Evaluation of immune exhaustion across the evolution of oligometastatic clear cell renal cell carcinoma using spatially resolved tissue immune profiling: Secondary analysis of a prospective trial.
Authors: Hara Kieko , Corredor-Alonso Guillermo E , Serrano Alejandra G , Hernandez Sharia D , Lu Wei , Shah Amishi Yogesh , Jonasch Eric , Karam Jose A. , Matin Surena F. , Tannir Nizar M. , Msaouel Pavlos , Sircar Kanishka , Solis Luisa Maren , Tang Chad 
Published: March 2023

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Background: In patients with clear cell renal cell carcinoma (ccRCC), progressive local dysfunction in the tumor immune microenvironment (TIM) has been shown to play a significant role in clinical outcomes and response to therapy. Patients with oligometastatic ccRCC may particularly benefit from localized treatment such as radiation therapy. However, immune cell dysfunction in TIM and its influence on patient outcomes in oligometastatic ccRCC has yet to be defined. To address this unmet need we conducted spatially resolved immune profiling on longitudinally collected patient samples from our ongoing prospective phase II trial (NCT03575611) of radiation therapy in oligometastatic ccRCC. This methodology offers a unique multidimensional evaluation of TIM in relation to histological architecture. Methods: Thirty-seven FFPE tumor samples from 32 patients with ccRCC (primary, 21; oligometastatic, 16) were obtained to construct a tissue microarray (TMA) and assess tumor immune activation biomarkers using the nanoString GeoMx Digital Spatial Profiler (DSP). We used the pan-cytokeratin, CD3, CD68, and SYTO13 as morphology biomarkers to profile, in each TMA core, a panel of 49 protein immune biomarkers in each of these compartments (DSP areas of illumination/AOI): tumor (panCK+), T-cell (CD3+), and macrophage (CD68+). In addition, DSP Immunofluorescence images were used to calculate cell density, and cell-to-cell distances of T cell and Macrophage. A median of 7 AOIs (range 3-9) were assessed in each tissue. Comparisons were made between primary and metastatic tissues by Mann-Whitney test with correction false discovery rate of 1%. Regression models were used to evaluate association between protein counts within each compartment and clinical outcomes. Results: In T-cells, CD127 was significantly higher in primary ccRCC tissue (p=0.00009) while FOXP3 and LAG3 were significantly increased in oligometastatic tissue (p=0.001). Macrophages in oligometastatic tissue expressed higher CD163 and ARG1 (p=0.01 and 0.02, respectively), compared to those in primary tissue. Average distances between macrophage and the nearest T cell were significantly shorter in oligometastatic tumor tissue compared to those in primary ccRCC (22.9 vs 28.8 µm, p=0.04). Higher PD-1 (R2=0.26) with lower CD127 (R2=0.22) and CD27 (R2=0.21) expression in T cells in primary tumor predicted earlier time to first diagnosis of metastases after nephrectomy (all p<0.05). Conclusions: Spatially resolved immune profiling revealed that the TIM in metastatic tumor harbored more T cells exhibiting markers of suppression with closer spatial interaction to macrophages compared with primary tumors. Furthermore, TIM with T cell exhaustion in primary tumors predicted earlier diagnosis of metastasis after nephrectomy.