Immune Heterogeneity in Breast Cancer Natural History: Benign Breast Disease to Breast Cancer

Benign breast disease (BBD), particularly lesions with atypical hyperplasia, confers an elevated risk of subsequent breast cancer; however, the majority of lesions do not progress, highlighting the need for biomarkers that distinguish indolent from high-risk disease. Emerging evidence suggests that the immune microenvironment may play a critical role in early breast carcinogenesis. We characterized immune heterogeneity across the spectrum of breast disease using Digital Spatial Profiling (DSP) of 21 immune-related proteins across 1,535 regions of interest (ROIs) from three cohorts: invasive breast cancers from the Carolina Breast Cancer Study (CBCS, n=58), a UNC BBD case series (n=20), and a Mayo Clinic BBD case–control cohort (n=177). Unsupervised clustering identified two reproducible immune states: a “High Immune” cluster enriched for adaptive immune markers and a “Quiet Immune” cluster characterized by lower immune activity. High Immune profiles predominated in tumors (77% of ROIs) but were less frequent in BBD (29% UNC; 42% Mayo). Notably, intra-lesional immune heterogeneity was substantially greater in BBD than in tumors, with over half of BBD lesions demonstrating mixed immune states across ROIs, whereas tumors more frequently exhibited immune concordance.