Orientador: Paulo Traiman / Coorientador: Andréa Teixeira de Carvalho / Banca: Anagloria Pontes / Banca: Rogério Dias / Banca: Walter Antonio Prata Pace / Banca: Ricardo Bassil Lasmar / Resumo: Introdução: O câncer epitelial de ovário (CEO) e endometriose apresentam alguns aspectos semelhantes, como infiltração tecidual e capacidade de produzir lesões em órgão adjacentes e a distância, no entanto, diferem de forma marcante em sua evolução. A análise das semelhanças e diferenças do sistema imune e resposta inflamatória relacionados a estas patologias podem permitir um melhor entendimento de seus mecanismos fisiopatológicos e consequentemente contribuir no aprimoramento do rastreamento, diagnóstico e estratégias terapêuticas. O objetivo desde estudo é avaliar e comparar a expressão gênica tecidual , citocinas, quimiocinas e micropartículas no plasma, líquido ascético e lavado peritoneal de pacientes com CEO e endometrioma. Métodos: Foram estudadas 60 pacientes, dividias em 3 grupos: Grupo 1 (CEO): 26 pacientes; Grupo 2 (endometriomas): 18 pacientes; Grupo 3 (controle): 16 pacientes. Todas as pacientes tiveram coletadas amostras de sangue, líquido ascítico ou lavado peritoneal e amostra tecidual das lesões. O material foi estocado para posterior análise. Citocinas e quimiocinas foram dosadas utilizando-se CBA, as amostras teciduais foram avaliadas com técnica de RT-qPCR e as micropartículas com técnica de imunoflorescência modificada para esta análise. Utilizou-se os teste de Mann-Whitney e Kruskal-Wallis quando apropriados. Para a identificação de associações entre dosagens de líquido ascítico e soro foi utilizado o coeficiente de correlação de Sperman. O valor de p<0,05 foi considerado significativo. Resultados: A idade média das pacientes no grupos CEO, endometrioma e controle foram respectivamente 62, 37 e 40,2 anos. Os pacientes foram estadiados segundo o critérios da FIGO para o câncer de ovário,10 pacientes nos estadios I e II, e 16 no estádio III/IV no grupo de CEO. O estadiamento dos pacientes com endometriomas foram realizados conforme os critérios da ASRM identificando-se ... / Abstract: Not available / Doutor

Purpose: Control of micrometastatic ovarian cancer in the peritoneal cavity remains a major objective in post-surgical treatment. The purpose of this project was to investigate the efficacy and toxicity of targeted alpha therapy (TAT) for ovarian cancer in vitro and in vivo in animal models and to select the optimal targeting vector for an ovarian cancer clinical trial. Animal models of ovarian, breast and prostate cancer were developed and for further TAT; a phase I melanoma clinical trial was supported, paving the way for an ovarian cancer clinical trial. Methods: The expression of the turnor-associated antigens (Her2, MUC1, uPAfuPAR) on cancer cell line, animal model xenografts and human ovarian cancer tissue was tested by immunostaining. MTS and TUNEL assays were used to evaluate cell killing of alpha conjugates in monolayer and spheroids. Toxicity and maximum tolerance doses for different vectors were tested and determined in vivo. Pharmacokinetics was studied for different time points and different parameters. The antiproliferative effect of 213Bi-C595 and 213Bi-PAI2 was tested at 9 days post-peritoneal cell inoculation of the ovarian cancer cell line OVCAR3. The treatment efficacy of 213Bi-Herceptin was tested at a 2 days post-subcutaneous breast cancer cell BT474 inoculation. Mice were injected (i.p) with various concentrations of alpha conjugates (AC). Changes in cancer progression were assessed by girth size and tumor size. Results: uPA/uPAR and MUCI are expressed on ovarian cancer cell lines and more than 45% ovarian cancer tissue, while HER2 was only positive in one cell line and was positive in less than 15% of ovarian cancer tissues. The ACs can target and kill cancer cells in vitro in a dose dependent fashion. TUNEL positive cells were found after incubation with the different ACs. PAI2 and C595 vectors were selected for in vivo ascites model study of OVCARJ cell with high expression. Delayed and acute toxicity in animal models showed that radiation nephropathy was the cause of body weight loss. Biodistribution studies showed that kidney was the major uptake organ. L-lysine can reduce kidney uptake for 213Bi-PAI2, but no significant differences were found. A single ip injection of 213Bi-C595 or 213Bi-PAI2 can inhibit ascites growth, whereas, 213Bi-Herceptin can inhibit breast cancer growth in a nude mice model. Conclusion: 213Bi labelled targeting vectors can specifically target ovarian cancer cells in vitro and inhibit tumor growth in vivo. These ACs may be useful agents for the treatment of ovarian cancer at the minimum residual disease stage.

Systems pathology attempts to introduce more holistic approaches towards pathology and attempts to integrate clinicopathological information with “-omics” technology. This doctorate researches two examples of a systems approach for pathology: (1) a personalized patient output prediction for ovarian cancer and (2) an analytical approach differentiates between individual and collective tumour invasion. During the personalized patient output prediction for ovarian cancer study, clinicopathological measurements and proteomic biomarkers are analysed with a set of newly engineered bioinformatic tools. These tools are based upon feature selection, survival analysis with Cox proportional hazards regression, and a novel Monte Carlo approach. Clinical and pathological data proves to have highly significant information content, as expected; however, molecular data has little information content alone, and is only significant when selected most-informative variables are placed in the context of the patient's clinical and pathological measures. Furthermore, classifiers based on support vector machines (SVMs) that predict one-year PFS and three-year OS with high accuracy, show how the addition of carefully selected molecular measures to clinical and pathological knowledge can enable personalized prognosis predictions. Finally, the high-performance of these classifiers are validated on an additional data set. A second study, an analytical approach differentiates between individual and collective tumour invasion, analyses a set of morphological measures. These morphological measurements are collected with a newly developed process using automated imaging analysis for data collection in combination with a Bayesian network analysis to probabilistically connect morphological variables with tumour invasion modes. Between an individual and collective invasion mode, cell-cell contact is the most discriminating morphological feature. Smaller invading groups were typified by smoother cellular surfaces than those invading collectively in larger groups. Interestingly, elongation was evident in all invading cell groups and was not a specific feature of single cell invasion as a surrogate of epithelialmesenchymal transition. In conclusion, the combination of automated imaging analysis and Bayesian network analysis provides an insight into morphological variables associated with transition of cancer cells between invasion modes. We show that only two morphologically distinct modes of invasion exist. The two studies performed in this thesis illustrate the potential of a systems approach for pathology and illustrate the need of quantitative approaches in order to reveal the system behind pathology.

by Cheung Kwok Kuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 111-130). / Chapter Chapter 1 --- General introduction of human ovarian cancer / Chapter 1.1 --- Epidemiology --- p.1 / Chapter 1.2 --- Symptoms and diagnosis --- p.4 / Chapter 1.3 --- Etiology --- p.5 / Chapter 1.3.1 --- Factors associated with decreased risks --- p.6 / Chapter 1.3.2 --- Factors associated with increased risks --- p.8 / Chapter 1.4 --- Classification of ovarian cancer --- p.12 / Chapter 1.5 --- Molecular basis of ovarian cancer --- p.18 / Chapter 1.6 --- Project aim --- p.29 / Chapter Chaper 2 --- "DOC-2, a differentially expressed gene in human ovarian cancer" / Chapter 2.1 --- Introduction --- p.32 / Chapter 2.2 --- Materials and Methods --- p.35 / Chapter 2.2.1 --- Expression of DOC-2 in human ovarian tissues --- p.35 / Chapter 2.2.1.1 --- Preparation of specimen --- p.35 / Chapter 2.2.1.2 --- Immunohistochemical studies of the expression of DOC-2 protein in human ovarian tissues --- p.35 / Chapter 2.2.1.3 --- Quantitation of immunoreactivity --- p.38 / Chapter 2.2.2 --- Effect of DOC-2 transfection on growth rate of the ovarian cancer cell lineSKOV3 --- p.39 / Chapter 2.2.2.1 --- Cell line --- p.39 / Chapter 2.2.2.2 --- Transfection of DOC-2 to SKOV3 ovarian carcinoma cell line --- p.39 / Chapter 2.2.2.3 --- Growth curve of the transfected ovarian carcinoma cell lines --- p.40 / Chapter 2.2.3 --- In vivo tumorigenicity study --- p.42 / Chapter 2.3 --- Results --- p.44 / Chapter 2.3.1 --- Expression of DOC-2 in human ovarian tissues --- p.44 / Chapter 2.3.2 --- Effects of DOC-2 transfected gene on the growth rate of the human ovarian cancer cell line SKOV3 --- p.46 / Chapter 2.3.2.1 --- Standard curves for calculating cell density from absorbance --- p.46 / Chapter 2.3.2.2 --- The effect of DOC-2 transfection on the growth rate of the human ovarian cancer cell line SKOV3 --- p.47 / Chapter 2.3.3 --- In vivo tumorigenicity --- p.48 / Chapter 2.4 --- Discussion --- p.50 / Chapter Chapter 3 --- DOC-2 expression in mouse embryonic development / Chapter 3.1 --- Introduction --- p.56 / Chapter 3.2 --- Materials and Methods --- p.60 / Chapter 3.2.1 --- Expression of murine homolog of DOC-2 (p96) during mouse embryonic development --- p.60 / Chapter 3.2.1.1 --- Preparation of paraffin-embedded mouse embryo sections --- p.60 / Chapter 3.2.1.2 --- Preparation of OCT-embedded mouse embryo sections --- p.61 / Chapter 3.2.1.3 --- Immunohistochemistry of murine homolog of DOC-2 (p96) on mouse embryos --- p.61 / Chapter 3.2.2 --- Effect of antibody blocking for DOC-2 protein on the growth of embryonic kidney in vitro --- p.62 / Chapter 3.3 --- Results --- p.64 / Chapter 3.4 --- Discussion --- p.69 / Chapter Chapter 4 --- Apoptosis / Chapter 4.1 --- Introduction --- p.72 / Chapter 4.1.1 --- Current methods for the detection of apoptosis --- p.74 / Chapter 4.1.1.1 --- Agarose gel electrophoresis --- p.75 / Chapter 4.1.1.2 --- Flow cytometric analysis --- p.76 / Chapter 4.1.1.3 --- 3-OH end labelling --- p.77 / Chapter 4.1.1.4 --- Nuclease assay --- p.78 / Chapter 4.1.2 --- Apoptosis in normal physiology and oncogenesis --- p.78 / Chapter 4.1.3 --- p53 and apoptosis --- p.80 / Chapter 4.1.4 --- bcl-2 and apoptosis --- p.83 / Chapter 4.2 --- Materials and Methods --- p.92 / Chapter 4.2.1 --- Expression of p53 and bcl-2 in human ovarian tissues --- p.92 / Chapter 4.2.1.1 --- Preparation of specimens --- p.92 / Chapter 4.2.1.2 --- Immunohistochemical studies of the expression of p53 and bcl-2 proteins in ovarian tissue --- p.92 / Chapter 4.2.2 --- In stiu terminal transferase-mediated dUTP nick and labelling (TUNEL) --- p.94 / Chapter 4.3 --- Results --- p.96 / Chapter 4.3.1 --- Expression of p53 and bcl-2 in human ovarian tissues --- p.96 / Chapter 4.3.2 --- Apoptosis in human ovarian tissues --- p.99 / Chapter 4.4 --- Discussion --- p.101 / Chapter Chapter 5 --- Concluding Remarks --- p.108 / References --- p.111 / Appendix --- p.131 / Figures and legend --- p.138

Ο καρκίνος εκ του επιθηλίου επιφανείας των ωοθηκών είναι η 5η πιο κοινή αιτία θανάτου σε γυναίκες στο Δυτικό κόσμο και είναι υπεύθυνος για τους περισσότερους θανάτους από ότι όλες οι γυναικολογικές κακοήθειες μαζί. Τόσο ο μηχανισμός του νεοπλαστικού μετασχηματισμού όσο και τα μοριακά μονοπάτια που οδηγούν στο ωοθηκικό επιθηλιακό καρκίνωμα δεν έχουν εξακριβωθεί πλήρως. Ένα πρόσφατα αναγνωρισμένο γονίδιο, η μετατχερίνη (MTDH), γνωστή και ως AEG-1 ή LYRIC ενοχοποιείται ως πιθανός σημαντικός διαμεσολαβητής κατά την καρκινογένεση, την μετάσταση και την αντίσταση στις χημειοθεραπείες. Ωστόσο, η κλινική σημασία και ο βιολογικός ρόλος της μετατχερίνης (MTDH), στο επιθηλιακό καρκίνωμα των ωοθηκών δεν έχουν γίνει αποσαφηνισθεί πλήρως. Η υπερέκφραση της MTDH/AEG-1 μπορεί να ενεργοποιήσει πολλά σηματοδοτικά μονοπάτια, όπως για παράδειγμα το NF-κΒ σηματοδοτικό μονοπάτι σε διάφορους καρκινικούς τύπους. Ο NF-κΒ έχει πρόσφατα συσχετισθεί με την ανάπτυξη και την εξέλιξη των όγκων και επιπλέον τα διμερή του NF-κΒ ενισχύουν την έκφραση ποικίλων γονιδίων που αφορούν την κυτταρική αύξηση, τη διαφοροποίηση, τις φλεγμονώδεις αντιδράσεις και τη ρύθμιση της απόπτωσης. Η παρούσα μελέτη σχεδιάστηκε προκειμένου να διερευνηθεί η έκφραση των πρωτεϊνών MTDH και NF-kB (p65/p50) σε επιθηλιακά ωοθηκικά καρκινώματα (καλοήθεις, οριακής κακοήθειας και διηθητικά καρκινώματα). Για το σκοπό αυτό μελετήθηκαν τμήματα επιθηλιακών νεοπλασμάτων ωοθηκών από 76 ασθενείς (15/46 οριακής κακοήθειας όγκοι, 30/46 διηθητικά αδενοκαρκινώματα και 31/76 κυσταδενώματα), μονιμοποιημένα σε ουδέτερη φορμόλη και εγκλεισμένα σε παραφίνη με τη μέθοδο της ανοσοϊστοχημείας για την έκφραση των πρωτεϊνών MTDH και NF-κB (p50/p65). Επίσης εκτιμήθηκε η σχέση της MTDH/AEG-1 με τον NF-κB και με κλινικοπαθολογοανατομικές παραμέτρους όπως ο βαθμός κακοήθειας του όγκου, το στάδιο, η μέγιστη διάμετρος του όγκου και η ηλικία της ασθενούς. Τα αποτελέσματα της ανοσοϊστοχημείας αναλύθηκαν με τη χρήση του στατιστικού πακέτου SPSS. Ο φυσιολογικός ωοθηκικός ιστός και τα κυσταδενώματα ήταν κυρίως αρνητικά για τις πρωτεΐνες MTDH/AEG-1 και NF-κB (p50, p65). Η έκφραση των MTDH/AEG-1 και NF-kappa B/ p50, πρωτεϊνών ήταν σημαντικά αυξημένες στα αδενοκαρκινώματα σε σχέση με τους οριακής κακοήθειας όγκους. Σε αντίθεση η έκφραση της NF-kappa B/ p65 πρωτεΐνης δεν έδειξε σημαντικές διαφορές μεταξύ των οριακής κακοήθειας όγκων και των αδενοκαρκινωμάτων. Σημαντική στατιστική συσχέτιση παρατηρήθηκε στην έκφραση των πρωτεϊνών MTDH/AEG-1, NF-kappa B/p50 και NF-kappaB/p65 στα αδενοκαρκινώματα. Καμία στατιστική συσχέτιση δεν παρατηρήθηκε στην έκφραση μεταξύ του NF-κB, της MTDH πρωτεΐνης και κλινικοπαθολογοανατομικών παραμέτρων. Συμπερασματικά, τα αποτελέσματα μας υποδεικνύουν ότι η MTDH/AEG-1 ίσως παίζει ένα σημαντικό ρόλο στην παθογένεια του ανθρώπινου ωοθηκικού καρκίνου, πιθανώς μέσω της ενεργοποίησης του σηματοδοτικού μονοπατιού του πυρηνικού μεταγραφικού παράγοντα NF-κB. Επειδή η MTDH συσχετίζεται σημαντικά με την αντίσταση στη χημειοθεραπεία, θα μπορούσε ενδεχομένως να αποτελέσει σημαντικό στόχο για θεραπεία, ενισχύοντας την αποτελεσματικότητα της χημειοθεραπείας στον επιθηλιακό ωοθηκικό καρκίνο. / Epithelial ovarian cancer is the fifth most common cause of cancer death in women in the Western world and the leading cause of death from gynaecological malignancies. Little is known about the mechanism of neoplastic transformation and the molecular events leading to epithelial ovarian cancer are poorly understood. A recently discovered gene, metadherin (MTDH, also known as AEG-1 or LYRIC) has emerged as a potentially crucial mediator of tumor progression, metastasis, and resistance to chemotherapies. The clinical significance and biological role of metadherin (MTDH), in epithelial ovarian carcinoma however, remains unclear. Overexpression of MTDH/AEG-1 can activate several downstream pathways, including the NFκB pathway in various types of cancer cells. Recently, NF-kB has been related to cancer development and progression and NF-kB dimers (p50/p65) could induce the expression of various genes regarding cell growth, differentiation, inflammatory responses and the regulation of apoptosis. This study was designed in order to determine the expression of the MTDH and NF-kB (p65/p50) proteins in epithelial ovarian tumors (benign, borderline and malignant). Formalin-fixed and paraffin embedded tissue blocks from 76 patients with epithelial ovarian neoplasms (15/46 borderline tumors, 30/46 invasive adenocarcinomas and 31/76 cystadenomas) were studied. Expressions of MTDH/AEG-1, NF-κB (p50, p65) were investigated immunohistochemically. The relationship of MTDH/AEG-1 with NF-κB and clinicopathological parameters such as tumor grade, stage, tumor maximal diameter and patient age were evaluated. The results of immunohistochemistry were analyzed with the SPSS statistic analyze protocol. Normal ovarian tissue and benign ovarian cystadenomas were mostly MTDH/AEG-1, NF-κB (p50, p65) negative. The expression of MTDH/AEG-1 and NF-kappaB p50, proteins were significantly higher in adenocarcinoma tissue in comparison with borderline tumors. In contast NF-kappaB p65 expession shows no significant differences between borderline tumors and adenocarcinomas. A statistical significant correlation was observed between MTDH/AEG-1 and NF-kappaB p50, p65 protein expression in adenocarcinomas. No statistical correlation was observed between the NF-Kb and MTDH protein expression and clinicopathological parameters. In conclusion our data indicate that the upregulation of MTDH/AEG-1 may play an important role in the pathogenesis of human ovarian cancer possibly through activation of Nuclear factor-κB signalling pathway. Since MTDH has also a significant correlation with chemoresistance could be an important therapeutic target enhancing chemotherapy efficacy in ovarian epithelial cancer.