Mass cytometry-based deep immune cell profiling of metastatic clear cell renal cell carcinoma patients refractory to combined immune checkpoint and tyrosine kinase inhibitor therapy.

576 Background: Combination of immune checkpoint inhibitors (ICIs) and tyrosine kinase inhibitors (TKIs) improved outcomes for patients with metastatic clear cell renal cell carcinoma (mccRCC). However, some patients experience rapid disease progression. Identifying specific immune phenotypes and predictive circulating biomarkers is essential for the early detection of patients who may be refractory to ICI-TKI therapy. Methods: Serum samples and peripheral blood mononuclear cells (PBMCs) were collected from patients classified as complete responders (CR, n=4), partial responders (PR, n=10), and non-responders (NR, n=8) after 15 days (T1) and 1 month (T2) of nivolumab (ICI) and cabozantinib (TKI) therapy. Serum cytokines were analyzed using LEGENDplex, while PBMC immunophenotyping was performed using CyTOF to assess 37 immune cell populations and T cell markers related to activation, migration, and exhaustion. Results: In both responding and non-responding patients a common significant 90% reduction of PD1+TM3-CD8+ cells (p=0.0096 and p=0.0001, in CR and NR, respectively) and dendritic cells (decrease of 47% p=0.0004 in CR, 70% p=0.0059 in PR and 62% decrease p=0.0396 in NR) following therapy was observed. PD1+ Treg cells decreased significantly (93% decrease, p=0.0068) only in the CR after therapy. Additionally, CRs exhibited higher levels of exhausted PD1+ Treg cells (2.88 fold, p=0.0084) at baseline and an increased number of pro-inflammatory CD4+ Th2 cells (2 fold, p=0.0170) after one month compared to the other groups. PRs and NRs showed lower levels of natural killer cells (2.2 fold, p=0.0069), and a significant decrease of monocytes populations after therapy (45% decrease p=0.0008 in PRs, 40% decrease p=0.0012 in NRs). The CRs exhibited elevated levels of IL-23 (18 fold higher vs. NRs and 3 fold higher vs. PRs, p= 0.0094; p=0.0011) and IL-27 at baseline , and higher IL-15 and IL-18 levels post-therapy compared to the PR and NR. Kaplan-Meier plot analysis indicated that higher IL-23 levels correlated with an improved probability of PFS in all cancer patients treated with nivolumab (2.97 vs. 29.7 months in low vs. high expression cohorts; HR= 0.53 [95% CI 0.33 - 0.85], log-rank P = 0.0074). Conclusions: Our results suggest that patient’s initial immune profile might be significant predictor of treatment success and the likelihood of developing resistance.