Patients with cancer are thought to have significant impairment of the immune system. One of the manifestations is believed to be skewing of the cytokine profile in favour of the immunosuppressive Th2 cytokine family as opposed to the immunofacilitatory Th1 cytokine family. In this MD thesis, a prospective study was undertaken to determine whether this occurred in breast cancer patients. IL-IO (representing the Th2 cytokine family) and IL-12 (representing the Th1 cytokine family) levels in the serum, IL-12 production capability of peripheral blood mononuclear cells (PBMC) along with the levels of IL-to and IL-12 in the tumour microenvironment as detected by immunohistochemistry were determined in breast cancer patients and compared with levels in healthy volunteers. No difference in serum IL-to and IL-12 levels or IL-12 production capability of PBMC was noted between breast cancer patients and controls. Among the known prognostic factors (tumour size, grade, lymph node involvement and oestrogen receptor status), grade was found to be inversely related to serum IL-12 levels (by univariate and multivariate analysis; P=O.045). No significant association between these prognostic factors and IL-IO levels were noted. Immunohistochemistry revealed mild to intense staining for IL-IO in the tumour vicinity, but in the case of IL-12, the staining was inconclusive. The effect of therapy on the levels of IL-IO and IL-12 was also investigated. Following surgical excision, serum IL-IO levels and IL-12 production capability of PBMC did not show significant change. However, serum IL-12 levels were significantly elevated after surgery raising the possibility of a partial skewing of the immune response in favour of the Th1 cytokine profile (Wilcoxon signed ranks test, P=O.OOl). After completion of adjuvant therapy (chemotherapy and/or radiotherapy), serum IL-IO levels again did not show any significant change, but serum IL-12 levels showed a downward trend which may be due to the immunosuppressive effect of the adjuvant therapy (Wilcoxon signed ranks test, P=O.06). In conclusion, this study did not find any evidence of Th2 bias as a manifestation of immune suppression in breast cancer patients. The change in serum IL-12 levels following surgery and adjuvant therapy is interesting and may reflect a change in immune status following the therapeutic interventions. The significance of this must be elucidated by studies with long term follow-up before definite conclusions can be made.

Breast cancer is one of the most common cancers in females in the western world, and despite the advances in diagnosis and treatment it is still associated with significant morbidity. Thus, improvements to existing treatment modalities remain a priority. Understanding the molecular mechanisms controlling tumour growth and its modulation will be key to developing new therapies. In recent years it has been shown that posttranslational modifications (PTMs) of histones and p53 are functionally important in the regulation of cellular processes such as proliferation, differentiation and DNA damage repair. Thus, this study assessed the incidence of histone and p53 PTMs in breast tumours, and investigated how small molecule inhibitors of acetyltransferases can manipulate the levels of these PTMs in tumour cells. Our initial study demonstrated that hypoacetylation of H4K16 is associated with higher grade breast tumours (Elsheikh et al., 2009). Therefore, the expression levels of enzymes that are known to modulate H4K16 acetylation in vivo was assessed using immunohistochemical staining of 880 human breast tumour tissue microarrays. This led to the identification of a cluster of biomarkers (hMOF, H4K16ac, H3K9me3 and SUV39H1) which are significantly associated with patient outcome. We also assessed in tumours the incidence of other potential biomarkers including selected p53 PTMs such as p53K373ac and p53 K386ac. These were also found to be associated with favourable patient outcome in Kaplan-Meier survival analysis. Other potential biomarkers were also assessed such as the histone variant H2A.Z and its hyperacetylated form. H2A.Z correlated with Estrogen Receptor status of the tumours, consistent with a report that the gene encoding this histone variant is estrogen-regulated. In summary, this study has revealed that histone and p53 PTMs in breast tumours are potentially useful biomarkers for the classification of tumour type and as prognostic indicators, for use in conjunction with other clinicopathological indicators, and other well established biomarkers such as estrogen receptor and HER2. In a second aspect of the study, we investigated the effects of the acetyltransferase inhibitors curcumin and garcinol on a breast cancer cell model (MCF-7 cells). Garcinol blocked transcription-related PTMs such as H3K18ac, but surprisingly induced hyperacetylation of H4K16. This was found to be correlated with increased TIP60 expression, and correlated with increased incidence of DNA damage and cell cycle arrest. Other changes in cancer -associated PTMs were also observed, including increased H4K20 trimethylation. Garcinol compounds also reduced colony formation by MCF-7 cells and augmented sensitivity to etoposide. In summary, the data shows that histone and p53 PTMs constitute novel biological prognostic markers in breast cancer, and that targeting the enzymes that regulate these events may provide new avenues to drug therapies. This version does not contain the previously published journal article reproduced in the print thesis.

Breast cancer is the most common malignancy among females. Although considerable progress in disease detection and treatment has greatly improved survival, this entails the endurance of sequential treatment combinations. Such treatments are associated with cancer related fatigue (CRF), a debilitating form of fatigue that often persists for months or years post treatment, and a reduction in cardiovascular health that increases the risk of cardiovascular disease.

Although global gene microarray studies have demonstrated the molecular heterogeneity of breast cancer (BC) and provided potential for clinical applications, the molecular subclassification of luminal/ER-positive tumours, which is the largest class of BC, remains unclear. Characterisation of luminal/ER-positive subtypes could have important implications in clinical decision-making and patient management. The patient study cohort is derived from a consecutive series of approximately 1902 cases of primary operable invasive breast carcinoma obtained from the Nottingham Tenovus Primary Breast Carcinoma Series, with patients presenting between 1986 and 1998. This is a well-characterized series of primary breast carcinoma that has been treated in a uniform way and previously used to study a wide range of proteins. Using gene microarray experiments in 128 frozen invasive BC derived from this series , 47,2 93 gene transcripts were analysed using a number of different bio-statistical models to identify a transcript signature for luminal/ER-positive BC, from which candidate genes were selected and that can be used to characterise ER-positive breast cancer. In addition, other biomarkers with strong relevance in ER-positive breast cancer were studied because the evidence strongly suggests an important role in the biology and molecular classification of ER-positive breast cancer. The selection criteria was based on published literature concentrating mainly on ER related pathways including ER coregulators (CARMI, PELPI), cellular proliferation (p27. TK1, cyclin B1), apoptosis (Bc12), Akt/PIK3 pathway (FOX03a), gene expression profiling (FOXA1, XBP1, TFF1) and endocrine resistance (CD71). Immunohistochemistry and high throughput tissue micro array technology were used to study the protein expression of 16 biomarkers with strong relevance to ER pathways in a well characterised consecutive series of invasive BC (n=1902) in addition to anther 9 markers that were available from the database of the breast cancer research group, University of Nottingham. The data were analysed using different clustering methods including K-means and Partitioning around Medoids. Kaplan Meier plots with Log-rank test (LR) were used to model clinical outcome. A transcript signature for ER positive BC was identified including RERG, GATA3 and other genes by a supervised classification analysis using 10-fold external cross-validation of the gene microarray data. Immunohistochemical validation studies confirmed their association with ER positive BC. Through a consensus approach using different clustering techniques applied to protein expression data 25 markers, three biological clusters (patient subclasses) in ER positive breast cancer showing significant difference in clinical outcome (LR= 28.185 & p<0.001) have been identified. Importantly, the poor prognosis cluster was significantly characterised by high tumour grade and frequent development of distant metastasis. In conclusion, our results emphasised the heterogeneity of luminal/ER-positive BC. Molecular profiling of breast cancer using protein biomarkers on TMAs can sub-classify ER-positive tumours into clinically and biologically relevant subgroups.

Aims The Sodium Iodide Symporter (NIS), which is located in the basolateral membrane of the thyroid follicular cell, catalyses an active transport mechanism allowing Iodide, an essential component of the thyroid hormones Tri-iodothyronine(T3) and Thyroxine(T4), to accumulate in the thyroid gland. Differentiated thyroid cancers retain the appearance and function of normal thyroid cells, and can, therefore, trap iodine. Radioiodide scintigraphy and therapy are based on the uptake of radioiodide by NIS in thyroid tissue, which can concentrate all isotopes of iodine. Breast cancer is the most common type of cancer in women in the UK, and is the second most common cause of cancer-related death in women in the UK. This situation remains true in Scotland, with breast cancer representing 27.5% of all female cancers. Despite a reduction in mortality figures between 1994 and 2004 of 18%, five year survival in Scotland remains only 80.2% for patients diagnosed between 1997 and 2001. When breast cancer recurs and becomes metastatic it is treatable, but not curable. Median overall survival of patients with metastatic disease remains poor, and is currently between one and two years. It is clear that metastatic breast cancer remains a challenging clinical issue and novel treatments are required. Radionuclides play an important part in the management of malignancy, including prostate cancer, neural crest tumours, and thyroid cancers. There is evidence that there may be a role for radionuclide therapy in breast cancer via the NIS mechanism. This was first suggested by Tazebay's landmark work in 2000, which demonstrated that a significant proportion (87%) of breast cancers contained detectable NIS protein by immunohistochemistry(IHC). Experimental design Tumour samples from patients with breast cancer were analysed by immunohistochemistry and ribonucleic acid(RNA) analysis, and this was correlated with functional activity of the NIS protein, by scintigraphic scanning of patients with metastatic breast cancer. Results Twenty-four patients had a Tc99m Pertechnetate scan; 20 patients had their RNA analysed; 22 patients had IHC performed on their tumour samples. NIS was detected only in the control Graves thyroid RNA and paraffin embedded cells extracted from a NIS expressing cell line. Breast tumour sample RNA did not demonstrate NIS. By IHC, 15 cases were defined as truly immunopositive, with the criteria for true positivity being a Histoscore of > 150, or demonstration of membrane staining. Seven cases were negative by the above criteria. Tc99m Pertechnetate uptake was observed in 11 of 24 patients who were scanned ie 46%. Of these 15 patients with positive expression of NIS in their breast cancer tissue sample, as measured by immunohistochemistry, 14 cases were also scanned. Of these, eight patients(57%)demonstrated uptake of Tc99m Pertechnetate on scintigraphy ie were true positives; six did not, ie their screening test (IHC) had incorrectly predicted positive uptake on subsequent scanning (false positive screening). Therefore, a positive predictive value of 57% was observed when using IHC as a screening test for detecting those patients who would go on to have a positive scan. In contrast, six patients had negative NIS expression by IHC, and no uptake on scintigraphy, ie true negatives. Only one patient had a false negative screening IHC result. A negative predictive value of 86% was seen when using immunohistochemistry as a screening test for detecting those patients who would have a negative scan. Sensitivity was 88%, with a specificity of 50%. Conclusions This data has shown that the prevalence of patients with metastatic breast cancer expressing NIS on IHC is relatively high (68%), when compared with one other published study of similar patients. Also, it has been demonstrated that a high proportion of NIS IHC positive patients who were scanned (57%) demonstrated uptake of Tc99m Pertechnetate in breast cancer primary or metastases. It can be concluded that IHC does act as a useful screening test for those who may ultimately benefit from radioisotope treatment. The most appropriate radioisotope for therapy remains to be determined.

Breast carcinoma is the most frequent type of cancer affecting women. Among the recently described molecular and phenotypic classes of breast cancer, human epidermal growth factor receptor 2 (HER2)-positive tumours are associated with a poor prognosis. HER2 status is currently assessed in routine breast cancer reporting using immunohistochemistry (IHC) in addition to in situ hybridisation (ISH) in borderline cases. The ability of HER2 gene status to predict response to targeted therapy (Trastuzumab) is well documented. However, prognostic information provided by IHC expression categories and prognostic value added by using ISH in borderline cases remains unclear. HER2 plays an important role in cancer progression being targeted to provide predictive and prognostic information. Moreover, HER2 is related to cancer resistance against a variety of therapies; however, trastuzumab has proved successful in treatment of this subgroup. Nevertheless, patients may acquire resistance to this drug after a period of treatment, which indicates that other molecular mechanisms might influence success of this therapy. Dimerisation between members of the HER family may contribute to resistance against treatments due to different combinations that trigger different downstream pathways. This is promoted by ligands, which are expressed as transmembrane precursor protein molecules and have a conserved epidermal growth factor-like domain. Through resistance to trastuzumab, other drugs are being developed to interact in different domains of HER2 protein. The study of interaction between receptors/ligands will characterise specifically their signalling pathway and understand which strategy to acquire. The main aim of this thesis was to assesses the status of HER2 protein (IHC), HER2 gene (chromogenic ISH) and HER heterodimers (in situ proximity ligation assay (PLA)), including HER2/EGFR, HER2/HER3 and HER2/HER4, in two BC series prepared in tissue microarray format; a series of consecutive primary operable BC cases (n = 1858) including HER2+ trastuzumab naïve cases (TrN, n = 221), the second series of HER2+ trastuzumab adjuvant treated cases (TrT, n = 143). Therefore determining the biological characterisation of these biomarkers by associating against clinicopathological parameters, survival outcome and understand the trastuzumab therapy value. There was excellent overall concordance between HercepTest negative (scores 0/1+) and positive (3+) with CISH positive/negative (defined as HER2/Chr17 copy number ratio of ≥ 2; p < 0.001). Kaplan-Meier analysis for breast cancer specific survival (BCSS) and disease free interval (DFI) revealed statistically significant differences between HER2 positive and negative cases detected by HercepTest and CISH (p < 0.001). Interestingly, it was identified that HercepTest 2+ non-amplified cases were not significantly different with those amplified 2+ or 3+ cases with respect to their behaviour (BCSS and DFI). The results revealed an inverse association between the HER heterodimerisation status and hormone receptor status (p < 0.001), and a significantly worse outcome amongst cases revealing high levels of all heterodimers (p < 0.001). Among ER+ cases, the heterodimers high levels were significantly associated with worse prognosis (p < 0.001) overall. However amongst the two HER2+ populations dimerisation status did not show an association with patient outcome. The overall concordance between HercepTest and CISH analysis for HER2 status was excellent. All HercepTest 2+ cases identified were observed to have poor outcomes similar to those HercepTest 3+ cases regardless of gene amplification status. In the current clinical environment cases exhibiting IHC 2+ and non-amplified gene HER2 status will not be offered targeted HER2 therapy but do exhibit aggressive clinical behavioural characteristics. Even though those patients with high levels of the HER2 truncated form, p95HER2, have shown poor outcome, this biomarker does not reveal any extra findings comparing with the HER2 expression results. Beside HER2, EGFR is the only monomer that reveals prognostic value amongst the breast cancer patients. Tumours exhibiting high levels of HER heterodimerisation have an adverse prognosis, however in the context of HER2+ breast cancer no association with clinical outcome was observed regardless of use of trastuzumab treatment. HER2/HER3 heterodimerisation status assessed in multivariate analyses has shown that this protein-protein interaction is associated with a poor prognostic outcome, which needs further investigation and assessment of clinical utility to use in the future in breast cancer treatment decision. Further quantification analysis of dimer/ligand complex using PLA of other HER family members may be useful to identify subset of patients associated with distinct outcome, response to treatment and relationships with HER signalling related biomarkers.

This thesis explores the experiences of 24 women who were treated for early-stage breast cancer (ESBC) or ductal carcinoma in situ (DCIS) in the UK between 6 months - 29 years previously. These experiences are important because better detection and treatments have resulted in increasing numbers of women surviving for longer. My own experience of ESBC, revealed in extracts from my research journal, brought both benefits and drawbacks to the research but ultimately resulted in a unique perspective. Participants accessed through a press release were interviewed in-depth. Data were then analysed using narrative and thematic analysis. Bury’s (1982) concept of illness as a biographical disruption was used as the theoretical framework but a consideration of the context revealed that ESBC/DCIS is not always experienced as disruptive. Analysis also revealed that the post-treatment period can present further challenges as loss of medical support can coincide with withdrawal of social support. Some participants reported feeling isolated and vulnerable post-treatment. Often their feelings were at odds with public expectations based on media representations of ‘successful’ breast cancer survivors, and the high profile of breast cancer charities which can suggest ‘pink fluffy’ imagery. Ongoing disruption to their bodies and relationships can mean that women feel in an ambiguous state which can be likened to a state of liminality (Turner, 1969). The current research combines the concepts of biographical disruption and liminality for the first time in a study of ESBC/DCIS, therefore enabling consideration of gendered aspects of the experience. In particular, the importance of breasts to femininity and sexuality can have implications for identity and relationships. I argue that, whilst women do not return to the ‘normality’ of pre-diagnosis, the wide range of experiences and emotions in the post-treatment period can be conceptualised as a ‘new normal’.

High quality population based data are essential to evaluate breast cancer. This study has improved data quality at the West Midlands Cancer Intelligence Unit through the development of a staging algorithm which significantly increased the proportion of cases with stage recorded. It has also demonstrated the benefits of combining cancer registry data with those from the NHS Breast Screening Programme (NHSBSP). This study identified stage as the most significant prognostic factor for breast cancer patients, and the favourable staging characteristics of screen detected cancers thus suggest that improved survival should result. The study also found that only 31.6% of cancers in eligible women were screen detected in the prevalent round. As 31% of breast cancers arose in women in the screening age range, only an estimated 10% of breast cancer patients benefit directly from screening. High interval cancer rates were identified, along with the need for clarification of the definitions used to identify interval cancers. This investigation identified changes in treatment over time across the region towards the King's Fund guidelines. However, treatment varied across the region and clear divergences from the guidelines were apparent. Furthermore, no association was identified between surgical caseload and survival, suggesting that the use of caseload as a proxy for specialist care may be questionable.

During severe hypoxia (<0.01% oxygen) the protein folding machinery becomes dysfunctional, resulting in the accumulation of unfolded proteins with consequent endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) and autophagy, a process involved in the physiological turnover of cytoplasmic components. The link between the UPR and autophagy is not clearly defined. The aim of this thesis is to investigate the role of the induction of UPR under severe hypoxia in tumour survival and resistance to therapy. The results of this research suggest that the activating transcription factor 4 (ATF4), a component of the PKR-like ER kinase (PERK) pathway, fundamental in the UPR, is required for the ER-stress induced upregulation of autophagy. Mechanisms other than hypoxia for UPR induction were investigated, using the proteasome inhibitor bortezomib (BZ). BZ treatment increased ATF4 protein levels in MCF7 cells, even transfected with short-interference RNA (siRNA) against the classical UPR activator PERK, suggesting that the proteasomal stabilization is likely the main mechanism for ATF4 protein accumulation. The induction of autophagy by BZ is dependent upon the upregulation of the microtubule-associated protein 1 light chain 3B (LC3B), an autophagy marker, by ATF4 and acts as a survival mechanism. Hypoxia, UPR and autophagy markers (such as Pimonidazole, carbonic anhydrases IX (CAIX), C/EBP homologous protein (CHOP) and LC3B) were evaluated by immunohistochemical approach in spheroids, xenografts models and breast cancer samples. CHOP immunohistochemical staining was performed in breast cancer sections from a series of patients. CHOP was expressed in cells surrounding necrotic areas. No correlation were found with clinical outcome and further studies are needed.

In developed countries, breast cancer is the commonest malignancy among women. Understanding the mechanisms involved in breast cancer progression and the influence of the microenvironment on cancer cell proliferation, results in better treatments. This study aimed to optimise breast cancer xenograft rates using a novel chamber developed for tissue engineering purposes. The established tumours were subjected to enzyme digestion, creating a single cell suspension, which was then injected into immunocompromised mice at primary, metastatic and intra-cardiac sites. The resulting tumours in the mammary fat pad (MFP) and bone were compared using species-specific reverse-transcription polymerase chain reaction (RT-PCR) and cDNA microarray, to examine the influence of the microenvironment on gene expression. The achieved xenograft graft rates of 25% were similar to those previously reported. The matrix metalloproteinase family of enzymes (MMPs) degrade extracellular matrix, influencing invasion and migration of malignant cells. RT-PCR results showed that the majority of the MMPs expressed in the cancers were stromal rather than tumour in origin. MT1-MMP, MMP-2 and MMP-11 had significantly higher expression levels in the MFP than in the bone, but MMP-9 was expressed more in the bone than MFP. There was also an up-regulation of stromal production of MT1-MMP and MMP-13 in the MFP in the presence of tumour. This may have significance when considering which MMPs are the most appropriate targets for inhibition during cancer treatment. The most significant of the differentially expressed genes on microarray analysis were trefoil factor 1 (TFF1) and insulin growth-factor binding protein 3 (IGFBP-3), both expressed significantly more in tumours from the MFP than the bone. The thesis presented demonstrates some of the complexities of tumour-stromal interactions and supports Paget’s seed-soil theory, confirming in several ways the variation in gene expression in breast cancer between primary and metastatic sites.