Ovarian cystectomy is commonly performed to treat benign ovarian cysts, but might cause inadvertent damage to normal ovarian tissue, thereby influencing a woman’s ovarian reserve. Ovarian reserve is defined as the existent quantitative and qualitative supply of follicles which are found in the ovaries that can potentially develop into mature follicles which in effect determine a woman’s reproductive potential. It is commonly quantified by the levels of serum FSH and recently by total antral follicle count (2.0-10.0 mm follicles in both ovaries) and AMH levels. These tests however have inherent biological variation in relation to menstrual cycle and ageing; and are also influenced by the intra- and inter-observer variations. The aim of this thesis was to develop a reliable method of examining the effect of ovarian cystectomy on ovarian reserve. I began by examining the ultrasound markers of ovarian reserve. AFC is measured using 2D ultrasound and there is some evidence that 3D ultrasound can make more reliable counts than 2D. I examined the reliability of these two methods and compared them to a new 3D assisted method, SonoAVC which is designed to make automated AFC. I found that the intra- and inter-observer reliability of SonoAVC in counting the number of antral follicles was superior to 2D and 3D manual methods. It however required post-processing of the counts by manually clicking on the antral follicles initially missed in the automated version, thereby making it a semi-automated method. I then compared 2D ultrasound to SonoAVC in measuring the size of antral follicles as there is increasing evidence that the small antral follicles might be more predictive of ovarian reserve. I found that SonoAVC measured the size of antral follicles significantly quicker than 2D and also that the number of small follicles measured by 2D were more than SonoAVC, thereby raising the possibility that 2D might overestimate the number of small antral follicles. I then studied the ability of antral follicle counts stratified by size in prediction of ovarian response and pregnancy. I found that the small antral follicles measuring between 2.0-4.0 mm were independent predictors of clinical pregnancy and ovarian response to assisted reproduction treatment. I then examined the AFCs of different sizes made by SonoAVC and 2D in bovine ovaries and compared to the follicles obtained by manually dissecting the follicles. I found that SonoAVC with post-processing significantly underestimated and 2D overestimated the number of antral follicles measuring 4.0mm or less, but both made comparable counts of follicles measuring more than 4.0mm when compared with the antral follicles dissected manually. However, the agreement with SonoAVC with post-processing was more than that with 2D. Having established that SonoAVC albeit with post-processing was the most reliable method in measuring the size of antral follicles, I began to examine the intra- and inter-cycle variation and compared to AMH. I found that the small antral follicle measuring 2.0-6.0 mm showed least intra-and inter-cycle variation and that it was comparable to AMH. The larger antral follicles showed significant intra-cycle variation but a non-significant inter-cycle variation in the early follicular phase of menstrual cycle. I also examined the inter-ovarian variation in the AFC’s and found that the small antral follicles measuring 2.0-6.0 mm again showed the least variation between ovaries within an individual. I was finally able to conclude that small antral follicles (≤6.0mm) measured using SonoAVC were the most reliable in prediction of ovarian reserve, and showed excellent correlation with AMH. Finally, I examined the effect of laparoscopic ovarian cystectomy on the ovarian reserve for up to 6 months post-operatively using AMH and small AFC measured by SonoAVC. I found that ovarian cystectomy significantly reduces ovarian reserve and that this effect may be more pronounced with cysts of endometriotic nature, followed by dermoid cysts. In summary, the effect of ovarian cystectomy on ovarian reserve is best quantified using AFC of small follicles measuring less than 6.0 mm as it provides reliable measures of ovarian reserve, has minimal biological variation and is comparable to AMH.

This thesis examines and explores women's mammography screening experiences and the factors influencing women's screening behaviours and choices. The sample consisted of Greek women who were recruited from women's organizations in Athens. 189 women completed a survey questionnaire about their values and beliefs in relation to mammography screening, 33 of whom subsequently underwent an in-depth interview to explore their experiences of mammography screening and their decision-making processes. According to the findings, women's mammography screening behaviour depended to a great extent on the quality and nature of their interactions within their social networks. Thus, the quality of information and meanings derived from these interactions was what determined women's behaviour. Family, close friends and doctors appeared to be important in the formation of women's beliefs, perceptions, emotions (fear of cancer) and behaviour in relation to breast screening. Interestingly, fear of cancer had an opposing effect towards their screening behaviour. A tentative model is presented, which attempts to explain the way these influences affect beliefs, perceptions and mammography screening behaviour, using elements of existing behavioural models. Further exploration of the influential factors and associations identified in this study is required.

The study was conducted following a systematic review of 38 articles (Collins et al 2004), which failed to find strong evidence for the optimum duration and frequency of follow-up, or the personnel best placed to provide it. Against this background of lack of evidence for ongoing follow-up, on the grounds of limited resources and an ever increasing population of breast cancer survivors; the level and nature of service provision for the patients attending a regional breast unit was changed. What was unknown was the impact this may have on the quality of the care and support and future health and well-being. Primary aim: To investigate the experience of being discharged from follow-up breast care services and evaluate the impact of the proposed changes from the patient’s perspective. This thesis reports on interviews with 20 women from the time they were discharged from the service to 2 years post discharge. In addition, 4 participants who developed recurrent disease gave interviews to discuss their experiences. 3 major themes emerged: 1. “Close shave” or “marked woman”? describes the approaches adopted by the women towards their disease, treatment and risk of recurrent disease; 2. A “blessing” or a “curse”? encompasses views of the value of follow-up care; 3. “freedom” or “isolation”? is the account of the impact of discharge from the service on future health, care and well-being. Creating a network of services and working collaboratively with other sectors may well provide the scope and quality of care that women in long-term recovery from breast cancer want and need from the survivorship care of the future.

Breast cancer, which is the most common cancer in women, is a complex disease characterised by multiple molecular alterations. Current routine clinical management relies on availability of robust clinical and pathologic prognostic and predictive factors, like the Nottingham Prognostic Index, to support decision making. Recent advances in highthroughput molecular technologies supported the evidence of a biologic heterogeneity of breast cancer. This thesis is a multi-disciplinary work involving both computer scientists and molecular pathologists. It focuses on the development of advanced computational models for the classification of breast cancer into sub-types of the disease based on protein expression levels of selected markers. In a previous study conducted at the University of Nottingham, it has been suggested that immunohistochemical analysis may be used to identify distinct biological classes of breast cancer. The objectives of this work were related both to the clinical and technical aspects. From a clinical point of view, the aim was to encourage a multiple techniques approach when dealing with classification and clustering. From a technical point of view, one of the goals was to verify the stability of groups obtained from different unsupervised clustering algorithms, applied to the same data, and to compare and combine the different solutions with the ones available from the previous study. These aims and objectives were considered in the attempt to fill a number of gaps in the body of knowledge. Several research questions were raised, including how to combine the results obtained by a multi-techniques approach for clustering and whether the medical decision making process could be moved in the direction of personalised healthcare. An original framework to identify core representative classes in a dataset was developed and is described in this thesis. Using different clustering algorithms and several validity indices to explore the best number of groups to split the data, a set of classes may be defined by considering those points that remain stable across different clustering techniques. This set of representative classes may be then characterised resorting to usual statistical techniques and validated using supervised learning. Each step of this framework has been studied separately, resulting in different chapters of this thesis. The whole approach has been successfully applied to a novel set of histone markers for breast cancer provided by the School of Pharmacy at the University of Nottingham. Although further tests are needed to validate and improve the proposed framework, these results make it a good candidate for being transferred to the real world of medical decision making. Other contributions to knowledge may be extracted from this work. Firstly, six breast cancer subtypes have been identified, using consensus clustering, and characterised in terms of clinical outcome. Two of these classes were new in the literature. The second contribution is related to supervised learning. A novel method, based on the naive Bayes classifier, was developed to cope with the non-normality of covariates in many real world problems. This algorithm was validated over known data sets and compared with traditional approaches, obtaining better results in two examples. All these contributions, and especially the novel framework may also have a clinical impact, as the overall medical care is gradually moving in the direction of a personalised one. By training a small number of doctors it may be possible for them to use the framework directly and find different sub-types of the disease they are investigating.

Introduction: Early diagnosis of breast cancer can result in less radical therapy and improved survival. Current screening and diagnostic tools have limitations, as do serum marker antigens due to their low sensitivity. We hypothesised that an immune response is an early event in cancer evolution. Autoantibodies, which are the amplified signals of cancer-derived antigens, can be detected in the peripheral blood of women with early breast cancer. This thesis is a continuation of previous work at the Nottingham Breast Unit aimed at developing new panel of assays for the detection of autoantibodies in breast cancer. The goal of this thesis was to investigate the use of a potentially more reproducible ELISA assay to measure serum autoantibodies to MUC1, p53 and c-myc either singly or in combination within a panel to further clarify a role of AAbs in screening, diagnosis or prognosis of primary breast cancer. Methods: Newly expressed, biotinylated and reconfigured p53 and c-myc antigens and purified MUC1 antigen were used to establish novel in-house ELISA. These were used to measure autoantibodies to the above 3 antigens in the serum of various populations which were collected over a two year period. These populations included an at-risk population (e.g. family history and atypical ductal hyperplasia) and a population of women who had just been diagnosed with primary breast cancer, either non-invasive ductal carcinoma in situ (DCIS) or invasive cancers. Cut-off values were established for each of the autoantibodies based on 2 or 3 standard deviations from the mean of a population of control samples. The control samples were obtained from a population of women who were either deemed ‘normal’ or who had a histological diagnosis of benign breast disease. The assay was validated by assessing effect of sample age as samples were of varying age, reproducibility using Bland Altman coefficient of reproducibility and reliability by establishing the assays ability to distinguish cancer from non-cancer. Results: Eight hundred and ninety eight samples were analysed in the study. One hundred and ten were Control samples. The remaining samples included 381 that were from an at-risk population and 407 that were from a primary breast cancer population. Mean ages of Control, at-risk and primary breast cancer populations were 58.8, 50 and 62.9 years respectively. Data establishing validity of assay confirmed that sample age did not affect signal strength for MUC1 and c-myc autoantibodies. Older samples for the p53 autoantibody had lower signal than recent ones. Reproducibility data was satisfactory and was best in the samples from the group of women with benign breast disease. Using either a 2 or 3 standard deviation cut-off value the assay was also able to distinguish cancer from non-cancer for both MUC1 and p53 autoantibodies. For the c-myc autoantibody, cancer samples showed increased signal compared to non-cancer although this did not reach significance. The at-risk population were routinely followed up in an outpatient clinic dedicated for women at increased risk of breast cancer. An individual positive marker was noted in up to 10% of at-risk patients. The panel of 3 assays showed a raised marker in 18.4%. This was significantly higher than that for the Control population whose panel detection was 9.1% whilst an individual marker was noted in up to 4.5% of samples. Only the c-myc autoantibody had similar prevalence in both Control and at-risk populations. There was no correlation between risk category and autoantibody detection. The specificity for MUC1, p53 and c-myc autoantibody serum tumour markers were 92.4%, 95.2% and 95% respectively. Specificity of the assay can be further increased if two or more markers were needed to be positive before a positive result is deemed for the assay. Thirteen women in the at-risk group developed breast cancer. The panel had a higher sensitivity to detect occult tumours compared to individual markers but at reduced specificity. Two of 13 at-risk patients (15.4%) who developed breast cancer had a raised marker (MUC1 & p53 autoantibodies) within the panel with a mean lead-time of 43.5 months. Further increasing the cut-off value to Mean + 4 standard deviation of Control population increased the specificity of the panel assay to 97.2% without altering the sensitivity to detect occult tumour (15.4%). Primary breast cancer population consisted of patients who were known to have DCIS or invasive breast cancer. The latter group was further subdivided into those who were detected via screening mammogram (screen-detected) and those who presented with a lump (symptomatic). Two of the 3 markers (p53 and c-myc autoantibodies) were significantly raised in the primary breast cancer population compared to the at-risk population as well as the Control group as detailed in earlier paragraph. Individual markers were detected in up to 20.9%, 10.3% and 9.8% for p53, c-myc and MUC1 autoantibodies respectively. The panel detection rate was 35.1%. The tumour markers showed limited use as a prognostic factor. Only the c-myc autoantibody correlated with a poorer survival due to distant metastasis in symptomatic breast cancers. Data for the screen-detected breast cancer cases showed that there were no correlation between any of the 3 serum marker detection and prognosis. Conclusion: Our data demonstrated the three autoantibody assays whether singly or in combination as a panel showed differences not only between cancer and non-cancer but also between Control and at-risk, as well as between at-risk and cancer. The panel showed that one or more assays were positive in 35% of breast cancers with a specificity of 83.6%. The specificity of the assay can be altered to meet clinical needs by either increasing the cut-off value or altering the markers within the panel. Current data in the literature suggests a number of markers that may be added or substituted into the panel to enhance the specificity and sensitivity. However a sensitivity of 15.4% for detection of occult tumour in the at-risk group makes any clinical application for screening in this group less cost effective using the version of the assays described in this thesis. The lead-time in the two patients who did show elevation of an autoantibody suggests that if the sensitivity and specificity can be improved that there is an in-vivo amplification signal, which might allow earlier identification of some breast cancers. Detection of c-myc autoantibodies indicates a poorer prognosis in the symptomatic group. The value of this information needs to be further determined in larger studies and within multivariate analysis. If the current results remain then there may be clinical implication to this early data. Comparison with previous data from the unit revealed that detection of cancer-associated autoantibodies in primary breast cancer and at-risk groups using this methodology appeared to be less sensitive. This may indicate that the current method has been successful in reducing background signal and hence reduce false positive results. It therefore appears that we have established a more reliable and reproducible assay compared to previous study to detect autoantibodies to tumour-associated antigens. However it is noted that this thesis reports single batches of antigens (MUC1, p53 and c-myc) used in the autoantibody assays. Investigation of differences in protein structure and immunogenicity between batches, which might also affect the sensitivity and specificity of these assays, was outside the scope of this thesis but is the subject of ongoing research by other members of the research group.

Introduction: Heavy menstrual bleeding is known to impact on health, wellbeing and social functioning. There is limited research examining women’s experiences of treatment for this complex condition. This longitudinal qualitative study explored women’s experiences of medical treatment for heavy menstrual bleeding with particular reference to women’s treatment preferences, quality of life and cultural variation. Methods: Data were generated by a series of two semi-structured interviews conducted with women who had either consented to participate in a randomised controlled trial (investigating effectiveness of levonorgestrel intra-uterine system [LNG-IUS] compared with standard medical treatments) or had declined due to an expressed treatment preference. The sample was purposefully selected to include a wide range of demographic characteristics and medical treatment options. Interviews were audiotaped and transcribed verbatim before being coded. A grounded approach to analysis was used with concepts emerging from coded data. Data generation and analysis were iterative and continued until theoretical saturation was reached. Findings: Twenty-seven women were selected and consented to the interview study, ten of whom expressed a treatment preference. A broad range of demographic characteristics and treatment options was achieved. In addition to physical aspects of heavy menstrual bleeding, most women described maintaining the societal norm of concealment of menstruation distressing. Expectations of positive treatment outcomes were high, but the experience of most treatment did not, in the initial stages, meet women’s expectations. After one year, women using LNG-IUS felt less restricted in their daily activities and described feeling less distress about their heavy menstrual bleeding. Many women who were using other treatments had discontinued them, several opting to manage their heavy menstrual bleeding by adapting coping behaviours rather than seeking alternative treatment. Treatment preferences appear to be influenced by knowledge gained from peers with LNG-IUS being the most preferred treatment. For many women in this sample the impact on quality of life was determined by their perceived ability to continue with their roles and responsibilities both at home and in the workplace. A conceptual model was developed when descriptive themes emerging from coded data were overlaid with recorded treatment outcomes. The model illustrates the complexity of heavy menstrual bleeding and highlights common feelings and experiences could be aligned to particular treatment trajectories. Conclusion: This study adds understanding to women’s experiences of medical treatment for heavy menstrual bleeding, preferences for treatment and how quality of life changes over time. The conceptual model developed highlights the complexity of this condition and might be used to improve communication between women and health professionals.

Over recent decades, translational ovarian cancer research has been impeded by its underappreciated molecular heterogeneity and five-year survival has remained poor. One strategy for addressing this problem is to search for molecular biomarkers that can better inform the development and targeting of novel treatments. The aim of this thesis was to construct and validate a tissue microarray of ovarian cancer cases and to survey the expression and prognostic capabilities of immunological molecular markers: specifically HLA class I and the membrane bound complement regulatory proteins CD46, CD55 and CD59. These are central to the efficacy of certain immunotherapies and while they have been shown to have prognostic power in breast and colorectal cancer, they have been investigated less in ovarian cancer. Five copies of a tissue microarray representing 339 cases of ovarian cancer which presented to Derby City General Hospital between 1982 and 1997 were made. The array was stained for CK7, CK20, CA125, CEA, p53 and Bcl-2 following a standard immunohistochemical protocol. A linked clinical database was adapted and assessed for data consistency and subsequently used to analyse the prognostic and clinicopathological associations of the expression data. The array was then stained for HLA class I, B2microglobulin and CD59 using commercial antibodies and for CD55 and CD46 using in-house antibodies. Retained expression of HLA class I molecules independently predicted improved prognosis. High expression of CD55 and CD59 were associated with worse prognosis, though not independently of other factors. CD55 expression was more widespread than previously appreciated. This thesis describes the discovery of a new independent marker of prognosis which suggests that immunoediting occurs in ovarian cancer, describes the distribution of markers known to have a negative impact on immunotherapy in ovarian cancer in a large series for the first time and documents the production of a valuable resource for future studies.

Breast cancer (BC) is the most commonly diagnosed cancer in the UK and the second leading cause of cancer-related deaths in women worldwide. Limited treatment is available to patients with metastatic BCs, which are resistant to therapy. There is a strong link between metastatic BC and Epithelial Mesenchymal Transition (EMT). There is a need to target EMT, in order to prevent metastasis for which the therapy is not effective. Therefore, there is a need for improved pre-clinical models that will allow studying EMT in real-time. This study aimed to refine current in vivo models of BC, by incorporating inducible bioluminescent reporter(s) that will allow real-time read-out of EMT. The cell-lines used were classified according to their expression of EMT markers and cancer stem cell (CSC) profile. Inducible bioluminescent EMT reporters based on the S100A4 or E-cadherin or N-cadherin promoter driving firefly luciferase were constructed and used to transduce the cell lines. The activity of the S100A4 reporter was validated in hypoxic conditions in MCF-7 and BT549 cells, where it was shown that induction of S100A4-generated bioluminescence was associated with upregulation in S100A4. In vivo it was shown that human mesenchymal stem cells promoted the tumour growth of MCF-7 cells and/or induced EMT-related traits, including downregulation/loss of E-cadherin, upregulation of Vimentin and/or upregulation of S100A4. The activity of the S100A4 reporter was tested in a similar in vivo model, where it was shown that S100A4-generated bioluminescence correlated with protein expression of S100A4. Taken all the data together, we generated an inducible bioluminescent EMT reporter that can detect the baseline levels and changes in the S100A4 expression. The model can be potentially used for giving a real-time read out of EMT/early stages of EMT and subsequently for testing novel drugs targeted at the onset of EMT.

The calpain system is a group of intracellular cysteine proteases. Dysregulated calpain activity, or mutation in calpain isoforms, as well as its endogenous inhibitor calpastatin, has been found to play an important role in tumorigenesis. The main aim of the current study is to explore the differential role of calpain family members in different breast cancer molecular subtypes; validate calpain-1 as a biomarker of trastuzumab response in HER2+ breast cancer patients; and explore the mechanisms by which calpain family regulates trastuzumab response in HER2+ cells. Three representative cell lines for different molecular subtypes were used: MDA-MB-231 (basal-like), MCF-7 (luminal) and SKBR3 (HER2+); and two additional HER2+ cell lines were used in the HER2+ study: acquired trastuzumab-resistant SKBR3 and inherent trastuzumab-resistant JIMT-1. The role of calpain in proliferation, signal transduction and apoptotic response was assessed using growth curves, phosphokinase arrays and Annexin V-FITC apoptosis assays, respectively. The effect of calpastatin knockdown, via shRNA, on cell migration was examined using Haptotaxis assay. The combined effect of calpeptin and trastuzumab on colony formation and cell cycle progression were examined using clonogenic survival and flow cytometry, respectively. Biomarker studies were conducted using standard immunohistochemistry. The results suggested that inhibition of calpain activity showed anti-proliferative effect on breast cancer cells across different subtypes. Knockdown of calpastatin in both MDA-MB-231 and MCF-7 cells did not have significant effects on migratory ability, either with or without calcium ionophore A23187. The study also showed that combining calpeptin and trastuzumab enhanced trastuzumab-induced anti-proliferative effects on SKBR3 and SKBR3/TR cells, but not in JIMT-1 cells. Combined treatment did not further reduce clonogenic survival either in SKBR3 or SKBR3/TR cells, compared with single agent alone. In all three HER2+ cells, combined treatment had no significant effect on trastuzumab-induced G0/G1 cell cycle arrest. Results from the immunohistochemical study suggested that high calpain-1 expression was significantly associated with adverse relapse-free survival in breast cancer patients who received adjuvant trastuzumab. Findings were validated in the expanded Nottingham and independent Newcastle patient cohort. Based on the previous in vitro study, suggesting a role of calpain in regulation of phospho-MSK1/2 expression; and because there is close link between calpain and caspase family. It was decided to explore the correlation between calpain system protein expression with MSK1 and two representative caspases (caspase-3 & -8), as well as their prognostic significance, in a large cohort of invasive breast cancer patients. Results demonstrated significant correlations between calpain-1 vs MSK1, and vs caspase-3; calpastatin vs MSK1, and vs caspase-8, however with low correlation coefficients. High MSK1 expression was significantly associated with improved breast cancer-specific survival. High caspase-3, but not caspase-8, was significantly associated with adverse breast cancer-specific survival. And combinatorial calpain-1 and caspase-3 expression provided additional prognostic values, especially in basal-like subtype. In conclusion, calpain system protein has been found to have roles in different breast cancer molecular subtypes. Calpain-1 is a potential biomarker for trastuzumab response in HER2+ breast cancer patients, and this study suggested MSK1 and caspase-3 could be potential biomarkers in breast cancer.

Background/Rationale: Breast cancer is the most common cancer in women worldwide. Recent findings found that self-help group is helpful for maintaining treatment regimens and coping with breast cancer and associated fears. However, there is a paucity of evidence on how a self-help group shapes women’s recovery, particularly in Jordan. Purpose of the Study/Setting: To investigate a breast cancer self-help group in Jordan. Study Design/Methods: Interpretative approach, with semi-structured interviews. A purposive sample of 34 participants was recruited from the King Hussein Cancer Centre comprising 15 women with breast cancer who participated in Sanad group; 13 non-participating women; and six health care professionals (two nurses and four social workers). Data Analysis: All qualitative data were analysed thematically using NVivo 9 software. Findings: The analysis revealed five categories and thirteen sub-categories. The first category was about decision-making: “finding other ways” involving two sub-categories focusing on the influences on making decisions and alternatives. The second category focused on the meaning of Sanad to women, involving three sub-categories, namely “getting out of the capsule”, “being part of a new family” and “learning from each other”. The third category focused on the perceived tensions in effective meetings involving two sub-categories: “it depends on who attends: behaviours and attributes in Sanad” and “from cohesion to disruption: dealing with loss in Sanad”. The fourth category was about ideas of recovery for Sanad members and involved three sub-categories, namely “from isolation to ‘recharging my battery’”, “from reluctance to acceptance” and “from hopelessness to hopefulness”. The fifth category addressed the ideas of recovery for non-attendees involving three sub-categories, from “being glued to my room to being attached to outside world”, “from hopelessness to depression” and “from hesitation to rejection of the treatment plan”. It was found that dominating the meetings, being sarcastic and underestimating the feelings of others play a major negative role in shaping discussions and create communication difficulties in Sanad meetings. Indeed, the current analysis shows a lack of hope among non-attendees of Sanad, feelings of helplessness, and negative attitudes towards treatment itself. A conceptual model involving three spheres (family, Sanad and recovery) was designed to assist nurses in identifying factors affecting women’s decisions about joining Sanad or not. Conclusion: The recovery process following attending Sanad is multifaceted, thus it is important to examine women’s values and needs, because the group is not homogeneous and commonality in terms of diagnosis is insufficient. Unless the components of the model proposed in this thesis and how they affect each other are understood by health professionals, addressing individual and social factors influencing women’s recovery is highly complex.