Abstract 6355: Mapping the genetic, epigenetic, and microenvironmental evolution of HER2-positive breast cancer during neoadjuvant therapy

Abstract Breast cancer, characterized by considerable heterogeneity, showcases genetic and epigenetic variability among individual tumors and their constituent cells. Notably, this intratumoral heterogeneity (ITH) can drive clonal evolution and therapy resistance, particularly in Human Epidermal Growth Factor Receptor 2 (HER2)-positive cancers. Despite the efficacy of HER2-targeted therapies, many patients develop resistance, resulting in poor prognosis. This study aims to elucidate the dynamic interplay between genetic alterations and epigenetic modifications during anti-HER2 therapy using advanced single-cell resolution techniques. Biopsy samples were collected at three time points from patients undergoing neoadjuvant HER2-targeted therapy as part of the TRIO-US B07 clinical trial. Utilizing methods such as low-pass whole-genome sequencing (WGS, n=47), single-nuclei WGS (snWGS, n=21), and matched single-nuclei RNA/ATAC-seq (n=22), we explored copy-number variations (CNVs), subclonal tumor dynamics, and alterations in the tumor microenvironment (TME). We also analyzed additional deep WGS (n= 205) to reconstruct the structure of HER2 amplicons. Our analysis revealed distinct CNV dynamics in HER2-positive breast cancers during treatment. We found that while broad-level CNVs remained stable, focal amplifications exhibited temporal variability, suggesting the presence of extrachromosomal DNA (ecDNA). Specifically, the structure of HER2 amplificons emerged as a pivotal determinant of aggressiveness, with higher numbers of co-amplified oncogenes correlating with non-pathological complete response (pCR) and metastasis. Single-cell resolution analyses unraveled significant subclonal diversity in a subset of patients exhibiting complex tumor architectures. Integration of transcriptomic and epigenomic profiling highlighted that pCR patients exhibited higher lymphocyte infiltration and dynamic immune shifts post-treatment. Conversely, non-pCR tumors displayed increased regulatory T cells. Furthermore, fibroblast state transitions characterized by altered extracellular matrix (ECM) gene expression suggested a potential role in modulating immune responses. Our findings provide novel insights into the genetic and epigenetic factors driving therapy resistance in HER2-positive breast cancer. We emphasize the importance of longitudinal, multimodal profiling to refine patient stratification and inform personalized treatment strategies. Overall, this study offers new avenues for understanding tumor evolution in response to therapy and highlights the need for comprehensive assessments of ITH and microenvironmental dynamics for improved clinical outcomes. Citation Format: Seongyeol Park, Eran Kotler, Zhicheng Ma, Kathleen Houlahan, Aziz Khan, Sophia Pribus, Christina Curtis. Mapping the genetic, epigenetic, and microenvironmental evolution of HER2-positive breast cancer during neoadjuvant therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6355.