Dissertations / Theses on the topic 'Gliomas'

Gliomas constitute 36% of all primary brain tumors and 81% of all primary malignant brain tumors. The overall prognosis in patients with gliomas depends mainly on the location and histologic grade of the tumor.

The World Health Organization classification of gliomas is the primary basis for guiding therapy and assessing overall prognosis in gliomas. This classification system, based on histological features, often falls short of predicting therapeutic response of individual tumors within the same histological grade. Yet, it still remains the grading method for both research and clinical prospects.

Unlike any other organ the brain has multiple protective layers such as the skull that ensure a homeostatic environment. The resulting reduced access to the brain and the absence of plasmatic brain tumor markers bring neuroimaging in a central position in diagnosis and management of brain tumors. Moreover, neuroimaging has evolved from a purely morphologic investigation into a diagnostic tool that allows characterization of particular physical alterations within brain tissue. Understanding the relationship between the physical characteristics of tumor tissue, studied by MR imaging, and biological characteristics of the tumor is therefore important for the appropriate integration of neuroimaging in brain tumor management. The general objective of this work is to define the relationship between physiologybased MR imaging and biological features of glial tumors. Diffusion and perfusionweighted imaging, physiologybased MR techniques provide the data based on physical characteristics of the tissues. Diffusion weighted imaging (DWI) allows the measurement of water molecules diffusivity within the brain tissue by means of apparent diffusion coefficient (ADC) measurements. Perfusion weighted imaging (PWI) is based on changes of MR signal during the passage of contrast material through the intravascular space and allows hemodynamic measurements such as those of cerebral blood volume (CBV)within the brain tissue.

Highgrade diffuse gliomas are currently differentiated from low grade diffuse gliomas by increased cellularity with nuclear atypia, mitotic activity, endothelial proliferation and necrosis. Components of the extracellular matrix and angiogenesis constitute some other features of gliomas, which have established links with oncogenic processes that influence the proliferative and infiltrative potentials of these tumors. We have specifically targeted these features in our comparative studies with the working hypothesis that physiologybased MR measurements, obtained in vivo, might provide information that is pertinent in terms of tumor malignancy.

We chose to approach the biology of brain tumors in two ways: in vivo, by means of metabolic imaging techniques such as positron emission tomography (PET) and ex vivo, by means of histological and immunohistochemical analyses of tumor specimens.

Many studies have investigated the relation between ADC values and cellularity in gliomas. The underlining theory is based on the premise that water diffusivity within the 9 extracellular compartment is inversely related to the content and attenuation of the constituents of the intracellular space. Therefore when cellularity increases, intracellular space volume increases with a relative reduction of the extracellular space, leading to restricted diffusion of water molecules. However other factors may affect the value of ADC in gliomas such as the extracellular matrix which contains various amounts of hydrophilic macromolecules susceptible to change water molecules diffusivity. Hyaluronic acid is one highly hydrophilic component of the extracellular matrix of gliomas and its level of expression changes significantly during the progression to anaplasia in gliomas. Our hypothesis was that hyaluronan may influence ADC values in high and low grade gliomas.

We demonstrated a positive correlation between ADC values and the immunohistochemical level of hyaluronan in glial tumors.

Previous studies have confirmed the utility of positron emission tomography using C11 Methionine (PETMET) as a prognostic tool in patients with gliomas. Higher MET uptake is associated with greater proliferative potential and a higher level of malignancy in gliomas.

The increased aminoacid uptake in gliomas seems to reflect increased transport mediated by aminoacid carriers located in the endothelial cell membrane. Our hypothesis was that CBV measurements, index of tumor vascularity, may be related to tumor aminoacid metabolism.

We found a positive correlation between maximum CBV values and maximum MET uptake values in gliomas.

A limitation to these preliminary studies was lack of direct correlation between MRbased measurements and histologic and metabolic data. Indeed, glial tumors are known for their remarkable tissue heterogeneity across different grades, within the same grade, and even within a single given tumor. Therefore we used image coregistration and stereotactic biopsies to further assess the relationship between MRbased imaging data and both metabolic and histologic analysis.

The second part of our studies was based on measurements at the exact same localization on both MR and PET images where biopsy specimens were performed. We found a local relationship between CBV and MET uptake values. Both measurements correlated with mitotic activity and endothelial proliferation; two features of tumor aggressiveness.

In order to quantify tumor cellularity and tumor angiogenesis, we respectively measured cell density and vessel density using immunohistochemical markers to identify vessels. We found a regional relationship between CBV and cell density, as well as vessel density in gliomas whereas no correlation was found regionally between ADC and cell density.

We concluded that CBV measurements may be used locally as indices of angiogenesis and cellularity in gliomas; whereas local ADC measurements are more variable and may not be used as a marker of cellularity in heterogeneous tumors such as gliomas.
Doctorat en Sciences médicales
info:eu-repo/semantics/nonPublished

Le gliome est le type le plus agressif et mortel de tumeur cérébrale. Ces tumeurs se caractérisent par la présence conjointe de phénotypes solides (de bas grade, moins invasif, hautement vascularisé) et diffus (haut grade, très envahissant et diffus) des glioblastomes multiformes. Les collagènes sont des composants majeurs de la MEC des cellules tumorales des gliomes, et sont également présents dans la membrane basale des vaisseaux sanguins, mais avec une composition différente entre vasculatures saine et tumorale. L'abondance et la typologie des collagènes dans la MEC des cellules tumorales et la vasculature représentent donc un marqueur potentiel de diagnostic pour la gradation des tumeurs gliales. Nous avons développé la spectro-imagerie infrarouge à transformée de Fourier pour déterminer les modifications morphologiques et moléculaires apparaissant dans les formes solides et diffuses de gliomes, ainsi que dans les vasculatures saine et tumorale. Nous avons d'abord mis en évidence les vasculatures saine et tumorale en utilisant des nanoparticules injectées dans le système sanguin. Ensuite, nous avons appliqué des méthodes de reconstruction spectrale pour distinguer les tissus sains vs. ceux des formes solide et diffuse de tumeurs sur la base de leurs contenus en collagène de la MEC. Enfin, nous avons déterminé les changements de types du collagène au cours de la progression tumorale, validant ainsi la notion que l’analyse de ces contenus est potentiellement un marqueur diagnostic pour la gradation des gliomes
The glioma is the most aggressive and lethal type of brain tumor. Such tumor is characterized both by solid (low grade, less invasive, highly vascularized) and diffuse (high grade, very invasive and diffuse) phenotypes in high-grades. Collagens are major components of ECM in glioma tumor cells, and are also present in basement membrane of blood vessels in vasculature, but with different composition between healthy and tumor capillaries. The abundance and typology of collagens in tumor cell ECM and vasculature is thus a potential diagnostic marker for grading glioma tumors. We developed Fourier transform infrared (FTIR) spectro-imaging as a functional technique to determine the morphological and molecular changes occurring in solid and diffuse form of tumor tissues as well as in healthy and tumor vasculatures. We first highlighted healthy and tumor vasculatures using nanoparticles injected in blood system. Then, we applied curve-fitting methods to distinguish between healthy tissue vs. solid and diffuse tumor tissues on the basis of the collagen contents found in ECM. Finally, we determined collagen typology changes during tumor progression, thus validating that collagen contents analysis is potentially a diagnostic marker for glioma grading

La caractérisation moléculaire multidimensionnelle des tumeurs et des tumeurs gliales en particulier est une étape importante pour l’identification de biomarqueurs (diagnostique, pronostique, théranostique et/ou de prédisposition), pour l’identification de cibles thérapeutiques et pour une meilleure compréhension de l’oncogénèse moléculaire.Nos travaux ont permis de confirmer et de consolider certaines données de la littérature comme par exemple : (i) la valeur pronostique favorable de la codélétion 1p/19q, (ii) la valeur pronostique favorable de la mutation IDH, (iii) le caractère mutuellement exclusive des mutations TP53 et de la codélétion 1p/19q et (iv) la rareté des altérations génétiques du PDGFRA dans les gliomes de bas grade (GDBG). De manière plus originale, nous avons identifié plusieurs sous-groupes génomiques de GDBG pertinents sur le plan clinico-biologique, notamment au sein des GDBG non 1p/19q codélétés : (i) 19q-délété ; (ii) 11p-délété, (iii) 7-gagné, (iv) 19-gagné et (v) inclassés. La perte du bras chromosomique 19q annule la valeur pronostique favorable de la mutation IDH dans les GDBG non 1p/19q codélétés. Nous avons également identifié des mutations géniques originales dans les GDBG (i.e. mutation TEP1 et RNF40) qui renforcent le rôle des télomères et du remodelage de la chromatine au sein des GDBG.Enfin, nous nous sommes concentrés sur la caractérisation des GDBG 11p-délétés qui sont de phénotype majoritairement astrocytaire et de moins bon pronostic. Ces GDBG surexpriment des gènes des cellules immunitaires (les GIM -Glioma infiltrating microglia-, les macrophages de type 1, les macrophages de type 2) et sont infiltrés par des cellules macrophagiques et microgliales. Ce microenvironnement dérégulé peut constituer une cible thérapeutique au sein des GDBG 11p-délétés. En conclusion, nos travaux participent à la dissection clinico-moléculaire des GDBG et à préciser la biologie d’un sous-type de GDBG caractérisé la perte du bras chromosomique 11p
Multildimensional molecular characterization of tumors and more specifically of gliomas is of pivotal importance to identify: (i) new biomarkers (i.e. diagnostic, prognostic, theranostic or predisposing), (ii) new therapeutic targets and (iii) to improve our understanding of molecular oncogenesis.Our work has confirmed and consolidated previous data published in the literature, for example that: (i) 1p/19q co-deletion is associated with better prognosis, (ii) IDH mutation is associated with better prognosis, (iii) TP53 mutations and 1p/19q codeletion are mutually exclusive and (iv) PDGFRA is rarely altered, at genomic level, in low-grade gliomas (LGG).More originally, we have identified several genomic groups, with clinical and biological relevances, in LGG and more specifically in LGG without 1p/19q co-deletion: (i) 19q-deleted, (ii) 11p-deleted, (iii) 7-gained, (iv) 19-gained and (v) unclassified. Interestingly, 19q deletion abrogates the positive prognostic value of IDH mutation in LGG without 1p/19q codeletion.We have also identified new recurrent somatic gene mutations in LGG (i.e. TEP1 and RNF40 mutations), supporting the critical role of telomeres and chromatin remodelling in LGG.Finally, we have characterized further 11p-deleted LGG that exhibit mostly astrocytic phenotype and poor prognosis. This subgroup includes LGG overexpressing genes of inflammatory/immune cells (GIM -Glioma infiltrating microglia-, M1 macrophages and M2 macrophages) and infiltrated by macrophagic/microglial cells. This peculiar microenvironment detected in 11p-deleted LGG might be used as a therapeutic target. In conclusion, our work participates to characterize clinico-biological portrait of LGG and to describe a singular genomic subgroup of LGG characterized by 11p loss

Gliomas are the most common primary brain tumours found in adults. The median survival of patients diagnosed with the most malignant form, glioblastoma multiforme (GBM), is 9-12 months and has changed little over the years despite advances in medical technology. Gene therapy may offer new solutions to treat this resistant disease. Hence, we tested three different gene therapy strategies. In our first study, we tested the efficacy of targeted therapy to correct common aberrations found in gliomas including amplification/mutation of receptor tyrosine kinases (RTK) and loss of PTEN, which result in an overactive PI3K/Akt pathway. Without PTEN, FOXO transcription factors are inactivated, and the cell becomes resistant to apoptosis and cell cycle arrest. By using an adenoviral vector (AdV) expressing an activated FOXO1 mutant (AdFOXO1;AAA), we restored apoptosis and cell cycle arrest, reduced tumour volume and prolonged survival in an intracerebral xenograft model. Secondly, we examined the therapeutic capacity of a novel replicating/non-disseminating AdV expressing the fusion protein of cytosine deaminase and uracil phosphoribosyltransferase (CU). CU can convert the non-toxic pro-drug, 5-fluorocytosine (5-FC) to the tissue diffusible chemotherapeutic drug, 5-fluorouracil (5-FU) to target dividing cells. In vitro, the replicating vectors were superior to the non-replicating vectors, but the fully replicating/disseminating vector did not perform considerably better than the replicating/non-disseminating vector suggesting that dissemination may not be advantageous. In vivo, the replicating/non-disseminating vector administered in conjunction with 5-FC prolonged survival in both an athymic and an immunocompetent mouse model. Moreover, an immune bystander effect in vivo was mediated by macrophages and T cells. Lastly, we investigated a method to harness a tool of the immune system, IFN-ß; this cytokine is known to have anti-angiogenic, anti-proliferative, and immunomo
Les gliomes sont des tumeurs primaires de cerveau les plus communes retrouvées dans les adultes. La survie médiane des patients diagnostiqués avec la forme la plus maligne, le glioblastome multiforme (GBM), est de 9 à 12 mois et a peu changé au cours des années en dépit des avances en technologie médicale. La thérapie génique peut offrir de nouvelles solutions pour traiter cette maladie résistante. Durant nos travaux, nous avons examiné trois stratégies différentes de thérapie génique Dans notre première étude, nous avons examiné l'efficacité de la thérapie visée à corriger des anomalies communes retrouvées dans les gliomes, comprenant l'amplification/mutation de récepteurs de type tyrosine kinase (RTK) et la perte de PTEN, qui mènent en conséquence à une voie activée de PI3K/Akt. Sans PTEN, les facteurs de transcription FOXO sont inactivés, et la cellule devient résistante à l'arrêt du cycle cellulaire et à l'apoptose. En utilisant un vecteur adénoviral (AdV) exprimant une protéine activée du mutant FOXO1 (AdFOXO1;AAA.), nous avons reconstitué les signaux pour l'arrêt du cycle cellulaire et l'apoptose in vitro ainsi que in vivo. Deuxièmement, nous avons examiné la capacité thérapeutique d'un nouveau vecteur adénovirale qui a la capacité de se répliquer sans provoquer de lyse cellulaire et qui exprime en plus la protéine de fusion uracile phosphoribosyltransférase/cytosine déaminase (CU). La protéine CU peut convertir le promédicament non-toxique, le 5-fluorocytosine (5-FC) à la drogue chimiothérapeutique diffusible, le 5-fluorouracile (5-FU) qui a comme cible des cellules en division cellulaire. In vitro, les vecteurs à capacité de répliquation étaient meilleurs que ceux qui ne pouvaient pas se répliquer. In vivo, le vecteur en présence du 5-FC a prolongé la survie de deux modès animaux (avec et sans sytèmes immunitaires). Dans un dernier temps, nous avons étudié une méthode pour exprimer l'IF

Les gliomes de bas-grade représentent la tumeur cérébrale la plus fréquente chez l’enfant. Elles sont caractérisées par un large spectre de sous-types tumoraux, très hétérogènes. Leur définition actuelle est principalement basée sur des critères histologiques ce qui représente une limite importante car ces classifications souffrent d’un manque de précision. Les progrès récents de la génomique nous permettent d’approfondir considérablement les connaissances sur la biologie de ces tumeurs afin d’enrichir leur classification actuelle. Ce travail présente une analyse approfondie des altérations génomiques de l’ADN et l’ARN des gliomes de bas-grade pédiatriques. Le premier niveau d’analyse se base sur l’analyse du séquençage à haut débit de 169 gliomes de bas-grade de l’enfant. Bien que les mutations des gènes BRAF et FGFR1 sont les plus fréquemment décrites dans ces tumeurs, nous avons identifié pour la première fois le réarrangement chromosomique MYB-QKI majoritairement associé aux gliomes angiocentriques. Dans un deuxième temps ce travail décrit l’analyse du transcriptome de 151 gliomes de bas grade extraits à partir de tissu conservé en paraffine. Nous avons observé des différences moléculaires en fonction de leur sous-type histologique, de la localisation tumorale et de leur statut BRAF. Dans le dernier volet de ce travail, nous avons testé la faisabilité d’isoler par cytométrie en flux une cellule unique en les distinguant selon un marqueur de différenciation glial (A2B5+ et A2B5-) et d’effectuer une analyse transcriptomique à haut-débit en séquençant l’ARN à l’échelle d’une cellule unique. Cette technique nous a permis de décrire des différences moléculaires intéressantes entre des cellules A2B5+ et A2B5-. Ces résultats soulignent l’intérêt d’exploiter des nouvelles technologies de pointe pour servir de base à l’étude des caractéristiques biologiques des cellules tumorales
Low-grade gliomas represent the most frequent brain tumor arising during childhood. They are characterized by a broad spectrum of tumor types.The definition of low-grade gliomas has been mainly based on morphology. This histological classification of pediatric low-grade gliomas (PLGG), suffers from the lack of reproducibility. The recent progress in molecular biology and genetics has brought new insights in the biology of those tumors and allows better understanding of their biology. This work provides a comprehensive analysis of two different genetic approaches in PLGGs. The first part is based on the description of somatic genetic alterations of the DNA. Using a large PLGG cohort, we have dissect the genome of those tumors and draw the landscape of their genetic alteration. Although BRAF and FGFR1 alterations are predominantly altered, we have discovered a new translocation, MYB-QKI, that is almost exclusively present in a specific histological subgroup; angiocentric gliomasThe second part of the thesis describes transcriptomic analysis of bulk PLGGs. This work describes molecular differences between PLGGs from distinct histologies and arising from different locations in the brain as well as different BRAF mutation status.We were also able to test single-cell expression analyses in three pilocytic astrocytomas (PAs) using RNA-sequencing. In this experimental work we have successfully tested the hypothesis that we can isolate single-cells from fresh PLGG tumors in order to analyze the trasncriptome at a large scale. We observed that single-cells expressing A2B5, a glial progenitor marker, isolated in pediatric PAs are characterized as a distinct biological population. These results underline the importance to improve the precision of the transcriptomic studies to capture the molecular signal of tumor cells and further understand the different pattern between normal cells and tumor cells

Reseksjon av høygradige gliomer Høygradige gliomer er den vanligste formen for primær hjernesvulst. Glioblastomer (Verdens Helseorganisasjon grad IV) og anaplastiske astrocytomer (Verdens Helseorganisasjon grad III) utgjør mellom 70 og 85 % av høygradige gliomer. Høygradige gliomer er assosiert med både høy morbiditet og mortalitet. Nærmest alle pasienter med høygradige gliomer opplever tilbakefall og dør som følge av sykdommen. Til tross for kirurgi, strålebehandling og cellegift, er median overlevelse for pasienter med glioblastom fremdeles under 12 måneder. For pasienter med anaplastisk astrocytom er median overlevelse 2 til 3 år. Høygradige gliomer infiltrerer omkringliggende hjernevev, og hensikten med kirurgi er, foruten å histopatologisk verifisere diagnosen, å fjerne så mye av svulsten som mulig uten å påføre pasienten nye eller økte nevrologiske utfall. I vår avdeling benytter vi et navigasjonssystem under operasjonene som nyttiggjør tredimensjonale preoperative MR-bilder og tredimensjonal ultralydavbildning under operasjonen. Dette navigasjonssystemet gjør at kirurgen til en hver tid kan se posisjonen til sine instrumenter i forhold til hjernen og svulsten. Ved hjelp av funksjonell MR (eller mer presist blood-oxygenation-level-dependent functional magnetic resonance imaging) og diffusjon tensor traktografi (DTT) kan en henholdsvis kartlegge viktige områder i hjernens grå og hvite substans før operasjonen. Disse undersøkelsene utføres som regel når svulster ligger i nær relasjon til ekstra følsomme områder av hjernen (for eksempel språkområder og viktige områder for bevegelse). Informasjon fra disse undersøkelsene kan også importeres i navigasjonssystemet som benyttes under operasjonen. I de to første studiene i denne avhandlingen ønsket vi å undersøke hvordan funksjonell MR og DTT ble brukt i preoperative vurderinger. Vi evaluerte om funksjonell MR og DTT i kombinasjon med tredimensjonal ultralydavbildning under operasjonen la forholdene til rette for skånsom fjerning av høygradige gliomer beliggende i ekstra følsomme områder av hjernen. I den tredje studien undersøkte vi konsekvensene av kirurgiske komplikasjoner og nevrologiske utfall som følge av kirurgi på glioblastompasienters funksjonsnivå og overlevelse. Videre gjorde vi volumetriske analyser for å beregne hvor mye svulstvev vi klarte å fjerne hos pasienter med primære glioblastomer behandlet i vår avdeling. I den fjerde studien undersøkte vi om det var noen sammenheng mellom overlevelse og fall i selvrapportert livskvalitet kort tid etter kirurgi hos pasienter med glioblastomer. Den femte studien var basert på data fra Kreftregisteret og undersøkte overlevelse og behandling blant eldre pasienter (≥66 år) med glioblastomer over en tyve års periode. Hovedfunnene i denne avhandlingen er: - Kombinasjonen av funksjonell MR, DTT og tredimensjonal ultralydavbildning kan være nyttig når en utfører kirurgisk reseksjon av høygradige gliomer beliggende i ekstra følsomme områder av hjernen. - Pasienter som opplevde komplikasjoner og nevrologiske utfall som følge av kirurgi hadde lavere sannsynlighet for å motta strålebehandling og kjemoterapi. - Tidlig fall i helserelatert livskvalitet etter kirurgi synes å være en sterk og uavhengig negativ prognostisk faktor for pasienter med glioblastom. - Økende alder er en sterk og uavhengig negativ prognostisk faktor for pasienter med glioblastom. Selv om det har vært en intensivering av behandling over tid, har gevinsten i den eldste aldersgruppen vært begrenset. Prognosen for de eldste er fremdeles svært dårlig til tross for multimodal behandling
High-grade gliomas are the most common primary brain tumour. Glioblastomas (World Health Organization Grade IV) and anaplastic astrocytomas (World Health Organization Grade III) account for 70-85% of high-grade gliomas. High-grade gliomas are associated with high morbidity and mortality. Virtually all patients with high-grade glioma will experience recurrence and will eventually die from progressing disease. Despite surgery, radiotherapy, and chemotherapy, median survival in patients with glioblastoma still does not exceed 12 months. The median survival for patients with anaplastic astrocytoma (AA) has been reported to be between 2 and 3 years. According to current guidelines, surgery is warranted to establish a histopathologic diagnosis and to achieve safe, maximal, and feasible resection. However, these aggressive tumours cannot be cured and overly aggressive resection is not recommended due to the risk of new neurological deficits. High-grade glioma surgery is a delicate balance between achieving maximal tumour resection and inducing new deficits. In our department a neuronavigation system based on preoperative 3D magnetic resonance imaging (MRI) and intraoperative 3D ultrasound is utilised when resecting high-grade gliomas. Blood-oxygenation-level-dependent functional magnetic resonance imaging (BOLD fMRI) and diffusion tensor tractography (DTT) are specialized MRI techniques for imaging eloquent cortices and neural tracts in grey and white matter, respectively. The neuronavigation system allows the integration of BOLD fMRI and DTT data if the tumours are located in eloquent regions. In the two first studies of this thesis we sought to investigate the use of BOLD fMRI and DTT for preoperative assessments and determine whether using these data together with 3D intraoperative ultrasound enabled safe resection of high-grade gliomas situated in eloquent regions. In the third study we wanted to explore the impact of surgical morbidity on functional outcome and survival in GBM patients. Further, we sought to determine extent of tumour resection achieved in a consecutive sample of primary GBM from our own department. In the fourth study we wanted to determine if changes in health related quality of life early after surgery could be a predictor for survival in patients with glioblastoma. The aims of the fifth study were to explore survival and the treatment provided to elderly patients (≥66 years) diagnosed with glioblastoma during a 20-year time period in a population-based cohort using the Norwegian Cancer Registry. This thesis investigated the role of surgical resection in the treatment of high-grade gliomas and the following conclusions can be drawn: - The combination of BOLD fMRI, DTT, and 3D intraoperative ultrasound may facilitate resection of high-grade gliomas harboured in eloquent areas while preserving motor and language function. - Functional neuronavigation combined with intraoperative 3D ultrasound can, in most patients, enable resection of brain lesions with general anaesthesia without jeopardizing neurological function. - Patients with perioperative complications and surgically acquired deficits were less likely to receive adjuvant therapy. - Early deterioration in HRQL after surgery was independently and markedly associated with impaired survival in patients with glioblastoma. - Advancing age remains a very strong and independent negative prognostic factor in glioblastoma. Although there has been an increase in the aggressiveness of treatment provided to elderly with glioblastoma, the gain for the oldest age group seems at best very modest. The prognosis of the oldest age group remains very poor, despite multimodal treatment.

The Best M.Phil Thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing Prize,1997-1999
published_or_final_version
Pathology
Master
Master of Philosophy

Orientador: Prof. Dr. Roberto Venegeroles Nascimento
Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Física, 2016.
Neste trabalho se estudaram aspectos básicos relacionados com a dinâmica de células cancerígenas do tipo B-Linfoma BCL1 e de gliomas fazendo ênfases neste último caso. O trabalho se iniciou revisando alguns modelos populacionais do câncer inspirados nos trabalhos de Lotka e Volterra o qual oferecem uma descrição muito simples da interação entre o câncer (presa) e o sistema imunológico (caçador). Posteriormente é revisado um modelo global espaço-temporal baseado nas equações de Fisher-Kolmogorov-Petrovsky- Piskounov (FKPP) [1] o qual permitiu aclarar a dicotomia entre proliferação e motilidade associada fortemente ao crescimento tumoral e à invasividade, respectivamente, das células cancerosas. A partir do modelo FKPP também se fez um estudo computacional mais detalhado aplicando diferentes protocolos de tratamentos para analisar seus efeitos sobre o crescimento e desenvolvimento de gliomas. O estudo sugere que um tratamento com maior tempo entre cada dose poderia ser mais ótimo do que um tratamento mais agressivo. Propõe-se também um modelo populacional local do câncer em que se tem em conta o caráter policlonal das células cancerígenas e as interações destas com o sistema imunológico natural e especifico. Neste último modelo se consegui apreciar fenômenos como dormancy state (estado de latência) e escape phase (fase de escape) para valores dos parâmetros correspondentes ao câncer de tipo B-Linfoma BCL1 [2] o qual explica os fenômenos de imunoedição e escape da imunovigilância [3] o qual poderia permitir propor novos protocolos de tratamentos mais apropriados.Depois se fez uma reparametrização do modelo baseado em algumas características mais próprias das células tumorais do tipo glioma e assumindo presença de imunodeficiência com o que se obtém coexistências oscilatórias periódicas tanto da população tumoral assim como das células do sistema imunológico o qual poderia explicar os casos clínicos de remissão e posterior reincidência tumoral. Finalmente se obtiveram baixo certas condições, uma dinâmica caótica na população tumoral o qual poderia explicar os casos clínicos em que se apresentam falta de controlabilidade da doença sobre tudo em pessoas idosas ou com algum quadro clinico que envolve alguma deficiência no funcionamento normal do sistema imunológico.
In this work we studied basic aspects of the dynamics of cancer cell type B-Lymphoma BCL1 and gliomas making strong emphasis in the latter case. We start reviewing some population models of cancer inspired in the work¿s of Lotka and Volterra, which offers a very simple description of the interaction between cancer (prey) and the immune system (Hunter). Subsequently revise a global model space-time based on the equations of Fisher-Kolmogorov-Petrovsky-Piskounov (FKPP) [1] which allowed elucidating the dichotomy between proliferation and strongly associated motility to tumor growth and invasiveness, respectively, of cancer cells. From the FKPP model also made a more detailed computer study applying different treatment protocols to analyze their effects on the growth and development of gliomas. The study suggests that treatment with longer time between each dose could be more optimal than a more aggressive treatment. Is studied also a local population cancer model that takes into account the polyclonal nature of cancer cells, and these interactions with the natural and specific immune system. In the latter model is able to appreciate phenomena as dormancy state and escape phase for values of parameters corresponding to lymphoma cancer BCL1 [2] which explains the phenomena of immunoediting and tumor escape immuno-surveillance [3] allowing elucidating treatments protocols more appropriate. A re-parameterization was made based on some features of tumor cells glioma type and assuming presence of immunodeficiency with that obtained coexistences periodic oscillatory both tumor populations as well as the immune system cells which could explain the clinical cases of remission and subsequent tumor recurrence. Finally obtained under certain conditions, a chaotic dynamics in tumor population which could explain the clinical cases that present lack of controllability of the disease on all in elderly or with some clinical picture involving some deficiency in the normal functioning of the immune system.

Uma variedade de agentes antitumorais é capaz de induzir danos no material genético e estimular respostas como o bloqueio do ciclo celular, reparo do DNA ou apoptose. A resposta inicial das células é o bloqueio no ciclo em uma tentativa de reparar o dano, no entanto, se esse dano for muito extenso ou comprometer o metabolismo celular, uma cascata de sinalização aciona mecanismos alternativos que inibem a proliferação das células e acionam vias de morte. Os astrocitomas malignos são os tumores cerebrais mais comuns que afetam o sistema nervoso central, compreendendo mais de 60% dos tumores cerebrais primários. O tratamento padrão é a radioterapia seguida de quimioterapia, no entanto, o prognóstico para pacientes portadores desse tipo de câncer ainda continua desanimador. A cisplatina é um agente genotóxico largamente empregado no tratamento de gliomas, além de outros tipos de câncer. Essa droga liga-se ao DNA, formando aductos, os quais levam a um bloqueio na duplicação e na transcrição, podendo induzir apoptose nas células dependendo da extensão do dano. No presente trabalho foram avaliadas as respostas celulares ao tratamento com a cisplatina em linhagem de glioma (U343 MG-a) e em fibroblastos normais transformados por SV40 (MRC-5). Foram avaliadas as respostas em termos de sobrevivência celular, indução de apoptose e expressão gênica em larga escala pela técnica de micro-arranjos de cDNA, sendo esta última realizada somente para a linhagem U343. A cisplatina causou uma redução acentuada na sobrevivência das células MRC-5 (~1%) e U343 (< 1%) após cinco dias de tratamento (teste de sobrevivência celular) com concentrações que variavam de 12,5 a 300 ?M. O tratamento por 24 h com iguais concentrações de cisplatina reduziu a sobrevivência das linhagens em cerca de 20-80% (teste de citotoxicidade). Ambas as linhagens sofreram apoptose após o tratamento com diversas concentrações de cisplatina (12,5, 25 e 50 ?M). A linhagem U343 apresentou uma freqüência máxima de apoptose de 20,4% após o tratamento com 25 ?M de cisplatina por 72 h, enquanto a linhagem MRC-5 apresentou 11,0% de apoptose após 50 ?M de cisplatina por 48 h. Os dados de expressão gênica analisados pelo método de micro-aranjos de cDNA, obtidos 48 h após o tratamento das células U343 com 25 ?M de cisplatina, mostraram genes significativamente (p ?0,05) reprimidos relacionados principalmente com alterações no citoesqueleto (TBCD, RHOA, LIMK2 e MARK1), apoptose ou sobrevivência celular (BCL2-XL, ING1, RHOA, VDP, TIMP2, DYRK3 e NFKBIE), invasão celular ou metástase (LIMK2, TIMP2 e CALU), reparo de DNA (SMC1L1) e metabolismo celular (DYRK3, MARK1, TBCD, LIMK2, VDP e P4HB), entre outros processos. Esses dados apontam para um sério comprometimento da maquinaria celular como um todo após os danos induzidos pela cisplatina. Embora o mecanismo de apoptose justifique cerca de 20% da extensão de morte celular, conforme foi comprovado nos ensaios de apoptose (induzida por 25 ?M de cisplatina), a maior parte das células são eliminadas em conseqüência da ação da droga em vários níveis do metabolismo e manutenção da integridade celular, visto o elevado grau de citotoxicidade da cisplatina, demonstrado nos testes de sobrevivência.
A variety of antitumoral agents is capable of inducing DNA damage and eliciting cell cycle arrest, DNA repair or apoptotic responses. The initial response is a cell cycle arrest in an attempt to repair the DNA damage, but under conditions of extensive DNA lesions and high drug cytotoxicity, a signaling cascade triggers alternative mechanisms that inhibit cell proliferation and activate cell death pathways. Astrocytomas are the most common neoplasm of the central nervous system, comprising more than 60% of primary brain tumors. The standard treatment for theses tumors are radiotherapy followed by chemotherapy, however, the prognostic for these patients is still very discouraging. Cisplatin is an efficient DNA-damaging antitumor agent employed for the treatment of various human cancers, including gliomas. This drug binds to DNA, producing diverse types of adducts, which can block replication, transcription, and lead to apoptosis induction. In the present work, we analyzed cellular responses to treatments with the anticancer agent cisplatin in MRC-5 (normal human fibroblasts SV40 transformed) and U343 MG-a (glioma cell line). The responses were evaluated in terms of cell survival, apoptosis induction and profiles of gene expression by the cDNA microarrays method (only for U343 cell line). Cisplatin treatment resulted in a pronounced reduction in MRC-5 cell survival (~ 1%) and U343 (< 1%) after five days of treatment (cell survival test) with several concentrations of cisplatin, ranging from 12.5 to 300 ?M. Following 24h of treatment under similar cisplatin concentrations the survival was reduced at about 20-80% (cytotoxicity test). Both cell lines underwent apoptosis after treatment with different concentrations of cisplatin (12.5; 25 and 50 ?M), but U343 cells presented a maximal frequency of 20.4% apoptosis (25 ?M cisplatin treatment for 72h), while MRC-5 cells presented 11.0% (50 ?M cisplatin treatment for 48h). Analysis of gene expression performed for U343 cells treated with 25 ?M cisplatin for 48h showed several genes that were found significantly (p ? 0,05) down-regulated, most of them related with cytoskeleton alterations (TBCD, RHOA, LIMK2 and MARK1), apoptosis or cell survival (BCL2-XL, ING1, RHOA, VDP, TIMP2, DYRK3 and NFKBIE), cell invasion or metastasis (LIMK2, TIMP2 and CALU), DNA repair (SMC1L1), and cell metabolism (DYRK3, MARK1, TBCD, LIMK2, VDP and P4HB), among others. As a whole, these data demonstrate a serious commitment of the cell machinery after cisplatin-induced cellular damage. About 20% of the cell death corresponds to apoptosis, as was showed by the present assays. However, most of the cells are eliminated by the action of the drug in various levels of the metabolism and maintenance of cell integrity, due to the elevated degree of cisplatin citotoxicity, as demonstrated in cell survival tests.

Novel strategies for the treatment of malignant gliomas are urgently needed. They are characterised by an inherent resistance to both chemo- and radiotherapeutics resulting in unrelenting tumour progression. While the exact mechanisms of treatment resistance remain undefined, it is now recognized that multiple components within the apoptotic pathway are heavily dysregulated in glioma cells and that the over-expression of anti-apoptotic proteins in patient samples correlates with inferior patient survival. The Inhibitor of Apoptosis Proteins (lAPs) represent the final molecular blockade preventing cellular apoptosis and have been identified as a potential rational therapeutic target in gliomas. The work described herein was focused on the development of novel therapeutic strategies that target the lAPs in malignant gliomas, that are readily translatable to the clinic, and that have the potential to improve patient outcomes. The first series of studies examined the hypothesis that targeting the lAPs in conjunction with other conventional and targeted therapies would overcome treatment resistance, and enhance anti-tumour activity. The novel, small molecule, lAP inhibitor LBW242 was shown to successfully target the lAPs in glioma cells and inhibit their ability to bind to and inactivate caspases. However when tested as a single agent in vitro, no stand alone anti-glioma activity of LBW242 was demonstrated. A screen of the activity of LBW242 in combination other pro-apoptotic compounds led to the discovery that lAP inhibition applied in combination with receptor tyrosine kinase (RTK) inhibition led to enhanced caspase activation and induction of apoptosis with a subsequent synergistic anti-glioma effect. The most profound effect was demonstrated with the specific combination of PDGFR and lAP inhibition both in vitro and in vivo as well as in primary patient derived glioma tumourspheres. While multiple RTKs have previously been validated as rational therapeutic targets, the clinical failure of RTK inhibitors in glioma patients has to date remained unexplained. The results in this thesis provide a novel explanation for the resistance of glioma cells to these targeted therapies, and more importantly offer a clinically tractable strategy of overcoming that resistance and improving patient outcomes. The second series of studies investigated the mechanism of synergy between lAP and RTK inhibition. The results showed that PDGFR inhibition does not stimulate apoptosis in glioma cells by previously described pathways. A screen of the entire apoptotic pathway revealed that treatment with imatinib modulates the expression of the anti-apoptotic protein NOL3/ARC. The results showed that imatinib treatment leads to down-regulation of NOL3 and that this effect is critical to the synergy between lAP and PDGFR inhibition. Further analysis suggested a critical role for NOL3 in gliomagenesis and treatment resistance NOL3 was found to be highly expressed in malignant gliomas and with expression levels that are inversely correlated with patient outcomes. A role for NOL3 has not previously been described in malignant gliomas. Finally, a series of studies were undertaken that tested the use of LBW242 in combination with the standard-of-care therapies of irradiation and temozolomide. In vitro assays demonstrated that LBW242 enhanced the pro-apoptotic activity of radiotherapy, and clonogenic assays showed that the combination therapy led to a synergistic anti-glioma effect in multiple glioma cell lines. Athymic mice bearing established human malignant glioma tumour xenografts treated with LBW242 plus radiation and temozolomide demonstrated a profound and synergistic suppression of tumour growth. Neurosphere assays revealed that the combination of radiation and LBW242 led to a pro-apoptotic effect in highly resistant glioma stem cells with a corresponding inhibition of tumour growth. The results indicate a potentially powerful strategy to enhance the therapeutic activity of standard-of-care therapies in glioma patients. Collectively, the findings of the studies in this thesis contribute to a better understanding of the mechanisms of treatment resistance in malignant gliomas, and demonstrate that the pro-apoptotic and anti-glioma effects of radiotherapy, chemotherapy and specific targeted therapies can be enhanced by the addition of a novel, small molecule lAP inhibitor. These results are readily translatable to clinical trial, and offer the potential for improved treatment outcomes for glioma patients.

Cytokines are proteins produced by cells of the immune system. The working hypothesis of the thesis is to show that the lymphocytes and glial cells in Glioblastoma Multiformae patients have an altered pattern of secretion of cytokines compared with low-grade and non-cancerous patients. Lymphocyte subset analysis was performed using flow cytometry. Serum specimens taken from both high and low grade glioma patients were analysed with samples taken from control patients with no history of cancer. Cerebro-spinal fluid was analysed from high-grade glioma patients. Comparisons of high grade pre-surgery serum samples, high grade post-surgery samples, control pre-surgery samples and control post-surgery samples were included. The analysis was completed utilising a luminex immunoassay. This technology is able to measure 34 cancer-associated analytes simultaneously. Immunohistochemistry of candidate biomarkers was done using primary tumour tissue. The results show that there were significant differences in several analytes in the sera and CSF of the different groups. These were follistatin, fibroblast growth factor (FGF), granulocyte – colony stimulating factor (G-CSF), soluble human epidermal growth factor receptor 2 neural (sHER2neu), soluble interleukin-6 receptor alpha (sIL-6R alpha), platelet-derived growth factor - AABB (PDGF-AABB), platelet and endothelial cell adhesion molecule (PECAM-1), stem cell factor (SCF), prolactin, soluble vascular endothelial growth factor receptor 1 (sVEGFR-1) and urokinase plasminogen activator (uPA). The data also show that tumour tissue revealed increased expression of follistatin and G-CSF with little expression of prolactin. In conclusion, these results suggest that potential candidate biomarkers can be used to enable diagnosis of glioma and moreso to distinguish between different grades of glioma using a panel of biomarkers.

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Glioblastoma multiforme (GMB) is the most malignant and common type of all astrocytic tumours. Current standard treatment for GBM patients involves maximum surgical resection of the tumour, followed by radiotherapy and chemotherapy, usually containing the alkylating agent Temozolomide (TMZ). Despite this aggressive combination therapy, the survival rate of GBM patients is still low. This work consisted in investigating the cytotoxic effects of Akt-inhibition by MK-2206 with irradiation (RT) and TMZ on in vitro human malignant glioma. Seven malignant glioma cell lines were cultured and tested for clonogenic survival, invasion inhibition, tumour spheroid growth and proliferation. The Akt-inhibitor MK-2206 and TMZ were added at different time treatments and in varying doses. Cultures were irradiated with single dose and with fractionated γ-irradiation. Cellular modulation of Akt and p-Akt were assessed by Western blot analysis. MK-2206 reduced the levels of phospho- Akt key protein in the PI3Kinase-Akt pathway, decreased cell survival, and inhibited invasion, proliferation and cell growth. The combination of MK-2206 and RT lead to enhanced inhibition of cell proliferation and invasion, which is not observed with RT alone. The radioenhancing effect of MK-2206 was most striking in inhibition of spheroid volume growth by fractionated RT; the radiosensitizing effect of MK-2206 was stronger than that of TMZ. MK-2206 enhanced the in vitro effects of RT and TMZ in terms of decreased cell survival, invasion, proliferation and growth in malignant glioma. Effects could be ascribed to inhibition of PI3K-Akt pathway.
O Glioblastoma multiforme (GBM) é o tipo mais maligno e mais comum de todos tumores astrocíticos. O tratamento atual para pacientes de GBM envolve máxima remoção cirúrgica, seguida de radio e quimioterapia, normalmente com o agente alquilante Temozolamida (TMZ). Apesar da agressividade da terapia combinada, o tempo de sobrevivência dos pacientes ainda é baixo. Este trabalho procurou investigar os efeitos citotóxicos do inibidor de Akt MK-2206 em combinação com irradiação (RT) e TMZ em um painel de células de gliomas humanos. Sete linhagens de glioma foram cultivadas e testadas em ensaio de sobrevivência clonogênica, inibição de invasão, e modelos de proliferação e crescimento de volume em esferóides. O inibidor MK-2206 e TMZ foram adicionados em diferentes tempos de tratamento e diferentes doses. As culturas foram irradiadas com doses únicas ou em terapias fracionadas com irradiação γ. A modulação celular de Akt e fosfo-Akt foi checada via Western Blot. O composto MK-2206 reduziu a fosforilação da proteína chave Akt na via PI3K, diminuindo a sobrevivência celular e inibindo invasão, proliferação e crescimento celular. A combinação de MK-2206 com RT levou a uma maior inibição de invasão e proliferação, o que não é observado somente com a RT. O efeito radiosensível de MK-2206 foi ainda maior na inibição do volume dos esferóides em terapia combinada com RT fracionada, sendo ainda maior do que o efeito combinado com TMZ. MK-2206 aumentou os efeitos in vitro de RT e TMZ em termos de redução de sobrevivência celular, invasão, proliferação e crescimento celular em gliomas malignos. Os efeitos podem ser atribuídos a inibição da via PI3KAkt.

The translocator protein (TSPO) is an 18 kDa molecule spanning the outer mitochondrial membrane. Structural predictions, reported physical interactions and experimental data from diverse models suggest that TSPO is involved in apoptosis, proliferation and steroidogenesis. TSPO is up-regulated in several human cancers and its high levels are frequently associated with advanced disease and poor outcome. TSPO was shown to be up-regulated in astrocytomas and this was correlated with high tumour grade and shorter patient survival. Here it is confirmed that TSPO is highly expressed in astrocytomas and this is correlated with tumour grade. TSPO expression is significantly higher in astrocytic tumours than in oligodendrogliomas and this differential expression is epigenetically driven. Low TSPO expression in oligodendrogliomas is associated with frequent promoter methylation, whereas in astrocytomas, where TSPO is expressed at high levels, the promoter typically remains unmethylated. As oligodendrogliomas and astrocytomas have very different biology and prognosis, and several TSPO ligands, including those enabling positron emission tomography (PET) scanning, are in development, we propose pre-operative PET imaging to differentiate these two histotypes. TSPO is not an independent prognostic factor in our patient population. The main cell population expressing TSPO within the tumour bulk are neoplastic cells. Consistent with no evidence for TSPO as a strong prognostic factor in gliomas, no alteration in proliferation, cell cycle progression and baseline or induced apoptosis following TSPO siRNA knock-down in an in vitro astrocytoma model is demonstrated. Furthermore, there was no evidence of cortisol production by several astrocytoma cell lines in vitro despite TSPO protein expression. Out of the examined MPTP-related binding and functional partners of TSPO (VDAC1, ANT, HK I and II) only HKII up-regulation appears be reproducibly related to patient outcome in gliomas. Consistently, targeting hexokinases in vitro in a glioma model inhibited proliferation and increased apoptosis.

Several biomarkers had been proposed as useful parameters to better define the prognosis or to delineate new target therapy strategies for glioblastoma (GBM) patients. MicroRNAs could represent interesting molecules, for their role in tumorigenesis and cancer progression and for their specific tissue expression. Although many studies have tried to identify a specific microRNAs signature for glioblastoma, by now an exhaustive GBM microRNAs profile is far to be well defined. In this work we set up a real-time qPCR, based on LNA primers, to investigate the expression of 19 microRNAs in brain tumors, focusing our attention on GBMs. MiRNAs expression in 30 GBM paired FFPE-Fresh/Frozen samples was firstly analyzed. The good correlation obtained comparing miRNAs results confirmed the feasibility of performing miRNAs analysis starting from FFPE tissues. This leads to many advantages, as a good disposal of archival tumor and normal brain specimens and the possibility to verify the percentage of tumor cells in the analyzed sample. In the second part we compared 3 non-neoplastic brain references to use as control in miRNAs analysis. Normal adjacent the tumor, epileptic specimens and a commercial total RNA were analyzed for miRNAs expression and results showed that different non-neoplastic controls could lead to important discrepancies in GBM miRNAs profiles. Analyzing 50 FFPE GBMs using all 3 non-neoplastic references, we defined a putative GBM miRNAs signature: mir-10b, miR-21 and miR-27a resulted upregulated, while miR-7, miR-9, miR-26a, miR-31, miR-101, miR-137, miR-222 and miR-330 were downregulated. Comparing miRNAs expression among GBM group and gliomas of grade I, II and III, we obtained 3 miRNAs (miR-10b, mir-34a and miR-101) showing a different regulation status between high grade and low grade gliomas. Intriguingly, miR-10b was upregulated in high grade and significantly downregulated in low grade gliomas, suggesting that could be a candidate for a GBM target therapy.

Several biomarkers had been proposed as useful parameters to better define the prognosis or to delineate new target therapy strategies for glioblastoma (GBM) patients. MicroRNAs could represent interesting molecules, for their role in tumorigenesis and cancer progression and for their specific tissue expression. Although many studies have tried to identify a specific microRNAs signature for glioblastoma, by now an exhaustive GBM microRNAs profile is far to be well defined. In this work we set up a real-time qPCR, based on LNA primers, to investigate the expression of 19 microRNAs in brain tumors, focusing our attention on GBMs. MiRNAs expression in 30 GBM paired FFPE-Fresh/Frozen samples was firstly analyzed. The good correlation obtained comparing miRNAs results confirmed the feasibility of performing miRNAs analysis starting from FFPE tissues. This leads to many advantages, as a good disposal of archival tumor and normal brain specimens and the possibility to verify the percentage of tumor cells in the analyzed sample. In the second part we compared 3 non-neoplastic brain references to use as control in miRNAs analysis. Normal adjacent the tumor, epileptic specimens and a commercial total RNA were analyzed for miRNAs expression and results showed that different non-neoplastic controls could lead to important discrepancies in GBM miRNAs profiles. Analyzing 50 FFPE GBMs using all 3 non-neoplastic references, we defined a putative GBM miRNAs signature: mir-10b, miR-21 and miR-27a resulted upregulated, while miR-7, miR-9, miR-26a, miR-31, miR-101, miR-137, miR-222 and miR-330 were downregulated. Comparing miRNAs expression among GBM group and gliomas of grade I, II and III, we obtained 3 miRNAs (miR-10b, mir-34a and miR-101) showing a different regulation status between high grade and low grade gliomas. Intriguingly, miR-10b was upregulated in high grade and significantly downregulated in low grade gliomas, suggesting that could be a candidate for a GBM target therapy.

Le glioblastome qui correspond au stade de malignité le plus élevé des gliomes est associé à un très mauvais pronostic car il envahit le parenchyme cérébral de manière diffuse, ce qui rend son exérèse complète généralement impossible, et il résiste aux traitements conventionnels en raison de sa résistance intrinsèque aux stimuli pro-apoptotiques. Le cancer de l’œsophage est également un cancer très agressif car invasif et résistant également aux stimuli pro-apoptotiques.

Les chémokines sont des cytokines chémotactiques responsables de la migration des leucocytes et exprimées en réponse à des cytokines inflammatoires, à des facteurs de croissance et à des stimuli pathogènes. De nombreux cancers possèdent un réseau complexe de ces chémokines. Les chémokines de type CXCL et plus particulièrement CXCL8 et CXCL12 sont impliquées dans la biologie des gliomes et du cancer de l’oesophage. Au cours de mon travail de thèse de doctorat, nous avons étudié l’expression des 15 chémokines CXCL et des 9 récepteurs aux chémokines CXCL dans divers modèles de gliomes et de cancers de l’œsophage. Cette étude menée par RT-PCR nous a permis de mettre en évidence la présence d’un patron d’expression complexe de ces chémokines CXCL dans les divers modèles analysés. Nous avons observé une expression plus importante des chémokines CXCL pro-angiogéniques par rapport aux chémokines anti-angiogéniques dans ces deux types de cancers. Nous avons également pu mettre en évidence une implication potentielle des chémokines CXCL2, CXCL3 et CXCL8 dans l’acquisition de la résistance au traitement par témozolomide des gliomes d’origine astrogliale.

Les glioblastomes et les cancers de l’œsophage étant deux types de cancers résistants aux stimuli pro-apoptotiques, et le témozolomide étant la seule molécule dotée de bénéfices thérapeutiques réels dans le cas du glioblastome, nous avons également testé le témozolomide dans nos modèles de cancer de l’œsophage in vitro et in vivo. Nous avons pu ainsi montrer un bénéfice thérapeutique réel apporté par cette molécule in vivo sur des animaux immunodéficients greffés avec des cellules humaines de carcinome épidermoïde de l’œsophage. Ce bénéfice thérapeutique peut être expliqué en partie par différents mécanismes d’action tels que l’induction de processus soutenus d’autophagie suivis par de l’apoptose mais également par des effets anti-angiogéniques. Enfin, nous avons pu montrer que la diminution d’expression même transitoire de la chémokine CXCL2 dans nos modèles in vitro de glioblastome et de carcinome épidermoïde de l’œsophage entraîne une diminution de la croissance de ces populations cellulaires cancéreuses, suggérant un rôle important de cette chémokine dans la biologie de ces deux types de cancers. Enfin, nous avons démontré un effet anti-angiogénique in vivo pour le témozolomide dans un modèle de xénogreffes de cancers oesophagiens humains chez la souris immunodéficiente.

En conclusion, l’ensemble de nos résultats suggèrent que le témozolomide, bien qu’il devienne bientôt un générique sous sa forme d’administration i.v. (la forme orale étant déjà générique), pourrait représenter une molécule d’intérêt pour combattre le cancer de l’œsophage, comme on le sait déjà depuis 2005 en ce qui concerne les glioblastomes. Nos résultats montrent ensuite l’importance du patron d’expression des chémokines CXCL dans la biologie des cellules gliales tumorales et des cellules cancéreuses de l’œsophage. Enfin, nos résultats montrent que le témozolomide détruit en partie ce réseau de chémokines CXCL au sein de ces deux types de cancers.

Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

El maneig dels gliomes de baix grau es basa en les característiques clíniques i radiològiques, incloent la classificació pronostica de Pignatti, que classifica als pacients com de baix o d'alt risc de recaiguda . Per determinar si les dades moleculars poden definir d’ una forma mes sensible el pronòstic dels pacient, hem examinat diverses alteracions moleculars en una cohort de 58 pacients. Hem registrat les dades clíniques i radiològiques que permeten obtenir la classificació pronòstica segons els criteris de Pignatti, i hem dut a terme l’estudi de les mutacions de IDH, les mutacions de TP53, la codeleció 1p / 19q, i la metilació del promotor de MGMT. Hem correlacionat les nostres troballes amb la supervivència lliure de progressió (SLP ) i la supervivència global (SG). L'edat mitjana dels pacients va ser de 45 anys; 69% de pacients es van classificar com de baix risc de recaiguda. Es van detectar mutacions de IDH en el 62% de casos, mutacions de TP53 en el 17%, codeletion 1p / 19q en el 46%, i metilació de MGMT en 40% dels pacients. Les anàlisis de supervivència es van realitzar en els 49 pacients que no presentaven captació de contrast a la resonància magnètica basal. En l'anàlisi univariant, les mutacions de IDH, la codeleció de 1p / 19q, i la combinació de les mutacions IDH amb la codeleció 1p / 19q es van associar amb una millor SLP (P = 0,006, P = 0,037 i P = 0,003, respectivament) i SG (P <0,001, P = 0,02 i P <0,001, respectivament). L'anàlisi multivariant va identificar la absència de mutacions de IDH com factor de risc en relació a la progressió ( [HR] = 3,1; P = 0,007) i a la mort (HR = 6,4; P <0.001). Podem concloure d’acord amb els nostres resultats, que l'estat mutacional de IDH es un marcador pronòstic independent a les variables clíniques, radiològiques i altres moleculars i per tant es planteja com a eina decisiva a l'hora de definir el tractament post-quirúrgic dels pacients amb gliomes de baix grau.
El manejo de los gliomas de bajo grado se basa en las características clínicas y radiológicas, incluyendo la clasificación pronóstica de Pignatti, que clasifica a los pacientes como de bajo o de alto riesgo de recaída. Para determinar si los datos moleculares pueden definir de una forma más sensible el pronóstico del paciente, hemos examinado varias alteraciones moleculares en una cohorte de 58 pacientes. Hemos registrado los datos clínicos y radiológicos que permiten obtener la clasificación pronóstica según los criterios de Pignatti, y hemos llevado a cabo el estudio de las mutaciones de IDH, las mutaciones de TP53, la codeleción 1p / 19q, y la metilación del promotor de MGMT. Hemos correlacionado nuestros hallazgos con la supervivencia libre de progresión (SLP) y la supervivencia global (SG). La edad media de los pacientes fue de 45 años; 69% de pacientes se clasificaron como de bajo riesgo de recaída. Se detectaron mutaciones de IDH en el 62% de casos, mutaciones de TP53 en el 17%, codeleción 1p / 19q en el 46%, y metilación de MGMT en 40% de los pacientes. Los análisis de supervivencia se realizaron en los 49 pacientes que no presentaban captación de contraste en la resonancia magnética basal. En el análisis univariante, las mutaciones de IDH, la codeleción de 1p / 19q, y la combinación de las mutaciones IDH con la codeleción 1p / 19q se asociaron con una mejor SLP (P = 0,006, P = 0,037 y P = 0,003, respectivamente) y SG (P <0,001, P = 0,02 y P <0,001, respectivamente). El análisis multivariante identificó la ausencia de mutaciones de IDH como factor de riesgo en relación a la progresión ([HR] = 3,1; P = 0,007) y la muerte (HR = 6,4; P <0.001). Podemos concluir de acuerdo con nuestros resultados, que el estado mutacional de IDH es un marcador pronóstico independiente de las variables clínicas, radiológicas y otras moleculares y por tanto se plantea como una herramienta decisiva a la hora de definir el tratamiento post- quirúrgico de los pacientes con gliomas de bajo grado.
Management of low-grade gliomas (LGG) is based on clinical and radiologic features, including the Pignatti prognostic scoring system, which classifies patients as low- or high-risk. To determine whether molecular data can offer advantages over these features, we have examined the prognostic impact of several molecular alterations in LGG. In a cohort of 58 patients with LGG, we have retrospectively analyzed clinical and molecular characteristics, including the Pignatti criteria, IDH mutations, TP53 mutations, the 1p/19q deletion, and MGMT methylation, and correlated our findings with progression-free survival (PFS) and overall survival (OS). Mean age of patients was 45 years; 69% were classified as low-risk by the Pignatti system. IDH mutations were detected in 62%, p53 mutations in 17%, the 1p/19q codeletion in 46%, and MGMT methylation in 40% of patients. Survival analyses were performed in the 49 patients without contrast enhancement. In the univariate analysis, IDH mutations, the 1p/19q codeletion, and the combination of IDH mutations with the 1p/19q codeletion were associated with both longer PFS (P = 0.006, P = 0.037, and P = 0.003, respectively) and longer OS (P < 0.001, P = 0.02, and P < 0.001, respectively). The multivariate analysis identified absence of IDH mutations as a factor for greater risk of progression (hazard ratio [HR] = 3.1; P = 0.007) and death (HR = 6.4; P < 0.001). We conclude that IDH mutations may be more effective than the Pignatti score in discriminating low- and high-risk patients with LGG.

Os gliomas representam mais de 70% dos tumores cerebrais primários. Os glioblastomas multiformes são gliomas malignos caracterizados por baixa incidência, mas altas taxas de mortalidade. Apesar da responsividade inicial ao tratamento padrão realizado com o quimioterápico alquilante temozolomida (TMZ), poucos foram os avanços para o prognóstico dos pacientes nos últimos 10 anos. Isso deve-se ao fato desses tumores serem raramente passíveis de ressecção cirúrgica e apresentarem alta taxa de recorrência. Além disso, a eficácia de seu tratamento encontra barreiras como efeitos colaterais indesejáveis e resistência quimioterápica. Nesse cenário, a descoberta de novas substâncias que possam atuar com efeito aditivo ou sinérgico e aumentem a sensibilização de células tumorais ao tratamento, torna-se uma estratégia terapêutica no campo da oncologia. O ditelureto de difenila (DTDF) é um composto orgânico contendo telúrio que apresenta interessantes efeitos biológicos in vitro, como antioxidante, quimioprotetivo, citotóxico e antitumoral. Sendo assim, o objetivo deste estudo foi avaliar os efeitos do DTDF e do quimioterápico TMZ, em regimes isolados e em associação. Para tal foram investigados seus efeitos citotóxicos, após exposição aguda e crônica, em culturas celulares de glioma não-resistente (M059J) e resistente à TMZ (GBM). No ensaio de viabilidade celular a TMZ apresentou citotoxicidade para as linhagens celulares testadas, com valor de IC50 maior na linhagem resistente do que quando comparado à linhagem não-resistente. Este dado foi confirmado pelo teste de duplicação cumulativa de população e, também, pela coloração com laranja de acridina, após o tratamento de 120 h, por observar-se um aumento na frequência de células positivas para a formação de organelas vesiculares ácidas em ambas as linhagens, sendo predominantemente em células GBM. Além disso, foi observado que o tratamento associando DTDF e a TMZ apresentou uma maior citotoxicidade quando comparado aos tratamentos isolados, após 120 h de tratamento. Portanto, o DTDF sensibilizou as células ao tratamento com TMZ. Essa sensibilização ocorreu em níveis aproximados para ambas as linhagens, sendo os efeitos do DTDF independentes do perfil de resistência à TMZ. Em conjunto, os dados desse trabalho sugerem o uso do DTDF em associação à TMZ como uma estratégia para reduzir as doses de TMZ empregadas na clínica e diminuir efeitos colaterais aos pacientes em tratamento de glioma
Gliomas represent more than 70% of primary brain tumors. Malignant gliomas are characterized by low incidence, but high mortality rates. Despite the initial responsiveness to the standard treatment with the chemotherapeutic alkylating temozolomide (TMZ), few advances have been made in the prognosis of patients in the last 10 years. This is due to the fact that these tumors are rarely amenable to surgical resection and have a high rate of recurrence. Moreover, the effectiveness of this treatment encounters barriers such as undesirable side effects and chemotherapeutic resistance. In this scenario, the discovery of new substances that may act with additive or synergistic effect and increase the sensitization of tumor cells to the treatment becomes a therapeutic strategy in the field of oncology. Diphenyl ditelluride (DPDT) is a derivative of tellurium used in various reactions of organic synthesis and has interesting in vitro biological effects, as antioxidant, chemoprotective, cytotoxic and antitumor agent. Therefore this work aimed to evaluate the cytotoxic effects of this organotellurium compound and the chemotherapeutic, TMZ, in isolated and in association regimens, after acute and chronic exposure, of non-resistant (M059J) and TMZ- resistant (GBM) glioma cells. Through the cell viability assay, it was shown that TMZ is cytotoxic for both cell lines tested, showing a higher IC50 value in the resistant line when compared to the other line. This data was confirmed by the cumulative population doubling test. In addition, by the acridine orange staining, it was verified that autophagy might favor the chemoresistance, although not being the main resistance mechanism in the lines tested. It was observed that DPDT clearly has a dose-dependent cytotoxic effect on the M059J and GBM cell lines, in a lower concentration range than that used with TMZ. DPDT sensitized the cells to TMZ treatment as evidenced by the decline in cell viability. It is important to point out that this sensitization occurred in low and approximate IC50 values after both 24 h and 120 h of treatment, being the effects of the DPDT independent of the resistance profile to TMZ. Taken together, data from this work suggest the use of DPDT in association with TMZ as an interesting strategy to reduce the doses of TMZ used in the clinic and to reduce side effects to patients under treatment of glioma.

O tratamento dos gliomas malignos cerebrais é um dos grandes desafios da medicina atualmente, pois, apesar do grande avanço no conhecimento destes tumores, o prognóstico de vida dos portadores desta doença é muito ruim. Foram estudados retrospectivamente 61 pacientes com diagnóstico de glioblastoma multiforme ou astrocitoma anaplásico, no período de 1998 a 2002, com o objetivo de avaliar o uso do Paclitaxel como potencializador do tratamento radioterápico destes tumores. Todos os pacientes foram tratados inicialmente com cirurgia para retirada ampla do volume tumoral (mínimo de 80%) seguido de tratamento com radioterapia fracionada e reforço com radiocirurgia estereotáxica. Em caso de crescimento tumoral, após o tratamento inicial dos pacientes com KPS > 70, novo tratamento cirúrgico e nova radiocirurgia foram indicados. Destes 61 pacientes, 32 receberam tratamento com Paclitaxel, na dose de 100mg/m2 e 29 pacientes não receberam nenhum tipo de quimioterápico. Os grupos foram comparados, em relação ao tipo histológico, faixa etária, sexo e localização tumoral, não havendo diferenças estatisticamente significantes entre os mesmos. Os pacientes de ambos os grupos tiveram acompanhamento laboratorial antes, durante e após o tratamento com paclitaxel e foram acompanhados até o óbito causado pela doença. Foram excluídos do estudo os portadores de tumor que foram a óbito por outras causas. A análise dos resultados mostrou que não houve diferenças estatísticas em relação à sobrevida média do grupo tratado com Paclitaxel e o grupo sem o tratamento (p=1,000). Da mesma forma, a comparação entre os pacientes com glioblastomas (p=0,8933) e com astrocitomas anaplásicos (p=0,5920) de ambos os grupos não mostrou diferença estatística em relação à sobrevida. O número de craniotomias( p=0,5268) e o número de radiocirurgias (p=0,3666) foram semelhantes estatisticamente. Os estudos laboratoriais realizados durante o tratamento no grupo que recebeu o paclitaxel, não mostraram alterações que levassem à suspensão do tratamento. A análise dos resultados deste estudo permitiu concluir que o uso do paclitaxel, concomitante ao tratamento radioterápico dos gliomas malignos cerebrais, não mostrou nenhum ganho adicional na sobrevida dos pacientes portadores destes tumores e, pela análise da necessidade de novo tratamento durante o curso da doença, não potencializou os efeitos da radioterapia. Palavras-chave: Paclitaxel, gliomas malignos, radioterapia
Nowadays, the treatment of the malignant cerebral gliomas is one of the greatest challenges for neurosurgons. Despite of the advance regarding these tumors? knowledge expectance of life for these patients is very bad. The main purpose of this study was to evaluate the use of paclitaxel to enhance the radiotherapy treatment in those tumors. Sixty-one patients with diagnosis for glioblastoma multiforme or anaplastic astrocytoma in the period of 1998 to 2002 were, retrospectively, studied. All patients were initially treated with surgery in order to remove a wide portion of the tumor?s volume (minimum of 80%). Then, the patients were treated with fractionated radiotherapy and reinforcement with stereotactic radiosurgery. If there was an increase in the tumor after the initial treatment, the patients with a KPS higher than 70 had new treatment with surgery as well as with radiosurgery. Among the 61 patients, 32 were treated with a 100 mg/m2 dose of paclitaxel, and 29 of then did not have any kind of chemotherapy treatment. Comparisons bettwen both regardhg to the histological type, age, gender and location of the tumor showed no differences . Patients of both groups had a laboratory follow-up before, during and after the treatment with paclitaxel. All of them were followed until their death, which was caused by the disease. Patients that died from other diseases were not included in the study. The analysis of the results indicated that there were no statistics differences regarding the mean survival time between the groups treated or nor treated with paclitaxel ( p=1,000). Likewise, a comparison between the glioblastomas (p=0,8933) and the anaplastic astrocytomas (p=0,5920) of both groups did not indicate any statistic difference regarding to the survival time. The was no statistic difference between the number of craniotomies (p=0,5268) as well as between the number of radiosurgeries. (p=0,3666). The laboratory studies held during the treatment of the group that received the paclitaxel did not show any changes which could lead to cease the treatment. Hence the results led to the conclusion that the treatment of malignant cerebral gliomas with paclitaxel and radiotherapy treatment at the same time did not give any additional gain in the patients?survival. Regarding to the demand for new treatment throughout the disease, there was no enhance of the radiotherapy effects with the Paclitaxel. Key words: Paclitaxel, malignant gliomas, radiotherapy.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-Graduação em Neurociências.
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Neste estudo foi avaliado o efeito da derrota social sobre a progressão tumoral de células de glioma C6 inoculadas no cérebro de ratos. Os animais foram divididos em 4 grupos: inoculados com salina (grupo S, controle), inoculados com glioma C6 (grupo I), inoculados com glioma e depois submetidos a dupla derrota social (grupo I/D) e derrotados e depois inoculados com glioma (grupo D/I). Após 15 dias da inoculação, os animais foram avaliados quanto à progressão tumoral, sobrevivência, atividade motora (teste de campo aberto) e parâmetros imunológicos (níveis séricos de IgG contra glioma C6 e contagem sanguínea de linfócitos, granulócitos e monócitos). A progressão tumoral nos grupos inoculado e depois derrotado (I/D) e derrotado e depois inoculado (D/I) aumentou 69% e 41%, respectivamente, em relação ao grupo inoculado (I); o grupo I/D apresentou uma progressão tumoral 19% maior do que o grupo D/I. A atividade motora dos animais dos grupos I/D e D/I foi reduzida em 52% e 26%, respectivamente. Os animais I/D e D/I sofreram uma redução da sobrevivência de 34% e de 16%, respectivamente, quando comparados ao grupo I. Os níveis séricos de IgG contra glioma C6 foram aumentados nos animais inoculados com glioma (grupos I, I/D e D/I); entretanto no grupo I/D este aumento foi menor. No grupo I/D também mostrou uma redução no número de linfócitos e de granulócitos. Em conclusão, os resultados indicaram que a derrota social em ratos aumentou a progressão tumoral de glioma e reduziu a sobrevivência e alguns parâmetros da resposta imune dos animais observadas no modelo experimental proposto.

Glioblastoma multiforme (GBM) is the most common form of primary brain tumor in humans. Its aggressive and infiltrative growth into the brain, and, at best, only partial sensitivity to radiotherapy and chemotherapy, renders it extremely difficult to treat and survival remains dismal.

Growth factors, such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), and their corresponding receptors are seen in glioma tissue, suggesting the presence of autocrine stimulatory loops. PDGFB and a mutated EGF receptor were also identified as cellular homologues of two oncoviruses, thereby indicating a role in tumorigenesis. This thesis presents a brain tumor model in mice, developed using a PDGFB coding retrovirus to induce overexpression of PDGF-B in neonatal mouse brain. Immature tumors, with histological characteristics of primary brain tumors developed at relatively high frequencies. Mice injected with a non-coding retrovirus did not develop tumors, indicating the crucial role of PDGF stimulation in this system. Tumor cells were also shown to continue to depend on PDGF stimulation when cultured in vitro.

In human glioblastomas, growth factor receptor signaling is present in conjunction with defects in cell cycle arrest pathways. When the PDGFB-virus model was used with p53 or Ink4a-Arf deficient mice, tumors arose with shorter latency and higher frequency. Loss of p53 or Ink4a-Arf seemed to facilitate signaling through the PI3K/Akt pathway. Thus, a functional role for the co-existence of p53 loss of function and PDGF signaling in a subset of gliomas is presented.

Human GBM samples were collected and analyzed with respect to expression and activation of the EGFR and PDGFRα. Most tumors expressed the both receptors at moderate to high levels, but high activation of either receptor seemed mutually exclusive.

Functional overexpression of the ATP binding cassette (ABC) transporters at the cell surface is thought to be responsible for clinical multidrug resistance (MDR) in tumours of the brain. Inhibition of ABC transporters by existing inhibitors has proven to be inconclusive. This research program hypothesized an alternative location for the ABC transporters in glioblastoma cells and also proposed to develop stationary phases for the identification of ABC transporters inhibitors. Expression profile investigation of P-glycoprotein (PGP), multidrug resistant protein 1 (MRP1), multidrug resistant protein 2 (MRP2) and the breast cancer resistant protein (BCRP) in glioblastoma multiforme cell lines and clinical patient specimens suggested varying levels of expression. Localisation studies by confocal microscopy confirmed cell surface expression and also indicated that BCRP was localised at the nucleus of the T98 and LN229 cells. Immunoblots of LN229 nuclear extracts indicated ~ 2 fold higher expression of BCRP as compared to cytoplasmic extracts. Immunohistochemistry studies with clinical samples confirmed the nuclear and perinuclear location of BCRP. IC50 value for Mitoxantrone (MTX); a BCRP substrate was calculated as 0.29 ± 0.020 μM for the LN229 cell line, and pre-treatment with the cell impermeant fumitremorgin C 3 (FTC, 5 μM) slightly reduced the IC50 value to 0.16 ± 0.087 μM. This refractoriness to FTC is in contrast with the literature showing a ~ 6-fold reduction in IC50 value of MTX upon pre-treatment with FTC in human breast cancer MCF-7 cell line with ectopic expression of BCRP. The results supported the notion that the nuclear presence of endogenously expressed BCRP actively extrudes MTX, and that because FTC is not able to inhibit the nuclear BCRP, significant reduction in the IC50 was not observed. The results suggest that the treatment of clinical MDR should be expanded to include inhibition of ABC transporters functioning at the nuclear membrane. Cellular membrane affinity chromatography columns were developed for the study of the MRP1, MRP2 and BCRP using Spodoptera frugiperda (Sf9) cells that had been stably transfected with human Mrp1, Mrp2 or Bcrp cDNA. The resulting columns and a control column were characterized using frontal affinity chromatography using [3H]-etoposide as the marker ligand and etoposide, benzbromarone and MK571 as the displacers on the CMAC(Sf9MRP1) column, etoposide and furosemide on the CMAC(Sf9MRP2) column and etoposide and fumitremorgin C on the CMAC(Sf9BCRP) column. The binding affinities obtained from the chromatographic studies were consistent with the data obtained using non-chromatographic techniques and the results indicate that the immobilized MRP1, MRP2 and BCRP transporters retained their ability to selectively bind known ligands. The results indicated that the CMAC(Sf9MRP1), CMAC(Sf9MRP2) and CMAC(Sf9BCRP) columns can be used for the study of binding to the MRP1, 4 MRP2 and BCRP transporters and that membranes from the Sf9 cell line can be used to prepare CMAC columns. This study expands our knowledge of the ABC transporters and makes a case for the finding that nuclear efflux proteins play a pivotal role in the overall MDR phenotype in CNS tumours. Also the CMAC columns developed and characterised provide a tool to study the binding of potential therapeutic candidates to ABC proteins.

This work examines the prognostic value of advanced MR at selected time points during the early stages of treatment in glioma patients. In this thesis, serial imaging of glioma patients was conducted using diffusion tensor imaging (DTI), dynamic contrast enhanced (DCE) and dynamic susceptibility contrast (DSC) MRI. A methodology for the processing and registration of multiparametric MRI was developed in order to simultaneously sample whole tumour measurements of multiple MR parameters with the same volume of interest. Differences between glioma grades were investigated using functional MR parameters and tested using Kruskal-Wallis tests. A 2-stage logistic regression model was developed to grade lesions from the preoperative MR, with the model retaining the apparent diffusion coefficient, radial diffusivity, anisotropic component of diffusion, vessel permeability and extravascular extracellular space parameters for glioma grading. A multi-echo single voxel spectroscopic sequence was independently investigated for the classification of gliomas into different grades. From preoperative MR, progression-free survival was predicted using the multiparametric MR data. Individual parameters were investigated using Kaplan-Meier survival analysis, before Cox regression modelling was used for a multiparametric analysis. Radial diffusivity, spin–lattice relaxation rate and blood volume fraction calculated from the DTI and DCE MRI were retained in the final model. MR parameter values were also investigated during the early stages of adjuvant treatment. Patients were scanned before and after chemoradiotherapy, with the change in MR parameters as well as the absolute values investigated for their prognostic information. Cox regression analysis was also performed for the adjuvant treatment imaging, with measures of the apparent diffusion coefficient, spin–lattice relaxation rate, vessel permeability and extravascular extracellular space, derived from the DTI and DCE datasets most predictive of progression-free survival. In conclusion, this thesis demonstrates multiparametric MR of gliomas during the early stages of treatment contains useful prognostic information relating to grade and progression-free survival interval.

Sobrevida após radioterapia em pacientes com glioma de alto grau Introdução: Gliomas de alto grau são os principais tumores primários do sistema nervoso central em adultos, cujo prognóstico permanece invariavelmente ruim, mesmo com a terapia atual: cirurgia e quimioirradiação. Na radioterapia utiliza-se de técnicas conformacionais (3DRT) e de intensidade modulada do feixe (IMRT). Objetivos: Avaliar a sobrevida dos pacientes tratados na instituição e os fatores que influenciam os resultados. Métodos: Análise retrospectiva dos tratamentos terapêuticos de pacientes diagnosticados com gliomas de alto grau entre 2009 e 2014 e tratados com radioterapia no Serviço de Radioterapia do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (HCRP-USP). Resultados: A sobrevida mediana obtida foi 16,63 meses, 29 pacientes (61,7%) foram à óbito e os demais encontravam-se em seguimento até o momento da análise. IMRT correspondeu à modalidade de tratamento em 68,1% dos casos, a duração média da radioterapia foi de 56 dias, com intervalo médio (demora) entre cirurgia e o início da radioterapia de 61,7 dias (27 a 123 dias). Observamos que idade, ressecção macroscópica total, tipo histológico e utilização de temozolomida adjuvante influenciaram a taxa de sobrevida (p<0,05). A sobrevida global estimada é de 18 meses (estimativa Kaplan Meyer). Nossa série avaliou os pacientes tratados e os dados corroboram os já relatados na literatura, respaldando o protocolo de tratamento institucional.
Introduction: High-grade gliomas are the main primary tumors of the central nervous system in adults, it`s prognosis remains invariably bad, even with current therapy: chemoradiation after surgery. Radiotherapy is currently employed as conformational techniques (3DRT) or intensity modulated beam (IMRT) with or without temozolomide. Objectives: To evaluate survival of patients treated with radiation and factors influencing results. Methods: Retrospective analysis of patients diagnosed with high-grade gliomas between 2009 and 2014 and treated with radiotherapy at the University Hospital of the Faculty of Medicine of Ribeirão Preto, University of São Paulo (HCRP-USP). Results: Median survival was 16.63 months, 29 patients (61.7%) died and the others were in follow-up at the time of analysis. IMRT was used in 68.1% of cases, the mean duration of radiotherapy obtained of 56 days, with a mean interval (delay) between the surgery/diagnosis and radiotherapy was 61.7 days (27-123 days). We found that age, total gross resection, histological type and use of adjuvant temozolomide influenced the survival rate (p <0.05). The estimated overall survival was 18 months (Kaplan Meyer estimator). Our data was similar to those reported in the literature, supporting the institutional treatment protocol.

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Glioblastoma multiforme (GMB) is the most malignant and common type of all astrocytic tumours. Current standard treatment for GBM patients involves maximum surgical resection of the tumour, followed by radiotherapy and chemotherapy, usually containing the alkylating agent Temozolomide (TMZ). Despite this aggressive combination therapy, the survival rate of GBM patients is still low. This work consisted in investigating the cytotoxic effects of Akt-inhibition by MK-2206 with irradiation (RT) and TMZ on in vitro human malignant glioma. Seven malignant glioma cell lines were cultured and tested for clonogenic survival, invasion inhibition, tumour spheroid growth and proliferation. The Akt-inhibitor MK-2206 and TMZ were added at different time treatments and in varying doses. Cultures were irradiated with single dose and with fractionated γ-irradiation. Cellular modulation of Akt and p-Akt were assessed by Western blot analysis. MK-2206 reduced the levels of phospho- Akt key protein in the PI3Kinase-Akt pathway, decreased cell survival, and inhibited invasion, proliferation and cell growth. The combination of MK-2206 and RT lead to enhanced inhibition of cell proliferation and invasion, which is not observed with RT alone. The radioenhancing effect of MK-2206 was most striking in inhibition of spheroid volume growth by fractionated RT; the radiosensitizing effect of MK-2206 was stronger than that of TMZ. MK-2206 enhanced the in vitro effects of RT and TMZ in terms of decreased cell survival, invasion, proliferation and growth in malignant glioma. Effects could be ascribed to inhibition of PI3K-Akt pathway
O Glioblastoma multiforme (GBM) ? o tipo mais maligno e mais comum de todos tumores astroc?ticos. O tratamento atual para pacientes de GBM envolve m?xima remo??o cir?rgica, seguida de radio e quimioterapia, normalmente com o agente alquilante Temozolamida (TMZ). Apesar da agressividade da terapia combinada, o tempo de sobreviv?ncia dos pacientes ainda ? baixo. Este trabalho procurou investigar os efeitos citot?xicos do inibidor de Akt MK-2206 em combina??o com irradia??o (RT) e TMZ em um painel de c?lulas de gliomas humanos. Sete linhagens de glioma foram cultivadas e testadas em ensaio de sobreviv?ncia clonog?nica, inibi??o de invas?o, e modelos de prolifera??o e crescimento de volume em esfer?ides. O inibidor MK-2206 e TMZ foram adicionados em diferentes tempos de tratamento e diferentes doses. As culturas foram irradiadas com doses ?nicas ou em terapias fracionadas com irradia??o γ. A modula??o celular de Akt e fosfo-Akt foi checada via Western Blot. O composto MK-2206 reduziu a fosforila??o da prote?na chave Akt na via PI3K, diminuindo a sobreviv?ncia celular e inibindo invas?o, prolifera??o e crescimento celular. A combina??o de MK-2206 com RT levou a uma maior inibi??o de invas?o e prolifera??o, o que n?o ? observado somente com a RT. O efeito radiosens?vel de MK-2206 foi ainda maior na inibi??o do volume dos esfer?ides em terapia combinada com RT fracionada, sendo ainda maior do que o efeito combinado com TMZ. MK-2206 aumentou os efeitos in vitro de RT e TMZ em termos de redu??o de sobreviv?ncia celular, invas?o, prolifera??o e crescimento celular em gliomas malignos. Os efeitos podem ser atribu?dos a inibi??o da via PI3KAkt

Purpose: The objective of this thesis is to evaluate clinical studies that have used oncolytic viruses as treatment and to compare their treatment-outcomes on patients with malignant glioma. Method: This thesis is a systematic literature review where PubMed has been used as the database for data collection. Two searches were done using the search phrases oncolytic virus AND Glioma and oncolytic virus AND brain tumor. Several of the articles showed up multiple times in different searches. After having applied the inclusion criteria, ten of the seventeen articles were removed. Remaining were seven articles used for the thesis. Results: The study conducted by Forsythe et al., using reovirus showed the median overall survival (OS) to be 21 weeks and the median time to progression (TTP) was 4.3 weeks. The study conducted by Kicielinski et al., using REOLYSIN showed the median OS to be 140 days. Median TTP was 61 days. The study conducted by Geletneky et al., 2017 was the first dose-escalating clinical trial for the use of H-1 parvovirus. The median TTP was 111 days and the median OS was 464 days. The study conducted by Lang et al., DNX-2401 was used and in group A the median OS time was 9.5 months. In group B the median OS in the group was 13 months. In another example of an oncolytic adenovirus is ONYX-015, the median TTP after treatment for all patients was 46 days. The median OS for patients diagnosed with glioblastoma multiforme was 4.9 months and for patients with anaplastic astrocytoma and anaplastic oligodendroglioma was 11.3 months across. In a study conducted by Freeman et al. using newcastle disease virus, the OS ranged from 3-66 weeks from the start of treatment and TTP ranged from 2-53 weeks. The study conducted by Markert et al., the median OS from treatment with G207 was 7.5 months. The median TTP was around 2.5 months. Conclusion: Oncolytic viruses are promising agents for treatment against malignant gliomas. No definite outcomes of the treatment could be concluded, however, the median survival was extended in certain cases. The patients tolerated the oncolytic viruses well with no adverse effects correlated with the treatments. There are currently more virus vectors being tested as new developments are needed in this field.

Background: Gliomas are the most frequent primary brain tumours in adults with two main histological subtypes: astrocytoma and oligodendroglioma. Translocator protein (TSPO) is a pro-inflammatory molecule over-expressed predominantly in activated microglia under pathological conditions. In astrocytoma samples, TSPO has also been found to be up-regulated and correlated with the malignancy of the tumours. [11C]-(R)PK11195 is a selective radioligand for the TSPO widely applied in clinical PET studies. We used [11C]-(R)PK11195 PET to investigate in vivo cerebral TSPO expression and microglial activation in patients with gliomas of different histological subtypes and grades. Methods: 24 glioma patients and 10 healthy volunteers underwent volumetric MRI and dynamic [11C]-(R)PK11195 PET scans. Tissue time-activity curves (TACs) were extracted from tumour regions and normal grey and white matter of the brains. Parametric maps of binding potential (BPND) were generated with the simplified reference tissue model. Co-registered MRI/PET was used to guide tumour biopsies. Tumour tissue was quantitatively assessed for TSPO expression and microglial infiltration by immunohistochemistry. Results: Three types of tumour TAC were observed in gliomas (grey matter-like kinetics, white matter-like kinetics and mixed kinetics), which differed between low-grade astrocytomas and low-grade oligodendrogliomas but were independent of the tumour grade. [11C]-(R)PK11195 BPND also differed between the two subtypes of low-grade gliomas, and low-grade gliomas demonstrated lower BPND than high-grade gliomas. 4 cases of high-grade glioma with minor or no contrast enhancement on MRI showed pronounced [11C]-(R)PK11195 binding. Immunohistochemistry confirmed that expression of TSPO correlated with [11C]-(R)PK11195 BPND of the tumour. It was related mainly to expression by neoplastic cells while the contribution from tumour-infiltrating microglia was minimal. When compared with control subjects, increased [11C]-(R)PK11195 BPND was found in patients’ normal appearing cerebral structures, being more prominent in the tumour-bearing than the tumour-free hemisphere. This extra-tumoral [11C]-(R)PK11195 binding was correlated with the duration of epileptic seizures, the symptom shared by the majority of our patients. Conclusions: Gliomas show differences in [11C]-(R)PK11195 kinetics and binding that are related to histological subtype and grade. Neoplastic cells rather than activated microglia are the main cellular sources expressing TSPO and determine the [11C]-(R)PK11195 binding within the tumours. [11C]-(R)PK11195 PET has the potential to detect malignant transformation of non-enhancing gliomas and facilitate the targeting of more aggressive areas in tumour biopsy. The high extra-tumoral [11C]-(R)PK11195 binding indicates widespread microglial activation in otherwise normal appearing cerebral structures of glioma patients. It is associated with epilepsy and could open up novel therapeutic perspectives for seizure control in this patient population.

Introduction: Fluorescein is widely used as a fluorescent tracer for many applications. Its capability to accumulate in cerebral areas with blood-brain barrier damage makes it an ideal dye for intraoperative visualization of high-grade gliomas (HGG). This study presents a new fluorescein-guided technique to remove HGG with a dedicated filter on the surgical microscope (FLUOGLIO trial). Methods: The FLUOGLIO study is a prospective phase II-trial to evaluate safety and obtain initial indications about efficacy of fluorescein-guided surgery for HGG. Since September 2011, 24 patients (age range 45-74 years) were enrolled in the study. Fluorescein was intravenous injected after intubation (5-10 mg/Kg). Tumor was removed with microsurgical technique and fluorescence visualization by BLU400 or YELLOW560 filters on the Pentero microscope (Carl Zeiss, Germany). Degree of tumor resection was calculated on an early (within 72 hours of surgery) postoperative MRI. In 11 patients, biopsies were performed at the tumor margin in order to evaluate sensitivity and specificity of fluorescein in tumor tissue identification. The study was approved by our Ethical Committee and registered on the European Regulatory Authorities website (EudraCT No. 2011-002527-18) Results: Median pre-operative tumor volume was 33.1 cm3 (1.3-87.8 cm3). No adverse reaction related to the administration of fluorescein was registered. Contrast-enhanced tumor was completely removed in 83% of the patients on early postoperative MRI. The remaining patients had a mean tumor resection of 92.6%. Median follow-up was 12 months. The biopsies at the tumor margins gave a preliminary estimation of sensitivity and specificity of fluorescein in identifying tumor tissue of 95% and 86% respectively. Conclusion: The presented data suggest that fluorescein-guided technique with a dedicated filter on the surgical microscope is safe and allows a high-rate of complete resection of HGG at the early post-operative MRI.

Grade 2 gliomas are malignant brain tumours affecting otherwise healthy adults. Although the long-term prognosis is poor, many patients are well and may have a high quality of life for several years. There is, however, a large variability in the natural course of the disease which makes it essential to identify patients who might benefit from early surgery or radio-therapy. The aim of the present thesis was to define new and clinically useful prognostic markers that may assist in the initial treatment decision and in patient follow-up.

A retrospective study of 189 patients with gliomas WHO grade 2 showed no advantage in survival of early tumour resection or radiotherapy, and confirmed that histological subtype and patient age are the most important predictors of survival (I). In 89 patients, the pre-treatment uptake of 11C-methionine (MET) measured with positron emission tomography (PET) was identified as a prognostic marker for survival (II). At the time of tumour progression, irradiated tumours demonstrated signs of a residual radiotherapeutic effect that correlated with the pre-treatment uptake of MET (III). Pre-treatment uptake of MET may, therefore, be important both in predicting the natural course of the disease and the response after treatment. Immunohistochemical staining of 40 tumour samples showed an inverse association between the number of tumour cells expressing platelet-derived growth factor alpha receptor (PDGFRa) and survival (IV). Also, a reduction was observed in the number of receptor-positive cells after malignant transformation, supporting the prognostic value of PDGFRa.

Lumbar puncture was performed in eight patients with newly diagnosed low-grade gliomas to identify three important growth factors in tumour development. Neither PDGF nor vascular endothelial growth factor (VEGF) were detected in the cerebrospinal fluid (CSF), and fibroblast growth factor 2 (FGF-2) was measurable at extremely low concentrations in two of the patients (V). A proteome screening of the CSF, using two-dimensional gel electrophoresis and mass spectrometry, detected alpha 2-HS glycoprotein at significantly higher concentrations than in a control group (VI). This glycoprotein emerges as a novel substance in glioma research and may be of great interest because of its suggested involvement in the embryonic development of the neocortex.

Existing methods of treating glioma are not effective for eradicating the disease. Therefore, new and innovative methods of treatment alone or in combination with existing therapies are necessary. Delivery of therapeutic agents through delivery carriers such as liposomes diminishes the harmful effects of the agent in healthy tissues and allows increased accumulation in the tumor. In addition, targeted chemotherapy using liposomes provides the opportunity for further increase in drug accumulation in tumor. However, the current targeting strategies suffer accelerated plasma clearance and are not advantageous in improving efficacy. The search for new tumor targets, novel ligands, new strategies for targeting, and particle stabilization will advance our ability to improve delivery at the tumor level while decreasing toxicity to normal tissues. The global objective of this thesis was to improve the status of current liposomal therapy to achieve higher efficacy in tumors. Here, we show a novel mechanism to increase targeting to tumor while uncompromising on the long circulation of stealth liposomes. Long circulation is essential for passive accumulation of the nanocarriers due to EPR effect, in order to see benefits of targeting. Using phage display technique, a variety of tumor specific peptides were identified for use as targeting moieties. One potential advantage of the approach proposed here is the rapid identification of patient tumor specific peptide that evades the RES. This could lead to the development of a nanocarrier system with high avidity and selectivity for tumors. Therefore, tumor accumulation of the targeted formulations will be higher than that of non‐targeted liposomes due to increased drug retention at the tumor site and uncompromised blood residence time.In addition, it has been shown that the distribution of nanocarriers, spatially within the tumor, is limited that might further hinder the distribution of the encapsulated drug, thereby limiting efficacy. It is necessary to release the drug from within the nanocarrier to promote increased efficacy. Here, we were able to address the problem of drug diffusion within the tumor interstitium using a combination therapy employing a remotely triggered thermosensitive liposomal chemotherapeutic. We fabricated a thermosensitive liposomal nanocarrier that maintained its stability at physiological temperature to minimize toxicity to healthy cells. We, then, showed a remote triggering mechanism mediated by gold nanorods heated via NIR can help in achieving precise control over the desired site for drug release. These strategies enabled increased drug availability at the tumor site and contributed to tumor retardation. Additionally, we show that the synergistic therapy employing gold nanorods and thermosensitive liposomes may have great potential to be translated to the clinic.

Malignant gliomas represent over 50% of the tumours which originate within the central nervous system. Current treatment modalities are in large part unsuccessful, and less than 10% of patients with high grade glioblastomas survive beyond 2 years. Understanding the cause of the rapid growth rate of malignant gliomas would be useful in devising new therapeutic strategies to treat this disorder. Previous work from this laboratory suggested that the enzyme protein kinase C (PKC) was a critical regulator of the hyperproliferative state of glioma cells. The results presented in this thesis demonstrate that (1) PKC activity was differentially increased in glioma cell lines when compared to other fast growing cell types of non-glial origin, (2) inhibitors of PKC (staurosporine, tamoxifen, CGP 41 251) block the proliferation rate of glioma cells at IC50 values that correspond to their respective IC50's for inhibition of PKC activity, (3) of the PKC isoforms, rat astrocytes and rat glioma cells contain $ alpha, beta, delta, varepsilon$ and $ zeta$, but not $ gamma$, (4) of the expressed isoforms, the activity and amounts of PKC$ alpha$ correlate with cellular growth and (5) an anti-sense oligonucleotide directed against PKC$ alpha$ decreases the proliferation rate of glioma cells. These observations suggest that aberrations in PKC signal transduction (particularly PKC$ alpha$) are important factors in the growth of glioma and have led to clinical trials utilizing PKC inhibitors as adjuncts in the therapy of patients harbouring these incurable neoplams.

Gliomas are the most common primary brain tumours in adults. Mutations in Isocitrate Dehydrogenase 1 and 2 (IDH1/2) define a glioma subtype with a better prognosis than its wildtype counterpart. Although IDH mutant low grade gliomas (LGGs) have a relatively indolent clinical course, recurrence and progression to a higher grade are inevitable. A major challenge in neuro-oncology is the management of LGGs which have progressed as there is no standard treatment of care. Monitoring glioma progression and response to treatment is critical for patient management and is currently performed by imaging. However, inflammatory responses can mimic progression, and this pseudoprogression hinders accurate prognostication. In this thesis, circulating tumour DNA (ctDNA) and RNA from tumour-educated platelets were assessed as biomarker sources for monitoring patients with IDH1 R132H gliomas. OMICs analysis of matched IDH mutant low grade and progressed astrocytomas was undertaken to identify factors driving glioma progression and recurrence. Identified factors were assessed in independent cohorts for their prognostic potential. Droplet digital PCR assays were established for detection of IDH1 wildtype and R132H transcript and DNA. While these exhibited high specificities, the sensitivities were not sufficient for robust detection of circulating mutant molecules from patients with IDH1 R132H gliomas. OMICs analysis revealed decreased differentiation and increased mesenchymal and immunosuppressive features in recurrent higher-grade IDH mutant astrocytomas. Carboxypeptidase E was validated as an independent positive predictor of overall survival (OS) for patients with IDH mutant astrocytomas. A 3-protein panel was validated as a strong independent negative predictor of OS in patients with astrocytomas. This research increased understanding of IDH mutant astrocytoma progression and identified novel biomarkers with the potential for improved prognostication and monitoring of astrocytomas.