Molecular Evaluation of RNA-Based Immune Checkpoint Profiling in Clear Cell Renal Cell Carcinoma

Background: Clear cell renal cell carcinoma is considered an immunologically "hot" tumor, with an abundance of tumor-infiltrating immune cells. This feature has a strong effect on therapeutic outcomes, especially in immune therapies. Within this context, immune checkpoint expression plays a central role in RCC immunotherapy, establishing new standards of care for patients with advanced RCC. However, most patients do not derive the maximum benefit from immune checkpoint inhibitors and may experience severe adverse effects. This may highlight the need to better understand the complexity of immunosuppressive mechanisms within the tumor microenvironment, which involves a variety of molecules. As such, this study aims to characterize the immune checkpoint status by investigating the co-expression (RNASeq) of the main checkpoints in a cohort of 16 ccRCC. Results: In the investigated cohort, the evaluation of immune checkpoint molecules revealed the complexity of immune regulatory mechanisms within the tumor microenvironment. The marked heterogeneity in the expression of eight major immune checkpoint actors, namely, PD1, PDL1, CTLA4, PDL2, VISTA, TIM3, TIGIT, and LAG3, is matched by multiple co-expressed immune checkpoint molecules, making the immune checkpoint burden score. Specifically, immune-cold tumors displayed the highest degree of co-expression, together with high levels of PDL2 and VISTA. Furthermore, the expression of immune checkpoints, both individually and collectively, was positively associated with intratumoral immune cytolytic activity, highlighting the coexistence of immune activation and immune suppression as tightly interconnected processes. Conclusions: The data presented here supports the concept that a ccRCC microenvironment may be driven by multiple modulatory mechanisms that allow immune escape to happen. Revealing the coordination of immune checkpoint molecules and the compensatory upregulation mechanisms may support future studies exploring multi-target immunotherapeutic strategies.