Extent and Patterns of MGMT Promoter Methylation in Glioblastoma- and Respective Glioblastoma-Derived Spheres.

PURPOSE: Quantitative methylation-specific tests suggest that not all cells in a glioblastoma with detectable promoter methylation of the O6-methylguanine DNA methyltransferase (MGMT) gene carry a methylated MGMT allele. This observation may indicate cell subpopulations with distinct MGMT status, raising the question of the clinically relevant cutoff of MGMT methylation therapy. Epigenetic silencing of the MGMT gene by promoter methylation blunts repair of O6-methyl guanine and has been shown to be a predictive factor for benefit from alkylating agent therapy in glioblastoma. EXPERIMENTAL DESIGN: Ten paired samples of glioblastoma and respective glioblastoma-derived spheres (GS), cultured under stem cell conditions, were analyzed for the degree and pattern of MGMT promoter methylation by methylation-specific clone sequencing, MGMT gene dosage, chromatin status, and respective effects on MGMT expression and MGMT activity. RESULTS: In glioblastoma, MGMT-methylated alleles ranged from 10% to 90%. In contrast, methylated alleles were highly enriched (100% of clones) in respective GS, even when 2 MGMT alleles were present, with 1 exception (<50%). The CpG methylation patterns were characteristic for each glioblastoma exhibiting 25% to 90% methylated CpGs of 28 sites interrogated. Furthermore, MGMT promoter methylation was associated with a nonpermissive chromatin status in accordance with very low MGMT transcript levels and undetectable MGMT activity. CONCLUSIONS: In MGMT-methylated glioblastoma, MGMT promoter methylation is highly enriched in GS that supposedly comprise glioma-initiating cells. Thus, even a low percentage of MGMT methylation measured in a glioblastoma sample may be relevant and predict benefit from an alkylating agent therapy. Clin Cancer Res; 17(2); 255-66. ©2010 AACR.

Encapsulation of packaging cell line results in successful retroviral-mediated transfer of a suicide gene in vivo in an experimental model of glioblastoma.

AIMS: Retroviral-mediated gene therapy has been proposed as a primary or adjuvant treatment for advanced cancer, because retroviruses selectively infect dividing cells. Efficacy of retroviral-mediated gene transfer, however, is limited in vivo. Although packaging cell lines can produce viral vectors continuously, such allo- or xenogeneic cells are normally rejected when used in vivo. Encapsulation using microporous membranes can protect the packaging cells from rejection. In this study, we used an encapsulated murine packaging cell line to test the effects of in situ delivery of a retrovirus bearing the herpes simplex virus thymidine kinase suicide gene in a rat model of orthotopic glioblastoma. MATERIALS AND METHODS: To test gene transfer in vitro, encapsulated murine psi2-VIK packaging cells were co-cultured with baby hamster kidney (BHK) cells, and the percentage of transfected BHK cells was determined. For in vivo experiments, orthotopic C6 glioblastomas were established in Wistar rats. Capsules containing psi2-VIK cells were stereotaxically implanted into these tumours and the animals were treated with ganciclovir (GCV). Tumours were harvested 14 days after initiation of GCV therapy for morphometric analysis. RESULTS: Encapsulation of psi2-VIK cells increased transfection rates of BHK target cells significantly in vitro compared to psi2-VIK conditioned medium (3 x 10(6) vs 2.3 x 10(4) cells; P<0.001). In vivo treatment with encapsulated packaging cells resulted in 3% to 5% of C6 tumour cells transduced and 45% of tumour volume replaced by necrosis after GCV (P<0.01 compared to controls). CONCLUSION: In this experimental model of glioblastoma, encapsulation of a xenogeneic packaging cell line increased half-life and transduction efficacy of retrovirus-mediated gene transfer and caused significant tumour necrosis.

Cilengitide combined with standard treatment for patients with newly diagnosed glioblastoma with methylated MGMT promoter (CENTRIC EORTC 26071-22072 study): a multicentre, randomised, open-label, phase 3 trial.

BACKGROUND: Cilengitide is a selective αvβ3 and αvβ5 integrin inhibitor. Data from phase 2 trials suggest that it has antitumour activity as a single agent in recurrent glioblastoma and in combination with standard temozolomide chemoradiotherapy in newly diagnosed glioblastoma (particularly in tumours with methylated MGMT promoter). We aimed to assess cilengitide combined with temozolomide chemoradiotherapy in patients with newly diagnosed glioblastoma with methylated MGMT promoter. METHODS: In this multicentre, open-label, phase 3 study, we investigated the efficacy of cilengitide in patients from 146 study sites in 25 countries. Eligible patients (newly diagnosed, histologically proven supratentorial glioblastoma, methylated MGMT promoter, and age ≥18 years) were stratified for prognostic Radiation Therapy Oncology Group recursive partitioning analysis class and geographic region and centrally randomised in a 1:1 ratio with interactive voice response system to receive temozolomide chemoradiotherapy with cilengitide 2000 mg intravenously twice weekly (cilengitide group) or temozolomide chemoradiotherapy alone (control group). Patients and investigators were unmasked to treatment allocation. Maintenance temozolomide was given for up to six cycles, and cilengitide was given for up to 18 months or until disease progression or unacceptable toxic effects. The primary endpoint was overall survival. We analysed survival outcomes by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00689221. FINDINGS: Overall, 3471 patients were screened. Of these patients, 3060 had tumour MGMT status tested; 926 patients had a methylated MGMT promoter, and 545 were randomly assigned to the cilengitide (n=272) or control groups (n=273) between Oct 31, 2008, and May 12, 2011. Median overall survival was 26·3 months (95% CI 23·8-28·8) in the cilengitide group and 26·3 months (23·9-34·7) in the control group (hazard ratio 1·02, 95% CI 0·81-1·29, p=0·86). None of the predefined clinical subgroups showed a benefit from cilengitide. We noted no overall additional toxic effects with cilengitide treatment. The most commonly reported adverse events of grade 3 or worse in the safety population were lymphopenia (31 [12%] in the cilengitide group vs 26 [10%] in the control group), thrombocytopenia (28 [11%] vs 46 [18%]), neutropenia (19 [7%] vs 24 [9%]), leucopenia (18 [7%] vs 20 [8%]), and convulsion (14 [5%] vs 15 [6%]). INTERPRETATION: The addition of cilengitide to temozolomide chemoradiotherapy did not improve outcomes; cilengitide will not be further developed as an anticancer drug. Nevertheless, integrins remain a potential treatment target for glioblastoma. FUNDING: Merck KGaA, Darmstadt, Germany.

Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial.

BACKGROUND: In 2004, a randomised phase III trial by the European Organisation for Research and Treatment of Cancer (EORTC) and National Cancer Institute of Canada Clinical Trials Group (NCIC) reported improved median and 2-year survival for patients with glioblastoma treated with concomitant and adjuvant temozolomide and radiotherapy. We report the final results with a median follow-up of more than 5 years. METHODS: Adult patients with newly diagnosed glioblastoma were randomly assigned to receive either standard radiotherapy or identical radiotherapy with concomitant temozolomide followed by up to six cycles of adjuvant temozolomide. The methylation status of the methyl-guanine methyl transferase gene, MGMT, was determined retrospectively from the tumour tissue of 206 patients. The primary endpoint was overall survival. Analyses were by intention to treat. This trial is registered with Clinicaltrials.gov, number NCT00006353. FINDINGS: Between Aug 17, 2000, and March 22, 2002, 573 patients were assigned to treatment. 278 (97%) of 286 patients in the radiotherapy alone group and 254 (89%) of 287 in the combined-treatment group died during 5 years of follow-up. Overall survival was 27.2% (95% CI 22.2-32.5) at 2 years, 16.0% (12.0-20.6) at 3 years, 12.1% (8.5-16.4) at 4 years, and 9.8% (6.4-14.0) at 5 years with temozolomide, versus 10.9% (7.6-14.8), 4.4% (2.4-7.2), 3.0% (1.4-5.7), and 1.9% (0.6-4.4) with radiotherapy alone (hazard ratio 0.6, 95% CI 0.5-0.7; p<0.0001). A benefit of combined therapy was recorded in all clinical prognostic subgroups, including patients aged 60-70 years. Methylation of the MGMT promoter was the strongest predictor for outcome and benefit from temozolomide chemotherapy. INTERPRETATION: Benefits of adjuvant temozolomide with radiotherapy lasted throughout 5 years of follow-up. A few patients in favourable prognostic categories survive longer than 5 years. MGMT methylation status identifies patients most likely to benefit from the addition of temozolomide. FUNDING: EORTC, NCIC, Nélia and Amadeo Barletta Foundation, Schering-Plough.

EORTC 26083 phase I/II trial of dasatinib in combination with CCNU in patients with recurrent glioblastoma.

The treatment of patients with recurrent glioblastoma remains a major oncologic problem, with median survival after progression of 7-9 months. To determine the maximum tolerated dose and dose-limiting toxicity (DLT), the combination of dasatinib and cyclonexyl-chloroethyl-nitrosourea (CCNU) was investigated in this setting. The study was designed as multicenter, randomized phase II trial, preceded by a lead-in safety phase. The safety component reported here, which also investigated pharmacokinetics and preliminary clinical activity, required expansion and is therefore considered a phase I part to establish a recommended dosing regimen of the combination of CCNU (90-110 mg/m(2)) and dasatinib (100-200 mg daily). Overall, 28 patients were screened, and 26 patients were enrolled. Five dose levels were explored. DLTs, mainly myelosuppression, occurred in 10 patients. Grade 3 or 4 neutropenia was recorded in 7 patients (26.9%) and thrombocytopenia in 11 patients (42.3%). No significant effect of CCNU coadministration on dasatinib pharmacokinetics was found. Median progression-free survival (PFS) was 1.35 months (95% confidence interval: 1.2-1.4) and 6-month PFS was 7.7%. In this phase I study of recurrent glioblastoma patients, the combination of CCNU and dasatinib showed significant hematological toxicities and led to suboptimal exposure to both agents.

Perfusion and diffusion MRI of glioblastoma progression in a four-year prospective temozolomide clinical trial

RESUME BUT Cette étude a été menée sur le suivi de patients traités pour un glioblastome nouvellement diagnostiqué. Son objectif a été de déterminer l'impact des séquences de perfusion et de diffusion en imagerie par résonance magnétique (IRM). Un intérêt particulier a été porté au potentiel de ces nouvelles techniques d'imagerie dans l'anticipation de la progression de la maladie. En effet, l'intervalle de temps libre de progression est une mesure alternative de pronostic fréquemment utilisée. MATERIEL ET METHODE L'étude a porté sur 41 patients participant à un essai clinique de phase II de traitement par temozolomide. Leur suivi radiologique a comporté un examen IRM dans les 21 à 28 jours après radiochimiothérapie et tous les 2 mois par la suite. L'évaluation des images s'est faite sur la base de l'évaluation de l'effet de masse ainsi que de la mesure de la taille de la lésion sur les images suivantes : T1 avec produit de contraste, T2, diffusion, perfusion. Afin de déterminer la date de progression de la maladie, les critères classiques de variation de taille adjoints aux critères cliniques habituels ont été utilisés. RESULAT 311 examens IRM ont été revus. Au moment de la progression (32 patients), une régression multivariée selon Cox a permis de déterminer deux paramètres de survie : diamètre maximal en T1 (p>0.02) et variation de taille en T2 (p<0.05). L'impact de la perfusion et de la diffusion n'a pas été démontré de manière statistiquement significative. CONCLUSION Les techniques de perfusion et de diffusion ne peuvent pas être utilisées pour anticiper la progression tumorale. Alors que la prise de décision au niveau thérapeutique est critique au moment de la progression de la maladie, l'IRM classique en T1 et en T2 reste la méthode d'imagerie de choix. De manière plus spécifique, une prise de contraste en T1 supérieure à 3 cm dans son plus grand diamètre associée à un hypersignal T2 en augmentation forment un marqueur de mauvais pronostic.

Alkylpurine-DNA-N-glycosylase confers resistance to temozolomide in xenograft models of glioblastoma multiforme and is associated with poor survival in patients.

Glioblastoma multiforme (GBM) is the most common and lethal of all gliomas. The current standard of care includes surgery followed by concomitant radiation and chemotherapy with the DNA alkylating agent temozolomide (TMZ). O⁶-methylguanine-DNA methyltransferase (MGMT) repairs the most cytotoxic of lesions generated by TMZ, O⁶-methylguanine. Methylation of the MGMT promoter in GBM correlates with increased therapeutic sensitivity to alkylating agent therapy. However, several aspects of TMZ sensitivity are not explained by MGMT promoter methylation. Here, we investigated our hypothesis that the base excision repair enzyme alkylpurine-DNA-N-glycosylase (APNG), which repairs the cytotoxic lesions N³-methyladenine and N⁷-methylguanine, may contribute to TMZ resistance. Silencing of APNG in established and primary TMZ-resistant GBM cell lines endogenously expressing MGMT and APNG attenuated repair of TMZ-induced DNA damage and enhanced apoptosis. Reintroducing expression of APNG in TMZ-sensitive GBM lines conferred resistance to TMZ in vitro and in orthotopic xenograft mouse models. In addition, resistance was enhanced with coexpression of MGMT. Evaluation of APNG protein levels in several clinical datasets demonstrated that in patients, high nuclear APNG expression correlated with poorer overall survival compared with patients lacking APNG expression. Loss of APNG expression in a subset of patients was also associated with increased APNG promoter methylation. Collectively, our data demonstrate that APNG contributes to TMZ resistance in GBM and may be useful in the diagnosis and treatment of the disease.

Cost-effectiveness of temozolomide for the treatment of newly diagnosed glioblastoma multiforme.

AIM: To perform a systematic review on the costs and cost-effectiveness of concomitant and adjuvant temozolomide with radiotherapy for the treatment of newly diagnosed glioblastoma compared with initial radiotherapy alone. METHODS: Electronic databases were searched for relevant publications on costs and cost-effectiveness until October 2008. RESULTS: We found four relevant clinical trials, one cost study and two economic models. The mean survival benefit in the radiotherapy plus temozolomide group varied between 0.21 and 0.25 life-years. Treatment costs were between 27,365 euros and 39,092 euros. The costs of temozolomide amounted to approximately 40% of the total treatment costs. The incremental cost-effectiveness ratios found in the literature were 37,361 euros per life-year gained and 42,912 euros per quality-adjusted life-year gained. However, the models are not comparable because different outcomes are used (i.e., life-years and quality-adjusted life-years). CONCLUSION: Although the models are not comparable according to outcome, the incremental cost-effectiveness ratios found are within acceptable ranges. We concluded that despite the high temozolomide acquisition costs, the costs per life-year gained and the costs per quality-adjusted life-year gained are comparable with other accepted first-line treatments with chemotherapy in patients with cancer.

Stem cell-related "self-renewal" signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma.

PURPOSE: Glioblastomas are notorious for resistance to therapy, which has been attributed to DNA-repair proficiency, a multitude of deregulated molecular pathways, and, more recently, to the particular biologic behavior of tumor stem-like cells. Here, we aimed to identify molecular profiles specific for treatment resistance to the current standard of care of concomitant chemoradiotherapy with the alkylating agent temozolomide. PATIENTS AND METHODS: Gene expression profiles of 80 glioblastomas were interrogated for associations with resistance to therapy. Patients were treated within clinical trials testing the addition of concomitant and adjuvant temozolomide to radiotherapy. RESULTS: An expression signature dominated by HOX genes, which comprises Prominin-1 (CD133), emerged as a predictor for poor survival in patients treated with concomitant chemoradiotherapy (n = 42; hazard ratio = 2.69; 95% CI, 1.38 to 5.26; P = .004). This association could be validated in an independent data set. Provocatively, the HOX cluster was reminiscent of a "self-renewal" signature (P = .008; Gene Set Enrichment Analysis) recently characterized in a mouse leukemia model. The HOX signature and EGFR expression were independent prognostic factors in multivariate analysis, adjusted for the O-6-methylguanine-DNA methyltransferase (MGMT) methylation status, a known predictive factor for benefit from temozolomide, and age. Better outcome was associated with gene clusters characterizing features of tumor-host interaction including tumor vascularization and cell adhesion, and innate immune response. CONCLUSION: This study provides first clinical evidence for the implication of a "glioma stem cell" or "self-renewal" phenotype in treatment resistance of glioblastoma. Biologic mechanisms identified here to be relevant for resistance will guide future targeted therapies and respective marker development for individualized treatment and patient selection.

Up-regulation of macrophage migration inhibitory factor gene and protein expression in glial tumor cells during hypoxic and hypoglycemic stress indicates a critical role for angiogenesis in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most malignant variant of human glial tumors. A prominent feature of this tumor is the occurrence of necrosis and vascular proliferation. The regulation of glial neovascularization is still poorly understood and the characterization of factors involved in this process is of major clinical interest. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine released by leukocytes and by a variety of cells outside of the immune system. Recent work has shown that MIF may function to regulate cellular differentiation and proliferation in normal and tumor-derived cell lines, and may also contribute to the neovascularization of tumors. Our immunohistological analysis of MIF distribution in GBM tissues revealed the strong MIF protein accumulation in close association with necrotic areas and in tumor cells surrounding blood vessels. In addition, MIF expression was frequently associated with the presence of the tumor-suppressor gene p53. To substantiate the concept that MIF might be involved in the regulation of angiogenesis in GBM, we analyzed the MIF gene and protein expression under hypoxic and hypoglycemic stress conditions in vitro. Northern blot analysis showed a clear increase of MIF mRNA after hypoxia and hypoglycemia. We could also demonstrate that the increase of MIF transcripts on hypoxic stress can be explained by a profound transcriptional activation of the MIF gene. In parallel to the increase of MIF transcripts, we observed a significant rise in extracellular MIF protein on angiogenic stimulation. The data of our preliminary study suggest that the up-regulation of MIF expression during hypoxic and hypoglycemic stress might play a critical role for the neovascularization of glial tumors.

Cilengitide: an RGD pentapeptide ανβ3 and ανβ5 integrin inhibitor in development for glioblastoma and other malignancies.

Cilengitide, a cyclicized arginine-glycine-aspartic acid-containing pentapeptide, potently blocks ανβ3 and ανβ5 integrin activation. Integrins are upregulated in many malignancies and mediate a wide variety of tumor-stroma interactions. Cilengitide and other integrin-targeting therapeutics have preclinical activity against many cancer subtypes including glioblastoma (GBM), the most common and deadliest CNS tumor. Cilengitide is active against orthotopic GBM xenografts and can augment radiotherapy and chemotherapy in these models. In Phase I and II GBM trials, cilengitide and the combination of cilengitide with standard temozolomide and radiation demonstrate consistent antitumor activity and a favorable safety profile. Cilengitide is currently under evaluation in a pivotal, randomized Phase III study (Cilengitide in Combination With Temozolomide and Radiotherapy in Newly Diagnosed Glioblastoma Phase III Randomized Clinical Trial [CENTRIC]) for newly diagnosed GBM. In addition, randomized controlled Phase II studies with cilengitide are ongoing for non-small-cell lung cancer and squamous cell carcinoma of the head and neck. Cilengitide is the first integrin inhibitor in clinical Phase III development for oncology.

Genomic copy number aberrations in glioblastoma, response to therapy and molecular machanisms

Résumé : Le glioblastome (GBM, WHO grade IV) est la tumeur cérébrale primaire la plus fréquente et la plus maligne, son pronostic reste très réservé et sa réponse aux différents traitements limitée. Récemment, une étude clinique randomisée (EORTC 26981/NCIC CE.3) a démontré que le traitement combiné de temozolomide et radiothérapie (RT/TMZ) est le meilleur dans les cas de GBM nouvellement diagnostiqués [1]. Cependant, seul un sous-groupe de patients bénéficie du traitement RT/TMZ et même parmi eux, leur survie reste très limitée. Pour tenter de mieux comprendre les réponses au traitement RT/TMZ, la biologie du GBM, identifier d'autres facteurs de résistance et découvrir de nouvelles cibles aux traitements, nous avons conduit une analyse moléculaire étendue à 73 patients inclus dans cette étude clinique. Nous avons complété les résultats moléculaires déjà obtenus par un profil génomique du nombre de copies par Array Comparative Genomic Hybridization. Afin d'atteindre nos objectifs, nous avons analysé en parallèle les données cliniques des patients et leurs profils moléculaires. Nos résultats confirment des analyses connues dans le domaine des aberrations du nombre de copies (CNA) et de profils du glioblastome. Nous avons observé une bonne corrélation entre le CNA génomique et l'expression de l'ARN messager dans le glioblastome et identifié un nouveau modèle de CNA du chromosome 7 pouvant présenter un intérêt clinique. Nous avons aussi observé par l'analyse du CNA que moins de 10% des glioblastomes conservent leurs mécanismes de suppression de tumeurs p53 et Rb1. Nous avons aussi observé que l'amplification du CDK4 peut constituer un facteur supplémentaire de résistance au traitement RT/TMZ, cette observation nécessite confirmation sur un plus grand nombre d'analyses. Nous avons montré que dans notre analyse des profils moléculaires et cliniques, il n'est pas possible de différencier le GBM à composante oligodendrogliale (GBM-O) du glioblastome. En superposant les profils moléculaires et les modèles expérimentaux in vitro, nous avons identifié WIF-1 comme un gène suppresseur de tumeur probable et une activation du signal WNT dans la pathologie du glioblastome. Ces observations pourraient servir à une meilleure compréhension de cette maladie dans le futur. Abstract : Glioblastoma, (GBM, WHO grade IV) is the most malignant and most frequent primary brain tumor with a very poor prognosis and response to therapy. A recent randomized clinical trial (EORTC26981/NCIC CE.3) established RT/TMZ as the 1St effective chemo-radiation therapy in newly diagnosed GBM [1]. However only a genetic subgroup of patients benefit from RT/TMZ and even in this subgroup overall survival remains very dismal. To explain the observed response to RT/TMZ, have a better understanding of GBM biology, identify other resistance factors and discover new drugable targets a comprehensive molecular analysis was performed in 73 of these GBM trial cohort. We complemented the available molecular data with a genomic copy number profiling by Array Comparative Genomic Hybridization. We proceeded to align the molecular profiles and the Clinical data, to meet our project objectives. Our data confirm known GBM Copy Number Aberrations and profiles. We observed a good correlation of genomic CN and mRNA expression in GBM, and identified new interesting CNA pattern for chromosome 7 with a potential clinical value. We also observed that by copy number aberration data alone, less than 10% of GBM have an intact p53 and Rb1 tumor .suppressor pathways. We equally observed that CDK4 amplification might constitute an additional RT/TMZ resistant factor, an observation that will need confirmation in a larger data set. We show that the molecular and clinical profiles in our data set, does not support the identification of GBM-O as a new entity in GBM. By combining the molecular profiles and in vitro model experiments we identify WIF1 as a potential GBM TSG and an activated WNT signaling as a pathologic event in GBM worth incorporation in attempts to better understand and impact outcome in this disease.

The biology of cancer stem cells : part 1: nuclear translocation of CD44 : part 2: IMP2 in glioblastoma cancer stem cells

SummaryCancer stem cells (CSC) are poorly differentiated, slowly proliferating cells, with high tumorigenic potential. Some of these cells, as it has been shown in leukemia, evade chemo- and radiotherapy and recapitulate the tumor composed of CSC and their highly proliferative progeny. Therefore, understanding the molecular biology of those cells is crucial for improvement of currently used anti-cancer therapies.This work is composed of two CSC-related projects. The first deals with CD44, a frequently used marker of CSC; the second involves Imp2 and its role in CSC bioenergetics. PART 1. CD44 is a multifunctional transmembrane protein involved in migration, homing, adhesion, proliferation and survival. It is overexpressed in many cancers and its levels are correlated with poor prognosis. CD44 is also highly expressed by CSC and in many malignancies it is used for CSC isolation.In the present work full-lenght CD44 nuclear localization was studied, including the mechanism of nuclear translocation and its functional role in the nucleus. Full-length CD44 can be found in nuclei of various cell types, regardless of their tumorigenic potential. For nuclear localization, CD44 needs to be first inserted into the cell membrane, from which it is transported via the endocytic pathway. Upon binding to transportinl it is translocated to the nucleus. The nuclear localization signal recognized by transportinl has been determined as the first 20 amino acids of the membrane proximal intracellular domain. Nuclear export of CD44 is facilitated by exportin Crml. Investigation of the function of nuclear CD44 revealed its implication in de novo RNA synthesis.PART 2. Glioblastoma multiforme is the most aggressive and most frequent brain malignancy. It was one of the first solid tumors from which CSC have been isolated. Based on the similarity between GBM CSC and normal stem cells expression of an oncofetal mRNA binding protein Imp2 has been investigated.Imp2 is absent in normal brain as well as in low grade gliomas, but is expressed in over 75% GBM cases and its expression is higher in CSC compared to their more differentiated counterparts. Analysis of mRNA transcripts bound by Imp2 and its protein interactors revealed that in GBM CSC Imp2 may be implicated in mitochondrial metabolism. Indeed, shRNA mediated silencing of protein expression led to decreased mitochondrial activity, decreased oxygen consumption and decreased activity of respiratory chain protein complex I. Moreover, lack of Imp2 severely affected self-renewal and tumorigenicity of GBM CSC. Experimental evidence suggest that GBM CSC depend on mitochondrial oxidative phosphorylation as an energy producing pathway and that Imp2 is a novel regulator of this pathway.RésuméLes cellules cancéreuses souches sont des cellules peu différentiées, à proliferation lente et hautement tumorigénique. Ces cellules sont radio-chimio résistantes et sont capable reformer la tumeur dans sont intégralité, reproduisant l'hétérogénéité cellulaire présent dans la tumeur d'origine. Pour améliorer les therapies antitumorales actuelles il est crucial de comprendre les mécanismes moléculaires qui caractérisent cette sous-population de cellules hautement malignes.Ce travail de thèse se compose de deux projets s'articulant autour du même axe :Le CD44 est une protéine multifonctionnelle et transmembranaire très souvent utilisée comme marqueur de cellules souches tumorales dans différents cancers. Elle est impliquée dans la migration, l'adhésion, la prolifération et la survie des cellules. Lors de ce travail de recherche, nous nous sommes intéressés à la localisation cellulaire du CD44, ainsi qu'aux mécanismes permettant sa translocation nucléaire. En effet, bien que principalement décrit comme un récepteur de surface transmembranaire, le CD44 sous sa forme entière, non clivée en peptides, peut également être observé à l'intérieur du noyau de diverses cellules, quel que soit leur potentiel tumorigénique. Pour passer ainsi d'un compartiment cellulaire à un autre, le CD44 doit d'abord être inséré dans la membrane plasmique, d'où il est transporté par endocytose jusqu'à l'intérieur du cytoplasme. La transportai permet ensuite la translocation nucléaire du CD44 via une « séquence signal » contenue dans les 20 acides aminés du domaine cytoplasmique qui bordent la membrane. A l'inverse, le CD44 est exporté du noyau grâce à l'exportin Crml. En plus des mécanismes décrits ci-dessus, cette étude a également mis en évidence l'implication du CD44 dans la synthèse des ARN, d'où sa présence dans le noyau.Le glioblastome est la plus maligne et la plus fréquente des tumeurs cérébrales. Dans ce second projet de recherche, le rôle de IMP2 dans les cellules souches tumorales de glioblastomes a été étudié. La présence de cette protéine oncofoetale a d'abord été mise en évidence dans 75% des cas les plus agressifs des gliomes (grade IV, appelés glioblastomes), tandis qu'elle n'est pas exprimée dans les grades I à III de ces tumeurs, ni dans le cerveau sain. De plus, IMP2 est apparue comme étant davantage exprimée dans les cellules souches tumorales que dans les cellules déjà différenciées. La baisse de l'expression de IMP2 au moyen de shRNA a résulté en une diminution de l'activité mitochondriale, en une réduction de la consommation d'oxygène ainsi qu'en une baisse de l'activité du complexe respiratoire I.L'inhibition de IMP2 a également affecté la capacité de renouvellement de la population des cellules souches tumorales ainsi que leur aptitude à former des tumeurs.Lors de ce travail de thèse, une nouvelle fonction d'un marqueur de cellules souches tumorales a été mise en évidence, ainsi qu'un lien important entre la bioénergétique de ces cellules et l'expression d'une protéine oncofoetale.

Radiation therapy and concurrent plus adjuvant temsirolimus (CCI-779) versus chemoirradiation with temozolomide in newly diagnosed glioblastoma without methylation of the MGMT gene promoter.

Background: Preclinical data indicate activity of mammalian target of rapamycin inhibitors and synergistic activity together with radiotherapy in glioblastoma. The aim of this trial is to assess the therapeutic activity of temsirolimus (CCI-779), an intravenous mTOR inhibitor, in patients with newly diagnosed glioblastoma with unmethylated O6 methlyguanine-DNA-methlytransferase (MGMT)promoter. Methods: Patients (n=257) with newly diagnosed glioblastoma after open surgical biopsy or resection fulfilling basic eligibility criteria underwent a central MGMT promoter analysis using quantitative methylation specific PCR. Patients with glioblastoma harboring an unmethylated MGMT promoter (n=111) were randomized 1:1 between radiotherapy (60 Gy; 5 times 2 Gy per week) plus concomitant and six cycles of maintenance temozolomide or radiotherapy plus weekly temsirolimus at 25 mg flat dose to be continued until progression or undue toxicity. Primary endpoint was overall survival at 12 months (OS12). Sample size of the investigational treatment arm required 54 patients to assess adequacy of temsirolimus activity set at 80%. More than 38 patients alive at 12 months in the per protocol population was considered a positive signal. A control arm of 54 patients treated with the standard of care was implemented to evaluate the assumptions on OS12. Results: Between December 2009 and October 2012, 111 pts in 14 centers were randomized and treated. Median age was 55 and 58 years in the temsirolimus and standard arm, respectively. Most patients (95.5%) had a WHO performance status of 0 or 1. Both therapies were properly administered with a median of 13 cycles of maintenance temsirolimus. In the per protocolpopulation, exactly 38 patients treated with temsirolimus (out of 54 eligible) reached OS12. In the intention to treat population OS12 was 72.2% [95% CI (58.2, 82.2)] in the temozolomide arm and 69.6% [95% CI (55.8, 79.9) in the temsirolimus arm [HR=1.16 95% CI (0.77, 1.76), p=0.47]. Conclusions: The therapeutic activity of temsirolimus in patients with newly diagnosed glioblastoma with an unmethylated MGMT promoter is too low.

MGMT methylation analysis of glioblastoma on the Infinium methylation BeadChip identifies two distinct CpG regions associated with gene silencing and outcome, yielding a prediction model for comparisons across datasets, tumor grades, and CIMP-status.

The methylation status of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene is an important predictive biomarker for benefit from alkylating agent therapy in glioblastoma. Recent studies in anaplastic glioma suggest a prognostic value for MGMT methylation. Investigation of pathogenetic and epigenetic features of this intriguingly distinct behavior requires accurate MGMT classification to assess high throughput molecular databases. Promoter methylation-mediated gene silencing is strongly dependent on the location of the methylated CpGs, complicating classification. Using the HumanMethylation450 (HM-450K) BeadChip interrogating 176 CpGs annotated for the MGMT gene, with 14 located in the promoter, two distinct regions in the CpG island of the promoter were identified with high importance for gene silencing and outcome prediction. A logistic regression model (MGMT-STP27) comprising probes cg1243587 and cg12981137 provided good classification properties and prognostic value (kappa = 0.85; log-rank p < 0.001) using a training-set of 63 glioblastomas from homogenously treated patients, for whom MGMT methylation was previously shown to be predictive for outcome based on classification by methylation-specific PCR. MGMT-STP27 was successfully validated in an independent cohort of chemo-radiotherapy-treated glioblastoma patients (n = 50; kappa = 0.88; outcome, log-rank p < 0.001). Lower prevalence of MGMT methylation among CpG island methylator phenotype (CIMP) positive tumors was found in glioblastomas from The Cancer Genome Atlas than in low grade and anaplastic glioma cohorts, while in CIMP-negative gliomas MGMT was classified as methylated in approximately 50 % regardless of tumor grade. The proposed MGMT-STP27 prediction model allows mining of datasets derived on the HM-450K or HM-27K BeadChip to explore effects of distinct epigenetic context of MGMT methylation suspected to modulate treatment resistance in different tumor types.