Abstract 202: The early immune landscape of breast cancer and the role of tertiary lymphoid structures.

Abstract Ductal carcinoma in situ (DCIS), stage 0 breast cancer (BC), is defined by tumor cells confined to the duct. Tumor infiltrating lymphocytes (TIL) are prognostic for invasive ductal carcinoma (IDC) of the breast; however, their role in DCIS remains unknown. Some DCIS studies have analyzed TIL subpopulation balances with a few identifying tertiary lymphoid structures (TLS) but their roles in progression and recurrence remains unclear due to contradictory findings. The aim of this study is to investigate the functionality and organization of TIL into TLS in pre-invasive BC lesions. Fresh tumor samples were obtained at surgery from patients with DCIS (n=19), IDC (n=19) and mammary reductions (MR; n=8 controls) for the prospective cohort. Tissues were dissociated without enzymes and immunophenotyped by flow cytometry. A retrospective cohort of FFPE tissue blocks (DCIS n=30, IDC n=30) were dual-stained by chromogenic immunohistochemistry (cIHC) for CD3+CD20 and PD-1+Ki67 and scored for TILs and TLS, with selected patients (DCIS n=10, IDC n=10) also stained by multiplex IHC (mIHC). We extended our analyses by re-analyzing public scRNA-seq datasets (DCIS n=6, IDC n=6) to further explore the immune landscape. Patients were matched by BC molecular subtypes to control variation between subtypes. In the prospective cohort, a higher proportion of B cell and T follicular helper (Tfh) TIL were detected in DCIS vs. MR, with the former paralleling IDC levels and potentially signaling a TLS presence. In the retrospective cohort, TLS were detected by dual cIHC in 75% of DCIS compared to 58% of IDC, the latter consistent with our previous analysis of 300 IDC patients. Spatial mIHC analysis of CD3, CD20, AID and CD23 revealed that while AID+ B cells were less frequent in DCIS-TLS the number of CD23+ mature FDC were comparable with IDC. Thus, DCIS-TLS have a mature stroma but limited Ig diversification, suggesting these structures are at an earlier stage of differentiation. Tfh cells play a crucial role in B cell maturation and antibody production. Flow cytometric data from the prospective cohort detected lower frequencies of functional CXCR5+PD1hiICOSint Tfh cells in DCIS vs IDC. scRNA-seq analysis revealed that DCIS Tfh were more naïve, whereas IDC Tfh had enhanced effector signatures. In the prospective cohort, CXCL13-producing CXCR5-PD1hiICOSint Tfh cells, known for their critical role in IDC-TLS formation, were also reduced in DCIS with CXCL13 expression primarily detected in podoplanin+ stromal cells within DCIS-TLS. Our data show that while high densities of TIL and TLS are observed in DCIS, adaptive immunity responses are less mature and therefore less functional than in IDC. This may partly explain why dense TIL and TLS in DCIS are not consistently associated with a good prognosis, unlike IDC. Our findings highlight the need to further dissect, beyond simple quantification, the immune composition and spatial organization across BC stages. Citation Format: Thila Vanhulst, Soizic Garaud, Alexandre De Wind, Anais Boisson, Pauline Delvaux, Doina Sofronii, Mireille Langouo Fontsa, Karen Willard-Gallo. The early immune landscape of breast cancer and the role of 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 202.