Abstract P2-01-14: The multi switching activity of neutrophils in the circulation of patients with early breast cancer
Authors: Anisha Ramessur, Bana Ambasager, Ilaria Malanchi, Raoul Charles Coombes
Journal: Cancer Research (2022)

Abstract Neutrophils (Ns) influence cancer cell survival and metastasis through the release of chemokines, cytokines and interactions within the microenvironment and at distal sites from the primary tumour. Background: Patients with Breast Cancer (BC) often display elevated levels of circulating blood neutrophils and an elevated neutrophil: lymphocyte ratio (NLR) is associated with a shorter disease-free survival in patients with Early Breast Cancer (EBC). Evidence from mouse models of BC shows that Ns can either have a tumour-promoting or tumour-inhibitory role. CD62L (L-selectin) is a cell adhesion molecule found on the surface of leukocytes which is important for helping circulatory Ns bind to the endothelium and migrate into tissues and is rapidly shed upon N activation. Our laboratory has found a lower proportion of Ns expressing CD62L in pre-clinical models of BC, which implies that Ns from tumour-bearing mice have less endothelial- adhesive capacity and hence will remain in the circulation for a longer time. However, there is limited knowledge regarding whether different N subpopulations are present within the circulation of patients with BC, how early this occurs in the disease process and whether this is influenced by BC phenotype. In order to address these questions, we phenotyped Ns from BC patients. Objectives: Our primary objectives were to investigate changes in the surface marker CD62L expression via flow cytometry in neutrophils from patients and HVs to see if the changes seen in the pre-clinical mouse models were also seen in patients. Since kinases govern many aspects of intracellular signalling and functions, we also assessed the Serine Threonine Kinase (STK) and Phosphotyrosine Kinase (PK) activity within circulatory neutrophils in patients with Breast Cancer and paired HVs using a Pamgene kinase assay. This evaluated the activity of kinases (n = 340 kinases) in N lysates to compare N kinase activity in EBC patients and age-matched HVs. Methods: We recruited 28 patients with Early BC (EBC) (10 Node +, 18 node -; 10 T1 and 16 T2 and 2 T3) and paired healthy volunteers (HVs) who were age and sex-matched to compare phenotypical features of Ns isolated from the blood. 21 Patients with Oestrogen receptor positive (ER+) and 7 with Oestrogen receptor negative (ER -) EBC were recruited between June 2018 and June 2021. No patient had overt metastatic disease and all were screened for exclusion factors, such as infection, autoimmune conditions and certain medication known to influence neutrophils. Results: We found that ER+ patients had a lower level of the CD62Llow neutrophil subset compared to HVs (P&amp;lt; 0.0375). In contrast there was a significant increase in the CD62Llow neutrophil subset (2-fold higher) for ER - patients compared to HVs (P&amp;lt; 0.0316).The Pamgene kinase assay showed that there was a general upregulation in both STK and PK kinase activity, especially the tyrosine family of kinases for patients with ER positive breast cancer (Median kinase score which is the combined sensitivity score and specificity score &amp;gt; 1.2) and a general downregulation in kinase activity, especially the CMGC family of kinases for patients with ER negative disease (Median kinase score &amp;gt; 1.2) compared to paired HVs. The functional implications of these differences in kinase activity are being actively investigated. Conclusion: We have detected changes in CD62L expression and neutrophil kinase activity within the circulation of patients with EBC and the nature of these changes appear to be intrinsically linked to ER subtype. These findings may have important implications for use as part of an early diagnostic multi-omic platform. Citation Format: Anisha Ramessur, Bana Ambasager, Ilaria Malanchi, Raoul Charles Coombes. The multi switching activity of neutrophils in the circulation of patients with early breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-01-14.