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

Introdução: Os ependimomas são tumores gliais raros e compreendem o terceiro tumor do sistema nervoso central mais frequente na infância. Apesar dos avanços terapêuticos, cerca de 50% dos pacientes desenvolvem recidiva local e 40% dos pacientes vão ao óbito. Uma das causas do insucesso das terapias é a alta heterogeneidade do tumor e a inconsistência do diagnóstico histológico. Em 2015, foi publicada pela primeira vez a caracterização molecular de ependimomas, sendo descrito nove subgrupos tumorais com perfis clínicos, demográficos e moleculares distintos. Objetivo: Estabelecer e padronizar a classificação molecular em amostras de ependimomas pediátricos e correlacionar a classificação com dados clínicos dos pacientes. Casuística e Métodos: Foram estudados 65 casos de ependimomas, diagnosticados no período entre 2001 a 2016 e provenientes do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto e de São Paulo e do Centro Infantil Boldrini-Campinas. Vinte e seis casos eram ependimomas supratentoriais, classificados com base na presença de fusões gênicas C11orf95-RELA, YAP1-MAMLD1 e YAP1-FAM118B utilizando RT-PCR seguida por sequenciamento de Sanger. Trinta e nove casos de fossa posterior foram classificados em Grupos A, B ou não A e B através do perfil de expressão proteica e gênica dos marcadores: LAMA2, NELL2 e TNC utilizando imuno-histoquímica e PCR quantitativo em tempo real, respectivamente. Resultados: Dentre os ependimomas supratentoriais foram identificadas três amostras primárias e cinco amostras recidivadas com presença de fusão RELA, média de idade de 7 anos (variação de 2,6-13,7 anos), predominância do sexo masculino e grau de ressecção cirúrgica completa. Já a fusão YAP1- MAMLD1 foi identificada em quatro casos, diagnosticados em crianças mais novas, média de idade de 0,9 anos (variação de 0,75-2 anos). Adicionalmente, foi encontrado um caso variante em ependimoma supratentorial, denominado fusão C11orf95-LOC-RELA. Dentre os casos de ependimoma de fossa posterior, foram identificadas 26 amostras primárias e sete recidivas sugestivas de pertencerem ao Grupo A (LAMA2+/ NELL2 -) e seis amostras do Grupo não-A e não-B (LAMA2 +/ NELL2 + e LAMA2 -/NELL2-). Entre os pacientes considerados Grupo A, 90% (24/28) apresentaram marcação positiva para TNC, indicando serem tumores de pior prognóstico. A expressão gênica de LAMA2 e NELL2 apresentou correlação negativa e os genes TNC e LAMA2 uma correlação positiva, p<0,01 e p<0,05, respectivamente. Em ependimoma de fossa posterior Grupo A, os pacientes submetidos às ressecções completa e incompleta apresentaram diferença significativa na sobrevida global (5 anos) de 71,2% ± 14,5% versus 21,4% ± 17,8%, p< 0,01 e na SLE (2 anos) 63,5% ± 14,8% versus 25% ± 15.3%, p <0.001. Conclusões: De acordo com os resultados obtidos foi possível estabelecer a classificação molecular em uma casuística brasileira, seguindo os padrões descritos na literatura. Dados gerados a partir dessa padronização serão de fundamental importância para melhoria da estratificação tumoral, contribuindo tanto para determinação de estratégicas terapêuticas subgrupo-específicas, quanto na busca de novos alvos terapêuticos. / Introduction: Ependymomas are rare glial cell-derived tumors and comprise the third most frequent central nervous system tumor in childhood. Despite the therapeutic advances, about 50% of patients develop local recurrence and 40% of patients go to death. One of the causes of the failure of the therapies is the high tumor heterogeneity and the inconsistency of the histological diagnosis. In 2015, the molecular characterization of ependymomas was published for the first time, and nine tumor subgroups with distinct clinical, demographic and molecular profiles were described. Aim: To establish and standardize molecular classification in pediatric ependymoma samples and to correlate the classification with clinical data of the patients. Methods: We studied 65 cases of ependymomas, diagnosed between 2001 and 2016, from the Clinics Hospital of the Medical School of Ribeirão Preto and São Paulo and the Boldrini Children\'s Center-Campinas. Twenty-six cases were supratentorial ependymomas, classified based on the presence of gene fusions C11orf95- RELA, YAP1-MAMLD1 and YAP1-FAM118B using RT-PCR followed by Sanger sequencing. Thirty-nine posterior fossa cases were classified into Groups A, B or non A and B through the protein and gene expression profile of the markers: LAMA2, NELL2 and TNC using immunohistochemistry and quantitative real-time PCR, respectively. Results: Among the supratentorial ependymomas, three primary samples and five relapsed samples with RELA fusion, mean age of 7 years (range of 2.6-13.7 years), male predominance, and degree of complete surgical resection were identified. The YAP1-MAMLD1 fusion was identified in four cases, diagnosed in younger children, mean age 0.9 years (range of 0.75-2 years). In addition, a variant case was found in supratentorial ependymoma, called fusion C11orf95- LOC-RELA. Twenty-six primary samples and seven recurrences suggestive of Group A (LAMA2 + / NELL2-) and six non-A and non-B Group samples (LAMA2 + / NELL2 + and LAMA2 - / NELL2-). Among the patients considered Group A, 90% (24/28) presented positive staining for TNC, indicating that tumors had a worse prognosis. The gene expression of LAMA2 and NELL2 presented negative correlation and the TNC and LAMA2 genes had a positive correlation, p <0.01 and p <0.05, respectively. In the group A posterior fossa ependymoma, patients submitted to complete and incomplete surgical presented a significant difference in overall survival (5 years) of 71.2% ± 14.5% versus 21.4% ± 17.8%, p <0 , 01 and in SLE (2 years) 63.5% ± 14.8% versus 25% ± 15.3%, p <0.001. Conclusions: According to the results obtained, it was possible to establish the molecular classification in a Brazilian cohort, following the standards described in the literature. Data generated from this standardization will be of fundamental importance for the improvement of tumor stratification, contributing both to the determination of subgroup-specific therapeutic strategies and to the search for new therapeutic targets.

Paediatric ependymomas are the third most common childhood brain cancer and represent a prognostic and therapeutic challenge. Previous evidence suggests that telomerase, a ribonucleoprotein critical in permitting limitless growth potential, may serve as both a prognostic marker and therapeutic target. Immunohistochemical analysis (n=198) and enzymatic detection (n=25) of telomerase was performed to determine prevalence and prognostic potential. The telomerase inhibitor Imetelstat was used to study telomerase inhibition in paediatric ependymoma cell lines, tumour initiating cells (TICs) and both subcutaneous and intracranial xenografts. Telomerase activity was detected in 76% of primary ependymomas and was associated with a reduced five-year progression-free survival (30% vs 75%). Telomerase inhibition in vitro resulted in shortened telomeres, increased senescence, growth inhibition and reduced self-renewal capacity. In vivo, Imetelstat shortened telomeres and reduced subcutaneous tumour volume by 40% compared to control mice. Therefore, telomerase may serve as an ideal prognostic marker and therapeutic target in paediatric ependymoma.

Paediatric ependymomas are the third most common childhood brain cancer and represent a prognostic and therapeutic challenge. Previous evidence suggests that telomerase, a ribonucleoprotein critical in permitting limitless growth potential, may serve as both a prognostic marker and therapeutic target. Immunohistochemical analysis (n=198) and enzymatic detection (n=25) of telomerase was performed to determine prevalence and prognostic potential. The telomerase inhibitor Imetelstat was used to study telomerase inhibition in paediatric ependymoma cell lines, tumour initiating cells (TICs) and both subcutaneous and intracranial xenografts. Telomerase activity was detected in 76% of primary ependymomas and was associated with a reduced five-year progression-free survival (30% vs 75%). Telomerase inhibition in vitro resulted in shortened telomeres, increased senescence, growth inhibition and reduced self-renewal capacity. In vivo, Imetelstat shortened telomeres and reduced subcutaneous tumour volume by 40% compared to control mice. Therefore, telomerase may serve as an ideal prognostic marker and therapeutic target in paediatric ependymoma.

Orientador: Helder Tedeschi / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T14:04:03Z (GMT). No. of bitstreams: 1 Santos_MarcosJulianodos_M.pdf: 1157265 bytes, checksum: d4e3bca14a9db06599e52397627cd980 (MD5) Previous issue date: 2013 / Resumo: Os ependimomas intramedulares são tumores de crescimento lento que acarretam déficits neurológicos progressivos e pode levar os pacientes a dependência funcional. Embora a remoção microcirúrgica seja o tratamento de escolha para a lesão, questionamento existe sobre se pacientes com apenas sinais clínicos leves devem ser submetidos a ressecções cirúrgicas radicais. Este estudo avaliou, segundo a escala funcional de McCormick, a evolução pós-operatória de uma série de vinte pacientes submetidos à ressecção microcirúrgica total para ependimomas intramedulares. A ressecção completa foi atingida em 19 dos 20 pacientes (95%), somente 1 paciente apresentou piora clínica (5%). Nos pacientes com independência funcional pré-operatória, com McCormick grau I e II, não houve piora clínica e todos os tumores foram ressecados completamente. No subgrupo de pacientes grau II, a média do status funcional pós-operatória apresentou melhora estatisticamente significativa. Nenhum paciente com grau IV melhorou após o tratamento cirúrgico. O tratamento cirúrgico foi eficaz para ressecar completamente os tumores sem agregar déficits neurológicos na maioria dos pacientes. Nos pacientes com McCormick graus I e II pré-operatório a cirurgia deve ser indicada no momento do diagnóstico / Abstract: Intramedullary ependymomas are slow-growing lesions that progressively lead to neurological compromise and functional dependence. Although surgical excision is the treatment of choice for such lesions, there is questioning as to whether patients with only subtle clinical findings should be subjected to radical surgical resections. This study has evaluated according to the McCormick functional scale the surgical outcome of a series of twenty patients with intramedullary ependymomas submitted to microsurgical resection. Total surgical resection was achieved in 19 of the 20 patients (95%). Only one patient experienced clinical worsening (5%). Patients classified as McCormick grade I and II who were independent pre-operatively remained so in the post-operative period and had their tumors completely removed. In grade II patients there was a significant improvement in their post-operative status. None of the grade IV patients improved after surgical treatment. Surgical treatment has proven to be efficient in completely removing tumors without adding neurological deficits in most patients. In patients McCormick grades I and II pre-operatively surgery should be indicated early in the diagnosis / Mestrado / Neurologia / Mestre em Ciências Médicas

Ce travail de thèse fait partie d’un effort pour le développement des biomarqueurs, actuellement largement inexistants, pour une meilleure classification, pour une détermination plus précise du pronostic, et pour la prédiction de la réponse au traitement des tumeurs gliales malignes de l’enfant (épendymomes et gliomes de haut grade). Dans certains cas, ces biomarqueurs peuvent aussi devenir des cibles thérapeutiques.Dans ces études, nous avons pu montrer que la surexpression fréquente des marqueurs neuronaux distingue les épendymomes supratentoriels des formes infratentorielles. Parmi les épendymomes supratentoriels, une forte expression de neurofilament 70 (NF) est corrélée avec une meilleure survie sans récidive. La tenascine C (TNC) est surexprimée dans les épendymomes infratentoriels. Une étude de collaboration européenne multi-institutionnelle a été mise en place, permettant d’analyser une cohorte pédiatrique de 250 patients atteints d’un épendymome, et de démontrer la forte immunoexpression de TNC comme un marqueur robuste, associé a des survies globale et sans récidive plus péjoratives, particulièrement parmi les enfants âgés de moins de trois ans. Ceci a été validé dans une cohorte indépendante. Des immunomarquages pour NF et TNC pourraient être utilisés en clinique pour aider à déterminer le pronostic des épendymomes chez l’enfant.Une analyse des marqueurs pour la prédiction de la réponse à une thérapie ciblée anti-EGFR (erlotinib) a été réalisée par imunnohistochimie et FISH. La perte fréquente de PTEN dans les gliomes infiltrants du tronc cérébrale et la confirmation des caractéristiques des certains sous groupes (avec une forte expression de EGFR ou avec une différentiation oligodendrogliale) nous a permis de dessiner le protocole pour la prochaine étude de phase III pour cette maladie dans le cadre d’un futur essai thérapeutique de phase I/II. Nous rapportons par ailleurs des mutations du gène PI3KCA dans certains gliomes infiltrants du tronc cérébral, qui comme la perte d’expression de PTEN pourrait entrainer une activation de la voie mTOR qui devient donc une cible thérapeutique majeure théorique dans cette maladie. Des études ultérieures seront nécessaires pour définir le rôle de l’interaction entre la perte de PTEN, la surexpression de EGFR, la différentiation oligodendrogliale, les mutations de PI3KCA et d’autres altérations récemment décrites, (gains et amplifications de PDGFRA/MET mutations de TP53) et leur relation avec le devenir des patients sous traitement ciblé et les thérapies ciblées les plus intéressantes dans cette maladie uniformément fatale.Ce travail de thèse nous a permis d’explorer le rôle de la pathologie moléculaire dans la prise en charge des épendymomes et gliomas de haut grade chez l’enfant. / Biomarkers for the classification, clinical management and prognosis of pediatric brain tumors (ependymoma and high grade glioma, (HGG)) are lacking. To address this, biomarkers were developed and explored in view of classification, prognostication, target identification and prediction of the efficacy of treatment for patients with such tumors.We show that overexpression of neuronal markers distinguishes supratentorial from infratentorial ependymoma, and among the former higher immunoexpression of neurofilament 70 (NEFL) is correlated with better progression free survival (PFS). Tenascin-C (TNC) is significantly overexpressed in infratentorial ependymoma. A multi-institutional European ependymoma collaboration group was established and analyses were performed in a pediatric cohort of 250 patients, where immunohistochemistry (IHC) for TNC showed to be a robust marker of poor overall survival (OS) and PFS, particularly among children under 3 years, this being further validated in an independent cohort. Techniques and scoring performed in different laboratories were highly reproducible. IHC for NEFL and TNC could be used for prognostication of pediatric ependymoma.The analysis of putative predictive markers for the response to targeted therapies in pediatric HGG in the setting of a clinical trial with the anti-EGFR agent erlotinib was performed by IHC and fluorescent in situ hybridization. The frequent loss of PTEN in diffuse intrinsic pontine glioma (DIPG) and the confirmation of the biological singularity of the certain subgroups (expressing EGFR, displaying oligodendroglial differentiation) which seem to be associated with better response to erlotinib have helped our group to establish the design of the next Phase III protocol for this disease at our institution. We report mutations in PI3KCA constituting the first identification of oncogene mutations in some DIPG, which further highlight their biological heterogeneity. Further studies are needed to define the interaction between PTEN loss, EGFR overexpression, oligodendroglial differentiation, PI3KCA mutations and other recent findings such as PDGFRA/MET gains/amplification and TP53 mutations in these heterogeneous lesions and their relationship to the outcome of patients under new targeted therapies for this largely fatal disease.This thesis has allowed us to explore the molecular pathology in the context of biology and clinical setting of pediatric brain tumors.

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

<p>Genetic and epigenetic variation is a key determinant of human diversity and has an impact on disease predisposition. Single nucleotide polymorphisms (SNPs) and copy number polymorphisms (CNPs) are the main forms of genetic variation. The challenge is to distinguish normal variations from disease-associated changes. Combination of genetic and epigenetic alterations, often together with an environmental component, can cause cancer. In paper I, we investigated possible alterations affecting the transcriptional regulation of PDGFRα in patients affected with central nervous system tumors by characterizing the haplotype combinations in the <i>PDGFRA</i> gene promoter. A specific over-representation of one haplotype (H2δ) in primitive neuroectodermal tumors and ependymomas was observed, suggesting a functional role for the ZNF148/PDGFRα pathway in the tumor pathogenesis. In paper II, 50 glioblastomas were analyzed for DNA copy number variation with a chromosome 22 tiling genomic array. While 20% of tumors displayed monosomy 22, copy number variations affecting a portion of chromosome 22 were found in 14% of cases. This implies the presence of genes involved in glioblastoma development on 22q. Paper III described the analysis of copy number variation of 37 ependymomas using the same array. We detected monosomy in 51.5% of the samples. In addition, we identified two overlapping germline deletions of 2.2 Mb and 320 kb (the latter designated as Ep CNP). In order to investigate whether Ep CNP was a common polymorphism in the normal population or had an association with ependymoma development, we constructed a high-resolution PCR product-based microarray covering this locus (paper IV). For this purpose, we developed a program called <i>Sequence Allocator</i>, which automates the process of array design. This approach allowed assessment of copy number variation within regions of segmental duplications. Our results revealed that gains or deletions were identical in size and encompassed 290 kb. Therefore, papers I-IV suggest that some SNPs and CNPs can be regarded as tumor-associated polymorphisms. Finally, paper V describes variation of DNA methylation among fully differentiated tissues by using an array covering ~9% of the human genome. Major changes in the overall methylation were also found in colorectal cancer cell lines lacking one or two DNA methyltransferases.</p>

Genetic and epigenetic variation is a key determinant of human diversity and has an impact on disease predisposition. Single nucleotide polymorphisms (SNPs) and copy number polymorphisms (CNPs) are the main forms of genetic variation. The challenge is to distinguish normal variations from disease-associated changes. Combination of genetic and epigenetic alterations, often together with an environmental component, can cause cancer. In paper I, we investigated possible alterations affecting the transcriptional regulation of PDGFRα in patients affected with central nervous system tumors by characterizing the haplotype combinations in the PDGFRA gene promoter. A specific over-representation of one haplotype (H2δ) in primitive neuroectodermal tumors and ependymomas was observed, suggesting a functional role for the ZNF148/PDGFRα pathway in the tumor pathogenesis. In paper II, 50 glioblastomas were analyzed for DNA copy number variation with a chromosome 22 tiling genomic array. While 20% of tumors displayed monosomy 22, copy number variations affecting a portion of chromosome 22 were found in 14% of cases. This implies the presence of genes involved in glioblastoma development on 22q. Paper III described the analysis of copy number variation of 37 ependymomas using the same array. We detected monosomy in 51.5% of the samples. In addition, we identified two overlapping germline deletions of 2.2 Mb and 320 kb (the latter designated as Ep CNP). In order to investigate whether Ep CNP was a common polymorphism in the normal population or had an association with ependymoma development, we constructed a high-resolution PCR product-based microarray covering this locus (paper IV). For this purpose, we developed a program called Sequence Allocator, which automates the process of array design. This approach allowed assessment of copy number variation within regions of segmental duplications. Our results revealed that gains or deletions were identical in size and encompassed 290 kb. Therefore, papers I-IV suggest that some SNPs and CNPs can be regarded as tumor-associated polymorphisms. Finally, paper V describes variation of DNA methylation among fully differentiated tissues by using an array covering ~9% of the human genome. Major changes in the overall methylation were also found in colorectal cancer cell lines lacking one or two DNA methyltransferases.

Prognostic factors come in a variety of forms and may be patient, tumour or environmental related. This thesis examines the interaction of prognostic factors for a variety of tumour types. It particularly focuses on single nucleotide polymorphisms (SNPs) of the vascular endothelial growth factor (VEGF) gene. The first section on meningiomas describes the frequency of sex steroid receptors in meningiomas. In this study, absence of progesterone receptors is associated with high tumour grade and male gender. Tumours that are progesterone receptor negative have an odds ratio for recurrence of 5.Choroid plexus carcinomas are aggressive malignant tumours generally occurring in young children. Gross total surgical resection has been shown to be a highly significant factor in tumour recurrence and survival. This study describes a treatment paradigm of neoadjuvant ICE chemotherapy in these children which decreases the vascularity and increase the chance of a complete removal. The operative blood loss with this regimen is reduced to 0.22 blood volumes from 1.11 blood volumes without neoadjuvant chemotherapy. The VEGF gene is highly polymorphic and SNPs of the region have previously been shown to influence VEGF protein expression. This study looks at cohorts of both adult gliomas and a variety of paediatric brain tumours; comparing them to controls. There are several associations described between the development of certain tumours and specific SNP genotypes. In addition to this, certain genotypes and haplotypes have an influence on survival of adult grade 2 astrocytomas and paediatric medulloblastomas and ependymomas. There are consistent themes to the prognostic genotypes throughout both the adult and the paediatric tumours.Prognostic factors come in a variety forms as described in this thesis. It is vital to understand the complex interaction between factors to best utilise them for the benefit of patients.