Abstract A085: Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary clear cell renal cell cancer
Authors: Giraldo Nicolas A. , Etienne Becht , Lacroix Laetitia , Vano Yann , Sanchez-Salas Rafael , Validire Pierre , Verkarre Virginie , Mejean Arnaud , Dieu-Nosjean Marie-Caroline , Oudard Stephane , Cathelineau Xavier , Fridman Wolf H. , Sautès-Fridman Catherine 
Published: May 2016

Abstract
This study aimed to decipher the potential mechanisms linking increased CD8+ T cell infiltration with an adverse clinical outcome in ccRCC.
Clear cell Renal Cell Carcinoma (ccRCC) appears to be one of the most sensitive tumors to checkpoint blockade therapies. However, contrary to most cancers, some studies suggest that high densities of infiltrating CD8+ T cells correlate with patients' poor prognosis. In this study we determined: 1) the prognosis associated with the expression of immune checkpoints and its coordination with dendritic cell (DC) and CD8+ cell infiltration, and 2) the major phenotypic traits of CD8+ tumor infiltrating lymphocytes (CD8+ TILs).
The prognosis associated with CD8+ and dendritic cell (DC) infiltrations, in addition to the expression of immune checkpoints were investigated in a cohort of 135 primary ccRCC by quantitative immunohistochemistry (IHC). We found that the densities of CD8+, PD-1+ and LAG-3+ cells were closely inter-correlated, and independently associated with decreased PFS and OS. In addition, patients whose tumors presented both high densities of PD-1+ infiltrating cells and PD-L1+ and/or L2+ tumor cells (&gt;5% positive cells), displayed the worst clinical outcome [9% 5-year disease-free survival (DFS) vs. 64% in the other group, P&lt;0.0001]. These parameters were modulated by the presence, phenotype and localization of DC in the tumour microenvironment. High densities of immature DC isolated in the tumour stroma (DC-Lamp+/CD83dim/HLA-DRdim) were associated with high expression of immune checkpoints and patients' poor clinical outcome. In contrast, the presence of mature DC (DC-Lamp+/CD83bright/HLA-DRbright) within Tertiary Lymphoid Structures (TLS) (TLS-DC) identified, among the tumours with high CD8+ TIL densities, those with low expression of immune checkpoints and prolonged survival (73% DFS at 5-years compared to 10%, P=0.001). Analysis of gene expression data from the TCGA (n=495) demonstrated an association between the over-expression of CD8 TIL-related genes (CD8A, TBX21, IRF1, GZMB and IFNG) and patients' poor clinical outcome, in addition to a strong correlation between the expression of CD8A, PD-1, LAG-3, PD-L2 and IFNG genes in ccRCC.
These results suggest that in the absence of an immune response orchestrated by TLS-DC, the CD8+ T cells in ccRCC display an inhibited phenotype, associated with more advanced tumor stages and poor clinical outcome.
To functionally characterize the CD8+ T cell infiltrates in ccRCC, we investigated the major phenotypic traits of freshly isolated lymphocytes from 21 primary tumors obtained by partial or radical nephrectomy. Non-supervised analyses of the CD8+ TIL phenotype revealed two different types of tumors: The first group or “inhibited group” (8/21) was characterized by the over-expression of inhibitory molecules (PD-1 and TIM-3) and activation molecules (CD69 and CD38), and the expansion of the effector memory cell subpopulation (CCR7-CD45RA-) in the CD8+ TIL; the second group (14/21) was characterized by the expansion of the CD8+EMRA subpopulation (CCR7-CD45RA+) in addition to a reduced expression of activation or inhibitory markers. Preliminary data suggest that the “inhibited group” of tumors is characterized by more advanced tumor stages, higher Fuhrman grades and increased CD8+ cell densities (as determined by IHC), and thus shares major characteristics with tumors associated with patients' worst clinical outcome in the retrospective cohort. The functional orientation and cytotoxic potential of TIL are under investigation in both groups of tumors to further characterize the exhaustion and/or suppressive features of CD8+ T cells associated with poor clinical outcome in primary ccRCC.
In summary, we demonstrated that the infiltration with CD8+ TILs in ccRCC is accompanied by the enhanced expression of immune checkpoints in a subgroup of tumors displaying more aggressive features and advanced tumors stages. This immune profile defines a poor prognosis group of patients that should be suitable to receive immune checkpoint inhibitors.
Citation Format: Nicolas A. Giraldo, Becht Etienne, Laetitia Lacroix, Yann Vano, Rafael Sanchez-Salas, Pierre Validire, Virginie Verkarre, Arnaud Mejean, Marie-Caroline Dieu-Nosjean, Stephane Oudard, Xavier Cathelineau, Wolf H. Fridman, Catherine Sautès-Fridman. Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary clear cell renal cell cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A085.