Pediatric Glioblastoma Research Articles

Evaluation of Histone 3 Lysine 27 Trimethylation (H3K27me3) and Enhancer of Zest 2 (EZH2) in Pediatric Glial and Glioneuronal Tumors Shows Decreased H3K27me3 in H3F3A K27M Mutant Glioblastomas

H3F3A mutations are seen in ∼30% of pediatric glioblastoma (GBMs) and involve either the lysine residue at position 27 (K27M) or glycine at position 34 (G34R/V). Sixteen genes encode histone H3, each variant differing in only a few amino acids. Therefore, how mutations in a single H3 gene contribute to carcinogenesis is unknown. H3F3A K27M mutations are predicted to alter methylation of H3K27. H3K27me3 is a repressive mark critical to stem cell maintenance and is mediated by EZH2, a member of the polycomb-group (PcG) family. We evaluated H3K27me3 and EZH2 expression using immunohistochemistry in 76 pediatric brain tumors. H3K27me3 was lowered/absent in tumor cells but preserved in endothelial cells and infiltrating lymphocytes in six out of 20 GBMs. H3K27me3 showed strong immunoreactivity in all other tumor subtypes. Sequencing of GBMs showed H3F3A K27M mutations in all six cases with lowered/absent H3K27me3. EZH2 expression was high in GBMs, but absent/focal in other tumors. However, no significant differences in EZH2 expression were observed between H3F3A K27M mutant and wild type GBMs, suggesting that EZH2 mediated trimethylation of H3K27 is inhibited in GBM harboring K27M mutations. Our results indicate that H3F3A K27M mutant GBMs show decreased H3K27me3 that may be of both diagnostic and biological relevance.

H3F3A K27M Mutation in Pediatric CNS Tumors

Brain tumors are one of the most common childhood malignancies. Diffuse high-grade gliomas represent approximately 10% of pediatric brain tumors. Exon sequencing has identified a mutation in K27M of the histone H3.3 gene (H3F3A K27M and G34R/V) in about 20% of pediatric glioblastomas, but it remains to be seen whether these mutations can be considered specific for pediatric diffuse high-grade astrocytomas or also occur in other pediatric brain tumors. We performed a pyrosequencing-based analysis for the identification of H3F3A codon 27 and codon 34 mutations in 338 pediatric brain tumors. The K27M mutation occurred in 35 of 129 glioblastomas (27.1%) and in 5 of 28 (17.9%) anaplastic astrocytomas. None of the other tumor entities showed H3F3A K27M mutation. Because H3F3A K27M mutations occur exclusively in pediatric diffuse high-grade astrocytomas, analysis of codon 27 mutational status could be useful in the differential diagnosis of these neoplasms.

Chromatin Remodeling Defects in Pediatric and Young Adult Glioblastoma: A Tale of a Variant Histone 3 Tail

Primary brain tumors occur in 8 out of 100 000 people and are the leading cause of cancer-related death in children. Among brain tumors, high-grade astrocytomas (HGAs) including glioblastoma multiforme (GBM) are aggressive and are lethal human cancers. Despite decades of concerted therapeutic efforts, HGAs remain essentially incurable in adults and children. Recent discoveries have revolutionized our understanding of these tumors in children and young adults. Recurrent somatic driver mutations in the tail of histone 3 variant 3 (H3.3), leading to amino acid substitutions at key residues, namely lysine (K) 27 (K27M) and glycine 34 (G34R/G34V), were identified as a new molecular mechanism in pediatric GBM. These mutations represent the pediatric counterpart of the recurrent mutations in isocitrate dehydrogenases (IDH) identified in young adult gliomas and provide a much-needed new pathway that can be targeted for therapeutic development. This review will provide an overview of the potential role of these mutations in altering chromatin structure and affecting specific molecular pathways ultimately leading to gliomagenesis. The distinct changes in chromatin structure and the specific downstream events induced by each mutation need characterizing independently if progress is to be made in tackling this devastating cancer.

H3.3 G34R mutations in pediatric primitive neuroectodermal tumors of central nervous system (CNS-PNET) and pediatric glioblastomas: possible diagnostic and therapeutic implications?

Pediatric glioblastomas recently have been exon sequenced with evidence that approximately 30 % of cases harbour mutations of the histone H3.3 gene. Although studies to determinate their role in risk stratification are on-going, it remains to be determined whether H3.3 mutations could be found in other tumors such as pediatric primitive neuroectodermal tumors of the central nervous system (CNS-PNETs) and whether the presence of H3.3 mutations in glioblastomas could be used as diagnostic tool in their differential diagnosis with CNS-PNETs. We performed a large mutational pyrosequencing-based screening on 123 pediatric glioblastomas and 33 CNS-PNET. The analysis revealed that 39/123 (31.7 %) glioblastomas carry H3.3 mutations. The K27M (AAG → ATG, lysine → methionine) mutation was found in 33 glioblastomas (26 %); the G34R (GGG → AGG, glycine → arginine) was observed in 6 glioblastomas (5.5 %). However, we also identified 4 of 33 cases (11 %) of CNS-PNETs harbouring a H3.3 G34R mutation. Multiplex ligation-dependent probe amplification analysis revealed PDGFR-alpha amplification and EGFR gain in two cases and N-Myc amplification in one case of H3.3 G34R mutated CNS-PNET. None of H3.3 mutated tumors presented a CDKN2A loss. In conclusion, because pediatric patients with glioblastoma and CNS-PNET are treated according to different therapeutic protocols, these findings may raise further concerns about the reliability of the histological diagnosis in the case of an undifferentiated brain tumor harbouring G34R H3.3 mutation. In this view, additional studies are needed to determine whether H3.3 G34 mutated CNS-PNET/glioblastomas may represent a defined tumor subtype.

Paediatric spinal glioblastoma: case report and review of therapeutic strategies

Although uncommon, there is significant morbidity and mortality associated with paediatric spinal glioblastoma. The paucity of cases makes treatment options difficult. The current recommended standard of care is biopsy followed by adjuvant chemo-radiotherapy, with emerging data supporting the role of safe gross total resection. The purpose of this paper is to provide a single-institution case study and to discuss current and future therapeutic treatment strategies. A 14-year-old boy presented with a 2-year history of intermittent back pain with recent progressively worsening motor and sensory deficits of the right side. Pre-operative MRI revealed an enhancing intra-medullary tumour extending from C2 to C7. During the operative case, no tumour-cord margin could be identified, and the patient underwent a subtotal excision. Histopathology confirmed glioblastoma. In the subsequent weeks, the patient's clinical condition deteriorated. Adjuvant therapy was declined by the family, and the patient died 9weeks after initial presentation. Despite major advances in surgical techniques, peri-operative neuro-imaging as well as chemo-radiotherapy, the prognosis of a paediatric intra-medullary high-grade spinal tumour remains poor. Detailed analysis of our understanding of tumour dynamics in this patient group is important in establishing future therapeutic strategies.

Giant cell glioblastoma in the pediatric age group: Report of two cases

Giant cell glioblastoma multiforme is a rare subgroup of glioblastoma multiforme. It constitutes about 5% of all glioblastoma cases. Pediatric giant cell glioblastoma is extremely rare. We report two such cases of giant cell glioblastoma in pediatric age group (≤18 years). The pertinent literature is reviewed regarding this uncommon entity.

Low Frequency of Molecular Alterations of H3.3-Atrx-Daxx Chromatin Remodeling Component Genes in Myelodysplastic Syndromes (MDS)

Abstract 3844Novel sequencing technologies have allowed identification of a group of highly recurrent genetic mutations in myelodysplastic syndromes (MDS). Of importance, it has been noticed that a majority of these mutated genes in MDS encode important components of epigenetic regulation, including both DNA methylation and histone modifications. This phenomenon highlights the importance of epigenetic mechanisms in the pathogenesis of MDS. Recently, highly recurrent somatic mutations in the Histone H3.3-ATRX-DAXX chromatin remodeling pathway have been documented in pediatric glioblastoma (Schwartzentruber et al. Nature and Wu et al. Nature Genetics 2012), further supporting the importance of epigenetic regulation for tumorgenesis. We therefore examined potential genetic and epigenetic alterations of the same pathway in MDS. First, in a cohort containing 80 samples of MDS whole bone marrow mononuclear cell DNA (representative of both lower and higher risk disease), we performed Sanger sequencing covering genomic areas of reported mutations of H3F3A, H3F3B, ATRX, and DAXX in glioblastoma. Sequenced genomic areas included reported mutations in pediatric tumors: Lys27 and Gly34 of H3F3A and H3F3B; sequences upstream of and within the helices domain of ATRXX; and the whole coding sequence of DAXX. Overall, we only detected one mutation of H3F3A (K27N) in one MDS case (76 year old male with RA; INT-1; diploid). No other reported mutation of H3F3B, ATRX and DAXX genes was detected in any other patients of this MDS cohort. Because of the potential of epigenetic deregulation, we then examined status of DNA methylation for the promoters of ATRX and DAXX in MDS patients by bisulfite pyrosquencing. While no DNA hypermethylation of DAXX promoter was detected, 8 out of 40 (20%) patients had hypermethylation of the CpG island in the promoter region of ATRX. However, six of these eight patients were females. Based on reports of ATRX methylation in healthy females, it is likely that the 6 cases in female patients represent physiological × chromosome inactivation. Finally, we performed RT-PCR analysis using cDNA samples isolated from CD34+ hematopoietic stem cells of 40 MDS patients. Results indicated that expression of ATRX and DAXX were increased by 2 fold (p-value 0.07) and 5.2 fold (p-value 0.0003) respectively compared to control CD34+ cells. The implications of this phenomenon need to be studied further. Taken together, these results suggest that genetic mutations of the H3.3-ATRX-DAXX chromatin remodeling do not play a role in the pathogenesis of MDS. Disclosures:No relevant conflicts of interest to declare.

Hotspot Mutations in H3F3A and IDH1 Define Distinct Epigenetic and Biological Subgroups of Glioblastoma

Glioblastoma (GBM) is a brain tumor that carries a dismal prognosis and displays considerable heterogeneity. We have recently identified recurrent H3F3A mutations affecting two critical amino acids (K27 and G34) of histone H3.3 in one-third of pediatric GBM. Here, we show that each H3F3A mutation defines an epigenetic subgroup of GBM with a distinct global methylation pattern, and that they are mutually exclusive with IDH1 mutations, which characterize a third mutation-defined subgroup. Three further epigenetic subgroups were enriched for hallmark genetic events of adult GBM and/or established transcriptomic signatures. We also demonstrate that the two H3F3A mutations give rise to GBMs in separate anatomic compartments, with differential regulation of transcription factors OLIG1, OLIG2, and FOXG1, possibly reflecting different cellular origins.

Molecular and Morphologic Correlates of the Alternative Lengthening of Telomeres Phenotype in High‐Grade Astrocytomas

Recent studies suggest that the telomere maintenance mechanism known as alternative lengthening of telomeres (ALT) is relatively more common in specific glioma subsets and strongly associated with ATRX mutations. We retrospectively examined 116 high-grade astrocytomas (32 pediatric glioblastomas, 65 adult glioblastomas, 19 anaplastic astrocytomas) with known ALT status using tissue microarrays to identify associations with molecular and phenotypic features. Immunohistochemistry was performed using antibodies against ATRX, DAXX, p53 and IDH1(R132H) mutant protein. EGFR amplification was evaluated by fluorescence in situ hybridization (FISH). Almost half of fibrillary and gemistocytic astrocytomas (44%) demonstrated ALT. Conversely all gliosarcomas (n = 4), epithelioid (n = 2), giant cell (n = 2) and adult small cell astrocytomas (n = 7) were ALT negative. The ALT phenotype was positively correlated with the presence of round cells (P = 0.002), microcysts (P < 0.0002), IDH1 mutant protein (P < 0.0001), ATRX protein loss (P < 0.0001), strong P53 immunostaining (P < 0.0001) and absence of EGFR amplification (P = 0.004). There was no significant correlation with DAXX expression. We conclude that ALT represents a specific phenotype in high-grade astrocytomas with distinctive pathologic and molecular features. Future studies are required to clarify the clinical and biological significance of ALT in high-grade astrocytomas.

Increased Microglia/Macrophage Gene Expression in a Subset of Adult and Pediatric Astrocytomas

Glioblastoma (GBM) is a highly malignant brain tumor with a dismal prognosis. Gene expression profiling of GBM has revealed clinically relevant tumor subtypes, and this provides exciting opportunities to better understand disease pathogenesis. Results from an increasing number of studies demonstrate a role for the immune response in cancer progression, yet it is unclear how the immune response differs across tumor subtypes and how it affects outcome. Utilizing gene expression data from The Cancer Genome Atlas Project and the Gene Expression Omnibus database, we demonstrate an enrichment of immune response-related gene expression in the mesenchymal subtype of adult GBM (n = 173) and pediatric high-grade gliomas (n = 53). In an independent cohort of pediatric astrocytomas (n = 24) from UCSF, we stratified tumors into subtypes and confirmed these findings. Using novel immune cell-specific gene signatures we demonstrate selective enrichment of microglia/macrophage-related genes in adult and pediatric GBM tumors of the mesenchymal subtype. Furthermore, immunostaining of adult GBM tumors showed significantly higher cell numbers of microglia/macrophages in mesenchymal versus non-mesenchymal tumors (p = 0.04). Interestingly, adult GBM tumors with the shortest survival had significant enrichment of microglia/macrophage-related genes but this was not true for pediatric GBMs. Consistent with an association with poor outcome, immune response-related genes were highly represented in an adult poor prognosis gene signature, with the expression of genes related to macrophage recruitment and activation being most strongly associated with survival (p<0.05) using CoxBoost multivariate modeling. Using a microglia/macrophage high gene signature derived from quantification of tumor-infiltrating cells in adult GBM, we identified enrichment of genes characteristic of CD4 T cells, granulocytes, and microglia/macrophages (n = 573). These studies support a role for the immune response, particularly the microglia/macrophage response, in the biology of an important subset of GBM. Identification of this subset may be important for future therapeutic stratification.

Pediatric glioblastoma: clinico-radiological profile and factors affecting the outcome

Glioblastoma in the pediatric age group is relatively rare. As a result, it has been difficult to deduce any consistent clinico-radiological and pathological profiles on these patients. Also, the prognostic factors affecting the survival in pediatric glioblastoma are not as well defined as in adults. In this retrospective series, 65 pediatric patients (age ≤ 18 years) from January 1995 to December 2011 with histopathologically proven diagnosis of intracranial glioblastoma were studied. Clinico-radiological, pathological, treatment, and follow-up data were collected. Progression-free and overall survivals were assessed using the Kaplan-Meier method. The male-to-female ratio was 2.6:1 with a mean age of 13.29 ± 4.53 years (range 2-18 years). Headache with or without vomiting (n = 51, 78 %), followed by seizures (n = 42, 65 %), and focal deficits (n = 31, 47 %) were the leading symptoms. Forty-nine (75 %) patients had tumors located superficially, whereas there were 16 patients with deeply located glioblastomas (25 %). Gross total tumor excision was achieved in 43 (66 %) patients, while the remaining patients had incomplete excision (n = 22, 34 %). Mean follow-up was 17.7 months (range 1.5-119 months). The median progression-free and overall survivals were 10 and 20 months, respectively. Extent of resection was found to be the independent predictor of survival (p value = 0.002). Pediatric glioblastomas are associated with longer progression-free as well as overall survivals. Extent of tumor resection is the strongest predictor of survival in pediatric glioblastoma. Hence, an aggressive surgical resection may fetch a better outcome in children with glioblastoma.

Frequent ATRX mutations and loss of expression in adult diffuse astrocytic tumors carrying IDH1/IDH2 and TP53 mutations

Gliomas are the most common primary brain tumors in children and adults. We recently identified frequent alterations in chromatin remodelling pathways including recurrent mutations in H3F3A and mutations in ATRX (α-thalassemia/mental-retardation-syndrome-X-linked) in pediatric and young adult glioblastoma (GBM, WHO grade IV astrocytoma). H3F3A mutations were specific to pediatric high-grade gliomas and identified in only 3.4 % of adult GBM. Using sequencing and/or immunohistochemical analyses, we investigated ATRX alterations (mutation/loss of expression) and their association with TP53 and IDH1 or IDH2 mutations in 140 adult WHO grade II, III and IV gliomas, 17 pediatric WHO grade II and III astrocytomas and 34 pilocytic astrocytomas. In adults, ATRX aberrations were detected in 33 % of grade II and 46 % of grade III gliomas, as well as in 80 % of secondary and 7 % of primary GBMs. They were absent in the 17 grade II and III astrocytomas in children, and the 34 pilocytic astrocytomas. ATRX alterations closely overlapped with mutations in IDH1/2 (p < 0.0001) and TP53 (p < 0.0001) in samples across all WHO grades. They were prevalent in astrocytomas and oligoastrocytomas, but were absent in oligodendrogliomas (p < 0.0001). No significant association of ATRX mutation/loss of expression and alternative lengthening of telomeres was identified in our cohort. In summary, our data show that ATRX alterations are frequent in adult diffuse gliomas and are specific to astrocytic tumors carrying IDH1/2 and TP53 mutations. Combined alteration of these genes may contribute to drive the neoplastic growth in a major subset of diffuse astrocytomas in adults.

Loss of ATRX, Genome Instability, and an Altered DNA Damage Response Are Hallmarks of the Alternative Lengthening of Telomeres Pathway

The Alternative Lengthening of Telomeres (ALT) pathway is a telomerase-independent pathway for telomere maintenance that is active in a significant subset of human cancers and in vitro immortalized cell lines. ALT is thought to involve templated extension of telomeres through homologous recombination, but the genetic or epigenetic changes that unleash ALT are not known. Recently, mutations in the ATRX/DAXX chromatin remodeling complex and histone H3.3 were found to correlate with features of ALT in pancreatic neuroendocrine cancers, pediatric glioblastomas, and other tumors of the central nervous system, suggesting that these mutations might contribute to the activation of the ALT pathway in these cancers. We have taken a comprehensive approach to deciphering ALT by applying genomic, molecular biological, and cell biological approaches to a panel of 22 ALT cell lines, including cell lines derived in vitro. Here we show that loss of ATRX protein and mutations in the ATRX gene are hallmarks of ALT–immortalized cell lines. In addition, ALT is associated with extensive genome rearrangements, marked micronucleation, defects in the G2/M checkpoint, and altered double-strand break (DSB) repair. These attributes will facilitate the diagnosis and treatment of ALT positive human cancers.

K27M mutation in histone H3.3 defines clinically and biologically distinct subgroups of pediatric diffuse intrinsic pontine gliomas

Pediatric glioblastomas (GBM) including diffuse intrinsic pontine gliomas (DIPG) are devastating brain tumors with no effective therapy. Here, we investigated clinical and biological impacts of histone H3.3 mutations. Forty-two DIPGs were tested for H3.3 mutations. Wild-type versus mutated (K27M-H3.3) subgroups were compared for HIST1H3B, IDH, ATRX and TP53 mutations, copy number alterations and clinical outcome. K27M-H3.3 occurred in 71 %, TP53 mutations in 77 % and ATRX mutations in 9 % of DIPGs. ATRX mutations were more frequent in older children (p < 0.0001). No G34V/R-H3.3, IDH1/2 or H3.1 mutations were identified. K27M-H3.3 DIPGs showed specific copy number changes, including all gains/amplifications of PDGFRA and MYC/PVT1 loci. Notably, all long-term survivors were H3.3 wild type and this group of patients had better overall survival. K27M-H3.3 mutation defines clinically and biologically distinct subgroups and is prevalent in DIPG, which will impact future therapeutic trial design. K27M- and G34V-H3.3 have location-based incidence (brainstem/cortex) and potentially play distinct roles in pediatric GBM pathogenesis. K27M-H3.3 is universally associated with short survival in DIPG, while patients wild-type for H3.3 show improved survival. Based on prognostic and therapeutic implications, our findings argue for H3.3-mutation testing at diagnosis, which should be rapidly integrated into the clinical decision-making algorithm, particularly in atypical DIPG.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-012-0998-0) contains supplementary material, which is available to authorized users.

A Prominin-1-Rich Pediatric Glioblastoma: Biologic Behavior Is Determined by Oxygen Tension-Modulated CD133 Expression but Not Accompanied by Underlying Molecular Profiles

Few studies on the biologic and molecular properties of pediatric glioblastoma have been performed. Until now, differential genomic analysis of CD133+ve and CD133-ve fractions has not been described in pediatric glioma. We hypothesize not only that the presence of CD133 could be the source of tumor resistance but also that maintenance of this molecule by hypoxia dictates cellular and molecular behavior. From a series of human glioblastoma biopsies investigated, only one, IN699 (from a pediatric glioblastoma), generated greater than 4% of the total cell volume as CD133+ve cells. Using this pediatric glioblastoma, containing unprecedented high levels of the putative brain tumor stem cell marker CD133, as a study model, we report biologic and molecular characteristics of the parent culture and of CD133+ve and CD133-ve populations derived therefrom under atmospheric and hypoxic culture conditions. Immunocytochemistry and flow cytometry were performed with antigenic markers known to characterize neural stem cells and associated glioma behavior. Behavioral analysis was carried out using proliferation, adhesion, migration, and invasion assays. Cell cycle analysis and array comparative genomic hybridization were used to assess copy number profiles for parental cells and CD133+ve and CD133-ve fractions, respectively. With regard to invasion and proliferation, CD133+ve and CD133-ve fractions were inversely proportional, with a significant increase in invasive propensity within the CD133-ve cells (P < .005) and a significant increase in proliferation within CD133+ve cells (P < .005). Our observations indicate identical genomic imbalances between CD133+ve and CD133-ve fractions. Furthermore, our research documents a direct link between decreasing oxygen tension and CD133 expression.

Novel Survivin Inhibitor YM155 elicits Cytotoxicity in Glioblastoma Cell Lines with Normal or Deficiency DNA-Dependent Protein Kinase Activity

Pediatric glioblastoma is a malignant disease with an extremely poor clinical outcome. Patients usually suffer from resistance to radiation therapy, so targeted drug treatment may be a new possibility for glioblastoma therapy. Survivin is also overexpressed in glioblastoma. YM155, a novel small-molecule survivin inhibitor, has not been examined for its use in glioblastoma therapy. The human glioblastoma cell line M059K, which expresses normal DNA-dependent protein kinase (DNA-PK) activity and is radiation-resistant, and M059J, which is deficient in DNA-PK activity and radiation-sensitive, were used in the study. Cell viability, DNA fragmentation, and the expression of survivin and securin following YM155 treatment were examined using MTT (methylthiazolyldiphenyl-tetrazolium) assay, ELISA assay, and Western blot analysis, respectively. YM155 caused a concentration-dependent cytotoxic effect, inhibiting the cell viability of both M059K and M059J cells by 70% after 48 hours of treatment with 50 nM YM155. The half-maximal inhibitory concentration (IC50) was around 30-35 nM for both cell lines. Apoptosis was determined to have occurred in both cell lines because immunoreactive signals from the DNA fragments in the cytoplasm were increased 24 hours after treatment with 30 nM YM155. The expression of survivin and securin in the M059K cells was greater than that measured in the M059J cells. Treatment with 30 nM YM155, for both 24 and 48 hours, significantly suppressed the expression of survivin and securin in both cell lines. The novel survivin inhibitor YM155 elicits potent cytotoxicity in glioblastoma cells in vitro via DNA-PK-independent mechanisms. YM155 could be used as a new therapeutic agent for the treatment of human glioblastomas.

Pediatric glioblastoma: Results with adjuvant chemoradiation using temozolomide.

2072 Background: Paediatric glioblastoma patients are underrepresented in major trials for this disease. The value of concurrent and adjuvant temozolomide is not known in this subset of patients. Methods: We retrospectively analysed our database between 2004 and 2010. All patients were treated with maximally safe surgical resection. This was followed by a uniform treatment schedule of post-operative radiation according to RTOG guidelines with concurrent daily temozolomide at 75 mg per meter square. The radiation dose was 60 Gy in 30 fractions planned by 3 dimensional conformal radiotherapy. 4 weeks later, adjuvant temozolomide was started at 150 mg per meter square, day 1 to 5 every 28 days and escalated to 200 mg per meter square if well tolerated. Log-rank test was used to compare survival distribution. The data was analysed using SPSS 16. Results: 23 patients were analysed. The median age was 11 years (range: 5 to 19 years). 15 males and 8 females were noted. 4 patients had seizures at presentation while 19 did not. 13 patients underwent a gross total resection while 10 underwent a subtotal resection. 5 patients received only concurrent temozolomide while 18 received concurrent and adjuvant temozolomide. The median number of adjuvant temozolomide cycles were 6 (range 2 to 12). The median follow up was 11.9 months (range: 1.6 to 30.1 months). 6 patients were dead and 17 were alive at the time of analysis. The median overall survival was not reached. The median survival for patients receiving only concurrent temozolomide was 13 months while that for patients receiving concurrent and adjuvant temozolomide was not reached and the difference was statistically significant (p=0.0009). The overall survival was not statistically different for age less or more than 10 years, gender, and type of surgery or the number of adjuvant cycles of temozolomide (less or more than 6). Conclusions: Temozolomide may have a role in paediatric Glioblastoma. Both concurrent and adjuvant temozolomide seem to be important for superior overall survival in this group of patients.

Abstract B61: Picropodophyllin (PPP) increases glucose metabolism and lactate production in paediatric glioblastoma cells

Abstract Introduction: Picropodophyllin (PPP) is of interest as an anti-cancer agent, as it has been shown to induce growth inhibition and apoptosis in a variety of human cancer cells and xenografts. Recently, PPP was found to have dramatic antitumor effects in human glioblastoma cells and in subcutaneous and intracerebral xenografts and reported to be an insulin-like growth factor-1 receptor inhibitor; however, its full mechanisms of action remain unclear. Aims: To examine the effect of PPP on glucose metabolism in cancer cells and to use hyperpolarized [1-13C]pyruvate 13C-magnetic resonance spectroscopy (MRS) and conventional 1H-MRS to measure lactate formation/production in real-time and in steady state, respectively, in order to develop a non-invasive biomarker of response for PPP treatment in a pediatric glioblastoma cell line. Methods: Pediatric glioblastoma KNS42 cells were treated for 24 hours with 2.4μM (5×IC50) of PPP. Real-time 13C exchange from hyperpolarized [1-13C] pyruvate to lactate was performed by DNP-13C MRS. Culture media and extracts from the PPP-treated and control KNS42 cells were analyzed by 1H-MRS to examine the effect of PPP on glucose metabolism and lactate production. Lactate dehydrogenase (LDH) expression was examined by western blot and LDH activity was also measured enzymatically. Cellular protein content and size were also evaluated. Results and Discussion: The rate of real-time 13C exchange from pyruvate to lactate measured by DNP-13C MRS showed a significant increase in the PPP-treated group (P = 0.027 versus control group). These DNP changes in PPP-treated KNS42 cells are consistent with the significant increase in intracellular lactate (P = 0.003) and lactate excretion to the culture media (P &amp;lt; 0.001). This increase in lactate production is also consistent with significantly elevated LDH activity (P = 0.003) in the PPP-treated group. However, no change in LDH expression was observed in the PPP-treated group. Significant increases in intracellular glucose (P = 0.006) and glucose uptake (P = 0.008) were also found in PPP-treated KNS42 cells. These changes indicate an up-regulation of glucose metabolism following PPP treatment and a subsequent increase in lactate production. Twenty-four hours PPP treatment caused a significant decrease in cell number when compared to controls (∼40%, P = 0.001). No significant difference in cellular protein concentration and size were found in PPP-treated cells when compared to controls, indicating that the observed increases in glucose metabolism, lactate production and LDH activity are due to changes in cellular metabolism following PPP treatment and not from changes in cellular size or protein concentration. The increase in lactate production following PPP treatment found in this study is an unusual observation, as most cancer targeting-treatments cause a decrease in lactate production. Hence, the use of DNP and 13C MRS to measure lactate production could provide a more specific non-invasive biomarker of response for PPP treatment. Conclusions: PPP treatment resulted in increased glucose metabolism and lactate production in KNS42 cells, as shown by both real time and steady-state measurements. These changes have potential as specific biomarkers of response for PPP treatment. We acknowledge the support received from the CRUK and EPSRC Cancer Imaging Centre with the MRC and Department of Health (England) grant C1060/A10334, NHS funding to the NIHR Biomedical Research Centre.

Distinct genetic alterations in pediatric glioblastomas

Pediatric high-grade tumors, especially glioblastomas (GBs), can be clinically devastating but are under-studied in comparison with adult GBs (aGBs). Molecular features of pediatric GBs (pGBs) are poorly understood and novel-targeted therapies have not been routinely used in pediatric patients with GBs. Twenty-four non-brainstem pGBs were studied. To compare pGBs with aGBs, immunohistochemical staining and fluorescent in situ hybridization were performed in paraffin-embedded tissues. Microarray gene expression analyses were performed in snap-frozen tissues of four primary pGBs, six primary aGBs, and one non-neoplastic brain. Immunohistochemial p16 loss was more frequent in pGBs, whereas p53, epidermal growth factor receptor, and phosphatase and tensin homolog loss were similar to that of aGBs. No case was isocitrate dehydrogenase (IDH)1 immunopositive or showed the IDH1 R132/IDH2 R172 mutation, suggesting primary GB. Microarray analysis revealed two pGB subtypes (A and B). Type B pGBs and aGBs had similar gene expression profiles; however, the profiles of type A pGBs differed from those of aGBs. In type A pGBs, we identified 90 up- and 63 down-regulated genes; platelet-derived growth factor receptor α polypeptide and CCND2 expression were significantly reduced, whereas they were up-regulated in aGBs. Our study found two distinct pGB gene expression profiles: one similar to that of aGBs and the other different. We identified significantly up- and down-regulated genes in pGBs that may provide better targets for diagnostic, prognostic, and therapeutic uses; however, more studies are required to determine the classification and optimal treatment of pediatric patients with GBs.

Abstract 3687: An integrative genomics approach identifies distinct molecular and epigenetic subgroups of pediatric glioblastoma

Abstract Pediatric glioblastoma (GBM) belongs to the comparably small group of childhood malignancies for which cure is still an exception. Histologically indistinguishable from their adult counterparts, they carry a similar dismal prognosis. Whereas genetic and epigenetic properties have been extensively studied in adult tumors, little is known about the molecular characteristics of pediatric GBM, although some reports indicate that it is likely a different entity in terms of tumor biology and molecular genetics. Thus, this study aimed to elucidate disease-defining molecular lesions by determining genomic, transcriptomic and epigenetic alteration profiles. Using an integrative genomics approach combining multiple screening strategies, we investigated primary tumor samples from 55 childhood GBM for copy-number aberrations (CNA), transcriptomic and epigenetic changes, complemented by sequencing analysis of TP53, IDH1/2 and further candidate genes. Methylome analysis revealed the existence of five separate clusters of childhood GBM with distinct molecular and clinico-pathological features. Methylation patterns correlated with novel recurrent, subgroup-specific driver mutations unique to the pediatric population, and with clearly distinguishable transcriptomic profiles. Integration of methylation and gene expression data suggested that different tumor subgroups are derived from at least two distinct precursor-cell populations, one of them without any signs of neural lineage commitment. Furthermore, distinct clusters were highly associated with the presence of balanced (13%) or aneuploid (33%) genomic profiles or with cases displaying highly-rearranged genomes (11%), or various high-level focal amplifications (43%) of known and novel oncogenes. Similar to adults, CNA frequently targeted GBM core signaling pathways such as RTK/PI3K, p53 and RB signaling. TP53 loss-of-function mutations were present in 46% of pediatric GBM. IDH1 mutations were detected in only six patients (11%), but these tumors displayed concerted hypermethylation at a large number of loci, resembling a CpG island methylator phenotype (CIMP). Relevant findings are being validated by immunohistochemistry or FISH analysis in an independent, large-scale cohort representing 130 uniformly-treated pediatric GBM. This study, one of the largest cohorts of pediatric GBM investigated for molecular alterations to date, describes frequent genetic and epigenetic features of this devastating disease and further emphasizes and differences between adult and pediatric GBM. The identification of distinct molecular subgroups and commonly altered pathways will help to characterize molecular biomarkers for improved prognostic assessment and risk-adapted treatment stratification, and may facilitate the development of suitable in vitro and in vivo models for defining novel therapeutic strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3687. doi:1538-7445.AM2012-3687