Gliomas constituem um grupo heterogêneo de tumores cerebrais. Eles podem ser divididos em duas principais categorias: astrocíticos e oligodendrogliais. Os astrocíticos, objetos deste estudo, são classificados patologicamente em quatro subtipos baseado na presença ou ausência de atipia nuclear, mitose, neovascularização e necrose. Esta classificação é útil para definir tratamento e prognóstico. A origem glial é comumente confirmada pela detecção imunoistoquímica para GFAP. Neste trabalho nós investigamos o imunoconteúdo das proteínas marcadoras astrocíticas GFAP e S100B por ELISA e a atividade da enzima glutamina sintetase (GS) em tumores gliais e tecidos peritumorais de sete pacientes submetidos à ressecção cirúrgica. Os resultados demonstram elevados níveis de S100B em tecidos peritumorais não correlacionados com o grau de malignidade do tumor. Esta elevação poderia contribuir para manifestações convulsivas observadas em alguns pacientes. Em contraste com os dados obtidos pela imunoistoquímica para filamentos gliais, encontrou-se uma aparente correlação positiva entre o conteúdo de GFAP e o grau de malignidade do glioma. É possível que este aumento no conteúdo de GFAP seja referente a GFAP total (frações solúvel e insolúvel), em contraste com o conteúdo insolúvel (fibrilar e granular) detectado pela imunoistoquímica. A alta taxa GFAP/S100B (GFAP elevada/S100B reduzida) está de acordo com a sua elevada atividade mitótica, que pela sua vez poderia ser inibida pela S100B. Além disso, houve uma correlação positiva entre a atividade da GS e o grau de malignidade do tumor.

Malignant glioma have poor prognosis resulting mainly from high level of cell proliferation and invasion and resistance to conventional therapy. Identification of novel targets that are critical elements in gliomagenesis may help improve therapeutic outcome. Using genome-wide explorations of a comprehensive glioma specimen population, we identified whole gain of chromosome 19 as one of the major chromosomal aberrations in high grade glioma that correlates to patients' outcomes. Our analysis revealed for the first time NOTCH3 as one of the most significant gene amplifications mapped to chromosome 19. This amplification is associated with worse outcome compared to tumors with non-amplified locus. NOTCH signaling pathway is essential for cell proliferation, stem cell maintenance and differentiation and its deregulation has been reported in several human cancers. NOTCHs are key positive regulators of cell-cell interactions, angiogenesis, cell adhesion and stem cell niche development which have been shown to play critical roles in gliomagenesis and glioma drug resistance. Our objective is to determine NOTCH3 molecular roles in glioma pathogenesis and aggressiveness. Here we show for the first time that NOTCH3 plays a role in glioma cell proliferation, cell migration, invasion and apoptosis. We also found a NOTCH3 glioma addiction phenomenon. Therefore, our study uncovers, for the first time, the prognostic value and the oncogenic function of NOTCH3 in gliomagenesis and supports NOTCH3 as a promising target of therapy in high grade glioma. Our studies allow the identification of a subset of population that may benefit from GSI-based therapies. This may lead to the design of novel strategies to improve therapeutic outcome of patients with glioma by establishing medical and scientific basis for personalized medicine.

Radiation therapy seeks to effectively irradiate the tumor cells while minimizing the dose to adjacent normal cells. Prior research found that the low success rates for treating brain tumors would be improved with higher radiation doses to the tumor area. This is feasible only if the target volume can be precisely identified. However, the definition of tumor volume is still based on time-intensive, highly subjective manual outlining by radiation oncologists. In this study the effectiveness of two automated Magnetic Resonance Imaging (MRI) segmentation methods, k-Nearest Neighbors (kNN) and Knowledge-Guided (KG), in determining the Gross Tumor Volume (GTV) of brain tumors for use in radiation therapy was assessed. Three criteria were applied: accuracy of the contours; quality of the resulting treatment plan in terms of dose to the tumor; and a novel treatment plan evaluation technique based on post-treatment images. The kNN method was able to segment all cases while the KG method was limited to enhancing tumors and gliomas with clear enhancing edges. Various software applications were developed to create a closed smooth contour that encompassed the tumor pixels from the segmentations and to integrate these results into the treatment planning software. A novel, probabilistic measurement of accuracy was introduced to compare the agreement of the segmentation methods with the weighted average physician volume. Both computer methods under-segment the tumor volume when compared with the physicians but performed within the variability of manual contouring (28% plus/minus12% for inter-operator variability). Computer segmentations were modified vertically to compensate for their under-segmentation. When comparing radiation treatment plans designed from physician-defined tumor volumes with treatment plans developed from the modified segmentation results, the reference target volume was irradiated within the same level of conformity. Analysis of the plans based on post- treatment MRI showed that the segmentation plans provided similar dose coverage to areas being treated by the original treatment plans. This research demonstrates that computer segmentations provide a feasible route to automatic target volume definition. Because of the lower variability and greater efficiency of the automated techniques, their use could lead to more precise plans and better prognosis for brain tumor patients.

<p>Glioblastoma multiforme is a malignant brain tumor characterized by heterogeneity.Interactions between heterogeneous tumor cells are supposed to affect the behavior of awhole tumor cell population. In this thesis an <i>in vitro</i> model system of clonal glioma celllines originating from one glioblastoma tumor was used, and the behavior of cells incocultures was studied and compared the behavior of cells grown separately. The resultsindicate the presence of two types of interactions. In one, paracrine signals acted via extra-cellular media. This was associated with increased growth of the whole co-culture followedby a selective force driving one clone to dominance. In the other type, the cell clones grewside by side without signs of paracrine signalling, in a balance resulting in an increasedterminal cell density. Further investigations focused on mechanisms of interactions in thiscombination.</p><p>Two cell clones were chosen, a GFAP⁺ and a GFAP^-, for further experiments. Withdifferential display PCR it was possible to investigate their specific gene expressionpatterns. Seventeen cDNA fragments were differentially expressed, among them twocorresponded to known transcription factors, ATF3 and prox-1, one to a cytoskeletal protein,α-tropomyosin. The collection also contained eight ESTs (Expressed Sequence Tags) wherethe corresponding genes are unknown at present. Expression of the isolated sequences werealso analyzed in a panel of 12 different glioma cell lines and the results illustrate thecomplexity of gene expression and of tumor heterogeneity. Genes, the expression levels ofwhich were modulated in co-cultures and/or were cell density dependent, were alsoidentified.</p><p>PDGF B is suggested to play a role in sarcomas. The gene codes for an mRNA transcriptwith long UTRs, parts of which are deleted in the homologous oncogene <i>v-sis</i>. The UTRs ofPDGF B mRNAs in human sarcomas were investigated for deletions similar to <i>v-sis</i> thatmight result in increased protein levels. A new transcript variant was identified, lacking a149 base region in the 3'UTR, but its presence was not associated with increased levels ofprotein. Alterations in the 5'UTR were found more likely to be associated with increasedprotein levels.</p>

<p>Tumour targeted radiotherapy is an appealing approach for treatment of disseminated tumour cells. A targeting agent that specifically binds to a structure on tumour cells is then used to transport therapeutically relevant radionuclides. The epidermal growth factor receptor, EGFR, is overexpressed on tumour cells in several malignancies, e.g. highly malignant gliomas. In this thesis, three types of radiolabelled EGF-conjugates, aimed for targeting to EGFR-expressing tumour cells, were developed and studied: EGF-dextran labelled with ¹²⁵I, EGF labelled with ²¹¹At, and two EGF-chelates, DTPA-EGF and Bz-DTPA-EGF, labelled with the radioactive metals ¹¹¹In and ¹⁷⁷Lu. </p><p>The targeting properties of radioiodinated EGF-dextran were first studied in cultured glioma cells. Radioiodine coupled to the dextran part of EGF-dextran was retained in cells appreciably longer than radioiodine coupled to EGF. This can give about 100 times increased radiation dose to tumour cells.</p><p>Targeting with ²¹¹At-EGF was investigated in combination with the tyrosine kinase inhibitor gefitinib (Iressa™, ZD1839). The uptake of ²¹¹At-EGF in EGFR-expressing tumour cells increased with increasing gefitinib concentrations. This was the case for both gefitinib-resistant and gefitinib-sensitive cell lines. The effect of the combined treatment on cell survival, however, differed between the cell lines in an unexpected way. In gefitinib resistant cells, combined treatment decreased cell survival approximately 3.5 times relative to ²¹¹At-EGF treatment alone. In gefitinib sensitive cells, however, combined treatment increased the cell survival (i.e. a protective effect).</p><p>The EGF-chelates studied ([¹¹¹In]DTPA-EGF, [¹¹¹In]Bz-DTPA-EGF and [¹⁷⁷Lu]Bz-DTPA-EGF) all bound specifically with high affinity (K_d≈2 nM) to EGFR on cultured glioma cells. They were internalised after binding, and the cellular retention of radionuclides was high (60% remained after 45 h). A biodistribution study in mice showed that liver and kidneys accumulated a majority of the radioactivity. The EGF-chelates bound EGFR specifically also <i>in vivo</i>. A tumour-to-blood ratio of 25 was achieved in a preliminary study.</p>

Malignant glioma consists of a group of diseases where the localisation and the nature of the disease makes treatment an extreme challenge. Two important biological features of malignant glioma cells are their infiltrative growth and their ability to induce angiogenesis. Glioma cells migrate extensively behind the blood-brain barrier and infiltrate the surrounding brain making radical treatment with surgery and radiotherapy almost impossible. The aims of this thesis were to investigate factors of importance for glioma cell migration and angiogenesis and to evaluate if an anti-angiogenesis approach alone or in combination with current treatment modalities could inhibit glioma growth. For this purpose we used the BT4C orthotopic rat glioma model and investigated treatment effects of the vascular endothelial growth factor (VEGF) receptor-2 and epidermal growth factor (EGF) receptor tyrosine kinase inhibitor ZD6474 alone or in combination with temozolomide or radiotherapy. Altered protein expression pattern after anti-angiogenesis treatment was measured using a mass-spectrometric proteomic method, followed by multivariate data-analysis. The tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and VEGF showed altered temporal and spatial mRNA expression during glioma progression. In early stages of tumour progression the expression was found throughout the tumour while in later stages, the expression was more predominant in the invasive tumour border. ZD6474 in monotherapy significantly inhibited tumour growth in the BT4C glioma model. The effect was further enhanced when combined with radiotherapy or temozolomide. Using mass-spectrometric methods an altered protein expression pattern after ZD6474 treatment was observed implicating the possibility to use proteomic methods for finding predictive biomarkers for anti-angiogenesis treatment. In conclusion, this thesis demonstrates a co-expression of factors important for glioma growth and angiogenesis and that treatment with an angiogenesis inhibitor has additive effects on glioma growth when combined with radiotherapy and chemotherapy. Finally, an altered protein expression pattern after anti-angiogenesis treatment is evident and detectable. Hopefully this work will contribute to and encourage further research to reach a better understanding of how to combine and evaluate different treatment approaches in malignant glioma.

Primary brain tumours, and especially the most common form malignant gliomas, usually display a pronounced resistance to other treatment modalities when surgery fails to cure. Growth factors, such as EGF and its receptor, frequently amplified and overexpressed in malignant gliomas, and factors associated with multidrug resistance have been suggested to at least partially explain the poor outcome. The aim of this thesis was to characterise factors in primary brain tumours associated with the development of resistance with focus on the epidermal growth factor receptor (ErbB) family, and multidrug resistance (MDR). Influences of irradiation on the expression and activity of P-glycoprotein (Pgp) in malignant gliomas was evaluated. The effects showed that irradiation increased the efflux activity of Pgp in rat brain vascular endothelial cells, but not in glioma cells. In the intracranial BT4C glioma model, Pgp was detected in the capillary endothelium in the tumour tissue but not in glioma cells. Expression of several factors coupled to MDR (Pgp, MRP1, LRP, and MGMT) in primary brain tumours were analysed and correlated to clinical data. In gliomas, Pgp and MRP1 were predominantly observed in capillary endothelium and in scattered tumour cells, whereas LRP occurred only in tumour cells. In meningiomas, expression of the analysed markers was demonstrated in the capillary endothelium, with a higher expression of Pgp and MRP1 in transitional compared to meningothelial meningiomas. A pronounced expression of MGMT was found independently of the histopathological grade or tumour type. Survival analysis indicated a shorter overall survival for patients suffering from low-grade gliomas with high expression of Pgp. To explore the importance of the epidermal growth factor receptor (EGFR), expression levels of the family members (EGFR, ErbB2-4) were analysed and their relations to various clinical parameters were evaluated in gliomas and meningiomas. In gliomas, the highest EGFR expression was observed in high-grade tumours, while ErbB4 expression was most pronounced in low-grade tumours. In meningiomas, expression of EGFR, ErbB2, and ErbB4 was observed in the majority of the tumours. An intriguing observation in low-grade gliomas was a significantly decreased overall survival for patients with high EGFR protein expression. The effects of different time schedules for administration of the selective EGFR inhibitor ZD1839 in relation to irradiation of glioma cells were analysed. The analyses showed a heterogeneity in the cytotoxic effects of ZD1839 between cell lines, and it was obvious that some of the cell lines showed sensitivity to ZD1839 despite no or low expression of EGFR. The study also demonstrated the importance of timing of ZD1839 administration when this agent is combined with irradiation. In conclusion, in order to enhance the efficacy of radiotherapy by various drugs in malignant gliomas it may be essential to inhibit drug efflux activity in endothelial cells and to deliver drugs in an optimal timing in relation to radiotherapy. The heterogeneity in expression of drug resistance markers, as well as the ErbB family reflects the complexity in classification of primary brain tumours, and indicates that subgroups of patients with low-grade gliomas expressing Pgp and EGFR might benefit from more aggressive and individualised treatment.

Background: Glioblastoma is the most common and aggressive type of glioma and associated with poor prognosis. Apart from ionizing radiation and some rare genetic disorders, few aetiological factors have been identified for primary brain tumours. Inverse associations to asthma and low IgG levels for varicella zoster virus have in previous studies indicated that the immune system may play a role in glioma development. Little is known about prognostic factors in glioma. Previous studies have shown an association between age, Karnofsky performance status, O6-methylguanine-DNA methyltransferase (MGMT) hypermethylation, and prognosis. Polymorphisms in different low penetrance genes have in some studies been associated with glioma prognosis. Material and methods: In paper I, we analysed IgG levels for four different viruses, Epstein-Barr virus (EBV), cytomegalovirus (CMV), varicella zoster virus (VZV) and adenovirus (Ad), in prediagnostic blood samples from 197 cases with glioma and 394 controls collected from three large cohorts: the Northern Sweden Health and Disease Study; the Malmö Diet and Cancer Study; and the Diet, Cancer and Health cohort from Copenhagen. ELISA was used to measure IgG levels and for EBV response to both the nuclear antigen (EBNA1) and the viral capsid antigen (VCA) was measured, for VCA using immunoflourescence. IgG levels were divided into quartiles and binary logistic regression was used to compare the quartiles in cases and controls. All odds ratios were adjusted for age, sex, and cohort. In paper II-IV, we studied 176 glioblastoma cases from Sweden and Denmark. We collected treatment and follow-up data on the cases. We genotyped 30 tagging SNPs in EGF, 89 in EGFR, 27 in VEGFR2, and 17 in VEGF. We also studied 1458 SNPs in 136 DNA repair genes. Hazard ratios were calculated using Cox regression; the major allele was set as categorical variable and all HR were adjusted for age, sex, country, and treatment. For the DNA repair gene results, we adjusted the p-values for multiple testing. Significant findings were confirmed in separate datasets. Results and Discussion: We found a trend towards higher IgG VZV levels in controls compared to glioma cases, especially when restricting the analyses to only include glioma cases with at least 2 years between blood sample and diagnosis. This finding might indicate that there is an aetiological and not a disease-related association. This confirms previous findings and support that a strong immune system can detect and inhibit growth of small cancer clusters. In EGF, we found seven SNPs in one haplotype block that were significantly associated with glioblastoma survival. Four of the SNPs were available for confirmation; however, none reached statistical significance. One explanation could be age differences in the different cohorts. In EGFR, four SNPs associated with survival were found; however, as 89 polymorphisms were tested this was the expected outcome by chance. In VEGF and VEGFR2, we found two SNPs associated with glioblastoma survival, but they could not be confirmed in the separate dataset, and due to multiple testing, were considered to be false positives. Among the DNA repair genes, we found nine SNPs in three genes-MSH2, RAD51L1 and RECQL4-associated with glioblastoma survival after confirmation and adjustment for age, sex, country, and treatment. After adjusting for multiple testing, one SNP in MSH2 and one in RECQL4 remained significant. Conclusions: Our studies provide additional knowledge to the aetiological and prognostic factors important for glioma, emphasising the possible importance of immune function mechanisms. We found limited evidence for the role of genetic variants in glioma progression genes, and some for DNA repair variants as prognostic factors for glioblastoma survival.

Glioblastoma multiforme is a malignant brain tumor characterized by heterogeneity.Interactions between heterogeneous tumor cells are supposed to affect the behavior of awhole tumor cell population. In this thesis an in vitro model system of clonal glioma celllines originating from one glioblastoma tumor was used, and the behavior of cells incocultures was studied and compared the behavior of cells grown separately. The resultsindicate the presence of two types of interactions. In one, paracrine signals acted via extra-cellular media. This was associated with increased growth of the whole co-culture followedby a selective force driving one clone to dominance. In the other type, the cell clones grewside by side without signs of paracrine signalling, in a balance resulting in an increasedterminal cell density. Further investigations focused on mechanisms of interactions in thiscombination. Two cell clones were chosen, a GFAP+ and a GFAP-, for further experiments. Withdifferential display PCR it was possible to investigate their specific gene expressionpatterns. Seventeen cDNA fragments were differentially expressed, among them twocorresponded to known transcription factors, ATF3 and prox-1, one to a cytoskeletal protein,α-tropomyosin. The collection also contained eight ESTs (Expressed Sequence Tags) wherethe corresponding genes are unknown at present. Expression of the isolated sequences werealso analyzed in a panel of 12 different glioma cell lines and the results illustrate thecomplexity of gene expression and of tumor heterogeneity. Genes, the expression levels ofwhich were modulated in co-cultures and/or were cell density dependent, were alsoidentified. PDGF B is suggested to play a role in sarcomas. The gene codes for an mRNA transcriptwith long UTRs, parts of which are deleted in the homologous oncogene v-sis. The UTRs ofPDGF B mRNAs in human sarcomas were investigated for deletions similar to v-sis thatmight result in increased protein levels. A new transcript variant was identified, lacking a149 base region in the 3'UTR, but its presence was not associated with increased levels ofprotein. Alterations in the 5'UTR were found more likely to be associated with increasedprotein levels.

Tumour targeted radiotherapy is an appealing approach for treatment of disseminated tumour cells. A targeting agent that specifically binds to a structure on tumour cells is then used to transport therapeutically relevant radionuclides. The epidermal growth factor receptor, EGFR, is overexpressed on tumour cells in several malignancies, e.g. highly malignant gliomas. In this thesis, three types of radiolabelled EGF-conjugates, aimed for targeting to EGFR-expressing tumour cells, were developed and studied: EGF-dextran labelled with 125I, EGF labelled with 211At, and two EGF-chelates, DTPA-EGF and Bz-DTPA-EGF, labelled with the radioactive metals 111In and 177Lu. The targeting properties of radioiodinated EGF-dextran were first studied in cultured glioma cells. Radioiodine coupled to the dextran part of EGF-dextran was retained in cells appreciably longer than radioiodine coupled to EGF. This can give about 100 times increased radiation dose to tumour cells. Targeting with 211At-EGF was investigated in combination with the tyrosine kinase inhibitor gefitinib (Iressa™, ZD1839). The uptake of 211At-EGF in EGFR-expressing tumour cells increased with increasing gefitinib concentrations. This was the case for both gefitinib-resistant and gefitinib-sensitive cell lines. The effect of the combined treatment on cell survival, however, differed between the cell lines in an unexpected way. In gefitinib resistant cells, combined treatment decreased cell survival approximately 3.5 times relative to 211At-EGF treatment alone. In gefitinib sensitive cells, however, combined treatment increased the cell survival (i.e. a protective effect). The EGF-chelates studied ([111In]DTPA-EGF, [111In]Bz-DTPA-EGF and [177Lu]Bz-DTPA-EGF) all bound specifically with high affinity (Kd≈2 nM) to EGFR on cultured glioma cells. They were internalised after binding, and the cellular retention of radionuclides was high (60% remained after 45 h). A biodistribution study in mice showed that liver and kidneys accumulated a majority of the radioactivity. The EGF-chelates bound EGFR specifically also in vivo. A tumour-to-blood ratio of 25 was achieved in a preliminary study.