Les gliomes sont les tumeurs primitives les plus fréquentes du système nerveux central. Cette dernière décennie, l’apport de la biologie moléculaire a permis de mieux appréhender leurs comportements et de mieux préciser leur origine. Nous avons montré que le profil moléculaire des glioblastomes (grade IV dans la classification de l’OMS) et celui des astrocytomes pilocytiques (grade I dans la classification de l’OMS) différait notamment par l’expression d’un gène nommé KIAA 0510. La caractérisation de sa séquence nous a mené à nous intéresser à la Ténascine R, une glycoprotéine de la matrice extracellulaire impliquée dans les processus de migration et de différenciation cellulaire. Par ailleurs, l’expression de la Ténascine R pendant le développement, suggère son implication au cours de la corticogenèse.Dans le but de mieux comprendre l’origine des astrocytomes pilocytiques, notamment ceux de la région des voies optiques, nous avons mis en évidence au niveau du chiasma optique en développement des cellules de la glie radiaire à partir desquelles les astrocytomes pilocytiques des voies optiques pourraient dériver. / Gliomas are the most frequently occurring primary tumors in the central nervous system. These last years, molecular biology technics allowed a better understanding of the gliomagenesis as well as behaviour of these tumors. We have previously shown that molecular profiling of glioblastomes (WHO grade IV) and pilocytic astrocytomas (WHO grade I) differed for KIAA 0510 gene expression. This sequence was fully characterized and shown to be part of the tenascin R gene encoding for an extracellular matrix glycoprotein involved in migration and cell differentiation. In addition, during development, Tenascin-R may be involved in corticogenesis.In parallel, in the developing optic chiasm, we evidenced cells with radial glial characteristics from which the hypothalamo-chiasmatic pilocytic astrocytomas could derive.

Astrocitomas constituem o tipo mais comum de tumor cerebral neuroepitelial primário apresentando grande heteogeneidade. De acordo com a Organização Mundial de Saúde, os astrocitomas podem ser histologicamente divididos em graus I- IV. Astrocitomas pilocíticos (grau I) são tumores circunscritos, de crescimento lento e bom prognóstico. Astrocitomas difusos (grau II) apresentam hipercelularidade, crescimento relativamente lento e propensão para invadir o tecido cerebral normaladjacente. Astrocitomas anaplásicos (grau III) apresentam aumento da celularidade, atipia nuclear e figuras mitóticas. Glioblastomas (GBMs - grau IV) representam o mais frequente e maligno tumor cerebral humano com crescimento extremamente agressivo, anaplasia, células altamente proliferativas, com frequente neoangiogênese e necrose. O comportamento altamente invasivo dos GBMs, caracterizado pela infiltração difusa para o parênquima cerebral normal adjacente, inviabiliza a remoção cirúrgica total do tumor. Além disso, as células dos GBMs são relativamente resistentes às terapias disponíveis. Analogamente a outros tipos de câncer, os GBMs demonstram comportamentos semelhantes às de células trofoblásticas, sugerindo vias de sinalização compartilhadas no controle dos processos tumorigênicos e de implantação da placenta. Em ambos os casos, o estabelecimento de um fenótipo invasivo compreende processos celulares que incluem aumento da proliferação, expressão ou repressão de moléculas de adesão celular específicas, produção de enzimas que digerem a matriz extracelular, expressão de produtos de proto-oncogenes, ativação da telomerase, evasão ou edição da resposta imune do hospedeiro e angiogênese. Com base nas características comuns entre células tumorais e trofoblastos, o presente trabalho teve como objetivo a busca in silico de genes expressos em placenta e tecidos tumorais e que podem contribuir para o estabelecimento e manutenção do fenótipo maligno, utilizando os bancos de dados de MPSS e SAGE. Dentre os 12 genes avaliados, CD99 foi o que apresentou o maior valor de expressão média nas amostras de GBM em comparação a amostras de tecido cerebral não neoplásico. Em uma casuística ampliada de astrocitomas , observou-se uma maior expressão relativa de CD99 em todos os graus de malignidade, sendo que os GBMs apresentaram os valores mais elevados. Esses achados foram confirmados em nível proteico por western blot e imunoistoquímica. Além disso, foi realizada a análise de imunolocalização de CD99 em amostras de tumores astrocíticos, com localização restrita a membrana ou citoplasma, em contraste ao tecido cerebral não neoplásico ou astrocitomas pilocíticos não infiltrantes, que não apresentaram marcação nestas estruturas. Ao compararmos três linhagens celulares derivadas de GBM, CD99 apresentou maior expressão na membrana e maior capacidade migratória nas linhagens A172 e U87MG, enquanto que a linhagem T98G apresentou menor expressão da proteína e ausência de capacidade migratória. O silenciamento da expressão de CD99 por siRNA diminuiu significativamente a migração das linhagens celulares A172 e U87MG. Além disto, anticorpo anti-CD99 apresentou maior marcação por em lamelipódios das células U87MG, possivelmente por reorganização do citoesqueleto de actina. Os resultados integrados de expressão gênica e proteica sugerem que a expressão de CD99 em astrocitomas de diferentes graus de malignidade pode contribuir para a capacidade infiltrativa destes tumores, ressaltando a importância desta proteína como um potencial alvo para a redução da capacidade infiltrativa dos astrocitomas nos processsos de migração e invasão / Astrocytomas are the most common type of primary neuroepithelial brain tumour and show great heterogeneity. According to World Health Organization criteria, astrocytomas can be histologically separated into grades I through IV. Pilocytic astrocytomas (grade I) are circumscribed, slow growing tumours with a good prognosis and mainly occur in children or young adults. Low-grade astrocytomas (grade II) show hypercellularity, relatively slow growth, and a propensity to invade surrounding normal brain tissue. Anaplastic astrocytomas (grade III) have increased cellularity, nuclear atypia, and mitotic figures. Glioblastomas (GBMs - grade IV), are the most common malignant and aggressive of all brain malignancies, exhibiting anaplastic, highly proliferative cells, with frequent neoangiogenesis and necrosis. GBM cells can escape complete resectability and are relatively resistant to the available therapies (radiation and chemotherapy). Similar to other cancer types, GBMs demonstrates behaviours that are analogous to trophoblastic cells, suggesting shared pathways to control tumourigenic processes and placental implantation. In both cases, the establishment of an invasive phenotype comprises cellular processes that include increased proliferation, the expression or repression of specific cell adhesion molecules, the production of enzymes that digest the extracellular matrix, the expression of proto-oncogene products, telomerase activation, evasion or edition of the host immune response, and angiogenesis. Based on the shared characteristics of tumour cells and trophoblasts, we searched in silico for genes that are in both placenta and tumour tissues using MPSS and SAGE databases and that could contribute to the establishment and maintenance of a malignant phenotype. Among 12 selected genes, CD99 exhibited the highest relative mRNA expression in GBM compared to non-neoplastic brain tissues. In a larger cohort of astrocytic tumours, we further demonstrated increased CD99 expression in all malignant grades, with GBMs showing the highest values. These findings were confirmed at the protein level by Western blotting and immunohistochemistry. Additionally, we demonstrated the CD99 localisation profile in astrocytic tumours. Interestingly, CD99 expression was confined to the cytoplasm or membrane in more malignant astrocytomas, in contrast to non-neoplastic brain tissue or non-infiltrative pilocytic astrocytoma, which showed no obvious staining in these structures. Comparison of three GBM cell lines revealed higher CD99 expression at the membrane and higher migratory capacity in the A172 and U87MG lines, but lower CD99 expression and no migratory ability in the T98 line. Knocking down CD99 expression by siRNA decreased significantly the migration of both A172 and U87MG cell lines. Additionally, a higher anti-CD99 antibody staining was observed in lamellipodia of U87MG, probably because of actin citoskeletal reorganization. These integrated CD99 gene and protein expression results suggest that CD99 expression in astrocytomas of different malignant grades might contribute to the infiltrative ability and support the importance of CD99 as a potential target to reduce infiltrative astrocytoma capacity in migration and invasion.

As mitocôndrias desempenham um papel fundamental na sobrevivência e morte celular. Alterações do DNA mitocondrial (DNAmt) - como, por exemplo, amplificação, mutação homoplásmica, deleção e depleção -, bem como suas implicações clínico-patológicas, tem sido analisadas em inúmeras neoplasias humanas. No intuito de se pesquisar alterações mitocondriais associadas à tumorigênese, o presente trabalho teve como objetivos analisar a expressão de genes implicados no metabolismo energético e envolvidos na replicação e transcrição mitocondriais, quantificar o número de organelas mitocondriais e de cópias de DNAmt e analisar a expressão dos genes em astrocitomas de diferentes graus de malignidade (23 OMS grau I, 26 grau II, 18 grau III e 84 grau IV ou GBM) em relação ao tecido cerebral não tumoral (22 amostras). As expressões relativas dos genes selecionados, bem como as quantificações relativa e absoluta do DNA mitocondrial, foram realizadas por PCR em tempo real. O aumento de expressão relativa de genes-chave da via glicolítica, alterações nos níveis de expressão dos genes do ciclo dos ácidos tricarboxílicos e hipoexpressão de genes da fosforilação oxidativa detectados corroboraram o efeito Warburg. Foi demonstrado que a redução do número de cópias do DNAmt está associada com o grau de malignidade dos astrocitomas difusamente infiltrativos, sendo GBM o mais depletado e independente do número de organelas. As médias observadas para tecido não tumoral, astrocitoma grau I, grau II, grau III e GBM foram, respectivamente, 1,28, 0,26, 0,45, 0,42 e 0,17. Níveis aumentados de expressão relativa dos genes dos fatores de transcrição mitocondriais A (TFAM), B1 (TFB1M), B2 (TFB2M) e da subunidade catalítica da polimerase mitocondrial (POLG) foram detectados em todos os graus de astrocitomas, exceto TFB2M em astrocitoma grau II. Embora exista forte correlação entre os fatores de transcrição mitocondriais, somente os níveis de expressão de POLG se correlacionaram inversamente com o número de cópias de DNAmt. A expressão elevada de TFAM está associada a uma maior sobrevida no grupo de pacientes com GBM, interpretada como compensatório. As hiperexpressões de TFAM e POLG estão relacionadas a um melhor prognóstico em pacientes com GBM. Embora nossos achados da disfunção do metabolismo intermediário e depleção do DNAmt em astrocitomas corroborem a literatura, ainda não está bem esclarecida sua implicação na iniciação e manutenção da transformação maligna. Investigações futuras são necessárias para o esclarecimento destas questões. / Mitochondria has a key role in cell survival and death. Mitochondrial DNA (mtDNA) alterations, for example, amplification, homoplasmic mutation, deletion and depletion, and their clinical and pathological implications have been analyzed in human malignancies. In order to search for mitochondrial alterations associated to tumorigenesis, this study aimed to analyze the expression levels of genes involved in energetic metabolism, and in mitochondrial replication and transcription, to quantify the number of mitochondrial organelle and mtDNA copy number in astrocytomas of different grades of malignancy (23 WHO grade I, 26 grade II, 18 grade III and 84 grade IV or GBM) related to non-neoplastic brain tissue (22 samples). The relative expression level of the selected genes as well as the relative and absolute quantification of mtDNA were performed by real-time PCR. Relative expression increase of glycolytic pathway key genes, change of citric acid cycle genes and hipoexpression of oxidative phosphorylation genes were detected, and confirmed the presence of Warburg effect. The reduced mtDNA copy number was associated to the grade of malignancy of diffusely infiltrating astrocytoma, being GBM the most depleted, and not related to parallel decrease in the number of organelle. The mean mtDNA copy number for non neoplastic tissue, astrocytoma grade I, grade II, grade III and GBM were respectively 1.28, 0.26, 0.45, 0.42 and 0.17. The increased relative gene expression of mitochondrial transcription factor A (TFAM), B1 (TFB1M), B2 (TFB2M) and the catalytic subunit of mitochondrial polymerase (POLG) were observed in all grades of astrocytoma, except TFB2M in grade II astrocytoma. Although a strong correlation was observed among the mitochondrial transcription factors, only the expression level of POLG correlated inversely to the mtDNA copy number. The overexpression of TFAM was associated with long-term survival in the GBM patients and interpreted as compensatory. TFAM and POLG overexpressions were related to better prognosis in GBM patients. Although our findings concerning the impairment of intermediary metabolism and depletion of mtDNA in astrocytomas confirmed previous reports, their role in initiation or maintenance of malignant transformation were not fully understood. Further investigations are needed to clarify these issues.

As mitocôndrias desempenham um papel fundamental na sobrevivência e morte celular. Alterações do DNA mitocondrial (DNAmt) - como, por exemplo, amplificação, mutação homoplásmica, deleção e depleção -, bem como suas implicações clínico-patológicas, tem sido analisadas em inúmeras neoplasias humanas. No intuito de se pesquisar alterações mitocondriais associadas à tumorigênese, o presente trabalho teve como objetivos analisar a expressão de genes implicados no metabolismo energético e envolvidos na replicação e transcrição mitocondriais, quantificar o número de organelas mitocondriais e de cópias de DNAmt e analisar a expressão dos genes em astrocitomas de diferentes graus de malignidade (23 OMS grau I, 26 grau II, 18 grau III e 84 grau IV ou GBM) em relação ao tecido cerebral não tumoral (22 amostras). As expressões relativas dos genes selecionados, bem como as quantificações relativa e absoluta do DNA mitocondrial, foram realizadas por PCR em tempo real. O aumento de expressão relativa de genes-chave da via glicolítica, alterações nos níveis de expressão dos genes do ciclo dos ácidos tricarboxílicos e hipoexpressão de genes da fosforilação oxidativa detectados corroboraram o efeito Warburg. Foi demonstrado que a redução do número de cópias do DNAmt está associada com o grau de malignidade dos astrocitomas difusamente infiltrativos, sendo GBM o mais depletado e independente do número de organelas. As médias observadas para tecido não tumoral, astrocitoma grau I, grau II, grau III e GBM foram, respectivamente, 1,28, 0,26, 0,45, 0,42 e 0,17. Níveis aumentados de expressão relativa dos genes dos fatores de transcrição mitocondriais A (TFAM), B1 (TFB1M), B2 (TFB2M) e da subunidade catalítica da polimerase mitocondrial (POLG) foram detectados em todos os graus de astrocitomas, exceto TFB2M em astrocitoma grau II. Embora exista forte correlação entre os fatores de transcrição mitocondriais, somente os níveis de expressão de POLG se correlacionaram inversamente com o número de cópias de DNAmt. A expressão elevada de TFAM está associada a uma maior sobrevida no grupo de pacientes com GBM, interpretada como compensatório. As hiperexpressões de TFAM e POLG estão relacionadas a um melhor prognóstico em pacientes com GBM. Embora nossos achados da disfunção do metabolismo intermediário e depleção do DNAmt em astrocitomas corroborem a literatura, ainda não está bem esclarecida sua implicação na iniciação e manutenção da transformação maligna. Investigações futuras são necessárias para o esclarecimento destas questões. / Mitochondria has a key role in cell survival and death. Mitochondrial DNA (mtDNA) alterations, for example, amplification, homoplasmic mutation, deletion and depletion, and their clinical and pathological implications have been analyzed in human malignancies. In order to search for mitochondrial alterations associated to tumorigenesis, this study aimed to analyze the expression levels of genes involved in energetic metabolism, and in mitochondrial replication and transcription, to quantify the number of mitochondrial organelle and mtDNA copy number in astrocytomas of different grades of malignancy (23 WHO grade I, 26 grade II, 18 grade III and 84 grade IV or GBM) related to non-neoplastic brain tissue (22 samples). The relative expression level of the selected genes as well as the relative and absolute quantification of mtDNA were performed by real-time PCR. Relative expression increase of glycolytic pathway key genes, change of citric acid cycle genes and hipoexpression of oxidative phosphorylation genes were detected, and confirmed the presence of Warburg effect. The reduced mtDNA copy number was associated to the grade of malignancy of diffusely infiltrating astrocytoma, being GBM the most depleted, and not related to parallel decrease in the number of organelle. The mean mtDNA copy number for non neoplastic tissue, astrocytoma grade I, grade II, grade III and GBM were respectively 1.28, 0.26, 0.45, 0.42 and 0.17. The increased relative gene expression of mitochondrial transcription factor A (TFAM), B1 (TFB1M), B2 (TFB2M) and the catalytic subunit of mitochondrial polymerase (POLG) were observed in all grades of astrocytoma, except TFB2M in grade II astrocytoma. Although a strong correlation was observed among the mitochondrial transcription factors, only the expression level of POLG correlated inversely to the mtDNA copy number. The overexpression of TFAM was associated with long-term survival in the GBM patients and interpreted as compensatory. TFAM and POLG overexpressions were related to better prognosis in GBM patients. Although our findings concerning the impairment of intermediary metabolism and depletion of mtDNA in astrocytomas confirmed previous reports, their role in initiation or maintenance of malignant transformation were not fully understood. Further investigations are needed to clarify these issues.

Astrocitomas constituem o tipo mais comum de tumor cerebral neuroepitelial primário apresentando grande heteogeneidade. De acordo com a Organização Mundial de Saúde, os astrocitomas podem ser histologicamente divididos em graus I- IV. Astrocitomas pilocíticos (grau I) são tumores circunscritos, de crescimento lento e bom prognóstico. Astrocitomas difusos (grau II) apresentam hipercelularidade, crescimento relativamente lento e propensão para invadir o tecido cerebral normaladjacente. Astrocitomas anaplásicos (grau III) apresentam aumento da celularidade, atipia nuclear e figuras mitóticas. Glioblastomas (GBMs - grau IV) representam o mais frequente e maligno tumor cerebral humano com crescimento extremamente agressivo, anaplasia, células altamente proliferativas, com frequente neoangiogênese e necrose. O comportamento altamente invasivo dos GBMs, caracterizado pela infiltração difusa para o parênquima cerebral normal adjacente, inviabiliza a remoção cirúrgica total do tumor. Além disso, as células dos GBMs são relativamente resistentes às terapias disponíveis. Analogamente a outros tipos de câncer, os GBMs demonstram comportamentos semelhantes às de células trofoblásticas, sugerindo vias de sinalização compartilhadas no controle dos processos tumorigênicos e de implantação da placenta. Em ambos os casos, o estabelecimento de um fenótipo invasivo compreende processos celulares que incluem aumento da proliferação, expressão ou repressão de moléculas de adesão celular específicas, produção de enzimas que digerem a matriz extracelular, expressão de produtos de proto-oncogenes, ativação da telomerase, evasão ou edição da resposta imune do hospedeiro e angiogênese. Com base nas características comuns entre células tumorais e trofoblastos, o presente trabalho teve como objetivo a busca in silico de genes expressos em placenta e tecidos tumorais e que podem contribuir para o estabelecimento e manutenção do fenótipo maligno, utilizando os bancos de dados de MPSS e SAGE. Dentre os 12 genes avaliados, CD99 foi o que apresentou o maior valor de expressão média nas amostras de GBM em comparação a amostras de tecido cerebral não neoplásico. Em uma casuística ampliada de astrocitomas , observou-se uma maior expressão relativa de CD99 em todos os graus de malignidade, sendo que os GBMs apresentaram os valores mais elevados. Esses achados foram confirmados em nível proteico por western blot e imunoistoquímica. Além disso, foi realizada a análise de imunolocalização de CD99 em amostras de tumores astrocíticos, com localização restrita a membrana ou citoplasma, em contraste ao tecido cerebral não neoplásico ou astrocitomas pilocíticos não infiltrantes, que não apresentaram marcação nestas estruturas. Ao compararmos três linhagens celulares derivadas de GBM, CD99 apresentou maior expressão na membrana e maior capacidade migratória nas linhagens A172 e U87MG, enquanto que a linhagem T98G apresentou menor expressão da proteína e ausência de capacidade migratória. O silenciamento da expressão de CD99 por siRNA diminuiu significativamente a migração das linhagens celulares A172 e U87MG. Além disto, anticorpo anti-CD99 apresentou maior marcação por em lamelipódios das células U87MG, possivelmente por reorganização do citoesqueleto de actina. Os resultados integrados de expressão gênica e proteica sugerem que a expressão de CD99 em astrocitomas de diferentes graus de malignidade pode contribuir para a capacidade infiltrativa destes tumores, ressaltando a importância desta proteína como um potencial alvo para a redução da capacidade infiltrativa dos astrocitomas nos processsos de migração e invasão / Astrocytomas are the most common type of primary neuroepithelial brain tumour and show great heterogeneity. According to World Health Organization criteria, astrocytomas can be histologically separated into grades I through IV. Pilocytic astrocytomas (grade I) are circumscribed, slow growing tumours with a good prognosis and mainly occur in children or young adults. Low-grade astrocytomas (grade II) show hypercellularity, relatively slow growth, and a propensity to invade surrounding normal brain tissue. Anaplastic astrocytomas (grade III) have increased cellularity, nuclear atypia, and mitotic figures. Glioblastomas (GBMs - grade IV), are the most common malignant and aggressive of all brain malignancies, exhibiting anaplastic, highly proliferative cells, with frequent neoangiogenesis and necrosis. GBM cells can escape complete resectability and are relatively resistant to the available therapies (radiation and chemotherapy). Similar to other cancer types, GBMs demonstrates behaviours that are analogous to trophoblastic cells, suggesting shared pathways to control tumourigenic processes and placental implantation. In both cases, the establishment of an invasive phenotype comprises cellular processes that include increased proliferation, the expression or repression of specific cell adhesion molecules, the production of enzymes that digest the extracellular matrix, the expression of proto-oncogene products, telomerase activation, evasion or edition of the host immune response, and angiogenesis. Based on the shared characteristics of tumour cells and trophoblasts, we searched in silico for genes that are in both placenta and tumour tissues using MPSS and SAGE databases and that could contribute to the establishment and maintenance of a malignant phenotype. Among 12 selected genes, CD99 exhibited the highest relative mRNA expression in GBM compared to non-neoplastic brain tissues. In a larger cohort of astrocytic tumours, we further demonstrated increased CD99 expression in all malignant grades, with GBMs showing the highest values. These findings were confirmed at the protein level by Western blotting and immunohistochemistry. Additionally, we demonstrated the CD99 localisation profile in astrocytic tumours. Interestingly, CD99 expression was confined to the cytoplasm or membrane in more malignant astrocytomas, in contrast to non-neoplastic brain tissue or non-infiltrative pilocytic astrocytoma, which showed no obvious staining in these structures. Comparison of three GBM cell lines revealed higher CD99 expression at the membrane and higher migratory capacity in the A172 and U87MG lines, but lower CD99 expression and no migratory ability in the T98 line. Knocking down CD99 expression by siRNA decreased significantly the migration of both A172 and U87MG cell lines. Additionally, a higher anti-CD99 antibody staining was observed in lamellipodia of U87MG, probably because of actin citoskeletal reorganization. These integrated CD99 gene and protein expression results suggest that CD99 expression in astrocytomas of different malignant grades might contribute to the infiltrative ability and support the importance of CD99 as a potential target to reduce infiltrative astrocytoma capacity in migration and invasion.

Η διαχείριση της πληροφορίας που προέρχεται από εικόνες ιστοπαθολογίας μικροσκοπίου (βιοψίες) αποτελεί διεργασία υψηλής πολυπλοκότητας που αξιοποιείται για την εξαγωγή διαγνωστικών και προγνωστικών συμπερασμάτων από τον ιστοπαθολόγο. Η πολυπλοκότητα αυτή πηγάζει από τον τεράστιο όγκο βιολογικών οντοτήτων που περιέχονται στο δείγμα βιοψίας αλλά και στις μεταξύ τους πολυσύνθετες αλληλεπιδράσεις. Οι πιο σύγχρονες μέθοδοι τεχνητής νοημοσύνης προτείνουν εναλλακτικές προσεγγίσεις για την επίλυση των προβλημάτων υψηλής πολυπλοκότητας αυτού του τύπου. Ανάμεσα όμως στην είσοδο (δεδομένα) και έξοδο (αποτέλεσμα) των ‘έξυπνων’ υπολογιστικών συστημάτων, κρύβεται η μεθοδολογία και στρατηγική επεξεργασίας και ανάλυσης της διαθέσιμης πληροφορίας. Κατά το στάδιο αυτό οι παράμετροι ελέγχου διαχωρίζονται και συσχετίζονται μεταξύ τους ΄τυφλά’ (π.χ. με νευρωνικά δίκτυα, ασαφή λογική) σύμφωνα με συγκεκριμένα μαθηματικά κριτήρια (π.χ. πιθανοκρατικά, ελάχιστων τετραγώνων κ.α.) χωρίς όμως να λαμβάνουν υπόψη την ‘ευρετική’ (heuristic) του ειδικού με αποτέλεσμα να παρουσιάζουν πεπερασμένη ακρίβεια, μεγάλο χρόνο υλοποίησης, αδυναμία γενίκευσης. Έτσι, η απόδοση των συστημάτων αυτών εξαρτάται από το μέγεθος και ποιότητα (θορυβώδη, ελλιπή δεδομένα κ.α.) των δεδομένων, το πλήθος των συνδυασμών των ποσοτικών χαρακτηριστικών που περιγράφουν τα δεδομένα, τον καθορισμό των πλούσιων σε πληροφορία χαρακτηριστικών, την σημαντικότητα των επιμέρους χαρακτηριστικών και των μαθηματικών κριτηρίων ταξινόμησης. Για παράδειγμα πολλά χαρακτηριστικά περιγράφουν καλύτερα την υπό μελέτη διεργασία αλλά η εξαγωγή των πλούσιων σε προγνωστική πληροφορία χαρακτηριστικών απαιτεί πολλούς συνδυασμούς και μεγάλη υπολογιστική ισχύ. Επίσης πολλά χαρακτηριστικά σημαίνει εξειδίκευση του συστήματος στα δεδομένα εκπαίδευσης και αδυναμία εφαρμογής σε άγνωστα δεδομένα. Η παρούσα διατριβή διαπραγματεύεται τον σχεδιασμό, ανάπτυξη και υλοποίηση νέων μεθόδων επεξεργασίας και ανάλυσης ιατρικών εικόνων, επικεντρώνοντας ειδικότερα στην εφαρμογή των μεθόδων αυτών σε υπολογιστικό σύστημα μικροσκοπίας για την διάγνωση όγκων εγκεφάλου τύπου αστροκυττώματος. / Even though histological diagnosis is fundamentally important for patient's management, the potential of diagnostic errors in astrocytomas grading still remains substantially high, ranging from 25% to 40% in routine conditions. Diagnostic errors originate mainly from the lack of experience of experts; rare cancers low prevalence and their biological complexity hinder the establishment of concrete criteria able to predict tumours' behaviour, and, thus, to administrate proper treatments. The latter might explain the fact that a/ although promising treatments have been proposed, death rates have not been yet reduced and b/ the cost of rare cancers management still remains one of the highest healthcare economic burdens in Europe and worldwide. The aim of this thesis was to design, develop and implement new computerized methods to improve manual and computer-assisted malignancy grading of astrocytomas. Scientific objectives comprised: a/ develop a reliable and accurate segmentation algorithm for nuclei detection in routinely stained with H&E histopathological images of astrocytomas, b/ investigate and quantify modifications in nuclei morphology and texture with respect to the degree of tumour abnormality of astrocytic tumours, c/ evaluate whether quantitative analysis of cell nuclei by computer-assisted image analysis could assist the routinely performed malignancy grading of astrocytomas using conventional means, d/ investigate potential modifications in chromatin distribution, which might be used to improve the diagnostic evaluation of cases that histopathologists have difficulty in reaching definite diagnosis (i.e. 'intermediate' grade tumours), e/ support more reliable separation of high grade tumours into clinically meaningful subgroups of patients with grade III and grade IV tumours. For realizing the above objectives, a computer-assisted microscopy system was designed, built and implemented. The system was developed using novel methodologies that integrated state-of-art pattern recognition algorithms for microscopy image segmentation and classification. In addition, new classification techniques have been introduced. The usefulness of the proposed methods has been validated experimentally.

Les gliomes malins sont caractérisés par une prolifération importante, une migration diffuse des astrocytes tumoraux dans le parenchyme cérébral et un taux important de néo-angiogenèse. La gastrine appartient à la famille des peptides apparentés à la cholécystokinine et cette dernière est présente en abondance dans le cerveau. De plus la gastrine est capable de modifier le comportement biologique d’un certain nombre de tumeurs. Le groupe de recherche au sein duquel j’ai réalisé mon travail de thèse fut le premier à suggérer le rôle potentiel de la gastrine au niveau des taux de prolifération et de migration des astrocytes tumoraux. Nous avons précisé dans le présent travail divers effets biologiques provoqués par la gastrine au niveau de gliomes et de gliosarcome expérimentaux.<p>Nous avons au préalable tenté de caractériser par une technique de RT-PCR l’expression d’ARN pour divers récepteurs à la gastrine au sein de tumeurs du système nerveux central et périphérique (comprenant des gliomes, des méningiomes et des schwannomes), au sein de gliomes et d’un gliosarcome expérimentaux, et au sein de cellules endothéliales humaines de veines ombilicales HUVEC et de manchons vasculaires obtenus par microdissection au laser d’un glioblastome humain. Nous avons également développé un modèle de neurochirurgie expérimentale chez le rat consistant en la résection microchirurgicale de la tumeur cérébrale après un bilan iconographique par IRM. Nous avons ainsi montré que l’administration de gastrine dans le foyer opératoire après résection tumorale augmente significativement la période de survie de rats immunodéficients porteurs du modèle de gliome humain U373 et de rats conventionnels porteurs du modèle C6 de rat. In vitro, nous avons montré grâce au test colorimétrique MTT que la gastrine induit une diminution significative du taux global de croissance de ces deux modèles avec une accumulation des astrocytes tumoraux dans la phase G1 de leur cycle cellulaire. Par la technique de Western blotting nous avons également montré que la gastrine induit une diminution significative des taux protéiques du complexe cycline D3-Cdk4 dans les deux modèles expérimentaux. Nous avons montré que la gastrine est capable de réduire significativement l’invasion des modèles C6 de rat, U373 humain et de gliosarcome 9L de rat au travers d’une matrice de collagène et de réduire l’invasion des cellules U373 en chambre de Boyden. La gastrine modifie également significativement la motilité des cellules C6 et U373 et l’organisation de leur cytosquelette d’actine.<p>Nous avons découvert que la gastrine administrée en intracérébral dans le foyer tumoral U373 augmente significativement le taux d’angiogenèse au sein de la tumeur. Nous avons alors investigué l’effet de la gastrine et des antagonistes des récepteurs à cholécystokinine sur le taux d’angiogenèse in vitro en utilisant le modèle des cellules HUVEC cultivées sur Matrigel. L’effet pro-angiogénique in vitro et in vivo de la gastrine est significativement contrecarré par le produit L365,260, un antagoniste relativement spécifique du récepteur CCK-B de la gastrine. La gastrine est chémoattractante sur les cellules HUVEC et augmente significativement leur sécrétion d’IL-8. Toutefois l’effet pro-angiogénique de la gastrine serait en partie dépendant de la modification du taux d’expression des sélectines par les cellules HUVEC, et non de la sécrétion d’IL-8. Nous avons réalisé une revue de la littérature pour tenter de comprendre pourquoi les astrocytes tumoraux migrants sont résistants à la chimiothérapie conventionnelle. A la fin du chapitre Discussion, dans le sous-chapitre intitulé « Quels sont les espoirs thérapeutiques dans le cas des gliomes dits diffus? », nous tentons d’analyser les implications thérapeutiques potentielles qu’il serait possible de tirer du présent travail. <p><p><p> / Doctorat en sciences médicales / info:eu-repo/semantics/nonPublished

Des études suggèrent que les cellules inflammatoires joueraient un rôle initiateur du cancer et contribueraient activement à son développement. Mes travaux se focalisent sur l’étude des gliomes humains et les mastocytes. Les gliomes sont les tumeurs les plus fréquentes du SNC. Les mastocytes constituent des cellules d’intérêt dans l’étude du microenvironnement inflammatoire des tumeurs. Grâce à une technique de coculture mastocytes/astrocytomes nous avons montré que les mastocytes induisent la prolifération des astrocytomes humains et n’ont pas d’effet sur les astrocytes. Cet effet prolifératif nécessite un contact direct entre les deux types cellulaires (ce qui suggère l’implication de molécules d’adhérence) et est dépendant de la sécrétion de l’IL6. Aussi avons-nous ciblé le couple CD47/SIRPα et le couple CD40/CD40L qui sont impliqués dans le contrôle de la balance prolifération/apoptose ou/et la sécrétion de médiateurs de l’inflammation. L’activation du récepteur CD47 dans les astrocytomes humains favorise leur prolifération. La voie de signalisation intracellulaire implique le dimère βγ des protéines G, une activation consécutive de la voie PI3K/Akt, une surexpression de la protéine UHRF1 accompagnée d’une diminution de l’expression du GST p16INK4A. Il semblerait également que l’activation du récepteur CD47 induise une translocation de NF-κB et l’expression de gènes de cytokines en particulier l’IL-6 qui contribuerait à la prolifération des astrocytomes. Cette voie de signalisation n’est pas activée dans les astrocytes. En coculture l’augmentation de la prolifération des astrocytomes est accompagnée d’une diminution de l’expression de CD47 et son ligand SIRPα. Ces effets sont accompagnés par une phosphorylation d’Akt et ERK. Nous avons également montré que l’activation du récepteur CD40 favorise la prolifération des astrocytomes via la voie de l’IL6. / Studies suggest that inflammatory cells play an initiating role of cancer and would contribute actively to its development. My work focused on the study of human glioma and mast cells. Gliomas are the most frequent tumors of the central nervous system (CNS). Mast cells are cells of interest in the study of Tumor inflammatory Microenvironment. Using the technique of coculture: mast cells/astrocytomas we have shown that mast cells induce the proliferation of human astrocytomas and have no effect on astrocytes. The proliferative effect requires a direct contact between the two cell types (which suggests the involvement of adhesion molecules) and is dependent on the secretion of the IL6. We also targeted the CD47/SIRPα and CD40/CD40L interactions who are involved in the control of the proliferation/apoptosis balance or / and the secretion of mediators of inflammation. Activation of the CD47 receptor in human astrocytomas enhances their proliferation. Intracellular signaling pathway involves the βγ dimer of G-protein and consecutive activation of the PI3K/Akt Pathway. Activation of CD47 induces overexpression of the UHRF1 protein, this increase of UHRF1 accompanied by a decrease in the expression of the tumor suppressor gene (p16INK4A). It would also appear that CD47 receptor activation induces a translocation of NF - κB and the expression of genes of cytokines particularly IL-6 which would contribute to the proliferation of astrocytomas. This signalling pathway is not enabled in astrocytes. In coculture, the proliferation of astrocytomas is accompanied by a decrease in CD47 expression and its ligand SIRPα. These effects are accompanied by a phosphorylation of Akt and ERK. We have also shown that activation of CD40 receptor promotes the proliferation of astrocytomas via the IL-6 dependent pathway.

[ES] Los tumores cerebrales son una de las enfermedades más devastadoras en la actualidad por el importante deterioro cognitivo que sufren los pacientes, la elevada tasa de mortalidad y el mal pronóstico. Los astrocitomas de grado 4 conllevan una supervivencia de cinco años en aproximadamente el 5% de los pacientes diagnosticados, siendo los tumores más agresivos y letales del Sistema Nervioso Central (SNC). Los astrocitomas de grado 4 siguen siendo un problema médico complejo aún sin resolver. A pesar de representar más del 60% de los tumores cerebrales malignos en adultos, estos tumores tienen una baja prevalencia relativa y se consideran una enfermedad huérfana, lo que dificulta el desarrollo de nuevos fármacos o tratamientos que puedan beneficiar a los pacientes. La agresividad de estos tumores se debe a diferentes características, como la fuerte angiogénesis, la necrosis, la microproliferación vascular, la capacidad de invasión e infiltración de las células tumorales y un microambiente inmunológico particular. Además, debido a la rápida progresión de los astrocitomas de grado 4, en la zona de la lesión coexisten diferentes regiones específicas que cambian con el tiempo. Esta naturaleza compleja, junto con la marcada heterogeneidad interpaciente, intratumoral y longitudinal, complica el éxito de un único tratamiento eficaz para todos los pacientes. La imagen de resonancia magnética (MRI) supone una técnica útil para caracterizar la morfología y la vascularidad del tumor. El uso de métodos avanzados y robustos para analizar las imágenes de MR recogidas en las fases iniciales del tratamiento de los pacientes permite la delimitación de las diferentes regiones de los astrocitomas de grado 4, convirtiéndose en herramientas útiles para investigadores, radiólogos y neurocirujanos. Además, el cálculo de biomarcadores vasculares de imagen, como los propuestos en esta tesis, facilitaría la caracterización del tumor, la estimación del pronóstico y los enfoques de tratamiento más personalizados. Esta tesis propone cuatro pilares fundamentales para avanzar en el manejo de los astrocitomas de grado 4. Estos incluyen I) la caracterización multinivel del tumor para mejorar las clasificaciones de los gliomas de alto grado del SNC; II) la búsqueda y desarrollo de biomarcadores robustos para estimar el pronóstico de los pacientes desde el momento prequirúrgico; III) así como para evaluar la respuesta a los tratamientos y la selección de los pacientes que pueden beneficiarse de terapias específicas; y IV) el diseño e implementación de estudios clínicos y protocolos para la recogida de datos a largo plazo de cohortes de pacientes notables a nivel internacional. Para abordar estos cuatro pilares, se ha utilizado un enfoque interdisciplinario que combina el análisis de imágenes médicas, técnicas avanzadas de inteligencia artificial y variables moleculares, histopatológicas y clínicas. En conclusión, hemos abordado la influencia de la heterogeneidad interpaciente e intratumoral del astrocitoma de grado 4 para la caracterización y clasificación del tumor, la estimación del pronóstico del paciente y la predicción de las respuestas al tratamiento. Además, se han diseñado e implementado diferentes estudios clínicos que permiten la recogida de datos multinivel de cohortes internacionales de pacientes con astrocitoma de grado 4. / [CA] Els tumors cerebrals són una de les malalties més devastadores en l'actualitat per la important deterioració cognitiva que pateixen els pacients, l'elevada taxa de mortalitat i el mal pronòstic. Els astrocitomes de grau 4 comporten una supervivència de cinc anys en aproximadament el 5% dels pacients diagnosticats, sent els tumors més agressius i letals del Sistema Nerviós Central (SNC). Els astrocitomes de grau 4 continuen sent un problema mèdic complex encara sense resoldre. Malgrat representar més del 60% dels tumors cerebrals malignes en adults, aquests tumors tenen una baixa prevalença relativa i es consideren una malaltia òrfena, la qual cosa dificulta el desenvolupament de nous fàrmacs o tractaments que puguen beneficiar als pacients. L'agressivitat d'aquests tumors es deu a diferents característiques, com la forta angiogènesis, la necrosi, la microproliferació vascular, la capacitat d'invasió i infiltració de les cèl·lules tumorals i un microambient immunològic particular. A més, a causa de la ràpida progressió dels astrocitomes de grau 4, en la zona de la lesió coexisteixen diferents regions específiques que canvien amb el temps. Aquesta naturalesa complexa, juntament amb la marcada heterogeneïtat interpacient, intratumoral i longitudinal fa que es complique l'èxit d'un únic tractament eficaç per a tots els pacients. L'imatge de ressonància magnètica (MRI) suposa una tècnica útil per a caracteritzar la morfologia i la vascularitat del tumor. L'ús de mètodes avançats i robustos per a analitzar les imatges de MR recollides en les fases inicials del tractament dels pacients permet la delimitació de les diferents regions dels astrocitomes de grau 4, convertint-se en eines útils per a investigadors, radiòlegs i neurocirugians. A més, el càlcul de biomarcadors vasculars d'imatge, com els proposats en aquesta tesi, facilitaria la caracterització del tumor, l'estimació del pronòstic i els enfocaments de tractament més personalitzats. Aquesta tesi proposa quatre pilars fonamentals per a avançar en el maneig dels astrocitomes de grau 4. Aquests inclouen I) la caracterització multinivell del tumor per a millorar les classificacions dels gliomes d'alt grau del SNC; II) la cerca i desenvolupament de biomarcadors robustos per a estimar el pronòstic dels pacients des del moment prequirúrgic; III) així com per a avaluar la resposta als tractaments i la selecció dels pacients que poden beneficiar-se de teràpies específiques; i IV) el disseny i implementació d'estudis clínics i protocols per a la recollida de dades a llarg termini de cohorts de pacients notables a nivell internacional. Per a abordar aquests quatre pilars, s'ha utilitzat un enfocament interdisciplinari que combina l'anàlisi d'imatges mèdiques, tècniques avançades d'intel·ligència artificial i variables moleculars, histopatològiques i clíniques. En conclusió, hem abordat la influència de l'heterogeneïtat interpacient i intratumoral del astrocitoma de grau 4 per a la caracterització i classificació del tumor, l'estimació del pronòstic del pacient i la predicció de les respostes al tractament. A més, s'han dissenyat i implementat diferents estudis clínics que permeten la recollida de dades multinivell de cohorts internacionals de pacients amb astrocitoma de grau 4. / [EN] Brain tumors are one of the most devastating diseases today because of the significant cognitive impairment suffered by patients, high mortality rates, and poor prognosis. Astrocytomas grade 4 bring five-year survival in approximately 5% of diagnosed patients, being the most aggressive and lethal tumors of the Central Nervous System (CNS). Astrocytomas grade 4 continue to be an unresolved complex medical problem. Despite accounting for more than 60% of malignant brain tumors in adults, these tumors have a low relative prevalence and are considered an orphan disease, making difficult developing new drugs or treatments that might benefit patients. The aggressiveness of these tumors is due to different characteristics, such as strong angiogenesis, necrosis, vascular microproliferation, the capacity of the tumor cells to invade and infiltrate, and a particular immune microenvironment. In addition, due to the rapid progression of astrocytomas grade 4, different specific regions coexist in the lesion area which change over time. This complex nature, along with the marked interpatient, intratumor, and longitudinal heterogeneity, makes complicate the success of a single efficient treatment for all patients. Magnetic Resonance Imaging (MRI) represents a useful technique to characterize tumor morphology and vascularity. Using advanced and robust methods to analyze MR images collected from initial stages of patient management allows the delineation of different regions of astrocytomas grade 4, becoming useful tools for researchers, radiologists and neurosurgeons. In addition, the calculation of imaging vascular biomarkers, such as those proposed in this thesis, would facilitate tumor characterization, prognosis estimation and more personalized treatment approaches. This thesis proposes four fundamental pillars to advance the management of astrocytomas grade 4. These include I) the multilevel characterization of the tumor to improve classifications of high-grade CNS gliomas; II) the search and development of robust biomarkers for estimating patient prognosis from the presurgical moment; III) as well as for evaluating the response to treatments and the selection of patients who may benefit from specific therapies; and IV) the design and implementation of clinical studies and protocols for long-term collecting data from internationally remarkable cohorts of patients. To address these four pillars, an interdisciplinary approach has been used that combines medical imaging analysis, advanced artificial intelligence techniques, and molecular, histopathological, and clinical variables. Concluding, we have addressed the influence of both interpatient and intratumor heterogeneity of astrocytoma grade 4 for tumor characterization and classification, patient prognosis estimation and predicting treatment responses. In addition, different clinical studies have been designed and implemented allowing the collection of multilevel data from international cohorts of patients with astrocytoma grade 4. / Álvarez Torres, MDM. (2022). Characterization of vascular heterogeneity of astrocytomas grade 4 for supporting patient prognosis estimation, and treatment response assessment [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/188957