研究背景及目的：EB病毒(EBV)相關性胃癌的發病率約占胃癌的10%。近年來，越來越多的研究表明， EBV相關性胃癌的腫瘤抑制基因發生異常甲基化。然而，EBV的感染對全基因組DNA甲基化的影響尚不清楚。本研究通過分析EBV感染的細胞中全基因組DNA甲基化的情況，篩選出因EBV感染而發生甲基化的基因，並闡明靶基因在胃癌發生過程的作用。 / 方法：本研究應用穩定轉染EBV的胃癌細胞AGS (AGS-EBV)和無EBV轉染的AGS細胞為模型。採用高解析度的甲基化DNA免疫共沉澱晶片技術(MeDIP-chip)比較AGS-EBV 和AGS全基因組DNA甲基化的變化，並根據基因本體論(GO)，對EBV誘導的甲基化基因進行分類。採用RT-PCR，去甲基化處理及亞硫酸氫鈉測序(BGS)等方法驗證EBV誘導的甲基化基因。同時，採用基因敲除和過表達方法體外研究靶基因的生物學功能：通過細胞活力實驗和集落形成實驗判斷靶基因對細胞增殖的影響；通过流式细胞技术、伤口愈合实验及侵袭实验研究筛选到的靶基因生长抑素受体1（SSTR1）的功能；此外，还通過腫瘤通路基因PCR晶片分析靶基因調控的下游腫瘤相關基因。 / 结果：EBV編碼的小RNA(EBER)原位雜交方法和EBV潛伏期膜蛋白(LMP2A)的表達均證實AGS-EBV細胞中確實存在EBV的感染。和AGS細胞相比，發現AGS-EBV細胞中DNA甲基轉移酶3b(DNMT3b)的表達和活性顯著增加。AGS細胞中， LMP2A過表達後，DNMT3b的表達和活性也顯著增加。通過MeDIP-chip篩選出AGS-EBV中1,065甲基化有差異的基因，其中886基因為高甲基化。GO分析結果表明這些高甲基化基因參與KEGG信號通路。其中，六個新的高甲基化基因(MDGA2, IL15RA, SCARF2, EPHB6, SSTR1 和 REC8)在AGS-EBV細胞中的表達低於AGS；經過去甲基化處理之後，這些基因的表達水準有顯著增加。 / 通過深入研究生長抑素受體1(SSTR1)的生物學功能，發現：敲除SSTR1 能促進胃癌細胞的增殖和集落形成；通過调节G1/S期的調節因子，加快細胞進入S期，顯著增加S期的細胞數目。此外，胰腺癌細胞PANC1細胞中，SSTR1的過表達，也進一步證實SSTR1確實是一種腫瘤抑制基因。腫瘤通路基因PCR晶片結果顯示SSTR1通過促進細胞週期抑制因數(包括p15，p16，p21和p27)的表達，同時抑制CDC25A 和Myc的表達，發揮抑制增殖作用，導致細胞週期停滯在G1期，減少細胞增殖。SSTR1也通過減少凋亡相關基因的表達參與細胞凋亡的過程。此外，SSTR1還能顯著下調遷移相關基因的表達。這些結果表明，在EB病毒相關胃癌的發生過程中，SSTR1通過調節細胞週期、凋亡及遷移的有關基因，進而抑制細胞增殖，減少細胞的遷移和轉移。 / 结论：AGS感染EBV後，通過LMP2A促進DNMT3b的表達，激活DNMT3b的活性，導致886個腫瘤相關基因发生甲基化。SSTR1是一種EBV誘導的新的甲基化基因，在胃癌中具有抑制腫瘤的特性。研究表明， 由EBV誘導的SSTR1所具有的表觀遺傳學抑制作用參與EB病毒相關性胃癌的發病機制。 / Background and Aims: Epstein-Barr virus (EBV)-associated gastric cancer (GC) accounts for about 10% of all GCs. Accumulating evidences revealed aberrant rmethylation of tumor suppressor genes in EBV-associated GCs. However, the effect of EBV infection on the genome-wide aberrant DNA methylation remains unclear. We aim to profile the genome-wide EBV-associated hypermethylation in EBV-infected cells, to identify EBV-associated methylated genes and to elucidate their function in gastric carcinogenesis. / Methods: The cell model of gastric cancer AGS cells with or without stable EBV infection was used in this study. Genome-wide DNA methylation profiles were compared between AGS-EBV and AGS cells by high resolution Methyl-DNA immunoprecipitation microarry (MeDIP-chip) assay. EBV-associated methylated genes were classified according to gene ontology (GO). The novel EBV-associated methylated candidates were validated using bisulfite genomic sequencing (BGS), RT-PCR, and demethylation treatment. Biological function of one of the candidate genes (Somatostatin Receptor 1, SSTR1) was studied in vitro using gene knockdown and over-expression approaches simultaneously. Effects of SSTR1 expression on gastric cancer cell was measured by cell viability assay, colony formation assay, flow cytometry, wound-healing assay and invasion assay. Gene modulation by SSTR1 in human cancer pathways was assessed by cancer pathway PCR array. / Results: EBV infection was confirmed by EBER in situ hybridization. LMP2A expression was detected in AGS-EBV cells but not in EBV negative AGS cells. Expression and activity of DNMT3b was found to be significantly increased in AGS-EBV cells compared to AGS cells. Ectopic expression of LMP2A in AGS enhanced the expression and activity of DNMT3b. MeDIP-chip profiling identified a total of 1,065 genes differentially methylated by EBV infection (fold changes ≥2, P < 0.05) (fold-changes 2.4365.2). Gene ontology analysis indicated the enrichment of hypermethylated genes involving in important KEGG pathways. Notably, in addition to higher methylation levels confirmed by BGS, six novel hypermethylated genes (MDGA2, IL15RA, SCARF2, EPHB6, SSTR1 and REC8) were down-regulated in AGS-EBV cells as compared with AGS cells. Furthermore, demethylation treatment increased transcription levels of the six genes in AGS-EBV cells. / The biological function of SSTR1 gene was further investigated. Knockdown of SSTR1 in GC cells increased cell proliferation (P < 0.05) and colony formation ability (P < 0.01), and markedly increased cells in S phase through regulating G1/S phase mediators. Overexpression of SSTR1 in PANC1 cell line further confirmed that SSTR1 indeed was a tumor suppressor gene. Analysis of SSTR1 regulation of cancer pathway demonstrated that SSTR1 exerted antiproliferative effect by inducing cyclin-dependent inhibitors (p15, p16, p21 and p27) and inhibiting cell divison cycle 25 homolog A (CDC25A) and Myc, resulting in cell cycle arrest in G1 phase and reduction of cell proliferation. SSTR1 also took part in proliferation by decreasing expression of apoptosis regulators. Moreover, SSTR1 significantly downregulated the expression of migration-related genes, including ITGA1, ITGA2, ITGA3, ITGB5, IL8, MMP1 and PLAUR. These findings suggest that SSTR1 inhibits proliferation and reduces cell migration/invasion in gastric cancer by deregulating genes invloved in the regulation of cell cycle, survival/apoptosis and migration. / Conclusions: EBV infection in AGS cells induces genome-wide aberrant hypermethylation of 886 genes which involved in important cancer-related pathways. EBV-associated methylation is mediated by activation of DNMT3b through LMP2A. We identified and functionally characterized a novel EBV-associated methylated gene SSTR1 which exerted anti-tumor properties in GC. Epigenetic silencing of SSTR1 associated with EBV infection contributes to the pathogenesis of EBV-associated GC. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhao, Junhong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 129-141). / Abstract also in Chinese. / ABSTRACT --- p.i / 摘 要 --- p.iv / Acknowledgements --- p.vi / Publications --- p.vii / Research articles --- p.vii / Conference abstracts --- p.viii / Table of contents --- p.x / list of tables --- p.xiii / list of figures --- p.xiv / list of abbreviations --- p.xvi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- Gastric Cancer --- p.3 / Chapter 1.2.1 --- Eipdemiology of Gastric Cancer --- p.4 / Chapter 1.2.2 --- Pathology of Gastric Cancer --- p.8 / Chapter 1.2.3 --- Risk Factors for Gastric Cancers --- p.11 / Chapter 1.3 --- Epstein-Barr Virus-associated Gastric Cancer (EBVaGC) --- p.15 / Chapter 1.3.1 --- Historical Discovery and Harm of EBV --- p.15 / Chapter 1.3.2 --- Molecular Biology of EBV --- p.16 / Chapter 1.3.3 --- Latent and Lytic Infection of EBV --- p.18 / Chapter 1.3.4 --- EBV Products --- p.18 / Chapter 1.3.5 --- EBV-associated gastric cancer (EBVaGC) --- p.28 / Chapter 1.4 --- EBV-induced Epigenetic Alteration in Gastric Carcinogenesis --- p.36 / Chapter 1.4.1 --- Cytosine Methylation and CpG Island --- p.36 / Chapter 1.4.2 --- DNA Methylation in Gastric Cancer --- p.39 / Chapter 1.5 --- How to identify EBV-induced promoter methylation in gastric cancer --- p.45 / Chapter 1.5.1 --- Methylated DNA Immunoprecipitation (MeDIP) --- p.48 / Chapter 1.5.2 --- Combined Bisulfite Restriction Analysis (COBRA) --- p.49 / Chapter 1.5.3 --- Bisulfite Genomic Sequencing --- p.49 / Chapter 1.5.4 --- Pyrosequencing --- p.49 / Chapter Chapter 2 --- Materials and Methods --- p.51 / Chapter 2.1 --- Materials --- p.51 / Chapter 2.1.1 --- Cancer Cell Lines and Culture Condition --- p.51 / Chapter 2.1.2 --- Primary GC Samples --- p.52 / Chapter 2.2 --- EBV Encoded Nuclear RNA (EBER) in situ Hybridization (EBER-ISH) --- p.52 / Chapter 2.3 --- Western Blot Analysis --- p.53 / Chapter 2.4 --- Plasmid and Transfection --- p.56 / Chapter 2.4.1 --- Plasmid Construction and Extraction --- p.56 / Chapter 2.4.2 --- Plasmid Transfection --- p.59 / Chapter 2.5 --- Gene Expression Analysis --- p.59 / Chapter 2.5.1 --- Purification of Total RNA (RNeasy Kit, Qiagen) --- p.59 / Chapter 2.5.2 --- cDNA Reverse Transcription --- p.60 / Chapter 2.5.3 --- Semi-Quantitative PCR --- p.62 / Chapter 2.5.4 --- Quantitative Real-Time PCR (qRT-PCR) --- p.64 / Chapter 2.6 --- DNMT1 and 3b Activity Assay --- p.64 / Chapter 2.7 --- DNA Methylation Analysis --- p.64 / Chapter 2.7.1 --- Genomic DNA Extraction --- p.64 / Chapter 2.7.2 --- Genome-wide Profiling of EBV-associated DNA Methylation by MeDIP-chip --- p.65 / Chapter 2.7.3 --- Bioinformatics Analysis --- p.66 / Chapter 2.7.4 --- CpG Island Prediction and Analysis of the Targets’ Promoter Region --- p.66 / Chapter 2.7.5 --- DNA Sodium Bisulfite Modification --- p.67 / Chapter 2.7.6 --- Target Gene Methylation in GC Cell Lines --- p.67 / Chapter 2.7.7 --- Bisulfite Pyrosequencing Analysis in GC Tissue Samples --- p.70 / Chapter 2.8 --- Biological Function Analysis of SSTR1 --- p.72 / Chapter 2.8.1 --- Cell Proliferation Assay for Stable Transfection --- p.72 / Chapter 2.8.2 --- Colony Formation Assay --- p.72 / Chapter 2.8.3 --- Cell Cycle Analysis Assay --- p.72 / Chapter 2.8.4 --- Cell Migration Analysis --- p.73 / Chapter 2.8.5 --- Invasion Analysis --- p.73 / Chapter 2.8.6 --- Human Cancer Pathway Finder RT2 Profiler PCR Array Analysis --- p.74 / Chapter 2.9 --- Statistical Analysis --- p.76 / Chapter Charpter 3 --- results --- p.77 / Chapter 3.1 --- EBV Infection in AGS-EBV Cell Model --- p.77 / Chapter 3.2 --- Activation of DNMT3b in AGS-EBV Cells --- p.80 / Chapter 3.3 --- LMP2A Induced DNMT3b Activity in AGS Cells --- p.82 / Chapter 3.4 --- Genome-wide Profiling of DNA Methylation Associated with EBV Infection Using MeDIP-chip --- p.84 / Chapter 3.5 --- EBV-associated Cancer Pathways Defined by EBV-associated Promoter Methylated Genes --- p.86 / Chapter 3.6 --- CpG Hypermethylation and Transcriptional Silencing of EBV-associated Methylated Genes in AGS-EBV Cells --- p.88 / Chapter 3.7 --- Bioinformatics Analysis of SSTR1 Using University of California Santa Cruz Genome Bioinformatics (UCSC) Database and CpG Island Searcher --- p.93 / Chapter 3.8 --- COBRA Analysis of SSTR1 Promoter Methylation in GC Cell Lines --- p.93 / Chapter 3.9 --- Frequent SSTR1 Hypermethylation was Associated with EBV Positive Primary Gastric Cancer --- p.96 / Chapter 3.10 --- SSTR1 was Down-regulated in GC Cell Lines through RNA Interference --- p.103 / Chapter 3.11 --- SSTR1 Knockdown Induced Cell Proliferation in GC Cell Lines --- p.105 / Chapter 3.12 --- SSTR1 Knock-down Promoted Cells to Enter into S Phase --- p.108 / Chapter 3.13 --- SSTR1 Knock-down Increased the Migration Ability of GC --- p.110 / Chapter 3.14 --- SSTR1 Knock-down Promoted Cell Invasion --- p.112 / Chapter 3.15 --- Ectopic Expression of SSTR1 Inhibited Proliferation and Clonogenicity in PANC1 Cancer Cells --- p.114 / Chapter 3.16 --- Identification of Genes Modulated by SSTR1 --- p.116 / Chapter Chapter 4 --- Discussion --- p.119 / Chapter Chapter 5 --- Limitation of the study --- p.127 / ConclusionS --- p.128 / Reference --- p.129

Selected prognostic and predictive factors of pituitary tumours in patients with acromegaly (abstract) Pituitary tumours in patients with acromegaly (somatotroph adenomas, pituitary neuroendocrine tumours - PitNETs) are a rare disease with significant morbidity and high impact on economic resources of the health care system. The surgery is the first line of treatment, and while it cures a subset of the patients, another therapeutical approach combining radiotherapy and pharmacotherapy is needed in the rest. This usually includes treatment with costly somatostatin analogues (SSA). On the opposite, the therapeutic response to SSA is not a rule. This work aimed to perform precise clinicopathological characterisation of available tissue samples of PitNETs in acromegalic patients (n=110) from REgistr SEllárních Tumorů (RESET registry) and further explore potential factors related to clinically aggressive disease phenotype (tumour size, invasion, biochemical parameters) and factors that might predict therapeutic response to the first generation SSA. An extensive immunohistochemical evaluation was performed: tumours were histologically subclassified (expression of cytokeratins, prolactin, TSH) and proliferative activity was assessed (Ki67, p53, mitotic count) as well as expression of possible pharmacological...

Introdução: A doença de Cushing apresenta elevada morbimortalidade. Seu tratamento de escolha é a cirurgia transesfenoidal que possui resultados satisfatórios em cerca de 70% dos casos. Na doença persistente ou recorrente, reabordagem cirúrgica, radioterapia e adrenalectomia bilateral podem ser realizadas, porém, essas opções apresentam como desvantagens o desenvolvimento de hipopituitarismo e a dependência de terapia de reposição. Até o momento, nenhuma droga tem se mostrado eficaz no tratamento do corticotrofinoma. Os esquemas terapêuticos mais eficazes são os inibidores da esteroidogênese que não atuam no tumor hipofisário. Objetivos: avaliar o efeito do octreotide e da cabergolina administrados isoladamente e em associação nas concentrações urinárias de cortisol e plasmáticas de ACTH em pacientes com corticotrofinomas; correlacionar esse efeito com a expressão tumoral dos receptores SSTR2, SSTR5 e DRD2; correlacionar a expressão tumoral desses receptores através de RT-PCR quantitativa e imunohistoquímica; avaliar se o uso prévio dessas drogas altera a expressão desses receptores. Casuística e Métodos: grupo controle composto por 11 pacientes (10 mulheres e 1 homem) entre 21 e 43 anos sem tratamento prévio à neurocirurgia e um grupo tratado formado por 11 pacientes (2 homens e 9 mulheres) entre 22 e 53 anos que receberam o seguinte tratamento antes da cirurgia: coleta de três amostras de cortisol urinário e ACTH plasmático, seguida da introdução de octreotide 100 g, subcutâneo, 8/8h durante 30 dias e nova coleta de três amostras de cortisol urinário e ACTH plasmático. Em seguida, iniciou-se a cabergolina 0,5 mg via oral 3 vezes na semana durante 30 dias com nova coleta de três amostras de cortisol urinário e ACTH plasmático. A seguir, o octreotide era associado por mais 30 dias com nova coleta de três amostras de cortisol urinário e ACTH plasmático. Resultados: Os valores de cortisol urinário apresentaram queda significante após o uso de cabergolina isolada (P = 0,016) e em associação ao octreotide (P = 0,012). A eficácia do tratamento combinado não foi maior que a da cabergolina isolada. Os valores de ACTH plasmático não revelaram diferença significante durante o tratamento e não se correlacionaram com os valores de cortisol. A média de expressão do mRNA do gene DRD2 foi maior no grupo tratado (1,170 ± 0,417) quando comparada ao grupo controle (0,776 ± 0,252) (P = 0,036). Houve dissociação entre os conteúdos de mRNA e da proteína desse receptor. Não foi possível analisar a expressão do mRNA do gene SSTR5, pois o tratamento das amostras com DNAse causou degradação do RNA. A imunorreatividade para SSTR5 esteve presente em todos os pacientes e não foi alterada pelo tratamento prévio. Não houve diferença estatística na expressão do gene SSTR2 entre os grupos controle (1,253 ± 0,511) e tratado (1,267 ± 0,386) bem como diferença significante da imunoexpressão do SSTR2 entre os grupos. Houve correlação entre os conteúdos de mRNA e da proteína desse receptor (P = 0,021). Não houve correlação entre a expressão dos receptores analisados e a resposta ao octreotide e à cabergolina isoladamente ou em associação. Conclusões: a cabergolina isolada representa opção terapêutica na doença de Cushing persistente ou recorrente. A associação de octreotide na dose estudada por 30 dias não foi mais eficiente em reduzir o cortisol urinário. A resposta a essas drogas não está relacionada à expressão dos receptores SSTR2, SSTR5 e DRD2 / Introduction: The Cushings disease presents high morbimortality. Its treatment of choice is transsphenoidal surgery which has satisfactory results in about 70% of cases. In persistent or recurrent disease, a second transsphenoidal surgery, radiotherapy and bilateral adrenalectomy can be carried through, however, these options present disadvantages as development of hypopituitarism and lifelong dependence on hormone replacement therapy. Presently, no drug has shown efficacy in corticotrophinomas treatment. The most efficient agents are the inhibitors of steroidogenesis which have no effect at pituitary tumor. Objectives: To evaluate isolated octreotide and cabergoline effects and their association on plasma ACTH and urinary cortisol in Cushings disease patients, to correlate this effect with tumoral expression of SSTR2, SSTR5 and DRD2 receptors; to correlate tumoral expression of these receptors by quantitative RT-PCR and immunohistochemistry; to evaluate whether these drugs modifies these receptors expression. Patients and methods: control group with 11 patients (10 women and 1 man) between 21 and 43 years who underwent pituitary surgery with no prior treatment and a treated group with 11 patients (2 men and 9 women) between 22 and 53 years that received the following treatment before surgery: : after three baseline urinary cortisol samples and one plasma ACTH sample, patients received octreotide 100 g, subcutaneous 8/8h for 30 days collecting three urinary cortisol samples and one plasma ACTH. After that, cabergoline was introduced 0,5 mg 3x/week for 30 days collecting three urinary cortisol samples and one plasma ACTH sample. Then, octreotide was associated to cabergoline for another 30 days followed by three urinary cortisol and one plasma ACTH sample. Results: Urinary cortisol concentrations significantly decreased after isolated and combined cabergoline use (P = 0,016 and P = 0,012, respectively). Combined treatment efficacy was not greater than isolated cabergoline administration. Plasma ACTH did not change statistically during treatment and did not correlate with urinary cortisol. The average of DRD2 gene expression was higher in control group (0,776 ± 0,252) in relation to treated group (1,170 ± 0,417) (P = 0,036). It had dissociation between mRNA and protein contents of this receptor. SSTR5 gene mRNA expression was not analyzed due to RNA degradation after DNAse tissue treatment. SSTR5 immunoreactivity was present in all patients and it was not modified by previous treatment. No statistic difference was observed between SSTR2 gene expression in control group (1,253 ± 0.511) and in treated group (1,267 ± 0,386). There was no significant difference in SSTR2 immunoexpression between groups. It had correlation between the mRNA and protein contents of this receptor (P = 0.021). No significant relationship was found between hormonal response to isolated and combined therapy and receptors mRNA expression levels. Conclusions: cabergoline represents therapeutical option in persistent or recurrent Cushings disease. Octreotide-cabergoline association in the studied dosage and for the period of 30 days was not more efficient in eliciting urinary cortisol reduction. The responsiveness to these drugs did not correlate to SSTR2 and DRD2 mRNA expression

Introdução: A doença de Cushing apresenta elevada morbimortalidade. Seu tratamento de escolha é a cirurgia transesfenoidal que possui resultados satisfatórios em cerca de 70% dos casos. Na doença persistente ou recorrente, reabordagem cirúrgica, radioterapia e adrenalectomia bilateral podem ser realizadas, porém, essas opções apresentam como desvantagens o desenvolvimento de hipopituitarismo e a dependência de terapia de reposição. Até o momento, nenhuma droga tem se mostrado eficaz no tratamento do corticotrofinoma. Os esquemas terapêuticos mais eficazes são os inibidores da esteroidogênese que não atuam no tumor hipofisário. Objetivos: avaliar o efeito do octreotide e da cabergolina administrados isoladamente e em associação nas concentrações urinárias de cortisol e plasmáticas de ACTH em pacientes com corticotrofinomas; correlacionar esse efeito com a expressão tumoral dos receptores SSTR2, SSTR5 e DRD2; correlacionar a expressão tumoral desses receptores através de RT-PCR quantitativa e imunohistoquímica; avaliar se o uso prévio dessas drogas altera a expressão desses receptores. Casuística e Métodos: grupo controle composto por 11 pacientes (10 mulheres e 1 homem) entre 21 e 43 anos sem tratamento prévio à neurocirurgia e um grupo tratado formado por 11 pacientes (2 homens e 9 mulheres) entre 22 e 53 anos que receberam o seguinte tratamento antes da cirurgia: coleta de três amostras de cortisol urinário e ACTH plasmático, seguida da introdução de octreotide 100 g, subcutâneo, 8/8h durante 30 dias e nova coleta de três amostras de cortisol urinário e ACTH plasmático. Em seguida, iniciou-se a cabergolina 0,5 mg via oral 3 vezes na semana durante 30 dias com nova coleta de três amostras de cortisol urinário e ACTH plasmático. A seguir, o octreotide era associado por mais 30 dias com nova coleta de três amostras de cortisol urinário e ACTH plasmático. Resultados: Os valores de cortisol urinário apresentaram queda significante após o uso de cabergolina isolada (P = 0,016) e em associação ao octreotide (P = 0,012). A eficácia do tratamento combinado não foi maior que a da cabergolina isolada. Os valores de ACTH plasmático não revelaram diferença significante durante o tratamento e não se correlacionaram com os valores de cortisol. A média de expressão do mRNA do gene DRD2 foi maior no grupo tratado (1,170 ± 0,417) quando comparada ao grupo controle (0,776 ± 0,252) (P = 0,036). Houve dissociação entre os conteúdos de mRNA e da proteína desse receptor. Não foi possível analisar a expressão do mRNA do gene SSTR5, pois o tratamento das amostras com DNAse causou degradação do RNA. A imunorreatividade para SSTR5 esteve presente em todos os pacientes e não foi alterada pelo tratamento prévio. Não houve diferença estatística na expressão do gene SSTR2 entre os grupos controle (1,253 ± 0,511) e tratado (1,267 ± 0,386) bem como diferença significante da imunoexpressão do SSTR2 entre os grupos. Houve correlação entre os conteúdos de mRNA e da proteína desse receptor (P = 0,021). Não houve correlação entre a expressão dos receptores analisados e a resposta ao octreotide e à cabergolina isoladamente ou em associação. Conclusões: a cabergolina isolada representa opção terapêutica na doença de Cushing persistente ou recorrente. A associação de octreotide na dose estudada por 30 dias não foi mais eficiente em reduzir o cortisol urinário. A resposta a essas drogas não está relacionada à expressão dos receptores SSTR2, SSTR5 e DRD2 / Introduction: The Cushings disease presents high morbimortality. Its treatment of choice is transsphenoidal surgery which has satisfactory results in about 70% of cases. In persistent or recurrent disease, a second transsphenoidal surgery, radiotherapy and bilateral adrenalectomy can be carried through, however, these options present disadvantages as development of hypopituitarism and lifelong dependence on hormone replacement therapy. Presently, no drug has shown efficacy in corticotrophinomas treatment. The most efficient agents are the inhibitors of steroidogenesis which have no effect at pituitary tumor. Objectives: To evaluate isolated octreotide and cabergoline effects and their association on plasma ACTH and urinary cortisol in Cushings disease patients, to correlate this effect with tumoral expression of SSTR2, SSTR5 and DRD2 receptors; to correlate tumoral expression of these receptors by quantitative RT-PCR and immunohistochemistry; to evaluate whether these drugs modifies these receptors expression. Patients and methods: control group with 11 patients (10 women and 1 man) between 21 and 43 years who underwent pituitary surgery with no prior treatment and a treated group with 11 patients (2 men and 9 women) between 22 and 53 years that received the following treatment before surgery: : after three baseline urinary cortisol samples and one plasma ACTH sample, patients received octreotide 100 g, subcutaneous 8/8h for 30 days collecting three urinary cortisol samples and one plasma ACTH. After that, cabergoline was introduced 0,5 mg 3x/week for 30 days collecting three urinary cortisol samples and one plasma ACTH sample. Then, octreotide was associated to cabergoline for another 30 days followed by three urinary cortisol and one plasma ACTH sample. Results: Urinary cortisol concentrations significantly decreased after isolated and combined cabergoline use (P = 0,016 and P = 0,012, respectively). Combined treatment efficacy was not greater than isolated cabergoline administration. Plasma ACTH did not change statistically during treatment and did not correlate with urinary cortisol. The average of DRD2 gene expression was higher in control group (0,776 ± 0,252) in relation to treated group (1,170 ± 0,417) (P = 0,036). It had dissociation between mRNA and protein contents of this receptor. SSTR5 gene mRNA expression was not analyzed due to RNA degradation after DNAse tissue treatment. SSTR5 immunoreactivity was present in all patients and it was not modified by previous treatment. No statistic difference was observed between SSTR2 gene expression in control group (1,253 ± 0.511) and in treated group (1,267 ± 0,386). There was no significant difference in SSTR2 immunoexpression between groups. It had correlation between the mRNA and protein contents of this receptor (P = 0.021). No significant relationship was found between hormonal response to isolated and combined therapy and receptors mRNA expression levels. Conclusions: cabergoline represents therapeutical option in persistent or recurrent Cushings disease. Octreotide-cabergoline association in the studied dosage and for the period of 30 days was not more efficient in eliciting urinary cortisol reduction. The responsiveness to these drugs did not correlate to SSTR2 and DRD2 mRNA expression

<p>Type 1 diabetes is resulting from the selective destruction of insulin-producing beta-cells within the pancreatic islets. Somatostatin acts as an inhibitor of hormone secretion through specific receptors (sst1-5).</p><p>All ssts were expressed in normal rat and mouse pancreatic islets, although the expression intensity and the co-expression pattern varied between ssts as well as between species. This may reflect a difference in response to somatostatin in islet cells of the two species.</p><p>The Non-Obese Diabetic (NOD) mouse model is an experimental model of type 1 diabetes, with insulitis accompanied by spontaneous hyperglycaemia. Pancreatic specimens from NOD mice at different age and stage of disease were stained for ssts. The islet cells of diabetic NOD mice showed increased islet expression of sst2-5 compared to normoglycemic NOD mice. The increase in sst2-5 expression in the islets cells may suggest either a contributing factor in the process leading to diabetes, or a defense response against ongoing beta-cell destruction.</p><p>Somatostatin analogues were tested on a human endocrine pancreatic tumour cell line and cultured pancreatic islets. Somatostatin analogues had an effect on cAMP accumulation, chromogranin A secretion and MAP kinase activity in the cell line. Treatment of rat pancreatic islets with somatostatin analogues with selective receptor affinity was not sufficient to induce an inhibition of insulin and glucagon secretion. However, a combination of selective analogues or non-selective analogues via co-stimulation of receptors can cause inhibition of hormone production. For insulin and glucagon, combinations of sst2 + sst5 and sst1 + sst2, respectively, showed a biological effect.</p><p>In summary, knowledge of islet cell ssts expression and the effect of somatostatin analogues with high affinity to ssts may be valuable in the future attempts to influence beta-cell function in type 1 diabetes mellitus, since down-regulation of beta-cell function may promote survival of these cells during the autoimmune attack. </p>

Type 1 diabetes is resulting from the selective destruction of insulin-producing beta-cells within the pancreatic islets. Somatostatin acts as an inhibitor of hormone secretion through specific receptors (sst1-5). All ssts were expressed in normal rat and mouse pancreatic islets, although the expression intensity and the co-expression pattern varied between ssts as well as between species. This may reflect a difference in response to somatostatin in islet cells of the two species. The Non-Obese Diabetic (NOD) mouse model is an experimental model of type 1 diabetes, with insulitis accompanied by spontaneous hyperglycaemia. Pancreatic specimens from NOD mice at different age and stage of disease were stained for ssts. The islet cells of diabetic NOD mice showed increased islet expression of sst2-5 compared to normoglycemic NOD mice. The increase in sst2-5 expression in the islets cells may suggest either a contributing factor in the process leading to diabetes, or a defense response against ongoing beta-cell destruction. Somatostatin analogues were tested on a human endocrine pancreatic tumour cell line and cultured pancreatic islets. Somatostatin analogues had an effect on cAMP accumulation, chromogranin A secretion and MAP kinase activity in the cell line. Treatment of rat pancreatic islets with somatostatin analogues with selective receptor affinity was not sufficient to induce an inhibition of insulin and glucagon secretion. However, a combination of selective analogues or non-selective analogues via co-stimulation of receptors can cause inhibition of hormone production. For insulin and glucagon, combinations of sst2 + sst5 and sst1 + sst2, respectively, showed a biological effect. In summary, knowledge of islet cell ssts expression and the effect of somatostatin analogues with high affinity to ssts may be valuable in the future attempts to influence beta-cell function in type 1 diabetes mellitus, since down-regulation of beta-cell function may promote survival of these cells during the autoimmune attack.

Neuroendocrine tumors (NETs), originally described as carcinoids, represent a rare and heterogeneous group of neoplasms associated with intensive secretion of hormones, bioactive peptides and amines. Most of the patients are diagnosed at a late stage of disease, often with liver metastases. Surgery remains the main treatment to control metastatic disease, but is not curative. Oncolytic virotherapy represents a promising approach to treat cancer and different strategies have been exploited to restrict viral replication to tumor cells. We developed an oncolytic adenovirus based on serotype 5, Ad5[CgA-E1A], where the chromogranin A (CgA) promoter controls expression of the E1A gene and thereby virus replication. We found that Ad5[CgA-E1A], selectively replicates in NET cells and it is able to suppress fast-growing human BON carcinoid tumors in nude mice. The activity of Ad5[CgA-E1A] was not completely blocked in liver cells. We further repressed virus replication in hepatocytes by targeting E1A with miR122, an miRNA specifically expressed in the liver. miRNAs bind to mRNA and induce its cleavage or translational blockage. By insertion of tandem repeats of miR122 target sequences in 3’UTR of E1A gene, we observed reduced E1A protein expression and replication arrest in miR122 expressing liver cells. The oncolytic potency of the miR122-targeted virus was not affected in NET cells. Since some NET and neuroblastoma cells express high levels of somatostatin receptors (SSTRs), we introduced in the virus fiber knob cyclic peptides, which contain four amino acids (FWKT) and mimic the binding site of somatostatin for SSTRs. The FWKT-modified Ad5 transduces midgut carcinoid cells from liver metastases about 3-4 times better than non-modified Ad5. Moreover, FWKT-modified Ad5 overcomes neutralization in an ex vivo human blood loop model to a greater extent than Ad5, indicating that the fiber knob modification may prolong the systemic circulation time. NETs represent a huge therapeutic challenge and novel diagnostic markers are needed for early detection and effective treatment of NETs. We have profiled primary tumors and liver metastases of ileocaceal NETs, using Affymetrix microarrays and advanced bioinformatics. We have identified six novel marker genes and show high similarity between primary lesions and liver metastases transcriptome by hierarchical clustering analysis.

Le carcinome de Merkel est un cancer cutané de différenciation neuroendocrine rare, mais agressif, dont le facteur étiologique principal est le polyomavirus de Merkel (MCPyV). L’objectif de ce travail a été d’identifier des marqueurs virologiques et cellulaires pronostiques ou théranostiques à l’aide d’une cohorte historicoprospective de patients ayant un carcinome de Merkel. Les patients ayant des titres élevés d’anticorps dirigés contre la protéine de capside VP1 du MCPyV ont un pronostic favorable, tandis les anticorps dirigés contre les oncoprotéines virales reflètent l’évolution tumorale. Par ailleurs, il existe une hétérogénéité d’expression des récepteurs à la somatostatine dans les carcinomes de Merkel. Ce marqueur cellulaire peut constituer un outil théranostique lors de thérapies ciblées utilisant les analogues de la somatostatine. Enfin, nos travaux actuels portent sur l’évaluation de l’immunité cellulaire chez ces patients, avec une étude ayant montré la valeur pronostique du ratio neutrophiles/lymphocytes sanguin. / Merkel Cell Carcinoma is a rare and aggressive neuroendocrine skin cancer. The Merkel cell polymavirus has been identified as the main etiological agent of such cancer. We aimed to identify viral and cellular markers relevant as prognostic and theranostic tools in patients with Merkel Cell Carcinoma. Using serological immunoassays, we observed that patients with high levels of serum antibodies against the MCPyV major capsid protein at baseline had better outcomes, whereas antibodies directed against the MCPyV oncoproteins reflected the tumor burden and course of disease. We also demonstrated that Merkel Cell Carcinoma tumors displayed a heterogeneous expression of receptors to somatostatin, which could constitute a theranostic tool for the use of targeted therapies using somatostatin analogs. Finally, current studies focus on the assessment of cellular immunity in Merkel Cell Carcinoma patients. Our results indicate that the blood neutrophil-to-lymphocyte ratio is an independent marker of mortality in Merkel Cell Carcinoma patients.

Os tumores carcinóides broncopulmonares típicos (CT) são proliferações de células neuroendócrinas. Foram consideradas como adenomas e acreditava-se que não tinham potencial para disseminação hematogênica e linfática. Porém, a ocorrência de metástase linfática e hematogênica acontece em um quinto dos indivíduos acometidos por essa patologia. A variação no comportamento clínico dos carcinóides broncopulmonares torna imperativa a realização de pesquisas que visem à melhor compreensão dessa doença. É fundamental determinar a agressividade e o risco individual da ocorrência de metástase linfática e hematogênica para que se possa oferecer um tratamento individualizado para cada binômio doente-doença. A classificação atual divide os tumores carcinóides, conforme o grau histológico de malignidade em típico e atípico, agrupando as neoplasias de acordo com o índice mitótico, relação volumétrica núcleo/citoplasma, presença ou ausência de necrose, pleomorfismo nuclear e invasão vascular. Receptores celulares na superfície externa da membrana plasmática podem ser ubiquamente expressos em diversos tipos celulares ou específicos para determinada população celular. Os receptores de somatostatina são específicos de células neuroendócrinas e também são expressos nas neoplasias desta natureza. Existem 5 tipos de receptores de somatostatina (SSTR). A interação da somatostatina (SST) com seu receptor específico provoca as inibições do ciclo celular e da angiogêsese, bem como estimula a apoptose. A meia-vida plasmática da SST é breve. Análogos com menor metabolização eram necessários. Foram desenvolvidos os análogos como octreotide e lanreotide. Estes análogos foram acoplados à radionuclídeos, possibilitando aplicação em diagnóstico, estadiamento e tratamento dos tumores neuroendócrinos. O SSTR do tipo 2 possui maior afinidade pela somatostatina. A expressão imunohistoquímica de SSTR-2 em carcinóide típicos ofereceria métodos adicionais de diagnóstico e tratamento para esta doença. Com o objetivo de demonstrar a expressão de SSTR-2 em carcinóides broncopulmonares típicos, bem como verificar se existia relação entre a expressão e ocorrência de metástase linfática 62 pacientes tiveram suas amostras de tumor submetidas ao método imunohistoquímico. Verificou-se, ademais, se a expressão de SSTR-2 e o índice de expressão imunohistoquímica eram variáveis independentes preditivas do risco de metástase linfática. A relação entre expressão de SSTR-2 e variáveis clínicas pré-operatórias também foi analisada. 36 pacientes tinham tumores que expressavam SSTR-2 (58,1%), enquanto 26 doentes tinham tumores que não expressavam SSTR-2 (41,9%). Não existiu diferença estatística significante entre a expressão de SSTR-2 e a ocorrência de metástase linfática (teste exato de Fisher, p=0,529). Também não existiram diferenças estatísticas significantes nas análises multivariadas que testaram se tanto o SSTR-2 quanto o índice de imunohistoquímica eram variáveis independentes preditivas do risco de metástase linfática. Neste estudo, os CT expressaram SSTR-2. Além disso, não existiu relação entre a ocorrência de metástase linfática e a expressão de SSTR-2. Por fim, o SSTR-2 e o índice imunohistoquímico não foram variáveis independentes do risco de metástase linfática / Typical pulmonary carcinoids are neuroendocrine cells proliferations and they were former considered lung adenomas with no hematogenic or lymphatic metastatic potential. However, it is known that up to 20% of patients develop metastatic disease. It is mandatory that new studies be developed due to the variation in clinical presentation of these patients. It is also required that the individual risk of lymphatic and hematogenic metastasis be determined in order to individualize the patients treatment. Pulmonary carcinoids are classified according to hystologic grade. The current classification includes hystologic grade, presence or absence of necrosis, nuclear pleomorphism, and vascular invasion. Somatostatin receptors (SSTR) are neuroendocrine cell specific receptors and can be detected in neuroendocrine tumors as well. There are 5 SSTRs subtypes. Somatostatin is a peptide that inhibits the cell cycle and angiogenesis as well as increases the apoptosis by binding to SSTR. The use of long-acting form of octreotide (a SST analogue) has been associated with treatment (radiolabeled somatostatin analogs) and diagnosis (OctreoScan®). Encouraging results have been obtained with the use of radiolabeled somatostain analogs yttrium-90 and Lu-177 to treat patients with neuroendocrine tumors. This study was designed to evaluate if typical bronchopulmonary carcinoid expressed Somatostatin receptor type 2 using the the immunohistochemical technique to identify Somatostatin receptor type 2. This study verified if there was relation between Somatostatin receptor type 2 expression and lymphatic metastasis. Futhermore, we verified if Somatostatin receptor type 2 and imunnohistochemistry score would be independent preditive markers to lymphatic metastasis. 62 patients were evaluated. 36 (58,1%) patients expressed Somatostatin receptor type 2 in their tumor samples whereas 26 (41,9%) patients did not express Somatostatin receptor type 2. This study did not verify significant statistical difference between SSTR-2 expression and lymphatic metastasis. Somatostatin receptor type 2 and imunnohistochemistry score were not independent preditive markers to lymphatic metastasis. There were no significant statistical differences on multivariate analyses. In conclusion this study verified that there was Somatostatin receptor type 2 expression on tumor samples studied but there was no relation between Somatostatin receptor type 2 and lymphatic metastasis. Futhermore, Somatostatin receptor type 2 and its imunnohistochemistry score were not independent preditive markers to lymphatic metastasis

La tomographie par émission de positons (TEP) est une technique d’imagerie moléculaire avec de meilleures performances que la tomographie par émission monophotonique. Son utilisation contribue à l’amélioration des prises en charge des patients. Dans les centres dépourvus de cyclotrons, le 68Ga disponible à partir d’un générateur constitue une alternative pour le développement de traceurs TEP. Pour pouvoir développer des 68Ga-traceurs, un travail de caractérisation de la qualité des éluats a été effectué. Des méthodes de marquage adaptées ont été mises en place et validées. Nous nous sommes intéressés à trois cibles moléculaires particulièrement intéressantes dans les pathologies cardiovasculaires: les récepteurs de la somatostatine (SSTR) surexprimés dans les tumeurs neuroendocrines (TNE) mais constituant aussi une cible d’intérêt dans les pathologies cardiovasculaires à composante inflammatoire ; la phosphatidylsérine (PS), un marqueur de l’apoptose cellulaire et de l’activation plaquettaire ; la P-sélectine, un marqueur des activations plaquettaire et endothéliale. Les traceurs suivants ont été développés: 1) Analogues de la somatostatine ciblant les SSTR: a)68Ga-DOTANOC validé pour l’imagerie des TNE-Gastroentéropancréatiques dans le cadre d’un essai clinique multicentrique. b) 68Ga-NODAGANOC testé in vitro sur des cellules d’adénocarcinome pancréatique. Cette validation initiale dans l’application la plus fréquente(oncologie) a pour objectif de faciliter le passage vers des applications cardiovasculaires futures (athérosclérose, myocardite...) ; 2) Un peptide ciblant la PS : le 68Ga-P04087 ; 3) Un polysaccharide ciblant la P-sélectine: 68Ga-NODAGA-Asphy. Les deux derniers traceurs ont été testés sur un modèle d’endocardite infectieuse chez le rat. / The Positron emission tomography (PET) is a molecular imaging technique with usually better performances than Single-Photon Emission Computed Tomography. Consequently, the use of PET and appropriate tracers could enable clinicians to make a better therapeutic decision, thus improving the management of patients. In centers without cyclotrons, 68Ga available from a generator is an alternative for the development of PET tracers. In order to develop 68Ga labeled-molecules, a characterization of the quality of the eluates was performed. Radiolabeling techniques adapted to the quality of the starting material were developed and validated. In this thesis we focused on three particularly interesting molecular targets in cardiovascular pathologies: somatostatin receptors (SSTR), overexpressed in neuroendocrine tumors (NETs) but also constituting a target of interest in cardiovascular diseases with an inflammatory component; phosphatidylserine (PS), a marker of cell apoptosis and platelet activation; P-selectin, a marker of platelet and endothelial activation.The following tracers have been developed: 1) Somatostatin analogues which target SSTR: a) 68Ga-DOTANOC validated for Gastroenteropancreatic-NETs imaging and used in a multicenter clinical trial. b) 68Ga-NODAGANOC tested in vitro on pancreatic adenocarcinoma cells. This initial validation in the most common application (oncology) aims to facilitate the transition to future cardiovascular applications (atherosclerosis, myocarditis ...) 2) A peptide for PS targeting: 68Ga-P04087; 3) A polysaccharide for P-selectin targeting: 68Ga-NODAGA-Asphy. The last two radiolabeled molecules were tested in a rat model of infective endocarditis.