Abstract 6553: Ex vivo investigation of estrogen receptor-related macrophages polarization in breast cancer and its prognostic significance

Breast cancer (BC) is the second most frequent cause of cancer-related deaths. In clinical practice, ER, PR and HER2 assessment in histopathological samples divides BC into biological subtypes and guides treatment decisions. While these subtypes reveal key insights on prognosis they crucially also exhibit distinct immune environments, with ER-positive disease being considered ‘immune-cold’. Recently, though immunotherapy has shown efficacy in treating ER-negative tumors, there is a lack of biomarkers that may select patients as good candidates for immunotherapy treatment in BC. In this study, we characterized the immune milieu across tumors of different ER-positivity, challenging the canonical breast cancer ER+/ER- dichotomy. A publicly available RNA-seq dataset (TCGA BRCA) with 481 breast cancer patients and complete information on ER percentage IHC status was analyzed in R Studio (v 4.4.0). We demonstrated that patients with ER IHC expression below 50% (ER-low: <10% and ER-intermediate: 10-49%) exhibit worse prognosis than those with ER-high (above 50%, p-value = 0.0082), throwing doubt onto the sufficiency of the contemporary therapy in ER+ disease for patients of lower ER expression. We have further investigated immune enrichment using the quantiseqr package, and observed gradual changes in immune fractions across three clusters of ER expression (<10%, 11-49%, >50%). While macrophages constitute the most numerous population in ER-high and ER-intermediate tumors, for ER-low patients’ cell proportions are more balanced: the fraction of M2 macrophages, B-cells and CD4+ T-cells is reduced whereas macrophages M1, neutrophils, and T-reg increase. Differences between mean abundances in high vs low clusters remain statistically significant for all measured cells except for NK cells. We also noticed effects for M1- and M2-like macrophages by underlying ER expression: the higher the ER expression the more relative abundance of M2 cells, with moderate positive correlation across the tumors with increasing ER deciles (Spearman correlation r = 0.4, p-value < 0.0001). Conversely, M1-like cells decrease at once. Median relative abundance of macrophages M1 had prognostic value in the entire cohort of breast tumors (log-rank test p-value = 0.041), what mostly originated from tumors of high ER levels (n = 151 vs 193, p-value=0.0089), and did not achieve statistical relevance in any other subgroup. While recent in-vitro research postulated existing difference of macrophages’ populations in cancers of distinct ER expression, and in environment of variable estrogen levels, this study demonstrates the fluctuation of ER-related M1- and M2-like macrophage proportions in clinical samples. We moreover provide evidence that M1-like macrophages have prognostic relevance in the ER-high subgroup. Further experiments detailing mechanisms of the observed phenomenon are underway. Citation Format: Daria Kalinska-Lysiak, Katarzyna Zielniok, Zak Kinsella, Radoslaw Zagozdzon, Darran O’Connor. Ex vivo investigation of estrogen receptor-related macrophages polarization in breast cancer and its prognostic significance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6553.