Immune Microenvironment Profiling of Breast Cancer Brain Metastases Using Multiplex Immunofluorescence.

2526 Background: Despite clinical implications, the complexity of brain metastases (BM) immune microenvironment in breast cancer (BC) patients is poorly understood. Multiplex immunofluorescence (mIF), a novel imaging technique allowing simultaneous visualization and quantification of several IF labeled proteins while maintaining spatial information, holds promise to comprehensively describe BCBM immune microenvironment, potentially providing valuable information to improve treatment. Methods: Clinical data and archival BM samples were collected for 60 BC patients undergoing neurosurgery (2003-2018) at three institutions. BM immune contexture was characterized using a custom mIF panel, including cell subtyping (CD4, CD8, FOXP3, CD68), activation (Granzyme B) and localization (keratin for tumor recognition) markers. Mean immune cell density (cells/mm2) for each sample was determined by digital image analysis and classified in tumor and stroma areas. Associations between immune subpopulations, BC subtype and overall survival from BM diagnosis (OS) were studied. Results: Up to date, 30 BCBM samples have been analyzed; 33% HR+/HER2-, 20% HR-/HER2+, 10% HR+/HER2+, 37% HR-/HER2-. At a median follow-up of 46 months, BC subtype was the only clinical variable associated with OS (longest for HER2+ and shortest for HR-/HER2-, log-rank p = 0.002). In the total sample area, no significant difference in immune cell densities was observed according to BC subtype. In the tumor area, HR+/HER2- tumors showed higher densities of CD8+ and CD68+ cells compared to other subtypes (p = 0.036 and p = 0.016, respectively). In stroma, HR-/HER2- tumors presented numerically higher densities of CD4+ and FOXP3+ cells and higher ratio of CD4/CD8 and FOXP3/CD8 ratio (not statistically significant). Higher CD4/CD8 and FOXP3/CD8 ratio in the stroma was significantly associated with worse OS, even after correction by BC subtype (Table). Conclusions: In BCBM, immune infiltrate differs according to BC subtype. Preliminary results suggest that a more tolerogenic immune microenvironment is associated with worse OS and might represent a target for optimization of immunotherapy for these patients. Updated results for all 60 patients will be presented. [Table: see text]