OR30-07 Reconfiguring the Tumor Microenvironment: Immune Dynamics and NET Response in ER⁺/HER2-Negative Breast Cancer

Abstract Disclosure: G. Oner: None. M. Praet: None. M. Morse: None. S. Altintas: None. S. Koljenovic: None. N. Canturk: None. P. van Dam: None. G.R. Devi: None. Background: Neoadjuvant endocrine therapy (NET) is widely used for ER⁺/HER2-negative breast cancer as it reduces tumor burden and modulates the tumor microenvironment. However, its effects on key immune cell populations remain unclear. Methods: In this retrospective study, pre- and post-treatment samples from 44 ER⁺/HER2-negative breast cancer patients treated at Antwerp University Hospital and Duke University(2006-2018) were analyzed by immunohistochemistry. We quantified stromal tumor-infiltrating lymphocytes (sTILs) along with markers for T cells, regulatory T cells (including the FOXP3/CD4 ratio), and macrophages (CD68), as well as immune checkpoint markers (PD-L1, PD-1, CTLA-4, TIM-3, and LAG-3). Treatment response was evaluated by changes in tumor size, Ki-67, and the Residual Cancer Burden (RCB) index. Results: NET significantly reduced tumor size (median decrease: 4.5 mm; p < 0.001) and Ki-67 levels (median reduction: 9%; p < 0.001) while sTIL levels remained stable (median: 5%; p = 0.08). CD4⁺ T-cell infiltration increased significantly post-treatment (p = 0.03). CD8⁺ T cells exhibited a heterogeneous response with a significant negative correlation between CD8⁺ counts and tumor size (Spearman r = -0.45, p = 0.003) indicating that higher CD8⁺ infiltration is associated with smaller tumors. FOXP3⁺ T-cell counts decreased from a median of 22.5% to 15% (p = 0.14) and the FOXP3/CD4 ratio declined significantly from a median of 2.307 to 1 (p = 0.04) suggesting a shift toward a less immunosuppressive tumor microenvironment. Higher pre-treatment CD68 expression correlated significantly with improved tumor NET response (p = 0.03). Immune checkpoint analysis revealed non-significant trends toward increased expression of PD-L1 (9% to 25%; p = 0.088), PD-1 (18% to 34%; p = 0.16), and LAG-3 (43% to 66%; p = 0.22) while CTLA-4 (23% to 27%; p = 0.69) and TIM-3 (64% to 52%; p = 0.53) remained largely unchanged. Conclusion: NET elicits profound alterations in the tumor microenvironment of ER⁺/HER2-negative breast cancer. The therapy reduces tumor proliferation and reconfigures immune cell dynamics, as evidenced by increased CD4⁺ T-cell infiltration, a beneficial reduction in the FOXP3/CD4 ratio, and a heterogeneous response of CD8⁺ T cells where higher CD8⁺ levels are linked to smaller tumors. In addition, elevated pre-treatment macrophage levels are predictive of improved tumor NET response. These results underscore the potential of integrating immune parameters as predictive biomarkers for NET efficacy and support further exploration of combined treatment strategies, including the use of immune checkpoint inhibitors, to optimize patient outcomes.Department of Defense Breast Cancer Breakthrough level 2 Award W81XWH2010153 (G.R.D.) Presentation: Monday, July 14, 2025