Abstract PD2-04: Tumor Heterogeneity in the transATAC Breast Cancer Spatial Single Cell Atlas

Abstract Aims: Primary breast cancer (BC) is highly heterogeneous and this drives divergent clinical outcomes. Many molecular features have significant prognostic value in hormone receptor positive (HR+) BC, and several commercial multigene prognostic assays currently guide chemotherapy decision making. The translational study from the monotherapy arms of the Arimidex, Tamoxifen, Alone or in Combination trial (transATAC), has been pivotal for the validation and regulatory approval of multiple prognostic assays. Spatial single cell analysis may lead to new insights into molecular features and tumor microenvironment (TME) that associate with prognosis and allow the future development of the next generation of prognostic assays. We present preliminary analysis of the transATAC spatial single-cell atlas of BC heterogeneity. We assess whether single cell analysis of a tissue micro-assay (TMA) core, sampling of ∼1000 cells per tumor, is sufficient to recapitulate the major features of the tumor. Methods: TMA blocks of 0.6mm diameter cores were generated from tumors of 1710 patients. Cores were taken from two or more distinct tumor regions in 27 patients. Every region/tumor had 3 spatially distant cores taken and spread across separate TMA blocks. We present initial analysis of single cell spatial transcriptomic analysis of 6108 genes using a CosMx spatial molecular imager from two TMAs. The preliminary analysis was generated data for 160 cores from 155 transATAC patients with data combined per patient to generate 155 tumor samples that would better represent molecular data used in prognostic assays. OncotypeDX recurrence scores on a surgery excision were available for 133 HR+ tumors. Cell type was determined by semi-supervised clustering with InSituType using BC specific reference profiles. Pseudo-bulk gene expression profiles were created by summing the total gene expression from all cells and separately all tumor cells. Large-scale chromosomal copy number alterations (CNA) in individual tumor cells were detected by InferCNV and used to understand tumor clonality. Results: We analysed 155 transATAC HR+ and HR- tumours. A mean of 952 cells were analysed, with 967 transcripts from 603 genes. Across the full dataset, cell typing assigned 59% of cells to tumor cells, 21% to immune cells (T-cells, B-cells and Myeloid cells) and 20% other cells (Endothelial, Fibroblast and Pericytes). Single cell CNA analysis of individual tumors cells suggested only one patient had multiple clones (with 50 tumors cells per clone) and this patient had cores taken from 2 regions of the tumor. Recurrence scores generated from single cell pseudo-bulk data were significantly correlated with Oncotype Dx assay recurrence scores generated from surgery excisions (Pearson r=0.76; p2.2e-16). Across all 155 tumors, 97% of ESR1 transcripts were expressed in tumor cells. Pseudo-bulk ESR1 expression from tumors cells was significantly correlated with ESR1 expression in the Oncotype assay (Spearman ρ=0.66, p2.2e-16) and with the H score of ER protein expression (ρ=0.5, p=1.3e-9) taken from excision specimens. There was a significant inverse correlation between ESR1 expression in tumor cells and the percentage of immune cells in a tumor across all 155 samples (ρ =-0.27, p=0.0006) and for the HR+ subset with OncotypeDX scores (ρ = -0.28, p=0.001). Conclusions: Single cell spatial analysis of transATAC TMA cores reproduced key features of tumor biology assessed in resection specimens. Single cell analysis revealed an inverse association between ESR1 expression in tumor cell cells and immune cell infiltration in the tumour. Analysis of transATAC TMAs is ongoing, and updated analysis will be presented. Single cell spatial analysis is a powerful and robust technique to analyse single cell gene expression and may build a better understanding of BC biology and prognosis. Citation Format: E. F. Schuster, I. T. Kleijn, K. Dunne, R. Sardinha, E. O'Neill, G. Latifi, C. Kennedy-Dietrich, N. B. Jamieson, J. Cuzick, A. R. Brentnall, M. Dowsett, S. R. Johnston, N. C. Turner. Tumor Heterogeneity in the transATAC Breast Cancer Spatial Single Cell Atlas abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PD2-04.