Spatial profiling of the inflamed tumor microenvironment identifies novel prognostic subclasses in nonmetastatic clear cell renal carcinoma patients

Abstract INTRODUCTION: Clear cell renal cell carcinoma (ccRCC) is highly vascularized and inflamed, and RNA signatures of angiogenesis/inflammation have been suggested as biomarkers for predicting progression and therapy benefit. Here, we explored as to how the spatial localization of endothelial cells and leukocytes and their mutual interactions contribute to disease progression. EXPERIMENTAL PROCEDURES: We applied multiplexed fluorescence IHC (mfIHC) to quantify CD45+ leukocytes, CD31+ endothelial cells, D2-40+ lymphatic cells, CD11b+ myeloid cells, CD3+ and CD20+ lymphocytes, as well as PDGFRB+ fibroblasts and epithelial cells (CK+/Ecadherin+/CAIX+) in two cohorts of non-metastatic primary ccRCC. Both the Helsinki (N=178) and Turku cohorts (N=241) contained TMA cores from tumor center, tumor border, and benign areas of each patient. mfIHC data were compared with clinical parameters, such as tumor stage (pT), Fuhrman grade, necrosis, and patient outcome (RFS, recurrence-free survival). RESULTS: CD31+ endothelial cells were associated with favorable (p<0.001) and CD45+ leukocytes with poor prognosis (p<0.001), high pT (p<0.05), high Fuhrman grade (p<0.05), and high necrosis (p<0.05) in TMA cores representing the tumor center and tumor border areas. CD45+ cells varied spatially with regards to CD31+ cells and EpiStain+ cells. For example, in tumor center cores, CD45+ cells co-localized with EpiStain+ cells ranging from 0.1 to 97% (median for both cohorts, 70.6% and 72.5%). The association of CD45+ leukocytes with poor prognosis was significant when counting the fraction of CD45+ cells not interacting with CD31+ or EpiStain+ cells, but not when including only the interacting cells. We then categorized patients into three classes based on the quantity of the cell types: 1) CD45+High/CD31+Low, 2) CD45+Low/CD31+High, and 3) Other. CD45+High/CD31+Low phenotype patients had a significantly lower 5-year RFS rate compared to that of CD45+Low/CD31+High patients (Helsinki: 37.9% vs. 92.0%; Turku: 50.0% vs. 93.5%; p<0.001, Log-rank test). CONCLUSIONS: In ccRCC, both the quantity and spatial localization of inflammatory cells with respect to vascular endothelial cells and tumor cells dictates its association with patient prognosis. Our results suggest that although CD45+ leukocyte infiltration associates with recurrent disease, the higher risk is only observed when CD45+ cells are not in contact with vasculature or tumor cells. This suggests how analysis of well-known prognostic biomarkers by IHC can be refined by using mfIHC and considering the spatial configurations of cells in the TME in ccRCC. Ongoing work is aimed to add further profiling of additional TME cell subset associations with inflammation, prognosis, and therapy response. Citation Format: Teijo Pellinen, Lassi Luomala, Kalle Mattila, Jenni Säilä, Annabrita Hemmes, Katja Välimäki, Oscar Bruck, Lassi Paavolainen, Harry Nisen, Petrus Järvinen, Olli Kallioniemi, Paula Vainio, Panu Jaakkola, Tuomas Mirtti. Spatial profiling of the inflamed tumor microenvironment identifies novel prognostic subclasses in non-metastatic clear cell renal carcinoma patients. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4459.