Distinct epigenetic alterations and accelerated mitotic aging define second primary breast cancers.

As cancer survivorship is steadily increasing, second primary breast cancers (SPBCs) are becoming a more common disease linked to poorer clinical outcomes compared to first primary breast cancers (FPBCs). While the molecular features of FPBCs are well characterized, the biological drivers of SPBCs remain largely unexplored. We performed genome-wide DNA methylation profiling and analysis of clinically and demographically paired ER-positive, HER2-negative SPBC (n = 13) and FPBC (n = 29). We validated our findings by integrating genomic, transcriptomic, proteomic, and epigenomic data from paired tumors from The Cancer Genome Atlas. Chromatin Immunoprecipitation sequencing maps from ENCODE were used to examine epigenetic alterations of transcription factor binding sites (TFBSs) linked to accelerated aging and malignancy traits in SPBC. Epigenetic aging was assessed using the mitotic clock epiTOC. Here, we show that SPBC tumors are associated with poorer prognosis compared to FPBC. Epigenetic profiling identified 11,321 differentially methylated CpG sites compared to FPBCs, with hypermethylation enriched at gene promoters involved in morphogenesis, hormone signaling, and cell adhesion processes. Concordantly, transcriptomic analysis confirmed a coordinated downregulation of hormone signaling pathways, including estrogen, progesterone, and androgen cascades. Beyond promoter-level changes, SPBC tumors exhibit altered DNA methylation at key TFBSs involved in accelerated aging and cancer traits and significantly elevated mitotic age. SPBC tumors exhibit recurrent molecular scars, defined by suppressed endocrine signaling and accelerated mitotic aging. These epigenetic hallmarks may reflect the cumulative effects associated with prior malignancy and its clinical context, potentially contributing to more aggressive disease behavior. Our findings provide a foundation for developing personalized risk stratification and therapeutic approaches for survivors at risk of SPBC.