Epigenetic determinants of an immune-evasive phenotype in HER2-low triple-negative breast cancer

Identifying molecular drivers of aggressive biology is crucial for patients with triple-negative breast cancer (TNBC). While HER2-low expression predicts response to novel antibody-drug conjugates, its impact on TNBC biology remains unclear. To address this, we generated genome-wide DNA methylation profiles of 58 patients from a multi-institutional TNBC cohort treated at Duke, UCLA, and Scripps Health, integrating our data with three independent cohorts: The Cancer Genome Atlas, the Sweden Cancerome Analysis Network – Breast, and the I-SPY2 clinical trial. TNBC cases were classified as HER2-zero (IHC 0) or HER2-low (IHC 1+ or 2+, ISH non-amplified), and their genomic, epigenomic, transcriptomic, and proteomic landscapes were compared. Among the 506 patients analyzed (n=288 HER2-low; n=218 HER2-zero), HER2-low TNBC exhibited a significantly lower tumor mutational burden (P = 0.02) and consistent hypermethylation of immune-related genes, particularly HLA. Transcriptomic analyses revealed significant downregulation of immune response pathways, including leukocyte activation, T-cell signaling, and adaptive immunity (adjusted P < 0.001), alongside reduced immune cell infiltration in the tumor microenvironment (P = 0.002). Notably, higher expression of five immune-related genes most downregulated in HER2-low TNBC correlated with improved relapse-free (HR = 0.52; P < 0.001) and overall survival (HR = 0.36; P < 0.001). These findings highlight distinct molecular features of HER2-low TNBC, suggesting an immune-evasive phenotype that should be considered when selecting therapies for this challenging subtype. We aimed to profile genome-wide DNA methylation of 58 patients from a multi-institutional TNBC cohort treated at Duke, UCLA, and Scripps Health. DNA methylome was analyzed using the Infinium Methylation EPIC v2.0 BeadChip (Illumina, Inc) following the manufacturer’s protocol.