Abstract A085: High infiltration of NK cells expressing elevated LAG-3 in a subgroup of renal cell carcinoma patients
Authors: Lee Moon Hee , Järvinen Petrus , Nisén Harry , Brück Oscar , Ilander Mette , Mustjoki Satu , Anna Kreutzman 
Published: February 2019

Abstract
Background: Renal cell carcinoma (RCC) is considered one of the most immunogenic cancers with the highest number of indel mutations, frequent infiltration of T-cells, presence of many antigen-specific T-cell clones, and high immuno-oncologic (IO) sensitivity. Since less is known about natural killer (NK) cells in RCC, our primary aim was to investigate the intratumoral phenotype of NK cells as well as further assess the overall immune landscape of the tumors, peripheral blood (PB) and adjacent healthy kidney tissue, which may be critical for patient prognosis and predictions to targeted immunotherapies. Methods: We used multi-parameter flow cytometry together with a comprehensive immunostaining panel containing a total of 56 fundamental markers to cancer immunology to immunophenotype the tumor, adjacent healthy kidney tissue and presurgical peripheral blood (PB) samples from 31 RCC patients who underwent partial or radical nephrectomies. To study the intratumoral T-cell clonalities, T-cell receptor beta (TCRβ) deep sequencing was carried out with eight tumors. Results: Using hierarchical clustering (Spearman correlation distance and Ward linkage) and correlation analyses (Spearman correlation), we discovered that our patient cohort clustered into two distinct subgroups defined by a high (NKhigh, n=11; mean 29.7%) and low (NKlow, n=20; mean 9.4%) percentage of NK cells among the intratumoral lymphocyte population. Accordingly, the NKhigh subgroup had a lower percentage of T-cells (mean 36.9%) than the NKlow group (mean 65.7%), and overall, a significant negative correlation between T and NK cells was discovered. Our TCRβ sequencing results revealed a positive correlation between T-cell clonality and the intratumoral T-cell percentage, whereas the higher proportion of tumor NK cells associated with low T-cell clonality, possibly due to a polyclonal T-cell population. When we compared the expressions of the most clinically relevant IO markers (LAG-3 and PD-1) on the NK cells, LAG-3 was more expressed in the NKhigh group than in the NKlow group (21.3% vs 9.8%; p=0.08). In contrast, no differences were observed with PD-1. Clinical parameters such as tumor grade (Fuhrman), weight, size (diameter), the presence of necrosis, gender, or age of the patients did not differ between the two subgroups. To examine the overall immune landscape of RCC, we compared the cells from the tumor, PB, and healthy kidney tissue of seven patients. Our results showed that tumors have more NK cells compared to their corresponding T-cell-rich PB and healthy tissue counterparts, supporting our findings that some tumors accumulate NK cells. Compared to the adjacent healthy tissue, PD-1 and LAG-3 expressions were higher in the intratumoral CD8 cells. The expressions of PD-1 and LAG-3 on PB CD8+ T-cells or NK cells did not correlate with their intratumoral counterparts, whereas a positive correlation was found between the PB and tumor CD4+ T-cells for both LAG-3 and PD-1. Conclusions: Our study has led to the discovery of two distinct RCC tumor subgroups with differential expressions in the clinically leading molecules. These results suggest that immunophenotyping RCC patients may effectively aid in selecting those who will benefit the most from immune checkpoint inhibition therapies such as anti-PD1 and -LAG3. Prospective analyses on spatial immunoprofiling by multiplexed immunohistochemistry and mutational load by exome sequencing will be assessed next in order to find answers to why some tumors are NK-dominant, with expectations of understanding further the biologic differences between the two tumor types.
Citation Format: Moon Hee Lee, Petrus Järvinen, Harry Nisén, Oscar Brück, Mette Ilander, Satu Mustjoki, Kreutzman Anna. High infiltration of NK cells expressing elevated LAG-3 in a subgroup of renal cell carcinoma patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A085.