THE PROGNOSTIC ROLE OF THE IMMUNE TUMOR MICROENVIRONMENT IN METASTATIC RENAL CELL CARCINOMA PATIENTS TREATED WITH IMMUNOTHERAPY (Meet-URO 18 I-TME study)

Background: Despite the plethora of new treatments available in metastatic renal cell carcinoma (mRCC), there is a pressing need to identify clinical and translational parameters that could select patients most likely to benefit from immunotherapy. Therefore, the personalization of cancer therapy is a crucial clinical issue which drives the preclinical and clinical assessment of prognostic and predictive factors in mRCC patients receiving immunotherapy. Clinical factors have the advantage of being easily assessable and applicable, but great interest and effort have been spent on translational research, especially on the investigation of the immune tumor microenvironment (I-TME). Methods: The Meet-URO 18 is a multicentric retrospective study assessing the I-TME of mRCC patients treated with ≥2nd line nivolumab, dichotomized into responders and non-responders according to clinical benefit [progression-free survival ≥12 months and ≤3 months, respectively]. The primary objective was to identify differential immunohistochemical (IHC) and molecular patterns between the two groups. The analyses were performed on tumor samples of primary tumor or metastases. Here, we report the preliminary IHC analyses, secondary IHC analyses and preliminary molecular analyses. The immunohistochemical analyses included the grading assessment, the histological revision and the digital multitarget analysis of immune infiltration and tumor cells. This latter included the assessment of the lymphocyte infiltration (CD3+, CD8+, CD4+ and FOXP3+ T cells, CD8+/CD4+ ratio and peri/intra-tumoral T cells), expression of specific molecules in tumor cells (CD56, CD15, CD68 and ph-mTOR) and PD-L1 expression. The molecular analyses were conducted using the analytical platform NanoString nCounter®, which analyzed the expression of a panel of 71 genes on microRNA (miRNA): 5 housekeeping transcripts and 66 endogenous miRNAs from the gene signature developed by D’Costa et al., which are involved in: Angiogenesis, Immunomodulation mediated by T-effector response, Mechanisms of tumor invasion, Mechanisms of calcium channel flows. Results: In the primary analyses, 116 tumor tissue samples from 84 patients (59% primary tumors, 41% metastases) were evaluated. Samples from responders (N = 55, 47%) were significantly associated with lower expression of CD4+ T lymphocytes (p = 0.015) and higher levels of ph-mTOR (p = 0.029) and CD56+ (p = 0.035) compared to samples from non-responders (N = 61, 53%). Responders showed also a higher CD3+ expression (p = 0.059) and CD8+/CD4+ ratio (p = 0.084). Non-responders were significantly associated with a higher percentage of clear cell histology (p = 0.005) and grading (p < 0.05). In the secondary analyses, 161 tumor tissue samples from 113 patients (57% primary tumors, 43% metastases) were evaluated. Responders’ tumor tissue (N = 90, 56%) was associated with lower CD4 expression (p = 0.014), higher CD56 expression (p = 0.046) and a higher CD8/CD4 ratio (p = 0.03). In the molecular analyses, correlating the mRNA levels of all 66 genes with response to therapy, the mRNA of the Fas Ligand gene (FASLG) was found to be significantly more expressed in responders’ tumor tissue. In our samples, we also identified the four molecular signatures developed by D’Costa et al. together with the dichotomization into two major pathways: the Angio-Invasion pathway and the Immuno-Invasion pathway. Considering the correlation between molecular signatures and the response to therapy, the Angiogenesis signature resulted significantly over-expressed in non-responder patients compared with responder ones (p = 0.035). Conclusions: Our study reported interesting results and differential IHC and molecular patterns between tumors of patients responder and non-responder to nivolumab were identified. Responder patients have a more immunological phenotype with a distinct T-lineage profile and an interesting role of ph-mTOR pathway, CD56 on tumor cells and FasL gene expression, while non-responder patients have a more angiogenic phenotype with higher clear-cell histology, grading and angiogenesis signature. Integration of IHC and molecular analyses are planned to deeply examine the prognostic role of I-TME. Moreover, further investigations on a larger population will be conducted to confirm our results.