Integrating receptor interactions and dynamics and Interference with endocrine disruptors in the mechanisms of action of PPAR nuclear receptors

Abstract Peroxisome Proliferator-Activated Receptors (PPARs) form a family of three nuclear receptors regulating important cellular and metabolic functions. PPARs control gene expression by directly binding to target promoters as heterodimers with the Retinoid X Receptor (RXR), and their transcriptional activity is enhanced upon activation by natural or pharmacological ligands. The binding of PPAR/RXR heterodimers on target promoters allows the anchoring of a series of coactivators and corepressors involved in promoter remodeling and the recruitment of the transcription machinery. The transcriptional output finally depends on a complex interplay between (i) the respective expression levels of PPARs, RXRs and of other nuclear receptors competing for DNA binding and RXR recruitment, (ii) the availability and the nature of PPAR and RXR ligands, (iii) the expression levels and the nature of the different coactivators and corepressors and (iv) the sequence and the epigenetic status of the promoter. Understanding how all these factors and signals integrate and fine-tune transcription remains a challenge but is necessary to understand the specificity of the physiological functions regulated by PPARs. The work presented herein focuses on the molecular mechanisms of PPAR action and aims at understanding how the interactions and mobility of the receptor modulate transcription in the physiological context of a living cell: Such observations in vivo rely on the use of engineered fluorescent protein chimeras and require the development and the application of complementary imaging techniques such as Fluorescence Recovery After Photobleaching (FRAP), Fluorescence Resonance Energy Transfer (FRET) and Fluorescence Correlation Spectroscopy (FCS). Using such techniques, PPARs are shown to reside solely in the nucleus where they are constitutively associated with RXR but transcriptional activation by ligand binding -does not promote the formation of sub-nuclear structures as observed with other nuclear receptors. In addition, the engagement of unliganded PPARs in large complexes of cofactors in living cells provides a molecular basis for their ligand-independent activity. Ligand binding reduces receptor diffusion by promoting the recruitment of coactivators which further enlarge the size of PPAR complexes to acquire full transcriptional competence. Using these molecular approaches, we deciphered the molecular mechanisms through which phthalates, a class of pollutants from the plastic industry, interfere with PPARγ signaling. Mono-ethyl-hexyl-phthalate (MEHP) binding induces the recruitment of a specific subset of cofactors and translates into the expression of a specific subset of target genes, the transcriptional output being strongly conditioned by the differentiation status of the cell. This selective PPARγ modulation induces limited adipogenic effects in cellular models while exposure to phthalates in animal models leads to protective effects on glucose tolerance and diet-induced obesity. These results demonstrate that phthalates influence lipid and carbohydrate metabolism through complex mechanisms which most likely involve PPARγ but also probably PPARα and PPARß, Altogether, the molecular and physiological demonstration of the interference of pollutants with PPAR action outlines an important role of chemical exposure in metabolic regulations. Résumé Les PPARs (Peroxisome Proliferator-Activated Receptors) forment une famille de récepteurs nucléaires qui régulent des fonctions cellulaires et métaboliques importantes. Les PPARs contrôlent l'expression des gènes en se liant directement à leurs promoteurs sous forme d'hétérodimères avec les récepteurs RXR (Retinoid X Receptor), et leur activité transcriptionnelle est stimulée par la liaison de ligands naturels ou pharmacologiques. L'association des hétérodimères PPAR/RXR avec les promoteurs des gènes cibles permet le recrutement de coactivateurs et de corépresseurs qui vont permettre le remodelage de la chromatine et le recrutement de la machinerie transcriptionnelle. Les actions transcriptionnelles du récepteur dépendent toutefois d'interactions complexes qui sont régulées par (i) le niveau d'expression des PPARs, des RXRs et d'autres récepteurs nucléaires entrant en compétition pour la liaison à l'ADN et l'association avec RXR, (ii) la disponibilité et la nature de ligands de PPAR et de RXR, (iii) les niveaux d'expression et la nature des différents coactivateurs et corépresseurs et (iv) la séquence et le marquage épigénétique des promoteurs. La compréhension des mécanismes qui permettent d'intégrer ces aspects pour assurer une régulation fine de l'activité transcriptionnelle est un défi qu'il est nécessaire de relever pour comprendre la spécificité des fonctions physiologiques régulées par les PPARs. Ce travail concerne l'étude des mécanismes d'action moléculaire des PPARs et vise à mieux comprendre comment les interactions du récepteur avec d'autres protéines ainsi que la mobilité de ce dernier régulent son activité transcriptionnelle dans le contexte physiologique des cellules vivantes. De telles observations reposent sur l'emploi de protéines fusionnées à des protéines fluorescentes ainsi que sur le développement et l'utilisation de techniques d'imagerie complémentaires telles que le FRAP (Fluorescence Recovery After Photobleaching), le FRET (Fluorescence Resonance Energy Transfer) ou la FCS (Fluorescence Corrélation Spectroscopy). En appliquant ces méthodes, nous avons pu montrer que les PPARs résident toujours dans le noyau où ils sont associés de manière constitutive à RXR, mais que l'ajout de ligand n'induit pas la formation de structures sub-nucléaires comme cela a pu être décrit pour d'autres récepteurs nucléaires. De plus, les PPARs sont engagés dans de larges complexes protéiques de cofacteurs en absence de ligand, ce qui procure une explication moléculaire à leur activité ligand-indépendante. La liaison du ligand réduit la vitesse de diffusion du récepteur en induisant le recrutement de coactivateurs qui augmente encore plus la taille des complexes afin d'acquérir un potentiel d'activation maximal. En utilisant ces approches moléculaires, nous avons pu caractériser les mécanismes permettant aux phtalates, une classe de polluants provenant de l'industrie plastique, d'interférer avec PPARγ. La liaison du mono-ethyl-hexyl-phtalate (NERF) à PPARγ induit un recrutement sélectif de cofacteurs, se traduisant par l'induction spécifique d'un sous-ensemble de gènes qui varie en fonction du niveau de différentiation cellulaire. La modulation sélective de PPARγ par le MEHP provoque une adipogenèse modérée dans des modèles cellulaires alors que l'exposition de modèles animaux aux phtalates induit des effets bénéfiques sur la tolérance au glucose et sur le développement de l'obésité. Toutefois, les phtalates ont une action complexe sur le métabolisme glucido-lipidique en faisant intervenir PPARγ mais aussi probablement PPARα et PPARß. Cette démonstration moléculaire et physiologique de l'interférence des polluants avec les récepteurs nucléaires PPAR souligne un rôle important de l'exposition à de tels composés dans les régulations métaboliques.

Prophylactic isolated limb perfusion for localized, high-risk limb melanoma: results of a multicenter randomized phase III trial. European Organization for Research and Treatment of Cancer Malignant Melanoma Cooperative Group Protocol 18832, the World Health Organization Melanoma Program Trial 15, and the North American Perfusion Group Southwest Oncology Group-8593.

PURPOSE: Patients with primary cutaneous melanoma > or = 1.5 mm in thickness are at high risk of having regional micrometastases at the time of initial surgical treatment. A phase III international study was designed to evaluate whether prophylactic isolated limb perfusion (ILP) could prevent regional recurrence and influence survival. PATIENTS AND METHODS: A total of 832 assessable patients from 16 centers entered the study; 412 were randomized to wide excision (WE) only and 420 to WE plus ILP with melphalan and mild hyperthermia. Median age was 50 years, 68% of patients were female, 79% of melanomas were located on a lower limb, and 47% had a thickness > or = 3 mm. RESULTS: Median follow-up duration is 6.4 years. There was a trend for a longer disease-free interval (DFI) after ILP. The difference was significant for patients who did not undergo elective lymph node dissection (ELND). The impact of ILP was clearly on the occurrence-as first site of progression - of in-transit metastases (ITM), which were reduced from 6.6% to 3.3%, and of regional lymph node (RLN) metastases, with a reduction from 16.7% to 12.6%. There was no benefit from ILP in terms of time to distant metastasis or survival. Side effects were higher after ILP, but transient in most patients. There were two amputations for limb toxicity after ILP. CONCLUSION: Prophylactic ILP with melphalan cannot be recommended as an adjunct to standard surgery in high-risk primary limb melanoma.

Analyse de la production thymique et de la prolifération périphérique des lymphocytes T CD4 et CD8 durant la phase chronique de l'infection à VIH-1

RESUME Dans le cadre de l'infection à VIH-1, deux mécanismes généraux, a) une destruction périphérique massive ou b) un défaut dans la production périphérique ou centrale de nouvelles cellules, pourraient être à l'origine de l'épuisement des lymphocytes T CD4. La question soulève une importante controverse. Dans cette étude, la production thymique et la capacité de prolifération de lymphocytes T ont été étudiées conjointement. La production thymique a été évaluée par l'analyse du contenu en cercles d'excision générés lors du réarrangement du récepteur aux cellules T (ou TRECs) des cellules T CD4 et CD8 périphériques, provenant de sujets sains VIH-1 négatifs (n=120) ou infectés par le VIH-1 (n=297), au stade précoce, intermédiaire et tardif de la phase chronique de la maladie. Au stade précoce, nous observons que le contenu en TRECs de la population CD4 est supérieur à celui de la population contrôle. Aucune différence n'est observée lors de la phase intermédiaire, alors que le contenu en TRECs est inférieur lors de la phase tardive, en comparaison avec le groupe contrôle. Pour les lymphocytes T CD8, le contenu en TRECs reste inférieur au groupe contrôle, à tous les stades de la maladie. Ainsi, au stade précoce, la production thymique chercherait à compenser la perte de lymphocytes T CD4 puis, avec l'évolution de la maladie, cette possibilité s'épuiserait. Les profils d'expression des gènes régulateurs du cycle cellulaire pour les cellules T CD4 et CD8 périphériques, obtenus par la méthode des biopuces d'ADNc (microarray), ont permis l'analyse de la capacité de prolifération périphérique des lymphocytes T. Trois populations cellulaires ont été comparées entre elles : lymphocytes provenant de sujets infectés par le VIH-1, lymphocytes provenant de sujets VIH-1-négatifs et lymphocytes activés in vitro provenant de sujets VIH-1-négatifs. Les résultats montrent, pour les cellules T CD8, un état d'activation et un profil d'expression des gènes régulateurs du cycle cellulaire comparables à ceux des cellules activées in vitro. Le profil d'expression génétique des cellules T CD4, par contre, montre une activation sub-optimale, conjointement à une forte expression de p53, ce qui pourrait amener à un bloc en phase G1 du cycle cellulaire ainsi qu'à une forte apoptose. En conclusion, cette perturbation de la progression du cycle cellulaire des lymphocytes T CD4 périphériques pourrait contribuer à l'échec de la restauration du nombre de lymphocytes T CD4 et ceci, malgré une production thymique conservée dans les stades précoces de la maladie, comme démontré par l'analyse du contenu en TRECs.

RNA interference screening identifies a novel role for autocrine fibroblast growth factor signaling in neuroblastoma chemoresistance.

Chemotherapeutic drug resistance is one of the major causes for treatment failure in high-risk neuroblastoma (NB), the most common extra cranial solid tumor in children. Poor prognosis is typically associated with MYCN amplification. Here, we utilized a loss-of-function kinome-wide RNA interference screen to identify genes that cause cisplatin sensitization. We identified fibroblast growth factor receptor 2 (FGFR2) as an important determinant of cisplatin resistance. Pharmacological inhibition of FGFR2 confirmed the importance of this kinase in NB chemoresistance. Silencing of FGFR2 sensitized NB cells to cisplatin-induced apoptosis, which was regulated by the downregulation of the anti-apoptotic proteins BCL2 and BCLXL. Mechanistically, FGFR2 was shown to activate protein kinase C-δ to induce BCL2 expression. FGFR2, as well as the ligand fibroblast growth factor-2, were consistently expressed in primary NB and NB cell lines, indicating the presence of an autocrine loop. Expression analysis revealed that FGFR2 correlates with MYCN amplification and with advanced stage disease, demonstrating the clinical relevance of FGFR2 in NB. These findings suggest a novel role for FGFR2 in chemoresistance and provide a rational to combine pharmacological inhibitors against FGFR2 with chemotherapeutic agents for the treatment of NB.

The prominent role of neutrophils during the initial phase of infection by Leishmania parasites.

Neutrophils are rapidly and massively recruited to the site of Leishmania inoculation, where they phagocytose the parasites, some of which are able to survive within these first host cells. Neutrophils can thus provide a transient safe shelter for the parasites, prior to their entry into macrophages where they will replicate. In addition, neutrophils release and synthesize rapidly several factors including cytokines and chemokines. The mechanism involved in their rapid recruitment to the site of parasite inoculation, as well as the putative consequences of their massive presence on the microenvironment of the focus of infection will be discussed in the context of the development of the Leishmania-specific immune response.

Premier épisode thymique: cas particulier de l'intervention dans la phase précoce des troubles bipolaires [First episode of mood disorders: an opportunity for early intervention in bipolar disorders].

While early intervention strategies have been developed for psychotic disorders, affective psychoses and bipolar disorders have been neglected by this movement. However, when considering that outcome of bipolar disorders is often not as favorable as previously thought and that delay between illness onset and introduction of an adequate treatment is often very long, such developments seem clearly justified. In this paper we briefly review arguments supporting early intervention in bipolar disorders, the practical and theoretical obstacles that still need to be overcome, the strategies that may already now contribute to decrease treatment delay, and we describe current state of research regarding identification of the prodromal phase of bipolar disorders.

Motivation des patients à participer à un essai clinique de phase I en oncologie

Chaque année en Suisse, 15000 personnes meurent du cancer, ce qui en fait la deuxième cause de mortalité [1]. Il est donc nécessaire d'introduire sur le marché de nouveaux traitements plus efficaces. Ces derniers doivent passer par une période d'essais cliniques qui comporte plusieurs phases, dont la phase I. Celle-ci sert principalement à déterminer la dose maximale tolérable d'un médicament, en exposant le patient à des doses croissantes. Déterminer les motivations des patients à participer à la phase I d'un essai clinique, alors que les bénéfices personnels sont relativement faibles [3].

Implications of the serpentine phase transition on the behaviour of beryllium and lithium-boron of subducted ultramafic rocks

The Totalp-Platta-Malenco ophiolites in the Eastern Central Alps offer a unique opportunity to study the behaviour of Li, Be and B in ultramafic rocks in response to serpentinization and to progressive Alpine metamorphism. These units represent the remnants of a former ocean-continent transition that was intensely serpentinized during exposure on the Jurassic seafloor of the Ligurian Tethys. From north to the south, three isograd reactions (lizardite double right arrow antigorite + brucite; lizardite + talc double right arrow antigorite; lizardite + tremolite double right arrow antigorite + diopside) have been used to quantify the evolution of the light element content of metamorphic minerals. We determined the Li, Be and B concentrations in major silicate minerals from the ultramafic bodies of Totalp, Platta and Malenco by secondary ion mass spectrometry. Mantle minerals have Be concentrations (e.g. <0.001-0.009 mu g/g in olivine) similar to the metamorphic minerals that replace them (e.g. <0.001-0.016 mu g/g in serpentine). The mantle signature of Be is thus neither erased during seafloor alteration nor by progressive metamorphism from prehnite-pumpellyite to epidote-amphibolite facies. In contrast, the Li and B inventories of metamorphic minerals are related to the lizardite-to-antigorite transition. Both elements display higher concentrations in the low-temperature serpentine polymorph lizardite (max. 156 mu/g Li, max. 318 mu g/g B) than in antigorite (max. 0.11 mu g/g Li, max. 12 mu g/g B). Calculated average B/Li ratios for lizardite (similar to 1395) and antigorite (similar to 115) indicate that Li fractionates from B during the lizardite-to-antigorite transition during prograde metamorphism in ultramafic rocks. In subduction zones, this signature is likely to be recorded in the B-rich nature of forearc fluids. Relative to oceanic mantle the Be content of mantle clinopyroxene is much higher, but similar to Be values from mantle xenoliths and subduction-related peridotite massifs. These data support previous hypothesis that the mantle rocks from the Eastern Central Alps have a subcontinental origin. We conclude that Be behaves conservatively during subduction metamorphism of ultramafic rocks, at least at low-temperature, and thus retains the fingerprint of ancient subduction-related igneous events in mantle peridotites. (C) 2010 Elsevier Ltd. All rights reserved.

A Multi-Center Phase II Study (SAKK 36/06) of Single Agent Everolimus (RAD001) In Patients with Relapsed or Refractory Mantle Cell Lymphoma

Introduction: Mantle cell lymphoma (MCL) accounts for 6% of all B-cell lymphomas and remains incurable for most patients. Those who relapse after first line therapy or hematopoietic stem cell transplantation have a dismal prognosis with short response duration after salvage therapy. On a molecular level, MCL is characterised by the translocation t[11;14] leading to Cyclin D1 overexpression. Cyclin D1 is downstream of the mammalian target of rapamycin (mTOR) kinase and can be effectively blocked by mTOR inhibitors such as temsirolimus. We set out to define the single agent activity of the orally available mTOR inhibitor everolimus (RAD001) in a prospective, multi-centre trial in patients with relapsed or refractory MCL (NCT00516412). The study was performed in collaboration with the EU-MCL network. Methods: Eligible patients with histologically/cytologically confirmed relapsed (not more than 3 prior lines of systemic treatment) or refractory MCL received everolimus 10 mg orally daily on day 1 - 28 of each cycle (4 weeks) for 6 cycles or until disease progression. The primary endpoint was the best objective response with adverse reactions, time to progression (TTP), time to treatment failure, response duration and molecular response as secondary endpoints. A response rate of 10% was considered uninteresting and, conversely, promising if 30%. The required sample size was 35 pts using the Simon's optimal two-stage design with 90% power and 5% significance. Results: A total of 36 patients with 35 evaluable patients from 19 centers were enrolled between August 2007 and January 2010. The median age was 69.4 years (range 40.1 to 84.9 years), with 22 males and 13 females. Thirty patients presented with relapsed and 5 with refractory MCL with a median of two prior therapies. Treatment was generally well tolerated with anemia (11%), thrombocytopenia (11%), neutropenia (8%), diarrhea (3%) and fatigue (3%) being the most frequent complications of CTC grade III or higher. Eighteen patients received 6 or more cycles of everolimus treatment. The objective response rate was 20% (95% CI: 8-37%) with 2 CR, 5 PR, 17 SD, and 11 PD. At a median follow-up of 6 months, TTP was 5.45 months (95% CI: 2.8-8.2 months) for the entire population and 10.6 months for the 18 patients receiving 6 or more cycles of treatment. Conclusion: This study demonstrates that single agent everolimus 10 mg once daily orally is well tolerated. The null hypothesis of inactivity could be rejected indicating a moderate anti-lymphoma activity in relapsed/refractory MCL. Further studies of either everolimus in combination with chemotherapy or as single agent for maintenance treatment are warranted in MCL.

Virological and Immunological Characterization of Novel NYVAC-Based HIV/AIDS Vaccine Candidates Expressing Clade C Trimeric Soluble gp140(ZM96) and Gag(ZM96)-Pol-Nef(CN54) as Virus-Like Particles.

The generation of vaccines against HIV/AIDS able to induce long-lasting protective immunity remains a major goal in the HIV field. The modest efficacy (31.2%) against HIV infection observed in the RV144 phase III clinical trial highlighted the need for further improvement of HIV vaccine candidates, formulation, and vaccine regimen. In this study, we have generated two novel NYVAC vectors, expressing HIV-1 clade C gp140(ZM96) (NYVAC-gp140) or Gag(ZM96)-Pol-Nef(CN54) (NYVAC-Gag-Pol-Nef), and defined their virological and immunological characteristics in cultured cells and in mice. The insertion of HIV genes does not affect the replication capacity of NYVAC recombinants in primary chicken embryo fibroblast cells, HIV sequences remain stable after multiple passages, and HIV antigens are correctly expressed and released from cells, with Env as a trimer (NYVAC-gp140), while in NYVAC-Gag-Pol-Nef-infected cells Gag-induced virus-like particles (VLPs) are abundant. Electron microscopy revealed that VLPs accumulated with time at the cell surface, with no interference with NYVAC morphogenesis. Both vectors trigger specific innate responses in human cells and show an attenuation profile in immunocompromised adult BALB/c and newborn CD1 mice after intracranial inoculation. Analysis of the immune responses elicited in mice after homologous NYVAC prime/NYVAC boost immunization shows that recombinant viruses induced polyfunctional Env-specific CD4 or Gag-specific CD8 T cell responses. Antibody responses against gp140 and p17/p24 were elicited. Our findings showed important insights into virus-host cell interactions of NYVAC vectors expressing HIV antigens, with the activation of specific immune parameters which will help to unravel potential correlates of protection against HIV in human clinical trials with these vectors. IMPORTANCE: We have generated two novel NYVAC-based HIV vaccine candidates expressing HIV-1 clade C trimeric soluble gp140 (ZM96) and Gag(ZM96)-Pol-Nef(CN54) as VLPs. These vectors are stable and express high levels of both HIV-1 antigens. Gag-induced VLPs do not interfere with NYVAC morphogenesis, are highly attenuated in immunocompromised and newborn mice after intracranial inoculation, trigger specific innate immune responses in human cells, and activate T (Env-specific CD4 and Gag-specific CD8) and B cell immune responses to the HIV antigens, leading to high antibody titers against gp140. For these reasons, these vectors can be considered vaccine candidates against HIV/AIDS and currently are being tested in macaques and humans.

Safety, pharmacokinetics, and preliminary clinical activity of inotuzumab ozogamicin, a novel immunoconjugate for the treatment of B-cell non-Hodgkin's lymphoma: results of a phase I study.

PURPOSE Inotuzumab ozogamicin (CMC-544) is an antibody-targeted chemotherapy agent composed of a humanized anti-CD22 antibody conjugated to calicheamicin, a potent cytotoxic agent. This was a phase I study to determine the maximum-tolerated dose (MTD), safety, and preliminary efficacy of inotuzumab ozogamicin in an expanded MTD cohort of patients with relapsed or refractory CD22(+) B-cell non-Hodgkin's lymphoma (NHL). PATIENTS AND METHODS Inotuzumab ozogamicin was administered intravenously as a single agent once every 3 or 4 weeks at doses ranging from 0.4 to 2.4 mg/m(2). Outcomes included MTD, safety, pharmacokinetics, response, progression-free survival (PFS), and overall survival. Results Seventy-nine patients were enrolled. The MTD was determined to be 1.8 mg/m(2). Common adverse events at the MTD were thrombocytopenia (90%), asthenia (67%), and nausea and neutropenia (51% each). The objective response rate at the end of treatment was 39% for the 79 enrolled patients, 68% for all patients with follicular NHL treated at the MTD, and 15% for all patients with diffuse large B-cell lymphoma treated at the MTD. Median PFS was 317 days (approximately 10.4 months) and 49 days for patients with follicular NHL and diffuse large B-cell lymphoma, respectively. CONCLUSION Inotuzumab ozogamicin has demonstrated efficacy against CD22(+) B-cell NHL, with reversible thrombocytopenia as the main toxicity.

RTOG 0525: A randomized phase iii trial comparing standard adjuvant temozolomide (tmz) with a dose-dense (dd) schedule in newly diagnosed glioblastoma (gbm)

Radiotherapy with concomitant and adjuvant TMZ is the standard of care for newly diagnosed GBM. MGMT methylation status may be an important determinant of treatment response. This trial, conducted by the RTOG, EORTC, and NCCTG, determined if intensified TMZ improves survival (OS) or progression free survival (PFS) in all patients or specific to MGMT status. Eligibility criteria included age . 18 yrs, KPS ≥ 60, and existence of a tissue block with . 1cm2 tumor for prospective MGMT and retrospective molecular analysis. Patients were randomized to Arm 1: standard TMZ (150-200 mg/m2 x 5 d) or Arm 2: dd TMZ (75-100 mg/m2 x 21 d) q 4 wks for 6-12 cycles. Symptom burden, quality of life (QOL), and neurocognition were prospectively and longitudinally assessed in a patient subset. 833 patients were randomized (1173 registered). Inadequate tissue (n ¼ 144) was the most frequent reason for nonrandomization.No statistical difference was observed between Arms 1 and 2 for median OS (16.6, 14.9 mo, p ¼ 0.63), median PFS (5.5, 6.7 mo, p ¼ 0.06), or methylation status. MGMT methylation was associated with improved OS (21.2, 14 mo, p , 0.0001), PFS (8.7, 5.7 mo, p , 0.0001), and treatment response (p ¼ 0.012). Cox modeling identifiedMGMT status and RPA class as significant predictors of OS; treatment arm and radiation technique (EORTC vs. RTOG) were not. There was increased grade ≥ 3 toxicity in Arm 2 (19%, 27%, p ¼ 0.008), which was mostly lymphopenia and fatigue. This study did not demonstrate improved efficacy for dd TMZ for newly diagnosed GBM regardless of methylation status. However, it confirmed the prognostic significance of MGMT methylation in GBM, demonstrated the feasibility of tumor tissue collection, molecular stratification, and collection of patient outcomes in a large transatlantic intergroup trial, thereby establishing a viable clinical trial paradigm. Support: NCI U10 CA 21661 and U10 CA37422.