Neuro-Oncology, Jun 2014

In contrast to most other malignant diseases, especially in children, up to now glioblastoma multiforma (GBM) is a lethal diagnosis for most of the patients. Operation and radiotherapy are very effective to reduce the tumor burden, however, a strong adjuvant treatment is lacking. To target glioblastoma cells more effectively, it is crucial to understand the cellular signaling and regulation, particularly in the EGF and VEGF dependent pathways. Since it has been shown that glioblastomas are extremely heterogeneous regarding genetic, epigenetic or signaling regulation, receptor expression etc., analysis of individual patient derived tumor cells is particularly important. We established a collection of well-characterized heterogeneous early-passage brain tumor cell lines. Since August 2009, more than 26 clinical samples from patients with WHO grade IV GBM and Anaplastic Astrocytoma, WHO grade III, were collected. Cell lines were established that were in depth analysed both for genetic and epigenetic regulation, receptor expression, and sensitivity to cytostatic or targeted drugs. We found that cells in monolayer and spheroid cultures, and cells grown using standard and stem cell selective culture medium, respectively, or, further, tumors grown in xenograft models behave differently with regard to the receptor dependent signaling pathways. Establishment of such models is crucial to design targeted therapy approaches that allow direct transfer from the laboratory system to the clinical application.

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HG- 0 3 5 6 8 11 18 25 27 30 35 40 43 50 59 70 71 84 87 91 96 109 . NEWCASTLE DISEASE VIROTHERAPY 0 3 5 6 8 11 18 25 27 30 35 40 43 50 59 70 71 84 87 91 96 109 A NOVEL IMMUNOTHERAPEUTIC APPROACH FOR HIGH GRADE GLIOMA Carolien Koks 0 3 4 5 6 8 10 11 17 18 25 27 29 30 34 35 40 42 43 49 50 58 59 70 71 75 84 86 87 90 91 95 96 108 109 Abhishek Garg 0 3 4 5 6 8 10 11 17 18 25 27 29 30 34 35 40 42 43 49 50 58 59 70 71 75 84 86 87 90 91 95 96 108 109 Michael Ehrhardt 0 2 3 5 6 8 9 11 15 18 25 27 28 30 33 35 40 41 43 48 50 56 59 70 71 73 84 85 87 89 91 93 96 107 109 Matteo Riva 0 3 4 5 6 8 10 11 17 18 25 27 29 30 34 35 40 42 43 49 50 58 59 70 71 75 84 86 87 90 91 95 96 108 109 Steven De Vleeschouwer 0 3 4 5 6 8 10 11 17 18 25 27 29 30 34 35 40 42 43 49 50 58 59 70 71 75 84 86 87 90 91 95 96 108 109 Patrizia Agostinis 0 3 4 5 6 8 10 11 17 18 25 27 29 30 34 35 40 42 43 49 50 58 59 70 71 75 84 86 87 90 91 95 96 108 109 Norbert Graf 0 2 3 5 6 8 9 11 15 18 25 27 28 30 33 35 40 41 43 48 50 56 59 70 71 73 84 85 87 89 91 93 96 107 109 Stefaan Van Gool 0 3 4 5 6 8 10 11 17 18 25 27 29 30 34 35 40 42 43 49 50 58 59 70 71 75 84 86 87 90 91 95 96 108 109 KU Leuven 0 3 5 6 8 11 18 25 27 30 35 40 43 50 59 70 71 84 87 91 96 109 Leuven 0 3 5 6 8 11 18 25 27 30 35 40 43 50 59 70 71 84 87 91 96 109 Belgium 0 2 3 5 6 8 9 11 15 18 25 27 28 30 33 35 40 41 43 48 50 56 59 70 71 73 84 85 87 89 91 93 96 107 109 University of Saarland 0 3 5 6 8 11 18 25 27 30 35 40 43 50 59 70 71 84 87 91 96 109 0 Medical School , Homburg , Germany 1 Pathology Department Alexandria University , Alexandria , Egypt 2 Pediatric Oncology Department Alexandria University , Alexandria , Egypt 3 HG-002. SURGICAL MANAGEMENT FOR PEDIATRIC INTRA-AXIAL BRAIN STEM TUMORS, SINGLE CENTER EXPERIENCE Ahmed Farhod 4 Neurosurgery Department Alexandria University , Alexandria , Egypt 5 HG-001. CULTURE CONDITION-DEPENDENT GROWTH OF EARLY PASSAGE PATIENT-DERIVED HUMAN GLIOBLASTOMA CELL LINES Carl Friedrich Classen, Doreen William, and Michael Linnebacher; University Medicine Rostock , Rostock , Germany 6 HG-005. TARGETING RESISTANCE PATHWAYS IN BRAF-MUTANT PEDIATRIC GLIOMAS Tsun-Wen Yao , Yasuyuki Yoshida, Jie Zhang, Tomoko Ozawa , David James, and Theodore Nicolaides; University of California San Francisco , San Francisco, CA , USA 7 Department of Radiology & Nuclear Medicine, VU University Medical Center , Amsterdam , The Netherlands 8 HG-009. MOLECULAR DRUG IMAGING: PIONEERING WORK IN CHILDREN WITH DIFFUSE INTRINSIC PONTINE GLIOMA Sophie Veldhuijzen van Zanten 9 Neuro-Oncology Research Group, VU University Medical Center , Amsterdam , The Netherlands 10 Department of Pediatrics, VU University Medical Center , Amsterdam , The Netherlands 11 HG-006. NIMOTUZUMAB CONTAINING REGIMEN FOR PEDIATRIC DIFFUSE INTRINSIC PONTINE GLIOMAS: SINGLE CENTER STUDY Rejin Kebudi 12 Department of Otolaryngology/Head and Neck Surgery, VU University Medical Center , Amsterdam , The Netherlands 13 Department of Nuclear Medicine & PET Research, VU University Medical Center , Amsterdam , The Netherlands 14 Division of Pediatric Hematology and Oncology, Department of Child and Adolescent Medicine, University Medical Center G o ̈ttingen , G o ̈ttingen , Germany 15 Department of Neuroradiology, University of W u ̈rzburg , W u ̈rzburg , Germany 16 Children's Hospital, University of Mainz , Mainz , Germany 17 Division of Pediatric Hematology and Oncology, Department of Pediatrics and Adolescent Medicine, Medical University of Graz , Graz , Austria 18 HG-011. CENTRALLY REVIEWED GLIOMATOSIS CEREBRI IN CHILDREN AND ADOLESCENTS: A RETROSPECITVE ANALYSIS FROM THE HIT-HGG DATABASE Martin Benesch 19 Department of Pediatric Hematology and Oncology, Saarland University , Homburg/Saar, Homburg/Saar , Germany 20 Department of Pediatric Hematology , Oncology and Neurooncology , University Children's Hospital W u ̈rzburg , Wu ̈ rzburg , Germany 21 Department of Radiation Oncology, University of Leipzig , Leipzig , Germany 22 Institute of Neuropathology, University of Bonn , Bonn , Germany 23 Children's Hospital Augsburg, Swabian Children's Cancer Center , Augsburg, Augsburg , Germany 24 Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf , Hamburg , Germany 25 HG-025. THE ROLE OF NG2 EXPRESSING PROGENITOR CELLS IN DIFFUSE INTRINSIC PONTINE GLIOMA Sridevi Yadavilli 26 European Molecular Biology Laboratory (EMBL) , Heidelberg , Germany 27 HG-024. MULTIPLE NOVEL FUSION GENES WITHIN THE RTK-RAS-PI3K SIGNALLING AXIS HIGHLIGHT ITS CENTRAL ROLE IN THE TURMORIGENESIS OF PEDIATRIC GLIOBLASTOMA Sebastian Bender 28 Duke University School of Medicine , Durham, NC , USA 29 Children's National Medical Center , Washington, DC , USA 30 HG-023. TREATMENT RESULTS OF DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) IN CHILDREN. ONE-CENTER EXPERIENCE Monika Drogosiewicz 31 University Hospital , Heidelberg , Germany 32 Department of Pathology and Molecular Medicine, 2nd Faculty of Medicine, Charles University , Prague , Czech Republic 33 Department of Radiotherapy and Oncology, University Hospital Motol , Prague , Czech Republic 34 Department of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University , Prague , Czech Republic 35 HG-034. HIGH-GRADE GLIOMAS - EXTENT OF RESECTION SIGNIFICANTLY IMPACTS THE PROGNOSIS OF PEDIATRIC PATIENTS TREATED IN PRAGUE CENTRE Michal Zapotocky 36 Department of Biology and Medical Genetics, 2nd Faculty of Medicine, Charles University , Prague , Czech Republic 37 Department of Neurosurgery, 2nd Faculty of Medicine, Charles University , Prague , Czech Republic 38 Department of Radiology, 2nd Faculty of Medicine, Charles University , Prague , Czech Republic 39 CHRU Hautepierre , Strasbourg , France 40 HG-039. THE ROLE OF ACVR1 MUTATIONS IN DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) Katy Taylor 41 Queensland Childrens Tumour Bank , Brisbane , Australia 42 Institute of Cancer Research , London , UK 43 HG-038. TARGETING MYC/MYCN BY INHIBITION OF CHECKPOINT KINASE 1 (CHK1) SENSITIZES PAEDIATRIC GLIOBLASTOMA (PGBM) CELLS TO TEMOZOLOMIDE Meera Nandhabalan 44 Structural Genomics Consortium , Oxford , UK 45 Hospital Sant Joan de De ́u , Barcelona , Spain 46 Stanford University , Palo Alto , USA 47 Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins Hospital , Baltimore, MD , USA 48 Johns Hopkins Department of Pediatric Oncology , Baltimore, MD , USA 49 Johns Hopkins Department of Pediatric Hematology/Oncology , Baltimore, MD , USA 50 HG-044. INCREASED 5-HYDROXYMETHYLCYTOSINE AND DECREASED 5-METHYLCYTOSINE ARE INDICATORS OF GLOBAL EPIGENETIC DYSREGULATION IN DIFFUSE INTRINSIC PONTINE GLIOMA Sama Ahsan 51 Division of Neuropathology, Johns Hopkins Hospital , Baltimore, MD , USA 52 Center for Genetic Medicine, Children's National Medical Center , Washington D.C. , USA 53 Laboratory of Pathology, National Institutes of Health , Bethesda, MD , USA 54 Pediatric Oncology Branch, National Institutes of Health , Bethesda, MD , USA 55 Texas Tech University , Lubbock, TX , USA 56 Institut Gustave Roussy et Universite ́ Paris-Sud , Villejuif , France 57 National Cancer Institute , Bethesda, MD , USA 58 Oregon Health & Science University , Portland, OR , USA 59 HG-047. FUNCTIONALLY-DEFINED THERAPEUTIC TARGETS IN DIFFUSE INTRINSIC PONTINE GLIOMA: A REPORT OF THE CHILDREN'S ONCOLOGY GROUP DIPG PRECLINICAL CONSORTIUM Catherine Grasso 60 Texas Children's Cancer Center , Houston, TX , USA 61 Pape ́ Family Pediatric Research Institute , Portland, OR , USA 62 Universite ́ d'Evry-Val d'Essone , Evry , France 63 Stanford University , Stanford, CA , USA 64 Johns Hopkins University , Baltimore, MD , USA 65 The First Affiliated Hospital of Soochow University , Suzhou , China 66 University of Toronto , Toronto, ON , Canada 67 Universite ́ Paris-Sud , Villejuif , France 68 VU University Medical Center , Amsterdam , The Netherlands 69 Cincinnati Children's Hospital Medical Center , Cincinnati, OH , USA 70 HG-051. HIGH FREQUENCY OF MISMATCH REPAIR GENE DEFICIENCY AMONG PEDIATRIC HIGH GRADE GLIOMAS IN JORDAN Nisreen Amayiri 71 HG-048. PRECLINICAL BIO-DISTRIBUTION OF 72 Division of Hematology/Oncology, The Hospital for Sick Children, Institute of Medical Sciences, The University of Toronto, Department of Pediatrics, University of Toronto , Toronto, ON , Canada 73 Division of Hematology/Oncology, The Hospital for Sick Children, Institute of Medical Sciences, The University of Toronto; Department of Pediatrics, University of Toronto , Toronto, ON , Canada 74 Department of Pathology, King Hussein Cancer Center , Amman , Jordan 75 Department of Pediatrics, King Hussein Cancer Center , Amman , Jordan 76 St. Jude Children's Research Hospital , Memphis, TN , USA 77 Department of Pediatrics, University of Toronto; Department of Pathology, Hospital for Sick Children , Toronto, ON , Canada 78 The Familial Gastrointestinal Cancer Registry at the Zane Cohen Centre for Digestive Disease and Department of Surgery, Mount Sinai Hospital , Toronto, ON , Canada; Division of Gastroenterology , Toronto, ON , Canada 79 The Familial Gastrointestinal Cancer Registry at the Zane Cohen Centre for Digestive Disease and Department of Surgery, Mount Sinai Hospital , Toronto, ON , Canada 80 Department of Radiology, King Hussein Cancer Center , Amman , Jordan 81 Department of Surgery, King Hussein Cancer Center , Amman , Jordan 82 ZR-BEVACIZUMAB IN DIPG AND SUPRATENTORIAL GBM XENOGRAFT MODELS USING POSITRON EMISSION TOMOGRAPHY (PET) Marc H. A. Jansen 83 Department of Radiation Oncology, Kantonsspital St. Gallen , St. Gallen , Switzerland 84 HG-055. AN INDIVIDUAL PATIENT DATA META-ANALYSIS ON CHARACTERISTICS, TREATMENT AND OUTCOME OF PATIENTS WITH METASTATIC HIGH-GRADE GLIOMAS Sophie Pietschmann 85 Institute for Medical Biostatistics, Epidemiology and Informatics, University of Mainz Medical Center , Mainz, Mainz , Germany 86 Department for Radiation Oncology, University of Leipzig Medical Center , Leipzig , Germany 87 HG-054. BI-ALLELIC MISMATCH REPAIR MSH6 GENE MUTATIONS IN A PATIENT SURVIVING A CHILDHOOD MALIGNANT BRAIN TUMOR Johanna Sandgren 88 Department of Pediatrics and Adolescent Medicine, Division of Pediatric Hematology and Oncology, University Medical Center Goettingen , Goettingen , Germany 89 Haematology/ Oncology Service, KK Women's and Children's Hospital, Singapore , Singapore 90 Yong Loo Lin School of Medicine, National University of Singapore , Singapore, Singapore 91 HG-056. HIGH GRADE GLIOMAS IN CHILDREN: REPORT FROM A CHILDREN'S CANCER CENTRE IN SINGAPORE Nicole Yu-Fang Sieow 92 National Institute of Health , Bethesda, MD , USA 93 Hospital for Sick Children , Toronto, ON , Canada 94 Seattle Children's Hospital , Seattle, WA , USA 95 Cincinnati Children's Hospital Medical Center , Cincinnati, OH , USA 96 HG-074. ESTABLISHMENT OF AN INTERNATIONAL DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) REGISTRY Maryam Fouladi 97 Institute of Cancer Research , Sutton , UK 98 Gustave Roussy Institute , Villejuif , France 99 Great Ormond Street Hospital for Children NHS Trust , London , UK 100 Royal Children's Hospital Brisbane , Brisbane , Australia 101 Lurie Children's Hospital , Chicago, IL , USA 102 University of College London, Institute of Child Health , London , UK 103 Hopital Sainte Anne , Paris , France 104 Children's National Medical Center , Washington, DC , USA 105 Radboud University Nijmegen Medical Center , Nijmegen , The Netherlands 106 Department of Pathology, University of California , San Francisco, CA , USA 107 Children's Hospital of Philadelphia, University of Pennsylvania , Philadelphia, PA , USA 108 Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center , New York, NY , USA 109 HG-077. REDUCTION IN H3K27ME3 IS A MOLECULAR AND PROGNOSTIC SURROGATE IN PEDIATRIC GLIOBLASTOMAS Siram Venneti 110 Department of Pathology and Laboratory Medicine, Children's Hospital Los Angeles, Keck School of Medicine University of Southern California , Los Angeles, CA , USA 111 Laboratory of Chromatin Biology and Epigenetics, The Rockefeller University , New York, NY , USA 112 Department of Biomolecular Chemistry, University of Wisconsin , Madison, WI , USA 113 NYU Genome Technology Center, New York University , New York, NY , USA In contrast to most other malignant diseases, especially in children, up to now glioblastoma multiforma (GBM) is a lethal diagnosis for most of the patients. Operation and radiotherapy are very effective to reduce the tumor burden, however, a strong adjuvant treatment is lacking. To target glioblastoma cells more effectively, it is crucial to understand the cellular signaling and regulation, particularly in the EGF and VEGF dependent pathways. Since it has been shown that glioblastomas are extremely heterogeneous regarding genetic, epigenetic or signaling regulation, receptor expression etc., analysis of individual patient derived tumor cells is particularly important. We established a collection of well-characterized heterogeneous early-passage brain tumor cell lines. Since August 2009, more than 26 clinical samples from patients with WHO grade IV GBM and Anaplastic Astrocytoma, WHO grade III, were collected. Cell lines were established that were in depth analysed both for genetic and epigenetic regulation, receptor expression, and sensitivity to cytostatic or targeted drugs. We found that cells in monolayer and spheroid cultures, and cells grown using standard and stem cell selective culture medium, respectively, or, further, tumors grown in xenograft models behave differently with regard to the receptor dependent signaling pathways. Establishment of such models is crucial to design targeted therapy approaches that allow direct transfer from the laboratory system to the clinical application. - INTRODUCTION: Intra-axial brain stem tumors belong to the most challenging neurosurgical problems. For decades the brain stem remained a no-man’s land, but this concept changed recently due to advances in the diagnostic and surgical tools. MATERIAL AND METHODS: We review the surgical outcome of 21 pediatric cases surgically treated for brain stem glioma at the Neurosurgery Department, Alexandria University between 2008 and 2013. They underwent 29 surgical procedures and include 10 boys and 11 girls. Age ranged from 2-18 years (mean 9 years), and follow up period ranged from 6 to 63 months. RESULTS: Among 29 surgical procedures, 9 procedures were carried out at the midbrain, 13 at the pons, and 5 at the medulla oblongata. The remaining 2 surgical interventions were for CSF diversion (V-P shunt and 3rd ventriculostomy). Patients with high grade glioma or DIPG were treated by radiotherapy 54 Gy/ 30 fractions, while those with low grade glioma were treated by carboplatin and vincristine for 18 months. 8 patients were operated twice; 4 for tumor regrowth, 2 as a planned second stage surgery, and 2 for CSF diversion. Intra-operative neurophysiological monitoring was available for 19 procedures, and all cases were operated without neuronavigation or intra-operative MRI. Total resection was achieved in 8 out of 27 procedures(29.6%), the most common pathology was pilocytic astrocytoma (14 cases). There was no postoperative mortality in this series, worsening of the pre-operative neurological status occurred in 12 patients but was permanent in only 2 cases. Six cases died during the follow up period; 3 cases had anaplastic astrocytoma (WHO G 3) and 3 other children suffered from diffuse pontine glioma. CONCLUSION: Microsurgery is the mainstay in the management of focal pediatric brain tumors. radiotherapy and/or chemotherapy are used in selected cases of focal tumors, however they represent the main treatment in diffuse glioma. The prognosis of children with relapsed malignant glioma remains poor. The results of new drugs, if available, on tumor control are disappointing. Active specific immunotherapy is a new promising treatment modality under development in several centres. We report an update of our experience. Children were treated with neurosurgery and vaccination with monocyte-derived mature dendritic cells loaded with tumor lysate. They had leukapheresis after weaning corticosteroids. Leukapheresis was performed via insert of a double-lumen inguinal venous access. Safety, feasibility and efficacy were endpoints of the HGGIMMUNO-2003 cohort comparison study. Forty five children (24 females) aged 2-17 years were included. Twelve patients had relapsed anaplastic astrocytoma, 2 anaplastic ependymoma, 2 Oligoastrocytoma grade III, 27 GBM, and 2 unspecified malignant glial tumors. They were treated with a median of 6 vaccines (4-17). The treatment was feasible. There were no treatment-related toxicities. Immunotherapy was given in ambulatory setting. Median PFS and OS of the total group were 3.5 and 12.1 months with 26.9% 2-year OS. Median PFS and OS of the subgroup with relapsed GBM were 2.7 and 10.4 months with 21.2% 2-year OS. The tail of the PFS curve remained stable at 17.7% after 16 months, with longest follow-up till 143.2 months. Similarly, the tail of the OS curve remained stable at 15.9% OS from 25 months. Because of low numbers, we could not detect any change in PFS or OS for the different cohorts in HGG-IMMUNO-2003. We could not detect any particular predictive parameter for long-term survival. Immunotherapy after neurosurgery provides a realistic second chance for children with relapsed malignant glioma, at least if the tumor can be again resected up to a subtotal extent. Collaborative efforts are urgently needed to implement this technology in a network of centres, so that randomized clinical trials can be initiated. In spite of current multimodal treatment the prognosis of Glioblastoma Multiforme patients remains poor. Here, we investigate oncolytic Newcastle disease virus (NDV), as a novel approach for glioma therapy in the murine orthotopic GL261 glioma model. Oncolytic viruses have the potential to lower tumor volume and viability, as well as to alter the immunosuppressive tumor microenvironment and stimulate antitumor immunity. This immune involvement has not been investigated in orthotopic glioma and the mechanisms involved remain to be unraveled. In vitro, GL261 cells were sensitive to NDV-mediated cytotoxicity in a dose- and time-dependent manner. Cell death showed features of necroptosis. NDV infection furthermore induced immunogenic cell death (ICD) in GL261 cells. The induced ICD route proved to be unique in that it lacks secreted ATP. In vivo, NDV increased median survival of glioma-bearing animals and 50% survived long-term, versus none of the controls. Treated animals had higher percentages of IFNg+ CD4+/CD8+ T cells within their brain, as well as lower percentages of myeloid derived suppressor cells. The importance of these activated T cells was demonstrated in immunodeficient animals, where NDV failed to induce long-term survival after glioma challenge, though it could prolong median survival slightly. Tumor rechallenge in long-term surviving immunocompetent animals resulted in 80% survival, demonstrating tumor-rejecting immune memory following ND virotherapy. For the first time, we describe the antiglioma activity of NDV in an orthotopic glioma model. Though NDV exerts a direct cytotoxic effect on glioma cells, the curative effect depends mainly on the induced cellular antitumor immune response. The mechanism behind this is a unique ICD route induced by viral infection in the tumor cells, which triggers the cells to activate the immune system. In ongoing work we are investigating the synergism between virotherapy and immunotherapy and expanding ND virotherapy to other brain tumor models such as DIPG. Mutational activation of BRAF is a common finding in pediatric gliomas. As many as 14% of pediatric high grade glioma and up to 66% of certain low grade pediatric glioma subtypes contain the BRAFV600E mutation. Small molecule inhibitors that selectively target BRAFV600E are FDA approved # The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: . for melanoma and have shown significant efficacy in treating BRAFV600E glioma in pre-clinical trials. Despite showing initial efficacy, drug resistance significantly limits the use of BRAFV600E inhibitors in clinical settings. Here, we identified the molecular mechanisms of BRAFV600E inhibitor resistance in glioma and also provide strategies to over come drug resistance. We have generated multiple BRAFV600E inhibitor resistant glioma cell lines and murine models to study the mechanisms of drug resistance in glioma. The BRAFV600E inhibitor resistant AM38 and DBTRG-05MG cells are insensitive to BRAF inhibitor induced MAP kinase inhibition, reduced cell viability and cell cycle arrest. Also, the BRAFV600E inhibitor resistant glioma xenografts do not respond to drug induced suppression of tumor growth, resulting in poor animal survival. By phospho-receptor tyrosine kinase array and gene expression array, we found that certain receptor tyrosine kinases, including EGF and Axl receptors, and pro-survival pathways such as Wnt signaling, are hyperactivated in BRAFV600E inhibitor resistant gliomas. We further showed that pharmacological and genetic inhibition of those pathways significantly reduces the viability of those drug resistant cells. In conclusion, we have discovered mechanisms of BRAFV600E inhibitor resistance in glioma. We showed that BRAFV600E inhibitor resistant cells acquire drug dependency by upregulating pro-survival pathway, such as Wnt, and hyperactivating receptor tyrosine kinases, including EGFR and Axl, to bypass the insult from the drug. Our data suggests novel therapeutic strategies to BRAFV600E -mutant pediatric glioma in which no effective treatment is available. AIM: Nimotuzumab is an IgG1 antibody that targets epidermal growth factor receptor (EGFR). Overexpression of EGFR is detected in diffuse intrinsic pontine gliomas (DIPGs). METHOD: In DIPG patients, since 2004, TMZ was used for 6 weeks concomitantly during RT, followed by TMZ for 5 days with a 28-day cycle after RT for 12 courses. After 2010 May, children with progressive disease (PD) after RT + TMZ, received Nimotuzumab + chemotherapy (CT). After 2012 May, Nimotuzumab + CT was given as primary treatment during and after RT. Nimotuzumab was administered 150mg/m2/ week for 6 weeks during RT and then bi-weekly with CT until PD. RESULTS: As of January 2014, Nimotuzumab was used in 15 children with DIPG, 7 with PD, 8 with primary diagnosis. Among PD patients, 4 had clinical improvement/stable disease for 18, 22 + , 8 and 6 months; one is alive with disease (AWD) at 42 months from initial diagnosis, 3 died of disease (DOD) at 24, 14 and 13 months from primary diagnosis. The other 3 had stable disease for 5, 4, and 1 month but progressed afterwards and all are DOD. In 8 patients who received Nimotuzumab + CT as primary treatment, all had clinical improvement after RT and Nimotuzumab as expected. Five had clinical improvement for a duration of 19, 14 and 13 + , 9+ and 2+ months, 3 are alive, 2 are DOD. The other 3 were stable for 5, 6, and 6 months; but all are DOD. Nimotuzumab + carboplatin/vinorelbine/TMZ was well tolerated with no major adverse effect. Only one patient had hypersensitivity to Carboplatinum after Nimotuzumab that started after a year of its use. The median survival is 14 months for PD patients. CONCLUSION: Nimotuzumab was well tolerated with RT and CT. Our results show that nimotuzumab containing regimens might be promising in patients with progressive disease. However, in primary DIPG patients, due to limited number and short follow-up, we cannot draw a conclusion on its efficacy. HG-007. SECOND IMMUNOTHERAPY IN PATIENTS WITH HIGH GRADE GLIOMA: IS IT USEFUL? Stefaan Van Gool and Steven De Vleeschouwer; KU Leuven, Leuven, Belgium Multimodal strategies are developed to treat patients with high-grade glioma (HGG). Active specific immunotherapy rapidly emerges as a new treatment modality. We provide immunotherapy for adults with primary diagnosis of GBM (HGG-2006) and for children/adults with relapsed HGG (HGG-IMMUNO-2003). In this retrospective analysis, we questioned whether second immunotherapy upon a new event was useful in patients who already got immunotherapy for their disease. 35 patients were treated with two vaccination treatments, 12 adults (26-69y) with primary diagnosis of GBM and 23 patients (7-55y) with relapsed HGG at time of first immunotherapy. At both times, leukapheresis was performed and DCs were loaded with lysate of the newly resected tumor tissue. HGG-2006 patients treated with two immunotherapies had a median OS of 41.8m versus 14.8m in HGG-2006 patients (n ¼ 68) treated with one immunotherapy program. The age distribution of the former was younger than of the latter group. Similarly, HGG-IMMUNO-2003 patients treated with two immunotherapies had a median OS of 32m versus 11m in HGG-IMMUNO-2003 patients (n ¼ 163) with one immunotherapy program. The age of the former patient group was younger, and their HGG-IMMUNO-RPA risk profile was better. The time interval between the first and second leukapheresis was longer in the HGG-2006 than the HGG-IMMUNO-2003 patients. All second immunotherapy approaches were similar. There were less injections during second immunotherapy as compared to the first immunotherapy. The number of injections was similar to the numbers given to HGG-IMMUNO-2003 patients who got first vaccination at time of relapse. The OS calculated from the second leukapheresis in re-vaccinated patients was similar as the OS observed in HGG-IMMUNO-2003 patients treated for the first time at relapse. Second immunotherapy was feasible, and no extra vaccine-related toxicities were observed. These retrospective results show that second immunotherapy is worth to be considered along the disease course of patients with HGG. HG-008. PEDIATRIC HIGH GRADE GLIOMA (HGG): A REVIEW OF DATA SET ON PRESENTATION, TREATMENT AND OUTCOME FROM KING FAISAL SPECIALIST HOSPITAL AND RESEARCH CENTER, RIYADH, SAUDI ARABIA Amani Al-Kofide, Essam Al-Shail, Yasser Khafaga, Hindi Al-Hindi, M. Dababo, Anwar Ul Haq, M Anas, Mary Grace Barria, Khawar Siddiqui, Maher Hassounah, and Mouhab Ayas; King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia Pediatric HGG is a heterogeneous group of tumors that represent a fraction of pediatric CNS tumors, but account for a significant amount of morbidity and mortality among pediatric brain tumor patients. From Jan 2005 to December 2012, a total of 17 pediatric patients with HGG were registered and treated at our institution with a median follow-up of 28.7 months. Mean age at diagnosis was 9.3 years (median:10.3,Min-Max:3-13.6) with 64.7% boys (11of17). 58.8%(10of17) were with Grade III with 70%(7) boys. Male preponderance was also seen in Grade IV as 57.1%. 88.2% presented with local disease while in one patient the disease had disseminated to spine and in other to brain. 13.3%(2) presented with neurofibromatosis and tuberous sclerosis. Headache and vomiting were the most frequently registered complaints at presentation (76.5%) followed by seizures and ataxia (17.6%). 47.1%(8of17) were treatment naive while one patient came to us after finishing his 1st line treatment. All of our patients underwent surgery as 1st line therapy combined with XRT (16) and chemotherapy (15). 82.4%(14of17) received adjuvant chemotherapy and only one (9.1%,1of11 evaluable) showed complete response. Of 12 evaluable at the end of the 1st line treatment, 1(8.3%) had CR, 6(50%) PD and 5(41.7%) with SD. Our 1st line Treatment failure rate (relapse/PD) was 82.4% (14of17). 2nd line treatment was offered to 42.8% (6of14); 2 chemotherapy, 5 surgery and 3 XRT. However, for all our patients the disease progressed. Of those evaluable (14of17), all had PD at the time of last evaluation. Our 3 years overall survival at the time of last update of the dataset in October 2013, is 0.602 + 0.129 (Median 37.3 + 12.7) with (0.762 + 0.148) for Grade III compared to (0.429 + 0.187) for Grade IV (p-value: 0.07). Current therapies for HGG are not yet sufficient. A need to find new modalities for treating HGG is highlighted. INTRODUCTION: We are the first to report the use of molecular drug imaging in children. We aim to determine the feasibility of the procedure. In addition we aim to analyze the therapeutic potential and toxic risks of bevacizumab in children with diffuse intrinsic pontine glioma (DIPG). METHODS: Three patients, aged 6, 7 and 17 years, diagnosed with DIPG were included after standard treatment with radiotherapy. Each patient received 0.1mg/kg bevacizumab, labeled with 0.9MBq/kg zirconium-89(89Zr). Whole body PET-CT scans were performed at 1, 72 and 144 hours post-injection. Researchers closely monitored the feasibility of the procedure. To determine the tumor uptake and biodistribution of 89Zr-bevacizumab, Standardized Uptake Values (SUVs) were calculated. RESULTS: No adverse events occurred during any of the study procedures and scans were of good quality. Tumor uptake was shown in two of three patients and was limited to the T1-MRI contrast-enhanced part of the tumor, with SUVs of 15.0 and 1.8 in the enhancing parts versus 0.4 and 0.6 in the non-enhancing parts, respectively. The highest 89Zr-bevacizumab uptake in organs was observed in the liver, followed by the kidneys, lungs, and bone marrow. CONCLUSIONS: Administration of 89Zr-bevacizumab followed by a repeated PET-scan procedure is feasible in children. The uptake of bevacizumab in organs was high in all three DIPG patients. Only two patients, however, showed significant uptake of bevacizumab in their tumor. Moreover, uptake was limited to the contrast-enhancing part, suggesting that blood-brain barrier disruption is required for bevacizumab accessibility. Molecular drug imaging shows great clinical relevance in DIPG, since it can not only indicate which patients are more likely to benefit from treatment, it can also quantify the drug uptake. With this first successful introduction of molecular drug imaging in children with cancer, we emphasize its importance in treatment decision-making. ACKNOWLEDGEMENT: Funded by the Semmy Foundation (Stichting Semmy). HG-010. ONCOLYTIC VIROTHERAPY FOR PAEDIATRIC HIGH GRADE GLIOMA; EVALUATION OF THE EFFECTS OF ONCOLYTIC VIRUS ON CELL VIABILITY, MIGRATION AND INVASION Julia Cockle1, Elizabeth Ilett1, Karen Scott1, Anke Bru¨ ning-Richardson1, Susan Picton2, Susan Short1, and Alan Melcher1; 1Leeds Institute of Cancer and Pathology, Leeds, UK; 2Yorkshire Regional Centre for Paediatric Oncology and Haematology, Leeds, UK Paediatric high grade gliomas (pHGGs) are devastating tumours with five year survival outcomes between 15-35%. Despite aggressive management, tumours inevitably recur due to their diffuse and invasive nature and novel therapeutics are needed. Oncolytic virotherapy, which uses viruses to selectively infect and destroy cancer cells as well as to stimulate an anti-tumour immune response, offers a novel treatment approach. Here we evaluate the in vitro cytotoxic effects of the oncolytic viruses, herpes simplex virus (HSV), measles, reovirus and vaccinia, on a panel of paediatric glioma cell lines. We also describe for the first time, the effects of oncolytic viruses on the migratory behaviour of pHGG cells. Cell viability was examined over 96 hours by MTT and FACS-based assays. Migratory behaviour was examined using both 2D scratch assays and 3D spheroid invasion assays in collagen. The sensitivity of pHGG lines to oncolytic viruses was demonstrated by a loss of cell viability over 96 hours. The cytotoxic effect was particularly marked for cell lines treated with vaccinia, while sensitivity to other viruses was more cell-line dependent. Analysis of migratory and invasion assays indicated that HSV at multiplicity of infection 10, resulted in a near total blockade of both migration at 24 hours and invasion at 72 hours, in all cell lines tested. Some reduction in migration/invasion was also seen following vaccinia virus treatment, but this was not as effective as HSV. Our results demonstrate that pHGG cell lines are sensitive to the cytolytic effect of a range of oncolytic viruses. Moreover, we have shown that oncolytic viruses, particularly HSV, can block the migration and invasion of glioma cells in vitro. We propose that oncolytic viruses may have therapeutic benefits for pHGG, not only as a cytotoxic and immuogenic therapy, but also as anti-invasive agents, improving outcome for this devastating disease. BACKGROUND: Gliomatosis cerebri (GC) is a rare, diffusely infiltrating glioma variant with little or no mass effect, predominantly WHO grade III/IV morphology and an almost uniformly fatal outcome. The aim of this retrospective analysis was to describe the clinical characteristics and outcome of children and adolescents with centrally reviewed GC registered in the HIT-HGG database between 2005 and 2013. PATIENTS AND METHODS: A total of nineteen patients (male, n ¼ 13; median age at diagnosis 11.8 years [range, 0.2-18.3]) were identified. The majority of patients were treated according to the prospective multicenter trial (HIT-HGG-2007; EudraCT 2007-000128-42) including local radiotherapy with simultaneous temozolomide (TMZ) followed by twelve 28-day TMZ cycles. Twelve patients underwent biopsy; five tumour debulking and two did not undergo surgery. Thirteen patients had WHO grade III, three patients WHO grade IV and 1 patient WHO grade II morphology (radiological diagnosis, n ¼ 2). Central pathological review was performed in 16 of 17 patients with histopathological diagnosis. Conventionally fractionated local radiotherapy was performed in sixteen patients (median tumor dose 54 Gy [range, 30.6-59.4 Gy]). All patients received temozolomide-based chemotherapy. RESULTS: Data on response evaluation were available in eighteen patients (partial remission, n ¼ 2, stable disease, n ¼ 13, progressive disease, n ¼ 3). With a median follow-up of 12 months (range, 2-35) fourteen patients died and five are alive with partial remission (n ¼ 1), stable (n ¼ 2) or progressive disease (n ¼ 2). Twelve patients developed local and four disseminated progression. Median time-to-progression was 11 months (range, 1-35). Survival rates at 1 and 2 years were 27.5 + 11.4% and 6.9 + 6.6% for event-free survival; 59.0 + 11.9% and 27.0 + 11.2% for overall survival, respectively. CONCLUSIONS: GC carries an extremely grave prognosis. Molecular biological studies are necessary to better characterize these rare neoplasms. HG-012. RADIATION THERAPY WITH TOPOTECAN FOLLOWED BY MAINTENANCE ANTI-ANGIOGENIC TRIPLE THERAPY FOR PEDIATRIC DIFFUSE INTRINSIC PONTINE GLIOMAS Mikaela Porkholm1, Leena Valanne2, Tuula Lo¨ nnqvist3, Stefan Holm4, Birgitta Lannering5, Pekka Riikonen6, Dorota Wojcik7, Astrid Sehested8, Niels Clausen9, Arja Harila-Saari10, Eckhard Schomerus11, Halldora K. Thorarinsdottir12, P a¨ivi La¨ hteenma¨ ki13, Mikko Arola14, Harald Thomassen15, Ulla M. Saarinen-Pihkala16, and Sanna-Maria Kivivuori17; 1Division of Hematology-Oncology and Stem Cell Transplantation, Children’s Hospital, Helsinki University Central Hospital, Helsinki, Finland; 2Helsinki Medical Imaging Center, Helsinki University Central Hospital, Helsinki, Finland; 3Division of Child Neurology, Helsinki University Central Hospital, Helsinki, Finland; 4Department of Pediatric Hematology-Oncology, Astrid Lindgren Children’s Hospital, Karolinska University Hospital, Stockholm, Sweden; 5Department of Pediatric Hematology-Oncology, The Queen Silvia Children’s Hospital, University of Gothenburg, Gothenburg, Sweden; 6Department of Pediatrics, Kuopio University Hospital, Kuopio, Finland; 7Haukeland University Hospital, Bergen, Norway; 8Department of Pediatric Hematology/Oncology, Rigshospitalet, Copenhagen, Denmark; 9Department of Pediatrics, Aarhus University Hospital, Aarhus, Denmark; 10Pediatric Oncology/Hematology Section, Department of Pediatrics, Oulu University Hospital, Oulu, Finland; 11Department of Pediatric Hematology and Oncology, H. C. Andersen Children’s Hospital, Odense University Hospital, Odense, Denmark; 12Pediatric Hematology-Oncology, The Children’s Hospital, Reykjavik, Iceland; 13Department of Pediatrics, Turku University Central Hospital, Turku, Finland; 14Department of Pediatrics, Tampere University Central Hospital, Tampere, Finland; 15Trondheim University Hospital, Trondheim, Norway; 16Division of Hematology-Oncology and Stem Cell Transplantation, Children’s Hospital, Helsinki University Central Hospital, Helsinki, Finland; 17Division of Hematology-Oncology and Stem Cell Transplantation, Children’s Hospital, Helsinki University Central Hospital, Helsinki, Finland Despite major treatment attempts, the prognosis for pediatric diffuse intrinsic pontine gliomas (DIPGs) remains dismal. Gliomas are highly vascularized tumors and very dependent on angiogenesis. Suppression of blood vessel growth by anti-angiogenic drugs might therefore be an effective therapy. From these findings, the Nordic Angiocomb protocol for treatment of pediatric DIPGs was created. The treatment starts with local radiotherapy combined with topotecan and continues with an anti-angiogenic oral triple medication consisting of thalidomide, etoposide and celecoxib. The drugs are administered metronomically, meaning long-lasting, low-dose therapy without prolonged drug-free periods. Forty-one patients were enrolled in this study. Eight patients treated with only radiotherapy were used as controls. Overall survival, radiological response, quality of life, requirement of corticosteroids and adverse effects were monitored. For study patients, the 12 months overall survival was 61% and the median overall survival 12 months (range 4 - 60 months). 17% of the patients were long-term survivors (overall survival ≥ 24 months). Four radiological complete responses were seen, of which two were transient. Radiologically, 56% of the tumors reduced in size and 78% in signal intensity. The quality of life was excellent for most Angiocomb patients, and they were able to visit school or daycare and walk for a significantly longer time compared to controls (Log Rank 0.036 and 0.008, respectively). There was a trend towards less consumption of corticosteroids among study patients. Adverse effects were generally minor, mostly neutropenia and infections. In conclusion, the Angiocomb protocol resulted in a noticeable share of long-term survivors and created four extra-ordinary complete radiological responses. The treatment was generally well tolerated with acceptable adverse effects and allowed a good quality of life of the patients. These results warrant more studies about antiangiogenic combination therapy for DIPGs in the future. brain slices and 3D whole brain cultures, thus allowing assessment of drug distribution upon release from polymer matrices. CONCLUSIONS: We have developed two PLGA-based systems to deliver multiple chemotherapy agents simultaneously. Modification of drug release kinetics was achieved through tailoring the matrix formulation. The spatio-temporal distribution of drugs delivered from PLGA/PEG matrices within ex vivo brain cultures was dependent upon drug chemistry and release mechanism. The use of MSI modalities to measure brain distribution ex vivo offers a laboratorybased screen for the selection of drug candidates with good distribution profiles and which are thus amenable for local delivery systems. HG-013. COMPREHENSIVE GENOMIC ANALYSIS OF DIFFUSE INTRINSIC PONTINE GLIOMAS IDENTIFIES THREE MOLECULAR SUBGROUPS AND A NOVEL CANCER DRIVER, ACVR1 Pawel Buczkowicz1, Christine Hoeman2, Patricia Rakopoulos1, Sanja Pajovic1, Andrew Morrison1, Eric Bouffet1, Ute Bartels1, Oren Becher2, and Cynthia Hawkins1; 1Hospital for Sick Children, Toronto, ON, Canada; 2Duke University Medical Center, Durham, NC, USA Diffuse intrinsic pontine glioma (DIPG) is a devastating paediatric brain tumour with no effective therapy and near 100% fatality. The failure of most therapies can be attributed to the delicate location of these tumours and choosing therapies based on assumptions that DIPGs are molecularly similar to adult disease. Recent studies have unravelled the unique genetic make-up of this brain cancer with nearly 80% harbouring a K27M-H3.3 or K27M-H3.1 mutation. However, DIPGs are still thought of as one disease with limited understanding of the genetic drivers of these tumours. Here we apply methylation profiling, whole genome sequencing, expression profiling, and copy number analysis to discover that DIPGs are three molecularly distinct diseases (H3-K27M, Silent, and MYCN) and uncover a mutations in a novel driver, ACVR1 in 20% of DIPGs. Mutations in ACVR1 were constitutively activating, leading to SMAD phosphorylation and increased expression of downstream activin signalling targets ID1 and ID2. The H3-K27M subgroup is highly K27M-H3 mutated and associated with additional hits including activating mutations in ACVR1, frequent RB1 and TP53 deletions, PVT-1/MYC or PDGFRA gains/amplifications, genomic instability and alternative lengthening of telomeres. The MYCN subgroup is not associated with histone mutations and is instead characterized by hypermethylation and chromothrypsis of chromosome 2p with high-level amplifications of MYCN and ID2. The Silent subgroup affects younger children, has genomes with minimal genomic instability and fewer mutations, overexpresses WNT pathway genes, as well as genes with known cancer association such as MDM2, MSMP and ADAM33. Our results show that this seemingly homogeneous entity in fact comprises three distinct diseases with different demographic and molecular features. This complexity needs to be considered when designing new therapeutic approaches in order to improve outcome for these children. HG-014. TAILORED RELEASE OF DUAL CHEMOTHERAPIES FROM A POLYMER PASTE DELIVERED INTERSTITIALLY Toby W.A. Gould, Cheryl V. Rahman, Stuart J. Smith, David A. Barrett, Kevin M. Shakesheff, Richard G. Grundy, and Ruman Rahman; University of Nottingham, Nottingham, UK INTRODUCTION: The neuro-surgical resection cavity offers an opportunity for adjuvant localized drug administration in the treatment of highgrade gliomas. Pasting a depot of chemotherapeutics that will set around the surgical cavity may effectively target residual disease cells whilst minimising systemic toxicity [PLoS One 8(10): e77435 (2013)]. Here we evaluate two moldable biodegradable polymer matrices of (i) poly(lactic-co-glycolic acid) (PLGA)/poly(ethylene glycol) (PEG) microparticles and (ii) PLGA/ PEG microparticles mixed with drug-loaded PLGA microspheres for sustained multiple drug release capability, tailored drug-release kinetics and modeling brain drug distribution using Mass Spectrometry Imaging (MSI). METHODS: PLGA/PEG microparticle matrices loaded with methotrexate (MTX), Etoposide (ETOP) and Temozolomide (TMZ) were prepared and dual drug release combinations measured using high performance liquid chromatography (HPLC). In vitro cytotoxicity of released drugs was assessed by exposure to tumor cells. Spatio-temporal distribution of drugs released from polymer matrices implanted into brain slices was measured using Liquid Extraction Surface Analysis Mass Spectrometry (LESA-MS). RESULTS: Dual drug combinations were simultaneously released from PLGA/PEG microparticles over several weeks in vitro. 100 mm diameter drug-loaded PLGA microspheres permitted encapsulation of drugs leading to reduced dual drug release rates and a prolonged period of drug release. LESA-MS discriminates drugs from native compounds prevalent in ex vivo HG-015. ROBOT-ASSISTED CONVECTION-ENHANCED DELIVERY OF CARBOPLATIN FOR PROGRESSIVE DIPG - A FEASIBILITY AND SAFETY REPORT OF 5 CASES Neil Barua1, David Cronin1, Steven Gill1, and Stephen Lowisl2; 1Frenchay Hospital, Bristol, UK; 2Bristol Childrens Hospital, Bristol, UK BACKGROUND: Convection-enhanced delivery (CED) describes a direct method of drug delivery to the brain through surgically implanted microcatheters. There has been significant research focus on applying CED to the treatment of DIPG, where the eloquent location of tumour usually precludes resective surgery. We report our initial experience of CED of carboplatin in 5 patients with DIPG. METHODS: 5 patients with clinical and/or radiological tumour progression after radiotherapy were selected. Up to 4 catheter trajectories per patient were planned using custom-made stereotactic planning software. Catheters were implanted using a robot-assisted guidetube directed implantation technique. Infusions of carboplatin (0.18 mg/mL) were performed on 2 consecutive days. Serial T2-weighted MR imaging was used as a proxy measure of drug distribution. RESULTS: Catheter implantations were well tolerated with satisfactory targeting accuracy. No patients suffered haemorrhagic complications. High volume (up to 9mL/day), high flow rate infusions (up to 10microL/min) were performed with real-time MRI guidance. During the infusions patients exhibited worsening of preexisting neurological dysfunction, which was transient in all except one patient who required a prolonged period of non- invasive respiratory support. One patient developed transient hydrocephalus which resolved without intervention. Analysis of T2-weighted imaging indicated drug distribution throughout the majority of the targeted tumour volume in 4 out of 5 patients. CONCLUSIONS: The robot-assisted catheter insertion technique was well-tolerated and achieved satisfactory targeting. No adverse events related to implantation of catheters were observed in this cohort. High volume, high flow rate infusions achieved drug distribution thoughout the majority of the targeted tumour volume in 4 out of 5. Infusions were associated with only transient worsening of neurological function in all but one patient, in whom CPAP was required in the post-infusion period. This initial experience represents a strong foundation for a clinical trial of CED of carboplatin for DIPG. HG-016. SPONTANEOUS MALIGNANT TRANSFORMATION OF A CERVICAL SPINAL CORD PILOCYTIC ASTROCYTOMA INTO GLIOBLASTOMA IN A CHILD Audrey Hochart1, Claude-Alain Maurage2, Nathalie Rocourt1, Matthieu Vinchon2, Olivier Kerdraon2, Fabienne Escande2, Jacques Grill3, Vale´rie Koubi Pick3, and Pierre Leblond1; 1Centre Oscar Lambret, Lille, France; 2CHRU, Lille, France; 3Institut Gustave Roussy, Villejuif, France Malignant transformation of pediatric low grade glioma is a very uncommon event. Radiotherapy seems to be the principal risk factor implicated. This has been widely described in adults with WHO grade 2 glioma and pediatric cases have also been reported. To our knowledge, spontaneous malignant transformation of WHO grade I to grade IV glioma has not yet been described. We report the case of a 7 year-old child, who presented with a pilocytic astrocytoma of spinal cord from C2 to C6 treated by surgical excision seemed complete only, then regulary followed with MRI. Cells were Olig2 positive, and CD 34 negative. 5% of nuclei were immunoreactive for Ki67, only rare nuclei exhibited a faint p53 labelleling. EGFR mutation was negative. Tumor was negative for the BRAF-KIAA fusion, and V600E BRAF mutation was not found. Ten years later, patient consulted for neck pain and neurological disorders. Spinal MRI revealed tumor "relapse" presenting as a lesion from C1 to T1. Surgical resection was partial and histopathological examination concluded to a WHO grade IV glioblastoma. Tumor cells were labelled by anti-Olig2 (70%) but not anti-IDH1 (R132H) antibodies, Ki67 index reached 30% of tumor cell nuclei, p53 immunoreactivity was strong, 20% of tumor cells were EGFR positive. CD34 remained negative. V600E BRAF mutation was not found and tumor presented a H3.3 K27M histone mutation. Treatment was completed by a spinal radiotherapy (40 Gy) followed by a tumor bed boost of 10 Gy, with concomitant temozolomide, and maintenance chemotherapy with temozolomide. Five months later, a tumor progression was treated with irinotecan and bevacizumab. Unfortunately, patient died of tumor progression 12 months after diagnosis. Malignant transformation can occur without radiotherapy in pediatric pilocytic astrocytoma, and we described the first case of spontaneous malignant transformation of a cervical spinal cord pilocytic astrocytoma into glioblastoma in a child. HG-017. LONG-TERM SURVIVAL (OVER 20 YEARS) AND PATHOLOGICALLY CONFIRMED COMPLETE RESPONSE IN PEDIATRIC ANAPLASTIC ASTROCYTOMA: A CASE REPORT Gregory Burzynski, Tomasz Janicki, Stanislaw Burzynski, and Ania Marszalek; Burzynski Clinic, Houston, Texas, USA Pediatric anaplastic astrocytoma (PAA) is a rare, malignant, brain tumor with annual incidence in the USA of approximately 330 cases. Despite surgical resection, radiation and chemotherapy, curative treatment of these tumors is not yet available. The authors describe the case of a successful treatment of PAA patient who survived for over 20 years. A 14-year-old female with newly-diagnosed AA presented with a 4.8 x 2.1 cm tumor involving the left temporal lobe, crossing the midline and compressing the pons. In November 1993 the patient underwent a craniotomy and a biopsy of the tumor, but did not have radiation or chemotherapy. Approximately one month later, she presented to Burzynski Clinic (BC) and was admitted for treatment with antineoplastons A10 and AS2-1 (ANP) based on protocol CAN-01. The treatment was given at the average dosage of 3.4 g/kg/d of A10 and 0.46 g/kg/d of AS2-1. Follow-up MRIs at 3, 8, 9, 14, and 19 months later did not show any significant changes; however, all symptoms, except seizures, disappeared by 13 months. MRI of the head at 32 months showed a complete response. Intravenous ANP continued until the 40th month and then the patient was taking maintenance treatment with A10 and AS2-1 capsules (0.14 g/kg/d each) until the 56th month. At that time she underwent resection of the scar tissue causing the seizures. The pathological examination did not demonstrate any presence of neoplastic process. The patient became asymptomatic and her follow-up MRIs between 6 and 15 years since the treatment start did not show tumor recurrence. She continues to live a normal life over 20 years later. This report indicates that it is possible to obtain long-term survival in PAA with a currently available investigational treatment. HG-018. TEMOZOLOMIDE, BEVACIZUMAB WITH OR WITHOUT IRINOTECAN IN PEDIATRIC BRAIN TUMORS- MD ANDERSON CANCER CENTER EXPERIENCE Nisha Ramani, Wafik Zaky, Geoffrey Kannan, Ajaykumar Morani, David Sandberg, Leena Ketonen, Osama Maher, Fernando Corrales-Medina, Heather Meador, and Soumen Khatua; MD Anderson Cancer Center, Houston, TX, USA OBJECTIVE: To evaluate the outcome of brain tumors in children treated with temozolomide, bevacizumab with or without irinotecan. BACKGROUND: Even with continuing advances in multimodality treatment strategies for high grade gliomas (HGG) and diffuse intrinsic pontine gliomas (DIPG), the prognosis remains dismal. METHODS: Retrospective chart review from January 2011 to December 2013 in children who received treatment with temozolomide, bevacizumab with or without irinotecan was performed. 7 patients were identified of which 2 had localized glioblastoma multiforme (GBM), 3 had features of gliomatosis cerebri (GC) and 2 patients had DIPG. They were treated with surgery and/or radiotherapy with temozolomide followed by maintenance chemotherapy with temozolomide, bevacizumab with or without irinotecan. RESULTS: The median progression free survival (PFS) was 41 weeks for localized GBM, 34 weeks for GC and 49 weeks for DIPG. A patient with GC and grade II pathology showing highgrade features on neuroimaging, continues to remain stable at 42 weeks. 1 patient with GC and GBM on pathology showed epidermal growth-factor receptor (EGFR) expression of the tumor by a combined genomic and protein transcription analysis (G/P approach). He progressed after 2 cycles of maintenance chemotherapy but is currently showing marked treatment response after 3 cycles of erlonitib targeting EGFR. CONCLUSIONS: Maintenance therapy with temozolomide, bevacizumab with or without irinotecan showed tolerable toxicity. Though modest improvement of PFS in DIPG was seen when compared to historical controls (median PFS of 24 weeks) such was not noted in the other tumors. Also preliminary data shows the feasibility and likely efficacy of the G/P approach currently pursued in our institution as is seen in our patient with EGFR expression. Larger prospective studies are warranted with this therapeutic regimen using temozolomide, bevacizumab and irinotecan in these tumors to evaluate clinical efficacy, and the validity of the G/P approach to profile targeted therapy. HG-019. IN VITRO AND IN VIVO TARGETING OF NFkB BY DEHYDROXYMETHYLEPOXYQUINOMICIN (DHMEQ) IMPAIRS GROWTH IN PEDIATRIC GLIOBLASTOMA Maria Brassesco1, Lara Delsin1, Gabriela Roberto1, Cleide Silva1, Lange Ana1, Eduardo Rego1, Carlos Scrideli1, Kazuo Umezawa2, and Luiz Tone1; 1University of Sao Paulo, Ribeirao Preto, Brazil; 2Aichi Medical University, Nagakute, Japan INTRODUCTION: Primary brain tumors are the leading cause of cancer death in children. Despite advances in neurosurgery and post-operative radiation therapy and/or multiagent chemotherapy, pediatric patients with glioblastoma (GBM) have a dismal outcome, with 5-year survival rates that have remained between 5 and 15% for the last four decades. Vast evidence has indicated that the nuclear factor NF-kB is constitutively activated in cancer, playing key roles in growth and survival. Recently, Dehydroxymethylepoxyquinomicin (DHMEQ) has shown to be a potent NF-kB inhibitor with anti-tumor properties in adult GBM, though there still no data on pediatric counterparts. METHODS: In the present study, the effects of DHMEQ on survival and its ability to surmount tumor´s invasive nature were explored in 2 pediatric GBM cell lines (SF188 and KNS-42) by means of qPCR, proliferation, clonogenicity, apoptosis and invasion assays on matrigel coated chambers. For in vivo testing 2 × 106 cells were implanted subcutaneously in the back of 6-weeks-old nude mice (BALB/c nu). After tumor establishment, the experimental group was treated intraperitoneally with 10 mg/kg DHMEQ 3 times per week during 3 weeks, and the control group was administered vehicle solutions. RESULTS: Treatment with DHMEQ substantially impaired cell growth in dose and timedependent manner when compared with control. Cell clonogenicity was also significantly diminished with increased apoptosis. Invasive potential was inhibited accordingly with lowered expression of invasion-related genes, such as MMP-12, MMP-14, MMP-23b, TIMP-2 and uPA. Moreover, subcutaneous tumors formed by SF188 or KNS-42 cells were reduced by 20% and 70% in size, respectively, by intraperitoneal administration of DHMEQ. CONCLUSION: Taken together, our results strengthen the potential usefulness of DHMEQ, in future therapeutic strategies for the treatment of pediatric GBM. HG-020. CLINICAL AND RADIOLOGIC RESPONSE FOLLOWING BEVACIZUMAB AND IRINOTECAN IN RECURRENT OR PROGRESSIVE DIFFUSE PONTINE GLIOMA IN CHILDREN Sung Jin Kim, Chae-Yong Kim, In-Ah Kim, Jung Ho Han, Byung-Se Choi, Hyo Seop Ahn, and Hyoung Soo Choi; Seoul National University Bundang Hospital, Seongnam, Republic of Korea BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) of childhood has a grave prognosis. Chemotherapy appears to be ineffective and the role of this treatment in the recurrent or progressive setting is not known. Bevacizumab and irinotecan have been reported to have shown radiographic response and improvement in progression-free survival for malignant supratentorial gliomas in adults. The aim of this study is to assess clinical and radiologic response in DIPG in children. PROCEDURE: We retrospectively reviewed the medical records of DIPG patients treated with bevacizumab and irinotecan at our institution. Four consecutive patients (1 boy, 3 girls, age 8 15yrs) with recurrent or progressive DIPG were enrolled. Bevacizumab (10 mg/kg) and irinotecan (125 mg/m2) were given every 2 weeks. None of the patients were on enzyme inducing antiepileptic medications that could have interfered with irinotecan metabolism. MRI FU was done every 2 4 cycles or at the time of symptom aggravation. RESULTS: Combination therapy with bevacizumab and irinotecan is well-tolerated without toxicity, such as hemorrhage, hypertension, or diarrhea. One patient with metastatic nodules showed clinical improvement and decreased size of pontine tumor. However, metastatic nodules in lateral ventricles were increased with new lesions. Other three patients were clinically stable for 1 6 months before symptom aggravated. MRI showed decreased T1 enhancing lesions but increased T2 nonenhancing infiltrative lesions. CONCLUSIONS: Bevacizumab and irinotecan suppressed enhancing tumor recurrence more effectively than nonenhancing, infiltrative tumor growth or metastatic lesion. It is necessary to elucidate the mechanism of this response pattern in recurrent or progressive DIPG. HG-021. MOLECULAR INSIGHTS INTO HISTONE H3.3 MUTATIONS IN DIPG AND PAEDIATRIC GLIOBLASTOMA Farhana Haque, Ruman Rahman, Robert Layfield, and Richard Grundy; University of Nottingham, Nottingham, UK Diffuse intrinsic pontine glioma (DIPG) is an aggressive malignant astrocytoma in children, with poor survival rate and few treatment options. Recently, K27M somatic mutations have been identified in histone H3.1 and H3.3 proteins in 80% of DIPGs and 20% of non-brain stem paediatric gliomas. In addition, 30% of paediatric high grade gliomas (pHGG) exhibit G34R or G34V H3.3 mutations. Several studies have highlighted the epigenetic changes associated with these mutations; however their role in tumourigenesis is still unknown. Here we describe a strategy for the generation of antibodies that can discriminate H3.3 K27M, G34R and G34V mutant proteins from wild-type counterparts. The antibodies were validated by western blotting against DIPG and pHGG histone 3 mutant cell lines, control cell lines (HeLa and wild type histone pHGG/DIPG) and recombinant H3.3 proteins. Their utility as tools to study the molecular consequences of single amino acid substitutions in H3.3, is considered. For example, in providing new insights into spatial/anatomical aspects of the mutations in DIPG, or for the rapid identification of H3.3 mutant vs. wild-type DIPG/ pHGG cases. These mutant-specific antibodies will also be utilized to identify novel downstream targets and pathways of the different mutants which, in turn may have a role in DIPG/pHGG genesis and progression. HG-022. REIRRADIATION OF DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) AT PROGRESSION AFTER FIRST LINE TREATMENT WITH NIMOTUZUMAB, VINORELBINE AND STANDARD RADIOTHERAPY Lorenza Gandola1, Emilia Pecori1, Veronica Biassoni1, Elisabetta Schiavello1, Chiara Chiruzzi1, Filippo Spreafico1, Piergiorgio Modena2, Ferdinand Bach3, Emanuele Pignoli1, and Maura Massimino1; 1Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy; 2Ospedale S. Anna, Como, Italy; 3Oncoscienze AG, Wedel, Germany A pilot phase 2 protocol was launched in our Institution in 2009 combining nimotuzumab with concomitant radiation and vinorelbine for children with a diagnosis of DIPG confirmed by central radiological review. A protocol amendment in July 2011 introduced re-irradiation at relapse. Vinorelbine was administered weekly, with nimotuzumab in the first 12 weeks of treatment; radiotherapy was delivered from weeks 3 to 9, for a total dose of 54 Gy conventionally fractionated. Then vinorelbine and nimotuzumab were given every other week until the tumor progressed or for up to two years. Re-irradiation consisted of 19.8 Gy in 11 fractions of 1.8 Gy. The re-irradiation target volume included all tumor grossly visible on MRI, embracing previously-irradiated areas and any new areas of progressive disease. A 3D conformal technique was always adopted, and beam geometry for re-irradiation was chosen so as to avoid the entrance beam paths of the firstline treatment wherever possible. 25 children (mean age 7.4 years) were enrolled from August 2009 (median follow-up 29 months). Eleven of 12 locally-relapsing patients were re-irradiated, all as out-patients, at a median of 9 months after first radiotherapy (range 5-19 months). In 10 children symptoms improved enough to enable steroid suspension while in 7 tumor shrinkage was documented. Survival after re-irradiation ranged from 6 weeks to 14 months (median 6 months). Median OS was 16 months for re-irradiated children, and 12 months for the 5 children with local relapse but not re-irradiated (P 0.03). One-year PFS and OS rates of the whole series were 30 + 10% and 76 + 9%, respectively; 2-year OS was 27 + 9%. In our experience, re-irradiation of DIPG children progressing locally, was feasible without unexpected side-effects or worsening of neurological symptoms. This approach points to a real chance of a longer life expectancy after relapse that warrants assessment in appropriate, larger clinical trials. AIM: Presentation of treatment results of children with DIPG treated in one center according to Polish Pediatric Neuro-Oncology Group (PPNOG) protocol. MATERIAL AND METHODS: Between 2005 – 2013 73 patients with DIPG diagnosed by MRI were treated. There were 47 girls and 27 boys, age range from 2yrs3mos to 17yrs11mos (mean-8yrs8mos, median-8yrs). The time from first symptoms of disease to diagnosis ranged from 5 to 24 weeks. Cranial nerve palsy (80%) and balance disorders (76%) were the most common symptoms. At diagnosis 33% of patients presented with classical brainstem syndrome.On MRI 35 tumors had no contrast enhancement, in 34 patients the tumor showed ring-like enhancement and in 4 enhancement was homogenous.All patients were treated with 2 courses of temozolomide and cisplatin followed by irradiation (54Gy). Post-irradiation chemotherapy was also administered. It’s type depended on tumor response to pre-irradiation chemotherapy. Patients with good response and stabilization received 6 courses of temozolomide/cisplatinum, if not other individualized protocols.Analysis included; tumor response to 2 courses of temozolomide/cisplatin and other chemotherapy protocols when given in case of progression. Good response was defined as CR, PR and minimal response. Treatment results are presented (1,2,5 and 7 yr PFS and OS) RESULTS: 8 patients showed good response, 37 stable disease and 28 progression. 19 patients completed the protocol, 3 are still treated. 51 patients had progression and were treated with second-line chemotherapy consisting of irinotecan/carboplatin(7pts), etoposide/dacarbazine (26 pts), doxorubicin(11pts), ‘Packer protocol’(7pts). Best responses were achieved with etoposide/dacarbazine (42,3% good responses, 30,8% stabilization,26,9% progression, with time to consecutive progression from 0 – 108 weeks median-18 weeks).PFS and OS were as follows; at 1 year - 21,1% and 56,3%, at 2 – years - 7% and 23,9%, at 5 – years 7% and 7,5%. Prolonged survival can be achieved with post-irradiation chemotherapy in children with DIPG. Oncogenic activation of receptor tyrosine kinases (RTKs) as well as deregulated downstream signalling through the RAS/RAF and PI3K pathways are hallmarks of glioblastoma (GBM) tumorigenesis, giving rise to the most frequent and aggressive malignant brain tumor in children and adults. Genomic alterations including somatic mutations, gene amplifications as well as structural aberrations affecting RTKs or downstream effectors are found in about 90% of all cases. In addition, genomic rearrangements leading to fusion genes have been described for some members within this signalling axis (e.g. EGFR, PDGFR and BRAF). Here we report on whole-genome sequencing of 45 GBMs and matched RNA sequencing (n ¼ 36) conducted in the context of the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. Interestingly, we identified fusion events disturbing the RTK-RAS-PI3K signalling pathway in a third of all cases. Several novel fusions were predicted to activate RTK-PI3K signalling, such as ETV6-NTRK2, FGFR2-CLIP2 and rearrangements of PIK3R1/2. A recurrent RTK fusion seen in 20% of H3F3A wildtype tumors also led to substantially increased MAPK signalling in vitro, which could be abolished by a specific pharmacologic inhibitor targeting this RTK. These results are now being further examined in a preclinical in vivo xenograft model. In conclusion, our results suggest that genomic rearrangements leading to fusion genes within the RTK-RAS-PI3K signalling axis are a common mechanism to activate this signalling pathway, which is known to play a major role in the development and progression of this deadly brain tumor. INTRODUCTION: Pediatric diffuse intrinsic pontine glioma (DIPG) is one of the most difficult cancers to treat. pLys27Met (K27M) driver mutation in the H3F3A gene of histone 3 variant 3 (H3.3) and HIST1H3B (H3.1) are correlated with a subgroup of DIPG patients. Other genomic aberrations of DIPGs include p53 mutations and amplification of signaling pathways including PDGFRa. We recently reported the involvement of Hedgehog (Hh) signaling pathway in a subset of DIPGs. Modulation of Hh and tyrosine kinase receptors may alter the self-renewal properties of cancer stem cells (CSC). NG2+ progenitor cells are proliferative precursor stem cells that contribute to the neoplastic transformation of glioma cells in adults. However, the role of NG2 that has not been studies in DIPG. METHODS: We examined NG2 expression using mRNA, proteomics and Western blot analyses. In vivo and in vitro assays were performed using human primary DIPG cells. RESULTS: We used brainstem specimens (14 DIPG and 10 adjacent normal) and validated significant NG2 expression in DIPGs [10 of 14 (71 %), (fold change ¼ 33, p , 0.05)]. NG2 expression was associated with histone 3K27M mutation [8 of 10 (80 %)]. Two mechanisms of NG2 regulation in DIPG were identified: i) H3K27me2 binds to NG2 promoter, and ii) microRNA miR129 negatively regulates NG2. miR129 is downregulated in 85.7% of DIPG tumors (FC ¼ -30.79, n ¼ 7 pairs). We inspected methylation patterns corresponding to miR129 promoter and found overall hypermethylation in 66.6 % of specimens (6 of 9). NG2 knockdown in vitro (shRNA, miR129, demethylating drugs) retard cellular migration. Orthotopic injection of NG2+ cells results in rapid tumor formation while NG2-KD cells fail to form tumors. NG2+ progenitor cells divide symmetrically in vitro leading to terminally undifferentiated daughter cells. CONCLUSION: We offer a potential model for the expansion of DIPG stem cells and their self-renewal properties. HG-026. CHARACTERISATION OF PSEUDOPROGRESSION IN CHILDREN AND ADOLESCENTS WITH HIGH GRADE GLIOMAS Fernando Carceller Lechon1, Lucy Fowkes1, Komel Khabra1, Lucas Moreno Martin-Retortillo1, Lynley Vanessa Marshall1, Sucheta Vaidya1, Dow-Mu Koh1, Martin O Leach2, Andrew DJ Pearson2, and Stergios Zacharoulis1; 1Royal Marsden NHS Foundation Trust, London, UK; 2Institute of Cancer Research, London, UK BACKGROUND AND OBJECTIVES: Pseudoprogression (PsP) in high grade gliomas (HGG) is a treatment-related local tissue phenomenon characterized by inflammation. PsP hinders response interpretation and limits the validity of progression free survival (PFS) as a major outcome endpoint. This study aimed to analyze the incidence and characteristics of PsP in childhood HGG, the impact of adjuvant temozolomide on PsP, and the impact of PsP on the outcome. METHODS: Demographic, clinical, and radiological data from patients aged 1-21 years with a histological diagnosis of non-brain stem HGG between 1995-2012 were retrospectively collected; assessments for evidence of radiological and/or clinical PsP were made and correlated with PFS and overall survival (OS). RESULTS: 53 patients were identified, male:female 37:16, median age 12yr (range, 1-20). Surgery: biopsy or subtotal resection, 38; gross-tumour resection, 15. Radiotherapy was completed in 46 cases (≤54 Gy, 21; .54 Gy, 25) and concomitant temozolomide was given in 28 cases (61%). Six patients presented radiological evidence of PsP at a median of 65.5 days (range, 20-203) after radiotherapy. Three had clinical symptoms. Radiographic resolution/stabilization was achieved at a median of 140.5 days (range, 65.0-189.0). The overall rate of PsP was 11.3% (95%CI, 4.9-22.9). Rate of PsP with radiotherapy-only, 15.8% (3/ 19); with chemoradiotherapy, 11.1% (3/27), p ¼ 0.680. 12-month PFS of patients without PsP, 38.3% (95%CI, 24.4-52.2); with PsP, 83.3% (95%CI, 53.5-100); hazard ratio 0.497 (95%CI, 0.195-1.226); p ¼ 0.143. 12-month OS of patients without PsP, 57.4% (95%CI, 43.3-71.5); with PsP, 100.0% (no 95%CI); HR 0.469 (95%CI, 0.166-1.324); p ¼ 0.153. CONCLUSIONS: The overall rate of PsP in children/adolescents with HGG was similar to that reported in adults. The combination of temozolomide with radiotherapy was not associated with a higher rate of PsP compared to radiotherapy alone. A trend towards better PFS and OS was shown for those HGG who experienced PsP. HG-027. CHARACTERISATION OF PSEUDOPROGRESSION IN CHILDREN AND ADOLESCENTS WITH DIFFUSE INTRINSIC PONTINE GLIOMAS Fernando Carceller Lechon1, Lucy Fowkes1, Komel Khabra1, Lucas Moreno Martin-Retortillo1, Lynley Vanessa Marshall1, Dominic Schrey1, Giuseppe Barone2, Sucheta Vaidya1, Dow-Mu Koh1, Andrew DJ Pearson2, and Stergios Zacharoulis1; 1Royal Marsden NHS Foundation Trust, London, UK; 2Institute of Cancer Research, London, UK BACKGROUND AND OBJECTIVES: Pseudoprogression (PsP) in diffuse intrinsic pontine gliomas (DIPG) is a treatment-related local tissue phenomenon characterized by inflammation. PsP hinders response interpretation, and limits the validity of progression free survival (PFS) as a valuable outcome endpoint. This study aimed to analyze the incidence and characteristics of PsP in childhood DIPG, the impact of adjuvant temozolomide on PsP, and the impact of PsP on the outcome. METHODS: Demographic, clinical, and radiological data from patients aged 1-21 years with a radiological diagnosis of DIPG between 1995-2012 were retrospectively collected; assessments for evidence of radiological and/or clinical PsP were made and correlated with PFS and overall survival (OS). RESULTS: 52 patients with DIPG were identified, male:female 28:24, median age 6yr (range 1-19yr). Radiotherapy was completed in 44 cases (dose not available, 5; ≤54 Gy, 32; .54 Gy, 7). Concurrent temozolomide was given in 9 cases (20%). Six patients presented evidence of PsP (4 radiological, 2 clinical) at a median of 46 days (range, (-1)-125) after radiotherapy. The mean until radiological resolution/stabilization, in three of them, was 80.0 days (range, 49.0-91.0). The overall rate of PsP was 11.5% (95%CI, 5.0-23.3). Rate of PsP with radiotherapy-only, 8.6% (3/35); with chemoradiotherapy, 33% (3/9); p ¼ 0.089. 12-month PFS of patients without PsP 21.7% (95%CI, 9.7-33.7); with PsP, 50.0% (10.0-90.0); hazard ratio 0.426 (0.168-1.082); p ¼ 0.073. 12-month OS of patients without PsP 30.4% (95%CI, 17.1-43.7); with PsP, 83.3% (53.5-100); HR 0.368 (0.131-1.031); p ¼ 0.057. CONCLUSIONS: The overall rate of PsP in children and adolescents with DIPG was comparable to that reported in adults with HGG. Interestingly, there was a trend towards a higher rate of PsP when radiotherapy was combined with temozolomide compared to radiotherapy alone, and better PFS and OS for those patients with PsP. HG-028. INTRATUMOR HETEROGENEITY IN PEDIATRIC DIFFUSE INTRINSIC PONTINE GLIOMA Eshini Panditharatna1, Mojca Stampar1, Alan Siu2, Heather Gordish-Dressman1, Joseph Devaney1, Madhuri Kambhampati1, Eugene I. Hwang1, Roger J. Packer1, and Javad Nazarian1; 1Children’s National Medical Center, Washington, DC, USA; 2George Washington University, Washington, DC, USA INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) is an understudied pediatric brain cancer. The validity of at-diagnosis tumor biopsy is currently under debate with no published studies investigating tumor heterogeneity within the same patient. METHODS: Two brains obtained at postmortem from patients with DIPG were studied. Frozen and formalin fixed paraffin embedded (FFPE) specimens were processed (H&E, GFAP, Ki67) and examined by a neuropathologist. Histologic analysis revealed a highgrade infiltrating astrocytoma with variable cellularity and mitotic activity. In each tumor, the cellularity was the highest in basis pontis with small patches of necrosis, a characteristic of glioblastoma. Core punches (n ¼ 24) representing different geographical tumor locations and tumor grades were obtained (brainstem and metastatic lateral ventricle). Each core punch was processed for mRNA (NanoString) and methylation (Illumina 450) profiling. RESULTS: We show that mRNA profiles (unsupervised clustering) correspond to tumor grade across the brainstem (n ¼ 12 per case). The expression pattern of seven genes is sufficient for accurate molecular grading (WHO II, III, IV) of tissue specimens when compared with neuropathological assessment. To investigate molecular disparities within each grade, specimens were reanalyzed based on geographic location and pathology. Each tumor grade was then designated as classic or non-classic (not fitting all criteria for a specific grade). We show detectable differential mRNA expression patterns within each grade, helping to discriminate between classic and non-classic specimens. Methylation analysis (n ¼ 24, 12/case) showed clustering of specimens based on their tumor grade. However, non-classic specimens clustered with the next higher grade (e.g. WHO III non classic with classic WHO IV). The largest molecular changes were observed in tumors from distinct geographic locations (brainstem versus lateral ventricles). CONCLUSIONS: Significant intratumoral heterogeneity may affect the accuracy of targeted diagnosis in small biopsy specimens. A better understanding of these differences will help in improving therapy and devising accurate molecular-based tumor grading. HG-029. ABCG2 AND ABCB1 PROMOTE BRAINSTEM GLIOMAGENESIS AND LIMIT THE EFFICACY OF DASATINIB IN A DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) MOUSE MODEL Alexander H. Chung1, Rajendar K. Mittapalli2, William F. Elmquist2, and Oren J. Becher1; 1Duke University Medical Center, Durham, NC, USA; 2University of Minnesota College of Pharmacy, Minneapolis, MN, USA BACKGROUND: DIPG is an incurable tumor that arises in the brainstem of children. Our laboratory generates murine DIPGs by expressing PDGF-B and deleting p53 using the RCAS/tv-a system. Dasatinib, an inhibitor of PDGFR-A activation, has been evaluated in clinical trials for children with DIPG. As dasatinib is a substrate of the ABC transporters ABCG2 and ABCB1, we hypothesized that these transporters may limit the efficacy of dasatinib in DIPG. METHOD: We generated murine DIPGs in WT (ABCG2 + / + ; ABCB1a + / + ; ABCB1b + /+) and KO (ABCG2-/-; ABCB1a-/-; ABCB1b-/-) mice. Next, we investigated whether dasatinib is efficacious in DIPG-bearing KO mice. Additionally, we treated DIPG-bearing WT mice with dasatinib alone and dasatinib in combination with elacridar (a dual ABCG2/ABCB1 inhibitor). We also measured the distribution of dasatinib in the brains of all tumor cohorts. Lastly, we evaluated the blood-brain-barrier (BBB) of our DIPG mouse model with Texas Red Dextran (TRD). RESULTS: Surprisingly, we observed a significant survival benefit in untreated KO compared to untreated WT mice with DIPG (median survival 43 days vs. 34 days, p ¼ 0.02). We observed that dasatinib almost doubled the survival in the KO model: 44 days vs. 80 days (p ¼ 0.0004). Co-treatment with elacridar significantly increased the efficacy of dasatinib in the WT model: 42 days vs. 59 days (p , 0.0001). Pharmacokinetic analysis demonstrated that genetic ablation or pharmacological inhibition of ABC transporters significantly increased dasatinib delivery into normal brain but not into tumor. TRD studies demonstrated a disrupted BBB in both DIPG models. CONCLUSIONS: (1) ABC transporters promote brainstem gliomagenesis. (2) ABC transporters regulate dasatinib delivery into normal brain but not into the tumor as BBB is disrupted in our DIPG mouse model. (3) ABC transporter activity limits the efficacy of dasatinib in vivo. Our results suggest that ABCG2 and ABCB1 inhibitors may have a therapeutic role in the treatment of DIPG. HG-030. THE TYPE OF HISTONE H3 VARIANT WITH LYS27MET (K27M) CHANGE IS A PROGNOSTIC MARKER ASSOCIATED WITH TWO DIFFERENT PHENOTYPES OF DIFFUSE INTRINSIC PONTINE GLIOMAS (DIPG) David Castel1, Marie-Anne Debily2, Cathy Philippe1, Nathalene Truffaux1, Katy Taylor3, Raphae¨l Calmon4, Nathalie Boddaert4, Ludivine Le Dret1, Patrick Saulnier5, Ludovic Lacroix5, Alan Mackay3, Chris Jones3, Stephanie Puget6, Christian Sainte-Rose6, Thomas Blauwblomme6, Pascale Varlet7, and Jacques Grill8; 1UMR CNRS 8203 - Institut Gustave Roussy, Villejuif, France; 2Universite d’Evry Val d’Essone, Evry, France; 3Institute of Cancer Research, Sutton, UK; 4INSERM U1000 - Hopital Necker, Paris, France; 5Translational Research Lab - Institut Gustave Roussy, Villejuif, France; 6Neurosurgery Department - Hopital Necker, Paris, France; 7Neuropathology Departement - Hopital Sainte-Anne, Paris, France; 8Department of Pediatrics - Institut Gustave Roussy, Villejuif, France BACKGROUND: Histone H3 variant K27M mutations are the most frequent alterations observed in DIPG. They impair trimethylation of the K27 repressive histone mark by interfering with the PRC2 complex together with a general hypomethylation of the DNA. The mutations are twice more frequent in the histone H3F3A gene than in the HIST1H3B gene. Since these two proteins have different functions on the chromatin organization, we studied if they were associated with different biological characteristics. PATIENTS AND METHODS: Fifty-one DIPG were characterized clinically, radiologicaly and biologically (mutations, gene expression, CGHarray) along with 90 non-brainstem high-grade gliomas. Tumor material was obtained from stereotactic biopsies performed at diagnosis. RESULTS: All DIPG showed a loss of the H3K27me3 mark by immunohistochemistry. HIST1H3B K27M mutations were exclusively seen in DIPG while H3F3A K27M ones were distributed in all the midline structures of the central nervous system. Median overall survival of H3.1 mutated tumors was longer (17 vs 11 months, p ¼ 0.006). HIST1H3B mutations were associated with ACVR1 mutations while H3F3A mutations were associated with TP53 mutations and PDGFRA amplifications. DIPG with H3.1 mutants showed more frequently large pseudo-necrotic contrast enhancing lesions while DIPG with H3.3 mutants showed a pattern of infiltration respecting the brainstem fiber tracts. Gene expression profiling of the H3.1 mutant tumors in contrast to the H3.3 ones showed the overexpression of hypoxia/angiogenesis genes as well as an astrocytic phenotype. CONCLUSION: Despite similar consequences on the PRC2 complex, HIST1H3B and H3F3A are associated with two different biological phenotypes. In addition to their prognostic impact, they may suggest distinct therapeutic approaches. HG-031. THE PITFALLS OF MSI-H DETECTION IN BRAIN CANCERS DIAGNOSED IN THE CMMR-D (CONSTITUTIONAL MISMATCH REPAIR DEFICIENCY) SYNDROME Natacha Entz-Werle´1, Christine Maugard1, Gaelle Bougeard3, Aurelia Nguyen1, Marie Pierre Chenard1, Anne Schneider1, and Marie Pierre Gaub1; 1CHU Hautepierre, Strasbourg, France; 2EA3430 - Universite´ de Strasbourg, Strasbourg, France; 3Inserm U614, Rouen, France CMMR-D syndrome is one of the inherited cancer predisposition syndromes. More than two-third of the patients belonging to a CMMR-D family are diagnosed in the first decade and more than 50% of them will present a high grade glioma as a first cancer. Few medulloblastomas are also described. This familial syndrome is due to bi-allelic germline mutations in the genes involved in the MisMatch Repair (MMR) pathway (MLH1, MSH2, MSH6 or PMS2). The lack of these proteins is giving frequent insertion-deletion mutations, especially in short repetitive DNA sequences called microsatellites.Therefore, to detect this MMR deficiency in a tumor, a standardized allelotyping method targeting 5 monocleotide and dinuclotide repeats is used to reveal a MSI-H (microsatellite instability high) status. This strategy seems to be accurate in the "standard" and heterozygote forms (e.g. Lynch Syndrome), whereas it has been published as poorly sensitive and specific for CMMR-D cancers. In our center, a CMMR-D family was diagnosed recently and carrier of a homozygous MLH1 mutation. As previously described, the standard allelotyping pentaplex did not seem to be contributive in the multiple kids presenting a brain cancers (one medulloblastoma, one high grade glioma and one gliomatosis cerebri). Nevertheless, we extended the analyses to 10 other dinucleotide repeats (TP53, D8S264, D5S2013, D20S255, D9S171, D13S173, D2S138, D10S191, D4S394, D1S197) to understand the mechanisms of this MMR deficiency. In the brain tumors, there were more than 7 MSI out those 10 repeats confirming the MSI-H status and defining a new standard MSI multiplex. Regarding closely to the mononucleotide repeats (BAT25, BAT26, NR21, NR22 and NR24) comparatively to the paired blood DNA, there were slight differences such as one base pair gap or different peak heights in the amplified alleles. This new approach has to be standardized and confirmed in the larger cohort of the French CMMR-D families. HG-032. TARGETING MITOCHONDRIA AND CELL METABOLISM AS A NOVEL THERAPEUTIC APPROACH IN THE TREATMENT OF DIFFUSE INTRINSIC PONTINE GLIOMA Maria Tsoli1, Anne Vanniasinghe1, Peter Luk2, Pierre Dilda2, Michelle Haber1, Phil Hogg2, and David Ziegler1; 1Children’s Cancer Institute Australia, Sydney, NSW, Australia; 2Lowy Cancer Research Institute, Sydney, NSW, Australia BACKGROUND: New treatments are urgently needed for Diffuse Intrinsic Pontine Gliomas (DIPG). Mitochondria are attractive cancer drug targets as they play a fundamental role in energy production as well as cell growth and apoptosis. One of the key proteins responsible for maintaining mitochondrial function is adenine nucleotide translocase (ANT). PENAO is a novel drug in Phase I trial that potently targets mitochondrial function via inhibition of ANT. We assessed the therapeutic potential of targeting mitochondrial metabolism in DIPG using PENAO as a single agent and with rational combination therapies. METHODS: The pro-apoptotic effect of PENAO in DIPG neurospheres was measured via Annexin/PI staining and caspase 3/7 activity assays. Cell proliferation was measured with an alamar-blue assay. Flow cytometric analysis of cytosolic superoxide production (dehydroethidium stain) and membrane depolarization (JC-1 stain) were performed for loss of mitochondrial integrity. Other agents known to target the metabolic pathways were assessed by alamar-blue assays and Annexin/PI staining for potential synergy with PENAO, including anti-metabolites, Hexokinase II (HKII) inhibitors, Fatty acid synthase (FAS) inhibitors, and mTOR inhibitors. RESULTS: Treatment of DIPG neurospheres with PENAO profoundly inhibited cellular proliferation with a half maximal inhibitory concentrations (IC50) range of 0.5-3 mM. PENAO caused loss of mitochondrial activity with significantly increased cytosolic levels of superoxide and depolarised mitochondria, profound induction of caspase 3/7 activity and subsequent apoptotic cell death. The mTOR inhibitor rapamycin, and the FAS inhibitor cerulenin were the two agents that showed most striking synergy with PENAO. While neither agent had significant single agent activity, they both enhanced the pro-apoptotic and anti-proliferative effect of PENAO at low micromolar concentrations with minimal effect on normal human brain astrocytes or MRC5 lung cells. Moreover, cerulenin alone enhanced the efficacy of radiation therapy. CONCLUSION: Targeting mitochondrial and cellular metabolism via multiple pathways is a promising novel therapeutic strategy for DIPG. HG-033. TARGETED INHIBITION OF THE FACT (FACILITATES CHROMATIN TRANSCRIPTION) COMPLEX IS A NOVEL AND EFFECTIVE THERAPEUTIC APPROACH IN DIPG Sandy Simon1, Maria Tsoli1, Anne Vanniasinghe1, Michelle Monje2, Katerina Gurova3, Andrei Gudkov3, Michelle Haber1, and David Ziegler1; 1Children’s Cancer Institute Australia, Sydney, NSW, Australia; 2Stanford Cancer Institute, Stanford, CA, USA; 3Roswell Park Cancer Institute, Buffalo, NY, USA BACKGROUND: Novel therapies are urgently needed for Diffuse intrinsic pontine gliomas (DIPG). We performed a high-throughput drug screen of over 3,500 FDA-approved clinically active compounds against DIPG neurospheres. Two antimalarial drugs quinacrine and mefloquine were amongst the 2% of compounds that demonstrated significant cytotoxic activity. Their anti-tumor activity was found to be related to the activation of the tumor suppressor protein p53 and induction of apoptosis. Curaxins are a new class of anti-cancer drug which are structurally related to quinacrine and activate p53 while suppressing NF-kB and HSF1. They are direct inhibitors of the chromatin remodeling complex FACT (Facilitates Chromatin Transcription). We have examined FACT expression in DIPG, as well as efficacy of the curaxin clinical candidate, CBL0137, as a novel DIPG therapy. METHODS: FACT expression was measured by PCR and Western blot in DIPG neurospheres compared with normal brainstem astrocytes, and in primary tumor specimens compared with normal brainstem or cerebellum. The anti-tumor effects of CBL0137 alone or in combination with radiotherapy were measured in DIPG neurospheres using alamar-blue assays, clonogenic assays, Annexin-V staining and caspase 3/7 activity assays. We used western blot to measure the effect of CBL0137 on p53 expression, NFkB activity and epigenetic methylation status. RESULTS: FACT expression was significantly increased in both DIPG neurosphere cultures and primary DIPG tumors. CBL0137 inhibited the proliferation of DIPG neurospheres in short term culture assays and clonogenic assays at nanomolar concentrations, and enhanced the activity of radiotherapy. Treatment with CBL0137 led to increased p53 levels and suppression of NFkB-mediated transcription, activating caspase 3/7 and inducing apoptosis. Initial results suggest that treatment with CBL0137 increases H3K27me3 status. CONCLUSIONS: Our findings indicate that CBL0137 represents a novel, potentially effective therapy for children with DIPG. A Phase I COG trial of CBL0137 is planned that will include a cohort of DIPG patients. High-grade gliomas (HGGs) encompass the WHO Grade III and Grade IV gliomas (glioblastoma multiforme, GBM). To evaluate outcome of children with HGG in our institution we analyzed 37 patients diagnosed with primary supra or infratentorial HGG (excluding DIPG) within years 2000-2012. Patients cohort consisted of 25 boys and 12 girls (M:F ¼ 2,1:1) with median of age 10.8 years (0,5 to 19.6 years). Only two patients were younger than 3 years. 17 patients were diagnosed with grade III HGG, 15 patients with grade IV and 5 patients with gliomatosis cerebri or multifocal HGG. Most of HGGs were localized in hemisphere (n ¼ 21), less frequently in thalamus or basal ganglia (n ¼ 5) and infratentorialy (cerebellum, brain stem) (n ¼ 6). Leptomeningeal spread in spine was detected in 3 patients. Hereditary cancer predisposition syndrome was known in 2 patients (Li-Fraumeni, NF-1). Initial neurosurgery achieved gross total resection (GTR) in 15 pts, subtotal resection (STR) in 9 pts and biopsy or partial resection in 13 pts. Therapy consisted of irradiation plus chemotherapy; 2xtemozolomide (TMZ), 2xTMZ + CCNU, 2xTMZ + bevacizumab, 5xCCNU + CDDP + VCR. Patients under 3 years were treated with chemotherapy alone. Probability of 5 years overall survival (OS) in all our patients was 29%, with long-term survivors over 10 years. All patients with gliomatosis or multifocal HGG died with median survival time 0.9 years (0.3 - 2.3 y) as well as patients with leptomenigeal disease. Resection over 90% of tumor volume (STR, GTR) was accompanied with significantly better prognosis compared to biopsied and partially resected HGGs (40% vs 8%, p ¼ 0.01). No significant difference in OS was observed between Grade III and Grade IV HGGs. Our results support the importance of complete resection in HGG patients. Long-term survivorship in pediatric HGG patients is achievable. Supported by MHCZ-DRO, University Hospital Motol, Prague,Czech Republic 00064203. HG-035. NOVEL ORTHOTOPIC PAEDIATRIC GLIOBLASTOMA XENOGRAFTS EVALUATED WITH MAGNETIC RESONANCE IMAGING MIMIC HUMAN DISEASE Jessica Boult1, Maria Vinci1, Katy Taylor1, Lara Perryman1, Gary Box1, Alexa Jury1, Sergey Popov1, Wendy Ingram2, Michelle Monje3, Suzanne Eccles1, Chris Jones1, and Simon Robinson1; 1The Institute of Cancer Research, London, UK; 2Queensland Children’s Tumour Bank, Brisbane, QLD, Australia; 3Stanford University, Stanford, CA, USA Paediatric glioblastoma (pGBM) has distinct underlying biology compared with adult disease, and genetic subtypes differ with anatomical location. Tumours arise either supratentorially, in the pons (diffuse intrinsic pontine glioma, DIPG) or in the thalamus. It is imperative to accurately model these tumours in vivo for the evaluation of emerging targeted therapeutics. pGBMs grow in a partially infiltrative manner, and co-option of host vasculature leaves the blood brain barrier (BBB) intact. Evaluation of novel pGBM models in situ is vital and requires sensitive functional imaging. Established paediatric GBM cell lines SF188 (H3F3A wild-type) and KNS42 (H3F3A G34V) produce well-defined tumours, with minimal local invasion, when implanted supratentorially in mice. Tumours are clearly detectable using anatomical T2-weighted MRI and enhance following Gd-DTPA contrast agent administration, indicative of impaired BBB integrity. Fluid-attenuated inversion recovery (FLAIR) hyperintensity is used clinically to identify infiltrative tumour. D212-MG cells derived from a supratentorial giant-cell GBM, serially propagated subcutaneously before intracranial inoculation, demonstrate FLAIR hyperintensity principally at the rim. Conversely, Gd-DTPA enhancement was prevalent at the tumour core. H&E staining confirmed the presence of masses with focal invasion at the periphery. SU-DIPG-VI cells (H3F3A K27M), derived from a DIPG autopsy specimen, grew diffusely in the mouse pons with evidence of oedema, detectable by T2-weighted MRI hyperintensity and FLAIR hypointensity. Diffusely hyperintense T2-weighted lesions with indistinct boundaries and limited Gd-DTPA extravasation, suggestive of a largely intact BBB, were observed in the diffuse infiltrative tumours produced when QCTB-R059 primary thalamic GBM cells (H3F3A K27M) were implanted in the thalamus. In vivo propagation of primary and serially xenografted pGBM cells results in tumours that are more representative of clinical disease than long-established cell lines. There is substantial potential for the use of these models to evaluate preclinical efficacy of novel therapeutics in a range of anatomical and biological subgroups of pGBM. HG-036. OUR EXPERIENCE WITH TWO DIFFERENT CHEMOTHERAPY REGIMEN IN PATIENTS WITH DIFFUSE INFILTRATING PONTINE GLIOMA Suna Emir1, Haci Ahmet Demir1, Cengiz Bayram1, Faik C¸ etindag2, Gu¨ lsah Bayram Kabac¸am1, and Ali Fettah1; 1Ankara Children’s Hematology Oncology Training and Research Hospital, Ankara, Turkey; 2Atat u¨rk Training and Research Hospital, Ankara, Turkey Central nervous system tumors (CNS) are the second most common malignancy in children after leukemias. Brain stem gliomas comprise 10%-15% of all pediatric CNS tumors and most commonly arise in the pons. Diffuse infiltrating pontine gliomas (DIPG) comprise about 80 % of all brain stem gliomas. Despite advances in the management of CNS tumors, DIPGs have still an almost uniformly poor prognosis. RESULTS: We retrospectively analysed the clinical data of children diagnosed with DIPG at our center between 2010 and 2013. Six children with a median age of seven years were included. Male to female ratio was 3/3. Presenting symptoms were the presence of multiple cranial nerve deficits and cerebellar ataxia. The diagnosis of DIPG was based on typical radiologic and clinical findings. All six patients with a defined clinicoradiologic diagnosis of DIPG received local radiotherapy (RT) with 5500 cGy to 6000 cGy. Five patients had concomitant temozolomide (75 mg//m2/day) with RT. Two weeks after RT-temozolomide treatment, chemotherapy was given to all patients. Three patients received CT regimen consisting of temozolomide ( 200 mg/m2 1-5 day), 13 cis Retinoic acid (100 mg/m2/d, 1-21 days). Nimotuzumab ( 150 mg/m2) plus vinorelbine (25 mg/m2) combination was used in three patients weekly. There were radiation induced necrosis in two patients and intracranial bleeding associated with RT in one patient. Most common adverse events were mucositis grade 1-2 and hepatoxicity in patients receiving nimotuzumab + vinorelbine. Myelosupression were seen in patients receiving temozolomide. All patients in our group transiently respond to the treatment. But, five of them ultimately succumb to disease progression within 18 months of diagnosis. Median survival was 15 months (range 8-18 months) in our group. CONCLUSION: Although use of new agents such as nimotuzumab, temozolomide may slightly cause a prolonged survival, DIPGs continue having a dismal prognosis, needing new approaches. HG-037. IMAGING THE TENSIONAL HOMEOSTASIS OF ORTHOTOPIC PAEDIATRIC GLIOMA XENOGRAFTS IN VIVO WITH MAGNETIC RESONANCE ELASTOGRAPHY Jessica Boult1, Jin Li1, Maria Vinci1, Alexa Jury1, Sergey Popov1, Yann Jamin1, Craig Cummings1, Suzanne Eccles1, Jeffrey Bamber1, Ralph Sinkus2, Chris Jones1, and Simon Robinson1; 1The Institute of Cancer Research, London, UK; 2King’s College London, St. Thomas’ Hospital, London, UK The local microenvironment of tumour cells plays an important role in cancer progression. Generally, elevated tissue stiffness is associated with tumour malignancy and invasion. Magnetic resonance elastography (MRE) is an emerging imaging modality used to directly visualise and quantify (kPa) tissue mechanical properties in vivo following application of a vibrational mechanical stress. Paediatric glioblastomas (pGBM) grow in a partially infiltrative manner, with boundaries that are often difficult to define by conventional MRI. MRE was utilised to assess the viscoelastic properties of D212-MG orthotopic pGBM xenografts. D212-MG cells were derived from a supratentorial giant-cell GBM, serially propagated subcutaneously before intracranial inoculation. MRI and MRE were performed using a 7T Bruker Micro-imaging system; tumours were identified on T2-weighted images and 3D steady-state MRE data were acquired using a vibration frequency of 1000Hz. Maps of Gd (elasticity) and Gl (viscosity) were reconstructed with 300 mm isotropic pixels. Tumour extent and growth characteristics were assessed on H&E stained sections. D212-MG tumours were less elastic (lower Gd), viscous (lower Gl), and therefore softer than the surrounding brain tissue. Tumours were most clearly distinguishable in maps of Gl. D212-MG tumours were significantly less elastic and viscous than wellcircumscribed U87-MG human adult GBM tumours in the mouse brain. However, D212-MG did not significantly differ from RG2 rat glioma xenografts, which also grew as well-defined masses with focally invasive growth at the periphery. Histopathological correlates of vascularity and extracellular matrix composition are being assessed to better understand the basis for the observed dissimilarities in the viscoelasticitic properties between intracranial tumours and the surrounding brain tissue, and the differences between the tumour models studied. We will extend this work to diffuse infiltrative pGBM models and identify whether local viscoelastic properties as assessed by MRE may provide accurate, non-invasive biomarkers to assess pGBM growth, delineate tumour margins, and monitor therapeutic response. Glioblastoma in children is driven in part by specific mutations in the gene encoding the histone H3.3 variant, H3F3A. We had previously determined that the G34R/V mutation, present in around 20-25% of cerebral hemispheric tumours, conferred differential genomic binding of trimethylated H3K36 resulting in the aberrant expression of numerous developmental transcription factors, stem cell regulators and the oncogene MYCN. Genomic amplification of MYC/MYCN is observed in 7% and 3% of paediatric GBM respectively, and is mutually exclusive with H3F3A G34R/V, thus representing an alternative mechanism of protein upregulation in these tumours. We sought to determine whether targeting MYC/MYCN stabilization through inhibition of checkpoint kinase 1 (CHK1) may be a useful novel therapeutic strategy in MYC/MYCN overexpressing tumours. We screened a series of paediatric GBM models including the G34V mutant KNS42, MYC-amplified SF188 as well as a number of primary patient-derived cultures, with a panel of CHK1 inhibitors including AZD7762, LY2603618 and the oral inhibitor CCT244747. Sensitivity was associated with MYC/MYCN protein levels, with MYC-amplified SF188 up to 10-fold more sensitive than MYCN-overexpressing KNS42 cells. We observed a dose-dependent degradation of MYC/MYCN protein in response to CHK1 inhibition in all instances, with GI50 values corresponding with significant protein degradation ( 40%), rather than inhibition of auto-phosphorylation of CHK1 on residue Serine296, which occurred at significantly lower doses. Combining CHK1 inhibitors with temozolomide resulted in significant synergy in cells resistant to the alkylating agent due to both MGMT-dependent and –independent mechanisms, with combination indices of 0.3-0.7. These data identify approximately a third of cortical paediatric GBM to be amenable to CHK1 inhibition by virtue of MYC/MYCN dependency, achieved either by gene amplification or histone mutation. In addition, CHK1 inhibition may play a role in chemosensitization of paediatric glioblastoma cells to temozolomide regardless of mechanism of alkylating agent resistance. There is an urgent need for novel therapeutic strategies for children with diffuse intrinsic pontine glioma (DIPG), as outcome remains dismal with a median overall survival of 9-12 months. We have recently identified recurrent mutations in the gene ACVR1, which encodes the receptor serine/threonine kinase ALK2, in 25% of DIPG patients. These somatic mutations are located at the same residues (R206H, Q207E, R258G, G328E/V/W, G356D) that when present in the germline give rise to the congenital malformation syndrome fibrodysplasia ossificans progressiva (FOP), and ALK2 inhibitors are currently in development for this disease of abnormal cellular differentiation. We sought to investigate the role of ACVR1 mutations in the context of DIPG and to evaluate preclinically the potential of targeting the receptor in these tumours. ACVR1 mutations mark a distinct subgroup of DIPG, as they strongly co-segregate with histone H3.1 (HIST1H3B) K27M mutations, younger age of onset, slightly improved clinical outcome, and a female predominance. ACVR1 mutant tumours have an enrichment of gene expression associated with an astrocytic lineage, a phenotype observed histologically, sometimes in conjunction with areas of cartilaginous differentiation. In studies of subclonal variation, ACVR1 mutations are always found to be present in the earliest common ancestral clone, implying an important role early in tumour development. ACVR1 mutant tumours are associated with a gene expression signature indicative of downstream pathway activation (ID2-4) and overexpression of the mutations into wild-type DIPG cells confers a weak activation of phospho-Smads1/5/8. Screening a series of DIPG primary cell cultures with a panel of ALK2 inhibitors (LDN-193189, LDN-212854, dorsomorphin, DMH1, K02288) revealed selective growth inhibitory effects in vitro. ACVR1/ALK2 appears to be an attractive therapeutic target in at least a subset of DIPG patients, not least due to it’s presence on the ‘trunk’ of an individual tumour’s branching evolutionary history. HG-040. PAEDIATRIC GLIOBLASTOMA (PGBM) AND DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) HARBOUR MULTIPLE COMPLEX SUBCLONAL POPULATIONS OF TUMOUR CELLS Mara Vinci1, Alan Mackay1, Anna Burford1, Katy Taylor1, Sergey Popov1, Wendy Ingram2, Michelle Monje3, Marta Alonso4, Nagore Olaciregui5, Carmen de Torres5, Ofelia Cruz5, Jaume Mora5, Angel Carcaboso5, and Chris Jones1; 1Institute of Cancer Research, London, UK; 2Queensland Childrens Tumour Bank, Brisbane, Australia; 3Stanford University, Palo Alto, USA; 4University Hospital of Navarra, Pamplona, Spain; 5Hospital Sant Joan de De´u, Barcelona, Spain A major challenge to improve outcomes in pGBM/DIPG is their extensive intratumoral heterogeneity, reflected by differing cellular morphologies and genomic imbalances present within an individual sample. We sought to define the subclonal diversity of pGBM/DIPG with a view to better understanding the evolutionary dynamics underlying this variation. Firstly, we used whole genome/exome sequencing data to explore the clonal structure of a series of pGBM/DIPG specimens by fitting density distributions to copy number-adjusted variant allele fractions of somatic mutations. Where present, histone H3.3/H3.1 mutations were found in the implied most recent common ancestor, suggestive of an early and fundamental role in tumour development. In most cases, we also observed single nucleotide variants present in only a fraction of tumour cells, revealing a complex architecture implying multiple heterogeneous subclones present in several clusters, rather than dominant clonal cell populations. Direct evidence was provided by studying tumours for which multiple topographically-distinct samples were available. This identified subclones driven by different oncogenic variants, including multiple PIK3CA mutations present in variable proportions in histologically-distinct regions of the same DIPG autopsy. The phenomenon of phenotypic convergence was also observed when cells were cultured in vitro, with different subclonal populations harbouring mutually exclusive PIK3CA C420R/H1047L mutations. To explore this further, we used a custom capture set of 435 genes recurrently mutated in pGBM/DIPG. Sequencing primary tumours at high depth (.1000x) alongside multiple single cell-derived colonies expanded in vitro, we observed private events in individual subclones including mutations in functionally relevant genes such as tyrosine kinases, histone methyltransferases and DNA repair enzymes. This genetic variation underpinned a phenotypic diversity including differential clonogenicity, growth rates and morphology, assessed using high-throughput image analysis. These data have profound consequences for targeted therapies of pGBM/DIPG, and suggest the importance of applying evolutionary biology principles to guide rational design of novel interventional strategies. HG-041. TREATMENT RESULTS OF PILOT STUDY WITH IRONOTECAN AND CARBOPLATIN IN CHILDREN WITH NEWLY DIAGNOSED HIGH GRADE GLIOMAS (HGG). ONE INSTITUTION EXPERIENCE Iwona Filipek1, Monika Drogosiewicz1, Marta Perek-Polnik1, Ewa Swieszkowska1, Bozenna Dembowska-Baginska1, Elzbieta Jurkiewicz2, and Danuta Perek1; 1The Children’s Memorial Health Institute, Oncology Department, Warsaw, Poland; 2The Children’s Memorial Health Institute, Radiology Department, Warsaw, Poland INTRODUCTION: Treatment results in children with high grade gliomas remain poor. Since responses to irinotecan in HGG have been reported we have introduced a combination of irinotecan and carboplatin at first as a second line/salvage treatment, then as preirradiation chemotherapy in patients with newly diagnosed HGG. AIM: To assess treatment outcome of pilot study with irinotecan and carboplatin in children with HGG in comparison to historical group treated according to own protocol. PATIENTS AND METHODS: 17 pts: 9 girls and 8 boys treated between 2010 and 2013 were assessable for response. Two were diagnosed with anaplastic oligoastrocytoma, 2 anaplastic oligodendroglioma, 3 anaplastic astrocytoma and 10- glioblastoma. 2 pts underwent complete tumour resection, 1- subtotal, 8-partial resection and 5- biopsy only. At the onset of treatment three patients had disseminated disease. Chemotherapy protocol consisted of 5-day courses of irinotecan 50 mg/m2 and carboplatin 250 mg/m2 given every 3 weeks. Response to chemotherapy was evaluated using RANO criteria, toxicity- according to CTC. RESULTS: 17 pts received a cumulative number of 60 cycles of chemotherapy. Out of 17 pts with newly diagnosed HGG 3 pts achieved CCR, 1 pt- CR, 7 pts- PR, SD was observed in 3 pts and 3 had PD. 64% of pilot study pts achieved good response (CCR, CR or PR) compared to 30 % in historical group. 3 yr OS was 46,5 % in pilot study vs 31,8 % in historical group, PFS- 22,8% vs 22,7% (3 yrs) and 49,9% vs 38,6 % (1 yr) consecutively. In patients after complete resection (2 with GB, 1 with anaplastic oligoastrocytoma) treated according to pilot study 3 yr OS and PFS were estimated at 100%. Myelosuppression and gastrointestinal toxicities were the most common and manageable. CONCLUSION: Irinotecan with carboplatin regimen shows activity against HGG in children and has acceptable toxicity. HG-042. HYPOXIC CONDITIONS TRANSFORM PEDIATRIC HIGH GRADE GLIOMA CELLS TO A HIGHLY RESISTANT PROGENITOR-LIKE PHENOTYPE IN VITRO Aurelia Nguyen1, Erwan Pencreach1, Alan Mackay3, Franc¸ois Marie Moussalieh1, Dominique Guenot2, Izzie Namer1, Marie Pierre Chenard1, Chris Jones3, and Natacha Entz-Werle´1; 1CHRU Hautepierre, Strasbourg, France; 2EA3430 - Universite´ de Strasbourg, Strasbourg, France; 3ICR, London, UK Intratumoral hypoxia plays a fundamental role in tumor progression and resistance to therapies. Tumor cell adaptation to a hypoxic environment is regulated partially by the Hypoxia Inducible Factor-1alpha (HIF-1a), which is involved in gene expression of energetic metabolism, angiogenesis or metastatic processes. HIF-1a accumulation in cells is in particular driven by oncogenic pathway activation, mainly PI3K-AKT-mTOR and RAS-MAPK-mTOR pathways. In high grade gliomas (HGG), both hypoxic and normoxic conditions are present during tumor development, allowing the selection adaptation of tumor cells to different local environments. To demonstrate the role of hypoxia in the HGG, we first, characterized in 25 paediatric HGG samples the expression of the proteins involved in the two main signaling pathways. Secondly, we studied in three wellestablished cell lines (UW479, SF188 and KNS42) the impact of 1% hypoxia on proliferation, gene expression, metabolomic profiles and hypoxia-induced protein modulations. Thirdly, targeting key proteins in the hypoxia pathways (e.g. mTOR and HIF-1a) was a dynamic approach in the three cell lines to understand the inhibition and the potential therapeutic possibilities in these poor outcome tumors. More than 75% of the tumors had activated PI3K-AKT-mTOR or RAS-MAPK-mTOR pathways, and this protein modulation seemed to confer specific hypoxic profiles. In each of the cell lines, hypoxia is associated with a unique metabolomic, protein and gene expression signature indicative of three specific adaptive processes. These signatures are linked to a specific response to mTOR/ HIF-1a inhibition and could be predictive profiles defining sensitive HGGs to therapies targeting hypoxia. In the more drug resistant lines, cells grown under hypoxic conditions appear to be transformed to a progenitor-like phenotype, with a reduced proliferation rate and induced HIF-1/HIF-2 expression. These data demonstrate the importance of tumor microenvironmental conditions in determining therapeutic response and provide clues towards a mechanistic understanding of the inherent resistance of pediatric HGG. HG-043. IMMUNE RESPONSES AND CLINICAL OUTCOME IN A PILOT STUDY OF VACCINATION WITH GLIOMA-ASSOCIATED ANTIGEN PEPTIDES AND POLY-ICLC IN CHILDREN WITH NEWLY DIAGNOSED MALIGNANT BRAINSTEM AND NON-BRAINSTEM GLIOMAS Ian Pollack, Regina Jakacki, Lisa Butterfield, Ronald Hamilton, Ashok Panigrahy, Douglas Potter, Angela Connelly, Sharon Dibridge, Theresa Whiteside, and Hideho Okada; University of Pittsburgh School of Medicine, Pittsburgh, PA, USA BACKGROUND: Diffuse brainstem gliomas (BSGs) and other high-grade gliomas (HGGs) of childhood are among the most deadly brain tumors of childhood, and new therapies are needed. Immunotherapy is a particularly promising approach in this regard. Having identified a series of glioma-associated antigens (GAAs) commonly overexpressed in pediatric gliomas, we initiated a pilot study of subcutaneous vaccinations with GAA epitope peptides in HLA-A2+ children with newly diagnosed BSG and HGG. METHODS: Children received subcutaneous vaccinations with synthetic peptides derived from EphA2, interleukin (IL)-13 receptor-a2, and survivin, emulsified in Montanide-ISA-51 and administered every 3 weeks for 8 doses, in conjunction with intramuscular poly-ICLC. Booster vaccinations were administered every 6 weeks for up to 2 years for children with at least stable disease following the initial phase of therapy. Primary endpoints were safety and T cell responses against vaccine-targeted GAAs. Treatment response was evaluated clinically and by MR imaging. RESULTS: Twenty-six children were enrolled in the initial pilot phase of the study, 14 with newly diagnosed BSG treated with irradiation, and 12 with newly diagnosed BSG or HGG treated with irradiation and concurrent chemotherapy. No regimen-limiting non-CNS toxicity was encountered. Five children had symptomatic pseudoprogression, which responded to corticosteroids and was associated with prolonged survival. Only two patients had progressive disease during the first two vaccine courses; 19 had stable disease, 2 had PRs, 1 had a minor response (25-50% reduction in tumor size), and 2 had a prolonged continuing complete response after surgery. Enzyme-linked immunosorbent spot (ELISPOT) analysis in 21 children, showed positive anti-GAA immune responses in 13: to IL13Ra2 in 10, EphA2 in 11, and survivin in 3. CONCLUSION: GAA peptide vaccination in children with gliomas is generally well tolerated, with preliminary evidence of immunological and clinical responses. Careful monitoring and management of pseudoprogression is essential. Diffuse intrinsic pontine glioma (DIPG) is a universally fatal tumor. Eighty percent of DIPG patients have a mutation in the H3F3A gene resulting in missense mutation at position 27 of Histone 3.3 (H3K27M) which inhibits methylation at position 27 and leads to global chromatin changes. DIPG is also associated with global DNA hypomethylation. Evidence suggests that 5-methylcytosine (5mC) can be converted to cytosine in a multistep enzymatic process with 5-hydroxymethylcytosine (5hmC) being the initial intermediate. 5hmC represents an important epigenetic mark and with 5mC and histone methylation plays a key role in neural development. Due to the global hypomethylation noted in DIPGs, we hypothesized that levels of 5mC, 5hmC and H3K27 trimethylation would be altered. We used formalinfixed paraffin embedded tumor samples in tissue microarrays to perform immunohistochemical studies, comparing DIPG to adult and pediatric glioblastoma (GBM) and normal brain. Tumors were scored for histone 3 lysine 27 trimethylation (H3K27me3), histone 3 lysine 9 trimethylation (H3K9me3), 5-hydroxymethylcytosine (5hmC), and 5-methylcytosine (5mC). H-score was derived by multiplying intensity (0-2) by percentage of positive tumor nuclei (0-100%). We identified decreased H3K27me3 in the DIPG cohort compared to pediatric GBM (p , 0.01), adult GBM (p , 0.0001) and normal brain (p , 0.0001). H3K9me3 was not significantly different between tumor types. Global DNA methylation as measured by 5mC levels was significantly lower in DIPGs compared to pediatric GBM (p , 0.001), adult GBM (p , 0.01), and normal brain (p , 0.01). Surprisingly, 5hmC levels were significantly higher in DIPGs compared to pediatric GBM (p , 0.0001) and adult GBM (p , 0.0001). This data indicates that DIPGs are different from their GBM counterparts with derangements in both histone and cytosine methylation. This may shift the neural development of hindbrain progenitors into a pathological state. The presence of highlevels of 5hmC in the context of global hypomethylation may provide an opportunity for therapeutic intervention. HG-045. MANGO GINGER: A CURCUMA SPECIES HIGHLY SYNERGISTIC WITH CHEMOTHERAPY AGAINST GLIOMA CELL LINES Chepapil Ramachandran2, Smitha Nair2, Karl-W Quirrin3, Ziad Khatib1, Enrique Escalon1, and Steven Melnick1; 1Miami Children’s Hospital, Miami, FL, USA; 2Dharma Biiomedical LLC, Miami, FL, USA; 3Flavex Naturextrakte GmbH, Rehlingen, Germany Pediatric brain tumors are the most common form of solid tumors in children accounting for about 20-25% of all pediatric cancers. Chemotherapy options for brain tumor treatment are very much limited because of the blood brain barrier and emergence of drug resistance in brain tumor cells. Combining nutraceuticals or botanical drugs with cancer drugs is one of the ways to improve the efficiency of chemotherapy and quality of life in an integrative oncology setting. In the present investigation, cytotoxicity of anticancer drugs [Etoposide(ETP), Temozolomide, TMZ)] and supercritical extracts of Curcuma amada (CA-CO), C. xanthorrhiza (CX-CO) and C. longa (CL-CO), curcumin and Turmeric ForceTM either as single agent or their combinations in glioma cell lines (U87MG, U188 MG) were analyzed MTT assay. Synergism, additiveness or antagonism between cancer drugs and supercritical extracts were determined using CompuSyn analysis of cytotoxicity data. Apoptosis and necrosis induced by different agents or their combinations were analyzed using Roche Annexin-V-FLUOS staining kit in a flow cytometer. The expression of genes associated with apoptosis and cell proliferation (p53, p21, Bcl-2, Bax, and P10) were determined by RT-PCR assay. Both glioma cell lines are generally resistant to cancer drugs such as TMZ. CA-CO showed superior cytotoxic effects as compared to CX-CO and CL-CO. CA-CO also had significantly better cytotoxic effects than curcumin and Turmeric ForceTM. Compusyn analysis of cytotoxic data showed that the combination of ETP and/or TMZ with CA-CO produced very high synergistic effects on cytotoxicity. The combination of cancer drugs with CA-CO induced higher percentage of apoptosis and necrosis than single agents. Gene expression studies showed that CA-CO down regulated the expression of P10 and P53 genes and increased the ratio of Bax/ Bcl-2 mRNA. These positive results suggest the need for continuous evaluation of CA-CO in xenograft models and clinical trials in brain tumor patients. HG-046. CILENGITIDE AND METRONOMIC TEMOZOLOMIDE FOR RELAPSED OR REFRACTORY HIGH GRADE GLIOMAS OR DIFFUSE INTRINSIC PONTINE GLIOMAS IN CHILDREN AND ADOLESCENTS – PRELIMINARY RESULTS OF THE GERMAN PHASE II STUDY HIT-HGG-CILMETRO Carl Friedrich Classen1, Marion Hofmann2, Irene Schmid3, Thorsten Simon4, Eberhard Maaß5, Alexandra Russo6, Gudrun Fleischhack7, Martina Becker8, Holger Hauch9, Annette Sander10, and Christof Kramm2; 1University Childrens Hospital, Rostock, Germany; 2University Childrens Hospital, G o¨ttingen, Germany; 3Dr.von Haunersches Kinderspital, M u¨nchen, Germany; 4University Childrens Hospital, K o¨ln, Germany; 5Olgahospital, Stuttgart, Germany; 6University Childrens Hospital, Mainz, Germany; 7University Childrens Hospital, Essen, Germany; 8University Childrens Hospital, Frankfurt, Germany; 9University Childrens Hospital, Gießen, Germany; 10Medical School Childrens Hospital, Hannover, Germany INTRODUCTION: Relapsed high-grade gliomas (HGG) in pediatric patients like glioblastoma multiforme (GBM), anaplastic astrocytoma (AA), diffuse intrinsic pontine glioma (DIPG) represent a dismal prognosis group currently without standard salvage therapy. In this population, combined cilengitide and metronomic temozolomide was investigated: Cilengitide 1800 mg/m2 twice weekly; temozolomide: 75-80 mg/m2/d for 6 weeks with one week rest, in total over one year. PATIENTS AND METHODS: Between 02/2012-03/2013 28 patients were recruited (3 AA, 9 GBM, 15 DIPG, one anaplastic oligoastrocytoma), 27 after first-line temozolomide radiochemotherapy (HIT-HGG-2007 protocol). RESULTS: Ten months after recruitment closure, 3/28 relapse patients are still alive and progression-free (all GBM) with a follow-up of 11 to 22 months. The following serious adverse events (SAE) were observed: Intracranial hemorrhage (n ¼ 4); neurological deterioration (n ¼ 4) and seizures (n ¼ 3), pneumonia (n ¼ 2), edema (n ¼ 1), severe obstipation (n ¼ 1), pain (n ¼ 1), and cellulitis (n ¼ 1). No suspected unexpected serious adverse reaction (SUSAR) occurred. However, recruitment was prematurely stopped due to an altered risk benefit assessment after two negative clinical trials employing cilengitide in pediatric and adult HGG had been published. Survival of the present trial cohort was compared to a historical control of HGG relapse patients from our database, all treated individually for their relapse (n ¼ 417 patients: 159 GBM, 158 DIPG, 100 other histologies). Median overall survival in HIT-HGG-CilMetro was 0,397 (0,181-0,613) vs. 0,507 (0,451-0,562) years (not significant), OS and EFS after relapse were nearly identical. However, the three GBM survivors in the HIT-HGG-CilMetro group might represent true responders. CONCLUSION: HIT-HGG-CilMetro offered a feasible salvage treatment approach with tolerable toxicity for pediatric patients with relapsed HGG and might induce in contrast to previous cilengitide monotherapy trials an increased progression-free survival in a small relapsed GBM subgroup. Diffuse Intrinsic Pontine Glioma (DIPG) is a universally fatal cancer with only a 9-month median survival. The present study represents an international, multi-institutional collaboration to pool DIPG tissue resources and evaluate comprehensive molecular and functional targets of DIPG therapy. We performed whole exome sequencing of 17 DIPG tumors, RNAseq expression analysis and a chemical screen against 14 DIPG cell lines along with integrated computational analysis to identify potentially effective therapeutic strategies. Whole exome sequencing underscores the relatively quiet mutational landscape of this pediatric malignancy, with demonstration of known genomic aberrations such as histone H3 K27M mutations and TP53 loss/mutations, as well as discovery of previously unrecognized recurrent aberrations in ACVR1, NTRK1 and EphB6. The chemical screen identified consensus sensitivity to two classes of agents, histone deacetylase inhibitors and CDK inhibitors, with additional agents demonstrating efficacy for subsets of DIPG cell lines. Integrative computational analysis of drug screen data and paired RNAseq expression data additionally identified multiple candidate genes that have the potential to yield an effective treatment, if targeted in combination. Among these, combinatorial targeting of HDAC 1, together with HDAC 2 or 4 is predicted to be an effective strategy. Panobinostat, among the more promising agents identified by the chemical screen, inhibits these three HDACs. Accordingly, in vivo testing of panobinostat demonstrated efficacy in a pontine orthotopic xenograft model of DIPG. RNAseq expression analysis before and after exposure to panobinostat highlighted normalization of gene expression patterns, including up-regulation of tumor suppressor gene levels (e.g. EphB6) and down-regulation of oncogenes (e.g. MYC). Taken together, these data suggest the potential utility of specific drug combinations and provides evidence of in vivo treatment efficacy of the multi-HDAC inhibitor panobinostat. INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) is a devastating therapy-resistant tumor and children diagnosed with this disease have a dismal prognosis. Bio-distribution of therapeutic agents in DIPG is unknown, and poor tumor penetration may be the reason for therapeutic failure. Bevacizumab is a monoclonal antibody that neutralizes VEGF, a growth factor involved in angiogenesis and cell proliferation, which is overexpressed in DIPG. The role of bevacizumab in the treatment of DIPG is unclear. The aim of this study is to investigate bio-distribution and tumor-uptake of zirconium-89 (89Zr) labeled bevacizumabin a xenograft model, in relation to VEGF expression. METHODS: Human E98FM glioma cells, known for their diffuse growth pattern in vivo, were injected in the subcutis, pons, or striatum of 30 athymic mice. A sub-therapeutic dose of 89Zr-bevacizumab was administered 18 or 35 days after xenografting. The location of injection and timing of treatment represented different tumor localizations and disease stages. Seventy-two to 96 hours after administration of 89Zr-bevacizumab, mice were imaged using Positron Emitting Tomography (PET) and biodistribution was analyzed ex-vivo. VEGF expression was confirmed by in situ hybridisation (ISH). RESULTS: There was no significant 89Zr-bevacizumab uptake in the mouse brain nor in the E98 gliomas located in the pons and striatum at either stage of disease. Instead, the subcutaneous tumors showed high accumulation of 89Zr-bevacizumab. VEGF expression was confirmed by ISH in the subcutaneous tumors, but was absent in the majority of intracranial tumors. CONCLUSIONS: The uptake of 89Zr-bevacizumab in intracranial E98FM tumors is poor, while 89Zr-bevacizumab accumulates in subcutaneous E98FM tumors. We hypothesized that this difference is due to a lack of VEGF expression or possibly an intact blood-brain-barrier in these diffusely growing intracranial xenografts. These results stimulate the use of PET studies in children with brain tumors, to investigate correct drug targeting and to personalize treatment. HG-049. HIGHLY SELECTIVE INTRA-ARTERIAL CHEMOTHERAPY FOR THE TREATMENT OF PROGRESSIVE DIFFUSE INTRINSIC PONTINE GLIOMAS (DIPG) Kenneth Cohen, Eric Raabe, and Monica Pearl; Johns Hopkins University School of Medicine, Baltimore, MD, USA BACKGROUND: DIPG remains a largely fatal diagnosis. Radiation therapy remains the only intervention that alters the natural history of the disease, but is not curative. Given the frequent imaging feature of a noncontrast enhancing mass at diagnosis, the extent of systemic delivery of chemotherapeutics to the pons is uncertain. Strategies to improve delivery include: convection-enhanced delivery utilizing intrapontine catheters, blood-brain barrier disruption, and intra-arterial (IA) administration of chemotherapy into the vertebral circulation. This pilot study explores the safety, tolerability, and initial efficacy of highly selective intra-arterial administration of melphalan via the basilar artery at the time of clinical progression following initial radiotherapy for DIPG. METHODS: Subjects with a radiologic or biopsy proven diagnosis of DIPG are eligible at the time of progression following initial irradiation. Subjects with a documented hypercoagulable disorder or vasculopathy or who have been re-irradiated are not eligible. Two cycles of IA chemotherapy are planned 4 weeks apart. Either vertebral artery is catheterized with a 4-French guide catheter. Following a baseline angiogram, a 1.5-French microcatheter is advanced into the mid basilar artery. IA delivery of 4 or 6 mg of melphalan is delivered over 30 minutes. Subjects are observed overnight. Subjects are monitored for toxicity between, and following, IA administrations. RESULTS: To date, 2 children, of a planned cohort of 5 children, have been treated. Both children tolerated the actual IA administrations without any procedural associated toxicity including the absence of stroke, hemorrhage, or other technical complications. The first child received one of two planned IA administrations but clinically deteriorated shortly before the second IA infusion was planned. The second child received both planned IA administrations and is in early follow-up. CONCLUSIONS: Highly selective IA administration of melphalan to the basilar artery is feasible and acutely tolerated. Updated experience and follow-up with enrolled patients will be presented. HG-050. INVESTIGATIONAL AURORA A KINASE (AAK) INHIBITOR MLN8237 (ALISERTIB) SUPPRESSES NEUROSPHERE PROLIFERATION OF PEDIATRIC GLIOBLASTOMA AND PROLONGS ANIMAL SURVIVAL IN PATIENT TUMOR-DERIVED ORTHOTOPIC XENOGRAFT MOUSE MODEL Mari Kogiso, Linna Zhang, Lin Qi, Holly Lindsay, Frank Lin, Stacey Berg, Xiao-Nan Li, and Jodi Muscal; Texas Children’s Cancer Center, Texas Children’s Hospital, Baylor College of Medicine, Houston, TX, USA BACKGROUND: Pediatric glioblastoma (GBM) is highly aggressive and development of novel therapy is needed. AAK is critical for mitosis and its overexpression results in malignant transformation of normal cells. MLN8237 (Alisertib) is an investigational orally available reversible selective inhibitor of the Aurora A serine/threonine kinase. We investigated the effects of MLN8237 on glioma stem cell proliferation and animal survival. METHODS: Levels of AAK expression were screened in a panel of patient tumor-derived orthotopic xenograft mouse models. Anti-tumor effects were examined by Cell Counting Kit-8 assay in paired monolayer cell lines (FBS-based media) and neurosphere cultures (serum-free media containing EGF/bFGF) from two xenograft mouse models, IC-4687GBM (previously untreated) and IC-R0315GBM (established from a therapy-resistant autopsy sample). Susceptibility to cancer stem cell population was analyzed by flow cytometric analysis. Activity was examined in vivo by orally administered MLN8237 (30 mg/kg/day for 12 days) in NOD/SCID mice two weeks post intra-cerebral tumor cell implantation. RESULTS: Overexpression of AAK mRNA (4-10 fold over normal control) was observed in our pediatric GBM models. Tumor growth in monolayer cells was suppressed in the previously untreated line (4687GBM) but not in the therapy-resistant line (R0315GBM). Cell growth of neurospheres was suppressed in both lines in a time- and dose-dependent manner. Tumor killing activity in neurospheres was confirmed in cancer stem cell marker (CD133/CD15) expressing cells in both lines. In IC-4687GBM, animal survival was significantly prolonged in the treatment group (p , 0.05). In the therapy-resistant model IC-R0315GBM, no significant prolongation was observed, which correlates with the in vitro lack of effect on the monolayer cells. CONCLUSION: We detected high levels AAK expression in pediatric GBM models and MLN8237 appears active in targeting GBM in vitro and in vivo. Our data in the therapy-resistant model IC-R0315GBM support the future research into potential combination regimens in therapy-resistant GBMs. BACKGROUND: Biallelic mismatch repair deficiency (BMMRD) is a cancer predisposition syndrome affecting individuals from consanguinous families with multiple childhood cancers; most commonly malignant brain tumors. The impact of the syndrome on malignant gliomas in countries where consanguinity is high is unknown. We determined the prevalence of clinical findings suggestive of BMMRD and mismatch repair dysfunction among pediatric patients diagnosed with these cancers in twocancer centres in Jordan and Canada. PATIENTS AND METHODS: All patients , 18 years and diagnosed with intracranial high grade glioma (HGG) between 2003 and 2013 were included. Clinical data regarding presence of cafe´ au lait spots (CAL), family history of cancer, consanguinity, diagnosis and treatment were collected. Tumors were centrally reviewed and tested for evidence of mismatch repair deficiency by immunohistochemistry of the MMR proteins. RESULTS: Thirty five patients fulfilled the inclusion criteria; twenty two of them (63%) had available clinical data. Fifteen tumors (41%) lost at least one MMR stain and 80% also lost MMR stain in the corresponding normal endothelium suggestive of BMMRD. Two patients developed two intracranial tumors each; with identical MMR gene loss in both tumors. P53 immunopositivity was highly enriched in MMR deficient tumors (p ¼ 0.0003).Clinical data suggestive of BMMRD was present in 36% of patients. Frequency of MMRD was significantly lower in the Toronto cohort (23%, p ¼ 0.03). Importantly, genetic testing confirmed BMMRD in 50% of these patients. Five year overall survival for patients with MMRD tumors in both cohorts was 17 + /-10% and the only long term survivors were patients with BMMRD whose tumors were discovered by surveillance. CONCLUSIONS: In Jordan, the frequency of clinical and immunohistochemical alterations suggestive of BMMRD in pediatric HGG is high. Genetic testing is needed to confirm an underlying BMMRD in order to perform counseling and cancer screening to improve survival of these patients. HG-052. INTRANASAL DELIVERY OF CHEMOTHERAPEUTIC AGENTS IN A NON-HUMAN PRIMATE MODEL Shaefali Shandilya, Cynthia McCully, Robert Murphy, Srivandana Akshintala, Diane Cole, Rhonda Pung Macallister, Rafael Cruz, Brigitte Widemann, and Katherine Warren; National Cancer Institute, Bethesda, MD, USA BACKGROUND: Achieving adequate tissue concentrations of chemotherapeutic agents for the treatment of brain tumors is limited by the blood-brain barrier (BBB) and systemic toxicities. We hypothesized that intranasal (IN) administration could overcome the BBB by providing direct access to the CNS. This study evaluated the feasibility and the plasma and cerebrospinal fluid (CSF) pharmacokinetics of IN-delivered temozolomide (TMZ) compared to intravenous (IV) administration in a non-human primate (NHP) model. METHODS: In separate experiments, four adult rhesus monkeys received TMZ IN (dose ¼ 20 mg, bolus) and IV (dose ¼ 7.5 mg/kg, 1 hr infusion). Following both IN and IV dosing, serial paired plasma and CSF samples were collected. TMZ was quantified using HPLC/tandem mass spectrometry. Pharmacokinetic (PK) parameters were estimated using noncompartmental methods. Exposures, determined by areas under the concentration × time curves (AUCs), were dose-corrected. RESULTS: No significant toxicities were observed after IN or IV administration. PK parameters are reported as median (range) values: IN (n ¼ 4): CSF peak concentration (Cmax) was 2.39 (2.03-2.72) mM; time to Cmax (Tmax) was 1.50 (1.00-3.00) hr; AUCCSF was 6.80 (4.36-7.53) mM†hr/mg/kg. Plasma Cmax was 11.73 (7.87-12.80) mM; Tmax was 0.28 (0.18-1.00) hr; AUCPL was 11.26 (6.52-15.06) mM†hr/mg/kg. Ratio of exposure (AUCCSF:AUCPL) was 57.05% (38.31-99.10). IV (n ¼ 3): CSF Cmax was 18.96 (14.60-23.10) mM; Tmax was 1.53 (1.50-2.00) hr; AUCCSF was 7.31 (7.04-11.42) mM†hr/mg/kg. Plasma Cmax was 86.20 (83.60-127.80) mM; Tmax was 1.00 (1.00-1.50) hr; AUCPL was 35.09 (34.16-54.42) mM†hr/mg/kg. Ratio of exposure (AUCCSF:AUCPL) was 20.99% (20.07-21.40). Ratio of CSF exposure (IN AUCCSF:IV AUCCSF) was 65.90% (59.68-91.80). Ratio of plasma exposure (IN AUCPL:IV AUCPL) was 27.68% (18.59-33.33). CONCLUSIONS: Compared to IV-administered TMZ, IN administration achieved a higher CSF:systemic exposure ratio, clinically relevant CSF exposure, and reduced overall systemic exposure, suggesting that IN dosing allows sufficient delivery of TMZ to the CNS, while potentially decreasing systemic exposure and toxicity. HG-053. PI-3-KINASE PATHWAY INHIBITION IN A GENETIC MOUSE MODEL FOR HIGH-GRADE ASTROCYTOMA Ralph Salloum1, Amanda Smith1, Michelle Glaunert1, Annmarie Ramkissoon1, Scott Peterson2, Suzanne Baker3, and Lionel Chow1; 1Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA; 2Oncothyreon, Seattle, WA, USA; 3St. Jude Children’s Research Hospital, Memphis, TN, USA BACKGROUND: High-grade astrocytomas (HGA) are aggressive brain cancers that affect thousands of patients each year and carry a dismal prognosis. The phosphatidylinositol-3-kinase (PI3K)/Akt pathway is a core mutated pathway in gliomagenesis and is therefore an attractive therapeutic target. METHODS: We evaluated the mTOR inhibitor rapamycin (5 days, 25 mg/kg ip) and the irreversible PI3K inhibitor PX-866 (5 days, 3mg/kg po) alone and in combination in a genetically engineered mouse model for HGA lacking PTEN, a negative regulator of the PI3K pathway. Alterations of the PI3K signaling pathway and indices of proliferation and apoptosis were assessed in HGA by immunohistochemistry and western blotting. RESULTS: Rapamycin treatment had little effect on signaling upstream of mTOR but led to decreased phospho-S6. The proliferative index in rapamycin treated tumors (22.8%) was significantly lower than in vehicle treated HGA (45.3%, p ¼ 0.003) whereas apoptosis was significantly higher in rapamycin treated (7.5%) versus control tumors (2.2%, p ¼ 0.03). Treatment with PX-866 effectively decreased p-S6 however effects on other signaling intermediates of the PI3K/AKT pathway and on proliferation and apoptosis were variable and modest. Combination treatment was well tolerated by mice and significantly decreased the level of p-AKT by 3 fold (p ¼ 0.017) and p-4EBP1 by two fold (p ¼ 0.019) compared to either drug alone. The proliferative index in combination treated tumors (16.3%) was significantly lower than in rapamycin treated HGA (22.8%, p ¼ 0.041). Interestingly, the apoptotic index was 37.1% in tumors treated with the combination, a significant increase in cell death compared to rapamycin treatment alone (7.5%, p ¼ 0.004). CONCLUSION: The combination of rapamycin and PX-866 demonstrates cooperative effects on downregulation of PI3K signaling, suppression of cell proliferation and induction of apoptosis in a genetically engineered mouse model for HGA. Studies to evaluate the survival benefit of drug treatments are ongoing. Pediatric gliomas comprise a diverse group of malignancies accounting for more than half of childhood CNS-tumors. Here we apply next-generationsequencing to the exome-profiling of an unusual highly malignant oligodendroglioma operated from a 13 years old child who achieved long term survival. Radical tumor extirpation was performed and the treatment included 8-drugs-in-1-day, followed by radiotherapy. Today, 24 years later, the patient is still healthy. Patient’s blood and tumor DNA were exome enriched and sequenced resulting in 26X target region coverage. Surprisingly, we detected .33000 somatic events (SNVs and indels). This high somatic mutation rate was similar to that observed in some sporadic colorectal tumors and tumors developing in autosomal dominant Lynch syndrome (LS). It is known that germline mutations in DNA mismatch repair (MMR) genes: MLH1/2, MSH6 and PMS2 lead to LS. Childhood cancers are though not part of the LS tumor spectrum. In rare occasions however, germline bi-allelic MMR genes mutations may lead to a condition called constitutive MMR deficiency. The syndrome is characterized mainly by colorectal cancer, hematological neoplasias and brain tumors. In contrast to LS, these malignancies occur in the childhood or adolescence. Interestingly, the search for germline mutations revealed compound heterozygous mutations in MSH6. One of the mutations, rs63750897 introduces a predicted damaging aa substitution at position 503. The second one is a novel, nonsense mutation at aa position 201. Immunohistochemistry for MSH6 revealed no protein expression in the tumor. Very few cases of brain tumors due to bi-allelic MMR genes defects have been reported, however, they might have clinical, pathological or molecular characteristics that allow early diagnosis. It has also been proposed that alkylating agents should be avoided in such cases and that radiation should be used with caution. It is therefore important to document cases like this particularly with regard to the excellent outcome. BACKGROUND AND PURPOSE: Dissemination of high-grade gliomas (WHO IV) is rare. The aim of the study was to delineate the patient characteristics, treatment, and outcome of patients with metastatic glioblastoma multiforme (GBM)/gliosarcoma (GS) reported in the literature until April 2013. METHODS: PubMed and Web of Science were searched for case series and cohort studies reporting on metastatic GBM and GS. RESULTS: 215 publications reporting on 357 patients met our inclusion criteria. There was a significant increase in the number of reported cases over the last decades. Median age at initial diagnosis was 44.5 years (range, 0.42 84 years). Thirty-four patients (9.8 %) were children or adolescents, aged , 18 years. We estimated a median overall survival (OS) from diagnosis of metastasis (in the majority of the patients at time of relapse; in 17% at time of initial diagnosis) of 3.0 + 0.4 months. Interestingly, OS was similar for patients with metastatic disease at initial diagnosis when compared to patients with disseminated disease at time of relapse (3-months OS: 42 + 10% versus 46 + 5%). By univariable analyses, gender, age (cutoff 18 years), the histological subtype and the time interval between initial diagnosis and the occurrence of metastases did not influence outcome. In contrast, metastatic disease restricted to areas outside the central nervous system was associated with longer survival. The overall survival after diagnosis of metastasis was independent of the date of publication. CONCLUSIONS: Metastatic GBM/ GS are associated with a poor prognosis both in adults and children. Due to their relative rare occurrence it seems to be still a challenge to define the optimal treatment approach for these patients. It remains to be shown whether the disseminated disease predisposes for an insufficient tumor control or whether biology might be the underlying reason for the poor prognosis. BACKGROUND: High grade gliomas (HGG) in children, while not as common as in adults, are also aggressive disease with very poor overall survival. We retrospectively review our series of paediatric patients with HGGs in KK Women’s and Children’s Hospital (KKH). METHODS: Data was collected from the Singapore Childhood Cancer Registry (SCCR) and hospital chart records. All patients aged 0-19 years with HGGs diagnosed and treated in KKH from July 1997-June 2012 (15 years) were included. Information regarding demographics, clinical presentation, diagnosis, treatment and outcomes were studied. RESULTS: 27 patients with HGG were identified of which 15 (56%) were boys. The age at diagnosis ranged from 3 months to 14.7 years (median 6.8 years; mean 7.2 years). All patients were of Asian ethnicities - 17 (63%) Chinese, 6 (22%) Malay and 4 (15%) Indian. Most common presentations included vomiting (50%), cranial nerve palsies (46%), headache (43%) and unsteady gait (36%). The tumour locations were supratentorial in 12 patients, infratentorial in 13, and multifocal in 2 (including one with multifocal gliomatosis cerebri). Biopsy and/or resection were done for 19 patients (70%). Of these, 10 (53%) were WHO Grade III and 9 were Grade IV. The most common histologies were anaplastic astrocytoma (26%), glioblastoma NOS (16%), glioblastoma multiforme (11%) and anaplastic ependymoma (11%). Of the 12 supratentorial HGG, 11 underwent resection, 7 underwent radiotherapy and 4 underwent chemotherapy. Of the 13 infratentorial HGG, 4 underwent resection, 10 underwent radiotherapy and 5 underwent chemotherapy. Median survival was 42 months for supratentorial HGG (range 4 months to 16 years), and 16 months for infratentorial HGG (range 1 - 54 months). The 5-year overall survivals were: 31.3% (supratentorial HGG), 7.7% (infratentorial HGG), 17.3% (entire cohort). CONCLUSION: Survival outcome for HGG remains poor, especially for infratentorial HGG. HG-057. TARGETING THE ATM-MPG AXIS OVERCOMES ALKYLATING AGENT RESISTANCE IN PEDIATRIC GLIOBLASTOMA Cynthia Hawkins1, Kelly Burrell1, Yevgen Chornenkyy2, Mark Remke1, Brian Golbourn1, Pawel Buczkowicz2, Mark Barzczyk2, Michael Taylor1, James Rutka1, Peter Dirks1, Gelareh Zadeh3, and Sameer Agnihotri1; 1The Hospital for Sick Children, Toronto, ON, Canada; 2The University of Toronto, Toronto, ON, Canada; 3University Health Network, Toronto, ON, Canada Pediatric glioblastoma (pGBM) is among the most lethal primary brain tumors in children. Standard treatment for both children and adults involves surgical resection followed by radiotherapy in combination with the alkylating agent temozolomide (TMZ). Despite this less than 5% of patients survive longer than five years. In adults, the addition of TMZ results in a modest improvement in survival with TMZ-resistance being attributed to expression of the DNA-repair protein O6-methylguanine-DNA methyltransferase (MGMT). In pediatric GBM, TMZ appears to have little effect despite a lack of MGMT expression in many cases. We hypothesized that alternate mechanisms were mediating alkylating agent resistance in pediatric GBM and that targeting these pathways would improve response to TMZ. Using an siRNA screen targeting DNA damage response genes we identified that loss of members of the base excision repair (BER) pathway, including DNA-3-methyladenine glycosylase (MPG), the first rate-limiting step in BER, sensitized pediatric GBM cells, but not normal glial cells, to TMZ. 67% of pediatric GBMs expressed MPG and this was associated with worse overall survival. Interestingly, we identified MPG as a direct substrate of Ataxia telangiectasia mutated (ATM), a master regulator of the DNA damage response pathway. ATM phosphorylation of MPG was essential for MPG function and loss of phospho MPG or combined loss of MPG and ATM resulted in increased TMZ-induced cytotoxicity in pediatric GBM cells both in vitro and in vivo, in both established and primary pediatric GBM cell lines. By directly linking ATM kinase with BER, we demonstrate that by targeting cross-talking DNA repair pathways we can increase cytotoxicity of alkylating agents and can overcome alkylating agent resistance in this devastating pediatric disease. HG-058. INCREASE HISTONE METHYLATION FOR THE TREATMENT OF H3.3 K27M MUTANT BRAINSTEM GLIOMAS Rintaro Hashizume1, Yuichiro Ihara1, Noemi Andor1, Xiaoyue Chen3, Robin Lerner1, Xi Huang1, Maxwell Tom1, David Solomon2, Sabine Mueller1, Claudia Petritsch1, Zhiguo Zhang3, Nalin Gupta1, Todd Waldman2, and David James1; 1University of California San Francisco, San Francisco, CA, USA; 2Georgetown University, Washington, DC, USA; 3Mayo Clinic, Rochester, MN, USA INTRODUCTION: Histone gene mutations occur in pediatric brainstem gliomas. The most common mutation, a substitution of methionine for lysine at position 27 (K27M) of H3.3 protein, one of the histone H3 family members, causes substantial reduction in histone H3 tri- and di-methylation of K27 in cellular chromatin. We hypothesize that increase histone methylation would be a unique therapeutic approach for treating this cancer. METHOD: Histone H3.3 lysine 27 (H3K27) methylation status was evaluated by western blotting. Cell proliferation assays were performed to assess the response to pharmacological inhibition with GSK-J4, a selective inhibitor of H3K27 demethylase JMJD3 and genetic suppression using JMJD3 siRNA. Gene expression changes and sequence association of K27me3, as a result of GSK-J4 treatment, were examined in cells with the K27M mutation using CHIP sequence and gene expression array. In vivo tumor growth and response to therapy were quantitatively measured by bioluminescence imaging and animal survival. RESULTS: H3.3 K27M mutant brainstem glioma cells showed global reduction of H3K27 methylation compared to H3.3 wild-type glioma cells. Pharmacologic inhibition and genetic suppression of the H3 K27 demethylase JMJD3, had anti-tumor activities that are specific to glioma cells with K27M mutation. Inhibition of JMJD3 opposed the suppressive effect of K27M mutation on H3 K27 tri- and di-methylation. GSKJ4 treatment reduced the growth rate of K27M brainstem glioma xenografts in mice, while promoted tumor cell death, and significantly extended animal survival. Among the 27,958 probes on the arrays, 25 genes showed significantly up regulated and 21 genes were down regulated by GSKJ4 (P , 0.001). CHIP sequence association of K27me3, as result of GSKJ4 treatment, will be presenting in the meeting. CONCLUSION: Our findings suggest that pharmacologic modulation of histone K27 methylation by inhibiting the JMJD3 demethylase is a viable approach for treating with pediatric gliomas with H3F3A mutations. HG-059. A CASE OF DIFFUSE INTRINSIC PONTINE GLIOMA IN A 5 YEAR OLD FEMALE Amanda Dujua, Jayson Co, Flerida Hernandez, and Dennis Doromal; University of Santo Tomas Hospital, Manila, The Philippines Brainstem tumors occur in approximately 10% to 15% of pediatric patients with central nervous system tumors. The median age at diagnosis is 7 to 9 years. Intrinsic diffuse pontine gliomas (DPGs) are a unique subset of brainstem tumors with a dismal prognosis, and accounts for 70-80% of all brainstem tumors. Current standard of care is primarily radiation treatment. We report a case of a 5 year old female diagnosed with DPG who has completed conventional radiotherapy and has shown marked clinical improvement. Initial presentation was ataxia, followed by diplopia 2 weeks later. She suddenly developed right-sided hemiparesis, decreased sensorium, headache and vomiting prompting consultation at our institution. She could follow commands with spontaneous eye-opening but inappropriate verbal responses and disarticulation. There was noteddiplopia with left lateral rectus palsy and a weak gag reflex, right upper and lower extremity weakness (MMT 1/5) with 100% sensory deficit, and loss of bowel and bladder control. Cranial MRI with contrast revealed a large necrotic mass in the pons with marked surrounding edema consistent with brainstem glioma with mild obstructive hydrocephalus. Patient was initially started on Mannitol 0.6 mg/kg and Dexamethasone 0.6 mkd. He received primary radiation treatment with 54Gy in 1.8Gy fraction. After two weeks of radiotherapy, her sensorium and articulation improved, headache and vomiting resolved, swallowing done with ease, complete bowel and urinary bladder control regained, and motor function of the right extremities improved to MMT 4/5. Improvement in clinical status is usually evident in 2 to 3 weeks of radiation therapy alone, but progression-free interval is short (median , 6 months), with median survival less than 1 year and survival rates at 2 years of less than 20%. Studies have shown that adjuvant chemotherapy does not improve overall survival, although there are still ongoing investigations in the role of newer chemotherapeutic agents. HG-060. A NOVEL BISPECIFIC CHIMERIC ANTIGEN RECEPTOR TARGETING HER2 AND IL13Ra2 ENHANCES CONTROL OF EXPERIMENTAL HIGH GRADE GLIOMA (HGG) Meenakshi Hegde1, Amanda Wakefield1, Vita Brawley1, Zakaria Grada1, Tiara Byrd1, Kevin Chow1, Simone Krebs1, Helen Heslop1, Stephen Gottschalk1, Eric Yvon2, and Nabil Ahmed1; 1Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, USA; 2MD Anderson Cancer Center, Houston, TX, USA Chimeric antigen receptor (CAR)-modified T cells have shown considerable efficacy in the treatment of cancer. Targeting a single glioma-restricted antigen, however, carries an inherent risk of creating antigen loss tumor cell variants owing to the significant intra- and inter-tumoral heterogeneity that exists in High Grade Glioma (HGG). We have demonstrated that simultaneous targeting of multiple glioma-restricted antigens offsets this escape mechanism leading to improved therapeutic efficacy. Using in silico modeling, we designed a novel bispecific CAR that incorporates a HER2-specific scFv and a mutated IL13 molecule, joined in tandem (TanCAR), to target the glioma-restricted antigens, HER2 and IL13Ra2, respectively. The intracellular T-cell activating domain consisted of a T-cell z-signaling chain and a signaling moiety from the co-stimulatory molecule CD28. T cells from HGG patients were genetically engineered to express the TanCAR using a retroviral system and the surface expression of the extracellular antigen-binding domain was confirmed by flowcytometry using HER2- and IL13Ra2-specific strategies. In vitro functional assays, TanCAR T cells distinctly recognized HER2 and IL13Ra2 antigens individually, and interestingly, exhibited enhanced functionality upon encountering both targets simultaneously. HER2/IL13Ra2 bispecific TanCAR T cells showed improved killing of HER2 and IL13Ra2 positive autologous HGG cells and glioma cell lines over HER2- or IL13Ra2- specific CAR T cells from the same patient. Further, adoptively transferred HER2/IL13Ra2 bispecific TanCAR T cells induced regression of established and vascularized human HGG xenografts and improved tumor control compared to their HER2-specific or IL13Ra2-specific counterparts. This demonstrates the potential of TanCAR T cells as a more effective therapeutic T-cell product for future clinical application. HG-061. PRECLINICAL EVALUATION OF DASATINIB ALONE AND IN COMBINATION WITH CABOZANTINIB FOR THE TREATMENT OF DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) IN CHILDREN Nathalene Truffaux1, Cathy Philippe1, Gaetan Cornilleau1, Janna Paulsson2, Felipe Andreiuolo1, Lea Guerrini-Rousseau1, Stephanie Puget1, Birgit Geoerger1, Gilles Vassal1, Arne Ostman2, and Jacques Grill1; 1CNRS UMR 8203 Vectorology and New Anticancer Treatments, University Paris Sud 11; Gustave Roussy Institut, Villejuif, France; 2Dept. of Oncology-Pathology; Karolinska Institutet, Stockholm, Sweden PURPOSE: The platelet-derived growth factor (PDGF) pathway and in particular its receptor A (PDGFRA) is a possible therapeutic target in DIPG. We explored in vitro the efficacy of a multi-tyrosine kinase inhibitor, dasatinib on new DIPG models derived from stereotactic biopsies performed at diagnosis. EXPERIMENTAL DESIGN: Gene expression profile were obtained from 41 DIPG biopsied before treatment and compared with the profiles associated with sensitivity/resistance to dasatinib. A panel of 12 new DIPG cell lines were established, serially passaged and characterized by gene expression microarray and immunohistochemistry. Effects of dasatinib on proliferation, invasion and cytotoxicity were determined on 4 of these cell lines with a range of concentrations from 1 nM to 10 mM using live-cell imaging and cytometric assays. Downstream signaling and receptor tyrosine kinases (RTK) were assessed by Western blot and phospho-RTK array. Combination with cabozantinib (inhibitor of MET and other RTKs) was studied according to the Chou-Talalay method. RESULTS: DIPG primary tumors and cell lines exhibited the signature of sensitivity to dasatinib previously described for lung and breast carcinoma cell lines. Dasatinib reduced cell growth (IC50 ¼ 10-100 nM), migration (30-50% reduction) and invasion (30-60% reduction) at 100 nM and induced apoptosis in 1 out 4 DIPG cell lines. These effects were similar to those seen in T98G cells, the prototypic dasatinib sensitive cell line. Activity of downstream effectors of dasatinib targets was strongly reduced. Multiple RTKs were activated simultaneously in DIPG cell lines, including MET, suggesting benefit of combinations with other RTK inhibitors. Since MET was shown to be altered (amplification/mutation) in DIPG, we tested combination treatment with dasatinib and cabozantinib. Interestingly, synergistic effects were observed in 3 out 4 cell lines. CONCLUSIONS: Dasatinib exhibits anti-tumor effects in vitro on DIPG cell lines that could be increased by the combination with another RTK inhibitor targeting MET. HG-062. WHOLE EXOME SEQUENCING OF PEDIATRIC HIGH GRADE GLIOMAS REVEALS RECURRENT TARGETABLE SOMATIC MUTATIONS AND FREQUENT PATHOGENIC ALTERATIONS IN CANCER SUSCEPTIBILITY GENES D. Williams Parsons1, Frank Lin1, Lisa R. Trevin˜ o2, Felicia Gao1, Xiaoyun Shen1, Oliver Hampton2, Holly Lindsay1, Mari Kosigo1, Lin Qi1, Patricia A. Baxter1, Jack M. Su1, Murali Chintagumpala1, Robert Dauser1, Adekunle Adesina1, Sharon E. Plon1, Xiao-Nan Li1, David A. Wheeler2, and Ching C. Lau1; 1Texas Children’s Cancer Center, Houston, TX, USA; 2Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX, USA Children with pediatric high grade gliomas (HGG) have a dismal prognosis and novel treatment approaches are needed. To further characterize the genetic landscape of HGG and provide insight into potential clinically-relevant targets, we performed whole exome sequencing (WES) and high density SNP array analysis of 21 tumor-blood pairs obtained from 16 patients with glioblastoma multiforme (GBM) and 5 patients with diffuse intrinsic pontine glioma (DIPG). WES revealed a median of 28 somatic mutations per tumor, including six "hypermutated" cases with .150 mutations. Recurrent somatic mutations were identified in known HGG genes (e.g. H3F3A, ATRX, PDGFRA, RB1, ACVR1), with TP53 most frequently targeted (11/21, 52%). Notably, somatic Ras/MAPK pathway mutations (NF1, BRAF, KRAS) were identified in 8/21 tumors (38%) and PI3K pathway mutations (PIK3CA, PIK3R1) found in 6/21 cases (29%). Analysis of germline WES data revealed homozygous inactivation of PMS2 and MSH6 in two children with Turcot syndrome that confirmed prior clinical genetic testing in those patients. Putative cancer susceptibility mutations were also identified in 4 additional children without known familial cancer syndromes, including two patients with missense TP53 mutations, one patient with a heterozygous frameshift in MSH6, and one patient with a heterozygous truncating PMS2 mutation. Overall, 6/ 21 (29%) of patients sequenced were found to harbor pathogenic germline mutations in cancer susceptibility genes associated with risk of HGG development, including 5 of the 6 patients with hypermutated tumors. Orthotopic xenograft models and cell lines have been developed from a subset of these cases and are currently being utilized to determine the functional effects of the somatic mutations identified. In summary, next-generation sequencing of pediatric HGG has identified recurrent somatic alterations in potentiallytargetable pathways and revealed pathogenic alterations in cancer susceptibility genes in a substantial minority of patients, emphasizing the need for consideration of familial cancer syndromes in children with these tumors. HG-063. HISTOPATHOLOGICAL GRADING ACCORDING TO THE WHO CLASSIFICATION OF CNS TUMORS AND H3.3K27 MUTATIONAL STATUS REPRESENT INDEPENDENT PROGNOSTIC MARKERS IN PEDIATRIC HIGH GRADE GLIOMAS Torsten Pietsch1, Gerrit Gielen1, Anja zur Muehlen1, Robert Kwiecien2, Johannes Wolff3, and Christof Kramm4; 1Institute of Neuropathology, University of Bonn, Bonn, Germany; 2Institute of Biostatistics and Clinical Research, WW University of Muenster, Muenster, Germany; 3Department of Pediatric Hematology/Oncology, Cleveland Clinic, Cleveland, OH, USA; 4Department of Pediatric Hematology/Oncology, University of Goettingen, Goettingen, Germany While the prognostic impact of histological grading is well established in adult high grade gliomas, this has not been shown in large series of pediatric HGG patients. In this context, the relation to K3.3K27 mutations as a typical alteration in pediatric HGG of the midline is unclear. We have analysed samples from 159 HGG pediatric patients (age range, 0-18 years; 31 patients ,3 years at diagnosis) treated with an age adapted postoperative (radio)chemotherapy according to the German HIT-HGG/GBM or HIT-SKK (infant) protocols for the presence of H3.3K27 mutations by pyrosequencing. Histopathological classification was performed according to the revised WHO classification of CNS tumors 2007. Histological analysis led to the diagnosis of 44 anaplastic astrocytoma (WHO grade III, AAIII) and 115 glioblastomas (WHO grade IV; GBMIV). 27.7 % of these HGGs carried H3.3K27 mutations, and these mutated cases were mostly located in the midline and occured in non-infants. A significant superior overall survival in diffuse pediatric HGG patients graded as AA III in contrast to GBM IV was detected (for all patients, p ¼ 0.002, for non-infants only, p ¼ 0.025, log-rank-test). The presence of H3.3K27 mutations was a significant adverse prognostic marker. Multivariable analysis showed that WHO grading and H3.3K27 mutational status were independent prognostic markers. By combining these two features in Kaplan-Meier analysis for grouping non-infant patients, overall survival was best for patients with H3.3K27 wildtype(wt) AAIII (n ¼ 19, 5y-OS, 30.5 +/- 13.6 %, followed by H3.3wt GBM IV (n ¼ 40, 5y-OS, 10.6 +/- 5.7%), and H3.3K27 mutated cases did worse with lowest survival rates, irrespective to WHO grading (AAIII H3.3K27 mutated, n ¼ 11, 5y-OS, 9.1 + /-8.7%; GBMIV H3.3 mutated, n ¼ 30, 5y-OS, 0%). These data suggest that WHO histological grading and H3.3 mutational analysis allows a three-tier prognostic stratification of pediatric HGG patients and risk-adapted therapeutic design. HG-064. MicroRNA PROFILING REVEALS TWO SUBGROUPS OF DIFFUSE INTRINSIC PONTINE GLIOMA Rishi R. Lulla1, Joseph Laskowski1, Stewart Goldman1, Vidya Gopalakrishnan2, and Jason Fangusaro1; 1Northwestern University Feinberg School of Medicine, Ann & Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, USA; 2University of Texas MD Anderson Cancer Center, Houston, TX, USA BACKGROUND: Progress in understanding the biology of diffuse intrinsic pontine glioma (DIPG) has been limited by a lack of tumor specimens. Recent work has focused on protein, mRNA and methylation profiles as well as mutations in the genes encoding Histone 3.3. MicroRNAs are dysregulated in cancer, including pediatric CNS tumors. We aim to describe microRNA expression patterns in DIPG and their relationship to H3F3A K27M mutation status. METHODS: Tumor samples from patients DIPG at autopsy (n ¼ 14) were obtained. RNA purification from FFPE was performed using RecoverAllTM RNA isolation kit was performed and used for cDNA synthesis. TaqManw miRNA assays were used to quantify the levels of 762 mature miRNAs from each sample using the Applied Biosystems 7900HT Fast Real-Time PCR system in 384-well low density arrays (TLDAs). Evaluation of H3K27me3 is underway by immunohistochemistry. Clinical and pathologic data were collected when available. RESULTS: Tumor tissue was available for 14 patients. Histologically, 13 samples were malignant glioma and 1 was described as a PNET. Unsupervised hierarchical clustering based on miRNA expression pattern revealed 2 statistically distinct groups with 4 tumors in one (Group A) and 10 in the other (Group B). Clinical features of both groups including age and time to progression are similar. Group A has significant overexpression of several miRNAs including miR-197, miR-661 and miR-328 (p ,0.001) that are associated with tumor development and progression. Validation of differentially expressed miRNAs as well stratification by H3F3A K27M mutation status will be presented. CONCLUSIONS: To our knowledge, this work represents the first microRNA profiling in DIPG and identified two distinct subgroups with differentially expressed microRNAs. HG-065. INTEGRATIVE ANALYSIS OF THE CODING AND NON-CODING GENOME REVEALS NOVEL TARGETS OF TRANSCRIPTIONAL REGULATION IN DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) Alan Mackay, Katy Taylor, Mara Vinci, and Chris Jones; Institute of Cancer Research, London, UK Whole genome sequencing of diffuse intrinsic pontine glioma (DIPG) has to date focused on somatic coding variants, revealing highly recurrent mutations in the histone variants H3.3 (H3F3A) and H3.1 (HIST1H3B). These K27M substitutions confer a transdominant negative effect on H3K27 trimethylation, and have profound consequences on gene expression. We sought to exploit the wealth of information inherent in genome sequencing data in both coding and non-coding regions to identify novel mutational processes which may play a significant role in DIPG tumorigenesis. The somatic mutation rate in DIPGs ranged from 0.18-1.57 single nucleotide variants (SNVs)/Mb (1511-4951 variants/sample), with a relatively small number of somatic non-synonymous coding variants compared to other cancers (8-22), and a mutational signature characterised by a prominence of C . T substitutions at NpCpG trinucleotides. 757 recurrent non-coding variants (NCVs) were identified, with 15% located in upstream or downstream regulatory regions, including 79 genes overexpressed in DIPG including HOXA3, FGFR3, and BMPR1B. A total of 3777 NCVs (9.2%) were identified within ENCODE consensus transcription factor binding sites (TFBS), with significant enrichment of 67 regulatory motifs (e.g. POU2F2, HDAC2, p300) and 35 bound transcription factors. These included components of the PRC2 complex (EZH2, SUZ12), which regulate H3K27me3-mediated transcriptional repression as well as ZNF274, TRIM28 and SETDB1 that combine to target H3 histones at Lys9. One enriched site was TCF7L2, which is itself subject to somatic mutation, overexpression and promoter hypomethylation in DIPG. Several alterations were observed in regulatory regions of genes which also harboured coding mutations. IGF2R carried mutually exclusive recurrent somatic coding variants, NCVs in a TFBS, and a germline coding variant in DIPG samples. Taken together, the mutually exclusive patterns of somatic mutations in protein coding genes and their regulatory regions can identify functionally recurrent pathways in DIPG which may guide future therapeutic intervention. HG-066. THE BATS DIPG STUDY: A NATIONAL CLINICAL TRIAL OF UPFRONT BIOPSY AND TREATMENT STRATIFICATION FOR NEWLY DIAGNOSED DIFFUSE INTRINSIC PONTINE GLIOMA (DFCI #10-321) Mark Kieran1, Adam Fontebasso3, Simon Papillon-Cavanagh3, Jeremy Schwartzentruber3, Hamid Nikbakht3, Noha Gerges3, Pierre-Oliver Fiset3, Denise Bechet3, Damien Faury3, Nicolas De Jay3, Lori Ramkissoon1, Aoife Corcoran1, David Jones4, Dominik Sturm4, Pascal Johann4, Tadanori Tomita5, Stewart Goldman5, Mahmoud Nagib6, Anne Bendel6, Liliana Goumnerova2, Daniel C. Bowers7, Jeffrey R. Leonard8, Joshua B. Rubin8, Tord Alden5, Arthur DiPatri5, Samuel Browd9, Sarah Leary9, George Jallo10, Kenneth Cohen10, Michael D. Prados11, Anuradha Banerjee11, Anne-Sophie Carret12, Benjamin Ellezam12, Louis Crevier12, Almos Klekner13, Laszlo Bognar13, Peter Hauser14, Miklos Garami14, John Myseros15, Zhifeng Dong16, Peter M. Siegel16, William Gump17, Kanyalakshmi Ayyanar17, John Ragheb18, Ziad Khatib18, Mark Krieger19, Erin Kiehna19, Nathan Robison19, David Harter20, Sharon Gardner20, Michael Handler21, Nicholas Foreman21, Barunashish Brahma22, Tobey MacDonald22, Hayley Malkin1, Susan Chi1, Peter Manley1, Pratiti Bandopadhayay1, Lianne Greenspan1, Azra Ligon1, Steffen Albrecht3, Stefan M. Pfister4, Keith L. Ligon1, Jacek Majewski3, Nalin Gupta11, and Nada Jabado3; 1Dana-Farber Cancer Institute, Boston, MA, USA; 2Boston Children’s Hospital, Boston, MA, USA; 3Montreal Children’s Hospital, Montreal, QC, Canada; 4German Cancer Research Center (DKFZ), Heidelberg, Germany; 5Ann & Robert H. Lurie Children’s Hospital, Chicago, IL, USA; 6Children’s Hospital and Clinics of Minnesota, Minneapolis, MN, USA; 7University of Texas Southwestern Medical Center, Dallas, TX, USA; 8Washington University School of Medicine, St. Louis, MO, USA; 9Seattle Children’s Hospital, Seattle, WA, USA; 10The Johns Hopkins Hospital, Baltimore, MD, USA; 11University of California, San Francisco, San Francisco, CA, USA; 12CHU Ste-Justine, Montreal, QC, Canada; 13Medical and Health Science Center, Debrecen, Hungary; 14Semmelweis University, Budapest, Hungary; 15Children’s National Medical Center, Washington, DC, USA; 16Rosalind and Morris Goodman Cancer Research Centre, Montreal, QC, Canada; 17University of Louisville, Louisville, KY, USA; 18Miami Children’s Hospital, Miami, FL, USA; 19Children’s Hospital Los Angeles, Los Angeles, CA, USA; 20New York University Medical Center, New York, NY, USA; 21Children’s Hospital Colorado, Aurora, CO, USA; 22Children’s Healthcare of Atlanta, Atlanta, GA, USA BACKGROUND: The outcome of diffuse intrinsic pontine glioma (DIPG), based on clinical symptoms and imaging appearance rather than tissue, has not improved in the last 30 years. With dramatic advances in MR-imaging, neurosurgical techniques and molecular profiling of very small samples, the dogma that DIPG patients should not be biopsied needs to be revisited. METHODS: A collaboration of 22 clinical institutions throughout the United States joined together into a DIPG treatment consortium called BATS (Biology and Treatment Strategies) DIPG. Each neurosurgeon involved in the protocol is required to undergo specific operative (or surgical) training and all patients are monitored with extensive clinical and regulatory oversight to ensure that biopsies can be performed safely in a multi-institutional practice. RESULTS: To date, 28 patients with newly diagnosed classic DIPG (as defined by ,3 months of clinical symptoms and diffuse expansion of the pons on MRI) have undergone biopsy. Tumor tissue for molecular analysis was successfully obtained in 27 of 28 cases. Only one patient experienced significant morbidity possibly associated with the biopsy. Immunohistochemical profiling and genomic sequencing has revealed high quality tumor tissue and has resulted in the identification of a number of altered signaling pathways (EGFR, PI3K, PDGRFa, FGFR1 and ACVR1), resistance pathways (p53, MGMT) and epigenetic alterations (histone H3 mutations). CONCLUSIONS: Initial results from the BATS DIPG multi-institutional consortium has confirmed the safety of upfront biopsy for patients with DIPG and has allowed us to tailor therapy to the individual patient. Data regarding the identification of important molecular pathways in this disease will be presented. HG-067. MUTATIONS IN ACVR1 ALONG WITH K27M H3.1 INDUCES ECTOPIC PROLIFERATING LESIONS IN VIVO Christine Hoeman1, Francisco Cordero1, Kyle Halvorson1, Cynthia Hawkins2, and Oren Becher1; 1Duke University, Durham, NC, USA; 2University of Toronto, Toronto, ON, Canada BACKGROUND: Diffuse intrinsic pontine gliomas (DIPG) are the leading cause of death for children diagnosed with brain tumors. As there are currently no effective treatments, it is important to develop a mouse model that mimics the human disease in order to understand the biology of these tumors. Recently it has been shown that activating mutations in ACVR1, which encodes for the Activin A receptor (ALK-2), are found in 20% of DIPG patients. However, how these mutations affect tumors remains unknown. METHODS: Through the use of the RCAS/t-va system, we were able to study the effects of ACVR1 mutations in vitro and in vivo. Neurospheres generated from p3 Nestin t-va p53 floxed (Np53fl/fl) mice were infected in vitro with mutated RCAS R206H-ACVR1, G328V-ACVR1, G328E-ACVR1 virus and compared to WT-ACVR1. Proliferation was measured by BrdU assay. For in vivo studies, DF1 cells expressing RCAS R206H-ACVR1, G328V-ACVR1, G328E-ACVR1, or WT-ACVR1 along with RCAS K27M H3.1 and RCAS Cre were injected intracranially into Np53fl/fl mice to study possible glioma formation. RESULTS: Neurospheres infected with R206H-ACVR1 or G328V-ACVR1 demonstrated increased proliferation in vitro as compared to WT-ACVR1. Likewise, mice that were injected with R206H-ACVR1 or G328VACVR1, K27M H3.1, and Cre exhibited ectopic proliferating lesions in the upper or middle brainstem in vivo. Interestingly, mice injected with RCAS R206H-ACVR1 and Cre did not exhibit such lesions, suggesting a cooperation with K27M H3.1. Characterization of these lesions reveals that they are Olig2 negative and Nestin positive. CONCLUSIONS: Based on our results, mutations in ACVR1, in particular R206H and G328V, lead to increased proliferation in vitro. More importantly, these mutations along with K27M H3.1 and p53 loss lead to the formation of ectopic proliferating lesions in vivo. Further characterization of these lesions and determining the mechanism of lesion formation is ongoing and will be useful in treating DIPG. HG-068. TARGETING mTOR AND NOTCH IN DIFFUSE INTRINSIC PONTINE GLIOMA Isabella Taylor1, Marianne Hutt1, Melanie Weingart1, Antoinette Price1, Javad Nazarian2, Charles Eberhart1, and Eric Raabe1; 1Johns Hopkins School of Medicine, Baltimore, MD, USA; 2Children’s National Medical Center, Washington, DC, USA Diffuse intrinsic pontine glioma (DIPG) is known to harbor mutations in upstream activators of the mammalian target of rapamycin (mTOR) such as PI3K, PDGFR and EGFR, and our high-throughput analysis of autopsyderived DIPG samples identified increased expression of Notch pathway effectors. However, while Notch is important for proliferation and selfrenewal of adult GBM, and mTOR controls metabolism and growth in multiple cell types, their roles in DIPG are unknown. We confirmed that both primary DIPG samples and DIPG cell lines expressed high levels of Notch using western blot and qPCR assays. Because mTOR and Notch pathways are known to interact in other contexts, we examined the effects of blocking them alone and in combination in the established DIPG neurosphere lines JHH DIPG1, SF7761, SUDIPG2 and SUDIPG4. Gamma-secretase inhibitor (GSI) treatment inhibited NOTCH activity in a dose dependent manner, as measured by decreased expression of pathway targets Hes1 and Hey1. We also detected significant reductions in cellular proliferation and growth. To verify that the Notch pathway was being targeted, short hairpin RNAs targeting the core binding factor (CBF) were utilized. CBF/p300 is the common transcriptional modulator essential to canonical Notch signaling. Western blots showed that CBF and Hes1 were both decreased compare to the scrambled control. shCBF also led to decreased proliferation as measured by BrdU (p ,0.05) and increased cell death as measured by cleaved caspase 3 (p ,0.05). We observed a similar decrease in proliferation and increase in apoptosis using the dual TORC1/TORC2 inhibitor PP242. Combination therapy with MRK003 and PP242 led to a significant 2-fold increase in apoptosis compared to either treatment alone. These data suggest that dual targeting of the mTOR and NOTCH pathways with GSI will decrease DIPG cell growth and proliferation, increase cell death, and reduce tumorigenicity. HG-069. NIMOTUZUMAB EXPERIENCE IN PEDIATRIC HIGH-GRADE GLIAL TUMOURS Mehmet Kantar1, Sebnem Onen1, Serra Kamer2, Tuncer Turhan3, Omer Kitis4, Yesim Ertan5, Nazan Cetingul1, Yavuz Anacak2, Taner Akalin5, and Yusuf Ersahin3; 1Ege University School of Medicine, Department of Pediatrics, Pediatric Oncology Unit, Izmir, Turkey; 2Ege University School of Medicine, Department of Radiation Oncology, Izmir, Turkey; 3Ege University School of Medicine, Department of Neurosurgery, Izmir, Turkey; 4Ege University School of Medicine, Department of Radiology, Neuroradiology Unit, Izmir, Turkey; 5Ege University School of Medicine, Department of Pathology, Izmir, Turkey Treatment of high-grade brain tumors, especially diffuse intrinsic pontine gliomas or recurrent gliomas is a major problem. Irradiation plus temozolomide (TMZ) is the current combination approach for high grade gliomas, but with poor results. We, therefore, need to have new treatment strategies with novel drugs. In this study we shared our experience with nimotuzumab in high-grade gliomas. We used nimotuzumab (150 mg/m2/w) in combination with vinorelbine (VNR) in 5 DIPG, one glioblastoma multiforme and one recurrent anaplastic ependymoma. In DIPG group median age was 9 years-old (3.5-17), there were 4 female and 1 male. Diagnosis of DIPG was made with MRG in 4 cases, except one who have had biopsy. All were irradiated after diagnosis. TMZ was used in 4 out 5 patients, prior to NMZ-VNR. Upon clinical or radiological progression, NMZ-VNR was started at median 7 (3-13) months. Median time of NMZ-VNR use was 6 months (1.5-15). One patient did not receive TMZ, but still on NMZ-VNR. She is alive for 17 months. Three out of other 4 patients died of progressive disease, but one is still alive for 30 months. We have also used NMZ-VNR in a patient with GBM who progressed at 13 months. Use of NMZ-VNR for 4 months unfortunetely failed to stop progression in this patient. Last patient was an anaplastic ependymoma who relapsed 2 years after inial diagnosis. After second surgery NMZ-VNR combination was started at 27 months. During 9 month-use of this combination she stayed progression-free, however she later progressed and died of disease. Nimotuzumab was very well tolerated. Nimotuzumab plus vinorelbine combination seems to have some benefit in high-grade gliomas. Our results with two patients are encouraging. In children, we need to have more clinical trials with these targeted therapies of high-grade gliomas or recurrent gliomas. HG-070. HISTOLOGICAL AND MOLECULAR ANALYSIS OF A DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) WITH UNUSUAL METASTATIC RECURRENCE IN A 9-YEAR OLD PATIENT Gary Mason, Javad Nazarian, Cherry Ho, Joseph Devaney, Mojca Stampar, Madhuri Kambhampati, Faith Crozier, Gilbert Vezina, Roger Packer, and Eugene Hwang; Children’s National Medical Center, Washington, DC, USA INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor with inadequate therapeutic options. Although recurrence for DIPG is typically localized, a small proportion of DIPGs will have metastases. Recent molecular analysis of DIPGs has led to an increase in understanding of the pathogenesis of the disease. We report a patient with an unusual synchronous intraventricular and local recurrence. We present post-mortem targeted histone sequencing and methylation analyses of both primary and metastatic lesions. CASE: A 9-year old female with localized DIPG was treated on the COG protocol ACNS0927 with radiation therapy and vorinostat. Seven months after completing radiation, despite symptomatic stability, her DIPG recurred synchronously both locally and along the undersurface of the anterior corpus callosum, septum pellucidum, and frontal horns. Her metastasis demonstrated rapid growth and even with subsequent therapy, she ultimately progressed and died. METHODS: Post-mortem tissue was obtained after consent. Brainstem and metastatic tumors were divided into subsections that were alternatively frozen or formalin-fixed. Slides were stained and reviewed by a neuropathologist. DNA was extracted from both primary and metastatic tumors and used for methylation and directed histone sequencing. Data were analyzed using Partek Genome software. RESULTS: The metastatic lesions were of a higher grade with an increased Ki67 proliferation index compared to the primary lesion (30% vs. 8%, respectively). Genomic analysis showed both tumors were H3.1 and H3.3 wild type. Methylation analysis showed the metastatic tumor exhibited a distinct methylation pattern from the brainstem tumor specimens. CONCLUSIONS: This case demonstrates that disseminated DIPG lesions may be more aggressive although similar in genetic makeup to the primary lesion. However, the markedly different methylation patterns may have implications regarding appropriate treatment options in the context of disseminated disease. HG-071. RE-IRRADIATION PLUS BEVACIZUMAB FOR PROGRESSIVE DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) ASSESSMENT OF FEASIBILITY AND TOLERABILITY OF A TREATMENT STRATEGY Stephen Gilheeney, Nathan Millard, Kevin DeBraganca, Yasmin Khakoo, Kim Kramer, Suzanne Wolden, Maria Donzelli, Cheryl Fischer, Mary Petriccione, and Ira Dunkel; Memorial Sloan Kettering Cancer Center, New York, NY, USA BACKGROUND: For DIPG patients, radiation is the only modality shown to impact the natural history of their disease, and patients universally succumb to their disease. Bevacizumab has been shown to sensitize tumor cells from radioresistant niches in pre-clinical models. We now summarize our institutional experience using a re-irradiation strategy and bevacizumab for patients with progressive DIPG. METHODS: With an IRB and Privacy Board waiver, a retrospective analysis of patients with DIPG who had undergone re-irradiation with concomitant bevacizumab was performed. Initial treatment, time to progression, field and modality of re-irradiation, details of bevacizumab dosing, and acute and post-treatment toxicities were assessed. RESULTS: Five patients fit selection criteria. All received initial RT, two with concomitant biological agents. One patient received post-RT convection enhanced delivery of radio-immunotherapy. All patients recurred with worsening clinical symptoms prompting MRI investigation. Median time to progression for re-RT + bev patients (completion of radiation to radiographic progression) was five months (range 5-6 months). Re-irradiation was to the entire tumor - including areas of radiographic progression - via intensity modulated RT (IMRT). Median dose was 2400 cGy (range 2000-2400cGy) in 200cGy fractions. Bevacizumab (10 mg/kg every two weeks) was initiated during the first week of re-irradiation, continuing until progression. Common acute toxicities of bevacizumab (headache, hypertension) were not severe enough to stop treatment. All patients experienced some palliation of their symptoms noted at progression. Two patients imaged post-re-RT had radiographic response. All patients again developed symptoms of clinical progression prompting MRI. Median radiographic/ clinical stability was five months (range 2-7 months). CONCLUSIONS: In our series, re-irradiation with bevacizumab was tolerable and led to clinical stability in all patients treated and with some evidence of radiographic response. Limited survival time precludes long term toxicity assessment. Further study is needed. HG-072. EPIDEMIOLOGY OF MALIGNANT PONTINE GLIOMAS (MPG) IN THE PAEDIATRIC POPULATION IN CANADA: A STUDY OF THE CANADIAN PAEDIATRIC BRAIN TUMOUR CONSORTIUM (CPBTC) Samina Afzal1, Anne-Sophie Carret2, Adam Fleming3, Valerie Larouche4, Shayna Zelcer5, Donna L Johnston6, Maria Kostova7, Chris Mpofu8, Jean-Claude De´carie2, Douglas Strother9, Lucie Lafay-Cousin9, David Eisenstat10, Chris Fryer11, Juliette Hukin11, Ute Bartels12, and Eric Bouffet12; 1IWK Health Centre, Halifax, NS, Canada; 2St Justine Hospital, Montreal, QC, Canada; 3Montreal Children’s Hospital, Montreal, QC, Canada; 4Centre Hospitalier Universitaire de Que´bec, Sainte-Foy, QC, Canada; 5Western University Children’s Hospital, London, ON, Canada; 6Children’s Hospital of Eastern Ontario, Ottawa, ON, Canada; 7Kingston General Hospital, Kingston, ON, Canada; 8Saskatoon Cancer Centre, Saskatoon, SK, Canada; 9Alberta Children’s Hospital, Calgary, AB, Canada; 10Stollery Children’s Hospital,, Edmonton, AB, Canada; 11British Columbia Children’s Hospital, Vancouver, BC, Canada; 12Hospital for Sick Children, Toronto, ON, Canada BACKGROUND: Little is known regarding the epidemiology of MPG in children. OBJECTIVE: To determine the epidemiology of children with MPG diagnosed between 2000 and 2010 in Canada. METHODS: All CPBTC centers were contacted to identify patients , 18 years diagnosed with MPG between 2000-2010. Data collected included postal code, date of diagnosis, age at diagnosis, year and month of diagnosis, gender, clinical symptoms, clinical signs at presentation, description of first MRI imaging, treatment, recurrence and current status. A copy of at least one axial and sagittal picture (T2 or FLAIR) was requested for each patient. RESULTS: 141 (70 males) patients were reported by 14 of 17 CPBTC centers representing more than 90% of the pediatric Canadian population (mean overall incidence: 2.63/1,000,000 children/year). 4 patients were under one year of age, 50(35.5%) were between 1-6 years, 75(53.2%) patients between 6-12 and 12(8.5%) .12 years of age. Large annual variations were observed with a minimum of 5 diagnoses in 2000 and 23 in 2009. There was no evidence of Seasonal variation [winter: 29(20.6%), spring: 38(27.0%), summer: 42(29.8%), autumn: 32(22.7%)]. MRI imaging at the time of diagnosis was typical of DIPG for 119(84.4%) patients and atypical in 22(15.6%). 21 patients underwent a biopsy/resection with histologically: 7 GBM, 7 anaplastic astrocytomas, 6 low grade glioma and 1 PNET. 131 patients received upfront radiation and 62 chemotherapy. Median survival was 10 months, 3 patients are alive and three lost to follow-up at the time of progression. 83% patients died within 18 months of diagnosis and 17% beyond 18 months. 2/3 survivors had typical clinical symptoms and MRI findings of DIPG. CONCLUSION: This first comprehensive epidemiology study of DIPG suggests large annual variations in incidence. Some children with typical features of DIPG can be long-term survivors. However, reasons for such unexpected outcomes are unknown. HG-073. ELEVATED PD-1 EXPRESSION IN TUMOR AND PERIPHERAL BLOOD LYMPHOCYTES OF PEDIATRIC AND ADULT GLIOMA PATIENTS Melody Hsu, Joseph Lasky, Theodore Moore, Linda Liau, Tom Davidson, and Robert Prins; University of California, Los Angeles, CA, USA INTRODUCTION: The dismal outcome for pediatric high-grade gliomas with conventional therapy highlights the need for additional treatment modalities. Immunotherapy through PD-1 directed monoclonal antibody blockade has produced durable responses in several adult malignancies. To investigate the potential role of PD-1 blockade in pediatric gliomas, we analyzed the immunologic profile of adult and pediatric patients in tumors and peripheral blood. METHODS: Flow cytometric analysis of tumor infiltrating lymphocytes (TILs) (n ¼ 4) and peripheral blood mononuclear cells (PBMCs) (n ¼ 8) isolated from glioma patients were compared to PBMCs of healthy donors (n ¼ 4). The percentage of T lymphocytes expressing CD4, CD8, CD69, PD-1 and Treg markers were compared between patient PBMCs, TILs and donor PBMCs. RESULTS: The proportion of TILs expressing PD-1 was significantly elevated in the CD3+CD4+ T cell population (mean +SE: 61.90% + 4.85%) as compared to patient PBMCs (33.10% + 8.74%, p , 0.05) or donor PBMCs (20.22% + 4.81%, p , 0.001). Similarly, the percentage of CD3+CD8+ cells expressing PD-1 was also higher in TILs (56.08% + 8.08%) than in patient PBMCs (24.39% + 5.73%; p , 0.05) or donor PBMCs (16.56% + 3.48%; p , 0.01). Activation marker CD69 expression on CD3+CD8+ cells was elevated in TILs compared to patient or donor PBMCs (57.28% + 15.91% vs. 3.67% + 2.63% vs. 2.25% + 0.65%) with p-values ,0.05. The CD4 to CD8 ratio was significantly lower in TILs (mean +SE: 0.49 + 0.22) than donor PBMCs (1.577 + 0.23, p , 0.01). CONCLUSION: TILs isolated from glioma patients express a significantly increased proportion of PD-1 as compared to donor PBMCs. A similar increase in PD-1, although not significant, was also observed between patient and normal donor PBMC. These results suggest that a significant population of T lymphocytes are functionally impaired in glioma patients and represents a verifiable target for checkpoint inhibitor blockade in these patients. BACKGROUND: The International Diffuse Intrinsic Pontine Glioma (DIPG) Registry is a collaborative effort by physicians and researchers from North America, Europe, and Australia to centralize and standardize the collection of clinical, radiographic, pathology and genomics data. The goal is to develop new approaches to diagnosis, response assessment, treatment and follow-up to improve patient outcome. METHOD: The International DIPG Registry website (dipgregistry.org) facilitates education, recruitment and enrollment. Data are collected by registry staff, working directly with patient families and institutions. Living patients are referred through the website. Institutions provide deceased patient records after institutional approval. Demographic, clinical, treatment, and outcome data are abstracted by DIPG Registry staff using standarized case report forms (CRFs), coded and stored in an Oncorew clinical data base. Anonymized MRIs are uploaded utilizing Amicasw PACS Research System. Studies at diagnosis, and each progression or response are requested. A pathology repository stores tissue samples for central review and future research and will be linked to a bioinformatics repository of DIPG genomics data (dipg.progenetix.org). Collectively these repositories form the DIPG Registry. Overseeing the strategic direction of the registry are scientific and steering committees who also review and approve proposals from investigators around the world. RESULTS: Since April 2012, 195 patients have been enrolled; with 530 additional patients committed from 27 participating institutions internationally. The radiology repository contains over 700 studies from 92 patients. The pathology repository contains tissue on 14 patients, (10 fresh-frozen), with 36 specimens pending submission. Central review of specimens and imaging is on-going. Two approved research proposals include a study of long-term DIPG survivors and an epidemiological study to determine DIPG incidence patterns in North America CONCLUSIONS: The International DIPG registry provides a highly collaborative, international, hypothesis-driven research infrastructure that can support a wide spectrum of interdisciplinary and translational projects in DIPGs for all investigators. HG-075. TUMOUR LYSATE PULSED DENDRITIC CELL VACCINATION FOR CHILDREN WITH HIGH GRADE GLIOMA Rebecca Wallace1, Barry Flutter1, Daniel Morgenestern2, Darren Hargrave2, Esther Blanco2, Karen Howe2, Mark Lowdell3, Edward Samuel3, Antony Michalski2, and John Anderson1; 1Institute of Child Health, UCL, London, UK; 2Great Ormond Street Hospital for Children NHS Trust, London, UK; 3Royal Free Hospital, UCL, London, UK We have developed a phase I trial will assess the safety and feasibility of delivering tumour lysate pulsed dendritic cell vaccines in paediatric patients with a primary diagnosis of high grade glioma. The principle of anti cancer immunotherapy is to generate potent anti tumour responses, resulting in the generation of effector cells capable of specifically killing tumour cells. In diseases such as melanoma this approach has been used successfully to generate responses against defined tumour associated antigens (TAA). An alternative approach, for tumours in which TAAs have either not been defined, or are not sufficiently immunogenic, is to generate responses against a number of different but undefined tumour antigens using a n autologous tumour lystate. We have evaluated immune responses following vaccination with alpha type 1 polarised dendritic cells matured with both type 1 and 2 interferons in serum free medium, which have previously been shown to generate high levels of IL-12. The trial also is also evaluating the strength of immune responses following maturation in the presence or absence of prostaglandin E2. Dendritic cell vaccines are given in relapse or as primary treatment in combination with temozolomide chemotherapy. Although dendritic cell vaccination has been used before in children with cancer including high grade glioma, the incorporation of vaccinations into the context of standard chemotherapy is relatively novel, Hence the trial is predominantly addressing the question of the feasibility of delivering such a vaccination strategy in this setting. Additionally the study will also provide data on the nature of the immune response against the tumour and is designed to answer questions regarding the optimal way to activate autologous dendritic cells in the laboratory. Immune response data from the first 4 patients will be presented. HG-076. EFFICACY OF BEVACIZUMAB PLUS IRINOTECAN IN CHILDREN WITH RECURRENT OR PROGRESSIVE MALIGNANT GLIOMA Yoshiki Arakawa1, Katsutsugu Umeda2, Ken-ichiro Watanabe2, Takashi Mizowaki3, Masahiro Hiraoka3, Hidefumi Hiramatsu2, Souichi Adachi2, Takeharu Kunieda1, Yasushi Takagi1, and Susumu Miyamoto1; 1Department of Neurosurgery, Kyoto University Graduate School of Medicine, Kyoto, Japan; 2Department of Pediatrics, Kyoto University Graduate School of Medicine, Kyoto, Japan; 3Department of Radiation Oncology and Image-Applied Therapy, Kyoto University Graduate School of Medicine, Kyoto, Japan BACKGROUND: Bevacizumab (BEV) has been reported to be effective for recurrent adult malignant glioma. However, its efficacy remains controversial in recurrent or progressive pediatric malignant glioma. Here we report two children with recurrent or progressive malignant glioma treated with bevacizumab plus irinotecan (CPT-11). PATIENTS AND METHODS: Case 1; 5-year-old boy with recurrent glioblastoma after radiotherapy with concomitant and adjuvant temozolomide following surgery. Case 2; 12-years-old boy with progressive anaplastic astrocytoma during radiotherapy with concomitant temozolomide following biopsy. These children were administered 10 mg/kg BEV and 125mg/m2 CPT-11 every 2 weeks until progressive disease, unacceptable toxicity. RESULTS: In Case 1, posterior reversible encephalopathy syndrome (PRES) associated with hypertension occurred after 13 cycles of BEV. A month cessation of BEV improved PRES. The intensity of his chemotherapy was reduced as 7 mg/kg BEV and 125mg/m2 CPT-11 every 3 weeks. The tumor has not recurred 2 years after the first recurrence. In Case 2, 10 cycles of BEV and CPT-11 were administered and the tumor has shown partial response. CONCLUSION: The combination of BEV and CPT-11 appears to produce sustained disease control in some children with recurrent or progressive malignant glioma. Pediatric glioblastomas (GB) are highly aggressive and lethal tumors. Recent sequencing studies have shown that 30% of pediatric GBM show mutations in the H3F3A gene, a variant encoding histone H3.3. H3F3A K27M mutations lead to global reduction in H3K27me3. Our goal was to evaluate the utility of this reduction in H3K27me3 as a clinical and histopathologic biomarker. To address this question we assessed H3K27me3 by immunohistochemistry in 160 (118 pediatric and 42 adult) brain tumors of various subtypes and grades and 45 non-neoplastic reactive brain tissues. Global reduction in H3K27me3 was specific to H3F3A K27M mutant GB and was not observed in all other examined conditions. Further, H3F3A K27M mutant GB with reduced H3K27me3 exhibited a significantly poor prognosis compared to H3F3A wild type tumors with preserved H3K27me3 (Log-rank (Mantel-Cox) test hazards ratio ¼ 2.9, p ¼ 0.0021). These results suggest that global reduction in H3K27me3 is a histopathologic molecular surrogate for H3F3A K27M mutant GB and defines a prognostically poor subset of pediatric GB.

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