Ce travail de thèse fait partie d’un effort pour le développement des biomarqueurs, actuellement largement inexistants, pour une meilleure classification, pour une détermination plus précise du pronostic, et pour la prédiction de la réponse au traitement des tumeurs gliales malignes de l’enfant (épendymomes et gliomes de haut grade). Dans certains cas, ces biomarqueurs peuvent aussi devenir des cibles thérapeutiques.Dans ces études, nous avons pu montrer que la surexpression fréquente des marqueurs neuronaux distingue les épendymomes supratentoriels des formes infratentorielles. Parmi les épendymomes supratentoriels, une forte expression de neurofilament 70 (NF) est corrélée avec une meilleure survie sans récidive. La tenascine C (TNC) est surexprimée dans les épendymomes infratentoriels. Une étude de collaboration européenne multi-institutionnelle a été mise en place, permettant d’analyser une cohorte pédiatrique de 250 patients atteints d’un épendymome, et de démontrer la forte immunoexpression de TNC comme un marqueur robuste, associé a des survies globale et sans récidive plus péjoratives, particulièrement parmi les enfants âgés de moins de trois ans. Ceci a été validé dans une cohorte indépendante. Des immunomarquages pour NF et TNC pourraient être utilisés en clinique pour aider à déterminer le pronostic des épendymomes chez l’enfant.Une analyse des marqueurs pour la prédiction de la réponse à une thérapie ciblée anti-EGFR (erlotinib) a été réalisée par imunnohistochimie et FISH. La perte fréquente de PTEN dans les gliomes infiltrants du tronc cérébrale et la confirmation des caractéristiques des certains sous groupes (avec une forte expression de EGFR ou avec une différentiation oligodendrogliale) nous a permis de dessiner le protocole pour la prochaine étude de phase III pour cette maladie dans le cadre d’un futur essai thérapeutique de phase I/II. Nous rapportons par ailleurs des mutations du gène PI3KCA dans certains gliomes infiltrants du tronc cérébral, qui comme la perte d’expression de PTEN pourrait entrainer une activation de la voie mTOR qui devient donc une cible thérapeutique majeure théorique dans cette maladie. Des études ultérieures seront nécessaires pour définir le rôle de l’interaction entre la perte de PTEN, la surexpression de EGFR, la différentiation oligodendrogliale, les mutations de PI3KCA et d’autres altérations récemment décrites, (gains et amplifications de PDGFRA/MET mutations de TP53) et leur relation avec le devenir des patients sous traitement ciblé et les thérapies ciblées les plus intéressantes dans cette maladie uniformément fatale.Ce travail de thèse nous a permis d’explorer le rôle de la pathologie moléculaire dans la prise en charge des épendymomes et gliomas de haut grade chez l’enfant. / Biomarkers for the classification, clinical management and prognosis of pediatric brain tumors (ependymoma and high grade glioma, (HGG)) are lacking. To address this, biomarkers were developed and explored in view of classification, prognostication, target identification and prediction of the efficacy of treatment for patients with such tumors.We show that overexpression of neuronal markers distinguishes supratentorial from infratentorial ependymoma, and among the former higher immunoexpression of neurofilament 70 (NEFL) is correlated with better progression free survival (PFS). Tenascin-C (TNC) is significantly overexpressed in infratentorial ependymoma. A multi-institutional European ependymoma collaboration group was established and analyses were performed in a pediatric cohort of 250 patients, where immunohistochemistry (IHC) for TNC showed to be a robust marker of poor overall survival (OS) and PFS, particularly among children under 3 years, this being further validated in an independent cohort. Techniques and scoring performed in different laboratories were highly reproducible. IHC for NEFL and TNC could be used for prognostication of pediatric ependymoma.The analysis of putative predictive markers for the response to targeted therapies in pediatric HGG in the setting of a clinical trial with the anti-EGFR agent erlotinib was performed by IHC and fluorescent in situ hybridization. The frequent loss of PTEN in diffuse intrinsic pontine glioma (DIPG) and the confirmation of the biological singularity of the certain subgroups (expressing EGFR, displaying oligodendroglial differentiation) which seem to be associated with better response to erlotinib have helped our group to establish the design of the next Phase III protocol for this disease at our institution. We report mutations in PI3KCA constituting the first identification of oncogene mutations in some DIPG, which further highlight their biological heterogeneity. Further studies are needed to define the interaction between PTEN loss, EGFR overexpression, oligodendroglial differentiation, PI3KCA mutations and other recent findings such as PDGFRA/MET gains/amplification and TP53 mutations in these heterogeneous lesions and their relationship to the outcome of patients under new targeted therapies for this largely fatal disease.This thesis has allowed us to explore the molecular pathology in the context of biology and clinical setting of pediatric brain tumors.

INTRODUÇÃO: O tronco encefálico é uma pequena estrutura com elevada concentração de núcleos e tratos. Historicamente, houve grande debate sobre indicações cirúrgicas às lesões no tronco encefálico. A despeito do desenvolvimento da microcirurgia, cirurgia da base do crânio e da neuronavegação, poucos grupos têm experiência no manejo daquelas lesões. Quando lesões no tronco encefálico não afloram à superfície pial, torna-se crucial o conhecimento de áreas de segurança de acesso ao tronco, as quais representam estreitos corredores em que há paucidade de estruturas eloquentes e ausência de vasos perfurantes. OBJETIVO: Quantificar a área de trabalho gerada pelos acessos cirúrgicos mais comumente utilizados ao tronco encefálico, além de definir as exposições angulares geradas pelos mesmos acessos às áreas de segurança por meio de dissecções cadavéricas. Adicionalmente, detalhamos a anatomia cirúrgica de treze acessos ao tronco encefálico, com fotografias passo a passo e descrições detalhadas para auxiliar na melhor difusão destas técnicas. MÉTODOS: Foi realizada dissecção anatômica de 10 cadáveres humanos para demonstração de 13 acessos cirúrgicos ao tronco encefálico e da anatomia das seguintes zonas de segurança: mesencefálica anterior, sulco mesencefálico lateral, intercolicular, peritrigeminal, supra-trigeminal, pontina lateral, supra-colicular, infra-colicular, sulco mediano do quarto ventrículo, sulco posteromediano e olivar. Os acessos estudados foram: orbitozigomático, subtemporal, subtemporal transtentorial, subtemporal transtentorial com petrosectomia anterior, suboccipital telovelar, supracerebelar infratentorial mediano, paramediano e lateral, retrossigmoideo, extremo lateral, petrosectomia anterior, retrolabiríntico, e combinado. A dissecção foi realizada entre Janeiro a Julho de 2010, no Laboratório de Base de Crânio do Barrow Neurological Institute, localizado em Phoenix, Arizona, EUA. Os espécimes fixados em formalina e com artérias e veias perfundidas com silicone colorido foram dissecados em suporte de Mayfield em mesa cirúrgica, com todo instrumental cirúrgico simulando um ambiente operatório. Após cada acesso, neuronavegador era utilizado para coletar coordenadas tridimensionais de pontos pré-definidos nas craniotomias e no campo operatório, os quais após análise em software específico, se traduziam em valores das seguintes variáveis: área de exposição, exposição angular e extensão de exposição. Os resultados obtidos foram comparados quando havia interseção de área ou zona de segurança. RESULTADOS: A área de exposição média do orbitozigomático no tronco foi de 164,7 ± 43,6 mm2. A exposição angular horizontal à zona mesencefálica anterior foi 37,9 ± 7,3o. A área média produzida pelo retrossigmoide foi 538,6 ± 161,0 mm2. As exposições angulares horizontal e vertical médias geradas por esse corredor para a zona pontina lateral foram 31,1 ± 6,7o e 49,3 ± 9,4o, respectivamente. A área média produzida pelo far-lateral foi 856,8 ± 139,7 mm2. As exposições angulares horizontal e vertical médias deste acesso para a zona olivar foram 40,8 ± 10,2o e 54,8 ± 6,8o. CONCLUSÃO: O acesso orbitozigomático oferece mínima área de exposição do tronco, mas melhor trajetória em relação à zona mesencefálica anterior que o subtemporal. O supracerebelar infratentorial extremo lateral oferece melhor trajetória e ângulos ao sulco mesencefálico lateral que o subtemporal. Não há diferença significativa entre as áreas de exposição e exposições angulares ao tronco entre o retrossigmoide e retrolabiríntico, mas este último oferece trajetória mais direta / INTRODUCTION: The brainstem is a small structure disposing of high concentration of nuclei and tract. Historically, there was enormous discussion on surgical indications to brainstem lesions. In spite of the evolution of microsurgery, skull base surgery, and neuronavigation, few groups bear experience managing this pathology. Whenever lesions do not surface on pia or ependyma, it is key using the safe entry zones, managing the brainstem, which represent tiny corridors where eloquent structures and perforators are sparse. OBJECTIVE: To quantify the working area provided by the main surgical approaches to brainstem, as well as angles of attack provided by the same approaches to the safe zones through cadaveric dissections. It was possible at the same time to detail the microanatomy of thirteen approaches, with stepwise images and descriptions, in order to aid spreading this knowledge in Portuguese. METHODS: Ten human cadavers were dissected in order to visually demonstrate 13 surgical approaches to brainstem and these safe zones: anterior mesencephalic, lateral mesencephalic sulcus, intercolicular, peritrigeminal, supratrigeminal, lateral pontine, supracollicular, infracollicular, median sulcus of the fourth ventricle, posteromedian sulcus and olivary. The following approaches were analyzed: orbitozigomatic, subtemporal, subtemporal transtentorial, subtemporal transtentorial with anterior petrosectomy, median suboccipital telovelar, median, paramedian and lateral supracerebellar infratentorial, retrossigmoid, far-lateral, anterior petrosectomy, retrolabyrinthine, and combined. Dissections were carried out from January to July 2010, at the Skull Base Laboratory in the Barrow Neurological Institute, Phoenix, Arizona, USA. The specimens were lightly fixed in formalin while arteries and veins were perfused with color silicone. They were dissected on a Mayfield head-holder, using a complete set of surgical instruments simulating an operative environment. Neuronavigation was utilized after every approach to collect tridimensional coordinates of predefined points on the craniotomy edges and within the surgical field. Using a specific software, these coordinates translate themselves into the following variables: areas of exposure, angles of attack and lengths of exposure. The variables were compared among them when 2 or more approaches addressed overlapped areas. RESULTS: The mean area of exposure provided by the orbitozygomatic on the brainstem was 164,7 ± 43,6 mm2. The horizontal angle of attack to the anterior mesencephalic zone was 37,9 ± 7,3o. Mean area delivered by the retrosigmoid was 538,6 ± 161,0 mm2. Mean horizontal and vertical angles of attack produced by this corridor aiming the lateral pontine zone were 31,1 ± 6,7o e 49,3 ± 9,4o, respectively. The farlateral approach produced a mean area of exposure of 856,8 ± 139,7 mm2. Mean horizontal and vertical angles of attack offered by this avenue aiming the olivary zone were 40,8 ± 10,2o e 54,8 ± 6,8o. CONCLUSION: The orbitozygomatic approach provides a minimum area of exposure, but a better trajectory concerning the anterior mesencephalic zone comparing to the subtemporal. The extreme lateral supracerebellar infratentorial yields better trajectory and wider angles to the lateral mesencephalic sulcus than the subtemporal. There is no significant difference regarding areas of exposure and angles of attack to the brainstem between the retrosigmoid and retrolabyrithine, but the latter produces a more direct trajectory

A imagem molecular oferece a perspectiva de detectar doenças bem antes de os sintomas surgirem. A vasculatura tumoral é vital no crescimento do tumor e na disseminação de metástases, sendo assim alguns radiofármacos são dirigidos para a angiogênese. O glioma, tumor cerebral de baixa incidência porém alta mortalidade, requer um diagnóstico precoce para favorecimento da abordagem terapêutica. O objetivo desse estudo foi o desenvolvimento de novo radiofármaco para diagnóstico por imagem de glioma, baseado em peptídeos angiogênicos (GX1 e GX1-RGD) marcados com o radioisótopo tecnécio-99m. O desempenho dos conjugados peptídicos mostraram-se bastante parecidos em diversas avaliações. Eles foram radiomarcados com alta pureza radioquímica (>96%) e estáveis em soro até pelo menos 4h. Ambos são hidrofílicos e com baixa ligação às proteínas plasmáticas. A biodistribuição em animais sadios demonstrou alta excreção renal e depuração sanguínea rápida para ambos os radiotraçadores. Nos estudos in vitro, o 99mTc-HYNIC-PEG4-c(GX1) apresentou picos de ligação aos 60 min e o 99mTc-HYNIC-E-[c(RGDfk)-c(GX1)]) aos 120 min, nas células endoteliais HUVEC, usadas como controle, e nas células tumorais das linhagens U87MG e T98G. A captação tumoral nos animais foi mais acentuada para células U87MG, especialmente para o 99mTc-HYNIC-E-[c(RGDfk)-c(GX1)]) (2,87 ± 0,53% DI/g) aos 60 min p.i., com boa visualização em imagens adquiridas por gama-câmara e micro-SPECT/CT. Estudos realizados com os peptídeos conjugados à nanopartículas magnéticas para visualização em ressonância magnética também demonstraram especificidade dos produtos em tecidos tumorais. O desempenho do 99mTc-HYNIC-E-[c(RGDfk)-c(GX1)]) foi superior que o do traçador GX1, quanto à captação no glioma humano, podendo ser considerado como um promissor radiofármaco para diagnóstico de gliomas. / Molecular imaging offers the prospect of detecting diseases well before the symptoms appear. The tumor vasculature is vital in the tumor growth and dissemination of metastasis, thus some radiopharmaceuticals are directed to angiogenesis. The glioma, a brain tumor of low incidence but high mortality, requires an early diagnosis for favoring therapeutic approaches. The aim of this study was the development of a new radiopharmaceutical for imaging diagnosis of glioma, based in angiogenic peptides (GX1 and RGD-GX1) radiolabeled with technetium-99m radioisotope. The peptidic conjugates showed very similar performance in several evaluation. They were radiolabeled with high radiochemical purity (>96%) and are stable in the blood serum at least for four hours. Both are hydrofilic and had minimal binding to plasma protein. The biodistribution in healthy mice at many times, showed high renal excretion and fast blood clearance for both radiotracers. At in vitro studies, the 99mTc-HYNIC-PEG4-c(GX1) exhibit binding peaks at 60 min and the 99mTc-HYNIC-E-[c(RGDfk)-c(GX1)]) at 120 min, at HUVEC endotelial cells, used as control, and at tumor cell lines U87MG and T98G. The animal tumor uptake was more pronounced for U87MG cells, specially for 99mTc-HYNIC-E-[c(RGDfk)-c(GX1)]) (2.87 ± 0.53% DI/g) at 60 min p.i., with good visualization in images acquired with gama-camara and micro-SPECT/CT. Studies performed with peptides conjugate to magnetonanoparticles for MRI visualization also demonstred the peptides specificity in tumor tissue.The 99mTc-HYNIC-E-[c(RGDfk)-c(GX1)]) performance was superior to the GX1 tracer, regarding the glioma uptake, and may be consider as a promisser radiopharmaceutical for glioma diagnosis.

A imagem molecular oferece a perspectiva de detectar doenças bem antes de os sintomas surgirem. A vasculatura tumoral é vital no crescimento do tumor e na disseminação de metástases, sendo assim alguns radiofármacos são dirigidos para a angiogênese. O glioma, tumor cerebral de baixa incidência porém alta mortalidade, requer um diagnóstico precoce para favorecimento da abordagem terapêutica. O objetivo desse estudo foi o desenvolvimento de novo radiofármaco para diagnóstico por imagem de glioma, baseado em peptídeos angiogênicos (GX1 e GX1-RGD) marcados com o radioisótopo tecnécio-99m. O desempenho dos conjugados peptídicos mostraram-se bastante parecidos em diversas avaliações. Eles foram radiomarcados com alta pureza radioquímica (>96%) e estáveis em soro até pelo menos 4h. Ambos são hidrofílicos e com baixa ligação às proteínas plasmáticas. A biodistribuição em animais sadios demonstrou alta excreção renal e depuração sanguínea rápida para ambos os radiotraçadores. Nos estudos in vitro, o 99mTc-HYNIC-PEG4-c(GX1) apresentou picos de ligação aos 60 min e o 99mTc-HYNIC-E-[c(RGDfk)-c(GX1)]) aos 120 min, nas células endoteliais HUVEC, usadas como controle, e nas células tumorais das linhagens U87MG e T98G. A captação tumoral nos animais foi mais acentuada para células U87MG, especialmente para o 99mTc-HYNIC-E-[c(RGDfk)-c(GX1)]) (2,87 ± 0,53% DI/g) aos 60 min p.i., com boa visualização em imagens adquiridas por gama-câmara e micro-SPECT/CT. Estudos realizados com os peptídeos conjugados à nanopartículas magnéticas para visualização em ressonância magnética também demonstraram especificidade dos produtos em tecidos tumorais. O desempenho do 99mTc-HYNIC-E-[c(RGDfk)-c(GX1)]) foi superior que o do traçador GX1, quanto à captação no glioma humano, podendo ser considerado como um promissor radiofármaco para diagnóstico de gliomas. / Molecular imaging offers the prospect of detecting diseases well before the symptoms appear. The tumor vasculature is vital in the tumor growth and dissemination of metastasis, thus some radiopharmaceuticals are directed to angiogenesis. The glioma, a brain tumor of low incidence but high mortality, requires an early diagnosis for favoring therapeutic approaches. The aim of this study was the development of a new radiopharmaceutical for imaging diagnosis of glioma, based in angiogenic peptides (GX1 and RGD-GX1) radiolabeled with technetium-99m radioisotope. The peptidic conjugates showed very similar performance in several evaluation. They were radiolabeled with high radiochemical purity (>96%) and are stable in the blood serum at least for four hours. Both are hydrofilic and had minimal binding to plasma protein. The biodistribution in healthy mice at many times, showed high renal excretion and fast blood clearance for both radiotracers. At in vitro studies, the 99mTc-HYNIC-PEG4-c(GX1) exhibit binding peaks at 60 min and the 99mTc-HYNIC-E-[c(RGDfk)-c(GX1)]) at 120 min, at HUVEC endotelial cells, used as control, and at tumor cell lines U87MG and T98G. The animal tumor uptake was more pronounced for U87MG cells, specially for 99mTc-HYNIC-E-[c(RGDfk)-c(GX1)]) (2.87 ± 0.53% DI/g) at 60 min p.i., with good visualization in images acquired with gama-camara and micro-SPECT/CT. Studies performed with peptides conjugate to magnetonanoparticles for MRI visualization also demonstred the peptides specificity in tumor tissue.The 99mTc-HYNIC-E-[c(RGDfk)-c(GX1)]) performance was superior to the GX1 tracer, regarding the glioma uptake, and may be consider as a promisser radiopharmaceutical for glioma diagnosis.

No description available.

Malignant gliomas are the most common primary brain tumors in adults. Patients with these highly malignant tumors have an extremely poor prognosis. The situation with a highly proliferative tumor in a non-proliferating tissue should favor cytostatic treatment but so far the role of conventional chemotherapy has been adjunctive. The concentrations of three sex steroids, estradiol, progesterone and testosterone, were analyzed by radioimmunoassay after celite chromatography in brain tumor samples. Some malignant gliomas had high tissue concentrations of estradiol. Low progesterone levels may suggest steroid consumption. Estramustine (EM), a conjugate of estradiol-17ß and nornitrogen mustard had a dose-dependent antiproliferative effect on several human malignant glioma cell lines. At equimolar concentrations the inhibitory effects of the EM complex were clearly more pronounced than those of estradiol and nornitrogen mustard given alone or in combination. A specific binding protein (EMBP) is important for the cytotoxic action of EM. Using a mouse monoclonal antibody and an indirect antibodyperoxidase technique, EMBP was demonstrated in human glioma cells. Significant amounts of EMBP were also detected in human brain tumor tissue by radioimmunoassay. The mean concentrations (ng/mg protein) in 16 astrocytomas (2.6) and 7 meningiomas (5.1) were higher (p&lt;0.001) than in 18 samples of normal brain (0.5). The presence of the specific binding protein may suggest a selective binding and effect of EM in human brain tumor tissue. Human glioma cells displayed significant uptake, retention and metabolism of estramustine phosphate (EMP). After incubation with ^H-EMP a progressive uptake of radioactivity was recorded during 24 hours. Metabolism of parent EMP into estramustine and estromustine, which is a well known part of the metabolic pathway in man, was also demonstrated. A dose- dependent increase in DNA strand breaks was recorded at EMP- concentrations ranging 10-40 yg/ml. The uptake of ®^Rb, used as a tracer for potassium to study ion transport and membrane permeability, was reduced after incubation with EMP. Scanning electron microscopy gave further evidence for membrane damage. According to flow cytometric analyses exponentially growing glioma cells were accumulated in the G2/M stage and the fraction of Gi/Gq was reduced. EM seems to attack malignant cells in a multifocal fashion on several vital functions including the microtubule, the nucleus, and the cell membrane. The intact EM complex may be important for effects related to microtubule function which add to the cytotoxic potential of its constituents. These experimental findings justify further investigations on the role of sex hormones in brain tumor growth and development and of hormone-linked cytostatics in clinical treatment. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1990, härtill 6 uppsatser.</p> / digitalisering@umu

Astrocytoma is defined as the neoplasia of astrocytes, the most abundant non-neuronal glial cells in brain. According to the WHO classification, the different grades of astrocytoma are- gradeI/pilocytic astrocytoma (benign form), grade II/diffuse astrocytoma (DA), grade III/anaplastic astrocytoma (AA) and grade IV/Glioblastoma (GBM). Patients with grade II astrocytoma have median survival time of 6-8 years after surgical intervention. While the more aggressive grade III has a median survival of 2-3 years. Grade IV is the most malignant form and has a median survival of 15 months approximately. In spite of all the progress in the fields of diagnosis and therapy, the prognosis of GBM still remains very poor. The aggressiveness and poor survival of GBM is due to the recurrence which is primarily because of intratumoral heterogeneity, presence of glioma stem cells and infiltration of the tumor cells into the normal brain parenchyma. Apart from the role of genetic mechanisms in triggering tumorigenesis, epigenetic modifications particularly the DNA methylation and histone modifications, are now recognized as frequent alterations playing a crucial role in the development and progression of human malignancies. There are two distinct DNA methylation abnormalities. The first is the reduction in genome-wide DNA methylation levels (global hypomethylation) and the second is the hypermethylation in the CpG island of specific gene promoters. Hypomethylation is believed to induce proto-oncogene activation and chromosomal instability, whereas hypermethylation is strongly associated with silencing of tumor suppressor genes. Thus, DNA methylation can function as a “switch” to activate or repress gene transcription, providing an essential mechanism for overexpressed or silenced genes involved in the regulation of cell cycle, DNA repair, growth signalling, angiogenesis, apoptosis, migration, invasion and thus in the initiation and progression of astrocytoma. Recent studies have identified biomarkers with prognostic impact which would include promoter methylation of O⁶-methyl guanine-DNA methyltransferase methylation (MGMT), isocitrate dehydrogenase 1(IDH1) mutation and a glioma CpG-island methylator (G-CIMP) phenotype. In the current study, we have characterized the DNA methylation profile for the different grades of astrocytoma and analysed the significance of methylation events occurring commonly in all the grades or uniquely only in grade IV. One of the GBM-specific hypomethylated and upregulated genes, Fibromodulin (FMOD), was extensively investigated in terms of its role in glioma pathogenesis and its regulation. FMOD was found to induce F-actin stress fibre formation and promote glioma cell migration. We also found that FMOD-mediated glioma cell migration is dependent on Integrin/FAK/Src/Small Rho GTPases signalling cascade. We further found that TGFβ pathway regulates FMOD expression through a process involving active demethylation and chromatin state transitions on FMOD promoter. This work has been divided into three parts: Part I: Characterization of DNA methylome during progression of Astrocytoma To investigate the aberrant methylation pattern on a genome-wide scale, 17 Grade II, 16 Grade III and 36 Grade IV tumor samples as well as 9 control brain tissues were analysed using Infinium Human Methylation 450K Bead Array on Illumina platform. The analysis was carried out in two parts. Firstly, we validated the dataset with already existing TCGA dataset. Upon comparison, the methylation profile of our dataset was highly correlated to the TCGA dataset with correlation coefficient of 0.99. In addition, we also checked the methylation status of few known hypermethylated and hypomethylated genes which showed the similar type of differential methylation. Then, we characterized the differentially methylated CpGs based on their spatial distribution in the human genome, for different grades of astrocytoma. CpG-rich regions show more of hypermethylation while the non-CpG rich regions, like open sea or gene body, are observed to be hypomethylated. Secondly, we also analysed the differentially methylated genes which contribute to physiological events in gliomagenesis. We hypothesized that the methylation specific events that occur in grade II and remain similarly methylated in grade IV are the ones probably contributing to the initial astrocyte transformation. However, the methylation specific events responsible for the aggressive nature of grade IV may occur as differentially methylated genes only in grade IV (and not in grade II). In this analysis, we have identified differentially methylated genes that play a role in initial transformation process (293 genes hypermethylated and downregulated while 23 genes were hypomethylated and upregulated) and also those that play a role in tumor aggressiveness (459 genes hypermethylated and downregulated while 350 genes were hypomethylated and upregulated). The differentially methylated genes that were common in both grade II and grade IV showed an enrichment of cell proliferation pathways while the differentially methylated genes uniquely present in grade IV showed enrichment in pathways related to the aggressiveness phenotype of tumorigenesis like cell motility and angiogenesis. Part II: Fibromodulin (FMOD), a GBM-specific hypomethylated and upregulated gene, is essential for glioma cell migration Among differentially methylated genes specifically in GBM, fibromodulin (FMOD) is one of the top most hypomethylated genes. FMOD is a member of leucine – rich repeat proteoglycan that is widely distributed in interstitial connective tissues. We found that FMOD is hypomethylated and upregulated only in grade IV/GBM, not in the grade II. FMOD promoter methylation status is significantly negatively correlated to its transcript levels.Towards identifying functions of FMOD in glioma cells, total RNA derived from U251 cells transfected with either non-targeting siRNA or FMOD siRNA was subjected to transcriptome profiling. There were 872 genes upregulated and 299 genes downregulated in FMOD silenced cells than in control cells. PANTHER pathway analysis using the differentially regulated genes identified several pathways to be associated with FMOD. Cytoskeleton regulation by Rho GTPase, which is known to be involved in cell motility and migration, is enriched with highest significance. In coherence with the pathway analysis, modulating FMOD levels in glioma cells affected in glioma cell migration. Upon FMOD overexpression, there was significant increase in migration than in control cells. Conversely, when FMOD is silenced, there was delay in migration than in control cells and the delayed migration was rescued by the addition of recombinant purified FMOD protein. Prior neutralization with FMOD specific antibody inhibited cell migration suggesting that secreted FMOD promotes glioma cell migration. Overexpression of FMOD in glioma cells induced actin stress fibre formation required for the migration of cells. On the contrary, FMOD silencing resulted in the loss of F-actin stress fibres which was restored upon addition of FMOD purified protein exogenously to the media. To investigate further the role and the requirement of specific Rho GTPase in FMOD-mediated migration, each of members of Rho GTPase family was silenced and their effect on FMOD-induced silencing was studied. FMOD mediated glioma cell migration was delayed when RhoA, Rac1 and Cdc42 were silenced. In order to understand whether FMOD activates Integrin mediated signalling pathway, we performed western blot analysis to check the levels of phospho-FAK in either FMOD overexpressing or knockdown condition. We observed phospho-FAK levels increased upon FMOD overexpression and decreased upon FMOD silencing compared to the respective controls. Additional experiments revealed that inhibitors to Integrin, FAK and Src were able to abrogate the FMOD induced glioma cell migration. These results suggest that FMOD utilizes a pathway that involves Integrins, FAK, Src and Rho GTPases in promoting glioma cell migration. To comprehend the effect of FMOD promoter methylation status and its expression in GBM patient scenario, we stratified the patients into either high or low FMOD expression and promoter hypermethylation or hypomethylation. The GBM patients with low FMOD transcript levels and promoter hypermethylation showed better survival than the other group. Part III: Regulation of FMOD expression through TGFβ-dependent epigenetic remodelling in glioma To study how FMOD is regulated in glioma, we investigated the promoter sequence of FMOD by MatInspector. Several Smad-binding sites were located in FMOD promoter which indicated that FMOD might be regulated via TGFβ signalling pathway. Firstly, we checked active TGFβ signalling in glioma cell lines – LN229, U87 and U251. TGFβ-dependent signalling was active in U251 and U87 cells compared to LN229 cells as seen by the levels of phospho-Smad2. Moreover, FMOD transcript level was found to be high in U251 compared to LN229 cells. Further, TGFβ treatment increased FMOD promoter luciferase activity as well as FMOD transcript level in LN229 cells. In contrast, U251 cells that were treated with TGFβ RI inhibitor showed a significant decrease in FMOD promoter luciferase activity as well as FMOD transcript level. We correlated these findings with Smad2 occupancy at FMOD promoter by chromatin immunoprecipitation (ChIP). Smad2 association at FMOD promoter is found to be relatively higher in U251 cells than in LN229 cells which suggested that TGFβ induced transcription factor, Smad2, drives FMOD expression in U251 cells. Next, we investigated the role of TGFβ in FMOD promoter demethylation and chromatin state transition. Upon TGFβ treatment in LN229 cells, we found that there was gradual demethylation of FMOD promoter in a time-dependent manner. TGFβ treatment also altered the chromatin state by increasing the active marks (H3K4me3 and H3K9Ac) and decreasing the repressive mark (H3K27me3) with a simultaneous increase in Smad2 occupancy in the FMOD promoter. In contrast, TGFβ RI inhibitor treatment of U251 cells resulted in methylation of FMOD promoter in a time-dependent manner. Further, we observed a significant enrichment of repressive histone marks (H3K27me3) and loss of active chromatin marks (H3K4me3 and H3K9Ac) with a concomitant decrease in Smad2 occupancy at FMOD promoter. DNMT3A/B and EZH2 enzymes play a key role in DNA methylation and H3K27 trimethylation respectively. Accordingly, we examined the transcript levels of DNMT3A/B and EZH2 in LN229 cells treated with TGFβ as well as U251 cells treated with TGFβ RI inhibitor. In presence of TGFβ, DNMT3A/B and EZH2 transcript levels were significantly downregulated than in untreated cells in a time-dependent manner. Conversely, in U251 cells treated with TGFβ RI inhibitor, there was a significant increase in DNMT3A/B and EZH2 transcript levels when compared to untreated cells. TGFβ is known to promote glioma cell migration. In order to understand whether TGFβ-mediated glioma cell migration occurs via FMOD, we performed migration assay in U251 cells with or without TGFβ RI inhibitor followed by addition of either BSA control or FMOD purified protein. Upon TGFβ RI inhibitor treatment, there was delay in the migration of U251 cells than in untreated control cells which was rescued when purified FMOD protein was added, indicating that FMOD is essential for TGFβ signalling cascade to induce glioma cell migration. Therefore, we conclude from these results that epigenetically regulated FMOD is essential for TGFβ mediated glioma cell migration.

Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2017-06-05T19:35:37Z No. of bitstreams: 2 Dissertação - Aline Monezi Montel - 2015.pdf: 4015331 bytes, checksum: 793195b96d26505a33dde69ed1f9eee3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-06-06T12:36:41Z (GMT) No. of bitstreams: 2 Dissertação - Aline Monezi Montel - 2015.pdf: 4015331 bytes, checksum: 793195b96d26505a33dde69ed1f9eee3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-06-06T12:36:41Z (GMT). No. of bitstreams: 2 Dissertação - Aline Monezi Montel - 2015.pdf: 4015331 bytes, checksum: 793195b96d26505a33dde69ed1f9eee3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-04-24 / Glioblastoma multiforme (GBM) is the malignant brain tumor most frequent in adults characterized by its high proliferative and invasive properties in addition of its high resistance to the available treatments. Therefore, the investigation of novel agents able to improve survival and overall life quality of GBM patients is extremely needed. Ruthenium compounds have revealed to be good candidates as antitumor drugs demonstrating antiproliferative activity in some types of cancer such as breast, prostate and lung. Besides the chemical properties specific for these compounds, ruthenium also presents a variety of radioisotopes that can be potentially used in radionuclide therapy (RT). In this work we investigated the antitumor effect of non-radioactive and radioactive Ruthenium II Fosfinic Complexes (RFCs) [Ru(pic)(bipy)(dppb)]PF6, [Ru(pic)(bipy)(dppe)]PF6, [Ru(pic)(bipy)(dppf)]PF6 and [Ru(pic)(bipy)(dppp)]PF6 on GBM cell lines and evaluated its potential use for RT. The GBM cell lines: U87 (expressing native p53 protein) and T98 (expressing mutant p53 protein), as well as human pulmonary fibroblast (MRC5) were treated with diffferent concentrations of non radioactive and radioactive RFCs. Radioactive ruthenium complexes containing 103Ru were produced by neutron activation at the TRIGA MARK-I IPR-RI with a thermal neutronic flux of 4.3x1012 n.cm-2.s-1. Cell viability were evaluated by MTT assay, morphological alterations and RFCs mechanism of action were analyzed by phase contrast and fluorescence microscopy with DAPI and double acridine orange/ethidium bromide staining. Generation of oxygen reactive species (ROS) by RFCs was detected by diclorofluorescein (DCF) assay and quantification of the produced fluorescence was analyzed in ImageJ software (NIH, Bethesda, MD, USA). All tested compounds were cytotoxic to the cells in a dose-dependent manner presenting IC50 in the range of 1.49 µM to 27.8 µM and between 2.3x10-4 µM to 6.2x10-4 µM for non-radioactive and radioactive RFCs respectively. The RFCs induced morphological alterations indicative of cell death by apoptosis and/or necrosis such as decreasing cell size, cell roundness, nuclear fragmentation, cromatin condensation and formation of apoptotic bodies. RFCs neutronic activated demonstrated higher cytotoxic activity than its non-radioactive counterparts suggesting that upon neutronic activation 103Ru,  particles emitter, showed sinergistic antitumor effect. Therefore, ruthenium based complexes can serve as a prototype for the development of new anticancer drugs, as well as, the use of its radioisotopes may be considered for RT. Based on the published literature and to the best of our knowledge so far this is the first report describing the potential use of radioactive ruthenium compounds in GBM. / O glioblastoma multiforme (GBM) é o tumor cerebral maligno mais frequente em adultos, caracterizado por uma alta capacidade proliferativa e invasiva, além da alta resistência aos tratamentos disponíveis. Por isso, a investigação de novos agentes que possam melhorar a sobrevida e a qualidade de vida dos pacientes com GBM é extremamente necessária. Os compostos de rutênio têm se revelado bons candidatos como drogas antitumorais, apresentando atividade antiproliferativa em alguns tipos de câncer, tais como: mama, próstata e pulmão. Além das propriedades químicas, específicas destes compostos, o rutênio apresenta também radioisótopos que podem ser potencialmente usados na terapia por radionuclídeos (TR). Neste trabalho investigou-se o efeito antitumoral dos complexos fosfínicos de Rutênio (II) (CFRs): [Ru(pic)(bipy)(dppb)]PF6, [Ru(pic)(bipy)(dppe)]PF6, [Ru(pic)(bipy)(dppf)]PF6 e [Ru(pic)(bipy)(dppp)]PF6, não radioativos e radioativos, sobre linhagens celulares de GBM e avaliou-se o seu potencial uso em TR. As linhagens celulares de GBM: U87 (proteína p53 nativa) e T98 (proteína p53 mutante), assim como, células de fibroblastos pulmonares humanos (MRC5) foram tratadas com diferentes concentrações dos CFRs não radioativos ou radioativos. Os CFRs radioativos contendo 103Ru foram ativados neutronicamente utilizando o reator nuclear TRIGA MARK-I IPR-RI, com fluxo neutrônico de 4,3x1012 n.cm-2.s-1. A viabilidade celular foi avaliada pelo ensaio de MTT, alterações morfológicas e mecanismo de morte induzido pelos CFRs nas células tratadas foram identificados por microscopia de contraste de fase e de fluorescência utilizando coloração com DAPI ou dupla coloração com Laranja de acridina/Brometo de etídeo. A capacidade de geração de espécies reativas de oxigênio (ROS) pelos CFRs foi detectada pelo ensaio com diclorofluoresceína (DCF) e a quantificação da fluorescência produzida foi analisada no software image J (NIH, Bethesda, MD, USA). Todos os compostos foram citotóxicos de maneira dose-dependente, apresentando valores de IC50 variando de 1,49 µM a 27,8 µM e entre 2,3x10-4 µM a 6,2x10-4 µM em células de GBM para os compostos não radioativos e radioativos, respectivamente. Os compostos induziram alterações morfológicas indicativas de morte por apoptose e/ou necrose, como encolhimento e arredondamento celular, fragmentação nuclear, condensação da cromatina, formação de corpos apoptóticos. Os CFRs ativados neutronicamente apresentaram maior atividade citotóxica que os CFRs não-radioativos, indicando que os isótopos 103Ru, emissores de partículas β apresentaram efeito antitumoral sinergístico. Portanto, os complexos a base de rutênio podem servir como protótipo para o desenvolvimento de novos antineoplásicos, assim como, a utilização de seus radioisótopos podem ser considerados para TR. Com base na literatura, até a presente data, este é o primeiro relato do uso de compostos radioativos de rutênio em GBM.

INTRODUÇÃO: O tronco encefálico é uma pequena estrutura com elevada concentração de núcleos e tratos. Historicamente, houve grande debate sobre indicações cirúrgicas às lesões no tronco encefálico. A despeito do desenvolvimento da microcirurgia, cirurgia da base do crânio e da neuronavegação, poucos grupos têm experiência no manejo daquelas lesões. Quando lesões no tronco encefálico não afloram à superfície pial, torna-se crucial o conhecimento de áreas de segurança de acesso ao tronco, as quais representam estreitos corredores em que há paucidade de estruturas eloquentes e ausência de vasos perfurantes. OBJETIVO: Quantificar a área de trabalho gerada pelos acessos cirúrgicos mais comumente utilizados ao tronco encefálico, além de definir as exposições angulares geradas pelos mesmos acessos às áreas de segurança por meio de dissecções cadavéricas. Adicionalmente, detalhamos a anatomia cirúrgica de treze acessos ao tronco encefálico, com fotografias passo a passo e descrições detalhadas para auxiliar na melhor difusão destas técnicas. MÉTODOS: Foi realizada dissecção anatômica de 10 cadáveres humanos para demonstração de 13 acessos cirúrgicos ao tronco encefálico e da anatomia das seguintes zonas de segurança: mesencefálica anterior, sulco mesencefálico lateral, intercolicular, peritrigeminal, supra-trigeminal, pontina lateral, supra-colicular, infra-colicular, sulco mediano do quarto ventrículo, sulco posteromediano e olivar. Os acessos estudados foram: orbitozigomático, subtemporal, subtemporal transtentorial, subtemporal transtentorial com petrosectomia anterior, suboccipital telovelar, supracerebelar infratentorial mediano, paramediano e lateral, retrossigmoideo, extremo lateral, petrosectomia anterior, retrolabiríntico, e combinado. A dissecção foi realizada entre Janeiro a Julho de 2010, no Laboratório de Base de Crânio do Barrow Neurological Institute, localizado em Phoenix, Arizona, EUA. Os espécimes fixados em formalina e com artérias e veias perfundidas com silicone colorido foram dissecados em suporte de Mayfield em mesa cirúrgica, com todo instrumental cirúrgico simulando um ambiente operatório. Após cada acesso, neuronavegador era utilizado para coletar coordenadas tridimensionais de pontos pré-definidos nas craniotomias e no campo operatório, os quais após análise em software específico, se traduziam em valores das seguintes variáveis: área de exposição, exposição angular e extensão de exposição. Os resultados obtidos foram comparados quando havia interseção de área ou zona de segurança. RESULTADOS: A área de exposição média do orbitozigomático no tronco foi de 164,7 ± 43,6 mm2. A exposição angular horizontal à zona mesencefálica anterior foi 37,9 ± 7,3o. A área média produzida pelo retrossigmoide foi 538,6 ± 161,0 mm2. As exposições angulares horizontal e vertical médias geradas por esse corredor para a zona pontina lateral foram 31,1 ± 6,7o e 49,3 ± 9,4o, respectivamente. A área média produzida pelo far-lateral foi 856,8 ± 139,7 mm2. As exposições angulares horizontal e vertical médias deste acesso para a zona olivar foram 40,8 ± 10,2o e 54,8 ± 6,8o. CONCLUSÃO: O acesso orbitozigomático oferece mínima área de exposição do tronco, mas melhor trajetória em relação à zona mesencefálica anterior que o subtemporal. O supracerebelar infratentorial extremo lateral oferece melhor trajetória e ângulos ao sulco mesencefálico lateral que o subtemporal. Não há diferença significativa entre as áreas de exposição e exposições angulares ao tronco entre o retrossigmoide e retrolabiríntico, mas este último oferece trajetória mais direta / INTRODUCTION: The brainstem is a small structure disposing of high concentration of nuclei and tract. Historically, there was enormous discussion on surgical indications to brainstem lesions. In spite of the evolution of microsurgery, skull base surgery, and neuronavigation, few groups bear experience managing this pathology. Whenever lesions do not surface on pia or ependyma, it is key using the safe entry zones, managing the brainstem, which represent tiny corridors where eloquent structures and perforators are sparse. OBJECTIVE: To quantify the working area provided by the main surgical approaches to brainstem, as well as angles of attack provided by the same approaches to the safe zones through cadaveric dissections. It was possible at the same time to detail the microanatomy of thirteen approaches, with stepwise images and descriptions, in order to aid spreading this knowledge in Portuguese. METHODS: Ten human cadavers were dissected in order to visually demonstrate 13 surgical approaches to brainstem and these safe zones: anterior mesencephalic, lateral mesencephalic sulcus, intercolicular, peritrigeminal, supratrigeminal, lateral pontine, supracollicular, infracollicular, median sulcus of the fourth ventricle, posteromedian sulcus and olivary. The following approaches were analyzed: orbitozigomatic, subtemporal, subtemporal transtentorial, subtemporal transtentorial with anterior petrosectomy, median suboccipital telovelar, median, paramedian and lateral supracerebellar infratentorial, retrossigmoid, far-lateral, anterior petrosectomy, retrolabyrinthine, and combined. Dissections were carried out from January to July 2010, at the Skull Base Laboratory in the Barrow Neurological Institute, Phoenix, Arizona, USA. The specimens were lightly fixed in formalin while arteries and veins were perfused with color silicone. They were dissected on a Mayfield head-holder, using a complete set of surgical instruments simulating an operative environment. Neuronavigation was utilized after every approach to collect tridimensional coordinates of predefined points on the craniotomy edges and within the surgical field. Using a specific software, these coordinates translate themselves into the following variables: areas of exposure, angles of attack and lengths of exposure. The variables were compared among them when 2 or more approaches addressed overlapped areas. RESULTS: The mean area of exposure provided by the orbitozygomatic on the brainstem was 164,7 ± 43,6 mm2. The horizontal angle of attack to the anterior mesencephalic zone was 37,9 ± 7,3o. Mean area delivered by the retrosigmoid was 538,6 ± 161,0 mm2. Mean horizontal and vertical angles of attack produced by this corridor aiming the lateral pontine zone were 31,1 ± 6,7o e 49,3 ± 9,4o, respectively. The farlateral approach produced a mean area of exposure of 856,8 ± 139,7 mm2. Mean horizontal and vertical angles of attack offered by this avenue aiming the olivary zone were 40,8 ± 10,2o e 54,8 ± 6,8o. CONCLUSION: The orbitozygomatic approach provides a minimum area of exposure, but a better trajectory concerning the anterior mesencephalic zone comparing to the subtemporal. The extreme lateral supracerebellar infratentorial yields better trajectory and wider angles to the lateral mesencephalic sulcus than the subtemporal. There is no significant difference regarding areas of exposure and angles of attack to the brainstem between the retrosigmoid and retrolabyrithine, but the latter produces a more direct trajectory

Orientador: Helder Tedeschi / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T19:44:14Z (GMT). No. of bitstreams: 1 Valadares_MarceloGomesCordeiro_M.pdf: 1238842 bytes, checksum: f4f19f2521d318218d181dd5e357d746 (MD5) Previous issue date: 2015 / Resumo: Introdução: O tratamento para gliomas de alto grau consiste em cirurgia seguida de radioterapia e quimioterapia. Mesmo com o melhor tratamento disponível a sobrevida em geral não atinge 2 anos para glioblastoma multiforme e 2 a 5 anos para outros gliomas anaplásicos. Estudos realizados em países em desenvolvimento sugerem que as limitações financeiras dos sistemas públicos de saúde destes países podem dificultar o acesso da população aos recursos necessários, podendo inclusive influenciar negativamente a sobrevida. Neste trabalho analisamos uma série de pacientes portadores de gliomas de alto grau tratados em um hospital público de uma grande região metropolitana brasileira. Comparamos o tratamento oferecido com o preconizado na literatura e verificamos se eventuais limitações nas diversas etapas do tratamento multidisciplinar afetam a qualidade do serviço prestado. Método: Analisamos retrospectivamente prontuários de pacientes portadores de gliomas de alto grau tratados no Hospital de Clínicas da Unicamp entre 2007 e 2013. Resultados: Foram incluídos 135 pacientes. A idade média foi de 57 anos e 2 meses e haviam 2 homens para cada mulher. A unidade de emergência foi a porta de entrada para 89% dos pacientes. No momento do diagnóstico 90.3% já apresentavam sintomas há mais de 2 semanas e 14,8% estavam sintomáticos há mais de 12 semanas. Quimioterapia e radioterapia foram prescritas para 56% dos pacientes. Radioterapia apenas foi o tratamento de escolha para 25% e outros 19% foram encaminhados para cuidados paliativos. Tromboembolismo venoso foi diagnosticado em 26%, infecções relacionadas ao procedimento cirúrgico em 16%. Após a cirurgia a mediana de tempo para início dos tratamentos subsequentes foi: 6.71 semanas para a primeira consulta com o radioterapêuta, 6.28 semanas para a primeira consulta com o oncologista e 12.57 semanas para iniciar a quimioterapia e radioterapia. Progressão tumoral com necessidade de nova abordagem cirúrgica foi observada em 27 pacientes no grupo de terapia tardia (>6 semanas) e em nenhum paciente no grupo de terapia precoce (?6 semanas) (p=0.002). Não foi observada correlação estatística entre a ocorrência de eventos tromboembólicos (p=0.09) ou infecciosos (p=0.057) e a demora para o início do tratamento adjuvante. A mediana de sobrevida de 100 pacientes portadores de glioblastoma multiforme submetidos a todas as formas de tratamento foi de 7.8 meses. Conclusão: Embora o tratamento cirúrgico seguido de radioterapia e quimioterapia esteja disponível no Hospital de Clínicas da Unicamp, apenas 56% dos pacientes são submetidos a todo o tratamento preconizado e apenas 12% iniciam a quimioterapia e radioterapia em menos de 6 semanas após a cirurgia. Estes números são inferiores aos observados em países desenvolvidos, mas compatíveis com outros estudos de países em desenvolvimento. A mesma comparação é feita em relação à sobrevida global dos pacientes diagnosticados com glioblastoma multiforme. Os dados apresentados reforçam a tese de que o tratamento de portadores de gliomas de alto grau é influenciado negativamente pelas limitações presentes na saúde pública de países em desenvolvimento / Abstract: Introduction: The general management of high-grade gliomas is based on surgery plus radiotherapy and chemotherapy. However, in developing countries, the lack of financial resources may delay the treatment, and probably affect patients¿ outcome. Based on this, the aim of this study is to review the clinical data of high-grade glioma patients treated in a brazilian referral public hospital. Methods: We retrospectively analyzed the clinical data with emphasis on the management options (surgery, radiotherapy and chemotherapy) of high grade glioma patients from 2007 to 2014 at the University of Campinas (Unicamp) hospital. Results: A total of 135 patients (27% of all brain tumors) had the diagnosis of high-grade glioma and were included. The mean age of high-grade glioma patients at the time of diagnosis was 57 years and 2 months. There were 90 men (67%) and 44 women (33%). The outpatient referral system was responsible for only 15 (11%) admissions with 120 (89%) patients being admitted through the emergency unit. Headache was the main complaint and 90.3% of the patients had been symptomatic for over 2 weeks and 14,8% for over 12 weeks. After surgery 56% were treated with chemotherapy and radiotherapy. Radiotherapy was the single therapy for 25% of the cases. Another 19% were referred to supportive care. After surgery patients took a median 6.71 weeks to the first radiotherapy consultation, 6.28 weeks to the first clinical oncology consultation and a median 12.57 weeks to start radiation. Only 12% started radiation in less than 6 weeks. Tumor progression requiring a second surgery was observed in 27 patients in the delayed therapy group (> 6weeks) compared with no patients in the early therapy (< 6 weeks) group (P=.002). Glioblastoma Multiforme patients overall survival was 7.8 months. Conclusion: Although surgery, radiation and temozolamide were available at our institution only 56% of the patients received all these treatments and only 12% started radiation in less than 6 weeks. These numbers fail in comparison to many studies published in developed countries but are similar to others published in developing nations. The same applies to the overall survival calculated for glioblastoma patients. This study supports that financial constrictions in public health care in developing countries have a negative impact on high-grade glioma treatment / Mestrado / Neurologia / Mestre em Ciências Médicas