Imaging Mass Cytometry Reveals Tertiary Lymphoid Structures in Breast Cancer Treated with Pelareorep

AWARE-1 is a window-of-opportunity study evaluating the effects of the oncolytic virus (OV) pelareorep in early-stage breast cancer patients. This analysis focuses on Cohort 2 (n = 8), consisting of HR+ HER2- patients who received pelareorep on days 1, 2, 8, 9; letrozole on days 1-21; and atezolizumab on day 3. Biopsies were collected pre-treatment, on day 3, and on day 21. Tissue was stained using an imaging mass cytometry (IMC) antibody cocktail targeting a variety of immune cell subsets to characterize changes over time in the tumor immune microenvironment (TiME) and the subsequent development of tertiary lymphoid structures (TLS) in select subjects. Unsupervised PhenoGraph clustering identified 18 distinct phenotypes, including T helper (Th), activated T helper (ATh), cytotoxic T cells (CTC), B cells (BC), differentiated B cells (DBC), and dendritic cells (DC). CTC’s were significantly increased from screening to day 21 samples (p = 0.04) and from day 3 to day 21 samples (p = 0.04). Further analysis of these cell types and how they interact is performed using cellular neighborhood analysis, which identifies common arrangements of certain cell types throughout the samples. Cellular neighborhood analysis yielded an interesting neighborhood cluster comprising CTC, DC, TH, BC, DBC, and M2 Mac cells - defined as the TLS neighborhood. Visualization of the TLS neighborhood showed progressing stages of T cell and B cell organization, with T cells and B cells being largely diffuse and unstructured at screening and concentrating into zones of BC - BC aggregates surrounded by CTC, TH, and ATH cells at day 21. Pairwise permutation-based interaction analysis was applied to the cells comprising the TLS neighborhood to validate significant biological organization. At day 21 BC - TH, DBC - BC, and DBC - DBC interactions were significantly more likely to occur than at screening. Other interactions of note were between tumor - ATH, tumor - CTC, tumor - DC, CTC - ATH, and CTC - CTC interactions were significantly increased at surgery. This study is the first of its kind to identify and spatially validate the unique cell-cell interactions in the TiME of early breast cancer patients treated with the OV pelareorep as developing TLS’s, highlighting coordinated TiME reorganization and novel cell-cell interaction dynamics associated with TLS formation. Citation Format: Julian P. Olea, Homa Dadrastoussi, Kaijin Wu, Harley Wu, Paniz Zarand, Claudia Villa Celi, Jennifer S. Carew, Steffan T. Nawrocki, Xuelian Chen, Richard Trauger, Thomas C. Heineman, Kevin R. Kelly. Imaging mass cytometry of pelareorep treated early breast cancer samples reveals developing tertiary lymphoid structures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1221.