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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.

Background and Aim: Chemotherapy is used for treatment of breast cancer. However, relatively little is known about the extent or the time course of immune dysfunction caused by it. The aim of this study was to evaluate the circulating lymphocytes and tetanus & pneumococcal antibody titers pre and at various time points post-chemotherapy and check for association between circulating lymphocytes and tumour infiltrating lymphocytes (TILs) and their correlations with patient outcome. Methods: Detailed immunophenotyping of peripheral blood lymphocytes was performed by flow cytometry in 88 patients with primary breast cancer before and at various time points up to 9 months after chemotherapy. Peripheral blood levels of anti-pneumococcal and anti-tetanus antibodies were assessed using ELISA. Immunohistochemistry was used to assess the presence of tumour infiltrating CD20+, CD4+, CD8+ and FoxP3+ positive lymphocytes in the tumour microenvironment. Results: There were significant depletions of circulating B, CD4+T, CD8+T and NK cells at 2 weeks post-chemotherapy (p < 0.001), with B cells showing maximum depletion. Levels of B cells and CD4+ T cells remained significantly low even at 9 months post-chemotherapy (p < 0.001). Repopulating B and CD4+ T cell phenotypes were different from the pre-chemotherapy profile. Titers of anti-pneumococcal and anti-tetanus antibodies were significantly reduced post- chemotherapy and did not return to normal even at 9 months post-chemotherapy (p < 0.001). Smoking and chemotherapy regimen had significant correlations with degrees of depletion and repopulation of B and T cells. Chemotherapy regimen and the extent of depletion of lymphocytes had a significant influence on overall and disease-free survival. Analysis of TILs showed significant correlations between the stromal and intra-tumoural levels of each of the lymphocytes and between different lymphocytes. TILs correlated with hormone negative, triple negative and grade 3 tumours. Only tumour infiltrating CD8+ lymphocytes correlated with its matched circulating levels, and this positive correlation was stronger in hormone negative and triple negative tumours. High CD4+ stromal infiltrate was associated with better disease-free survival and overall survival. High stromal CD8+ T cells were associated with better disease-free survival in ER/PR negative patients, while high intra-tumoural CD8+ T cell and FoxP3+ infiltrate was associated with poor overall survival in the ER/PR positive cohort. Conclusion: Breast cancer chemotherapy has significant long-term effects on the immune parameters and this should be taken into consideration during clinical management. TILs are associated with poor prognostic features and also show some correlation with circulating lymphocytes. The site and type of lymphocyte infiltrate in the tumour microenvironment influences outcome.

Three main xenobiotic transporters have been implicated in modulating breast cancer response to chemotherapy. These are Pgp (P-glycoprotein), MRP1 (multidrug resistance-associated protein 1), and BCRP (breast cancer resistance protein). My first aim was to investigate expression of these proteins before and after neoadjuvant chemotherapy (NAC) for breast cancer to determine whether their levels define response to NAC or subsequent survival. Immunohistochemistry was performed for Pgp, MRP1, and BCRP on paraffin embedded tissue representing matched pairs of core biopsy (pre-NAC) and resection specimens (post-NAC) from 39 breast cancer patients. Pgp and MRP1 were found to be significantly up-regulated after chemotherapy but levels did not relate to response or survival. High post-NAC BCRP expression independently predicted for poorer disease free survival (hazard ratio of 4.04; p=0.013). Evidence within the literature suggested that MRP1 up-regulation after chemotherapy may be driven by activated Notch1. My second aim was to determine whether activated Notch1 expression correlated with MRP1 expression in the same patient samples. Further immunohistochemistry to determine activated Notch1 expression revealed a significant correlation between post-NAC activated Notch1 and MRP1 expression (rho coefficient 0.6; p=0.0008). The hypothesis that inhibition of Notch signaling enhances killing of breast cancer cells by chemotherapeutics was developed. MTT assays were performed after treatment of breast cancer cells with combinations of doxorubicin and the Notch inhibitor DAPT. Minor additive inhibition of survival/proliferation was seen in the combination treatment, failing to provide strong support for the hypothesis. The BCRP promoter has an oestrogen response element. My third aim was to investigate whether BCRP expression within breast tumours is regulated by oestrogen and whether this impacts on cancer outcome. Immunohistochemistry was performed for BCRP in tumour samples from 51 patients receiving neoadjuvant endocrine therapy (NAET); matched core biopsy (pre-NAET) and resection specimens (post-NAET) were investigated. BCRP expression was significantly up-regulated after exposure to NAET (p<0.0001). High pre-NAET BCRP expression independently predicted for poorer DFS (hazard ratio of 17; p=0.014). Subsequent methylation analysis of cancer cell lines showed that the degree of methylation in the BCRP promoter region was potentially inversely correlated to the BCRP protein expression observed on immunoblotting. DNA was extracted from clinical sample and pyrosequencing analysis was performed. No such inverse correlation was observed in the clinical samples. My work demonstrates that analysis of tumour samples pre- and post-neoadjuvant therapies provides a powerful way of investigating therapy-dependent changes in expression of molecules of interest, and may be critical for determining the prognostic or predictive value of some markers. Given the relatively small sample size of the cohort examined, future higher powered studies are required to determine the prognostic significance of BCRP expression.

Background: Inactivation of wild type P53 by its main cellular inhibitors, MDM2 and MDMX, is a well-recognised feature of tumour formation in liposarcomas (LS). MDM2 over-expression has been detected in approximately 80% of liposarcomas, but only limited information is available about MDMX expression levels. On commencing this work, we were not aware of any study that had described the patterns of MDM2 and MDMX co-expression in liposarcomas. Such information has become more pertinent as various novel MDM2 and / or MDMX single and dual affinity antagonist compounds have emerged as alternative approaches for potential targeted therapeutic strategies in LS. Methods: After appropriate optimisation and confirmation of experimental techniques, a case series of 64 pathologically characterised liposarcomas of various sub-types was analysed by immunohistochemistry, to simultaneously assess the expression levels of P53, MDM2 and MDMX. P53 mutation status was investigated in cases that over-expressed P53. Results: 83% of cases over-expressed MDM2 and 69% co-expressed MDMX at varying relative levels. The relative expression levels of the two proteins with respect to each other were subtype-dependent. This apparently affected the detected levels of P53 directly, in two distinct patterns. Diminished levels of P53 were observed when MDM2 was significantly higher in relation to MDMX, suggesting a dominant role for MDM2 in the degradation of P53. Higher levels of P53 were noted with increasing MDMX levels, suggesting an interaction between MDM2 and MDMX that resulted in reduced efficiency of MDM2 when degrading P53. No increased incidence of P53 mutations was detected in cases that over-expressed P53 compared to the general population of LSs. Conclusions: The results of the study indicated that complex dynamic interactions between MDM2 and MDMX proteins may directly affect the cellular levels of P53 in human liposarcomas. This suggests that careful characterisation of all these markers will be necessary when considering in vivo evaluation of novel MDM blocking compounds as a therapeutic strategy to restore wild type P53 functions.