Background: Humoral immune responses have previously been associated with improved outcomes, with B cell infiltrates able to independently predict pathologic complete response to neoadjuvant chemotherapy (NACT). B cells represent a diverse population of cells and the complex interplay between specific B cell subsets in the context of chemotherapy treated breast cancers remains unclear. Here, we investigate the dynamic changes in the B cell immune landscape before and after NACT treatment across different breast cancer subtypes. Methods: Treatment naïve, mid-treatment and post-NACT breast tumour tissue samples were dissociated into single cells, stained with two panels of B cell-specific antibodies recognising a total of 24 target proteins, and analysed by flow cytometry. In addition, PMBC before and after NACT were also profiled. B cell subsets were classified as either naïve (CD27—IgD+), class-switched memory (CD27+IgD—), unswitched memory (CD27+IgD+) or double negative (DN)(CD27—IgD—). DN B cells were further characterised into DN1 (CXCR5+CD21+) and DN2 (CXCR5—CD21—) subsets. In vitro co-cultures of breast cancer cell line spheroid and PBMC were carried out. Results: In both treatment naïve and chemotherapy treated samples, we observed a significant expansion in the DN B cell population within the TME compared to the periphery. DN B cells represented on average 40.96% of B cells in treatment naïve tumours vs 9.48% in PBMCs (p< 0.0001), and 71.80% vs 6.34% of B cells in post-NACT tumour vs PBMC samples respectively (p< 0.05). Interestingly, in treatment naïve PBMC and tumour tissue samples, the largest proportion of the DN subset consisted of DN1 cells, 69.35% and 64.11% respectively. In contrast, following NACT, DN2 cells constituted the majority of the DN population both within the TME (86.30%) and in the periphery (50.44%). Although the specific functions of these B cell subsets remain unclassified, deeper phenotyping suggests DN1 cells more closely resemble the phenotype of class-switched memory cells, whilst DN2 cells are thought to have antibody-secreting properties and more closely resemble the plasmablast phenotype. scRNA sequencing of B cells pre- and post NACT is currently underway. Conclusion: To our knowledge, this work is the first to identify an expanded population of DN B cells in breast tumour tissue and highlights the requirement for further investigation into these cells to decipher their role in the context of chemotherapy treatment and resistance in breast cancer. Citation Format: Esme Carpenter, Thanussuyah Alaguthurai, Farhana Hossain, Rosalind Graham, Helen Kakkassery, Sean Keane, Sheeba Irshad. Enrichment of atypical memory double negative (CD27— IgD—) tumour infiltrating B cells following neoadjuvant chemotherapy for early-stage breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-02.

Breast cancer patients with advanced metastatic disease can exhibit rapid disease progression, disease stabilisation or partial responses of varying duration. However, for reasons that are not fully elucidated, a small fraction of patients will elicit an exceptional durable response to standard anticancer treatments or survive significantly longer than patients with clinically comparable tumours. Here, we investigate the drivers of immune surveillance mechanisms across breast cancer subtypes in stage IV exceptional survivors (n=13) with matched control cohorts of stage IV typical responders (n=6), early breast cancer patients (n=5) and healthy volunteers (n=17). Peripheral blood mononuclear cells (PBMCs) from patients were stained with 8 panels providing 241 non-redundant immune parameters for flow cytometry analysis. Principle Component Analysis (PCA) showed distinct segregation of the exceptional survivors from the other control groups with an immune signature in exceptional survivors constituting of activated NK, CD8 T cells and gamma delta (gd) T cells pointing towards higher innate immunogenicity in these individuals. Specifically, although these metastatic exceptional responder patients possessed comparable NK cell frequencies, the proportion of NKG2D+CD56dimCD16+ NK cells were significantly enriched compared to the typical responders. Additionally, proportions of CD8+ central memory (CD45RA- CD27+) and effector memory (CD45RA- CD27-) gd T cells, were also seen to be significantly increased. Functional in vitro validation of these findings along with scRNA sequencing of lymph node and tumour tissue is currently underway. To our knowledge, this work is the first to explore in depth the immune signatures in the peripheral blood of exceptional survivors with metastatic breast cancer. Elucidating the immunological reasons for favourable atypical responses alongside functional tumour microenvironment analysis offers unique insights for predictive biomarker identification and discovery of axes that could be exploited therapeutically to benefit those with less favourable responses. Written informed consent was obtained from all individuals in accordance with the Declaration of Helsinki under the following research ethics committee; London-Chelsea approved study (REC ID 13/LO/1248). Citation Format: Helen Kakkassery, Thanussuyah Alaguthurai, Rosalind Graham, Esme Carpenter, Farhana Hossain, Sean Keane, Sheeba Irshad. Local and lymphoid immune surveillance mechanisms in “exceptional survivors” of stage 4 cancers following standard of care chemo- and targeted therapies [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD2-01.