Abstract LB309: Differential DNA methylation analysis and functional insights in luminal A and triple-negative breast cancer using FFPE samples

Background: Epigenetic modifications, such as DNA methylation, play a critical role in breast cancer heterogeneity, influencing tumor progression and therapeutic responses. This study investigates differential methylation patterns between luminal A (LumA) and triple-negative breast cancer (TNBC) subtypes using formalin-fixed, paraffin-embedded (FFPE) samples. Functional insights are derived through gene ontology and pathway analyses to understand subtype-specific epigenetic mechanisms. Methods: Genome-wide methylation profiling was performed on FFPE samples of LumA (n=3) and TNBC (n=5) cases using the Illumina Infinium EPIC array. Differentially methylated CpG sites were identified, and gene ontology analysis and pathway enrichment analyses were conducted on genes annotated to differentially methylated CpG sites to identify biological processes and pathways driving subtype differences. Results: Among the 937,690 original probes, 319,514 probes failed in at least one sample and were excluded from further analysis, thus, 618,176 probes were kept for analysis. A total of 28,801 differentially methylated CpG sites were identified between LumA and TNBC cases, with a threshold on methylation differences of 10%. A total of 12,772 genes were associated with the 28,801 significant differentially methylated probes identified between LumA and TNBC. Key enriched gene oncology terms included "small GTPase-mediated signal transduction ", " regulation of trans-synaptic signaling”, and “actin filament organization”, highlighting subtype-specific epigenetic regulation of tumor biology. Pathway analysis revealed significant enrichment of pathways such as "RHO GTPase cycle" and “RAC1 GTPase cycle” in TNBC, consistent with their distinct tumor microenvironments. Conclusions: This study highlights distinct methylation patterns in LumA and TNBC subtypes, reflecting their divergent biological and clinical behaviors. The identified pathways provide insights into potential therapeutic targets and biomarkers for precision oncology and the ability to obtain DNA methylation data from formalin-fixed, paraffin-embedded (FFPE) samples. Future work will validate these findings in larger, independent cohorts and explore their clinical applicability. Citation Format: Yangbo Sun, Khyobeni Mozhui, Jay H. Fowke, Saunak Sen, Karen C. Johnson. Differential DNA methylation analysis and functional insights in luminal A and triple-negative breast cancer using FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited s); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2): nr LB309.