Abstract P2-09-06: Quantitative spatial profiling of tumor associated macrophages and the PD-1/PD-L1 interaction in breast cancer
Authors: Pelekanou V , Neumeister V , Pusztai L , Rimm DL 
Published: February 2018

Abstract
Background: Although immunotherapy approaches are being successfully administered in some breast cancer (BC) patients (pts), biomarkers of response remain elusive. Tumor associated macrophages (TAMs) are the most prominent immune cells in breast tumors, mediating the cross-talk between tumor cells and tumor infiltrating lymphocytes (TILs). Specific biomarkers of breast TAMs' functional status remain to be defined. CSF-1R, is a TAMs regulator and key target across cancers clinically tested, alone or combined with anti-PD-1 checkpoint inhibitors.
The goals of the study were: 1) to objectively measure CSF-1R expression within all CD68+ and M2-like CD163+TAMs, as well as PD-L1/PD-1 spatial interaction and 2) to determine whether objectively quantifying these key immune mechanisms related to TAMs immunomodulation within the tumor microenvironment can predict outcome and potentially response to immunotherapy.
Methods: Tissue Microarrays (TMAs) from two Yale BC cohorts (Cohort A, all breast cases, n=320) (Cohort B, TNBC, n=132) were assessed by quantitative immunofluorescence (QIF) for CSF-1R/CD163/CD68; PD-1/PD-L1 interaction score (proportion of PD-1+ cells co-localized with PD-L1) and co-expression of the multiplexed biomarker panels. Biomarker positive cells and their co-localization were objectively measured using the AQUA method of QIF. QIF scores were compared by linear regression coefficients (R2). Overall and recurrence-free survival (OS and RFS) were assessed. Our protein data were compared with transcriptome data from the METABRIC study obtained from www.cbioportal.org.
Results: CSF-1R expression was associated with expression of both CD68 and CD163 in both cohorts (A: R2=0.64, B: R2=0.49). CSF-1R in CD163/CD68 was higher in TNBC Cohort B (p&lt;0.01) and in ER- cases of Cohort A (p=0.004). In Cohort A high CSF-1R expression (top 10%) in CD163+/CD68+ cells was associated with worse OS and RFS (All cases, p=0.015 and p=0.0005, respectively). After ER-status adjustment, high CSF-1R (in CD68 and CD163) was associated with worse OS and RFS only in ER- cases. High CSF-1R/CD68 (top 10%) was associated with ER- (p=0.0078), PR- (p=0.004) and increased recurrence rate (p=0.009). High CSF-1R/CD163 (top 10%) was also associated with increased recurrence rate (p=0.004). In TNBC, high CSF-1R correlated with worse OS (p=0.01) only in CD163+ TAMs. High CSF-1R scored as a continuous variable was related with worse RFS in both CD68+ (p=0.0026, RR 1.00/2.86) and CD163+ TAMs (p=0.006, RR 1.00/2.76). However, in multivariate analysis CSF-1R was not an independent prognostic factor for OS or RFS.
PD-L1 mostly co-localized with CD68 TAMs (R2=0.7). Tumor PD-L1 tended to be mutually exclusive of CSF-1R. PD-L1/PD-1 colocalization was higher in TNBC (p&lt;0.01) and associated with better OS (p=0.01). CSF-1R in TAMs tended to be higher when PD-1/PD-L1 colocalization was low.
The trend of mutual exclusivity between CSF-1R in TAMs and PD-1/PD-L1 was confirmed by expression (mRNA) data from METABRIC study.
Discussion: This novel multiplexed method profiling key tumor-immune suppression pathways could identify BC pts likely to respond to anti-PD-1/anti-CSF-1R therapy. This method could help stratify pts for mono- or combined therapy in future clinical trials.
Citation Format: Pelekanou V, Neumeister V, Pusztai L, Rimm DL. Quantitative spatial profiling of tumor associated macrophages and the PD-1/PD-L1 interaction in breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-09-06.