⁺PMN-MDSCs may be proposed as non-invasive biomarkers to predict clinical outcomes of (R/M) HNSCC patients developing resistance to immunotherapy, improving patient selection and suggesting novel personalized therapies.