Abstract P4-11-09: Epigenetic Alterations Affecting Cell Morphogenesis and Tumor Plasticity Pathways in Primary Tumors of Breast Cancer Patients with High Nodal Involvement

Abstract Introduction: Survival rates for patients with breast cancer have improved significantly with widespread screening and effective systemic therapies. However, patients who present with advanced nodal stage at diagnosis have higher rates of distant relapse and require more aggressive therapies than patients with pathologic N1 disease (pN1). Understanding the molecular alterations that occur in primary breast cancer (BC) that drive progression to a higher nodal stage (>pN1) could uncover potential therapeutic targets for patients with regionally metastatic BC. Given that epigenetic mechanisms represent that crucial layer of dynamic change that regulates genome-wide gene expression, we assessed the DNA methylation profiles of primary BC at various nodal stages. Methods: We conducted an exploratory study using a well-annotated, retrospective cohort of ER+/HER2- treatment-naive primary BC from patients with clinically positive nodes and complete pathologic nodal data treated at Duke University (n=49, 30 cases pN1 vs. 19 cases >pN1). DNA was extracted from paraffin-embedded samples, and DNA methylation profiling was performed using the Illumina EPIC BeadChip v1 arrays. The ChAMP package was utilized to correct batch effect, and the Wilcoxon test was used to identify Differentially Methylated Sites (DMS). Tumor content purity was assessed using the LUMP algorithm and Gene Ontology enrichment of the DMSs was examined with the GOmeth tool. Data analysis and visualization were performed using the R packages ggplot2 (v. 3.4.4), circlize (v. 0.4.16), and gplots (v. 3.1.3.1), with data management facilitated by the tidyverse (v. 2.0.0) collection. Results: Tumor purity was similar between the two groups (p>0.05). The comparison revealed distinct DNA methylation patterns in 962 genomic regions between pN1 and >pN1 tumors (differential methylation > 15%, p<0.01); 683 regions were hypermethylated and 279 were hypomethylated in the >pN1 group. There was a significant enrichment of biological processes associated with morphogenesis among the hypermethylated sites, with 22 of the 43 affected molecular pathways linked to cellular morphogenesis. Additionally, epigenetic alterations were predominantly found in pathways related to the response to bone morphogenic protein (BMP). Expression-methylation Quantitative Trait Loci (emQTLs) were linked to epithelial to mesenchymal transition (EMT) genes in >pN1 tumors. Finally, higher methylation of the protocadherin cluster genes (PCDHGA) and regions encompassing DUSP6 and KRT7 genes were identified in tumors of higher nodal stage. These findings suggest inherent or acquired epigenetic alterations in primary BC associated with higher nodal stage. Conclusions: Understanding the mechanisms underlying lymph node invasion in breast cancer progression is crucial for improving the clinical management of patients with BC. This study indicates that ER+/HER2- primary tumors from patients with higher nodal involvement have an epigenetic predisposition to activate morphogenic differentiation and EMT more efficiently than patients with lower nodal involvement. Deeper insight into these alterations could contribute to more accurate prognostic tools, the development of novel therapeutic approaches, and potentially help avoid more aggressive axillary procedures. Citation Format: Sookyung Ahn, Andrés F. Bedoya-López, Miquel Ensenyat-Mendez, Pere Llinàs-Arias, Diego M. Marzese, Maggie L. DiNome. Epigenetic Alterations Affecting Cell Morphogenesis and Tumor Plasticity Pathways in Primary Tumors of Breast Cancer Patients with High Nodal Involvement [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P4-11-09.