The M2/Alternatively Activated Macrophage Phenotype Correlates with Aggressive Histopathologic Features and Poor Clinical Outcome in Early Stage Breast Cancer.
Authors: Khramtsova G. , Liao C. , Khramtsov A. , Li S. , Gong C. , Huo D. , Nanda R. 
Published: December 2010

Abstract
Background: Angiogenesis is critical for breast cancer progression. Within tumors, non-neoplastic cells assist tumor growth by producing growth factors and pro-angiogenic cytokines. Studies have demonstrated that tumor-associated macrophages (TAMs) are recruited to tumors before malignant conversion and are essential for promoting angiogenesis. We sought to study the role that macrophage phenotype—classically activated (M1) and alternatively activated (M2)—plays in the subsequent activation of the angiogenic pathway, with the goal of understanding the mechanisms underlying angiogenesis in the different molecular subtypes of breast cancer.Methods: 128 matching breast tumors, DCIS and normal tissues were obtained from the University of Chicago Breast Cancer SPORE tissue bank under IRB approved protocols. Tissue microarrays were constructed and molecular subtype was assigned based on immunohistochemical (IHC) staining into the following groups: luminal A (ER+, PR+, HER2-), luminal B (ER+, HER2+ or ER+, PR-), HER2-like (ER-, HER2+) and basal-like (ER-, HER2-, EGFR+ and/or CK5/6+). Macrophage phenotype was determined using double staining with CD68/CD163 (M2) and CD68/CD80 (M1). Microvessel density (MVD) was measured by IHC staining using anti-CD34. Staining quantification was performed independently by two pathologists. To control for intra-individual correlation, linear mixed-effects models were used to compare differences in % of M1 and M2 with disease progression. To evaluate the association of MVD with % of M1 and M2, bivariate plots were generated and Pearson's correlation coefficients were calculated. Spearman's correlation coefficients were used for the correlation between macrophage phenotype and tumor stage and grade. The Kaplan-Meier method was used to calculate overall survival.Results: Of the tumors studied, 88% were stage I-II. 17% were grade 1, 39% grade 2 and 44% grade 3. 70 were luminal A, 36 basal-like, 9 HER2-like and 6 luminal B. The ratio of M2:M1 increased with disease progression from normal breast to DCIS to invasive cancer (p&lt;0.001). Increased M2% was associated with high tumor grade, increased MVD and decreased overall survival (all p&lt;0.001). M1% was associated with low tumor grade (&lt;0.001), but was not significantly associated with MVD or overall survival. Both the HER2-like and basal-like subtypes have significantly higher % M2 as compared to the luminal A subtype (p&lt;0.001).Discussion: There are several studies which suggest that activated TAMs are responsible for the secretion of pro-angiogenic cytokines which stimulate neovascularization. To our knowledge, this is the first study that has correlated macrophage phenotype to breast molecular subtype and MVD in human breast tumors. Our findings suggest that the M2 macrophage phenotype is associated with aggressive histopathologic features and poor clinical outcome. Inhibiting the M2 macrophage may prevent the release of pro-angiogenic factors, and might be an effective approach at preventing neovascularization and improving patient outcomes.
Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 107.