Background: The JAK-STAT pathway is a cellular signaling pathway, which acts normally in humans and animals in the control of multiple important functions. Dysregulation of this pathway has been identified in human cancers, as well as a limited number of veterinary cancers. Objectives: The aims of this study were to identify the presence and tentative activity of components of the JAK-STAT pathway in selected canine tumors. Methods: Formalin-fixed, paraffin-embedded samples from mast cell tumors (MCT), hemangiosarcomas (HSA), thyroid carcinomas, and apocrine gland anal sac adenocarcinomas (AGASACA) were obtained from the Diagnostic Histopathology Laboratory at the Virginia Maryland College of Veterinary Medicine. Immunohistochemistry was performed to evaluate protein levels of JAK1, phospho-JAK1, JAK2, phospho-JAK2, STAT3, and phospho-STAT3. Signalment, treatment information, and survival information was obtained from the medical record for each case. Results: Tumor samples were scored for percent positive neoplastic cells. Positive staining was seen for all antibodies in all tumor types, with expression of JAK1, STAT3, and pSTAT3 being highest overall for all tumor types. Significant associations were seen between JAK1 and survival time in MCT (p = 0.03), pJAK1 and survival time in HSA (p = 0.009) and MCT (p = 0.04), and pSTAT3 and metastasis in MCT (p = 0.0008). Conclusions: The finding of positive staining for the components of the JAK-STAT pathway in the tumor samples evaluated indicates presence and tentative activity of this pathway in the studied cancers. Further study of JAK1, pJAK1, and pSTAT3 should be pursued to evaluate their potential as therapeutic targets. / MS / The JAK-STAT pathway is a cellular signaling pathway which acts in humans and animals to control functions, such as development of the immune system, and development of the mammary glands during pregnancy. This pathway can become dysregulated, and contribute to development of cancer in both humans and animals. Development of cancer drugs that can target this pathway when dysregulated may aid in controlling further growth of spread of cancer, and may help to prolong survival in patients affected. In our study, four different cancer types were investigated in dogs for the presence and activity of components of the JAK-STAT pathway. Evidence of presence and activity was identified in the cancers evaluated, suggesting that more work should be done to determine if the JAK-STAT pathway is activated in other canine tumor types, and whether the pathway can be targeted as a cancer treatment.

Genetic causes of medullary thyroid carcinoma and Hirschsprung's disease Abstract Medullary thyroid carcinoma (MTC) and Hirschsprung's disease (HSCR) are classified as simple neurocristopathies, i.e. diseases linked to neural crest-derived cells. MTC is derived from parafollicular cells of the thyroid and HSCR is characterized by absence of enteric ganglia in the gastrointestinal tract. The RET proto-oncogene is only expressed in neural crest-derived cells, including parafollicular cells and enteric neurons. The RET encodes a transmembrane tyrosinekinase receptor that plays an important role during proliferation, differentiation and cell survival, and activates many signaling pathways. If the strictly regulated activation fails, e.g. due to mutations in the specific gene locations, the RET becomes a highly effective oncogene. Activating germline mutations in the RET proto- oncogene lead to hereditary forms of MTC, whereas sporadic forms of MTC are caused by somatic mutations in the tumor tissue. On the contrary, inactivating mutations induce migration failure of ganglion cell precursors during the development of enteric nervous system and result in the development of HSCR. In rare cases, the coexistence of both diseases is caused by mutations with a dual gain-of-function and loss-of-function character....

nÃo hà / INTRODUÃÃO: Estudos epidemiolÃgicos sugerem que o carcinoma de tireoide seja a neoplasia maligna mais frequente nos pacientes acromegÃlicos. Atà este momento nÃo hà relatos de estudos avaliando marcadores moleculares do carcinoma papilÃfero de tireoide (CPT) nessa populaÃÃo. OBJETIVOS: Avaliar a associaÃÃo entre acromegalia, presenÃa da mutaÃÃo BRAFV600E, marcadores imunohistoquÃmicos (galectina-3 e IGF-1) e caracterÃsticas clÃnico-patolÃgicas em pacientes acromegÃlicos com CPT. MATERIAL E MÃTODOS: Trata-se de um estudo transversal realizado no perÃodo de janeiro/09 a dezembro/2011, onde 11 pacientes acromegÃlicos com CPT, provenientes de 5 centros brasileiros de referÃncia no tratamento da acromegalia foram comparados com 45 pacientes com CPT sem acromegalia. Foram estudadas variÃveis clÃnicas e histopatolÃgicas do CPT. Utilizou-se cortes histolÃgicos de CPT emblocados em parafina para o estudo da mutaÃÃo BRAFV600E e para a anÃlise imunohistoquÃmica dos marcadores IGF-1 e galectina-3. Na anÃlise utilizou-se os testes t de student e do qui-quadrado (software SPSS, versÃo 13.0 para Windows) (p<0,05). RESULTADOS: A idade mÃdia dos pacientes acromegÃlicos com CPT foi de 61,5  6,02 anos, sendo 72,7% do sexo feminino. O tempo mÃdio de diagnÃstico da acromegalia foi de 7,7  3,90 anos, sendo o intervalo entre o diagnÃstico da acromegalia e do CPT, em mÃdia, 3,4  2,71 anos. Os nÃveis sÃricos de IGF-1 dos acromegÃlicos ao diagnÃstico do CPT foi de 417,0 ng/mL. NÃo houve diferenÃa quanto ao estadiamento TNM (Tumor, Nodule, Metastasis) e Ãndice prognÃstico AMES (Ages, Metastasis, Extent, Size) entre os grupos. Houve maior prevalÃncia da mutaÃÃo BRAFV600E (90,9% vs 55,6%; p=0,039) e de forte imunoexpressÃo para IGF-1 (88,9% vs 38,1%; p= 0,017) nos acromegÃlicos. NÃo houve diferenÃa na expressÃo de galectina-3 entre os grupos. CONCLUSÃO: Neste trabalho, pela primeira vez se mostrou uma alta prevalÃncia da mutaÃÃo BRAFV600E em CPT de acromegÃlicos, muito superior à descrita na populaÃÃo com CPT neste e em estudos anteriores (cerca de 40%). Contudo essa mutaÃÃo nÃo se mostrou associada a um fenÃtipo mais agressivo do tumor, o que diverge dos achados em populaÃÃo nÃo acromegÃlica com CPT. Conclui-se que a acromegalia à possivelmente associada à mutaÃÃo BRAFV600E em pacientes acromegÃlicos com CPT. Novos estudos serÃo necessÃrios para definir os mecanismos responsÃveis por tal associaÃÃo. / INTRODUCTION: Epidemiological studies suggest that thyroid carcinoma is the most common malignant neoplasm in acromegalic patients. At this moment there are no reports of studies evaluating molecular markers of papillary thyroid carcinoma (PTC) in this population. OBJECTIVES: The present work aimed to evaluate the association between acromegaly, expression of the mutation BRAFV600E, immunohistochemical markers (galectin-3 and IGF-1), and clinical-pathological characteristics in acromegalic patients with PTC. MATERIALS AND METHODS: This is a cross-sectional study conducted from January/09 to December/2011, where 11 acromegalic patients with CPT, from 5 Brazilian centers of reference in the treatment of acromegaly were compared with 45 patients with acromegaly without PTC. We evaluated clinical and histopathological variables of PTC. We used histological PTC embedded in paraffin for mutation study BRAFV600E and immunohistochemical analysis of markers IGF-1 and galectin-3. In the analysis we used the Student t test and chi-square test (SPSS software, version 13.0 for Windows) (p <0.05). RESULTS: The average age of acromegalic patients with PTC was 61.5  6.02 years and 72.7% were female. The average time of diagnosis of acromegaly was 7.7  3.90 years, and the interval between diagnosis of acromegaly and PTC was an average 3.4  2.71 years. The serum levels of IGF-1 in the diagnosis of acromegaly PTC was 417.0 ng / mL. There was no difference in the TNM (Tumor, Nodule, Metastasis) and AMES prognostic index (Ages, Metastasis, Extent, Size) between groups. There was a higher prevalence of the BRAFV600E mutation (90.9% vs 55.6%, p = 0.039) and stronger immunohistochemical expression for IGF-1 (88.9% vs 38.1%, p = 0.017) in acromegaly. There was no difference in the expression of galectin-3 between the groups. CONCLUSION: This work for the first time showed a high prevalence of mutations in BRAFV600E in PTC of acromegalic patients superior to those described in the population with PTC in this and previous studies (approximately 40%). However, this mutation was not associated with a more aggressive tumor phenotype, which differs from the findings in acromegalic population without PTC. We conclude that acromegaly is possibly associated to a mutation BRAFV600E in acromegalic patients with CPT. Further studies are needed to define the mechanisms responsible for this association.

Erhöhte Inzidenzen eines Schilddrüsenkarzinoms bei Hashimoto-Thyreoiditis (HT) wurden 1955 erstmals publiziert. Ähnliche Arbeiten liegen für Morbus Basedow (MB) vor. Die Beobachtungen sind weiter Gegenstand kontroverser Diskussionen. Die Ätiopathogenese ist ungeklärt. Aus einem Zeitraum von sieben Jahren wurden 112 Patienten mit MB und 77 Patienten mit HT als histologische Diagnose identifiziert und retrospektiv untersucht. Die Karzinominzidenz bei HT (16,9%) und MB (7,1%) ist gegenüber einer normalen Struma multinodosa deutlich erhöht. Multifokale Befunde fanden sich bei HT häufiger (38,5% vs 0%; p<0,05). Patienten mit MB wurden häufiger primär total thyreoidektomiert als Patienten mit HT (72,3% vs 42,9%; p<0,05). Dementsprechend waren bei Patienten mit HT mehr zweizeitige Verfahren notwendig als bei MB (11,7% vs 3,6%; p<0,05). Präoperativ wurde kein Karzinom definitiv gesichert. Eine präoperative richtig-positive Diagnose der Autoimmunerkrankung lag bei MB häufiger vor als bei HT (97,3% vs 68,6%; p<0,05). Eine Optimierung der Diagnostik, z.B. durch gezielten Einsatz der Feinnadelpunktion bei suspekten Knotenbefunden ist zu diskutieren. Weiterführende Untersuchungen zu Tumormarkern, u.a. im Gewebe, werden in Zukunft vermehrt im Fokus stehen.

Small cell variant of medullary thyroid carcinoma is an extremely rare histologic entity with a paucity of data. As such, there is a lack of literature and clinical experience regarding this disease. In this report, we examine a case of small cell variant of medullary thyroid carcinoma that presented with intractable nausea, vomiting and diarrhea. While these symptoms were essentially refractory to the standard symptomatic treatment, further laboratory analysis revealed dramatically elevated calcitonin levels and mildly raised thyroid-stimulating hormone levels. Interestingly, repletion of thyroid hormone and treatment with lanreotide resulted in an abatement of our patient's symptoms. This temporal clinical improvement highly suggests a potential role involving thyroid-stimulating hormone and calcitonin levels in the pathogenesis of this disease, and consequently suggests a role for thyroxine in treating the associated gastrointestinal symptoms.

Le cancer papillaire de la thyroïde (PTC) représente 70-80% des cas de cancers de la thyroïde. Il est principalement caractérisé par des réarrangements chromosomiques affectant le gène RET. Le réarrangement RET/PTC1, dans lequel RET est réarrangé avec un gène proapoptotique H4, représente 30% des cas sporadiques et jusqu’à 60% des cas survenus après irradiation. Afin d’inhiber l’oncogène de fusion RET/PTC1, nous avons utilisé un siRNA ciblant la zone de jonction RET/PTC1 (siRNA RET/PTC1) au niveau de l’ARN messager des cellules tumorales et montré sa spécificité et son efficacité. Néanmoins, le développement des siRNAs comme molécule d’intérêt thérapeutique se heurte in vivo à des difficultés liées à leur administration. Sous forme libre, ces molécules sont, en effet, très vite dégradées par les nucléases extracellulaires et leur pénétration intracellulaire est limitée. C’est la raison pour laquelle il est nécessaire de les vectoriser. Nous avons choisi de le faire par la méthode de « squalénisation » et avons couplé d’une manière covalente le squalène, un lipide naturel précurseur de la biosynthèse du cholestérol, au siRNA RET/PTC1. Le bioconjugué formé s’autoassemble spontanément en milieu aqueux sous forme de nanoparticules stables de 170 nm de diamètre. L’efficacité et la toxicité des nanoparticules siRNA RET/PTC1-squalène ont été étudiées in vitro dans deux lignées de PTC exprimant RET/PTC1 (BHP10-3 et TPC-1) et l’activité antitumorale a été évaluée in vivo sur des souris athymiques xénogreffées par BHP10-3 puis traitées en i.v. par ces nanoparticules. Les nanoparticules siRNA RET/PTC1-squalène ont montré une bonne efficacité antitumorale. En revanche, aucune activité inhibitrice n’a été retrouvée in vitro. En conclusion, nous avons réussi à vectoriser le siRNA RET/PTC1 par la méthode de squalénisation. Cette étude ouvre des perspectives thérapeutiques pour certains patients atteints de PTC et réfractaires au traitement conventionnel. / The papillary thyroid carcinoma (PTC) is the most common type of thyroid malignancy. This tumour is associated with somatic mutations of the RET proto-oncogene, due to gene rearrangements of the proto-RET. RET/PTC1 rearrangement is the most common genetic alteration identified to date, it is formed by an intra chromosomic rearrangement which leads to the juxtaposition of the RET Tyrosine Kinase domain of the proto-RET with the gene H4. The fusion RET/PTC1 oncogene represents an interesting target for small interfering RNA (siRNA) strategies since it is present only in the tumour cells and not in the surrounding normal cells. However, the biological efficacy of the siRNAs is hampered by their short plasma half-life due to poor stability in biological fluids and low intracellular penetration. In order to protect siRNA from degradation, and to improve their intracellular capture, we applied the concept of “squalenoylation”, ie. The bioconjugation of a drug substance to squalene, for the delivery of siRNA targeted toward the RET/PTC1 fusion oncogene. The acyclic isoprenoid chain of squalene was covalently coupled with RET/PTC1 siRNA at the 3’-terminus of the sense strand via a stable thioether linkage. The linkage of RET/PTC1 siRNA to squalene leads to an amphiphilic molecule that self-organise in water as RET/PTC1 siRNA-SQ nanoassemblies of 170 nm and Zeta potential of -26.4 mV. These RET/PTC1 siRNA-SQ NPs did not showed any cytotoxicity in vitro. Interestingly, in vivo, in a mouse xenografted RET/PTC1 experimental model, RET/PTC1 siRNA-SQ nanoparticles inhibited tumour growth, RET/PTC1 oncogene and oncoprotein expression, after intravenous injections of 2.5 mg/kg cumulative dose. In the last of this work, GALA-cholesterol combination with siRNA-SQ NPs further enhanced nucleic acid internalization, promoted their escape into the cytosol and consequently their gene silencing efficiency in vitro. In conclusion, these results showed that the “squalenoylation” offers a new non cationic plate-form for the siRNA delivery.

Le cancer médullaire de la thyroïde (CMT), qui représente 5 à 8% des cancers de la thyroïde, est issu de la transformation maligne des cellules C du parenchyme thyroïdien. Ce cancer, sporadique dans 70 à 80% des cas et familial pour les 20 à 30% restants, est essentiellement lié à des anomalies du proto-oncogène RET, codant un récepteur à activité tyrosine kinase. La fréquence élevée des mutations activatrices de RET ont permis d’identifier ce récepteur comme une cible thérapeutique majeure. Si la chirurgie est le traitement de référence pour les formes localisées, les formes localement avancées ou métastatiques, de pronostic plus péjoratif étaient avant le développement des thérapies moléculaires ciblées, dans une impasse thérapeutique. La meilleure compréhension de la biologie des tumeurs a permis le développement de ces thérapies plus rationnelles et plus spécifiques, en particulier des inhibiteurs d’activité tyrosine kinase (ITK). L’optimisation de leur utilisation en clinique nécessite de mieux comprendre leurs mécanismes d’action.Dans ce contexte, les objectifs de cette thèse ont été à la fois cognitifs et cliniques, visant à améliorer la compréhension de la réponse moléculaire à deux ITKs, le sunitinib et la vandetanib,dans le CMT, et à identifier de nouveaux biomarqueurs de suivi thérapeutique. Dans un premier temps, nous avons montré les effets antiprolifératifs, antitumoraux et antiangiogéniques du sunitinib et du vandetanib dans un modèle de CMT muté RETC634W, mettant en évidence des profils d’activité proches entre les deux inhibiteurs. Puis, les principales voies de signalisation mises en jeu lors de la réponse à ces ITKs ont été explorées par Reverse-Phase Protein Array(RPPA). Par une approche transcriptomique haut-débit menée sur des modèles précliniques, les principales fonctions cellulaires impliquées dans la réponse au sunitinib et au vandetanib ont été identifiées. Le rôle de gènes participant à l’invasion tissulaire et au pouvoir métastatique a été mis en évidence. De nouveaux biomarqueurs potentiels de réponse au vandetanib et au sunitinib, tels que l’IL-8 et le TGF-2 dont les taux sériques sont significativement plus élevés chez les patients atteints de CMT, ont été identifiés. Enfin, l’intérêt de trois approches méthodologiques dans lesuivi de la réponse antitumorale chez les patients a été évalué. Ainsi, le développement d’une méthode de dosage du vandetanib par spectrométrie de masse a permis de suggérer un lien entre des taux sériques élevés et l’apparition de toxicités sévères. L’évaluation de biomarqueurs dans le sérum de patients traités par le vandetanib a souligné l’intérêt de l’IL-8 comme marqueur pronostic potentiel dans cette pathologie. Enfin les résultats préliminaires, évaluant la réponse au sunitinib par échographie doppler sur un modèle préclinique de souris xénogreffées, ont confirmé l’intérêt de l’imagerie fonctionnelle dans ce domaine. / Medullary thyroid carcinoma (MTC) accounts for 5-8% of all thyroid cancers and occurs as either a sporadic form or in a familial context (25% of cases). Mutations which activate the RET proto-oncogene, encoding a tyrosine kinase receptor, are responsible for familial forms and are also detected in one-third of sporadic tumors. MTC patients with local disease may be cured after initial surgery, but persistent or recurrent disease occurs in half of cases, and distant metastases are the major cause of tumor related death. Up to now there is no effective systemic treatment and new therapeutic strategies are needed for locally advanced or metastatic MTC patients. The constitutive activation of RET is crucial in MTC pathogenesis and led to the development of small compounds targeting its tyrosine kinase activity (TKI). Gaining an understanding of how cancer cells respond to drugs is challenging to improve clinical use of these new therapeutic agents. In this context, we aimed to characterize molecular mechanisms of action of sunitinib and vandetanib, two TKIs currently evaluated in MTC patients. Our results, in in vitro as well as in vivo MTC models based on the RETC634W TT cell line, demonstrate that sunitinib and vandetanib has similar antiproliferative, antitumoral and antiangiogenic properties. Using the Reverse Phase Protein Array (RPPA) large-scale technology, we identified major signalling pathways inhibited after TKIs’ treatment. Expression microarrays allowed us to investigate signaling pathways modified after sunitinib and vandetanib treatment and to show that TKIs’ treatment induced major changes in the expression of genes involved in tissue invasion and metastasis. We identified encoding secreted proteins as candidate soluble biomarkers of response and, among them, we demonstrated that metastatic MTC patients presented increased serum levels of IL-8 and TGF-2. Three modalities for determining early responses to targeted agents in MTC patients were evaluated. First, a sensitive mass spectrometry assay was developed for the quantitation of vandetanib, and applied in MTC patients, showing a potential relationship between toxic side-effects and vandetanib serum levels and suggesting that therapeutic drug monitoring may be a useful tool for MTC patients’ follow-up. Then, candidate biomarkers were investigated in MTC patients underlying the potential use of IL-8 as prognostic marker. Finally, using doppler imaging in xenografted mice model, we confirmed the utility of imaging techniques in clinical evaluation of TKI’s response.

Os nódulos de tireoide são frequentemente encontrados na prática clínica e, com o auxílio de ultrassonografia de alta resolução, podem ser identificados em 17 a 67% da população. A alta prevalência desses nódulos causa preocupação frequente aos pacientes e aos clínicos devido ao risco de malignidade, o que, por sua vez, leva a investigações laboratoriais de alto custo, invasivas e, eventualmente, a cirurgias desnecessárias. A punção aspirativa com agulha fina (PAAF) é o método diagnóstico pré-operatório mais preciso para identificação de um nódulo maligno de tireoide, mas não consegue excluir malignidade nos casos de citologia inadequada, nódulos com diagnóstico citológico de lesão folicular ou atipia de significado indeterminado e nas citologias sugestivas de neoplasia folicular. O objetivo deste estudo foi analisar as características clínicas, laboratoriais, ultrassonográficas e citológicas de uma população de pacientes com nódulos de tireoide submetidos a tireoidectomia e a relação entre estes achados e o risco de malignidade. Além disto, em relação aos nódulos malignos, verificar se o valor de TSH esteve associado a um estadiamento mais avançado da doença. Foram avaliados prontuários de 353 pacientes submetidos a tireoidectomia, acompanhados no Hospital das Clínicas da FMUSP de São Paulo, no período de fevereiro de 2002 a abril de 2010. O número total de nódulos nestes pacientes foi 392. As características clínicas e laboratoriais analisadas em cada paciente foram idade, sexo, valores séricos de TSH e T4 livre, presença de anticorpo anti-tireoperoxidase (anti- TPO) e anti-tireoglobulina (anti-TG). Foram avaliadas a presença de características ultrassonográficas sugestivas de benignidade (presença de halo periférico hipoecoico, aparência espongiforme, aspecto isoecóico ou hiperecoico) e de malignidade (nódulo sólido hipoecoico, contornos irregulares, presença de microcalcificações). Baseados nestas características, os nódulos foram classificados em benignos, indeterminados e suspeitos para malignidade. O diagnóstico citológico foi classificado em benigno, indeterminado, suspeito e maligno, e a análise combinada das características ultrassonográficas e citológicas também foi avaliada. Ao exame histopatológico, 200 nódulos eram malignos e 192 nódulos eram benignos. Os nossos resultados mostraram que sexo, idade, valores séricos de TSH e T4 livre e presença de anticorpo anti-TPO e anti-TG não estiveram associados a uma maior chance de malignidade. O valor de TSH sérico também não esteve associado a maior risco de recorrência ou estadiamento mais avançado nos pacientes com câncer Os nódulos maiores estiveram mais associados a benignidade quando avaliamos toda amostra. Na análise multivariada de toda amostra, após regressão logística, apenas a citologia maligna, hipoecogenicidade e presença de microcalcificações foram associados a malignidade. Já, a classificação ultrassonográfica que não se baseia em apenas uma característica mas em um conjunto de características, apresentou um alto valor preditivo de benignidade e foi útil na identificação de nódulos com citologia indeterminada. A classificação ultrassonográfica tem o potencial de reduzir o número de cirurgias para nódulos com citologia indeterminada / Thyroid nodules are often encountered in clinical practice, and with the use of high-resolution ultrasound may be identified in 17 to 67% of the population. The high prevalence of these nodules cause frequent concern to patients and clinicians due to the risk of malignancy, wich in turn leads on costly investigations, use of invasive diagnostic methods and sometimes unnecessary surgeries. Fine needle aspiration biopsy (FNAB) is the most accurate preoperative diagnostic method to identify a malignant thyroid nodule. However, FNAB cannot rule out malignancy in cases of inadequate cytology, follicular lesions, atypia of undetermined significance, and in cytology suggestive of follicular neoplasm. The aim of this study was to analyze clinical, laboratory, ultrasound and cytopathologic characteristics of a group of patients with thyroid nodules undergoing thyroidectomy and the relationship between serum levels of TSH and the risk of malignancy. In nodules found to be malignant in this cohort, we analyzed the association of TSH levels with advanced disease stage and risk of recurrence. We analyzed the records of 353 patients who were followed at Hospital das Clínicas - São Paulo Medical School, between February 2002 and April 2010, and who subsequently underwent thyroidectomy. The total number of nodules in these patients was 392. The clinical and laboratory characteristics included in the analysis were age, gender, serum levels of TSH and free T4, and presence of serum thyroid anti-peroxidase (anti-TPO) and anti-thyroglobulin (anti-TG) antibodies. We evaluated the presence of ultrasonographic features suggestive of benignity (isoechoic or hyperechoic appearance, presence of hypoechoic peripheral halo, spongiform appearance) and malignancy (hypoechoic appearance, irregular border, presence of microcalcifications). Based on these ultrasonographic characteristics, we classified the nodules as benign, indeterminate or suspicious for malignancy. According to the FNAB cytology, we also subdivided the nodules into benign, indeterminate, suspicious and malignant. The combined analysis of ultrasonographic features and cytopathology was also evaluated. On histopathology, 200 nodules were malignant and 192 were benign. Our results showed that gender, age, serum levels of TSH and free T4, as well as the presence of anti-TPO and anti-TG were not associated with increased risk of malignancy. Similarly, serum TSH value was not associated with increased risk of recurrence or more advanced stage in patients with thyroid cancer. A large nodule size was associated with benignity. In multivariate analysis, after logistic regression, only malignant cytology, hypoechoic appearance and presence of microcalcifications were associated with malignancy. Furthermore, the ultrasonographic classification, wich was not based in only one feature but in a set of characteristics, showed a high predictive value for benignity and seems to be useful in identifying nodules with indeterminate cytology at risk for malignancy. The use of ultrassonographic classification has the potential to reduce the number of surgeries for nodules with indeterminate cytology

<p>Molecular mechanisms in the development and progression of thyroid carcinomas are still not fully understood. In the present thesis the highly malignant anaplastic thyroid carcinoma (ATC) was used to study regulation of apoptosis and tumor interstitial fluid pressure (IFP).</p><p>Addition of a natural estrogen metabolite, 2-Methoxyestradiol (2-ME), induced a G2/M cell cycle arrest and apoptosis in five out of six human ATC cell lines. Treatment with 2-ME induced DNA-fragmentation as well as activation of caspase-3. Inhibitors of JNK and p38 MAPKs activity decreased the effect of 2-ME suggesting involvement in the induction of apoptosis.</p><p>Solid tumors have an elevated IFP. High IFP forms or reflects a barrier for exchange of molecules between microvessels and surrounding tissue. The mechanisms for the generation of the high IFP were investigated using a specific TGF-β inhibitor in an ATC model in athymic mice. Tumor IFP was lowered in TGF-β inhibitor-treated compared to control mice. Affymetrix microarray analysis showed a decreased expression of macrophage-associated genes in treated tumors. Furthermore, the number and activity of tumor-associated macrophages was reduced after TGF-β inhibition. A decreased protein leakage together with an increased coverage of α-smooth-muscle actin (SMA)-expressing cells indicated vessel normalization. An adjuvant treatment with the TGF-β inhibitor resulted in an increased treatment efficacy of doxorubicin. Thus, TGF-β inhibitor-treatment suggests improved microvessel function which results in a lowering of tumor IFP and increased tumor drug uptake.</p><p>To create a model for specific inactivation of genes in the thyroid, a transgenic mouse with a thyrocyte-specific expression of Cre recombinase was generated. The thyroglobulin promoter together with an inducible Cre recombinase (<i>creER</i><i>T2</i>) was used. Two transgenic founder lines were identified expressing cre mRNA solely in the thyroid. Functional activity of the CreER^T2 protein was demonstrated by using a ROSA26-LacZ reporter mouse.</p>

Molecular mechanisms in the development and progression of thyroid carcinomas are still not fully understood. In the present thesis the highly malignant anaplastic thyroid carcinoma (ATC) was used to study regulation of apoptosis and tumor interstitial fluid pressure (IFP). Addition of a natural estrogen metabolite, 2-Methoxyestradiol (2-ME), induced a G2/M cell cycle arrest and apoptosis in five out of six human ATC cell lines. Treatment with 2-ME induced DNA-fragmentation as well as activation of caspase-3. Inhibitors of JNK and p38 MAPKs activity decreased the effect of 2-ME suggesting involvement in the induction of apoptosis. Solid tumors have an elevated IFP. High IFP forms or reflects a barrier for exchange of molecules between microvessels and surrounding tissue. The mechanisms for the generation of the high IFP were investigated using a specific TGF-β inhibitor in an ATC model in athymic mice. Tumor IFP was lowered in TGF-β inhibitor-treated compared to control mice. Affymetrix microarray analysis showed a decreased expression of macrophage-associated genes in treated tumors. Furthermore, the number and activity of tumor-associated macrophages was reduced after TGF-β inhibition. A decreased protein leakage together with an increased coverage of α-smooth-muscle actin (SMA)-expressing cells indicated vessel normalization. An adjuvant treatment with the TGF-β inhibitor resulted in an increased treatment efficacy of doxorubicin. Thus, TGF-β inhibitor-treatment suggests improved microvessel function which results in a lowering of tumor IFP and increased tumor drug uptake. To create a model for specific inactivation of genes in the thyroid, a transgenic mouse with a thyrocyte-specific expression of Cre recombinase was generated. The thyroglobulin promoter together with an inducible Cre recombinase (creERT2) was used. Two transgenic founder lines were identified expressing cre mRNA solely in the thyroid. Functional activity of the CreERT2 protein was demonstrated by using a ROSA26-LacZ reporter mouse.