Abstract A006: Copy number analysis of single-cell methylomes reveal distinct subclone methylation in early breast cancer
Authors: Mulqueen Ryan M , Mosaad Mariam , Bai Shanshan , Li Jiahong , Sei Emi , Lim Bora , Krishnamurthy Savitri , Thompson Alastair , Navin Nicholas 
Published: February 2025

Ductal carcinoma in situ (DCIS) harbors genomic copy number changes reflecting early events in advancing invasive breast cancer, and changes in specific cell states related to cancer initiation and progression. DNA methylation is a covalent modification to cytosines with cell type specificity, is stable in archival samples, and provides most of the specificity in multiple noninvasive pan-cancer screens. However, the creation of single-cell DNA methylomes is technically challenging and analysis is immature, leaving cell type and state level interpretation understudied. This has led to a gap in knowledge: it is not understood how the methylome reacts to gene dosage changes brought on by early copy number events, and it is not known how long these potential methylation changes persist. To this end, we have created and optimized a high throughput assay to measure these methylation changes at single-cell level, pairing our analysis with a multimodal view of these early copy number events. Here we describe three major steps in our work on elucidating methylome response to early breast cancer events: 1) We have generated methylome libraries for breast cancer cell lines with known large scale copy number alterations and validated our copy number calling in methylomes with those of gold-standard single-cell whole genome libraries. 2) We have generated thousands of single cell methylomes on normal breast samples attained from reduction surgeries paired with our groups’ work on the Human Breast Cell Atlas project. We were able to identify all major cell types of the human breast using methylation data for the first time. We used this data set to generate high-quality pseudobulk cell type specific methylomes. 3) We have profiled 5 DCIS and 1 synchronous invasive ductal carcinoma sample with subclonal copy number events, paired with single-cell transcriptomics. Through this we identified subclones in our methylome libraries and validated these through inferred copy number changes in our single-cell transcriptome libraries. We have uncovered thousands of hypomethylated regions enriched for specific transcription factor motifs, and widespread methylation changes across DCIS subclones. Notably many differential methylation changes occur outside of the genomic boundaries of copy number effects, suggesting distal changes by transcription factor gene dosage, chromatin perturbation, or tumor microenvironment interactions. These data will serve as a basis for future analyses into long-term follow through of archival samples and expand our understanding of this understudied epigenomic mark and its importance in early cancer evolution.

Citation Format: Ryan M Mulqueen, Mariam Mosaad, Shanshan Bai, Jiahong Li, Emi Sei, Bora Lim, Savitri Krishnamurthy, Alastair Thompson, Nicholas Navin. Copy number analysis of single-cell methylomes reveal distinct subclone methylation in early breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr A006.