Immune-associated alternative splicing signatures define molecular subtypes in breast cancer.

Alternative splicing (AS) shapes tumor biology by generating mRNA isoforms that regulate oncogenic signaling, immune modulation, and therapeutic response. However, its immune-related role in breast cancer (BRCA) remains insufficiently defined. Using TCGA-BRCA RNA-seq and clinical data, we quantified percent-spliced-in values with SpliceSeq and identified 1,063 differentially expressed AS events across 861 genes. These genes were enriched in cancer- and immune-related pathways, including focal adhesion, VEGF signaling, and cytokine-receptor interactions. Prognostic analyses revealed immune-associated AS events linked to overall survival and progression-free interval. Consensus clustering defined three AS-based subtypes with distinct immune landscapes and outcomes. C3 showed high immune infiltration, immune-activating molecule expression, and favorable prognosis, consistent with an immune-hot phenotype, whereas C1 displayed immunosuppressive features. Genomic alterations in splicing factors were associated with elevated tumor mutation burden, suggesting genomic instability may contribute to immune-related splicing dysregulation in BRCA.