Introduction:

Clear cell renal cell carcinoma (ccRCC) tumors have comparatively low frequencies of genetic alterations, yet very high levels of immune cell infiltration and favorable response rates to immunotherapy (IT) relative to other malignancies. Currently, the interplay between specific ccRCC somatic mutations and immune infiltration pattern is unclear. Identification of significant associations between common ccRCC somatic mutations and immune cell infiltration within the tumor immune microenvironment (TIME) could be impactful in both the research and clinical settings. Our primary objective is to analyze the associations between the most frequent somatic mutations in metastatic ccRCC and immune infiltration patterns within the TIME.

Methods:

Tumor samples were obtained from patients with metastatic ccRCC. Targeted sequencing was used to identify the most frequent recurrent somatic mutations. Multiplex immunofluorescent (IF) tissue staining was used to assess TIME infiltration patterns within three distinct regions of interest (ROI): the tumor-core, adjacent stroma, and the tumor-stroma interface. Slides were sequentially stained in two panels, one for lymphoid and one for myeloid markers. Quantitative image analysis was utilized to generate counts for each cell by IF marker. For each tissue sample, cell density (cell count / total cell count) was determined for each IF marker and a subset of dual-positive markers. A linear mixed model analysis was performed to test associations between immune cell density for each IF marker at each of the three ROIs, and mutation status. Log-rank testing and multivariable Cox regression were used to analyze survival outcomes (Covariates: Age, gender, and IMDC risk score at diagnosis).

Results:

48 tumor specimens (24 primary, 24 metastatic) were obtained from patients with metastatic ccRCC. The most frequent somatic mutations were VHL (79.1%), BAP1 (41.7%), SETD2 (41.7%), PBRM1 (35.4%), TP53 (18.8%), and KDM5C (16.7%). For all sampled tumors, SETD2 mutations correlated with significantly decreased of FOXP3+ T cell density in the tumor-core (p=0.046) at the tumor-stroma interface (p=0.037), and in the stroma (p=0.046) (Figure 1).  Increased CD206+ and CD68+/CD206+ macrophage densities were found within the tumor-core in tumors with KDM5C mutations (p=0.013 and p=0.047, respectively). PBRM1 mutants were found to have decreased tumor-core FOXP3+ T regulatory cell density (p=0.022). VHL mutants had significantly decreased stromal CD3+/CD8+ and CD8+ cytotoxic T cell density (p=0.046 and p=0.033, respectively). On Cox multivariable regression analysis, patients with SETD2 mutations had significantly worse overall survival (p<0.01) and survival following immunotherapy treatment (p<0.01).

Conclusion:

This study provides evidence that common somatic mutations in ccRCC, such as SETD2, PBRM1, and KDM5C, may be associated with distinct immune infiltration patterns within the TIME. These novel associations have the potential to inform precision research and immunotherapeutic treatment strategies.

Funding: Urology Care Foundation Research Scholar Award Program and Society for Urologic Oncology (to BJM); the United States Army Medical Research Acquisition Activity Department of Defense (KC180139 to BJM);