Background 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 are associated with poor survival in ccRCC patients.
Methods Tumor specimens were obtained from 41 patients with metastatic ccRCC who received immunotherapy (IT). Sections from the tumor core underwent multiplex immunofluorescence staining for CD68, CD163, and CD206. Digital pathologic analysis was used to convert the digital images to spatial point pattern plots (PPP). Ripley’s K function, the current standard metric for spatial heterogeneity, was utilized. Novel metrics were developed using a probability density function (PDF) for distances between cells, assuming that cells can be located anywhere with equal probability. Empirical histograms were generated from the PPPs. Deviation from the PDF demonstrates a non-random distribution. Deviations were quantified with the Kolmogorov-Smirnov (KS) test and Cramér-von Mises (CVM) criterion. Overall survival (OS) was assessed between groups stratified by the median value for each metric using Kaplan-Meier and log-rank analysis. Figure 1A.
Results 75 slides were analyzed from the 41 patients. The three metrics for measuring spatial heterogeneity had moderate and statistically significant correlation with each other (Spearman’s R: Ripley/KS=0.68, p<0.01; Ripley/CVM=0.54, p<0.01; KS/CVM=0.47, p<0.01; figure 1B). Using CVM, increasingly non-random distribution of the Tumor-CD68+ cell relationship was associated with worse OS (p<0.01, figure 1C), and increasingly non-random distribution of CD163+ cells suggested an association with worse OS without reaching statistical significance (p=0.06, figure 1C). No statistically significant associations were identified using the KS or Ripley’s K metrics.
Conclusions We describe CVM and KS as novel metrics for measuring spatial heterogeneity of immune cells. Increased spatial heterogeneity of CD68+ TAMs and tumor cells was associated with worse OS in patients with metastatic ccRCC who received IT. These findings corroborate prior reports of TAMs eliciting an immunosuppressive effect on the tumor-immune microenvironment, and demonstrate the novel finding of a clinically significant effect of TAM spatial clustering on OS.
This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/licenses/by/4.0/.
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.