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Title: A flow cytometric analysis of B and T-lymphocyte phenotypes in breast cancer : implications for prognosis, treatment and recovery
Author: Strachan, Caroline Louise
ISNI:       0000 0004 9358 3232
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2020
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Introduction: It is widely understood that the T-lymphocyte is closely linked to prognosis and treatment response in certain solid organ malignancies including breast. B-Lymphocytes have, however, received comparatively little attention in this field and it remains unclear what role, if any, B-lymphocyte subtypes play in carcinogenesis or prognosis in breast cancers. Likewise, it is unknown what effect systemic treatments such as chemotherapy have on B-cell immunity, or the role these lymphocyte subtypes may have in the side-effect profile of such therapies. Cancer related fatigue (CRF) is one such side-effect of cancer and its treatments, the pathogenesis of which has been linked to impaired immunity including altered lymphocyte phenotypes. My aim in this work was to perform detailed phenotypical analyses of B- and Tlymphocytes in breast cancer, as well as phenotypical alterations driven by cancer treatments, to provide insight into the function of B-lymphocytes in this disease process, adding to the growing body of knowledge driving immunotherapeutic development. Methods: 43 primary breast cancer patients, scheduled to receive adjuvant chemotherapy, were recruited following resection surgery alongside 10 age- and sex-matched healthy controls. 27 Chronic fatigue syndrome (CFS) patients were recruited at diagnosis, as a comparator group for assessments relating to CRF. 15 further patients with primary breast cancer were recruited prior to resection surgery for analyses of fresh tumour tissue and peripheral blood. Phenotypes of circulating lymphocytes were analysed using multicolour flow cytometry at various time-points in the larger breast cancer cohort and its associated comparator groups (CFS and healthy controls), along with general health and well-being screening questionnaires as used in the diagnosis of CFS. For the fresh tissue study, tumour tissue was biopsied and analyses of tissue-resident B- and T-lymphocytes were performed, along with circulating analyses as previously, focusing on regulatory B and T subtypes. Results were analysed using paired T-tests, 2-way ANOVA, and correlation analysis. Results: Lymphocyte phenotype was vastly altered by chemotherapy. Within the Tcell pool, the naïve subset is diminished with proportional increases to the memory cell pool and the overall expansion of HLA-DR surface expression on CD4+ T-cells. Regulatory T-cells were unchanged. Within the B-cell pool, memory cells were 5 largely reduced by chemotherapy, the resulting phenotype being naïve and transitional dominant. Pro-inflammatory cytokine expression by regulatory B-cells was diminished, yet IL-10 expression remains unchanged. The CD20+CD27+ regulatory Memory B-cell subset is demonstrated as a critical Bcell phenotype in breast cancer prognosis and in fatigue. CD27+ regulatory memory B-cell dominates the phenotype in tumour tissue where these subsets, in addition to naïve T-cells, correlated positively with poor prognostic indicators in breast cancer both in peripheral blood and tumour tissue. Additionally, expression of effector regulatory cytokines IL-10 and TNF-α from the memory B-cell subsets correlated with prognosis and fatigue. Conclusions: Detailed B-lymphocyte phenotypes have been clearly demonstrated in breast cancer and in fatigue, with the regulatory CD20+CD27+ memory B-cell subset prevailing as the dominant cell type relating to breast cancer prognosis, both in tumour and peripheral blood, as well as fatigue scores. This first insight into the phenotype and function of B-cells and B-regs in breast cancer highlights the memory B-cell subset as a driver in the pathogenesis of this disease, and provides a target for further research and potential novel lines of investigation in the on-going search for immunotherapies.
Supervisor: Hughes, Tom ; Carter, Clive ; Velikova, Salina ; Horgan, Kieran Sponsor: Not available
Qualification Name: Thesis (M.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available