Abstract 3897: Investigating the prevalence of ecDNA in breast cancer
Authors: Georgia Stavrou, Chris Bailey, Jeanette Kittel, Mariam Jamal-Hanjani, Charles Swanton, Nnennaya Kanu
Journal: Cancer Research (2025)

Abstract Extrachromosomal DNA (ecDNA) has been described in multiple cancer types. ecDNAs are circular and are usually greater than 500 base pairs in size. ecDNA structures lack centromeres and thus do not follow the Mendelian mode of inheritance, segregating unequally during mitosis. This can subsequently result in high copy number gene amplifications and enhance tumor heterogeneity, promoting tumor evolution. ecDNA structures have a dynamic nature whereby they can be maintained extrachromosomally in their circular form but can also reintegrate randomly back into the genome, in the form of Homogeneously Stained Regions (HSR). Earlier studies using TCGA reported that approximately 25% of breast samples contained ecDNA. We have recently reported that over 46% of HER2+ breast cancers have ecDNA amplifications. Moreover, ecDNA have been found in both pre-cancerous lesions, such as in tissues obtained from patients with Barret’s esophagus, but also following both targeted treatment and chemotherapy. This highlights the ability of the structures to be detected at any disease stage and treatment phase although exact mechanisms of ecDNA formation and maintenance have not been described fully. In the present study we use a tumor-informed in silico approach in conjunction with in vitro readouts to elucidate these mechanisms. 2981 Whole Genome Sequencing data from the Genomics England breast cancer cohort and cell line data from the Cancer Cell Line Encyclopedia were analyzed using Amplicon Suite. In parallel, a panel of eight ERBB2 amplified breast cancer cell lines was used to detect in vitro presence of ecDNA by metaphase FISH and Optical Mapping. Genes that were found to be differentially mutated according to the presence of ERBB2 focal amplifications were silenced in vitro, then gene expression of ERBB2 and target genes was evaluated using qPCR and the effect of ERBB2 signal was assessed using FISH. In the cohort analyzed, 22.8% of breast tumors had ecDNA amplifications. Enrichment analysis was conducted to assess the presence or absence of specific mutations in ERBB2-amplified vs non-amplified tumors. Several genes were identified as being positively or negatively enriched in the cohort. Similarly, we identified 9 breast cancer cell lines containing up to 5 different species of ecDNA. In a subset of cell lines computationally predicted to harbor ERBB2 on ecDNA, ERBB2 amplification was in the form of HSR. We selected HCC1569 as our candidate model and silenced candidate epigenetic regulators. Our preliminary data suggests this leads to a measurable and significant change on ERBB2 signal. At present, we highlight the propensity of ERBB2 to be amplified on ecDNA in both breast cancer patients and breast cancer cell lines. These cell lines can serve as models for the further study of the mechanisms of maintenance of ecDNA in vitro which will shed light on some of the key unresolved questions in the field. Citation Format: Georgia Stavrou, Chris Bailey, Jeanette Kittel, Mariam Jamal-Hanjani, Charles Swanton, Nnennaya Kanu. Investigating the prevalence of ecDNA in breast cancer [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 3897.