Abstract PO-034: Non-random spatial clustering of tumor associated macrophages is associated with poor survival in metastatic RCC patients receiving immunotherapy
Authors: Kimmel Gregory J. , Chakiryan Nicholas H. , Kim Youngchul , Hajiran Ali J. , Aydin Ahmet M. , Nguyen Jonathan V. , Moran-Segura Carlos M. , Dhillon Jasreman , Altrock Philipp , Manley Brandon J. 
Published: December 2020

Abstract
INTRODUCTION Tumor associated macrophages (TAM) stimulate tumor proliferation and facilitate immune escape via production of immunosuppressive cytokines. We hypothesize that non-random spatial clustering of TAMs within the tumor is associated with poor survival in RCC patients. METHODS Primary and metastatic tumor specimens were obtained from 39 patients with metastatic RCC who received immunotherapy (IT). 29 patients (74.3%) also received targeted therapy (TT) at some point during their disease course. Sections from the tumor compartment underwent immunohistochemical (IHC) staining for pan-macrophage marker CD68, and M2 markers CD163 and CD206. Digital pathologic analysis was used to convert the digital images to spatial point pattern plots (PPP). A probability density function (PDF) for the distance between cells was calculated under the assumption that cells can be located anywhere in the slide with equal probability. The PDF was then compared to empirical histograms of cells derived from the PPP. Deviations from the PDF, the expected random distribution, reveal evidence of cell clustering or dispersion. We assessed deviations with the Kolmogorov-Smirnov (KS) test. Patients were stratified by the median value into high and low KS groups, and survival outcomes were compared between the two groups using a Cox regression. A high KS statistic indicates a good fit between the two curves (in this case, randomness), and a low KS statistic indicates a poor fit (in this case, dispersion or clustering). RESULTS Among primary tumor specimens, spatial clustering of CD68+ cells was associated with worse overall survival (OS) (HR -1.7, p=0.015), worse OS from IT initiation (HR -1.5, p=0.025), and no difference in OS from TT initiation (HR -0.4, p=0.48). Among metastatic tumor specimens, spatial clustering of CD68+ cells was not associated with any of the three survival outcomes (OS: HR 0.9, p=0.11; OS from IT: HR 0.7, p=0.20; OS from TT: HR 0.0, p=0.97). Spatial clustering of CD163+ cells was not associated with any of the three survival outcomes for either primary and metastatic sites. Spatial clustering of CD206+ cells was not associated with any of the three survival outcomes for either primary and metastatic sites. CONCLUSION Non-random spatial clustering of CD68+ TAMs in the primary tumor specimen was associated with worse survival, driven primarily by worse survival from initiation of immunotherapy. These findings corroborate prior reports of TAMs eliciting an immunosuppressive effect on the tumor-immune microenvironment, introduce novel methodology for quantifying spatial heterogeneity, and demonstrate the novel finding of a clinically significant effect of TAM spatial clustering on overall survival.
Citation Format: Gregory J. Kimmel, Nicholas H. Chakiryan, Youngchul Kim, Ali J. Hajiran, Ahmet M. Aydin, Jonathan V. Nguyen, Carlos M. Moran-Segura, Jasreman Dhillon, Philipp Altrock, Brandon J. Manley. Non-random spatial clustering of tumor associated macrophages is associated with poor survival in metastatic RCC patients receiving immunotherapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-034.