Estudo de marcadores de diagnóstico e prognóstico em gliomas
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2010
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Os gliomas são os tumores cerebrais primários mais frequentes, representando mais de 80% de todas as neoplasias do Sistema Nervoso Central e são, actualmente, classificados, pela Organização Mundial de Saúde, em astrocitomas, oligodendrogliomas, tumores mistos e ependimomas. O diagnóstico e a classificação destes tumores são baseados nas características histopatológicas que, por vezes, são difíceis de definir. Apesar dos avanços na neurocirurgia, quimioterapia e radioterapia, o prognóstico dos doentes com gliomas é ainda muito reservado.
Nos últimos anos, estudos em gliomas têm identificado marcadores de diagnóstico, de prognóstico e preditivos, susceptíveis de ajudarem a definir a classificação histológica. A codelecção 1p/19q em tumores oligodendrogliais, a presença de mutações somáticas em genes que codificam isocitrato desidrogenase 1 e 2 (IDH1 e IDH2) em astrocitomas, oligodendrogliomas e oligoastrocitomas e a hipermetilação da região promotora do gene MGMT em glioblastomas são, actualmente, considerados os três marcadores mais interessantes em gliomas.
Estudos prévios sugerem que a codelecção dos braços dos cromossomas 1p e 19q tem provado ser um bom marcador de diagnóstico e prognóstico. A perda combinada de 1p e 19q tem sido associada com a morfologia de oligodendrogliomas e com o aumento da sobrevida nestes tumores. Contudo, em outros tumores, a componente oligodendroglial pode também conferir um melhor prognóstico. Assim, um dos objectivos deste Projecto, foi a avaliação de 1p/19q em relação ao diagnóstico e prognóstico dos doentes.
Estudos recentemente publicados sugerem que mutações nos genes IDH1 e IDH2, podem ser marcadores úteis de diagnóstico e prognóstico, no entanto, esta relação nem sempre é consensual. Os doentes com mutação nos genes IDH1 ou IDH2 parecem ter um melhor prognóstico do que os doentes sem mutação Deste modo, pretendeu-se identificar e caracterizar as mutações encontradas para a população em estudo, e correlacionar a sua presença com a sobrevida dos respectivos doentes. Também, foi objectivo de estudo avaliar se a presença de mutações permite distinguir glioblastomas primários (de novo) de glioblastomas secundários (que progridem de gliomas de baixo grau ou anaplásicos).
A proteína de reparação de DNA, MGMT, é um factor implicado na quimioresistência em gliomas. O valor preditivo e de prognóstico da hipermetilação da região promotora do gene MGMT permanece, contudo, controverso. Assim, foi também objectivo deste estudo, avaliar a importância da hipermetilação no prognóstico de doentes com glioblastomas.
No total, analisaram-se 141 amostras de doentes seguidos nos Hospitais da Universidade de Coimbra, com astrocitomas (n = 111), oligoastrocitomas (n = 18), oligoastrocitomas (n = 8) e ependimomas (n = 4). No estudo realizado a perda alélica 1p/19q foi avaliada por Fluorescence In Situ Hybridization (iFISH) (n = 130), as mutações somáticas nos genes IDH1 e IDH2 foram analisadas por Sequenciação directa (n = 128) e a análise de hipermetilação foi efectuada por Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA) (n = 38). Os dados obtidos sugeriram que a codelecção 1p e 19q está associada com a morfologia de oligodendrogliomas (p<0,0001), independentemente do grau do tumor. Contudo, esta alteração não foi associada com a quimiosensibilidade e aumento da sobrevida destes doentes.
Mutações IDH1 foram detectadas em 17 dos 128 gliomas estudados, e encontraram-se 3 mutações diferentes no gene IDH1. Este estudo sugeriu que as mutações IDH1 são frequentes em glioblastomas secundários, mas raros em glioblastomas primários (100% vs 1,1%, p<0,0001). Além disso, mutações IDH1 parecem ser também um factor de prognóstico mais favorável. Nenhum dos 128 gliomas analisados era, todavia, portador de mutações IDH2.
A região promotora do gene MGMT apresentou-se metilada em 67% dos doentes e não metilada em 33% dos doentes com glioblastomas. Verificou-se que os doentes com a região promotora hipermetilada, quando submetidos a radioterapia ou quimioterapia, apresentaram um aumento de sobrevida relativamente àqueles que também apresentavam metilação, mas que não foram submetidos a qualquer tratamento (p<0,001).
Assim, através da realização deste Projecto, a implementação dos três marcadores em estudo reveste-se da maior importância, devido ao seu valor no diagnóstico e à sua utilidade para prever e controlar melhor o prognóstico de alguns destes doentes. Tem ainda, um poder preditivo da resposta ao tipo de tratamento, o qual poderá ser optimizado e ajustado para alguns doentes previamente seleccionados.
Gliomas are the most common primary brain tumors, accounting for more than 80% of all neoplasms arising in the Central Nervous System (CNS) and are currently classified by the World Health Organization (WHO) into astrocytomas, oligodendrogliomas, mixed gliomas and ependymomas. The diagnosis and classification are based on their histopathological appearance, which sometimes may be difficult. Despite advances in neurosurgery, chemotherapy and radiotherapy, the prognosis of most glioma patients remains dismal. In recent years, extensive molecular studies have identified diagnostic, prognostic and predictive markers in gliomas that reinforce the WHO histological classification. The 1p19q coleletion in oligodendroglial tumors, the presence of somatic mutations in the genes enconding isocitrate dehydrogensase 1 and 2 (IDH1 and IDH2) in astrocytomas, oligodendrogliomas e oligoastrocytomas and the hypermethylation of the promoter of O6-methylguanine DNA methyltransferase (MGMT) gene in glioblastomas are currently the three most pertinent markers in diffuse gliomas. Previous studies suggest that codeletion of chromosome arms 1p and 19q has proven to be a powerful diagnostic and prognostic marker. Combined loss of 1p and 19q has been associated with morphology of oligodendrogliomas and with enhanced survival in these tumors. However, in other glial tumors, an oligodendroglial component can mean a better prognostic. Therefore, one of the aims of this Project is the 1p/19q evaluation concerning the patients’ diagnostic and prognostic. Recent published studies considered that mutations in IDH1 and IDH2, are an important diagnostic and prognostic marker but that is still unclear. Also, patients with tumors harboring mutation of IDH1 or IDH2 genes seem to have a better outcome than patients with non mutated tumors. Thus, another goal of this study focus on the identification and characterization of mutations, difference in survival at a population level and to assess whether they allow reliable discrimination between primary (de novo) glioblastomas and secondary glioblastomas (that progressed from low-grade or anaplastic gliomas). DNA repair protein, MGMT, is one implicated factor in glioma chemoresistance. The prognostic and predictive value of MGMT promoter hypermethylation however remains controversial. An additional aim of this study regards the evaluation of the prognostic significance of MGMT in patients with glioblastomas. In total, we analysed 141 patient samples for a series of patients followed in the University Hospital of Coimbra with astrocitomas (n = 111), oligodendrogliomas (n = 18), mixed gliomas (n = 8) and ependymomas (n = 4). In our study, the 1p/19q status was assessed by interphase Fluorescence In Situ Hybridization (iFISH) (n = 130), somatic mutations analysis of IDH1 and IDH2 genes was directed sequenced (n = 128) and MGMT hypermethylation analysis was performed by using Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA) on the glioma samples (n = 38). Our data suggested that deletions of chromosome arms 1p and 19q are associated with oligodendrogliomas morphology (p<0,0001), independently of tumor grade. However, this alteration is not associated with chemosensitivity and enhanced survival. IDH1 mutations were detected in 17 of 128 gliomas, in which 3 different mutations were identified. This study indicated that IDH1 mutations are frequent in secondary glioblastomas but rare in primary glioblastomas (100% vs 1,1%, p<0,0001). Therefore, IDH1 mutations also seem to be a more favorable prognostic factor. None of the 128 gliomas analyzed contained an IDH2 mutation. The MGMT gene promoter was methylated in 67% and unmethylated in 33% of glioblastoma patients. This marker can be a predictor factor of response to chemotherapy because the patients who had the promoter hypermethylation and underwent chemotherapy had increased their survival in relation to the patients who did not undergo the therapy (p<0,001). Therefore, according to these Project results, we emphasized the importance of the implementation of these three markers, in routine bases, due to their value concerning the diagnosis leading to a better understanding of the prognosis as well as the clinical course and management and also due to their predictive value of the response to chemotherapy treatments.
Gliomas are the most common primary brain tumors, accounting for more than 80% of all neoplasms arising in the Central Nervous System (CNS) and are currently classified by the World Health Organization (WHO) into astrocytomas, oligodendrogliomas, mixed gliomas and ependymomas. The diagnosis and classification are based on their histopathological appearance, which sometimes may be difficult. Despite advances in neurosurgery, chemotherapy and radiotherapy, the prognosis of most glioma patients remains dismal. In recent years, extensive molecular studies have identified diagnostic, prognostic and predictive markers in gliomas that reinforce the WHO histological classification. The 1p19q coleletion in oligodendroglial tumors, the presence of somatic mutations in the genes enconding isocitrate dehydrogensase 1 and 2 (IDH1 and IDH2) in astrocytomas, oligodendrogliomas e oligoastrocytomas and the hypermethylation of the promoter of O6-methylguanine DNA methyltransferase (MGMT) gene in glioblastomas are currently the three most pertinent markers in diffuse gliomas. Previous studies suggest that codeletion of chromosome arms 1p and 19q has proven to be a powerful diagnostic and prognostic marker. Combined loss of 1p and 19q has been associated with morphology of oligodendrogliomas and with enhanced survival in these tumors. However, in other glial tumors, an oligodendroglial component can mean a better prognostic. Therefore, one of the aims of this Project is the 1p/19q evaluation concerning the patients’ diagnostic and prognostic. Recent published studies considered that mutations in IDH1 and IDH2, are an important diagnostic and prognostic marker but that is still unclear. Also, patients with tumors harboring mutation of IDH1 or IDH2 genes seem to have a better outcome than patients with non mutated tumors. Thus, another goal of this study focus on the identification and characterization of mutations, difference in survival at a population level and to assess whether they allow reliable discrimination between primary (de novo) glioblastomas and secondary glioblastomas (that progressed from low-grade or anaplastic gliomas). DNA repair protein, MGMT, is one implicated factor in glioma chemoresistance. The prognostic and predictive value of MGMT promoter hypermethylation however remains controversial. An additional aim of this study regards the evaluation of the prognostic significance of MGMT in patients with glioblastomas. In total, we analysed 141 patient samples for a series of patients followed in the University Hospital of Coimbra with astrocitomas (n = 111), oligodendrogliomas (n = 18), mixed gliomas (n = 8) and ependymomas (n = 4). In our study, the 1p/19q status was assessed by interphase Fluorescence In Situ Hybridization (iFISH) (n = 130), somatic mutations analysis of IDH1 and IDH2 genes was directed sequenced (n = 128) and MGMT hypermethylation analysis was performed by using Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA) on the glioma samples (n = 38). Our data suggested that deletions of chromosome arms 1p and 19q are associated with oligodendrogliomas morphology (p<0,0001), independently of tumor grade. However, this alteration is not associated with chemosensitivity and enhanced survival. IDH1 mutations were detected in 17 of 128 gliomas, in which 3 different mutations were identified. This study indicated that IDH1 mutations are frequent in secondary glioblastomas but rare in primary glioblastomas (100% vs 1,1%, p<0,0001). Therefore, IDH1 mutations also seem to be a more favorable prognostic factor. None of the 128 gliomas analyzed contained an IDH2 mutation. The MGMT gene promoter was methylated in 67% and unmethylated in 33% of glioblastoma patients. This marker can be a predictor factor of response to chemotherapy because the patients who had the promoter hypermethylation and underwent chemotherapy had increased their survival in relation to the patients who did not undergo the therapy (p<0,001). Therefore, according to these Project results, we emphasized the importance of the implementation of these three markers, in routine bases, due to their value concerning the diagnosis leading to a better understanding of the prognosis as well as the clinical course and management and also due to their predictive value of the response to chemotherapy treatments.
Descrição
Dissertação de Mestrado em Biotecnologia para as Ciências da Saúde
Palavras-chave
Glioma , 1p/19q deletion , Chromosome , Isocitrate dehydrogenase 1 and 2 , Mutation , O-6-methylguanine-DNA methyltransferase , Hypermethylation , Biomarkers