Abstract 5133: End-stage breast cancer metastases manifest as two subtypes with distinct dissemination patterns, proliferation/EMT signatures, and immune microenvironments

End stage breast cancer often presents with oligometastatic tumors and lacks effective treatment options. Previous studies leveraged post-mortem tissue procurement (also called rapid autopsy) programs to collect tumors shortly after death for profiling by bulk whole exome sequencing (WES). This has revealed substantial heterogeneity in the genomes of metastatic estrogen receptor-positive (ER+) and triple-negative breast cancers. In order to understand the heterogeneity of both the genotypes, transcriptional phenotypes, and evolution of these tumors, we collected multi-site metastatic tumors from two end-stage ER+ breast cancer patients enrolled in the LEGACY rapid post-mortem tissue procurement trial. Metastatic tumors were profiled using WES and single-nuclei RNA sequencing (snRNA-seq). We reconstructed high resolution phylogenies for each patient using the mutation called from the snRNA-seq reads to determine the subclonal architecture of cancer cells. Based on the mutation patterns, the gene expression profiles, and the subclonal architecture, we found that the tumors in each patient were divided into two distinct subtypes. A majority of the tumors were grouped into a subtype characterized by a high intra-tumor heterogeneity, with many cell lineages shared widely across metastasis, indicating extensive and undirected dissemination. The cancer cells of this subtype exhibited an EMT gene expression signature. In contrast, the second tumor subtype observed was rare and demonstrated little intra-tumor heterogeneity, a proliferative gene expression signature, and an interferon response signature present widely across cells in the microenvironment. Across all tumors, macrophages comprised the majority of the immune cells. Low diversity tumor macrophages exhibited strong activation of multiple interferon signaling pathways compared to high diversity tumor macrophages. Interestingly, these macrophages expressed a mixture of M1 and M2 polarization markers as STAT1, SOCS1, ISG15, ISG20, MX1, MX2, OAS1, OAS3 that did not correspond to classical M1 or M2 polarization states. This contrast demonstrates that multi-site metastatic tumors and their microenvironment adopt dichotomous states, which may contribute to the difficulties in treating progressive disease. It also underscores the need for a comprehensive and personalized late-stage therapeutic strategy to target the diversity of existing tumor phenotypes. Citation Format: Isaac Bishara, Xuan Liu, Jason l. Griffiths, Patrick A. Cosgrove, Jasmine R. McQuerry, Jiayi Liu, Kena K. Ihle, Eliza R. Bacon, Feng Chi, Pierre Wallet, Vince Grolmusz, Diana Simons, Benjamin Copeland, Lance Pflieger, JinFeng Chen, Sumana Majumdar, Terron T. Crowder, Rena Emond, Rachel Factor, David D. Bowtell, Adam L. Cohen, Daniel D. Schmolze, Peter P. Lee, Lusine Tumyan, James R. Waisman, Andrea Bild, Aritro Nath, Jeffrey T. Chang. End-stage breast cancer metastases manifest as two subtypes with distinct dissemination patterns, proliferation/EMT signatures, and immune microenvironments [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 5133.