Dissertations / Theses on the topic 'Glioma diagnosis'

Les gliomes, et particulièrement les glioblastomes, sont, de par leur pronostic défavorable, un véritable problème de santé publique. La difficulté à les classifier correctement et le manque d'efficacité des thérapies anti-cancéreuses reflètent la méconnaissance de ces tumeurs. Afin d'améliorer le diagnostic, et de mieux comprendre le processus conduisant à la formation des gliomes et donc de trouver de nouveaux traitements, nous avons choisi d'étudier l'immuno-réactivité des sérums de patients. Nous avons ainsi pu découvrir une centaine de protéines dont le statut immunitaire change entre la condition normale et celle pathologique. En identifiant et étudiant ces protéines, nous pouvons mettre en lumière certains mécanismes importants pour la cancérogenèse et définir des clibles clés pour la thérapie. Parmi cette centaine de protéines, nous avons étudié l'immuno-réactivité d'une dizaine qui nous ont permis de définir deux types d'antigènes : ceux dont le statut immunitaire change avec l'apparition de la tumeur et ceux dont le statut change selon la sensibilité de la tumeur vis-à-vis des traitements. Nous avons alors choisi 3 de ces protéines pour les analyser plus en détail : eef1a1, crhsp24, mark3. Ces trois natigènes se trouvent être surexprimés dans les gliomes. L'inhibition de leur expression par si RNA induit une baisse de la prolifération des cellules tumorales, indiquant qu'ils sont impliqués dans la régulation de ce mécanisme. Cette fonction essentielle fait de ces protéines de potentielles cibles thérapeutiques. Les premiers tests de vaccination contre mark3 montrent que l'étude de cet antigène est intéressante même si les résultats ne sont pas ceux espérés. Nous montrons ici que des auto-anticorps associés à la présence des tumeurs gliales peuvent être trouvés dans le sérum. Ces anticorps ont clairement un intérêt diagnostique mais peuvent aussi avoir un intérêt pronostique. Leur analyse biologique peut amener à les considérer comme des cibles thérapeutiques potentielles
Gliomas, particulary glioblastoma, are public health problem because of their dark pronostic. Difficulties to classify and lack of anti-tumor therapy efficiency are due to ignorance about these tumors. To improve diagnosis and also to understand the process leading to glioma formation and so to find new treatment, we choose to study patient sera immuno-reactivity. So we discovered about a hundred of proteins which immune status change between normal and pathological condition. Identifying and studying these proteins, we highlight important mechanisms for cancer and define key targets for therapy. Among these proteins, we studied immuno-reactivity to ten which leads us to define two types of antigens : those which immune status changes with tumor appearance and those who change depending on the sensibility to treatment of the tumor. We then choose three of these proteins for detail analysis : eef1a1, crhsp24, mark3. These three antigens are overexpressed in gliomas. Expression inhibition by siRNA induced a diminution of tumoral cell proliferation, indicating that they are implicated in this mechanism regulation. This essential function makes these potential therapeutic targets. First tests of vaccination toward mark3 show that studying this antigen is of particular interest despite results are not those wished. We show here that auto-antibody associated to glioma presence can be found in sera. These antibodies clearly have diagnosis interest but also can have prognostic interest. Their biological analysis could lead to consider them like potential therapeutic targets

Le travail de thèse est dédié à la caractérisation de fusions spécifiques oncogéniques entre les gènes FGFR et TACC dans les gliomes. Nous avons analysé 907 gliomes pour la présence du gène de fusion FGFR3-TACC3. Nous avons montré que les fusions FGFR3-TACC3 ne touchent que les gliomes IDH wild-type (3%), sont mutuellement exclusives avec l'amplification de EGFR et avec la forme tronquée EGFRvIII et inversement, sont associées à l'amplification de CDK4 et de MDM2 et à la délétion du 10q. Les fusions FGFR3-TACC3 sont associées à une expression intense et diffuse de FGFR3 en immunohistochimie (IHC) et l'IHC pour FGFR3 est un marqueur prédictif très sensible de la présence des fusions FGFR3-TACC3. Les patients porteurs d'une fusion FGFR3-TACC3 ont une survie globale significativement plus longue comparés aux patients avec gliome IDH wild-type. Nous avons traité deux patients porteurs d'un gène de fusion FGFR3-TACC3 avec un inhibiteur tyrosine-kinase (TK) spécifique pour FGFR et nous avons observé une stabilisation de maladie et une réponse mineur chez un patient. Dans la deuxième section nous avons optimisé une nouvelle séquence de spectroscopie différentielle-MEGA-PRESS-pour la détection de l'oncometabolite 2-hydroxyglutarate (2 HG) qui s'accumule de manière spécifique dans les gliomes IDH mutés. Nous avons analysé de façon prospective une cohorte de 25 patients avant chirurgie pour probable gliome de grade II et grade III. Nous avons trouvé que la MEGA-PRESS est hautement spécifique (100%) et sensible (80%) dans la prédiction de la présence de la mutation IDH. Son taux est corrélé aux concentrations de 2 HG mesurés sur tissu congelé par spectrométrie de masse (GC-MS/MS)
This work is devoted to the characterization of a specific oncogenic fusion between FGFR and TACC genes in gliomas. Overall, we screened 907 gliomas for FGFR3-TACC3 fusions. We found that FGFR3-TACC3 fusions exclusively affect IDH wild-type gliomas (3%), and are mutually exclusive with the EGFR amplification and the EGFR vIII variant, whereas it co-occurs with CDK4 amplification, MDM2 amplification and 10q loss. FGFR3–TACC3 fusions were associated with strong and homogeneous FGFR3 immunostaining. We show that FGFR3 immunostaining is a sensitive predictor of the presence of FGFR3-TACC3 fusions. FGFR3-TACC3 glioma patients had a longer overall survival than those patients with IDH wild-type glioma. We treated two patients with FGFR3–TACC3 rearrangements with a specific FGFR-TK inhibitor and we observed a clinical improvement in both and a minor response in one patient. In the second section, we developed a non-invasive diagnostic tool by 1H-magnetic resonance spectroscopy in IDH mutant gliomas. We optimized a uniquely different spectroscopy sequence called MEGA-PRESS for the detection of the oncometabolite 2-hydroxyglutarate (2 HG) that specifically accumulates in IDH mutant gliomas. We analysed a prospective cohort of 25 patients before surgery for suspected grade II and grade III gliomas and we assessed specificity and sensitivity, correlation with 2 HG concentrations in the tumor and associations with grade and genomic background. We found that MEGA-PRESS is highly specific (100%) and sensitive (80%) for the prediction of IDH mutation and correlated with 2 HG levels measured by gas chromatography-tandem mass spectrometry (GC-MS/MS) in frozen tissue

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Tese (Doutorado em Tecnologia Nuclear)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP
FAPESP:11/12405-0

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Fundação Oswaldo Cruz, Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil
ntrodução e objetivos: O glioblastoma multiforme é um glioma de alto grau que apresenta um prognóstico ruim. O diagnóstico definitivo é estabelecido pela avaliação histológica, porém este pode apresentar conflitos na classificação, com isso surge à necessidade de ferramentas que auxiliem o patologista em sua análise. Atualmente, maior ênfase tem sido dada a alterações na glicosilação, pois estão associadas a neoplasias, e a descoberta da capacidade de lectinas em reconhecer tais alterações fez destas, ferramentas aplicáveis para o diagnóstico biomédico. Dessa forma, o objetivo deste trabalho é analisar a marcação das lectinas CpL, WGA e Con A em células da linhagem C6 e astrócitos. Métodos: As células foram cultivadas em condições estéreis, a 37ºC em atmosfera com 5 % de CO2 até atingirem confluência. Em seguida, foram lavadas com PBS e marcadas com as lectinas CpL, WGA e Con A numa concentração de 1 mg/ml, o controle negativo foi obtido com adição do carboidrato inibidor das lectinas (D-galactose, β-N-acetilglucosamina e glicose), respectivamente, numa concentração de 0,1 M. A incubação se deu por uma hora com proteção da luz, a análise foi realizada em microscópio de fluorescência. Para a quantificação em citometria de fluxo, as células foram marcadas obedecendo ao mesmo protocolo anterior, com exceção do tempo de incubação que se deu por 15 minutos. Posteriormente, as células foram lavadas, centrifugadas, transferidas para tubos e ressuspensas em PBS para a realização da leitura em citômetro. Resultados: A lectina CpL apresentou melhor marcação para os astrócitos, porém, ainda assim, mostra baixo desempenho comparado com as demais lectinas. Já a lectina WGA apresentou marcação eficiente tanto para astrócitos quanto para as células C6, esta última apresentou o dobro de emissão. Desta forma, é possível inferir que as lectinas CpL e WGA não são capazes de reconhecer diferenças importantes no perfil de glicoconjugados nas membranas das células C6 e dos astrócitos. Entretanto, a lectina Con A revelou marcação eficiente em relação às células C6 capaz de definir a forma celular, mostrando que há uma distribuição quase uniforme destes carboidratos ao longo da superfície da membrana, e ainda exibiu mediana de fluorescência cerca de 99 vezes superior em relação aos astrócitos. Assim, a Con A mostrou ser um marcador capaz de diferenciar as células da linhagem de glioma murino das células de cultura primária. Conclusão: Com base nestes resultados podemos inferir que a lectina Con A pode auxiliar numa identificação mais eficiente com possibilidade de um diagnóstico mais seguro.
Introduction and objectives: Glioblastoma multiforme is a high-grade glioma that has a poor prognosis. The definitive diagnosis is established by histological assessment. However, this can present conflicts in grading gliomas, which justifies new tools to assist the pathologist in his analysis. Currently, it is known that there are changes in glycosylation pattern of molecules associated with cancer, and the discovery of the ability of lectins to recognize these changes made these tools applicable for biomedical diagnosis. Thus, the aim of this study is to analyze the labelling of C6 and astrocyte lineage cells with fluorescent lectins CpL, WGA and Con A. Methods: The cells were cultured under sterile conditions at 37°C in an atmosphere with 5% CO2 until they reached confluence. They were then washed with PBS and labeled with CpL, WGA, or Con A lectins in a concentration of 1 mg/ml. Negative controls were incubated with the carbohydrate that competitively inhibits the reaction (D-galactose, β-N-acetylglucosamine and glucose, respectively), at a concentration of 0.1 M. The incubation occurred for one hour with protection from the light, the analysis was performed on a fluorescence microscope. For flow cytometry quantitation, cells were labeled following the same previous protocol, except that the incubation time was 15 minutes. Subsequently, the cells were washed, centrifuged, transferred to tubes and resuspended in PBS to carry out the reading on a cytometer. Results: The CpL lectin better labeled astrocytes. However, it showed a poor performance compared to other lectins. On the other hand, the WGA lectin efficiently marked both astrocytes and C6 cells; the latter presented the double emission compared to the former. Thus, it is possible to infer that the CpL and WGA lectins are not able to recognize important differences in the glycosylation profile in the membranes of C6 cells and astrocytes. However, the lectin Con A efficiently marked C6 cells, defining their morphology and showing that there is a nearly uniform distribution of glucose along the surface of the membrane, which was not observed in astrocytes. This also exhibited a median fluorescence about 99 times greater than that obtained for astrocytes. Thus, Con A showed to be a marker capable of differentiate cells of murine glioma lineage from primary astrocytes. Conclusion: Based on these results we can infer that the Con A lectin may be a tool for a more efficient identification of glioma cells in histopathological analysis.

Brain and central nervous cancer presents a significant clinical burden, accounting for 2.4% of all cancer deaths. High grade glioma is particularly deadly, with 5 year survival times of 35% or less. Traditional treatment includes tumor resection followed by radiation therapy or chemotherapy. Aggressive resection is essential in order to prolong patient life. In fact, several studies have shown that life expectancy increases with increased extent of resection. Extent of resection is burdened by the fact that surgeons must be careful not to remove functional brain tissue. Resection is incomplete more often than not due to lack of visual cues for the surgeon. He must rely on tactile sensation to distinguish tumor from healthy tissue. Methods such as intraoperative MRI and CT exist, but these require expensive equipment and special training that is not available in all surgical environments. Some laboratories have proposed small molecule dyes to solve this problem, but these are insufficient when used in an invasive tumor model. It was the goal of this research to provide an objective cue in the form of a nanoencapsulated visible dye without the need for additional equipment of changes to the surgery process itself other than injection of the dye. We hypothesized that the nanocarrier would allow staining of the tumor through passive targeting by taking advantage of the enhanced permeability and retention effect. Once the nanocarriers have reached the desired target, they would not diffuse out into healthy tissue due to their large size compared to small molecule dyes, which readily diffuse out and stain healthy tissue. To test this hypothesis, we prepared and characterized a liposomal nanocarrier encapsulating Evans blue dye. The nanocarrier was tested for safety in vitro and in vivo, then used to delineate tumor margins in an invasive rat glioma model in vivo. Microscopic analysis was then conducted to ensure only tumor tissue was stained by the nanocarrier. This thesis presents a successful method of tumor border delineation to provide surgeons with positive visual cues without the need for changes in surgical environment or techniques.

Introduction Cerebral glioma is the most prevalent primary brain tumour, of which the majority are high grade gliomas. High grade gliomas possess a poor prognosis, and glioblastoma patients survive less than one year after diagnosis. To date, histological grading is used as the standard technique for diagnosis and survival prediction. Previous studies using advanced techniques such as MR Perfusion have achieved a high sensitivity but a low specificity in identifying high grade gliomas. Moreover, they have failed to distinguish glioblastoma from anaplastic glioma. The purpose of the study presented here is to assess the diagnostic and prognostic value for cerebral glioma of cerebral blood volume maps derived from MR perfusion. Methods This retrospective study was approved by the local research ethics committee and clinical audit office. This study included 123 patients with newly diagnosed cerebral glioma, of all grades. Histological diagnosis was used as the standard reference for all potential patients. The relative tumour blood volume (rTBVmax) derived from MR perfusion was used for radiological grading of cerebral glioma. Receiver operating characteristics (ROC) were used to define the best threshold value in distinguishing the glioma grades and in determining the accuracy values (sensitivity, specificity, and positive and negative predictive values). For survival analysis, Kaplan-Meier was used to illustrate and compare the discriminatory value of the histological and radiological classifications. A multiple Cox regression model was used to assess the prognostic value of both classifications in addition to other tested demographic and clinical variables. Finally, the influence of potential moderators was assessed using ANOVA, to assess whether the variation in rTBVmax was only due to the difference in tumour grades. Results A model data set (n = 50) produced a 7-fold increase of TBVmax in tumour versus white matter and provided sensitivity and specificity of 97% and 94%, respectively, in distinguishing high versus low grade glioma. Moreover, a threshold value of 9.6 provided sensitivity and specificity of 100% and 56% in differentiating glioblastoma within the group of high grade gliomas. These threshold values were applied to the second group (n = 73) and provided sensitivity and specificity of 96% and 95% in distinguishing high versus low grade glioma, and 97% and 73% in differentiating, within the high grade gliomas, glioblastoma from anaplastic glioma. Using these two thresholds for a three-tier radiological classification, both the Kaplan-Meier plots and the multiple Cox regression showed that radiological classification was the most independent predictor of survival and tumour progression. The proposed radiological classification system was better than histological classification in predicting glioma patients survival especially noted in a group of moderately hyperaemic rTBVmax. Conclusion MR perfusion is a non-invasive and robust technique in glioma grading and survival prediction. The diagnostic value of rTBVmax derived from MR perfusion in differentiating high versus low grade glioma is promising. It may have a role in the future in defining the appropriate treatment. However, the proposed radiological classification was inferior in differentiating anaplastic glioma from glioblastoma multiforme. In the future, a more advanced multimodal MR, such as MR spectroscopy and MR diffusion, may be studied, besides MR perfusion, in order to improve this diagnostic accuracy.

Los tumores cerebrales constituyen menos del 2% de los tumores primarios, pero son uno de los peores tipos de cáncer en cuanto a “años de vida perdidos”. Los gliomas son los tumores cerebrales más prevalentes, con una esperanza de vida inferior a 15 meses para los casos de alto grado, como los glioblastomas (GBM). El método no invasivo más utlilizado para diagnóstico y seguimiento de la respuesta a la terapia en tumores cerebrales es la Resonancia Magnética (MR), en forma de imagen (MRI) y espectroscopía (MRS) o imagen espectroscópica (MRSI). Sin embargo, debido a restricciones éticas relacionadas a la participación de pacientes humanos en investigación, la optimización de los métodos de diagnóstico y seguimiento de la terapia requieren modelos preclínicos que reproduzcan las patologías humanas. Para este tipo de estudios, se utilizan principalmente modelos murinos, que pueden dividirse en modelos genéticamente modificados (GEM) con desarrollo espontáneo de tumor y los modelos de generación de tumores por inyección estereotáxica de líneas celulares. En la presente tesis, se llevó a cabo una detallada caracterización de dos colonias GEM, S100β-v-erbB/inK4a-Arf (+/-) y GFAP-V12 HA-ras B8. Se observó una baja penetrancia de desarrollo de tumores (16% y 1% respectivamente), haciendo de éstos una herramienta poco útil para estudios de respuesta a la terapia. La escasez de modelos preclínicos de grado bajo/intermedio sirvió de motivación para desarrollar un modelo de tumor glial transplantable con esas características, por disgregación de tumores provenientes de la colonia GEM. Ello nos debería permitir obtener una mayor incidencia tumoral en comparación con las colonias GEM. Se generaron gliosferas a partir de tumores GEM de grado III y se logró más de un 60% de penetrancia cuando estas células fueron inyectadas estereotácticamente en el estriado de ratones C57BL/6. Sin embargo, la aplicación de protocolos de descongelación y cultivo a éstas células ocasionó una progresión a grado IV (GBM), lo que sugiere que el modelo generado constituye, potencialmente, un modelo murino de GBM secundario. Además, este modelo transplantable fue ampliamente caracterizado por métodos de MRI/MRSI, así como por métodos de perturbación del patrón espectral con MRSI (PE-MRSI) para una posible aplicación en el desarrollo de clasificadores de respuesta a la terapia en tumores. También se llevó a cabo una evaluación genética restringida de los modelos murinos seleccionados (p.e. tumores GL261, línea celular GL261, GEM y tumores derivados de GEM), utilizando el método de secuenciación de Sanger para comprobar la presencia de mutaciones normalmente presentes en gliomas (en los genes IDH1, IDH2 y p53). Finalmente, esta tesis describe la estrategia desarrollada para estudios longitudinales de detección de respuesta temprana a la terapia/recidiva en tumores preclínicos, utilizando métodos de imagen molecular basados en MRSI. Así ratones implantados con tumores GL261 (glioblastoma) recibieron tratamiento con temozolamida (TMZ), basándonos en protocolos previamente establecidos. La detención del crecimiento tumoral (respuesta a la terapia) fue detectada por MRI. Tanto animales tratados como animales control fueron estudiados por MRSI y técnicas de reconocimiento de patrones (extracción de fuentes en sistema semi-supervisado). Las fuentes extraídas de regiones de interés fueron capaces de distinguir entre tumores GL261 proliferando activamente y tumores respondiendo a la terapia, basándose en cambios de patrón del metaboloma registrado por MRSI. Se obtuvieron mapas nosológicos codificados en tipo e intensidad de color durante y después de la terapia, lo que permitió un seguimiento de la respuesta, así como la detección de heterogeneidad intratumoral en dicha respuesta, siendo capaces de detectar la detención del crecimiento tumoral y la recidiva antes de los cambios observados en el volumen tumoral por MRI. Esta metodología fue ratificada por análisis histopatológico y cálculo de tasas de proliferación, apoptosis y de índice mitótico.
Brain tumours account for less than 2% of all primary tumours, but are one of the most lethal cancers when “life lost” years are considered. Gliomas are the most prevalent type with a median life expectancy below 15 months for the high grade ones, such as glioblastomas (GBM). The most common non-invasive medical technique used for tumour diagnosis and therapy monitoring of brain tumours patients is Magnetic Resonance (MR), in the form of imaging (MRI) and spectroscopy (MRS) or spectroscopic imaging (MRSI). However, due to the ethical restrictions regarding the use of human patients for research study, the improvement of diagnostic and therapy follow-up protocols requires reliable models that mimic human disease. In this regard, mainly murine models are used and can be divided into the genetically engineered model (GEM) of spontaneous tumour development and the engrafted tumour model. In this thesis, a comprehensive MR characterization of two GEM colonies, namely S100β-v-erbB / inK4a-Arf (+/-) and GFAP-V12 HA-ras B8, was carried out. A low tumour penetrance found (16% and 1%, respectively) together with stochastic onset of GEM tumours, made them impractical for use in therapy response studies. The latter and the scarcity of low/intermediate grade brain tumour preclinical models motivated us to attempt to develop a transplantable glial tumour model of low/intermediate grade by disaggregation of a tumour mass from GEM. This should allow us to obtain an increased tumour incidence rate in comparison to GEM animals. Gliospheres from a grade III GEM tumour were successfully generated and displayed more than 60% penetrance, when stereotactically injected into the striatum of C57BL/6 mice. However, the application of freezing and cell culture protocols produced a progression to grade IV GBM, which made the developed transplantable model qualify as potential secondary GBM model in mice. Additionally, this transplantable model was widely characterized using MRI/MRS methods, as well as perturbation-enhanced MRSI (PE-MRSI) for a possible application in the future in therapy strategies and development of tumour therapy response detection classifiers. A restricted genetic evaluation of selected murine tumour models (i.e. GL261 tumours, GL261 cell line, GEM and GEM-derived tumours) was carried out using the Sanger method to check for a possible presence of particular driver mutations commonly occurring in gliomas (IDH1, IDH2 and p53). Finally, the work describes the strategy followed for longitudinal therapy studies follow-up and early response/relapse detection in preclinical brain tumours, through molecular imaging methods based in MRSI. GL261 (glioblastoma) tumour bearing mice were treated with temozolomide (TMZ), based on previously established protocols. The expected transient growth arrest (response to therapy) was detected by MRI. Animals subjected to therapy and control animals were followed up by MRSI and pattern recognition techniques (semi-supervised source extraction) were applied. The sources extracted from the region of interest were able to discriminate between GL261 tumours actively proliferating and tumours responding to therapy, based on their metabolome pattern changes recorded by MRSI. Colour-coded nosological images produced throughout and after the course of therapy allowed convenient tracking of response changes and differentiated the intratumoural heterogeneity of response, hinting the growth arrest and relapse, before changes in tumour volume were observed by MRI. The methodology was validated with histopathological analysis and calculation of proliferation and apoptotic rates and mitotic index.

Le complexe Shelterin, composé de 6 protéines (POT1 / TRF1 / TRF2 / TIN2 / RAP1 et ACD) joue un rôle majeur au niveau des télomères. Ainsi, il permet la protection de l’extrémité simple brin par la formation de la D-loop, la régulation de la signalisation des voies de dommages à l’ADN ; il participe à la réplication des télomères et contrôle l’accessibilité et la processivité de la télomérase, unique enzyme permettant l’allongement des télomères. Au cours de cette thèse, mon travail s’est organisé autour de 2 principaux axes, le premier, fondamental s’est intéressé aux effets extra-télomériques de la protéine ACD (anciennement appelée TPP1). Le deuxième, plus transversal s’est attardé sur les processus de maintenance des télomères dans le cas des gliomes. Concernant le premier aspect, il est maintenant connu que la protéine ACD fait le lien entre TIN2 et TERT (sous unité catalytique de la télomérase) aux télomères. Ces deux protéines peuvent aussi partiellement se localiser à la mitochondrie et y possèdent alors divers effets sur le métabolisme, la régulation du stress oxydant ou encore la mitophagie. Ainsi, et suite à des prédictions in silico de potentiel MTS pour ACD, nous avons émis l’hypothèse qu’ACD pourrait être le partenaire manquant de TIN2 et TERT à la mitochondrie. Dans ce cas il restait alors à identifier ses fonctions mitochondriales. Après avoir démontré la localisation partielle d’ACD à la mitochondrie par différentes méthodes, nous avons pu mettre en évidence son influence dans la protection contre le stress oxydatif. Ainsi la surexpression d’ACD réduit la production secondaire de radicaux oxygénés mitochondriaux et la perte d’ADN mitochondrial. Le stress oxydatif causant la réduction des foci mitochondriaux d’ACD. Dans un second temps, nous nous sommes intéressés aux mécanismes de maintenance des télomères (TMM) que les cellules cancéreuses acquièrent afin d’outrepasser la sénescence réplicative. Dans ce sens, les tumeurs peuvent réactiver la télomérase (95% des cas) ou utiliser un processus alternatif (ALT) basé sur la recombinaison homologue (5% des cas). Pour les gliomes, jusqu’à 25% des tumeurs utilisent le processus ALT, associé à la perte d’ATRX, les autres gliomes utilisent la télomérase et présentent classiquement une mutation du promoteur de TERT (TERTmt). Ces deux marqueurs moléculaires ont par ailleurs une valeur diagnostique et pronostique et font parties des critères de classification histo-moléculaire de l’OMS . Or, de 4 à 28% des gliomes (selon les sous-types) ne possèdent ni altération d’ATRX ni mutation de TERT suggérant une activation de l’un des TMM par d’autres altérations voire d’autres voies. Dans ce sens, nous avons développé un test mesurant le TMM vrai en nous basant sur la recherche des c-circle (un marqueur du ALT) et proposé un algorithme breveté (TeloDiag) prenant en compte ce TMM, les mutations d’IDH et le grading histologique. Le TeloDiag permet de re-classer 38% des gliomes atypiques (au niveau moléculaire). Il a généré une nouvelle catégorie de tumeurs de haut grade IDHwt et ALT+, n’existant pas dans la classification OMS et montrant une tendance à un meilleur pronostic que les glioblastomes IDHwt (TERTmt). Enfin, nous avons apporté la preuve de concept de la faisabilité de ce test en circulant, pour les astrocytomes IDHmt
The Shelterin complex, made of 6 proteins (POT1 / TRF1 / TRF2 / TIN2 / RAP1 and ACD) plays a major role in telomeres. Thus, it allows the protection of the telomeric single-stranded end by the formation of the D-loop, the regulation of DNA damage signaling pathways; it participates in telomere replication and controls the accessibility and processivity of the telomerase, the unique enzyme allowing telomere lengthening. During this thesis, my work was organized in 2 main axes, the first, fundamental, was interested in the extra-telomeric effects of the ACD protein (also called TPP1). The second, more transversal, focused on the processes of telomere maintenance in gliomas. Concerning the first aspect, it is now known that the ACD protein makes the link between TIN2 and TERT (catalytic subunit of telomerase) in the telomeres. These two proteins can also partially localize to the mitochondria and then have various effects on mitochondrial metabolism, on the oxidative stress regulation or on the mitophagy process. Thus, and following in silico predictions of a putative MTS for ACD, we hypothesized that ACD could be the missing partner of TIN2 and TERT in the mitochondria. In this case, it then remained to identify its mitochondrial functions. After demonstrating the partial localization of ACD in the mitochondria by different methods, we were able to demonstrate its influence in the protection against oxidative stress. Thus overexpression of ACD reduces secondary production of mitochondrial oxygen radicals and loss of mitochondrial DNA. Oxidative stress causing reduction of ACD mitochondrial foci. Secondly, we looked at the telomere maintenance mechanisms (TMM) that cancer cells acquire in order to override replicative senescence. In this sense, tumors can reactivate telomerase (95% of cancer) or use an alternative process (ALT) based on homologous recombination (5% of cancer). In the case of gliomas, up to 25% of tumors use the ALT process, associated with the loss of ATRX, the other gliomas use telomerase and typically have a mutation of the TERT promoter (TERTmt). These two molecular markers also have diagnostic and prognostic value and are part of the WHO histo-molecular classification criteria. But, 4 to 28% of gliomas (depending on the subtypes) do not have an ATRX alteration or TERT mutation suggesting activation of one of the TMM by other alterations or even other pathways. In this sense, we have developed a test measuring the true TMM based on the detection of c-circles (a marker of ALT) and proposed a patented algorithm (TeloDiag) taking into account this TMM, IDH mutations and the histological grading. The TeloDiag makes it possible to re-classify 38% of atypical gliomas (at the molecular level). It generated a new category of high grade IDHwt and ALT + tumors, not found in the WHO classification and showing a tendency for a better prognosis than IDHwt glioblastomas (TERTmt). Finally, we provided the proof of concept of the feasibility of this circulating test for IDHmt astrocytomas

DNA microarrays represent an ultra-high throughput gene expression assay employed to study the transcriptomic profiles of biological tissues. These devices are increasingly being used to study many aspects of gene regulation, and there is growing interest in the biotechnology and pharmaceutical industries for developing such devices in efforts toward rational product/drug design. The DNA microarray also provides a unique and objective means for diagnosis and prognosis of human diseases based on patterns of gene expression. This is especially important in cancer research and the thrust toward personalized medicine. This dissertation details the design and development of oligonucleotide microarrays and the design and execution of a gene expression study conducted using human glioma specimines. Chapter 2 details the design and development a ~10,000 gene human oligonucleotide microarray. This device consisted of a 21,168 features, each composed of a particular human gene-probe and was applied to the challenge of diagnostic and prognostic estimation for human gliomas (chapter 3). Gliomas are the most frequent and deadly neoplasms of the human brain characterized by a high misdiagnosis rate and low survival. The study in chapter 3 demonstrated that the specified design and development parameters were appropriate for conducting gene expression analysis and that this platform can be used successfully to predict malignancy grade and survival for glioma patients.

Les tumeurs gliales du cerveau et en particulier les glioblastomes sont des tumeurs de très mauvais pronostic. Les paramètres qui contrôlent des phénotypes comme l'agressivité, la migration, ou la chimio-résistance de ces tumeurs sont mal connus. Dans ce contexte tumoral, il est envisagé que les microARN (ARN non-codants d'une vingtaine de bases) soient des acteurs essentiels des phénomènes de modification phénotypique parce qu'ils sont capables d'orchestrer l'expression de nombreux gènes. Nous avons montré que les microARN sont des marqueurs tissulaires précieux pour le diagnostic permettant de différencier les deux types principaux de gliomes à partir de prélèvements tumoraux. Nous avons aussi observé que plusieurs microARN sont, en outre, sécrétés par les cellules gliales saines ou cancéreuses au sein de microvésicules appelées exosomes. Le contenu en ARN de ces exosomes a été caractérisé par analyse moléculaire transcriptomique (ARN messagers et microARN) par techniques d'hybridation sur puces à ADN Affymetrix. Les profils ARN exosomaux sains et cancéreux sont distincts, mais ils ne reflètent pas intégralement le profil ARN des cellules dont ils sont issus. Des conditions de stress hypoxique ou l'utilisation de composés pharmacologiques (GW4869 et 5-aza-2'-désoxycitidine) n'affectent pas la quantité d'exosomes produite par la lignée de glioblastome (U87) en culture. Les profils ARN sont cependant modifiés, et le contenu des exosomes produits semble donc être un mécanisme actif et régulé. Enfin, des exosomes cancéreux incubés avec des cellules saines ont très peu d'effet sur le phénotype de celles-ci. Les microARN tissulaires et exosomaux seraient donc des acteurs importants de la physiopathologie du gliome et de sa progression, dont les rôles restent encore à préciser.

L’intégration des paramètres moléculaires dans la classification des gliomes par l’OMS en 2016, particulièrement la mutation IDH1R¹³²H, a un réel impact diagnostique. Cette mutation est associée à un meilleur pronostic mais les mécanismes physiopathologiques sous-jacents à son expression sont encore mal connus. Ce travail propose une approche par imagerie multimodale intégrant de l’IRM multiparamétrique et de la TEP multitraceurs multiparamétrique pour la caractérisation non invasive de cette mutation dans les gliomes. Des cellules de gliome de haut grade humain (U87-MG) exprimant ou non la mutation IDH1R¹³²H (IDH1+ ; IDH1-) par la méthode CRISP/Cas9 ont été étudiées en culture cellulaire (in vitro), dans un modèle préclinique de rat après une greffe orthotopique (in vivo) et après dissection (ex vivo). In vitro, les tumeurs IDH1+ ont montré de faibles niveaux d’expression des intégrines αvβ3 et des récepteurs TSPO considérés comme des marqueurs biologiques d’agressivité dans les voies de l’angiogenèse et de l’inflammation tumorale. In vivo, ces tumeurs IDH1+ présentaient une néo-vascularisation plus dense mais davantage fonctionnelle, similaire à un réseau vasculaire cérébral sain en IRM (4.7T). Les variations des spectres par résonance magnétique corroboraient ces résultats faisant état d’un profil en métabolites moins agressif pour ces tumeurs exprimant la mutation IDH1R¹³²H. La combinaison des paramètres statiques et dynamiques en TEP avec une diminution de la fixation et une pente plus prononcée en TEP au [⁶⁸Ga]NODAGA-(RGDyK)₂, ainsi qu’une diminution de la fixation en TEP à la [¹⁸F]DPA-714 dans les tumeurs IDH+, ciblant respectivement les intégrines αvβ3 et les récepteurs TSPO permettait d’obtenir de bonnes performances diagnostiques dans la discrimination de la mutation IDH1R¹³²H. Ex vivo, l’analyse spectroscopique retrouvait un profil de métabolites moins agressif. Cette étude translationnelle, démontrant l’intérêt de l’imagerie multimodale et multiparamétrique, associe l’expression de la mutation IDH1R¹³²H à un profil tumoral de moindre agressivité dans des cellules de gliome de haut grade humain. Cette étude pilote, après validation sur des cultures de cellules primaires humaines, pourrait conduire à une étude clinique d’imagerie multimodale pour la caractérisation non invasive de la mutation IDH1R¹³²H
The integration of molecular parameters in the classification of gliomas by the WHO in 2016, particularly IDH1R¹³²H mutational status, has significant diagnostic impact. This mutation is associated with a better prognosis but the physiopathological mechanisms underlying its expression remain poorly understood. Our work evaluates a multimodal imaging approach integrating multiparametric MRI and multiparametric multitracer PET to non-invasively characterize this mutation in gliomas. High grade human glioma cells (U87-MG) expressing or not the IDH1R¹³²H mutation (IDH1+ ; IDH1-), constructed using CRISP/Cas9 were studied in cell culture (in vitro), in a preclinical rat model after stereotactic grafting (in vivo) and after autopsy (ex vivo). In vitro, IDH1+ tumors expressed low levels of integrins αvβ3 and TSPO receptors which are considered to be biological markers of aggressiveness involving angiogenesis and tumor inflammation pathways. In vivo, MRIs (4.7T) of these IDH1+ tumors showed areas of high vascular densities which were characterized by functional neo-vascularizations, comparable to healthy cerebral vascular networks. Magnetic resonance spectra variations confirmed these results and revealed a less aggressive metabolite profile for these IDH1+ tumors. The combination of static and dynamic PET parameters with decreased uptake and a pronounced decrease in the PET slope of [⁶⁸Ga]NODAGA-(RGDyK)₂, as well as decreased PET uptake of [¹⁸F]DPA-714 in IDH1+ tumors, targeting αvβ3 integrins and TSPO receptors, yielded good diagnostic performances to discriminate the IDH1R¹³²H mutation. Ex vivo, spectroscopic analyses indicated less aggressive metabolic profile. This translational study demonstrates the benefits of multimodal and multiparametric imaging, and associates expression of the IDH1R¹³²H mutation, in high-grade human glioma cells with a less aggressive tumor profile. Validation of results from this pilot study in human primary cell cultures, may lead to a clinical multimodal imaging study to non-invasively characterize the IDH1R¹³²H mutation

Les tumeurs cérébrales malignes sont la première cause de mortalité par cancer chez l’enfant avec une survie médiane de 12 à 14 mois et une survie globale à 5 ans de 20%, pour les gliomes de haut grade. Ce travail de thèse propose des méthodes innovantes pour l’analyse de blocs de données de génomiques, dans le but d’accroître les connaissances biologiques sur ces tumeurs. Les méthodes proposées étendent les travaux de Tenenhaus et al (2011), introduisant le cadre statistique général : Regularized Generalized Canonical Correlation Analysis (RGCCA). Dans un premier temps, nous étendons RGCCA à la gestion de données en grande dimension via une écriture duale de l’algorithme initial (KGCCA). Dans un deuxième temps, la problématique de la sélection de variables dans un contexte multi-Blocs est étudiée. Nous en proposons une solution avec la méthode SGCCA, qui pénalise la norme L1 des poids des composantes. Dans un troisième temps, nous nous intéressons à la nature des liens entre blocs avec deux autres adaptations. D’une part, la régression logistique multi-Blocs (multiblog) permet de prédire une variable binaire, comme la réponse à un traitement. D’autre part, le modèle de Cox multi-Blocs (multiblox) permet d’évaluer, par exemple, le risque instantané de rechute. Enfin, nous appliquons ces méthodes à l’analyse conjointe des données de transcriptome et d’aberrations du nombre de copies, acquises sur une cohorte de 53 jeunes patients avec un gliome de haut grade primaire. Les résultats sont décrits dans le dernier chapitre du manuscrit
Cerebral malignant tumors are the leading cause of death among pediatric cancers with a median survival from 12 to 14 months and an overall survival of 20% at 5 years for high grade gliomas. This work proposes some innovative methods for the analysis of heterogeneous genomic multi-Block data, with the main objective of increasing biological knowledge about such tumors. These methods extend works of Tenenhaus and Tenenhaus (2011), who introduce Regularized Generalized Canonical Correlation Analysis (RGCCA) as a general statistical framework for multi-Block data analysis. As a first step, we extended RGCCA to handle large-Scale data with kernel methods (KGCCA). As a second step, SGCCA for variable selection within the RGCCA context is studied and leads to an additional constraint on the L1-Norm of the weight vectors. Then, as a third step, we focused on the nature of the links between blocks, with 2 other developments. On one hand, multi-Block logistic regression (multiblog) enables to predict a binary variable, such as response to treatment. On the other hand, the Cox model for multi-Block data (multiblox) enables the assessment of the instant risk, for instance, of relapse. We applied these methods to the joint analysis of Gene Expression and Copy Number Aberrations, acquired on a cohort of 53 young patients with a primary High Grade Glioma. Results are detailed in the last chapter of this work

Purpose: The prognosis for malignant gliomas is poor, and overall survival remains less than 15 months despite improved diagnostic and treatment techniques, underscoring the need for novel therapeutic options. This multicenter, single-arm Phase Ib/II trial was conducted to assess the safety and efficacy of AdV-TK, an adenoviral vector containing the herpes simplex virus thymidine kinase gene, in patients newly diagnosed with high grade glioma. Patients & Methods: Patients were recruited between 2005 and 2010 at four clinical sites. In addition to the standard of care (SOC), which included surgery, adjuvant XRT + temozolomide (TMZ), AdV-TK was administered to the tumor bed at the time of resection followed by valacyclovir prodrug. A total of 48 patients completed therapy per protocol, and were stratified based on extent of resection (total vs. subtotal) and pathological diagnosis (GBM vs. AA/AO). Data from 134 matched patients, who underwent similar SOC at a fifth clinical site during the same time period, were used for descriptive comparison. Results: There were no dose-limiting toxicities or significant adverse events considered related to AdV-TK. Median overall survival (OS) was 17.05 months, with 67% and 35% at 1- and 2- years respectively. Median OS for patients with total resection was 25 months, compared with 13.5 months for patients with subtotal resection. This difference was more pronounced in patients with a pathological diagnosis of GBM (25.05 vs. 10.6 months). In the comparison group median OS was 13.5 months, with 57% and 22% at 1- and 2- years respectively. Median OS for those with total resection was 16.9 months and 12.47 months for subtotal resection. Progression free survival (PFS) was also improved in the AdV-TK group (8.3 vs. 6.43 months). Conclusions: Results from this multicenter Phase Ib/II trial demonstrate that AdV-TK plus valacyclovir can be safely delivered at the time of surgical resection without added toxicity to patients with newly diagnosed high grade glioma. The median OS and the 1- and 2-year survival rates of AdV-TK study patients compare favorably to historical reports and a matched comparison set. Survival outcomes are significantly better in patients having undergone total resections versus subtotal resections. These data strongly support launching a statistically powered randomized clinical study of AdV-TK for the treatment of high grade glioma.

Tumour targeted radiotherapy is an appealing approach for treatment of disseminated tumour cells. A targeting agent that specifically binds to a structure on tumour cells is then used to transport therapeutically relevant radionuclides. The epidermal growth factor receptor, EGFR, is overexpressed on tumour cells in several malignancies, e.g. highly malignant gliomas. In this thesis, three types of radiolabelled EGF-conjugates, aimed for targeting to EGFR-expressing tumour cells, were developed and studied: EGF-dextran labelled with ¹²⁵I, EGF labelled with ²¹¹At, and two EGF-chelates, DTPA-EGF and Bz-DTPA-EGF, labelled with the radioactive metals ¹¹¹In and ¹⁷⁷Lu.

The targeting properties of radioiodinated EGF-dextran were first studied in cultured glioma cells. Radioiodine coupled to the dextran part of EGF-dextran was retained in cells appreciably longer than radioiodine coupled to EGF. This can give about 100 times increased radiation dose to tumour cells.

Targeting with ²¹¹At-EGF was investigated in combination with the tyrosine kinase inhibitor gefitinib (Iressa™, ZD1839). The uptake of ²¹¹At-EGF in EGFR-expressing tumour cells increased with increasing gefitinib concentrations. This was the case for both gefitinib-resistant and gefitinib-sensitive cell lines. The effect of the combined treatment on cell survival, however, differed between the cell lines in an unexpected way. In gefitinib resistant cells, combined treatment decreased cell survival approximately 3.5 times relative to ²¹¹At-EGF treatment alone. In gefitinib sensitive cells, however, combined treatment increased the cell survival (i.e. a protective effect).

The EGF-chelates studied ([¹¹¹In]DTPA-EGF, [¹¹¹In]Bz-DTPA-EGF and [¹⁷⁷Lu]Bz-DTPA-EGF) all bound specifically with high affinity (K_d≈2 nM) to EGFR on cultured glioma cells. They were internalised after binding, and the cellular retention of radionuclides was high (60% remained after 45 h). A biodistribution study in mice showed that liver and kidneys accumulated a majority of the radioactivity. The EGF-chelates bound EGFR specifically also in vivo. A tumour-to-blood ratio of 25 was achieved in a preliminary study.

La néoangiogenèse tumorale est un élément pronostique de l’évolution de nombreux cancers. L’intégrine alphaVbeta3 ainsi que la métalloprotéase matricielle 9 (MMP-9), sont des marqueurs de ce processus. Leur ciblage offre la perspective d’une information diagnostique pour la détection précoce, l’évaluation de l’agressivité de pathologies et la sélection de patients répondeurs aux nouvelles thérapies anti-angiogéniques. Dans ce contexte, notre travail s’attèle à mettre au point les techniques nécessaires à la caractérisation de radiotraceurs. Des modèles de tumeurs richement néovascularisées ont été sélectionnés : le mélanome malin et le gliome malin. Nous nous sommes dans un premier temps intéressés à la détection de l’intégrine alphaVbeta3. Un traceur technétié, le 99mTc-DTPA-bis-c(RGDfK) a servi de support à la validation de nos techniques d’analyse. Cette méthodologie d’évaluation a ensuite été adaptée à des projets collaboratifs. L’étude du 18F-ribofuranose-RGD est réalisée avec le Centre de Recherche en Cancérologie de Toulouse (INSERM UMR 1037) et l’Institut des Sciences Moléculaires (CNRS UMR 5255). Un radioligand de la MMP-9, l’111In- DOTA-F3B, fait l’objet d’un partenariat avec l’ARNA (ARN : Régulations Naturelle et Artificielle, INSERM UMR 869) et l’Institut Lumière Matière (CNRS UMR 5306). Le composé technétié a démontré une bonne affinité et spécificité pour alphaVbeta3. In vivo, chez l’animal, les radioligands technétiés et fluorés ont permis l’identification de tumeurs alphaVbeta3 positives. L’111In-DOTA-F3B a, quant à lui, permis la visualisation de tumeurs chez l’animal et sur coupes tissulaires. Ces traceurs constituent une piste intéressante pour l’imagerie de la néoangiogenèse tumorale
Tumor neoangiogenesis is a predictive element of the evolution of numerous cancers. AlphaVbeta3 integrin and matrix metalloprotease 9 (MMP-9) are markers of tumor neoangiogenesis. Their targeting appears of great interest either for early detection, aggressiveness staging of the disease or for selection of responders to new-targeted therapies. In this context, our objective is to develop methodologies needed for radiotracers characterization. Tracers have been investigated in different tumor models for which vascularization is very important: melanoma and glioma. First of all 99mTc-DTPA-bis-c(RGDfK) has been assessed in our laboratory and helped us to develop analytical methods. These methodologies were used in different partnership, the evaluation of 18F-ribofuranose-RGD targeting alphaVbeta3 with INSERM UMR 1037 and CNRS UMR 5255, and 111In-DOTA-F3B for molecular imaging of MMP-9 with INSERM UMR 869 and CNRS UMR 5306.The technetium peptide has demonstrated good affinity and specificity for alphaVbeta3. In vivo analysis in mice showed that both tracers were able to identify some alphaVbeta3-positive tumors. 111In-DOTA-F3B allowed us to detect hMMP-9 positive tumors in mice and in tumor tissue sections. In conclusion, these tracers still require to be investigated but represent promising tracers for tumor neoangiogenesis

La présence de lipides mobiles a été mise en évidence par spectroscopie RMN du proton dans plusieurs tumeurs animales et humaines. Le but de cette étude était de déterminer la localisation des lipides mobiles dans un modèle expérimental de gliome chez le rat, et de comprendre leur intérêt et leur signification pour le diagnostic des tumeurs cérébrales. La mesure du coefficient de diffusion des lipides mobiles a montré que ces lipides sont inclus dans des compartiments de large diamètre compatible avec les gouttelettes lipidiques. Ces gouttelettes sont principalement localisées dans la nécrose et dans quelques cellules qui l'entourent. Une étude effectuée à différents stades du développement tumoral par spectroscopie 1D et 2D J-résolu couplée à une étude histologique a montré que le signal des lipides mobiles n'est détecté qu'en fin de croissance tumorale, quand la nécrose et les gouttelettes lipidiques sont présentes. Une étude immunohistologique a démontré que les cellules situées en périphérie de nécrose sont en hypoxie. Mais, la présence de gouttelettes lipidiques n'a été détectée que dans les cellules hypoxiques adjacentes à la nécrose. La mesure des temps de relaxation a montré que ces lipides sont caractérisés par des T1 et T2 relativement courts. Enfin, une étude immunohistologique a permis de mettre en évidence la présence de corps apoptotiques au sein de la nécrose. Ce travail démontre que le signal des lipides mobiles détectés par SRM 1H in vivo dans un gliome chez le rat provient des gouttelettes lipidiques dont la présence est liée à une hypoxie sévère, à la nécrose, et à l'apoptose
Mobile lipids have been detected by in vivo proton NMR spectroscopy (1H MRS) in several animal and human tumors. The aim of this study was to determine the location of mobile lipids in a model of rat glioma and to understand their interest and their meaning for the diagnosis of brain tumors. The measurement of mobile lipid diffusion coefficient showed that they are contained in large sized compartments compatible with lipid droplets (. . . ) The mobile lipid relaxation times T1 and T2 are relatively short. Finally we showed through an immunohistological study that apoptotic bodies can be found in the necrosis of tumors. This study allowed us to show that mobile lipid signal detected by in vivo 1H MRS in the model of rat glioma arises from lipid droplets related to severe hypoxia, necrosis, and apoptosis

L'objectif de cette thèse est de décrire et comprendre les processus interactionnels qui gouvernent les consultations d'annonce de diagnostic de glioblastome par une équipe pluridisciplinaire à une personne adulte. Le glioblastome est une tumeur cérébrale grave qui engage le pronostic vital à court et moyen terme. L’annonce de son diagnostic constitue un acte interlocutoire fondateur dans le processus de soin qui va sceller une relation entre deux individus, un qui sait mais qui a du mal à dire, et l’autre qui veut savoir tout en redoutant de découvrir et comprendre une réalité sombre qui le concerne. Cette révélation faite au patient et à son entourage est un acte engageant pour celui qui annonce la mauvaise nouvelle, ce dernier ne pouvant se détacher ni des émotions qu’il va provoquer en l’autre ni des siennes propres. Pour ce travail de recherche, un dispositif original a été mis en place pour l’annonce du diagnostic faite à quatre patients entourés de leur famille et personne de confiance. Deux équipes de professionnels étaient composées de médecins, psychologue et infirmiers. Les analyses de quatre consultations d’annonce constituées chacune de cinq entretiens polylogiques ont mis en évidence les attentes parfois surprenantes des patients vis-à-vis des médecins quant à la manière d’annoncer, ainsi que les effets psychiques de cette annonce. Les stratégies discursives utilisées par les interlocuteurs en présence, dans une visée inconsciente probablement protectrice pour la plupart, sont également dévoilées et permettent de donner des repères précis, notamment en ce qui concerne la formation des médecins mais également des autres acteurs impliqués dans l’annonce de diagnostic
This thesis aims at describing and understanding the processes of interaction that are at stake during the announcement of the diagnosis of glioblastoma to an adult by a team encompassing various fields of action. Glioblastoma is a serious brain tumor that can be life-threatening in a short or mid- term. The announcement of the diagnosis is of paramount importance in the healing process that is going to seal a relationship between two persons, one who knows but who has some troubles telling it and another one who wants to know while fearing to discover and understand a dark reality about himself. This revelation that is said to the patient and his/her relatives is an engaging act for the person announcing the bad news, the latter being unable not to feel concerned with the impact of his words or to deny his own emotions. For this research, an original scheme was put into practice to announce this diagnosis to four patients who were surrounded by their families and friends. Two teams of professionals were composed of doctors, psychologists and nurses. The analyses, resulting from four consultations of announcement, each one constituted of five philological interviews, showcased the sometimes surprising expectations of the patients concerning the doctors as for their way of announcing as well as the psychic effects of this revelation. The discursive strategies used by the persons present, in an unconscious perspective and even protective for most of them, are also unveiled and enable to give thorough landmarks, especially about the training of the doctors but also the other actors involved in the announcement of the diagnosis

Il Sistema Nervoso Centrale (SNC), possiede una limitata capacità di rigenerarsi spontaneamente in seguito a traumi o malattie; le poche strategie rigenerative rivolte a pazienti con traumi o malattie al SNC oggi disponibili, sono tuttavia limitate. Strategie rigenerative dei tessuti neurali, come la somministrazione di cellule e/o farmaci, hanno condotto a risultati sperimentali promettenti nei modelli animali; l’estensione degli esiti all’ambito clinico risulta però difficoltosa. L’efficacia della terapia cellulare, che adopera il trapianto di cellule staminali nel SNC al fine di sostituire i tessuti danneggiati, si è dimostrata limitata a causa della bassa sopravvivenza cellulare e delle criticità di integrazione cellulare che si presentano durante il trapianto. La somministrazione di molecole terapeutiche al SNC tramite metodi convenzionali, come la somministrazione orale o endovenosa, viene invece ostacolata dalla difficoltosa diffusione attraverso la barriera ematoencefalica. Per favorire la sopravvivenza e l’integrazione cellulare post-trapianto, così come per somministrare localmente e in modalità sostenibile agenti biologici ai siti danneggiati del SNC, oggi si ricorre attivamente all’utilizzo di biomateriali. Poiché i trattamenti farmacologici attualmente presenti si limitano a ritardare la progressione delle malattie del SNC, risultano urgenti metodi di medicina rigenerativa che siano in grado di prevalere sul progredire della malattia, promuovendo quindi la rigenerazione tissutale. Questo lavoro di tesi vuole essere uno studio sui recenti biomateriali impiegati come veicoli per la trasmissione di cellule e farmaci per la riparazione di danni acuti e/o cronici del SNC, con particolare attenzione ai casi di lesioni traumatiche encefaliche e del midollo spinale, e a malattie degenerative come la degenerazione maculare legata all’età e la retinite pigmentosa, che determinano degenerazione dei fotorecettori e dell’epitelio pigmentato retinico.

Medical imagery is increasingly evolving towards higher resolution and throughput. The increasing volume of data and the usage of multiple and often novel imaging modalities necessitates the use of mathematical and computational techniques for quicker, more accurate and more robust analysis of medical imagery. The fields of computer vision and machine learning provide a rich set of techniques that are useful in medical image analysis, in tasks ranging from segmentation to classification and population analysis, notably by integrating the qualitative knowledge of experts in anatomy and the pathologies of various disorders and making it applicable to the analysis of medical imagery going forward. The object of the proposed research is exactly to explore various computer vision and machine learning methods with a view to the improved analysis of multiple modalities of brain and cardiac imagery, towards enabling the clinical goals of studying schizophrenia, brain tumors (meningiomas and gliomas in particular) and cardiovascular disorders. In the first project, a framework is proposed for the segmentation of tubular, branched anatomical structures. The framework uses the tubular surface model which yields computational advantages and further incorporates a novel automatic branch detection algorithm. It is successfully applied to the segmentation of neural fiber bundles and blood vessels. In the second project, a novel population analysis framework is built using the shape model proposed as part of the first project. This framework is applied to the analysis of neural fiber bundles towards the detection and understanding of schizophrenia. In the third and final project, the use of mass spectrometry imaging for the analysis of brain tumors is motivated on two fronts, towards the offline classification analysis of the data, as well as the end application of intraoperative detection of tumor boundaries. SVMs are applied for the classification of gliomas into one of four subtypes towards application in building appropriate treatment plans, and multiple statistical measures are studied with a view to feature extraction (or biomarker detection). The problem of intraoperative tumor boundary detection is formulated as a detection of local minima of the spatial map of tumor cell concentration which in turn is modeled as a function of the mass spectra, via regression techniques.

Ce travail a pour objectif de mettre en place des techniques de spectroscopie rmn du proton localisee, et de developper des techniques a deux dimensions frequentielles (2d), en vue d'etudier un modele de gliome intracerebral chez le rat. Une technique de localisation mono-voxel (press) a ete implementee afin de mesurer les temps de relaxation longitudinaux et transversaux des principaux metabolites visibles par rmn dans le cerveau de rat. L'implementation d'une sequence d'imagerie spectroscopique multi-tranches multi-echos a ensuite permis d'obtenir la distribution des principaux metabolites presents dans le cerveau de rat tumoral avec une resolution spatiale digitale d'environ 1l et dans un temps d'experience d'un peu plus d'une heure. Une seconde technique d'imagerie spectroscopique a permis d'obtenir des cartes du ph extracellulaire sur le meme modele animal. Ces cartes ont ete correlees avec la distribution de plusieurs metabolites detectes par rmn. Une sequence 2d j-resolu localisee a ete developpee en vue d'augmenter le nombre de metabolites identifiables par rapport a une technique 1d. Parallelement, une methode a ete proposee pour obtenir les cartes 2d sans suppression du signal de l'eau. Ces methodes ont permis d'etudier le signal des lipides mobiles presents dans les tumeurs intracerebrales chez le rat. Enfin, une sequence 2d j-resolu, spatialement resolue, utilisant les techniques d'apodisation a l'acquisition et multi-echos, a ete developpee. Des cartes 4d j-resolu ont ete obtenues sur le cerveau de rat tumoral, avec une resolution spatiale nominale de 12 l et dans un temps d'experience d'une heure environ.

BACKGROUND: Gliomas are the most common primary brain malignancy, with more than 16,000 patients diagnosed every year (Ostrom, et al., 2015). Outcomes vary widely depending on tumor grade and treatment, and have been steadily improving with the advent of new therapeutics. Glioma patients frequently undergo chemotherapy to remove residual tumor after surgery, and many of these cytotoxic therapies are known to affect rapidly dividing cells such as ovarian follicles (Vassilakopoulou et al., 2016). The negative effects of chemotherapy on fertility have been demonstrated in patients with breast and colorectal cancer (Bines, et al., 1996; Avastin Prescribing Information). Additionally, infertility has been linked with decreased quality of life, primarily in women (O’Moore et al., 1983; Greil, 1997). Fertility treatments are available for women undergoing cancer treatment, however it is unknown whether these treatments are routinely discussed with glioma patients before initiating chemotherapy. OBJECTIVE: The primary goal of this study was to assess whether female glioma patients are being effectively counselled on their possible loss of fertility and their choices for fertility treatment prior to beginning chemotherapy. To this end, it was also important to understand the barriers preventing patients from obtaining information related to their fertility. Another principle goal of this study was to describe the effects of chemotherapy on a sample of women with glioma. Finally, this study sought to understand the priorities of women with glioma in regards to family planning, and to address these priorities in the context of a comprehensive fertility preservation discussion. METHODS: To assess these endpoints, a survey was designed and delivered to patients being treated at the Neuro-oncology clinic of the University of California, San Francisco. Eligible candidates were identified prior to a clinic visit, and patients were asked whether they would like to participate in the survey. Consenting patients then completed the survey at home or in the clinic. Seventy two women completed the survey. Data was analyzed using STATA Software Version 10.0. RESULTS: Analysis of the survey results showed that only 35% of women receiving chemotherapy reported having a discussion regarding fertility preservation prior to beginning treatment. Of those who reported having this discussion, only 80% were aware that chemotherapy could negatively affect their fertility. Many women reported that while fertility preservation was not important to them at the time of diagnosis, it was a priority for them at the time of survey completion. Most women surveyed expressed a desire to have a fertility preservation discussion with a reproductive specialist. CONCLUSIONS: The data obtained in this study suggest a lack of understanding of the negative effects of chemotherapy which may be addressed with a more comprehensive fertility discussion with glioma patients prior to beginning treatment. Although interest in having children tends to decrease after cancer treatment, the majority of respondents still report wanting a child after treatment. The priorities of women in the study reflect a concern for the health of their future offspring which may be best addressed prior to beginning treatment in order to increase their chances of conceiving at a later date.

Dissertação de Mestrado em Biologia Celular e Molecular, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra.
Os gliomas são os tumores cerebrais primários mais comuns que surgem no sistema nervoso central (SNC). Segundo a Organização Mundial de Saúde (OMS) a sua classificação é baseada na célula de origem, células gliais, e são classificados de acordo com seu grau histológico de malignidade, do menos agressivo (Grau I) para o mais agressivo (Grau IV), também conhecido como Glioblastoma (GBM). Este último tem origem em astrócitos e é considerado o tumor cerebral mais letal na idade adulta, representando 20% de todos os tumores cerebrais primários. Infelizmente o prognóstico da maioria dos doentes com gliomas, e especialmente com glioblastomas, continua a ser muito reservado. E há décadas que a sobrevida se mantém entre 9 a 15 meses. Apesar de todos os avanços na compreensão dos mecanismos moleculares envolvidos na biologia destes tumores cerebrais, é urgente a descoberta de estratégias eficazes no diagnóstico, prognóstico e tratamentos terapêuticos. Ao longo dos últimos anos, foram descritos alguns marcadores moleculares promissores para o diagnóstico, prognóstico e resposta à terapêutica. O presente estudo focou-se na investigação do valor do marcador IDH1 no diagnóstico e prognóstico de doentes com glioma. Além disso, também teve como objectivo explorar os níveis da progranulina (PGRN) em doentes com glioblastomas correlacionando-os com a sua sobrevida. Várias mutações têm sido descritas no gene IDH1, no entanto, a mais frequente (> 95%) é p.R132H. Noventa e seis doentes seguidos no Centro Hospitalar e Universitário de Coimbra (CHUC) foram estudados para a presença de mutações somáticas no gene IDH1 usando em paralelo dois métodos diferentes, sequenciação de Sanger e imunohistoquímica (IHQ). Dos resultados obtidos podemos concluir que a combinação das duas técnicas aumenta a especificidade e sensibilidade na detecção de mutações. Assim, no dia-a-dia aconselhamos o uso de IHQ como método de primeira linha, seguida de sequenciação para casos em que a IHQ tenha sido negativa ou que apresentem características clínicas/morfológicas duvidosas. Além disso, doentes com mutações no IDH1 apresentaram um melhor prognóstico do que os doentes com tumores não mutados. Recentemente, foi demonstrado que a PGRN e expressão do mRNA estão aumentadas em vários tipos de tumores. Também, este ano foi descoberto que a PGRN torna as células de glioblastoma resistentes à temozolomida, agente quimioterapêutico de primeira linha no tratamento destes tumores. O nível da PGRN foi avaliado em 40 doentes com glioblastoma através da técnica de ELISA. Surpreendentemente, plasmas de doentes com GBM apresentaram níveis da PGRN significativamente diminuídos quando comparados com os indivíduos controlo (p˂0.001). E ainda, os doentes com níveis diminuídos de PGRN apresentaram uma sobrevida menor em relação aos doentes com níveis normais. Estes resultados sugeriram que a PGRN poderá ser usada como um possível marcador de prognóstico. Contudo, estudos adicionais deverão ser realizados de modo a estabelecer uma correlação entre o nível da PGRN no plasma e a sua expressão no tecido tumoral.
Gliomas are the most common primary brain tumours that arise in the central nervous system (CNS). According to the World Health Organization (WHO) their classification, is based on cell origin, glial cells and are graded according to their histological degree of malignancy, ranging from the least aggressive (Grade I) to the most aggressive (Grade IV), also known as Glioblastomas (GBM). This latter one, arise from astrocytes and are considered the most lethal adult brain tumour, representing 20% of all primary brain tumours. Unfortunately, the prognosis of most gliomas, especially glioblastoma, continues to be dismal and the median survival has remained at 9 to 15 months for decades. Despite all the progresses in understanding the molecular mechanisms involved in biology of this brain tumours, the discovery of successful strategies for diagnosis, prognosis and therapeutic treatments are urgently needed. Over the past few years, a few promising molecular markers have been described conferring diagnostic, prognostic and predictive value to therapy response. This study focused on the investigation of the value of IDH1 as diagnostic and prognostic marker in glioma patients. In addition, we also aim to explore the progranulin (PGRN) levels in glioblastoma patients to further correlate with their overall survival. Several mutations have been described in IDH1 gene, however the most frequent (>95%) is p.R132H. Ninety-six patients assisted in the Centro Hospitalar e Universitário de Coimbra (CHUC) were screened for the presence of somatic mutations in IDH1 gene using two different methods in parallel, Sanger sequencing and Immunohistochemistry (IHC). Gathering the results, we concluded that the combination of the two techniques increased the specificity and sensitivity of mutation detection. Furthermore, in a daily practice, we advocate the use of IHC as a first line method, followed by sequencing in IHC-negative cases with dubious clinical or morphological features. Moreover, the patient’s tumour harbouring IDH1 mutations showed a better outcome than patients with non-mutated tumours. Recently, it has been shown that PGRN and PGRN mRNA expression is highly increased in several human tumour types. In addition, this year it was demonstrated that PGRN promotes temozolomide resistance, the current first-line chemotherapeutic agent for glioblastoma. The level of PGRN has been assessed in 40 glioblastoma patients by ELISA technique. Surprisingly, the plasma levels of PGRN in the patients with GBM were significantly decreased compared with healthy control (p˂0.001). Furthermore, patients with decreased levels of PGRN show a shorter survival than patients with normal levels. These results suggested that PGRN might be used as a potential prognostic marker. However, further studies should be performed in order to establish a correlation between plasma level and tumour tissue PGRN expression as well as patient prognosis.

The clinical management of glioma remains a challenge. The prognosis is poor—for glioblastoma multiforme, the most virulent of these brain cancers, survival is only ~1 year. Surgical resection of the tumor is the first line of defense. Several studies demonstrate a survival advantage in patients who undergo near-complete tumor resection; however, achieving complete resection is limited by the difficulty of visualizing residual tumor after de-bulking. Intraoperative fluorescence guidance is a promising candidate to better visualize residual tumor. The most clinically developed form uses protoporphyrin IX fluorescence, the precursor to heme in its biosynthesis which preferentially accumulates in tumor cells after the administration of 5-aminolevulinic acid. Challenges remain in quantitatively assessing the fluorescence to reduce variability of outcome and improve tumor detection specificity, and in observing sub-surface tumor fluorescence. To these ends, this work outlines the development of intraoperative techniques to 1) quantify tissue fluorescence using a handheld fiberoptic probe and 2) improve detection by reconstructing the depth-resolved fluorescence topography of sub-surface tumor. As a critical component to achieve these objectives, a technique to measure the tissue optical properties was developed. This technique used diffuse reflectance measurements mediated by a handheld fiberoptic probe to derive the tissue optical properties. The handheld fiberoptic probe was further developed to include fluorescence spectroscopy. A novel algorithm to combine the fluorescence measurement and the tissue optical properties was derived in order to extract the quantitative fluorescence spectrum, i.e. fluorescence without confounding effects of tissue optical properties. The concentration of fluorescent tumor biomarker can then be extracted. The quantitative fluorescence work culminated in deployment of the fiberoptic probe in clinical trials for the resection of intracranial tumors. The quantitative fluorescence probe out-performed a state-of-the-art fluorescence surgical microscope for a broad range of brain tumor pathologies. A novel technique for depth-resolved fluorescence detection was developed utilizing multi-excitation fluorescence imaging. An algorithm to extract depth information from the multi-excitation images was derived, with validation in phantoms and a rat brain tumor model. This demonstrates the potential for depth-resolved fluorescence imaging, which there is a clear need for in tumor resection guidance.

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2017
Primary brain tumours are a critical cause of morbidity and mortality in both adults and children, representing around 1-2% of all newly diagnosed tumours and accounting for about 2% of all cancer-related deaths. Gliomas are the most prevalent primary malignant brain tumour representing 80% of these. Over the past years, distinctive genetic profiles have been identified in several glioma types which have led to the WHO 2016 new classification of CNS tumours that incorporates molecular parameters into the tumour classification criteria, breaking with the previous approach based entirely on histological features. This refines tumour diagnostics and can also provide important prognostic and therapeutic response information. The aim of this study was to apply and verify if Next-Generation Sequencing (NGS) can effectively replace the classical methods – pyrosequencing and Sanger sequencing - in establishing the molecular diagnostics of gliomas. Thus, a glioma-tailored gene panel covering 518 amplicons of 19 genes frequently aberrant in gliomas was designed and applied to assess 30 glioma samples. This targeted NGS approach was carried out by Illumina® TruSeq® technology in Illumina® MiSeq System. DNA libraries preparation showed a success rate of 90%. Data analysis was performed using several bioinformatical software and filtering parameters of variants were optimized to reduce sequencing artefacts in the NGS run. Better DNA quality libraries presented more reliable results showing less DNA sequence changes. The sensitivity and specificity of the 19-gene panel for detection of DNA sequence variants were verified by single-gene analyses which showed to be substantial concerning hotspot mutations but not so trustworthy concerning new-mutations detected by NGS. The presented findings showed that, even though NGS application in routine glioma molecular diagnostics can’t be yet implemented, further investigation of this technology is promising since NGS showed to be a resourceful tool for glioma genetic profiling, displaying its potential as diagnostic method which would facilitate the integrated histological and molecular glioma classification.
Os tumores cerebrais primários são uma causa crítica de morbilidade e mortalidade em adultos e crianças, representando cerca de 1-2% de todos os tumores recém-diagnosticados e cerca de 2% de todas as mortes relacionadas com o cancro. Os gliomas são o tipo de tumor cerebral maligno primário mais prevalente representando 80% destes. Ao longo dos últimos anos, foram identificados perfis genéticos distintivos em vários tipos de glioma o que levou à nova classificação de tumores do SNC de 2016, pela OMS, que incorpora os parâmetros moleculares nos critérios de classificação do tumor, quebrando com a abordagem anterior inteiramente baseada nas características histológicas. Esta nova abordagem veio aperfeiçoar o diagnóstico dos tumores e a capacidade de fornecer, também, informações importantes quanto ao prognóstico e resposta à terapêutica. O objetivo deste estudo foi aplicar e verificar se a Sequenciação de Nova Geração (NGS) pode efetivamente substituir os métodos clássicos - pirosequenciação e sequenciação de Sanger - no estabelecimento do diagnóstico molecular dos gliomas. Assim, foi desenhado e aplicado um painel genético adaptado a gliomas que cobriu 518 amplicões de 19 genes frequentemente aberrantes nestes, para analisar 30 amostras de gliomas. Esta abordagem direcionada da NGS foi realizada pela tecnologia TruSeq da Illumina®, no seu sistema MiSeq. A preparação das bibliotecas de DNA mostrou uma taxa de sucesso de 90%. A análise dos dados foi realizada recorrendo a vários softwares bioinformáticos e os parâmetros de filtração das variantes obtidas foram otimizados para reduzir os artefactos da sequenciação resultantes da execução da NGS. As bibliotecas de DNA de melhor qualidade apresentaram resultados mais confiáveis exibindo menos alterações nas sequências de DNA. A sensibilidade e a especificidade do painel de 19 genes para a deteção de mutações foram verificadas por análise individual dos genes, indicando ser substanciais em relação às mutações hotspot, mas não tão confiáveis em relação às novas mutações detetadas pela NGS. Os resultados apresentados mostraram que, apesar de não ser possível implementar para já a NGS na rotina do diagnóstico molecular de gliomas, é promissora uma investigação adicional nesta tecnologia, uma vez que a NGS mostrou ser uma ferramenta rica em recursos para delinear o perfil genético dos gliomas, ilustrando o seu potencial como método de diagnóstico que facilitaria a classificação histológica e molecular integrada dos gliomas.