Abstract Background: Chemotherapy-free regimens combining HER2-targeted therapy with immune checkpoint blockade are emerging as promising strategies in HER2-Enriched breast cancer. In our Phase II trial of durvalumab, trastuzumab, and pertuzumab (DTP), ER-negative, PR-negative, HER2-Enriched tumors (BluePrint®) achieved a 49% Pathologic Complete Response (pCR) rate, suggesting an immunologically responsive subset. To define the cellular mechanisms underlying response and resistance to this chemotherapy-free approach, we performed single-nucleus RNA sequencing and spatial transcriptomic profiling on paired tumor samples. Methods: Paired pre-treatment and surgical biopsies (n=37) underwent snRNA-seq to profile immune composition, functional states, and lineage dynamics. Patients were stratified by Residual Cancer Burden (RCB), with RCB-0 considered Responders (n=18) and RCB-2/3 as Nonresponders (n=6). Cell identities were label-transferred to Xenium 5K spatial transcriptomic data to assess cellular localization and interactions within the tumor microenvironment. Results: We recovered 470, 997 high quality nuclei, including 168,516 immune cells. Pre-treatment Responder samples were enriched for CD4+ T follicular helper cells (CD4+CXCL13+PD-1+ Tfh), CD8+ tissue-resident memory T cells (CD8+ Hobit+ Trm), and CD8+ exhausted T cells (CD8+Tim3+PD-1+ Tex), along with higher frequencies of LAMP3+ mature dendritic cells (mDCs), and naïve B-cells. Spatial mapping revealed co-localization of Tfh, mDCs, and B-cell subsets within tertiary lymphoid structures (TLS) spanning multiple maturation stages. CellChat analyses demonstrated Responders exhibiting a coordinated Tfh-mDC-B-cell communication network enriched in CXCL13-mediated B-cell recruitment, CD40 and CD28 costimulation, and cytokine programs supporting TLS maturation. In contrast, Nonresponders engaged predominantly inhibitory signaling pathways, marked by CD22-mediated suppression, BTLA-HVEM immune dampening, and reduced chemokine guidance, consistent with abortive B-cell activation. Conclusions: Integrated single-nucleus and spatial transcriptomic profiling identifies coordinated Tfh-B-cell and dendritic-B-cell interactions, spatially organized within TLS, as dominant determinants of response to DTP therapy in HER2-Enriched early breast cancer. Citation Format: Mailin Li, Xen Ping Hoi, Jenying Deng, Junjun Zheng, Rabia Hashmani, Recep Bayraktar, Wei Qian, Jianying Zhou, Jian Guan, Kai Sun, Hanh Mai, Tiffany Sheu, Susan Haley, Mary Schwartz, Sunil Mathur, Stephen T. Wong, Fotis Nikolo, Keith Syson Chan, Polly Niravath, Jenny C. Chang. Tertiary lymphoid structure (TLS)-associated immune circuits define response to durvalumab, trastuzumab, and pertuzumab (DTP) in HER2-enriched early breast cancer 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 6741.
Li et al. (2026) studied this question.