Uncovering T-cell Receptor Clones and Immunogenic Targets in HER2DX-defined HER2-positive Breast Cancer

To explore the role of the immune system in early-stage HER2+ breast cancer (HER2+ BC), focusing on how T-cell receptor (TCR) dynamics relate to the prognostic 14 B-cell gene/IgG immune signature (IGG) included in the clinically available HER2DX genomic test. This study aims to clarify how TCR diversity and targeting of tumor-associated antigens (TAA) contribute to patient outcomes and could inform potential therapeutic strategies. TCR/BCR clones were identified by PCR amplification and deep sequencing (ImmunoSEQ) in 41 early-stage HER2+ BC samples. CDR3 sequences were cross-referenced with the VDJ database, excluding inconclusive matches (VDJdb score 0-1). Protein expression was analyzed by digital spatial profiling (GeoMx) in 23 samples. In 6 samples, TCR identification was performed through single-cell RNA sequencing (scRNAseq; Chromium). IGG expression in each sample was evaluated using the HER2DX assay and correlated with bulk RNA data from TCGA and MTBC datasets. Spearman’s correlation and Wilcoxon tests were used for statistical analysis (R software). Of the 12,575 TCR clones with predicted targets, 759 (5.9%) showed reliable matches (score 2-3). 53 of them (7%) recognized TAA, primarily MART1 (18.9%), followed by gp100, ABCD3, MAGEA6, KRAS, NY-ESO1, p53, TERT, and others. Most non-human epitopes belonged to common viruses such as Influenza A (31.2%), EBV (30.7%), and CMV (20%). IGG expression was correlated with the number of TCR templates (Cor: 0.47, p<0.01), TCR entropy (Cor: 0.60, p<0.001), and shared TCR clonotypes between samples (Cor: 0.48, p<0.01). IGG was higher in samples with TCR clones against TAA (p50 75.6 vs. 61.1, p=0.044). A positive correlation was observed between the number of clones targeting human and viral epitopes (Cor: 0.44, p=0.013). The scRNAseq data confirmed that IGG-high samples exhibit greater TCR polyclonality and a higher fraction of cytotoxic CD8+ T cells (p<0.05), with upregulated perforin and granzyme A/B expression. IGG correlated with CD27 (Cor: 0.47, p=0.025) and CD3 (Cor: 0.52, p=0.011) protein levels, as well as the IFN-γ signature in TCGA (Cor: 0.56, p<0.01) and MTBC (Cor: 0.71, p<0.01) data. Fibronectin correlated with PD1 (Cor: 0.61, p<0.01) and CTLA4 (Cor: 0.81, p<0.001) levels, and inversely with IGG (Cor: -0.50, p=0.017), indicating that IGG-low tumors might have a denser stroma and a more exhausted, less active immune infiltrate. Early-stage HER2+ BC with high IGG expression is characterized by a robust, polyclonal immune response, with TCR clones targeting both tumor and viral antigens. These findings suggest enhanced immune fitness and may explain IGG favorable prognostic value. The discovery of shared TCR clones across tumors may help identify immunogenic targets, providing new opportunities for developing immune-based treatments or engineered T-cell therapies. Víctor Albarrán-Fernández, Carlota Rubio-Pérez, Patricia Galván, Oleguer Castillo, Paula Blasco, Esther Sanfeliu, Anabel Martínez-Romero, Mercedes Marín, Patricia Villagrasa, Francisco Pardo, Laia Paré, Isabel García-Fructuoso, Raquel Gómez, Elia Seguí, Bárbara Adamo, Benjamin Walbaum, Olga Martínez-Saez, Tomás Pascual, María Vidal, Montserrat Muñoz, Sònia Guedan, Fara Brasó, Laura Angelats, Aleix Prat. Uncovering T-cell receptor clones and immunogenic targets in HER2DX-defined HER2-positive 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 5867.