Thesis (Ph.D.)--University of Nebraska-Lincoln, 2008. / Title from title screen (site viewed Feb. 17, 2009). PDF text: iv, 173 p. ; 687 K. UMI publication number: AAT 3325857. Includes bibliographical references. Also available in microfilm and microfiche formats.

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Orientador: Debora Aparecida Pires de Campos Zuccari / Coorientador: Eduardo Alves Almeida / Banca: Angelo Gustavo Zucca Matthes / Banca: Eny Maria Goloni Bertollo / Banca: Luiz Octávio Regasini / Banca: Sebastião Roberto Taboga / Resumo: O câncer de mama é a principal causa de mortes relacionadas ao câncer em mulheres e pesquisas têm sido focadas em identificar e validar biomarcadores que podem ser utilizados para confirmar o diagnóstico e determinar o prognóstico. Mudanças no ritmo circadiano podem contribuir para o desenvolvimento do câncer e sendo assim, a melatonina, um hormônio sintetizado pela glândula pineal à noite, na ausência de luz e seus metabolitos AFMK e AMK são sugeridos como potenciais biomarcadores. A melatonina pode atuar através do receptor MT1 regulando cinases e a expressão de genes específicos relacionados a proliferação, angiogênese, diferenciação celular e transporte de glicose. A expressão elevada de GLUT1 (Glucose Transporter-1) está associada ao prognóstico ruim no câncer. Os objetivos deste estudo foram comparar os níveis de melatonina, AFMK e AMK em mulheres recentemente diagnosticadas com câncer de mama, mulheres em quimioterapia adjuvante, enfermeiras que trabalham à noite em comparação com mulheres saudáveis e hábitos normais e avaliou-se a expressão do receptor MT1 e GLUT1 em tumores de mama e correlacionou com os subtipos moleculares, características patológicas e prognóstico. Foi coletado sangue de 53 mulheres com câncer de mama sendo 47 sem tratamento e 6 em quimioterapia adjuvante, 19 mulheres saudáveis sendo 1 O enfermeiras de turno noturno e 9 mulheres de hábitos normais. Os compostos foram quantificados por espectrometria de massas. Para a expressão de MT1 e GLUT1 foi... / Abstract: Breast cancer is the Jeading cause of cancer-related deaths in women and researches has been focused on identify and validate biomarkers that can be used to confirm the diagnosis and determine the prognosis. Changes in circadian rhythm may contribute to the development of cancer and thus melatonin, a hormone synthesized by the pineal gland at night, in the absence of light and its metabolites AFMK and AMK are suggested as potential biomarkers. Melatonin can act through the MT1 receptor by regulating kinases and the expression of specific genes related to proliferation, angiogenesis, ce/1 differentiation and glucose transport. High GLUT1 (Glucose Transporter-1) expression is associated with poor prognosis in cancer. The objectives of this study were to compare the leveis of melatonin, AFMK and AMK in women newly diagnosed with breast cancer, women on adjuvant chemotherapy, health nurses which work at night compared to healthy women and normal habits and the expression of MT1 receptor and GLUT1 in breast tumors and correlated with molecular subtypes, pathological features and prognosis. Blood from 53 women with breast cancer was colletcted, 47 of them without treatment and 6 in adjuvant chemotherapy, 19 healthy women, 1 O of them night shift nurses and 9 women of normal habits. Compounds were quantified by mass spectrometry. For the expression of MT1 and GLUT1, immunohistochemistry was performed in 42 breast tumors. The results showed that women with breast cancer had lower ... / Doutor

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Survival from breast cancer has significantly improved over the past years. Therefore, survivorship issues are an area of crucial importance to be addressed as early as possible by all health care providers dealing with breast cancer patients. Fertility and pregnancy-related issues represent a priority area of concern for young women with breast cancer. Despite a growing attention towards these issues has been given over the past years and is currently provided to young breast cancer patients, several grey zones persist in many domains of this field and some physicians are still uncomfortable to deal with this topic. In this thesis, we aimed at providing evidence on several unmet fertility and pregnancy-related issues faced by young breast cancer patients with the ultimate goals to further improve their quality of life and to help physicians during the oncofertility counseling of these women.In Chapter 1 of this thesis, we addressed questions related to the factors with a potential negative or protective impact on the ovarian function of young breast cancer patients. First, we assessed the influence of carrying a germline BRCA mutation on the reproductive potential of young breast cancer patients and the performance of fertility preservation strategies in this setting. We conducted a retrospective analysis within two prospective studies that involved young women with newly diagnosed breast cancer who underwent oocyte cryopreservation or ovarian tissue cryopreservation for fertility preservation at CUB-Hôpital Erasme in Brussels (Belgium) between January 2006 and December 2016. A total of 101 patients were included, of whom 29 had germline deleterious BRCA mutations. We observed a consistent trend for reduced reproductive potential and performance of both oocyte and ovarian tissue cryopreservation in BRCA-mutated patients.Second, we evaluated the gonadotoxicity of anticancer treatments focusing particulary on the risk of treatment-related amenorrhea (TRA) with the addition of taxanes to anthracycline-based chemotherapy and with the administration of chemotherapy plus the anti-human epidermal growth factor receptor 2 (HER2) agents trastuzumab and/or lapatinib. This analysis was conducted in 2,862 premenopausal patients with early-stage HER2-positive breast cancer enrolled in the ALTTO randomized trial (NCT00490139) by assessing menopausal status at week 37 visit following the initiation of anti-HER2 treatment. Addition of taxanes to anthracycline-based chemotherapy led to a statistically significantly higher risk of TRA. No difference in TRA rates was observed between the four anti-HER2 treatment arms (trastuzumab alone, lapatinib alone, their sequence or their combination); the absence of higher TRA rates in the dual blockade arm as compared to single agent arms may suggest the gonadal safety of these agents. TRA was associated with statistically significant improved disease-free survival (DFS) and overall survival (OS) in patients with hormone receptor-positive/HER2-positive disease. Third, we provided evidence on the efficacy and safety of temporary ovarian suppression with gonadotropin-releasing hormone agonists (GnRHa) during chemotherapy as an option for ovarian function preservation in young breast cancer patients undergoing (neo)adjuvant chemotherapy. We performed a meta-analysis including individual patient-level data from the 5 major trials (PROMISE-GIM6, POEMS/SWOG S0230, Anglo Celtic Group OPTION, GBG-37 ZORO, Moffitt-led trial) that investigated the role of this strategy. A total of 873 breast cancer patients were included. Concurrent administration of GnRHa and chemotherapy significantly reduced the risk of developing chemotherapy-induced premature ovarian insufficiency (POI), and was associated with a higher number of subsequent pregnancies. Similar DFS and OS were observed between groups irrespective of the estrogen receptor (ER) status of the disease suggesting the safety of administering GnRHa concurrently with chemotherapy.In Chapter 2 of this thesis, we aimed to address questions related to the safety of pregnancy following prior history of breast cancer including the impact of timing of pregnancy, induction of abortion or breastfeeding, as well as to investigate the pregnancy outcomes in these patients. First, we assessed if there were differences in survival outcomes between patients who conceived following breast cancer diagnosis as compared to those who did not have a subsequent pregnancy with a particular focus to women with ER-positive tumors. In addition, we investigated the potential impact of timing of pregnancy, induction of abortion and breastfeeding on patients’ prognosis. For this purpose, a multicenter retrospective case-control study was conducted. A total of 1,207 breast cancer patients with known ER status were included in the analysis, of whom 333 conceived after prior breast cancer and 874 had no subsequent pregnancies. Long-term results from this study confirmed that pregnancy after breast cancer can be considered safe irrespectively of ER status and should not be discouraged. This was observed independently of pregnancy outcome, pregnancy interval, and breastfeeding status. Second, we assessed the safety of pregnancy following prior history of HER2-positive breast cancer, and the pregnancy outcomes in women who conceived during or after chemotherapy plus anti-HER2 treatment. We collected all the pregnancy events that occurred in the NeoALTTO (NCT00553358) and ALTTO (NCT00490139) trials. These are two randomized studies that explored the role of trastuzumab and/or lapatinib in patients with HER2-positive early breast cancer as neoadjuvant and adjuvant treatment, respectively. A total of 92 patients had at least one pregnancy after inclusion in the two trials, of whom 12 patients conceived during anti-HER2 targeted therapy (exposed group) while 80 after the end of treatment (unexposed group). We observed that having a pregnancy after prior history of HER2-positive early breast cancer did not appear to impact on DFS. A high rate of induced abortion was observed among women in the exposed group; nevertheless, despite only 5 live births being described in this group, unintentional exposure to trastuzumab and/or lapatinib during gestation did not seem to affect newborn outcomes. Patients in the unexposed group appeared to have normal pregnancy outcomes. Therefore, overall, having a pregnancy following completion of chemotherapy plus anti-HER2 therapy showed to be safe without compromising fetal outcome or maternal prognosis.In conclusion, taken together, we believe that our findings can serve as a rational basis to improve the oncofertility counseling of young breast cancer patients facing concerns related to TRA risk, preservation of ovarian function and/or fertility as well as for those willing to consider a future pregnancy. Several ongoing and upcoming projects from our group have been started based on the results presented in this thesis and are expected to provide further evidence on these crucial topics. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

The Wilms' Tumour Suppressor gene 1, WT1, encodes for a complex protein which is essential in mammals throughout life. Its roles vary with the developmental stages: in the embryo, it regulates the epithelial-mesenchymal balance required for a correct organogenesis and acts as a tumour suppressor; in the adult, it is involved in the maintenance of tissue homeostasis and has been controversially considered as an oncogene. Breast cancer is one of the adult tumours in which WT1 oncogenic function was first hypothesised. This malignancy is the most common in women, with more than one million cases being diagnosed worldwide every year, and represents the leading cause of cancer related deaths. Because of its major health burden, this disease has been extensively studied and special attention has also been paid to normal mammary gland biology: several works have shown that breast cancer can be divided into many molecular subtypes, which may reflect the cell of origin of the tumour; moreover, many genes involved in the normal development of the mammary gland have been proven to also play a role in breast tumorigenesis. WT1 expression has been previously reported in both healthy mammary glands and breast cancer samples, however, its function in this context is not well understood and the evidence gathered so far is extremely contradictory. This thesis aimed to investigate the exact role played by WT1 in both mammary gland and breast cancer biology, using a combination of in vivo and in vitro techniques. Following flow cytometry isolation, Wt1 mRNA expression was detected in the myoepithelial and stem cell subpopulations of the healthy gland. To investigate the effects of WT1 loss, Wt1 conditional mice were crossed with two different mammary specific Cre lines: the knockout animals developed, bred and lactated normally, however, they showed a significant increase of ductular branches during pregnancy, suggesting that WT1 may be involved in the regulation of branching morphogenesis. In order to study WT1 role in mammary tumours, the gene was knocked out in a breast cancer mouse model and knocked down in several breast cancer cell lines, using both constitutive and inducible lentivirus-based systems. WT1 loss did not seem to affect cell proliferation, but resulted in a significant increase in cell migration in vitro and in the upregulation of mesenchymal markers. Furthermore, bioinformatics analysis showed that the WT1-positive tumours mainly belong to the luminal/ER-positive subtypes and express lower levels of mesenchymal markers than the WT1-negative tumours. As a whole, the findings of this thesis characterise WT1 expression in the healthy mammary gland and provide the first evidence of its possible function in this organ; moreover, this work seems to rule out an oncogenic role for WT1 in breast cancer, while suggesting that it could be an upstream regulator of cell migration. Additional experiments are required to confirm this result in vivo and verify whether it could lead to any clinical application.

The interpretation of medical images is one of the most difficult tasks in computer vision, largely because of the high degree of variability associated with normal and abnormal appearances. This thesis introduces a systematic method for the detection of microcalcifications as one of the most important signs of early breast cancer. It involves a four step procedure. The first step is blob detection to detect regions of microcalcification size range. The second step involves a specially designed directional region growing method to find the best fitting boundaries for each blob region. A newly developed combination of classifiers is then applied to label each region as a microcalcification or background. The final processing step involves a search for the existence of clusters of microcalcifications using a hierarchical nearest mean clustering method. The contributions of the work to the field of image processing are; a new blob detection system; a novel region growing method and a theoretical framework for combining classifiers which use a combination of shared and distinct representations. Here specifically, we present a blob detection method with the capability of detecting any suspected blob of specific size range. Then a new region growing method is developed based on a unique directional growing process providing predictable behaviour for the method. The application of two discontinuity measures is considered for the extraction of two fitting boundaries representing information about the region and its local background. The information conveyed by the boundaries and their associated regions is used to compute reliable representations for labelling each blob region. The robustness of the region growing method to the choice of a starting point and to Gaussian noise is examined on real images. We demonstrate that commonly used classifiers provide reliable results in labelling the suspected regions. In spite of achieving an acceptable performance using different individual classifiers, a decision fusion rule involving a weighted combination of classifiers is developed and its performance on the problem is investigated. The combination rule is applicable when mixed mode representations (some shared and some individual features) are used. A comparative study of the individtial classifiers and also of conventional classifier combination techniques with the weighted combiner is performed on independent test sets. The results achieved with the presented algorithm are very promising and approaching a level where a clinical pilot evaluation for screening purposes would be warranted.

The RAB guanosine triphosphates (RAS-related in brain) belong to the Ras superfamily of GTPases, and loss of RAB 25 expression has been reported in a number of breast cancer cases containing H-Ras point mutations, particularly triple negative breast cancers (TNBC), one of the most aggressive subtypes of breast cancer and associated with a poor prognosis. The mechanism involved in the progression of these tumors is poorly understood. In this study, we are trying to understand if loss of RAB25 expression in Human Mammary Epithelial Cell (HMEC) lines co-operates with H-Ras mutations and contributes to tumorigenesis. HMEC were immortalized by transduction with LXSN CDK4 R24C, a mutant form of cyclin-dependent kinase, followed by transduction with hTERT, catalytic subunit of the telomerase enzyme that permits the cells to exceed the Hayflick Limit and become immortal. We have found that with loss of RAB25 and over expression of mutant H-Ras61L, immortal HMEC undergo transformation. We have looked into the co-operativity between loss of Rab25 and H-Ras61L mutant by in-vitro studies to show their anchorage independent growth and increased ability to migrate. Furthermore, cells express low CD24, high CD44, and very low levels of Claudin indicating that cells acquire stem-like properties upon transformation. Loss of RAB25 and over-expression of H-ras61L resulted in increased expression of transcription markers Snail and Slug that drive these cells to lose E-cadherin and undergo Epithelial Mesenchymal Transition (EMT). This study shows that loss of RAB25 and over-expression of mutant H-Ras can transform HMEC and give rise to mesenchymal stem-like tumors. Our findings reveal that RAB25 functions as a tumor suppressor gene, and loss of RAB25 could serve as a novel biomarker in the prognosis of Claudin-low type of TNBC.

<b>Introduction:</b> Almost 7,000 people are diagnosed with ductal carcinoma in situ (DCIS) in the United Kingdom each year, but there remains uncertainty regarding its natural history and optimal management. The aim of this thesis was to evaluate factors contributing to the epidemiology of DCIS and its outcomes. <b>Methods:</b> 1) A cohort study comparing risk factors for DCIS and invasive breast cancer (IBC) using UK Biobank; 2) A cohort study examining the accuracy of preoperative biopsy in DCIS using clinical records from the Netherlands Cancer Institute; 3) A cohort study examining the rate of invasive breast cancer following treatment for screen-detected DCIS in England using the National Health Service Breast Screening Programme (NHSBSP) audit; 4) A methodological study to develop an algorithm to identify invasive breast cancer recurrences, which in the future may used to identify DCIS recurrences, using all relevant routinely collected data stored within Public Health England (PHE). <b>Results:</b> (1) For both DCIS and IBC, postmenopausal BMI was associated with an increased risk of developing disease, and the number of live births was associated with a decreased risk of developing disease. However, the magnitude of the effect differed between DCIS and IBC. The increased risk from postmenopausal BMI &GE;35 kg/m² was larger for DCIS than for IBC (RR 2.35, 95% CI 1.14-4.82), and the trend of reduction in risk with each additional live birth was greater for DCIS than for IBC (p for trend = 0.03). (2) Consideration of mammographic lesion size and the absence of necrosis on biopsy may be helpful in selecting low-risk women for non-operative management of DCIS in the future, as may use of the 9G vacuum-assisted method of biopsy. (3) The cumulative risks of IBC at 5, 10 and 15 years after screen-detected DCIS in England were 3.5%, 7.1%, and 9.4% respectively. Women with clear surgical margins of 1-2 mm had a higher IBC rate than women with clear margins of 5+ mm (RR 1.85, 95% CI 1.20-2.84). Women given breast-conserving surgery (BCS) without radiotherapy had a higher ipsilateral IBC rate than women given BCS with radiotherapy (RR 1.63, 95% CI 1.27-2.10). Women given hormone therapy had a lower rate of any IBC compared with oestrogen receptor (ER) positive women not given hormone therapy (RR 0.76, 95% CI 0.63-0.93). (4) There was good agreement between the number of recurrences indicated by the developed algorithm using routinely collected data sources and the number of recurrences recorded in the test dataset. This finding supports the potential value of compiling recurrence information on a nationwide basis from routinely collected data, for use in future descriptive and epidemiological studies and in follow-up for randomised trials. <b>Conclusions:</b> Using a variety of methods these studies have all succeeded in adding to knowledge about the epidemiology of DCIS. This knowledge can be used to help the future management of women with DCIS. In addition, each of the studies has planned extensions and will continue to contribute further knowledge periodically into the future.

Optoacoustic (OA) imaging is an emerging low-cost hybrid imaging investigation/technique currently in clinical feasibility studies for breast cancer diagnosis and staging. The technique applies pulsed light to the tissue of interest where molecules absorb the light photons and generate acoustic pressure waves. The resulting acoustic responses are detected using ultrasound transducers and converted into images. Image contrast within a pixel is dependent on the relative concentration and absorption characteristics (i.e. spectrum) of the chromophores within the illuminated tissue. Thus, tissue responses from illumination using multiple wavelengths, chosen to reflect the differential absorption of oxy-/deoxy- and total haemoglobin, can be measured. In turn, these signals can be regarded as surrogate measures of tissue hypoxia and neoangiogenesis, hallmarks of cancer associated with adverse outcomes. The aim of this PhD was to translate optoacoustic imaging into the breast clinic to try and fulfil some of the unmet clinical needs in breast cancer imaging using the imaging biomarker roadmap by O'Connor et al. Translation of this new technology to the clinical environment required extensive preparatory work, including the procurement and installation of a scanner prototype, liaison with UK regulatory bodies to secure ethical and MHRA approval, as well as several technical developments (performed during the course of the PhD) to make the technology suitable for breast cancer imaging. The first chapter of the thesis reviews the unmet needs of breast cancer imaging, being followed by a summary of recent techniques and technologies that may potentially fulfil gaps in knowledge and address some of the specific diagnostic challenges in breast cancer imaging. The capabilities of optoacoustic imaging are then discussed in the context of this evolving landscape of new imaging techniques and technologies with a particular focus on the tumour biology (neoangiogenesis and hypoxia) that can be measured in humans using multimodality and multi parametric imaging. Chapter 2 reviews of the current state of clinical translation of optoacoustic imaging, highlighting the particular areas in which clinical translation has advanced the most (breast cancer, melanoma and inflammatory bowel disease). Chapter 3 discusses the logistical, regulatory and technical challenges and solutions involved in translating optoacoustic imaging to the clinic and setting up a clinical service. Chapter 4 presents a series of validation experiments of oxygen saturation aimed at establishing the relationship between the optoacoustic signal and invasive pO2 measurements with an OxyLite probe in a porcine kidney model. This work was conducted in close collaboration with leading clinicians from the local transplant team. The following chapter describes the results of the first stage of our clinical work in the breast, namely the healthy volunteer study. This part had several aims: to perform qualitative assessment of the optoacoustic features of the normal breast under physiological conditions; to establish a robust scanning technique and identify technical and image interpretation pitfalls; and to perform qualitative evaluation of the hormonal changes that occur during the menstrual cycle and menopause, which, in turn, were used to validate surrogate measures of oxy-, deoxy and total haemoglobin. Chapter 6 then focuses on the qualitative assessment of benign and malignant breast lesions and their appearances on optoacoustic imaging. The patient study was divided into three phases. Phase 1 created a feature set to differentiate benign from malignant lesions, while Phase 2 was a transition between the prototype scanner and the installation of the first-generation clinical scanner. In Phase 3 the feature set created in Phase 1 was validated in a reader study. The sensitivity and specificity of optoacoustic imaging for lesion detection and differentiation of benign from malignant lesions was compared with mammography and ultrasound. Chapter 7 then deals with the quantitative analysis of the Phase 1 and Phase 3 data acquired in Chapter 6, assessing the relationships between the use of single wavelengths, spectral unmixing, vascularity versus receptor status, heterogeneity of signal intensity in relation to tumour stage and grade. This chapter also discusses the potential and limitations of quantifying the optoacoustic signal and leads to the final chapter, a discussion of future directions in optoacoustic imaging in breast cancer. At the end of this thesis, chapter 8 briefly discusses the potential future directions for the use of optoacoustic imaging as a clinical and scientific tool.