Abstract P4-03-04: Tumor infiltrating macrophages, lymphocytes and matrix metalloproteinase 9 (MMP-9) expression in breast cancer
Authors: Pelekanou V , Brown JR , Rimm DL 
Published: March 2017

Background: Immune therapy has been highly successful in tumors with high lymphocytic infiltrate, but they only represent the minority of breast neoplasms. Macrophages rather than lymphocytes, are more prominent in mammary development and disease. Specific markers of breast tumor associated macrophages (TAMs) remain to be defined. Local interactions define their plasticity and activity, rendering in situ investigation important in their characterization. MMP-9 is an important regulator of breast cancer microenvironment that could mediate cross-talk between TAMs and tumor cells. Here we objectively measure CD68 and CD163 and also MMP-9 within each macrophage subtype to determine the relationship between macrophage expression, tumor infiltrating lymphocytes (TILs) and molecular subtypes in breast cancer.

Methods: Using a multiplexed quantitative immunofluorescence (QIF)-based assay for simultaneous detection of DAPI (all cells), Cytokeratin (epithelial cells, clone CK8/CK18), CD163 (M2 Macrophages, clone CD163-L-U), CD68 (pan macrophage marker, clone PG-M1), and MMP-9 (Matrix Metalloproteinase 9, clone D6O3H XP). We measured the levels of protein expression in breast carcinomas on two sets of Yale tissue microarrays (TMA) [YTMA201 (all breast cases, n=399) and YTMA149 (Triple Negative, n=160)]. Markers were measured using the AQUA method of QIF on TMAs at two-fold redundancy. Linear regression coefficients (R2) were used to compare antibody QIF scores within cores from different areas of the tumors. Median cut-point was used to stratify patients for overall and disease specific survival (OS and DSS).

Results: Cases with high TILs, as shown by assessment of CD3, 8 and 20, generally show an inverse relationship with both CD68 and CD163, especially in ER+ cases. MMP-9 was then measured in both subtypes of macrophages. In ER+ tumors MMP-9 was expressed in CD163+ macrophages (p=0.007), while in TNBC it was found in CD68+/CD163- macrophages (p&lt;0.001). In all cases MMP-9 was significantly higher in ER- cases (CD68+/CD163- p=0.0001), (CD163+ p=0.01). In ER+ cases high MMP-9 expression was associated with shorter OS (p&lt;0.0001 in CD163+ cells). On the contrary, in TNBC high MMP-9 was associated improved DSS in the CD68 compartment (p=0.007).

Discussion: Using an objective, quantitative multiplex assay for synchronous measurement in tumor and microenvironment, we found an inverse relationship between TILs and macrophage infiltration, suggesting immune modulation by different cellular elements. Within the macrophage population, we found that MMP9 expression is a function of the breast cancer molecular phenotype. Most significantly, the ER status of the tumor is correlated with the macrophage subtypes that express MMP9. Efforts to determine the clinical value of these observations are underway to better determine the balance between pro- and antitumor immunity in breast cancer.

Citation Format: Pelekanou V, Brown JR, Rimm DL. Tumor infiltrating macrophages, lymphocytes and matrix metalloproteinase 9 (MMP-9) expression in breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-03-04.