Abstract PS2-09-17: Comprehensive peripheral immune profiling in Triple Negative Breast Cancer (TNBC) links immune cell dynamics to therapeutic response and irAEs: insight from the IRIS Study

Abstract Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype with limited targeted options. Immune checkpoint inhibitors (ICIs) have reshaped treatment approaches in both metastatic and early-stage TNBC. In the neoadjuvant setting, ICIs combined with chemotherapy improve pathological complete response (pCR) rates over chemotherapy alone. Despite their benefits, ICIs pose risks of immune-related adverse events (irAEs), requiring careful patient selection and monitoring. Comprehending the immune context may be fundamental for tailoring therapy and predicting outcomes in TNBC. Methods: Between March 2021 and April 2025, peripheral blood samples were collected at baseline from patients with TNBC treated with ICIs and enrolled in the translational, prospective and multicentre (12 Italian sites) IRIS study investigating circulating immune cells as predictive or prognostic biomarkers. In 60 patients (49 in the neoadjuvant and 11 in the metastatic setting), immunophenotype was performed by flow cytometry (FACS) to characterize circulating B cells, T cells, dendritic cells (DC), and Natural Kiler cells (NK) by a manual gating strategy. To capture immune cell heterogeneity, high-dimensional analysis was applied, including t-distributed stochastic neighbour embedding (t-SNE) for dimensionality reduction and FlowSOM for unsupervised clustering. For consistency, datasets were down sampled to 6000 T cells and 1600 B cells per sample, excluding sample below this cut-off (FlowJoTMv10). Statistical analysis included Mann-Whitney U test, univariate and multivariate Cox models for time to first irAE (TTirAE), defined as the interval between the first ICI cycle and onset of irAE (any grade), and Logistic regression (R software v.4.4.3). Results: In patients with TNBC, those in the metastatic setting exhibited significantly lower percentages of circulating unswitched and switched memory B cells (p = 0.0004 and p = 0.035) and higher frequencies of naive B cells (p = 0.0046). Additionally, dysfunctional central memory (CM) Th1-like cells and dysfunctional transitional CD4+ T cells were enriched in the metastatic group. Focusing on TTirAE (No irAEs: n = 34; irAEs: n = 13), a higher baseline percentage of peripheral plasmacytoid dendritic cells (pDCs) was associated with a lower risk of irAEs (HR = 0.89; 95% CI: 0.80 - 0.99; p = 0.040), whereas increased frequencies of CM Th1 cells were linked to a higher risk (HR = 2.10; 95% CI: 1.05 - 4.20; p = 0.036). These associations remained statistically significant after adjusting for age at diagnosis and treatment setting. Considering only patients with early-stage TNBC, baseline circulating immune profiling revealed distinct differences between patients achieving pCR (n = 29) and those with residual disease (RD, n = 11). Patients with pCR showed higher percentages of dysfunctional CD4+CD28-Tregs (p = 0.014) and NKT cells (p = 0.038), whereas those with RD had increased frequencies of mature plasma cells (p = 0.015), transitional Th1/Th17 cells (p = 0.031), and CM Tc1 cells (p = 0.043). Although not statistically significant, higher baseline frequency of CD8+ naive T cells showed a trend toward a positive association with pCR (OR = 1.05; 95% CI: 1.00-1.11; p = 0.068). In contrast, CM Tc1 cells, plasma cells, and transitional marginal zone-like B cells were negatively associated with pCR (OR < 1.0; 0.070 < p < 0.080). Conclusion: Our preliminary data highlight the potential utility of peripheral immunophenotyping in TNBC as a non-invasive tool to identify predictive biomarkers and guide patient stratification in immunotherapy-based approaches. Citation Format: L. Negrini, V. Martini, B. Conte, I. Taglialatela, T. L. Landolfo, S. Gobbato, B. Ruffilli, S. Nardin, F. D'Avanzo, V. Rossi, C. Branni, J. Gennari, A. Turki, G. Di Foggia, M. Bitrus, A. Giacobino, E. Rota Caremoli, I. Romaniello, A. M. Vandone, V. Prati, V. Guarneri, A. Gennari. Comprehensive peripheral immune profiling in Triple Negative Breast Cancer (TNBC) links immune cell dynamics to therapeutic response and irAEs: insight from the IRIS Study [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 PS2-09-17.