33P Breast cancer brain metastases present a suppressed immune microenvironment enriched in macrophagic components as compared to paired primary tumors

Despite potential clinical implications, the complexity of tumor immune microenvironment (TIME) of breast cancer (BC) brain metastases (BMs) is still poorly understood. We previously reported that a higher CD4+FoxP3+/CD8+ cell ratio in the stromal compartment and a higher density of CD163+ M2-polarized macrophages are associated with worse overall survival in BCBMs. We here assess the evolution of TIME from primary BC to paired BMs using multiplex immunofluorescence. TIME profiling of archival FFPE specimens of paired primary BC and BMs from 25 patients was carried out using two multiplex immunofluorescence panels (CD4, CD8, Granzyme B, FoxP3, CD68, pan-cytokeratin, DAPI; CD3, PD-1, PD-L1, LAG-3, TIM-3, CD163, pan-cytokeratin, DAPI). Variations in immune cell densities were assessed using a paired Wilcoxon test. In both the tumor and stromal compartment, BMs exhibited a more immunosuppressive TIME with significantly lower lymphocytic (CD3+), T cell (CD8+), and cytotoxic cell (GranzymeB+) infiltration (p<0.001), higher CD4+FoxP3+ infiltration (p<0.001), and a higher CD4+FoxP3+/CD8+ ratio (p<0.01) as compared to primary tumors. Moreover, the lymphocytic infiltrate presented a more immune exhausted phenotype, with higher levels of CD3+PD-1+ (p<0.01) and CD3+PD-1+LAG3+ (p<0.05) cells, in BMs as compared to primary BCs. PD-L1 expression in the tumor compartment was also reduced in BMs compared to primary BCs (p<0.001). In addition, TIM3+ cells were significantly less represented in both the tumor and stromal compartment (p<0.01) On the other hand, BMs presented significantly higher levels of macrophagic (CD68+) infiltration in the tumor compartment (p=0.008) and of CD163+ M2-polarized macrophagic infiltration in the stromal compartment (p<0.001), which was associated with a significant decrease in the CD3/CD163+ ratio in both compartments (p<0.001). Our results suggest that BCBMs present a more immunosuppressed TIME as compared to primary tumors, comprising immune phenotypes (e.g. immunosuppressive tumor-associated macrophages) that may represent a potential target for immunotherapeutic strategies for BCBMs.