Os tumores do sistema nervoso central representam aproximadamente 2% de todos os tipos de cânceres. Embora a incidência dos tumores do SNC seja pequena, comparada com outras neoplasias, estes tumores estão entre as mais graves malignidades humanas, pois afetam o órgão responsável pela coordenação e integração de todas as atividades orgânicas. Os gliomas são os tumores mais comuns do SNC. Apesar do progresso marcante na caracterização da patogênese molecular dos gliomas, esses tumores permanecem incuráveis e, na maioria dos casos, refratários aos tratamentos, devido à sua heterogeneidade molecular. O aparecimento desses tumores ocorrem a partir do acúmulo de alterações genéticas nas células. Para entender o mecanismo molecular de formação e progressão tumoral é indispensável identificar os genes que acumulam essas alterações. Um polimorfismo de base única (SNP Single Nucleotide Polymorphism) é geralmente definido como uma substituição estável de apenas uma base na molécula de DNA com frequência maior que 1%, em pelo menos uma população Os SNPs são reconhecidos como importantes ferramentas na genética humana e médica e têm sido amplamente utilizados nos estudos de associação genética de várias doenças complexas, como por exemplo: distúrbios cardiovasculares, psiquiátricos e autoimunes, obesidade, osteoporose, diabetes e câncer Sendo assim, este trabalho teve como objetivo analisar polimorfismos entre populações caso e controle na intenção de identificar associações destes genótipos na suscetibilidade aos tumores. A técnica utilizada para a análise de polimorfismos foi de PCR-RFLP onde observamos diferenças nas distribuições genotípicas entre pacientes e controles nos SNPs EGF+61, GSTP-1Ile 105 Val, XRCC1 Arg 194 Trp, Pro 206 Pro, Arg 280 His, Arg 399 Gln, Gln 632 Gln, XRCC2 Arg 188 His, XRCC3 Thr 241 Met e XRCC4 G1394T, onde as variantes EGF G61, Trp194, Val105, Pro206, His280, Gln632, His188, Met241 e XRCC4 T1394 foram observados com maior freqüência entre os portadores de gliomas. Dessa forma, estas variantes podem ser fatores de susceptibilidade para o desenvolvimento dos tumores. / The Central nervous system tumors represent about 2% of all cancers. Although the incidence of CNS tumors is small compared with other cancers, these tumors are among the most serious human malignancies, because they affect the body responsible for coordination and integration of all organic activities. Gliomas are the most common tumors of the CNS. Despite remarkable progress in characterizing the molecular pathogenesis of gliomas, these tumors remain incurable and, in most cases, refractory to treatment, due to its molecular heterogeneity. The appearance of these tumors occurs from the accumulation of genetic changes in cells. To understand the molecular mechanism of tumor formation and progression is essential to identify genes that accumulate these changes. A single base polymorphism (SNP Single Nucleotide Polymorphism) is generally defined as a stable replacement of only one base in the DNA molecule often greater than 1% in at least one population SNPs are recognized as important tools in human genetics and medical and have been widely used in genetic association studies of various complex diseases, such as: cardiovascular, psychiatric and autoimmune diseases, obesity, osteoporosis, diabetes and cancer. Thereby, the objective of this study was to analyze the polymorphisms between cases and control the intention to identify associations of these genotypes in susceptibility to tumors. The technique used for the analysis of polymorphisms were PCR-RFLP where we observe differences in genotype between patients and controls in the EGF +61, GSTP-1 Ile105Val, XRCC1 Arg 194 Trp, Pro 206 Pro, Arg 280 His, Arg 399 Gln, Gln 632 Gln, XRCC2 Arg 188 His, XRCC3 Thr 241 Met and XRCC4 G1394T, where the variants EGF G61, Trp194, val105, Pro206, His280, Gln632, His188, Met241 and XRCC4 T1394 were observed more frequently among patients with gliomas. Thus, these variants can be important factors of susceptibility to the tumor development.

Glioblastoma multiforme is the most common and lethal primary brain tumour in adults. Its aggressive and invasive phenotype makes it resistant to current standards of care, with a patient median survival following treatment of only 14 months. Potent and safe therapeutics are necessary to improve patient prognosis. Globally, efforts are being made in immunotherapies to combat such deleterious tumours. Preliminary work in the Stojdl lab has developed a novel oncolytic virus platform for brain cancer therapy that is non-toxic and exhibits potent anti-tumour efficacy. This platform is based on the rhabdovirus Farmington, identified for its potent oncolytic properties and engineering malleability. Herein, we begin to show our capability to discover and vaccinate against immunogenic neoepitopes derived from a mouse cancer mutanome. Engineering Farmington virus to express neoepitopes, allows for robust tumour specific immune proliferation following a prime vaccination. Overcoming problems of targeting self-antigen and antigen loss variants, a multi-neoepitope vaccine, presented here, is one of many alternative approaches to help combat cancer resistance. Despite achieving robust anti-tumour immunity by vaccination, selectivity of the tumour microenvironment remains an enormous challenge. Cumulative efforts in immunotherapy research will help drive novel therapeutics, like Farmington, into clinic and, ultimately, improve patient’s prognosis and quality of life.

The study evaluated the survival of 364 glioblastoma multiforme (GBM) patients who received different modalities of treatment in two Canadian tertiary care centres. Retrospective and prospective databases were utilized to do a retrospective population based cohort study. The thesis question was among treated GBM patients in Edmonton and Halifax; does the survival rate differ with introduction of concomitant temozolomide and radiation therapy (RT) versus non concomitant treatment? Our results indicate that concomitant temozolomide with radiation therapy and surgery was associated with longer survival in comparison to radiation therapy with surgery. We also found that age; surgical resection and shorter time to radiation therapy are important factors for longer survival. / Clinical epidemiology

Glioblastoma multiforme (GBM) is the most common primary brain tumor. Given the current standard of care, the prognosis for patients diagnosed with this disease is still poor. There consequently exists a need to improve current treatments, as well as to develop new ones. Many obstacles however need to be overcome to facilitate this effort and one of these involves the development of improved methods to monitor treatment effects. At present, the effects of treatment are typically assessed by radiological means several months after its initiation, which is unsatisfactory for a fast growing tumor like GBM. It is however likely that treatment effects can be detected on a molecular level long before radiological response, especially considering many of the targeted therapies that are currently being developed. Biomarkers for treatment efficacy may be of great importance in the future individualization of brain tumor treatment. The work presented herein was primarily focused on detecting early effects of GBM treatment. To this end, we designed experiments in the BT4C rat glioma model in which we studied effects of both conventional radiotherapy and an experimental angiogenesis inhibitor, vandetanib. Brain tissue samples were analyzed using a high throughput mass spectrometry (MS) based screening, known as Surface Enhanced Laser Desorption/Ionization - Time of Flight - Mass Spectrometry (SELDI-TOF-MS). The vast amounts of data generated were subsequently analyzed by established multivariate statistical methods, such as Principal Component Analysis (PCA), Partial Least Squares (PLS), and Orthogonal Partial Least Squares (OPLS), developed for analysis of large and complex datasets. In the radiotherapy study we detected a protein spectrum pattern clearly related to tumor progression. We notably observed how this progression pattern was hampered by radiotherapy. The vandetanib study also revealed significant alterations of protein expression following treatment of different durations, both in tumor tissue and in normal brain contralateral to the tumor. In an effort to further elucidate the pathophysiology of GBM, particularly in relation to treatment, we collected extracellular fluid (ECF) samples from 11 patients diagnosed with inoperable GBM. The samples were collected by means of stereotactic microdialysis, both from within the contrast enhancing tumor and the brain adjacent to tumor (BAT). Samples were collected longitudinally from each patient in a time span of up to two weeks, during which the patient received the first five fractions of radiotherapy. The ECF samples were then analyzed by Gas Chromatography Mass Spectrometry (GC-MS) to screen them with respect to concentrations of low molecular weight compounds (metabolites). Suitable multivariate analysis strategies enabled us to extract patterns of varying metabolite concentrations distinguishing between samples collected at different locations in the brain as well as between samples collected at different time points in relation to treatment. In a separate study, we also applied SELDI-TOF-MS and multivariate statistical methods to unravel possible differences in protein spectra between invasive and non-invasive WHO grade I meningiomas. This type of tumor can usually be cured by surgical resection however sometimes it grows invasively into the bone, ultimately causing clinical problems. This study revealed the possibility to differentiate between invasive and non-invasive benign meningioma based on the expression pattern of a few proteins. Our approach, which includes sample analysis and data handling, is applicable to a wide range of screening studies. In this work we demonstrated that the combination of MS screening and multivariate analyses is a powerful tool in the search for patterns related to treatment effects and diagnostics in brain tumors.

The GATA transcription factors consist of six family members that bind the consensus DNA binding element W-GATA-R, and are poorly characterized in the central nervous system (CNS). In this thesis we identify GATA4 to be expressed in the neurons and glia of normal murine and human embryonic and adult CNS with significant loss in Glioblastoma Multiforme (GBM). GBM is the most common and lethal primary brain tumour and exhibits multiple molecular aberrations. Here we report that loss of the transcription factor GATA4, a negative regulator of normal astrocyte proliferation, is a driver in glioma formation and fulfills the hallmarks of a tumour suppressor gene. Although GATA4 was expressed in normal brain, loss of GATA4 was observed in GBM operative samples and was a negative survival prognostic marker. GATA4 loss occurred through promoter hypermethylation or novel somatic mutations. Loss of GATA4 in normal human astrocytes promoted high-grade astrocytoma formation, in cooperation with other relevant genetic alterations such as activated Ras or loss of TP53. Loss of GATA4 with activated Ras in normal astrocytes promoted a progenitor like phenotype, formation of neurospheres and the ability to differentiate into astrocytes, neurons and oligodendrocytes. Re-expression of GATA4 in human GBM cell lines, primary cultures and brain tumour initiating cells suppressed tumour growth in vitro and in vivo through direct activation of the cell cycle inhibitor P21CIP1, independent of TP53. Re-expression of GATA4 also conferred sensitivity of GBM cells to temozolomide, a DNA alkylating agent currently used in GBM therapy. This sensitivity was independent of MGMT, the DNA repair enzyme often implicated in temozolomide resistance. Instead GATA4 reduced expression of APNG, a DNA repair enzyme poorly characterized in GBM mediated temozolomide resistance. Identification and validation of GATA4 as a tumour suppressor gene and its downstream targets in GBM may yield promising novel therapeutic strategies.

Aberrations in Epidermal Growth Factor Receptor (EGFR/ErbB1) signalling are the most common oncogenic stimuli in human glioblastoma multiforme (GBM). Interactions between mutant and wildtype ErbB family members in GBMs are of biological and potential therapeutic importance. In this thesis, we describe our work developing and optimizing a novel in situ proximity ligation assay (PLA) for dimerization and activation analysis of EGFR mutants prevalent in GBMs. Utilizing this novel in situ platform for EGFR dimerization analysis, we seek to systematically interrogate the dimerization capacity and activation status amongst EGFR and EGFR mutants. Our in vitro analysis using this platform demonstrates the aberrant homo-/hetero-dimeric properties of EGFRvIII and EGFRc958 mutants, the two most common mutants associated with EGFR amplification in GBMs. In addition, dimer phospho-activation status can be detected using in situ PLA with ≥ 16-fold sensitivity and ≥ 17-fold signal-to-noise than phospho-EGFR measurements currently undertaken with IHC or IF. These aberrant features are not overexpression dependent but appear independent of cellular expression levels, suggesting inherent properties of the mutant receptors. This EGFR dimerization/activation detection platform may also be useful for evaluating novel anti-EGFR therapeutics. Our data suggests that various EGFR monoclonal antibody therapies have unique dimerization blocking abilities and that certain mutant EGFR dimer configurations can evade blockage by anti-EGFR treatments. Furthermore, we report for the first time the detection of wt- and EGFRvIII dimerization in GBM specimens, in keeping with our prior cell line data, and a potential feature of prognostic or diagnostic value in GBMs harbouring them. Additionally, we demonstrate the utility of this platform for measuring pEGFR and total EGFR expression on tissue samples, which has not been efficacious to date with conventional antibody-mediated techniques. Results from this thesis may therefore provide novel insights into the interaction and activation characteristics of EGFR mutants prevalent in GBMs, as well as the efficacy of current anti-EGFR therapies to target these mutants. In summary, these findings demonstrate the successful application of a novel in situ EGFR molecular detection platform which may have clinical utility in diagnostic evaluation or stratification of GBM patient subgroups for prognosis and treatment. Furthermore, since PLA allows specimen assessment of not only expression and activation, but also dimerization, which is not evaluated by current IHC techniques, it will likely serve as a way to evaluate promising anti-EGFR strategies directed at preventing EGFR dimerization and activation.

The GATA transcription factors consist of six family members that bind the consensus DNA binding element W-GATA-R, and are poorly characterized in the central nervous system (CNS). In this thesis we identify GATA4 to be expressed in the neurons and glia of normal murine and human embryonic and adult CNS with significant loss in Glioblastoma Multiforme (GBM). GBM is the most common and lethal primary brain tumour and exhibits multiple molecular aberrations. Here we report that loss of the transcription factor GATA4, a negative regulator of normal astrocyte proliferation, is a driver in glioma formation and fulfills the hallmarks of a tumour suppressor gene. Although GATA4 was expressed in normal brain, loss of GATA4 was observed in GBM operative samples and was a negative survival prognostic marker. GATA4 loss occurred through promoter hypermethylation or novel somatic mutations. Loss of GATA4 in normal human astrocytes promoted high-grade astrocytoma formation, in cooperation with other relevant genetic alterations such as activated Ras or loss of TP53. Loss of GATA4 with activated Ras in normal astrocytes promoted a progenitor like phenotype, formation of neurospheres and the ability to differentiate into astrocytes, neurons and oligodendrocytes. Re-expression of GATA4 in human GBM cell lines, primary cultures and brain tumour initiating cells suppressed tumour growth in vitro and in vivo through direct activation of the cell cycle inhibitor P21CIP1, independent of TP53. Re-expression of GATA4 also conferred sensitivity of GBM cells to temozolomide, a DNA alkylating agent currently used in GBM therapy. This sensitivity was independent of MGMT, the DNA repair enzyme often implicated in temozolomide resistance. Instead GATA4 reduced expression of APNG, a DNA repair enzyme poorly characterized in GBM mediated temozolomide resistance. Identification and validation of GATA4 as a tumour suppressor gene and its downstream targets in GBM may yield promising novel therapeutic strategies.

Aberrations in Epidermal Growth Factor Receptor (EGFR/ErbB1) signalling are the most common oncogenic stimuli in human glioblastoma multiforme (GBM). Interactions between mutant and wildtype ErbB family members in GBMs are of biological and potential therapeutic importance. In this thesis, we describe our work developing and optimizing a novel in situ proximity ligation assay (PLA) for dimerization and activation analysis of EGFR mutants prevalent in GBMs. Utilizing this novel in situ platform for EGFR dimerization analysis, we seek to systematically interrogate the dimerization capacity and activation status amongst EGFR and EGFR mutants. Our in vitro analysis using this platform demonstrates the aberrant homo-/hetero-dimeric properties of EGFRvIII and EGFRc958 mutants, the two most common mutants associated with EGFR amplification in GBMs. In addition, dimer phospho-activation status can be detected using in situ PLA with ≥ 16-fold sensitivity and ≥ 17-fold signal-to-noise than phospho-EGFR measurements currently undertaken with IHC or IF. These aberrant features are not overexpression dependent but appear independent of cellular expression levels, suggesting inherent properties of the mutant receptors. This EGFR dimerization/activation detection platform may also be useful for evaluating novel anti-EGFR therapeutics. Our data suggests that various EGFR monoclonal antibody therapies have unique dimerization blocking abilities and that certain mutant EGFR dimer configurations can evade blockage by anti-EGFR treatments. Furthermore, we report for the first time the detection of wt- and EGFRvIII dimerization in GBM specimens, in keeping with our prior cell line data, and a potential feature of prognostic or diagnostic value in GBMs harbouring them. Additionally, we demonstrate the utility of this platform for measuring pEGFR and total EGFR expression on tissue samples, which has not been efficacious to date with conventional antibody-mediated techniques. Results from this thesis may therefore provide novel insights into the interaction and activation characteristics of EGFR mutants prevalent in GBMs, as well as the efficacy of current anti-EGFR therapies to target these mutants. In summary, these findings demonstrate the successful application of a novel in situ EGFR molecular detection platform which may have clinical utility in diagnostic evaluation or stratification of GBM patient subgroups for prognosis and treatment. Furthermore, since PLA allows specimen assessment of not only expression and activation, but also dimerization, which is not evaluated by current IHC techniques, it will likely serve as a way to evaluate promising anti-EGFR strategies directed at preventing EGFR dimerization and activation.

Thesis (M.S.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains vii, 78 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 70-78).

Glioblastoma multiforme (GBMs) are the most common and severe form of malignant brain tumors. Despite recent advancement in the fields of surgical resection, radiotherapy and chemotherapy, the prognosis for patients with GBMs remains poor with the median survival rate of approximately a year. Recently, our laboratory has demonstrated the oncogenic role of cell cycle-related kinase (CCRK), a 42-kD protein kinase responsible for regulating cell growth in GBM carcinogenesis, suggesting that CCRK is a candidate oncogene in GBMs. Nevertheless, the regulation of CCRK expression and the cellular mechanism for its overexpression in GBMs remain elusive. Understanding the regulation of human CCRK expression in GBMs should therefore shed light on the development of better prognostic and therapeutic methods for this deadly disease. This study aims to characterize the human CCRK gene and the regulation of its expression in GBMs. We first characterized the 5’ upstream sequence of CCRK by in silico analysis, which revealed the absence of TATA box but the presence of three potential transcription factor binding sites for Sp1, c-Myc and CREB, and identified the transcription start site by 5’-RACE at 240 bp upstream of the start codon. In vitro analysis of the CCRK promoters revealed the presence of nucleotide polymorphisms in three high-grade glioblastoma cell lines U-87 MG, U-138 MG and U-373 MG, and the control fibroblast cell line CCD19Lu. Furthermore, three CpG islands within the CCRK promoter were identified and the CCRK promoter was hypomethylated in these cell lines. Sp1, c-Myc and CREB binding sites as well as the nucleotide polymorphisms on the CCRK promoter were further investigated. The results from electrophoretic mobility shift assay showed that these transcription factors interacted with the corresponding cis-regulatory elements on the CCRK promoter, removal of the potential Sp1 and c-Myc binding sites lowered the CCRK promoter activity by 46 – 66 % in vitro. In addition, mutations introduced to the nucleotide polymorphisms reduced the CCRK promoter activity by 62 – 81 % in U-87 MG cells and enhanced the CCRK promoter activity by 1.35-fold in U-138 MG cells, suggesting their importance in regulating CCRK expression. c-Myc is a proto-oncogene with its overexpression associated with a large variety of tumors. As c-Myc binding site was identified in the CCRK promoter, therefore, the effect of c-Myc on CCRK expression was examined. c-Myc overexpression resulted in significant enhancements in the CCRK promoter activity by 30.74-fold in U-87 MG cells, 26.5-fold in U-373 MG cells and 6.09-fold in U-138 MG cells. On the contrary, c-Myc knockdown reduced the CCRK promoter activity by 49 % in U-87 MG cells, 35 % in U-373 MG cells and 17% in U-138 MG cells. In summary, this study represents the first molecular characterization of the human CCRK gene and findings of this study would prime others for future research on the molecular pathogenesis of CCRK-mediated GBMs and for developing CCRK as a potential therapeutic and diagnostic target for GBMs and possibly other cancers. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy