Approximately 80% of all breast cancer cases are estrogen receptor-positive (ER+). This subtype is known to have distant recurrences in a subset of patients after adjuvant endocrine therapy, partially due to the heterogeneity of the disease. ER+ breast cancer has been generally classified as an immune-cold disease. Thus, to better inform treatment decisions and to consider the prospect of immunotherapy, the immune microenvironment needs to be thoroughly characterized. In this study, the proteome and transcriptome of the tumour and tumour microenvironment (TME) were characterized using the GeoMx Digital Spatial Profiler (DSP) and NanoString’s BC360 gene expression panel. Spatially resolved tumour and TME across a patient's lumpectomy demonstrated substantial heterogeneity in the expression of commonly targeted immune and tumour proteins. Results from this study demonstrated heterogeneity across the tumour and TME in ER+ breast cancer, which may be reflective of a variable immune response.

Background: Neoadjuvant partial breast irradiation using stereotactic body radiotherapy (SBRT) has emerged as a strategy to induce tumor and immune responses in early-stage, low-risk breast cancer. While prior studies have demonstrated encouraging response rates and evidence of immune modulation, the optimal radiotherapy regimen for immune priming remains unclear. SIGNAL 2.0 is a randomized phase II trial designed to compare the biological and immunological impact of a single-fraction versus three-fraction neoadjuvant SBRT. Materials and Methods: Sixty-one postmenopausal patients ≥ 50 years with unifocal, hormone positive, node-negative invasive ductal carcinoma < 3 cm were randomized 1:1 to receive either 21 Gy in one fraction or 30 Gy in three fractions, delivered to the tumor in the prone position. Core biopsies were collected pre-SBRT and 14–20 days post-SBRT at the time of surgery. Immune markers were assessed using tumor-infiltrating lymphocyte (TIL) scoring, NanoString nCounter PanCancer Immune Profiling, and NanoString GeoMx Digital Spatial Profiling (DSP). Results: Available tumor samples from 47 patients underwent paired tissue analysis. Three-fraction SBRT induced 200 differentially expressed genes, including enrichment of pathways related to adaptive immune activation, with significant increases in expression levels of macrophages, dendritic cells, neutrophils and CD8 T-cells. Proteomic profiling also identified a significant increase in the expression levels of neutrophils, Treg cells, macrophages, and NK cells in the tumor microenvironment of the samples from patients receiving the three-fraction regimen. Conclusions: Neoadjuvant SBRT induces measurable immune activation, with three-fraction regimens generating more extensive transcriptional, proteomic, and cellular immune changes than a single fraction. Three-fraction neoadjuvant SBRT may provide superior immune priming, providing a foundation for future trials integrating neoadjuvant radiotherapy with immunomodulatory therapies.