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In the past decades our knowledge concerning breast cancer progression from ductal carcinoma in situ (DCIS) to invasive cancer has grown rapidly. However, molecular factors driving the progression are still largely unknown. In the first study, we investigated tumor evolution in breast cancer by analyzing TP53 mutation status in tumors from various stages of the disease. Presence of the same TP53 mutations in both DCIS and invasive components from the same tumor indicates same cellular origin. The role of mutant TP53 in the progression of breast cancer is less clear and may vary between subtypes. In the second study, we studied the prognosis of basal-like DCIS in a large population-based cohort. Basal-like DCIS was associated with about doubled but not statistically significant risk for local recurrence compared with the other molecular subtypes. Molecular subtype was a better prognostic parameter than histopathological grade. In the third study, we studied markers in primary DCIS in relation to type of recurrence. Interestingly, recurrences after an ER-/HER2+, ER negative or EGFR positive primary DCIS were more often of the in situ type. The molecular subtype ER+/HER2+, FOXA1 positivity and FOXC1 positivity were risk factors for any recurrence. In the fourth study, we proposed a histological classification system for a new entity: neoductgenesis. We also evaluated histologic criteria for neoductgenesis. According to our criteria, good agreements among pathologists were achieved. Neoductgenesis was related to more aggressive tumor biology and to mammographic features. The result indicates potential benefits for women earlier considered having pure DCIS but later diagnosed as breast carcinoma with neoductgenesis, suggesting a need to develop appropriate treatment regiments. Our findings have to be repeated and the relation to prognosis warrants further studies.

<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.

Made available in DSpace on 2014-06-11T19:26:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2001Bitstream added on 2014-06-13T20:34:10Z : No. of bitstreams: 1 lopes_mc_me_botfm.pdf: 427624 bytes, checksum: 0bc1ebccf90a1df7c064aa28076686ba (MD5) / O grau nuclear é considerado um dos principais fatores prognósticos do carcinoma invasivo de ductos mamários, porém não é frequentemente utilizado em Punção aspirativa por agulha fina (PAAF) na rotina diária de muitos patologistas. Para avaliar a concordância citohistológica da graduação nuclear foi realizado estudo retrospectivo de 90 casos de carcinoma ductal invasivo da mama, inicialmente diagnosticados por PAAF e com subsequente confirmação histológica. As leituras dos preparados citológicos foram realizadas em esfregaços corados pela Hematoxilina-Eosina por dois patologistas independentemente, utilizandose a graduação nuclear de Black modificada por Fisher (BM) e simplificada por Cajulis (BS) e posteriormente confrontadas com a graduação de Bloom&Richardson na histologia. O índice de concordância com a histologia variou de 64,4% a 68,8% na classificação de BM e de 86,6% a 88,8% na classificação de BS. Na graduação pelo sistema de Black modificado, a reprodutibilidade intra-observador foi de 68,8%, enquanto que a reprodutibilidade inter-observador variou de 56,6% a 68,8%. No sistema de Black simplificado, a reprodutibilidade intraobservador foi de 93,3%, enquanto que a reprodutibilidade interobservador variou de 82,2% a 84,4%. Constatou-se que a graduação nuclear na PAAF utilizando o sistema de Black simplificado (BS) teve maior concordância... / Nuclear grading is one of the major prognostic factors of invasive ductal carcinoma of the breast. However, many pathologists do not routinely use it in fine-needle aspiration biopsy (FNAB). A retrospective study was performed on 90 cases of invasive breast ductal carcinoma, diagnosed by FNAB and confirmed by histology, to evaluate the cyto-histological agreement of nuclear grading. Cytological readings were made from smears stained with Hematoxilin-Eosin by two independent pathologists, using Black’s nuclear grading a) modified by Fisher (BM) and b) simplified by Cajulis (BS). This was then compared with Bloom & Richardson’s histological grade. The histological agreement rate was between 64.4% and 68.8% for BM classification and between 86.6% and 88.8% for BS. Grading using BM classification showed intraand inter-observer reproducibility rates of 68.8% and from 56.6% to 68.8% respectively, while BS showed 93.3% and from 82.2% to 84.4% respectively. Nuclear grading in FNAB using Black’s simplified system (BS) gave... (Complete abstract click electronic access below)

Orientador: Ulisses Frederigue Júnior / Resumo: O grau nuclear é considerado um dos principais fatores prognósticos do carcinoma invasivo de ductos mamários, porém não é frequentemente utilizado em Punção aspirativa por agulha fina (PAAF) na rotina diária de muitos patologistas. Para avaliar a concordância citohistológica da graduação nuclear foi realizado estudo retrospectivo de 90 casos de carcinoma ductal invasivo da mama, inicialmente diagnosticados por PAAF e com subsequente confirmação histológica. As leituras dos preparados citológicos foram realizadas em esfregaços corados pela Hematoxilina-Eosina por dois patologistas independentemente, utilizandose a graduação nuclear de Black modificada por Fisher (BM) e simplificada por Cajulis (BS) e posteriormente confrontadas com a graduação de Bloom&Richardson na histologia. O índice de concordância com a histologia variou de 64,4% a 68,8% na classificação de BM e de 86,6% a 88,8% na classificação de BS. Na graduação pelo sistema de Black modificado, a reprodutibilidade intra-observador foi de 68,8%, enquanto que a reprodutibilidade inter-observador variou de 56,6% a 68,8%. No sistema de Black simplificado, a reprodutibilidade intraobservador foi de 93,3%, enquanto que a reprodutibilidade interobservador variou de 82,2% a 84,4%. Constatou-se que a graduação nuclear na PAAF utilizando o sistema de Black simplificado (BS) teve maior concordância... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Nuclear grading is one of the major prognostic factors of invasive ductal carcinoma of the breast. However, many pathologists do not routinely use it in fine-needle aspiration biopsy (FNAB). A retrospective study was performed on 90 cases of invasive breast ductal carcinoma, diagnosed by FNAB and confirmed by histology, to evaluate the cyto-histological agreement of nuclear grading. Cytological readings were made from smears stained with Hematoxilin-Eosin by two independent pathologists, using Black's nuclear grading a) modified by Fisher (BM) and b) simplified by Cajulis (BS). This was then compared with Bloom & Richardson's histological grade. The histological agreement rate was between 64.4% and 68.8% for BM classification and between 86.6% and 88.8% for BS. Grading using BM classification showed intraand inter-observer reproducibility rates of 68.8% and from 56.6% to 68.8% respectively, while BS showed 93.3% and from 82.2% to 84.4% respectively. Nuclear grading in FNAB using Black's simplified system (BS) gave... (Complete abstract click electronic access below) / Mestre

Topoisomerases são enzimas nucleares que participam na regulação da estrutura do DNA nas células eucarióticas. A topoisomerase III é o mais novo membro da família das topoisomerases. Seu papel no desenvolvimento dos tumores mamários ainda necessita ser investigado. O objetivo do presente estudo foi avaliar a imunoexpressão da topoisomerase III nos carcinomas mamários, e comparar sua expressão com dados clinicopatológicos e marcadores imunohistoquímicos clássicos de importância prognóstica nos carcinomas mamários. Utilizando-se tissue microarrays contendo 171 casos de carcinomas ductais mamários primários, foi analisada a expressão imunohistoquímica de topoisomerase III, receptor de estrógeno, receptor de progesterona, HER-2, Ki67, p53 e BRCA-1. Positividade para topoisomerase III foi encontrada em 33,9% dos casos, e sua expressão relacionou-se com metástases à distância (p=0.036) e óbito (p=0.006). Negatividade para topoisomerase III relacionou-se com negatividade para HER=2 (p<0.001), p53 (p<0.001) e BRCA-1 (p=0.001), e com baixa expressão de Ki-67 (p<0.001). Na Análise de Riscos Múltiplos de Cox, a expressão de topoisomerase III foi um significante preditor de sobrevida [razão de risco 3.006 (intervalo de confiança a 95%: 1.582-5.715); p=0.001]. Concluindo, a topoisomerase III pode ser útil na avaliação do prognóstico de pacientes com câncer de mama, além de ser um fator independente de predição da sobrevida. / Topoisomerases are ubiquitous nuclear enzymes that regulate DNA structure in eukaryotic cells. The role of topoisomerase III, the newest member of the topoisomerase family, in the clinical outcome of breast cancer is still poorly understood. This study aims to investigate the immunoexpression of topoisomerase III in breast cancer and its relationships with clinicopathological features and immunohistochemical markers of prognostic significance in breast pathology. Using tissue microarrays containing 171 cases of primary invasive breast cancer, we analyzed the immunoexpression of topoisomerase III, estrogen receptor, progesterone receptor, HER-2, Ki67, p53 and BRCA-1. Immunostaining for topoisomerase III was found in 33.9% of breast carcinomas, and immunopositivity was related with distant metastasis (p=0.036) and death (p=0.006). Decreased expression of topoisomerase III was related with negativity with HER-2 (p<0.001), p53 (p<0.001) and BRCA1 (p=0.001) and low expression of Ki67 (p<0.001). In the multivariate analysis, topoisomerase III expression was a significant predictor of survival [hazard ratio 3.006 (95% confidence interval 1.582-5.715); p=0.001]. In conclusion, topoisomerase III expression can be a useful marker in assessing the prognosis of patients with breast cancer and is an independent predictor of survival.

Alterations to the tumour microenvironment is a common feature of many cancers, including breast cancer, and there is increasing evidence that alterations to the microenvironment, including; increased integrin expression, ECM deposition and protease activity, promote cancer progression. Most invasive breast cancers arise from a preinvasive stage, ductal carcinoma in situ (DCIS). Previous work in our laboratory has shown the microenvironment of DCIS is altered, such that myoepithelial cells (MECs) switch to a tumour-promoting phenotype, associated with upregulation of integrin αvβ6 and fibronectin (FN) expression. Mechanisms by which integrin αvβ6 and FN expression are regulated is unclear. We show DCIS progression into invasion is accompanied by an increase in MEC expression of integrin αvβ6 and periductal FN deposition, and their expression were associated in DCIS. These findings were modelled in isolated primary DCIS-MECs, primary normal MECs and MEC lines, with and without integrin αvβ6 expression, where integrin αvβ6-positive MECs upregulating FN expression. We identified integrin αvβ6-positive DCIS ducts were larger than integrin αvβ6-negative DCIS ducts, and mechanical stretching of primary normal MECs and a normal MEC line led to upregulation of integrin αvβ6 expression and FN deposition in a TGFβ-dependent manner. We further show upregulation of integrin αvβ6 and FN by MECs mediate TGFβ-dependent upregulation of MMP13 which promotes breast cancer cell invasion in vitro. These data show altered tissue mechanics in DCIS and MEC expression of integrin αvβ6 and FN deposition are linked, and implicate TGFβ in their activation. These findings suggest integrin αvβ6 and FN may be used as markers to stratify DCIS patients.

Topoisomerases são enzimas nucleares que participam na regulação da estrutura do DNA nas células eucarióticas. A topoisomerase III é o mais novo membro da família das topoisomerases. Seu papel no desenvolvimento dos tumores mamários ainda necessita ser investigado. O objetivo do presente estudo foi avaliar a imunoexpressão da topoisomerase III nos carcinomas mamários, e comparar sua expressão com dados clinicopatológicos e marcadores imunohistoquímicos clássicos de importância prognóstica nos carcinomas mamários. Utilizando-se tissue microarrays contendo 171 casos de carcinomas ductais mamários primários, foi analisada a expressão imunohistoquímica de topoisomerase III, receptor de estrógeno, receptor de progesterona, HER-2, Ki67, p53 e BRCA-1. Positividade para topoisomerase III foi encontrada em 33,9% dos casos, e sua expressão relacionou-se com metástases à distância (p=0.036) e óbito (p=0.006). Negatividade para topoisomerase III relacionou-se com negatividade para HER=2 (p<0.001), p53 (p<0.001) e BRCA-1 (p=0.001), e com baixa expressão de Ki-67 (p<0.001). Na Análise de Riscos Múltiplos de Cox, a expressão de topoisomerase III foi um significante preditor de sobrevida [razão de risco 3.006 (intervalo de confiança a 95%: 1.582-5.715); p=0.001]. Concluindo, a topoisomerase III pode ser útil na avaliação do prognóstico de pacientes com câncer de mama, além de ser um fator independente de predição da sobrevida. / Topoisomerases are ubiquitous nuclear enzymes that regulate DNA structure in eukaryotic cells. The role of topoisomerase III, the newest member of the topoisomerase family, in the clinical outcome of breast cancer is still poorly understood. This study aims to investigate the immunoexpression of topoisomerase III in breast cancer and its relationships with clinicopathological features and immunohistochemical markers of prognostic significance in breast pathology. Using tissue microarrays containing 171 cases of primary invasive breast cancer, we analyzed the immunoexpression of topoisomerase III, estrogen receptor, progesterone receptor, HER-2, Ki67, p53 and BRCA-1. Immunostaining for topoisomerase III was found in 33.9% of breast carcinomas, and immunopositivity was related with distant metastasis (p=0.036) and death (p=0.006). Decreased expression of topoisomerase III was related with negativity with HER-2 (p<0.001), p53 (p<0.001) and BRCA1 (p=0.001) and low expression of Ki67 (p<0.001). In the multivariate analysis, topoisomerase III expression was a significant predictor of survival [hazard ratio 3.006 (95% confidence interval 1.582-5.715); p=0.001]. In conclusion, topoisomerase III expression can be a useful marker in assessing the prognosis of patients with breast cancer and is an independent predictor of survival.

Breast cancer is the most common cancer in Taiwanese women and the invasive ductal carcinoma (IDC) is the most common type. Increasing evidence shows that cancer stem cells (CSCs) have been implicated in tumorigenesis, tumor progression, and drug-resistance. In addition, four reprogramming factors (Octamer-binding Protein 4 (Oct4), Sex-determining Region Y (SRY)-related Box 2 (Sox2), Nanog and Lin28) employed to induce induced pluripotent stem (iPS) cells are associated with CSCs formation. The purpose of this study was to investigate the relationship of the protein expression levels of the reprogramming factors (Oct4, Sox2, Nanog and Lin28) with the tumorigenesis, clinicopathologic outcomes and prognosis of breast IDC patients. Immunohistochemistry (IHC) assay of tissue microarrays, made by 309 IDC and 20 breast fibrosis paraffin embedded samples, were used to examine the protein expression levels of Oct4, Sox2, Nanog and Lin28 in normal mammary ductal tissues, tumor adjacent normal mammary ductal tissues, ductal carcinoma in situ (DCIS), IDC and recurrence tissues. Our IHC results showed that Sox2 and Lin28 were expressed in half of breast IDC patients¡¦ tumor tissue (49.6% and 49.7%, respectively), but Oct4 and Nanog are less expressed (13.5% and 24.7%, respectively). The protein expression levels of the four proteins were positively correlated with each other. In addition, the expression levels of the four proteins were upregulated in tumor adjacent normal tissue as compared to breast fibrosis pateints¡¦ normal mammary ductal tissue. To compare the expression levels of the four proteins in different tissues; such as tumor adjacent normal, DCIS, IDC and recurrence tissues, the expression levels of the four protiens gradually decreased when tumor developed and progressed. However, their expression levels were comparable between IDC and recurrence tissues. Additionally, the high expression levels of four proteins were high in two good clinicopathological characteristics and a biomarker of breast cancer; such as nuclear Sox2 and Lin28 in those with pathology stage I; nucleus expression of the four proteins in those with well and moderate cell differentiation; and Sox2 in those with positive estrogen receptor. However, the four proteins¡¦ expression levels were not correlated with IDC patients¡¦ survival. In conclusion, the reprogramming factors: Oct4, Sox2, Nanog and Lin28 may play an important role in tumorigenesis of breast IDC, but their impacts on tumor progression were quite small.

Elevated level of lipogenic enzymes and overall lipogenesis have been reported in a wide variety of cancers and blocking the lipogenic pathway by chemical inhibitors or RNA interference causes tumor cell death by apoptosis which provides a strong rationale for targeting lipogenic pathway for the treatment and prevention of cancer however the exact role of lipogenesis as a cause, facilitator or consequence is not yet clearly understood. Therefore in this dissertation research, we set up to determine the mechanism of tumor cell death by inhibiting lipogenesis and to determine the role of increased lipogenesis in the breast cancer progression. In the first part of this study, we investigated the status of fatty acid synthase (FAS) gene which is regarded as the key lipogenic gene in fatty acid biosynthetic pathway and is responsible for the synthesis of lipid molecules by facilitating the condensation reaction between acetyl-CoA and malonyl-CoA in the presence of NADPH. We observed that normal breast epithelial cells MCF10A cells have very low level of FAS expression whereas breast cancer cell lines MCF7, MDA MB231 and MDA MB231 LM have significant overexpression. Next, we observed the similar trend of FAS overexpression in breast cancer stem-like cells (CSCs) isolated from the MCF7, MDA MB231 and MDA MB231 LM cell lines using cell surface markers (CD24-/CD44+/ESA+). These cells were previously transplanted into the mammary fat pad of nude mice and the results of our limiting dilution analysis indicate that CSCs had a significantly higher ability of forming breast cancer in the injected animals which explains our rationale to use CSCs in our research. In order to exploit this lipogenic pathway for the treatment and chemoprevention of breast cancer, we then examined the effects of resveratrol on breast cancer cells. Resveratrol is a natural polyphenolic compound and has been shown to exhibit cardio-protective as well as anti-neoplastic effects on various types of cancers. However, the exact mechanism of its anti-tumor effect is not clearly defined. We observed that resveratrol significantly reduced the cell viability by inducing apoptosis in parental cells as well as in CSCs. Resveratrol also inhibited mammosphere formation which is an inherent property of CSCs. This inhibitory effect of resveratrol is accompanied by a significant reduction in lipid synthesis which is caused by the down-regulation of the FAS gene followed by up-regulation of pro-apoptotic genes, DAPK2 and BNIP3. The activation of apoptotic pathway in the cancer stem-like cells was suppressed by FAS overexpression suggesting that resveratrol-induced apoptosis is indeed through the modulation of FAS-mediated cell survival signaling. Importantly, resveratrol was able to significantly suppress the growth of CSC in an animal model of human breast cancer xenograft without showing apparental toxicity. Taken together, our results indicate that resveratrol is capable of inducing apoptosis in the CSCs through suppression of lipogenesis by modulating FAS expression, which highlights a novel mechanism of anti-tumor effect of resveratrol. Taken together, our results indicate that resveratrol is capable of inducing apoptosis in the cancer stem-like cells through suppression of lipogenesis by modulating FAS expression, which highlights a novel mechanism of anti-tumor effect of resveratrol. In the second part of research, we tried to determine the role of elevated level of lipogenesis in normal to ductal carcinoma in situ (DCIS) progression. For this, we first analyzed the expression profile of various lipogenic genes using an expression microarray and found that CSCs from DCIS.com showed significantly higher level of ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC) and FAS than the normal non-tumorigenic stem-like cells obtained from MCF10A. The result was also confirmed by qRT-PCR and Western blot as well as in clinical specimens of DCIS by immunohistochemistry. In the next step, we detected that SREBP1, the master regulator of lipogenic genes, is also upregulated in DCIS and further identified that SREBP1 regulates the co-ordinate expression of ACLY, ACC and FAS ultimately resulting in the elevation of lipogenesis. In order to determine the role of SREBP1 overexpression in normal to DCIS transition, we overexpressed the SREBP1 in MCF10A cells which induced a significant increase in the downstream key lipogenic genes ACLY, ACC1 and FAS which resulted in the clear upregulation of total lipid content in the cells. Furthermore, we found that this elevation of lipogenesis in MCF10A-SREBP1 stem-like cells confers proliferative advantage as well as a significant increase in mammosphere forming ability and anchorage independent growth (3D culture). Thus, our results showed a possibility that increased lipogenesis in normal stem-like cells may be responsible for providing oncogenic transformation properties which can be confirmed at least in our in vitro model. We then examined the effects of resveratrol on CSCs sorted from DCIS.com. We found that resveratrol decreased the cell viability and increased apoptosis by reducing the total lipid content by inhibiting the expression of SREBP1 and downstream lipogenic genes. Resveratrol also hindered the stemness of the DCIS CSCs by inhibiting its mammosphere forming ability. When DCIS CSCs were transplanted into mammary fat pad of nude mice which were on resveratrol treatment, we observed that resveratrol significantly suppressed the formation of DCIS by downregulating lipogenic genes and by upregulating pro-apoptotic genes, DAPK2 and BNIP3. Collectively, our results indicate that lipogenic genes SREBP1 co-ordinately regulates the overexpression of ACLY, ACC1 and FAS in DCIS CSCs at an early stage of breast tumorigenesis and thus confer proliferative and survival advantages. Anti-growth effect of resveratrol on DCIS CSCs also provides us with a strong rationale to use this agent for chemo-prevention against DCIS.