published_or_final_version / Public Health / Master / Master of Public Health

Extensive research and clinical observations in the past 20 years confirmed that the cessation of ovarian function at menopause does not stop the process of sex steroid hormone synthesis in females. Indeed, multiple extra-ovarian tissues contain the same enzymatic machinery the ovary uses which can maintain a significant rate of local hormonal synthesis sufficient to cause pathological outcomes. This is commonly termed “intracrine”. However, two obstacles face intracrinology. Firstly, wide clinical appreciation of this mechanism in causing disease and in targeting it with therapy does not currently exist. Secondly, blood hormonal assays are used in the clinic to diagnose and manage intracrine based disorders. This could be entirely misleading, since hormonal synthesis, action and metabolism occur within the tissue and, therefore, measuring blood levels is not reflective of the actual disease environment. This thesis presents evidence of significant intracrine based disorders in menopausal women that could be effectively managed by tackling the core intracrine mechanism. Three protocols are investigated emphasizing the usefulness of menopausal intracrine estrogen inhibition. The first presents a joint objective of treating menopausal symptoms using estrogenic replacement therapy while reducing breast cancer risk using long-term aromatase inhibitors. Aromatase inhibition is used to suppress the local estrogen synthesis in the breast. The second protocol is a new method of acute inhibition of breast estrogens to improve the accuracy of breast imaging techniques. This method showed a benefit in reducing the benign parenchymal enhancement during breast MRI indicating a potential improvement in specificity. The third protocol involves using aromatase inhibitors in the treatment of severe endometriosis that did not respond to oophorectomy. The pathogenesis of breast cancer, endometriosis and fibroids are believed to involve intracrine estrogen activity. Another significant contribution presented in this thesis is the development of a new technique that enables minimally invasive tissue assays of hormones in their genuine site of synthesis rather than indirectly in the blood. The new assay requires only microliter volumes of sample and employs a novel digital microfluidics technology. Estrogen and other sex steroids were extracted from droplet-scale breast tissue and blood samples.

Two studies were designed to characterize the effects of a non-steroidal aromatase inhibitor, letrozole, on ovarian function in cattle. The specific objective was to test the hypothesis that letrozole will arrest dominant follicle growth resulting in emergence of a new follicular wave at a predictable interval post-treatment. In a first experiment, postpubertal beef heifers were assigned randomly to four treatment groups and given phosphate-buffered saline (controls; n=10), or letrozole at a dose of 500 (n=9), 250 (n=10), or 125 (n=10) µg/kg intravenously 4 days after follicular ablation (~2.5 days after wave emergence). In a second study, postpubertal beef heifers were assigned randomly to four treatment groups. One group received no treatment (control; n=17) and the other groups (n=9-10) were given 85 µg/kg of letrozole per day (250 µg/kg total dose), from Days 1 to 3, Days 3 to 5, or Days 5 to 7 (Day 0 = pre-treatment ovulation,) corresponding to the periods before, during and after selection of the dominant follicle, respectively. Follicular dynamics were monitored ultrasonically and blood samples were collected for endocrine assays. Follicle diameter profiles and plasma LH, FSH, and estradiol concentrations were analyzed. Additionally, during the second trial, CL diameter profiles and plasma progesterone concentrations were measured. In both studies, the diameter profile of the dominant follicle was larger in heifers treated with letrozole than in control heifers (P<0.05) and the intervals to new wave emergence and onset of regression of the extant dominant follicle were longer (P<0.05) in heifers treated with letrozole than in controls, regardless of the dose (high, medium, or low; single vs multiple) and the stage of the follicle wave in which treatments were initiated. Furthermore, during the second experiment, the mean CL diameter was larger in letrozole-treated heifers, although there were no differences in plasma progesterone concentrations between treated and control animals. The effects on dominant follicle and CL diameter profiles appeared to be the result of the significantly increased plasma LH concentrations observed in letrozole-treated animals during both treatment approaches (single vs multiple dose). Incomplete and inconsistent inhibition of estradiol production and the lack of a surge on FSH observed in both experiments may be a result of insufficient circulating levels of letrozole during the treatment period. In summary, a single or multiple dose of letrozole did not induce regression of the extant dominant follicle, nor did it directly affect FSH release. Conversely, letrozole extended the lifespan of the dominant follicle, in association with increased endogenous levels of LH, thereby delaying the next FSH surge and subsequent follicular wave emergence. Results suggest that letrozole has potential as a non-steroidal method for controlling ovarian function in cattle, but further studies are warranted to clarify dosage and timing of treatment to predictably affect follicular wave dynamics in cattle.

Extensive research and clinical observations in the past 20 years confirmed that the cessation of ovarian function at menopause does not stop the process of sex steroid hormone synthesis in females. Indeed, multiple extra-ovarian tissues contain the same enzymatic machinery the ovary uses which can maintain a significant rate of local hormonal synthesis sufficient to cause pathological outcomes. This is commonly termed “intracrine”. However, two obstacles face intracrinology. Firstly, wide clinical appreciation of this mechanism in causing disease and in targeting it with therapy does not currently exist. Secondly, blood hormonal assays are used in the clinic to diagnose and manage intracrine based disorders. This could be entirely misleading, since hormonal synthesis, action and metabolism occur within the tissue and, therefore, measuring blood levels is not reflective of the actual disease environment. This thesis presents evidence of significant intracrine based disorders in menopausal women that could be effectively managed by tackling the core intracrine mechanism. Three protocols are investigated emphasizing the usefulness of menopausal intracrine estrogen inhibition. The first presents a joint objective of treating menopausal symptoms using estrogenic replacement therapy while reducing breast cancer risk using long-term aromatase inhibitors. Aromatase inhibition is used to suppress the local estrogen synthesis in the breast. The second protocol is a new method of acute inhibition of breast estrogens to improve the accuracy of breast imaging techniques. This method showed a benefit in reducing the benign parenchymal enhancement during breast MRI indicating a potential improvement in specificity. The third protocol involves using aromatase inhibitors in the treatment of severe endometriosis that did not respond to oophorectomy. The pathogenesis of breast cancer, endometriosis and fibroids are believed to involve intracrine estrogen activity. Another significant contribution presented in this thesis is the development of a new technique that enables minimally invasive tissue assays of hormones in their genuine site of synthesis rather than indirectly in the blood. The new assay requires only microliter volumes of sample and employs a novel digital microfluidics technology. Estrogen and other sex steroids were extracted from droplet-scale breast tissue and blood samples.

Two studies were designed to characterize the effects of a non-steroidal aromatase inhibitor, letrozole, on ovarian function in cattle. The specific objective was to test the hypothesis that letrozole will arrest dominant follicle growth resulting in emergence of a new follicular wave at a predictable interval post-treatment. In a first experiment, postpubertal beef heifers were assigned randomly to four treatment groups and given phosphate-buffered saline (controls; n=10), or letrozole at a dose of 500 (n=9), 250 (n=10), or 125 (n=10) µg/kg intravenously 4 days after follicular ablation (~2.5 days after wave emergence). In a second study, postpubertal beef heifers were assigned randomly to four treatment groups. One group received no treatment (control; n=17) and the other groups (n=9-10) were given 85 µg/kg of letrozole per day (250 µg/kg total dose), from Days 1 to 3, Days 3 to 5, or Days 5 to 7 (Day 0 = pre-treatment ovulation,) corresponding to the periods before, during and after selection of the dominant follicle, respectively. Follicular dynamics were monitored ultrasonically and blood samples were collected for endocrine assays. Follicle diameter profiles and plasma LH, FSH, and estradiol concentrations were analyzed. Additionally, during the second trial, CL diameter profiles and plasma progesterone concentrations were measured. In both studies, the diameter profile of the dominant follicle was larger in heifers treated with letrozole than in control heifers (P<0.05) and the intervals to new wave emergence and onset of regression of the extant dominant follicle were longer (P<0.05) in heifers treated with letrozole than in controls, regardless of the dose (high, medium, or low; single vs multiple) and the stage of the follicle wave in which treatments were initiated. Furthermore, during the second experiment, the mean CL diameter was larger in letrozole-treated heifers, although there were no differences in plasma progesterone concentrations between treated and control animals. The effects on dominant follicle and CL diameter profiles appeared to be the result of the significantly increased plasma LH concentrations observed in letrozole-treated animals during both treatment approaches (single vs multiple dose). Incomplete and inconsistent inhibition of estradiol production and the lack of a surge on FSH observed in both experiments may be a result of insufficient circulating levels of letrozole during the treatment period. In summary, a single or multiple dose of letrozole did not induce regression of the extant dominant follicle, nor did it directly affect FSH release. Conversely, letrozole extended the lifespan of the dominant follicle, in association with increased endogenous levels of LH, thereby delaying the next FSH surge and subsequent follicular wave emergence. Results suggest that letrozole has potential as a non-steroidal method for controlling ovarian function in cattle, but further studies are warranted to clarify dosage and timing of treatment to predictably affect follicular wave dynamics in cattle.

Tamoxifen and aromatase inhibitors are treatment options for women with breast cancer and evidence on the risk of stroke is important in choosing between these two options. A systematic review of two randomized controlled trials and their nine related trial reports showed different methods for adverse event reporting and inconsistent estimates of stroke risk. In an observational cohort study of 5443 Ontario women, aged 66 years or older with early stage breast cancer, 86 ischemic stroke events (1.6%) occurred during follow-up of 5 years. There was no statistically significant difference in the risk of stroke between the hormone therapy groups [adjusted HR for tamoxifen compared to AI 1.330 (0.810, 2.179)]. Results were similar across cardiovascular disease risk groups and were robust to different follow up periods and analytic methods. This study suggests that there is no significant difference in stroke between these treatment options.

Vaginal atrophy symptoms such as dryness, irritation, and itching, are common after menopause. Vaginal estrogen therapy is the most effective treatment but not appropriate for all women. Women with estrogen-responsive breast cancer treated with aromatase inhibitor (AI) treatment, suppressing estrogen levels, often suffer from more pronounced vaginal atrophy symptoms. However, vaginal estrogen treatment is not recommended, leaving them without effective treatment options. The aim of this thesis was to study the effect of long-term anti-estrogen therapy on circulating estrogen levels and biochemical factors in vaginal mucosa in relation to morphological changes and clinical signs of vaginal atrophy. Circulating estrogen levels were analyzed by use of mass spectrometry and radioimmunoassay. Immunohistochemistry was used to study vaginal proliferation and steroid hormone receptors in vaginal mucosa. Vaginal gene expression was studied by use of microarray technology and bioinformatic tools, and validated by use of quantitative real-time PCR and immunohistochemistry. An estrogenic regulation of aquaporins and a possible role in vaginal dryness was investigated in vaginal mucosa and in Vk2E6E7 cells. Aromatase inhibitor-treated women had higher than expected estradiol and estrone levels but still significantly lower than other postmenopausal women. Aromatase was detected in vaginal tissue, the slightly stronger staining in vaginal mucosa from AI-treated women, suggest a local inhibition of vaginal aromatase in addition to the systemic suppression. Vaginal mucosa from AI-treated women had weak progesterone receptor, and strong androgen receptor staining intensity. Low estrogen levels lead to low expression of genes involved in cell adhesion, proliferation, and differentiation as well as weak aquaporin 3 protein immunostaining. The higher than expected estrogen levels in AI-treated women suggest that estrogen levels might previously have been underestimated. Systemic estrogen suppression by treatment with AIs, and possibly also by local inhibition of vaginal aromatase, results in reduced cell adhesion, proliferation, differentiation, and weak aquaporin 3 protein staining. Low proliferation and poor differentiation leads to fewer and less differentiated superficial cells affecting epithelial function and possibly also causing vaginal symptoms. Aquaporin 3 with a possible role in vaginal dryness, cell proliferation, and differentiation should be further explored for the development of non-hormonal treatment options for vaginal symptoms.

Tamoxifen and aromatase inhibitors are treatment options for women with breast cancer and evidence on the risk of stroke is important in choosing between these two options. A systematic review of two randomized controlled trials and their nine related trial reports showed different methods for adverse event reporting and inconsistent estimates of stroke risk. In an observational cohort study of 5443 Ontario women, aged 66 years or older with early stage breast cancer, 86 ischemic stroke events (1.6%) occurred during follow-up of 5 years. There was no statistically significant difference in the risk of stroke between the hormone therapy groups [adjusted HR for tamoxifen compared to AI 1.330 (0.810, 2.179)]. Results were similar across cardiovascular disease risk groups and were robust to different follow up periods and analytic methods. This study suggests that there is no significant difference in stroke between these treatment options.

Introduction Recent advances in microarray technology have allowed more understanding of the complex molecular biology of breast cancer. The traditional prognostic information afforded by hormone receptor status and pathology variables is being supplemented and superseded by gene signatures predictive of risk of recurrence and response to treatments. Approximately 75% of breast cancers are oestrogen receptor positive (ER+) and can be treated by drugs that block oestrogen production such as letrozole. However not all ER+ tumours respond and even those that initially respond can develop resistance. Treating patients with neoadjuvant letrozole affords a unique opportunity to sample the same tumour in vivo at different time points reducing any potential inter-patient and inter-tumour variability. The molecular effects of drugs can be assessed long before clinical outcome is apparent. Underlying genetic differences or characteristics of the patient, tumour or sample may affect the molecular response to treatment. This project set out to use sequential patient-matched samples to evaluate molecular changes in breast tumours in the presence or absence of endocrine treatment in different subtypes, defined by histology or mutation status and to assess molecular variation between primary tumour and nodal metastasis. Methods RNA was extracted and processed to generate whole transcriptome Illumina Beadarray gene expression data from four unique cohorts of patients. Clinical data on treatments, recurrence and survival was collected from medical records. The first cohort compared 25 breast cancer patients with matched samples at diagnosis and at surgery, 14-35 (median 23) days later, with no intervening treatment; with 36 patients treated with neoadjuvant letrozole. A PCR assay to detect 8 known PIK3CA mutations and assessment of PTEN status was performed at both the primary and secondary event in a second cohort of 120 patients with endocrine treated disease who relapsed with either recurrence, lymph node metastases, a new second primary or progression of disease on primary endocrine therapy. The third cohort compared the molecular response to neoadjuvant letrozole in 14 patients with invasive lobular cancer (ILC) and 14 patients with invasive ductal cancer (IDC). A fourth cohort of women with node positive disease at diagnosis were assessed for variations in gene expression profiles between primary tumour and synchronous metastatic axillary lymph nodes (68 samples from 31 patients). Results The genomic profile of the no intervening treatment cohort did not differ significantly. Some changes in inflammatory genes were evident. This reassures us that changes seen during treatment are truly due to drug effect. This validates the use of a second biopsy to explore prediction of response. PIK3CA mutation status is maintained in the majority of patients with endocrine resistant disease and changed in only 15.7%. Where there was a change in PIK3CA this was significantly more likely to be a second primary breast cancer rather than a recurrence or progression of the primary cancer. PTEN status was also maintained in most patients. This does not support the theory that acquisition of a PIK3CA mutation is responsible for developing endocrine resistance. Novel PI3K inhibitor drugs may still be suitable in endocrine-resistant disease if activation of the pathway develops by other mechanisms. Consistent with previous studies, significant molecular differences were observed between ILC and IDC pre-treatment. Over half of these molecular differences were maintained after 3 months of letrozole. However, changes over time in individual tumours in response to letrozole were highly consistent in both ILC and IDC. When comparing primary with synchronous metastatic nodes only 39% of tumours clustered together with their matched primary or node. The molecular subtype of the node was often a poorer prognosis than the primary. There were also differences in subtype between nodes in a small cohort of patients with 2 involved nodes. Conclusions We have demonstrated that neoadjuvant window studies are a valid model for assessment of drug effects and evaluated differences in histology and mutation status. Endocrine resistance in breast cancer is rarely related to acquisition of PIK3CA mutations. Synchronous lymph node metastases can differ greatly from their matched primary. These findings are highly relevant when considering prescribing (neo)/adjuvant therapy and have significantly improved our understanding of breast cancer as we strive towards personalised medicine.

Submitted by Juliana Viezel null (juliana_viezel@hotmail.com) on 2018-03-16T14:03:07Z No. of bitstreams: 1 PPGCMI-Dissertação Juliana Viezel_final.pdf: 2082649 bytes, checksum: 013b5b944459dbceae8cb3436ae205bc (MD5) / Approved for entry into archive by Claudia Adriana Spindola null (claudia@fct.unesp.br) on 2018-03-16T14:35:51Z (GMT) No. of bitstreams: 1 viezel_j_me_prud.pdf: 2082649 bytes, checksum: 013b5b944459dbceae8cb3436ae205bc (MD5) / Made available in DSpace on 2018-03-16T14:35:51Z (GMT). No. of bitstreams: 1 viezel_j_me_prud.pdf: 2082649 bytes, checksum: 013b5b944459dbceae8cb3436ae205bc (MD5) Previous issue date: 2018-02-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Câncer é considerado atualmente uma das principais causas de mortalidade. O câncer de mama é o tipo de câncer que mais afeta mulheres em todo o mundo e a prática de exercício físico pode melhorar diversos aspectos afetados pela doença ou pelo seu tratamento como: composição corporal, perfil lipídico e glicêmico e nível de atividade física. No entanto, há necessidade de mais estudos que avaliem as respostas individuais ao treinamento físico de mulheres sobreviventes de câncer de mama que estão sob tratamento hormonal. Objetivo: Analisar o efeito do treinamento combinado e a resposta individual de mulheres pós menopausa em tratamento para o câncer de mama com inibidores de aromatase ao treinamento nas variáveis de composição corporal, perfil metabólico e nível de atividade física habitual. Métodos: Participaram do estudo 36 mulheres sobreviventes de câncer de mama que fazem uso de inibidores de aromatase, randomizadas em grupo exercício (n=18) e controle (n=18). As avaliações foram realizadas nos momentos pré e após 24 semanas de treinamento combinado. Foram realizadas medidas antropométricas, composição corporal por meio do DEXA, exames bioquímicos sanguíneos (colesterol total e frações, glicemia e triglicérides) e pratica de atividade física por meio de acelerômetros. O treinamento combinado consistiu de três sessões semanais com duração de aproximadamente 1h40 minutos. As participantes do grupo exercício foram distribuídas em dois grupos de acordo com o percentual de mudança na massa corporal gorda para análise da responsividade. As análises estatísticas foram realizadas no programa SPSS, versão 21 e significância estabelecida em 5%. Resultados: O treinamento foi efetivo para reduzir a gordura corporal e aumentar a pratica de atividade física por semana (p<0,05). O grupo todo apresentou grande variação nas respostas ao treinamento em todas as variáveis estudadas. Quanto a responsividade, quando separadas pela mudança percentual na massa corporal gorda, as participantes que apresentaram respostas positivas ao treinamento foram as que apresentaram melhora nas variáveis de IMC (-0,03%), massa total (-4,1%) e de composição corporal quando comparadas ao grupo não responsivo e grupo controle (p<0,05). Conclusão: O treinamento combinado é efetivo na melhora da composição corporal, bem como tem potencial para aumento do nível de atividade física de mulheres que tomam inibidores de aromatase para tratamento do câncer de mama e variáveis de composição corporal podem interferir na resposta ao treinamento combinado dessa população. / Cancer is currently considered a major cause of mortality. Breast cancer is the type of cancer that more affects women in the world and the practice of physical exercise can improve several aspects affected by the disease or its treatment such as body composition, lipid and glycemic profile and level of physical activity. However, more studies are needed to assess individual responses to physical training of women who are undergoing hormone treatment. Objective: To analyze the effect of combined training and the responsiveness of postmenopausal women on treatment for breast cancer with aromatase inhibitors to training in variables of body composition, metabolic profile and habitual physical activity level. Methods: Thirty-six survivors of breast cancer using aromatase inhibitors, randomized in group exercise (n = 18) and control (n = 18) participated in the study. The evaluations were performed in the pre and post 24 weeks of combined training. It were evaluated anthropometric measurements, body composition through DEXA, blood biochemical tests (total cholesterol and fractions, glycemia and triacylglycerol) and physical activity through accelerometers. The combined training consisted of three weekly sessions lasting approximately 1h40 minutes. Participants in the exercise group were divided into two groups according to the percentage change in body fat mass for the analysis of responsiveness. Statistical analyzes were performed in the SPSS program, version 21 and significance was set at 5%. Results: The training was effective in reducing body fat and increasing physical activity per week (p<0.05). The whole group presented a wide variation in the responses to training in all variables studied. Regarding responsiveness, when participants were separated by the percentage change in fat body mass, participants who presented positive responses to training were those who showed improvement in BMI (-0.03%), total mass (-4.1%) and body composition when compared to nonresponsive group and control group (p<0.05). Conclusion: Combined training is effective in improving body composition, as well as has the potential to increase the level of physical activity of women taking aromatase inhibitors for breast cancer treatment and body composition variables may interfere in the response to combined training of this population.