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Combining CGH and high-resolution allelotyping study for ependymoma.January 2001 (has links)
Zheng Ping-pin. / Thesis submitted in: December 2001. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 118-159). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACT(ENGLISH/CHINESE) --- p.iii / CONTENTS --- p.viii / LIST OF TABLES --- p.xi / LIST OF FIGURES --- p.xii / PUBLICATION --- p.xiii / Chapter CHAPTER I --- INTRODUCTION / Chapter I.1. --- Preface --- p.1 / Chapter I.2. --- Overview of Carcinogenesis --- p.2 / Chapter I.3. --- Oncogene --- p.5 / Chapter I.4. --- Tumor Suppressor Genes (TSGs) --- p.6 / Chapter I.5 --- Detection of Oncogene and Tumor Suppressor Genes --- p.9 / Chapter I.5.1 --- Detaction of Oncogene --- p.9 / Chapter I.5.2. --- Detection of Tumor Suppressor Genes --- p.11 / Chapter I.6. --- Profiles of Oncogenes/TSGs and Molecular Subtype about Astrocytic Tumors --- p.17 / Chapter I.7. --- Intratumoral Heterogeneity and Microsatellite Instability --- p.20 / Chapter I.8. --- Outline of Ependymoma --- p.20 / Chapter I.9. --- Clinicopathological Factors and Prognosis --- p.22 / Chapter I.9.1. --- Histology and Grading (2000) --- p.22 / Chapter I.9.2. --- Prognosis Factors --- p.23 / Chapter I.9.2.1. --- Age/Sex/Location --- p.23 / Chapter I.9.2.2. --- Extent of Resection --- p.25 / Chapter I.9.2.3. --- Radiotherapy and Chemotherapy --- p.25 / Chapter I.9.2.4. --- Histology --- p.26 / Chapter I.10. --- "Cytogenetic, Molecular Genetic and Molecular Studies" --- p.27 / Chapter I.11. --- Advantages and Disadvantages of The Research Methods --- p.34 / Chapter CHAPTER II --- AIM OF STUDY --- p.36 / Chapter CHAPTER III --- MATERIALS AND METHODS --- p.37 / Chapter III.1. --- Tumor Samples and DNA Preparations --- p.37 / Chapter III.1.1. --- Tumor Samples --- p.38 / Chapter III.1.2. --- DNA Preparation --- p.38 / Chapter III.2. --- Comparative Genomic Hybridization --- p.42 / Chapter III.2.1. --- Metaphase Preparation --- p.42 / Chapter III.2.2. --- "DNA Labeling, Hybridization, and Detection" --- p.43 / Chapter III.2.3. --- Digital Image Analysis --- p.45 / Chapter III.3 --- High-Resolution Allelotying (Microsatellite Analysis) --- p.46 / Chapter III.3.1 --- General Outline --- p.46 / Chapter III.3.2 --- Multiplex PCR --- p.47 / Chapter III.3.3 --- Pooling of PCR Products --- p.49 / Chapter III.3.4 --- Electrophoresis --- p.50 / Chapter III.3.5. --- Assessment of Allelic Imbalance by Calculating Allelic Ratio --- p.52 / Chapter III.3.6 --- Standards of Evalution --- p.53 / Chapter III.3.7 --- Separating Allelic Loss from Allelic Duplication --- p.54 / Chapter III.3.8 --- Statistical Analyses --- p.54 / Chapter CHAPTER IV --- RESULTS --- p.54 / Chapter IV.1. --- CGH Study --- p.54 / Chapter IV.1.1 --- Overview --- p.54 / Chapter IV.1.2 --- Common Deletion Regions --- p.58 / Chapter IV.1.3 --- Common duplication Regions --- p.60 / Chapter IV.2. --- High-Resolution Allelotyping (Microsatellite Analysis) --- p.60 / Chapter IV.2.1. --- Overview of Results --- p.60 / Chapter IV.2.2. --- LOH profile of Individual Chromosome --- p.93 / Chapter IV.2.3. --- Overlapping Small Deletion Regions --- p.95 / Chapter CHAPTER V --- DISCUSSION --- p.97 / Chapter V.1. --- . General Outline --- p.98 / Chapter V.2. --- Chromosome 22 --- p.99 / Chapter V.3. --- Chromosome 17 --- p.102 / Chapter V.4. --- Chromosome 6 --- p.104 / Chapter V.5. --- Chromosome 16 --- p.105 / Chapter V.6. --- Chromosome 19 --- p.107 / Chapter V.7. --- Chromosome 20 --- p.108 / Chapter V.8. --- Chromosome 7 --- p.109 / Chapter V.9. --- Chromosome 12 --- p.110 / Chapter V.10. --- Chromosome 9 --- p.111 / Chapter V.11. --- Chromosome 5 --- p.112 / Chapter V.12. --- Chromosome 4 --- p.112 / Chapter V.13. --- Correlation of CGH with Allelotyping in the Study --- p.112 / Chapter V.14. --- Conclusion --- p.114 / Chapter CHAPTER VI --- LIMITATIONS OF THE STUDY --- p.115 / Chapter CHAPTER VII --- FUTURE STUDY --- p.116 / REFERENCES --- p.118
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Molecular genetic studies of oligodendroglial and ependymal tumors.January 1998 (has links)
by Tong Yuen Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 124-141). / Abstract also in Chinese. / acknowledgements --- p.i / Abstract (English/Chinese) --- p.ii / contents --- p.vi / list of tables --- p.viii / ost of figures --- p.x / Chapter I. --- introduction --- p.1 / Chapter I.1. --- Tumors of the Central Nervous System --- p.1 / Chapter I.2. --- Histopathological Classification of Human Glial Tumors --- p.3 / Chapter I.2.1. --- Histopathology of Astrocytic Gliomas --- p.3 / Chapter I.2.1.1. --- Diffuse Astrocytomas --- p.3 / Chapter I.2.1.2. --- Others --- p.6 / Chapter I.2.2. --- Histopathology of Non-Astrocytic Gliomas --- p.6 / Chapter I.2.2.1. --- Oligodendroglial Tumors --- p.6 / Chapter I.2.2.2. --- Ependymal Tumors --- p.9 / Chapter I.3. --- Tumor Suppressor Genes --- p.14 / Chapter I.3.1. --- p53 --- p.14 / Chapter I.3.1.1. --- Historical Perspectives --- p.14 / Chapter I.3.1.2. --- Structure of p53 Gene and Protein --- p.15 / Chapter I.3.1.3. --- Functions of Wild-Type p53 Protein --- p.18 / Chapter I.3.1.4. --- Regulation and Modulation of the Functions of p53 --- p.21 / Chapter I.3.1.5. --- Mechnism of p53 Inactivation --- p.23 / Chapter I.3.1.6. --- p53 Mutation Profiles in Human Tumors --- p.25 / Chapter I.3.2. --- Novel Genes --- p.28 / Chapter I.3.2.1. --- PTEN/MMAC1 --- p.28 / Chapter I.3.2.2. --- DMBT1 --- p.31 / Chapter I.4. --- Cytogenetic and Molecular Genetic Studies in Gliomas --- p.34 / Chapter I.4.1. --- Astrocytic Gliomas --- p.34 / Chapter I.4.2. --- Non-Astrocytic Gliomas --- p.39 / Chapter I.4.2.1. --- Oligodendroglial Tumors --- p.39 / Chapter I.4.2.2. --- Ependymal Tumors --- p.46 / Chapter II. --- objectives of study --- p.49 / Chapter III. --- materials and methods --- p.52 / Chapter III.l. --- Patients and Materials --- p.52 / Chapter III.2. --- Collection of Samples --- p.57 / Chapter III.3. --- DNA Extraction --- p.58 / Chapter III.3.1. --- Extraction of Genomic DNA from Formalin-Fixed Paraffin Embedded Tissues --- p.58 / Chapter III.3.2. --- Extraction of Genomic DNA from Blood --- p.60 / Chapter III.4. --- Loss of Heterozygosity (LOH) Analysis on Chromosome 10q --- p.61 / Chapter III.4.1. --- Microsatellite Markers --- p.62 / Chapter III.4.2. --- Amplification of Target Sequences by PCR --- p.63 / Chapter III.4.3. --- Denaturing Polyaerylamide Gel Electrophoresis --- p.64 / Chapter III.4.4. --- Detection of Loss of Heterozygosity (LOH) --- p.64 / Chapter III.5. --- Mutational Analysis of p53 and PTEN/MMAC1 --- p.66 / Chapter III.5.1. --- Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) Analysis --- p.66 / Chapter III.5.1.1. --- PCR Primers --- p.66 / Chapter III.5.1.2. --- PCR Amplification of Target Sequences --- p.68 / Chapter III.5.1.3. --- Non-denaturing Polyacrylamide Gel Electrophoresis --- p.71 / Chapter III.5.2. --- Direct DNA Sequencing Analysis --- p.72 / Chapter III.5.2.1. --- Cycle Sequencing --- p.72 / Chapter III.5.2.2. --- Denaturing Gel Electrophoresis --- p.73 / Chapter III.6. --- Differential PCR for Detection of MDM2 Amplification --- p.74 / Chapter III.6.1. --- DNA Amplification by PCR --- p.74 / Chapter III.6.2. --- Polyacrylamide Gel Electrophoresis --- p.75 / Chapter III.6.3. --- Detection of Gene Amplification --- p.75 / Chapter IV. --- Results --- p.77 / Chapter IV.1. --- LOH Analysis of Chromosome l0q --- p.77 / Chapter IV.2. --- Mutational Analysis ofp53 and PTEN/MMAC1 --- p.92 / Chapter IV.3. --- Differential PCR Analysis of MDM2 Amplification --- p.103 / Chapter V. --- discussion --- p.109 / Chapter V.l. --- p53 Gene Inactivation Studies --- p.110 / Chapter V.2. --- Molecular Genetic Studies on Chromosome l0q --- p.113 / Chapter V.3. --- Microsatellite Instability in Non-Astrocytic Gliomas --- p.117 / Chapter V.4. --- Significance of This Study --- p.118 / Chapter V.5. --- Limitations of This Study --- p.119 / Chapter V.6. --- Future Studies --- p.122 / Chapter VI. --- REFERENCES --- p.124
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Estudo das alterações na expressão gênica dos ependimomas / Study of gene expression alterations in ependymomasAndrade, Fernanda Gonçalves de 11 June 2014 (has links)
Ependimomas são tumores gliais raros. Podem ser encontrados em qualquer localização do sistema nervoso central e, apesar de histologia similar, parecem apresentar alterações genômicas distintas. As variáveis clínicas são intercorrelacionadas e, geralmente, incapazes de predizer o curso da doença. O objetivo do presente estudo foi analisar a expressão aumentada de genes e proteínas em ependimomas e correlacionar com dados clínicos dos pacientes. Foram estudados casos de pacientes com ependimoma submetidos à ressecção cirúrgica no Hospital das Clínicas, Universidade de São Paulo, no período entre 1996 e 2011 (33 amostras de tecido congelado para análise de expressão gênica por PCR quantitativo em tempo real e 149 amostras com tecido incluído em parafina, correspondentes a 121 casos devido a recidivas, para análise de proteína por imuno-histoquímica de tissue microarrays). As reações de imuno-histoquímica foram analisadas semiquantitativamente e graduadas com um índice de marcação calculado pelo produto da porcentagem de núcleos marcados pela intensidade de marcação. Oitenta e um casos eram adultos (média de 27,2 anos). Havia 60 casos intracranianos e 61 intramedulares, dos quais 10 eram mixopapilares, 92 grau II e 19 grau III. Ressecção completa foi possível em 62% dos casos e recidiva foi confirmada em 41,1%. Observou-se menor tempo para recidiva em crianças e tumores intracranianos, supratentoriais (p < 0,001 em ambos), histologia anaplásica e ressecções incompletas (p < 0,05 em ambos). Os seguintes genes foram selecionados em dados públicos de SAGE e literatura: ARMC3, CCND1, CHST5, DNALI1, FGFRL1, GNA13, IGF2, MSX1, NOTCH1 e RSPH3. ARMC3, RSHL3, CHST5 e DNALI1 apresentaram maiores níveis de expressão em ependimomas intramedulares (p < 0,05), e FGFRL1, NOTCH1 e CCND1 nos casos supratentoriais (p < 0,01). IGF2 apresentou maiores níveis de expressão em crianças e CHST5 em adultos (p < 0,05 em ambos). Foram observados maiores níveis de expressão de FGFRL1 (p < 0,05), CCND1 e IGF2 (p < 0,01 em ambos) em casos com histologia anaplásica. Nenhum dos genes analisados apresentou impacto no tempo livre de progressão ou na sobrevida. A expressão da proteína codificada por CCND1, ciclina D1, também foi avaliada por imuno-histoquímica, por ser uma proteína com expressão aumentada em diversos tipos de neoplasias e não ter ainda um valor prognóstico bem estabelecido em ependimomas. Houve correlação entre expressão de ciclina D1 a nível de mRNA e da proteína (p < 0,0001). As correlações entre ciclina D1 e histologia anaplásica e localização supratentorial foram confirmadas pela análise proteica (p < 0,0001 em ambos). Adicionalmente, foi também observada maior expressão de ciclina D1 em pacientes mais jovens (p < 0,01). A maior expressão de ciclina D1 em tumores com localização supratentorial foi independente do grau histológico e da idade do paciente. Recidiva foi mais frequente em casos com maiores níveis de expressão de ciclina D1 (p < 0,05), embora uma maior correlação com tempo livre de progressão foi observada apenas em casos com ressecção completa (p < 0,001). Os ependimomas apresentaram expressão gênica diferencial de acordo com idade, localização do tumor e grau histológico nesse estudo. A determinação dos níveis da expressão de ciclina D1 pode ser útil para guiar o seguimento e tratamento dos casos supratentoriais com ressecções completas / Ependymomas are rare glial cell-derived tumors. They can be found in any central nervous system localization and despite the histological similarity, they seem to display distinct genomic abnormalities. Clinical variables are intercorrelated and they are usually unable to predict the disease course. We aimed to analyze increased gene and protein expression in ependymomas and to correlate with patients\' clinical data. We studied patients with ependymoma submitted to surgical resections at Hospital das Clinicas, University of São Paulo, from 1996 to 2011 (33 fresh-frozen samples for gene expression analysis by quantitative real-time PCR and 149 formalin-fixed, paraffin-embedded samples, relative to 121 patients due to relapses, for protein analysis by tissue microarray immunohistochemistry). Immunohistochemical reactions were analyzed semi-quantitatively and scored with a labeling index (LI) calculated as the product of the percentage of the positively stained nuclei by the intensity of staining. Eighty-one cases were adults (mean 27.2 years). There were 60 intracranial and 61 spinal cases, of which 10 tumors were myxopapillary, 92 were grade II and 19 were grade III. Gross total resection was achieved in 62% of cases and relapse was confirmed in 41.4% of cases. We observed a shorther time to relapse in children and supratentorial intracranial tumor localization (p<0.001 for both), anaplastic histology and incomplete resections (p<0.05 for both). The following genes were selected based on public SAGE database and literature: ARMC3, CCND1, CHST5, DNALI1, FGFRL1, GNA13, IGF2, MSX1, NOTCH1 and RSPH3. ARMC3, RSHL3, CHST5 and DNALI1 presented higher expression levels in intramedullary ependymomas (p < 0.05) and FGFRL1, NOTCH1 and CCND1 in supratentorial cases (p < 0.01). IGF2 presented higher expression levels in pediatric cases and CHST5 in adults cases (p < 0.05 in both). Higher expression levels of FGFRLI1 (p < 0.05), CCND1 and IGF2 (p < 0.01 for both) were observed in anaplastic histology cases. None of the genes impacted in progression free survival or overall survival of patients. The expression of protein codified by CCND1, cyclin D1, was also evaluated by immunohistochemistry, because its overexpression has been related with several types of neoplasias and its prognostic value has not yet been fully established in ependymomas. There was a correlation of cyclin D1 expression at mRNA and protein levels (p < 0.0001). Correlations between cyclin D1 and anaplastic histology and supratentorial localization were confirmed by protein analyses (p < 0.0001 for both parameters). Additionally, high expression of cyclin D1 was observed in younger patients (p < 0.01). The higher cyclin D1 expression in supratentorial tumor localization was independent of histological grade and age of patient. Relapse was more frequent in cases with higher cyclin D1 expression levels (p < 0.05) although correlation with progression free survival was just observed in gross total resection cases (p < 0.001). Ependymomas presented differential gene expression according to age, tumor localization and histological grade in our study. Determination of cyclin D1 expression levels may be useful to guide follow-up and treatment in supratentorial cases with gross total resection
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Estudo das alterações na expressão gênica dos ependimomas / Study of gene expression alterations in ependymomasFernanda Gonçalves de Andrade 11 June 2014 (has links)
Ependimomas são tumores gliais raros. Podem ser encontrados em qualquer localização do sistema nervoso central e, apesar de histologia similar, parecem apresentar alterações genômicas distintas. As variáveis clínicas são intercorrelacionadas e, geralmente, incapazes de predizer o curso da doença. O objetivo do presente estudo foi analisar a expressão aumentada de genes e proteínas em ependimomas e correlacionar com dados clínicos dos pacientes. Foram estudados casos de pacientes com ependimoma submetidos à ressecção cirúrgica no Hospital das Clínicas, Universidade de São Paulo, no período entre 1996 e 2011 (33 amostras de tecido congelado para análise de expressão gênica por PCR quantitativo em tempo real e 149 amostras com tecido incluído em parafina, correspondentes a 121 casos devido a recidivas, para análise de proteína por imuno-histoquímica de tissue microarrays). As reações de imuno-histoquímica foram analisadas semiquantitativamente e graduadas com um índice de marcação calculado pelo produto da porcentagem de núcleos marcados pela intensidade de marcação. Oitenta e um casos eram adultos (média de 27,2 anos). Havia 60 casos intracranianos e 61 intramedulares, dos quais 10 eram mixopapilares, 92 grau II e 19 grau III. Ressecção completa foi possível em 62% dos casos e recidiva foi confirmada em 41,1%. Observou-se menor tempo para recidiva em crianças e tumores intracranianos, supratentoriais (p < 0,001 em ambos), histologia anaplásica e ressecções incompletas (p < 0,05 em ambos). Os seguintes genes foram selecionados em dados públicos de SAGE e literatura: ARMC3, CCND1, CHST5, DNALI1, FGFRL1, GNA13, IGF2, MSX1, NOTCH1 e RSPH3. ARMC3, RSHL3, CHST5 e DNALI1 apresentaram maiores níveis de expressão em ependimomas intramedulares (p < 0,05), e FGFRL1, NOTCH1 e CCND1 nos casos supratentoriais (p < 0,01). IGF2 apresentou maiores níveis de expressão em crianças e CHST5 em adultos (p < 0,05 em ambos). Foram observados maiores níveis de expressão de FGFRL1 (p < 0,05), CCND1 e IGF2 (p < 0,01 em ambos) em casos com histologia anaplásica. Nenhum dos genes analisados apresentou impacto no tempo livre de progressão ou na sobrevida. A expressão da proteína codificada por CCND1, ciclina D1, também foi avaliada por imuno-histoquímica, por ser uma proteína com expressão aumentada em diversos tipos de neoplasias e não ter ainda um valor prognóstico bem estabelecido em ependimomas. Houve correlação entre expressão de ciclina D1 a nível de mRNA e da proteína (p < 0,0001). As correlações entre ciclina D1 e histologia anaplásica e localização supratentorial foram confirmadas pela análise proteica (p < 0,0001 em ambos). Adicionalmente, foi também observada maior expressão de ciclina D1 em pacientes mais jovens (p < 0,01). A maior expressão de ciclina D1 em tumores com localização supratentorial foi independente do grau histológico e da idade do paciente. Recidiva foi mais frequente em casos com maiores níveis de expressão de ciclina D1 (p < 0,05), embora uma maior correlação com tempo livre de progressão foi observada apenas em casos com ressecção completa (p < 0,001). Os ependimomas apresentaram expressão gênica diferencial de acordo com idade, localização do tumor e grau histológico nesse estudo. A determinação dos níveis da expressão de ciclina D1 pode ser útil para guiar o seguimento e tratamento dos casos supratentoriais com ressecções completas / Ependymomas are rare glial cell-derived tumors. They can be found in any central nervous system localization and despite the histological similarity, they seem to display distinct genomic abnormalities. Clinical variables are intercorrelated and they are usually unable to predict the disease course. We aimed to analyze increased gene and protein expression in ependymomas and to correlate with patients\' clinical data. We studied patients with ependymoma submitted to surgical resections at Hospital das Clinicas, University of São Paulo, from 1996 to 2011 (33 fresh-frozen samples for gene expression analysis by quantitative real-time PCR and 149 formalin-fixed, paraffin-embedded samples, relative to 121 patients due to relapses, for protein analysis by tissue microarray immunohistochemistry). Immunohistochemical reactions were analyzed semi-quantitatively and scored with a labeling index (LI) calculated as the product of the percentage of the positively stained nuclei by the intensity of staining. Eighty-one cases were adults (mean 27.2 years). There were 60 intracranial and 61 spinal cases, of which 10 tumors were myxopapillary, 92 were grade II and 19 were grade III. Gross total resection was achieved in 62% of cases and relapse was confirmed in 41.4% of cases. We observed a shorther time to relapse in children and supratentorial intracranial tumor localization (p<0.001 for both), anaplastic histology and incomplete resections (p<0.05 for both). The following genes were selected based on public SAGE database and literature: ARMC3, CCND1, CHST5, DNALI1, FGFRL1, GNA13, IGF2, MSX1, NOTCH1 and RSPH3. ARMC3, RSHL3, CHST5 and DNALI1 presented higher expression levels in intramedullary ependymomas (p < 0.05) and FGFRL1, NOTCH1 and CCND1 in supratentorial cases (p < 0.01). IGF2 presented higher expression levels in pediatric cases and CHST5 in adults cases (p < 0.05 in both). Higher expression levels of FGFRLI1 (p < 0.05), CCND1 and IGF2 (p < 0.01 for both) were observed in anaplastic histology cases. None of the genes impacted in progression free survival or overall survival of patients. The expression of protein codified by CCND1, cyclin D1, was also evaluated by immunohistochemistry, because its overexpression has been related with several types of neoplasias and its prognostic value has not yet been fully established in ependymomas. There was a correlation of cyclin D1 expression at mRNA and protein levels (p < 0.0001). Correlations between cyclin D1 and anaplastic histology and supratentorial localization were confirmed by protein analyses (p < 0.0001 for both parameters). Additionally, high expression of cyclin D1 was observed in younger patients (p < 0.01). The higher cyclin D1 expression in supratentorial tumor localization was independent of histological grade and age of patient. Relapse was more frequent in cases with higher cyclin D1 expression levels (p < 0.05) although correlation with progression free survival was just observed in gross total resection cases (p < 0.001). Ependymomas presented differential gene expression according to age, tumor localization and histological grade in our study. Determination of cyclin D1 expression levels may be useful to guide follow-up and treatment in supratentorial cases with gross total resection
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