A single-cell spatial proteomic analysis of the TNBC microenvironment defines genotype-specific features

Breast cancer in women with germline BRCA1/2 pathogenic variants (g BRCA1 / 2 ) are generally treated with platinum-based therapies and PARP inhibitors (PARPi) with resistance commonly emerging. As the tumor microenvironment (TME) in gBRCA1 triple-negative breast cancer (TNBC) is enriched with tumor-infiltrating lymphocytes (TILs) and CD8 T cells, treatment trials have been done combining PARPi and immune checkpoint inhibitors (ICIs) in BRCA1 TNBC. This combination has not been shown to be more effective than PARPi alone. Evaluating the TME in gBRCA1 / 2 TNBC may help identify tumors most likely to benefit from PARPi/ICI therapy. We performed a detailed spatial proteomic analysis to characterize tumor-immune cell interactions in patients with gBRCA1/2 and wild-type (WT) TNBC with spatial tissue multiplexing (PhenoCycler) in 101 g BRCA1 , 24 g BRCA2 , and 30 WT TNBCs with matched RNAseq for 34 g BRCA1 , 8 g BRCA2 , and 16 WT TNBCs. A 43-plex antibody panel was developed featuring markers of DNA damage and repair, immune subtypes and exhaustion. We detected single tumor cells (PANCK+) in S/G2 phase (Geminin+) with double-stranded DNA breaks (yH2AX+) and DNA repair capacity (RAD51+) across all three cohorts. g BRCA1/2 TNBC patients exhibited a significantly lower proportion of tumor cells with homologous recombination proficiency (HRP) (g BRCA1 p = 0.006; g BRCA2 p = 0.007) compared to WT TNBC. CD4 & CD8 T cells, and CD20 B cells had intact DNA repair in WT and g BRCA1 / 2 TNBC. The frequency of CD8+ T (p=0.016) and CD20 B (p=0.003) cells was significantly higher in g BRCA1 compared to WT TNBC; BRCA2 and WT TNBC showed no differences. A detailed characterization of CD8 T cells revealed significantly increased numbers of potentially dysfunctional CD8 T cells in BRCA1 (TOX, p<0.0001; LAG-3, p=0.028; PD-1, p=0.033) and BRCA2 (LAG-3, p=0.033) compared to WT TNBC. We observed two types of TMEs in g BRCA1 TNBC: 1) CD8 low (mean<9.38%) with 1.4-fold increased immune checkpoint (PD-1) expression (mean: 15.4%) and high DNA damage in tumor cells; and 2) CD8 high (>9.38%) with reduced PD-1 and low DNA damage in tumor cells. Our findings suggest that although g BRCA1/2 variants lead to DNA damage and impaired repair in tumor cells, T cells (CD4, CD8) and B cells (CD20) retain intact DNA repair mechanisms. We also found that g BRCA1/2 TNBCs exhibit higher levels of immune checkpoint proteins LAG-3 and PD-1 on CD8 T cells compared to WT TNBC. This finding suggests the potential utility of additional ICI (LAG-3, PD-1) beyond PD-L1 blockade. Importantly, patients with g BRCA1 -associated TNBC exhibit two different TMEs, suggesting that the response to ICI- and DNA-damaging-based therapies may differ between tumors, and anticipated prior to treatment. Defining treatment-naïve TME is crucial for designing personalized, targeted ICI strategies for individuals with BRCA -mutated TNBC.