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. 2013 Nov;15(Suppl 3):iii173–iii177. doi: 10.1093/neuonc/not186

PEDIATRICS LABORATORY RESEARCH

PMCID: PMC3823901
Neuro Oncol. 2013 Nov;15(Suppl 3):iii173–iii177.

PL-001. NEURO-GLIAL INTERACTIONS IN THE PONTINE MICROENVIRONMENT

Viola Caretti 1, Alyssa Noll 1, Pam Woo 1, Michelle Monje 1

Abstract

Diffuse intrinsic pontine glioma (DIPG) is the pediatric brain malignancy with the worst prognosis. Our group has found a strong mitogenic effect of neuronal activity on normal neural stem and glial precursor cells. Neuronal activity may have a similar mitogenic effect in the DIPG microenvironment. We thus hypothesize that an interaction between ventral pontine neurons and glioma cells is pivotal to DIPG growth. We have investigated neuron-glioma cell interactions in vitro and found that human DIPG cells preferentially migrate towards human cortical areas rich in mature neurons compared to other regions poor in mature neurons (e.g. white matter tracts and the walls lining the lateral ventricles). These data may explain the histopathological hallmark of perineuronal satellitosis in DIPG. To test the role of physiological neuronal activity on DIPG growth in the ventral pons of freely moving mice, we are employing in vivo optogenetic techniques in our well-established orthotopic DIPG xenograft models. Utilizing these in vivo optogenetic neuronal stimulation models we will present data describing the effect of neuronal activity on glioma in vivo. If proven, this concept of neuronal-activity dependent glioma growth could not only provide fundamental insight into the pathogenesis of DIPG and other gliomas but also elucidate novel therapeutic targets for this devastating disease.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii173–iii177.

PL-002. CELL MIGRATION IN PAEDIATRIC GLIOMA; CHARACTERISATION AND POTENTIAL THERAPEUTIC TARGETING

Julia Cockle 1,2, Anke Bruning-Richardson 1, Susan Picton 2, Jane Levesley 1, Elizabeth Ilett 1, Susan Short 1, Alan Melcher 1, Sean Lawler 3

Abstract

Paediatric high grade gliomas are devastating tumours with five year survival outcomes between 15–35%. Despite aggressive management, tumours inevitably recur due to their diffuse and invasive nature. Novel therapeutics that effectively block brain tumour invasion are badly needed, however no previous studies have specifically examined paediatric glioma cell migration. Here we describe for the first time the migratory behaviour of four paediatric cell lines (SF188, Res186, KNS42, UW479) in vitro. Migratory characteristics were examined using 2D (transwell membrane, immunofluorescence, live cell imaging) and 3D (migration on nanofibre plates and spheroid invasion assays in collagen) assays. Each cell line was able to migrate, but there were differences in morphology, actin structure and migration rates. We then investigated the effectiveness of treatment with the small molecule glycogen synthase kinase-3 (GSK3) inhibitors lithium chloride (20 mM) and the indirubin derivative BIO (5 µM) which have previously been shown to block migration of adult glioma cells. The addition of LiCl and BIO reduced migration in all assays and all cell lines and resulted in cytoskeletal rearrangement of stress fibres and focal adhesions when viewed by immunofluorescence. Loss of polarity and differences in cellular movement were observed by live cell imaging in the presence of drugs. This is the first study of its kind to describe and demonstrate the migratory characteristics of paediatric glioma cell lines in 2D and 3D models and to exhibit the ability of two known adult glioblastoma inhibitors, LiCl and BIO to inhibit migration in paediatric cell lines. We propose that LiCl, BIO and its derivatives may be suitable novel chemotherapeutic candidates for migration inhibition in paediatric gliomas and warrant further investigation in this devastating disease.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii173–iii177.

PL-003. MEMBRANE DEPOLARIZATION IS A MEDULLOBLASTOMA TUMOR SUPPRESSOR

Livia Garzia 1, Adrian Dubuc 1, Graham Pitcher 1, Paul Northcott 5, Adrian Mariampillai 2, Stephen Mack 1, Kory Zayne 1, Tiffany Chan 1, Patryk Skowron 1, Xiaochong Wu 1, Anath Lionel 4, Sorana Morrisy 1, Cynthia Hawkins 1, Paul Kongkham 3, James Rutka 1, Annie Huang 1, Anna Kenney 4, Victor Yang 2, Michael Salter 1, Michael Taylor 1

Abstract

The extensive studies of medulloblastoma (MB) genomics revealed that only a few genetic events are highly recurrent, and the mutated genes do not appear to converge on well-known signalling pathways, and do not suggest targets that could be used to treat a meaningful percentage of patients. Therefore we present a genome-wide investigation of MB DNA hypermethylation in an effort to determine the genes driving tumor pathogenesis. We treated MB cell lines with a DNA demethylating agent and an histone deacetylase, performed genome wide methylated DNA immunoprecipitation on both MBs and normal cerebella then narrowed the analysis to genes that were transcriptionally silenced. Pathway analysis revealed that genetic and epigenetic events converge on synaptic electrical activity and neuronal membrane homeostasis. We performed pharmacological and optogenetic modulation of membrane potential to functionally assess its role in MB, we found a decrease in cellular proliferation and increased differentiation in response to depolarization. We also show that membrane depolarization is tumor suppressive in vivo as depolarized MB cells are unable to transplant the disease in immuncompromised mice. MB cells evade differentiation in response to membrane depolarization through selection of clones with somatic genetic or epigenetic events affecting genes involved in synaptic formation and function. Membrane depolarization therefore represents a novel form of non-genic tumor suppressor and represents an attractive therapeutic avenue for the treatment of children with malignant brain tumors.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii173–iii177.

PL-004. METASTATIC RECURRENT MEDULLOBLASTOMA IS HIGHLY DISTINCT FROM ITS MATCHED PRIMARY TUMOR

Livia Garzia 1, Sorana Morrisy 1, Patryk Skowron 1, Salomeh Jelveh 2, Patricia Lindsay 2, David Largaespada 3, Lara Collier 3, Adam Dupuy 4, Richard Hill 2, Michael Taylor 1

Abstract

Medulloblastoma (MB) is the most common paediatric malignant brain tumor. By the way of optimal surgery, radiation, and chemotherapy, medulloblastoma can be treated in a good fraction of children but despite the best therapy the disease recurs. Many factors composing the tumor niche can also influence radiosensitivity. For this reason we developed a protocol to study the genetic differences between primary and recurrent (radioresistant) tumors in vivo, using our transposon mutagenesis driven mouse model. We developed a novel murine model of metastatic MB, which is highly penetrant, has a short latency, and involves random secondary genetic events. The model is based on mobilizing the Sleeping Beauty transposon in the cerebella of Ptch + /− mice. We performed surgical removal of the murine tumors, and then treated the mice by multi-fraction CT-guided craniospinal irradiation. 70% of the mice treated with surgery and CSI recurred locally, a smaller fraction recurred distally with disease on the spinal cord. By the way of next generation sequencing followed by gCIS prediction or convergence analysis we found that recurrences are highly genetic divergent from their matched primary tumor. We have identified several potential synthetic lethal genes in the primary tumors, and relapse drivers, which are only found mutated in the recurrences. We selected actionable targets and performed in vitro radiosensitization assays with small molecules inhibitors of the predicted driver genes, showing a reversal of radiation resistance. Highly targetable events in genes known to play a role in cell-cycle, apoptosis and proliferation, like Trp53 and ALK, are potential drivers of local and distal MB recurrence. Recurrent medulloblastoma can be modelled in mice by the way of surgery followed by CSI irradiation. Murine primary MBs are highly genetically different from the recurrences, urging the scientific community to develop different therapeutic approaches to efficiently target primary and recurrent human tumors.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii173–iii177.

PL-005. SMALL RNA SEQUENCING DISCLOSES THE Oct4-miR199a/214 PATHWAY CONTRIBUTES IN CHEMORESISTANCE AND TUMOR MALIGNACY OF PEDIATRIC INTRACRANIAL GERM CELL TUMORS

Tsung-Han Hsieh 1, Hsei-Wei Wang 1, Wei-Chung Cheng 2, Tai-Tong Wong 2

Abstract

Pediatric central nervous system germ cell tumors (GCTs) are rare and heterogeneous neoplasms and vary in histological differentiation, prognosis and clinical behavior. The reported incidence of GCTs is significantly higher in Taiwan, Japan and Korea compared to Western countries. Primary GCTs can be divided into germinomas and non-germinomatous malignant GCTs (NGMGCTs, includes all GCTs except for germinoma & benign mature teratoma) according to degrees of malignancy: germinomas have best prognosis and survival rate than NGMGCTs. The mechanisms underlying this difference are still unclear. Here we used small RNA sequencing (smRNA-seq) to distinguish miRNA expression between germinomas and NGMGCTs. We found that the expression of certain miRNAs, including miR-199b-5p, miR-199b-3p, miR-214-3p, miR-214-5p and miR-199a-5p, are up-regulated in NGMGCTs. Mir-214-3p and miR-199a-5p are transcribed from the same miRNA cluster chromosome region, we hypothesized that the miR-199a/214 cluster plays a crucial role in NGMGCT malignancy and pathogenesis. Overexpressing miR-214-3p and miR-199a-5p in an embryonic carcinoma cell line led to the enhancement of cisplatin resistance and malignacy. We found that important stemness genes, Oct4 and Nanog, were over-expressed in germinomas and possibly represses miR-199a/214 expression. When we knocked down the expression of Oct4, but not another stemness gene Nanog, miR-199a-5p and miR-214-3p levels were up-regulated. Our data suggest a new pathway, Oct4-miR-199a/214, contribute in chemo-resistance and tumor malignancy of GCTs.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii173–iii177.

PL-006. FUNCTIONS OF VOLTAGE-GATED POTASSIUM CHANNEL EAG2 IN MEDULLOBLASTOMA AND MECHANISM-BASED THERAPY USING A FDA-APPROVED DRUG

Xi Huang 1, Ye He 1, Adrian Dubuc 2, Rintaro Hashizume 3, Wei Zhang 1, Samantha Stehbens 4, Susan Younger 1, Suzanne Barshow 1, Sijun Zhu 1, Xiaochong Wu 2, Michael Taylor 2, Sabine Mueller 5, William Weiss 3, David James 3, Marc Shuman 6, Yuh Nung Jan 1, Lily Jan 1

Abstract

INTRODUCTION: Medulloblastoma (MB) is the most common pediatric brain cancer. Standard of care with surgery, radiation and chemotherapy typically results in serious cognitive and neuroendocrine deficits that substantially impact quality of life. It is critically important to identify novel targets and develop targeted therapy. Here we report the functions of the voltage-gated potassium channel EAG2 in MBs and mechanism-based therapy using a FDA-approved drug. METHODS: We conducted microarray, qPCR, IHC and bioinformatic analyses to study the expression of EAG2 in MBs. We studied the functions of EAG2 using electrophysiology and tumor biology approaches following RNAi knockdown or genetic knockout. We investigated the role of eag channel in the brain tumors of Drosophila melanogaster. Finally, we studied the efficacy of a FDA-approved drug in blocking EAG2 channel and inhibiting MB growth in mouse models. RESULTS: We identify that EAG2 expression is elevated across different molecular subtypes of MBs. EAG2 knockdown or knockout impairs MB growth and metastasis while Eag2 function is largely dispensable for normal mouse development. Mechanistically, we show that EAG2 controls MB cell proliferation and migration by regulating cell volume dynamics via effect on the p38 MAPK pathway. We find that the brain tumor-specific function of eag potassium channel is evolutionarily conserved in Drosophila melanogaster. We discover that a FDA-approved drug potently blocks EAG2 channel and inhibits MB growth and metastasis in multiple preclinical mouse models. CONCLUSION: Our study reveals the evolutionarily conserved functions of EAG2 potassium channel in promoting MB tumor growth and metastasis by regulating cell volume dynamics, the perturbation of which acts through the p38 MAPK pathway. We identify a novel EAG2 blocker that is potent in inhibiting in vivo MB growth and metastasis. The fact that the drug under consideration is already FDA-approved for other uses should accelerate the time necessary to initiate clinical trials.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii173–iii177.

PL-007. DEVELOPMENT OF A DIPG ORTHOTOPIC MODEL IN NUDE MICE USING AN IMPLANTABLE GUIDED-SCREW SYSTEM

Miguel Marigil 1, Patricia Jauregi 1, Miguel Angel Idoate 1, Enric Xipell 1, Guillermo Aldave 1, Marisol Gonzalez-Huarriz 1, Sonia Tejada-Solís 1, Ricardo Diez-Valle 1, Angel Montero-Carcaboso 2, Jaume Mora 2, Marta M Alonso 1

Abstract

Diffuse intrinsic pontine gliomas (DIPG) constitute one of the most aggressive pediatric tumors due to its poor prognosis and shortage of effective treatments. New therapeutic approaches for the treatment of such deleterious tumors are most needed. Unfortunately, there is a paucity of DIPG animal models that allow the evaluation of agents in a consistent and reproducible fashion. In this study we aim to develop a DIPG tumor model in nude mice to test the effectiveness of different therapeutic choices. To overcome the problems associated with using stereotactic techniques to establish DIPGs we used an implantable guide-screw system based in a previous one described for supratentorial gliomas. The system consists of a 2.6-mm guide screw with a central 0.5-mm diameter hole that accepts the 26-gauge needle of a Hamilton syringe. We localized our entry point 1.5 mm right to the lamda and just posterior to the lambdoid suture. Next, we performed a small mark in this point with a 15-Gauge needle which was subsequently widened with a hand-controlled twist drill. A stylet is used to cap the screw between treatments. DIPGs tumor cells and/or therapeutic agents are injected in a freehand fashion by using a Hamilton syringe and a 26-gauge needle fitted with a cuff to determine the depth of injection. To test this system, guide screws were successfully implanted in 10 mice. After 3 days recovery, mice were inoculated with a DIPG cell line. All the mice develop severe neurologic symptoms by day 35 after inoculation. Pathologic examination demonstrated that all these animals have tumors in the pons that infiltrated the surrounding structures. In the fullness of time our DIPG orthotopic model recapitulates the invasive phenotype shown by this tumor in the children. Moreover, this model constitutes a promising in vivo readout to measure the efficacy of therapeutic agents.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii173–iii177.

PL-008. RECURRENT ACTIVATING ACVR1/ALK2 MUTATIONS IN DIFFUSE INTRINSIC PONTINE GLIOMA

Katy Taylor 1, Alan Mackay 1, Nathalene Truffaux 2, Olena Morozova 3, Yaron Butterfield 3, Cathy Phillipe 2, Maria Vinci 1, Carmen de Torres 4, Ofelia Cruz 4, Jaume Mora 4, Darren Hargrave 5, Michelle Monje 6, Stephanie Puget 7, Stephen Yip 3, Chris Jones 1, Jacques Grill 2

Abstract

Diffuse intrinsic pontine glioma (DIPG) have a universally dismal prognosis (median 9–12 months), with neither chemotherapeutic nor targeted agents showing any substantial survival benefit in clinical trials in children with these tumours. Recent high-throughput sequencing approaches have revealed a striking prevalence of K27M mutations in the genes encoding the histone variants H3.3 (H3F3A) or H3.1 (HIST1H3B) in this disease, though approaches for specific novel therapeutic interventions are not clear, and little has been reported of the secondary mutations accompanying these changes. We carried out whole genome sequencing on a series of 20 pre-treatment biopsy samples of DIPG, for which the patients underwent a safe stereotactic procedure. We identified mutually exclusive recurrent alterations in the PI3-kinase pathway targeting PIK3CA, PIK3R1 and PTEN through SNVs and microdeletion, in addition to amplification of MET as previously described, and truncating mutation of NF1. In total, 9/20 (45%) DIPG cases harboured some form of alteration predicted to activate the RTK/PI3K/MAPK pathways. We also report the novel identification of recurrent activating mutations in the ACVR1/ALK2 gene, which encodes a type I activin receptor serine/threonine kinase, in 18% of a wider series of DIPG samples (18/57). Strikingly, these somatic mutations (R206H, R258G, G328E/V/W, G356D) have not been reported previously in cancer, but are identical to those found in the germline of patients with the congenital childhood developmental disorder fibrodysplasia ossificans progressiva (FOP), and have been shown to constitutively activate the BMP/TGF-b signalling pathway. They co-segregated with H3.1 K27M mutations and wild-type p53, and patients with this mutation had a longer overall survival and PI3-kinase pathway activation. These mutations represent novel targets for therapeutic intervention in this otherwise incurable disease.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii173–iii177.

PL-009. DEFINING THE CELL TYPE- AND BRAIN REGION-SPECIFIC DETERMINANTS UNDERLYING KIAA1549:BRAF-INDUCED MURINE GLIOMA IN VITRO AND IN VIVO

Aparna Kaul 1, Yi-Hsien Chen 1, Sonika Dahiya 1, Ryan Emnett 1, Scott Gianino 1, David Gutmann 1

Abstract

Tandem duplications involving the BRAF kinase gene have been identified as the most frequent genetic alteration in sporadic pediatric low-grade glioma (pilocytic astrocytoma), creating a novel fusion protein (KIAA1549:BRAF; f-BRAF) with increased BRAF and MEK activity. To define the role of f-BRAF in murine gliomagenesis, we demonstrate that f-BRAF regulates proliferation in a cell type- and brain region-specific manner. For these experiments, we leverage both in vitro primary cell culture systems as well as a novel genetically-engineered mouse (GEM) strain in which f-BRAF expression is controlled by both Cre recombinase activity and doxycycline treatment. First, we show that f-BRAF expression increases mouse NSC, but not astrocyte, proliferation in vitro. Second, we demonstrate that f-BRAF expression increases cerebellar and third ventricle NSC proliferation in vitro, but has no effect on cortical or lateral ventricle NSC growth. Third, we show that f-BRAF-expressing cerebellar NSCs induce low-grade glioma-like lesions when implanted into the cerebellum in vivo. Fourth, we demonstrate that while f-BRAF expression leads to increased MEK activation in astrocytes and NSCs, f-BRAF-driven NSC proliferation results from tuberin/Rheb-mediated mTOR hyperactivation. Lastly, we use a conditional f-BRAF-expressing knock-in mouse strain to demonstrate that f-BRAF expression in BLBP+ NSCs in vivo specifically leads to increased astrocyte number in the cerebellum. Collectively, these results establish an obligatory role for cell type- and brain region-specific determinants in defining the spatial predilection for pilocytic astrocytomas to form in the cerebella of young children. Furthermore, we show that mTOR pathway activation represents a key growth regulatory mechanism common to both sporadic and familial neurofibromatosis type 1-associated low-grade gliomas in children. With the development of two potential preclinical model (explant and GEM) systems, further mechanistic explorations as well as therapeutic drug testing now become possible.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii173–iii177.

PL-010. NONE OR LITTLE NUMBER OF CHROMOSOMAL ABERRATIONS DETECTED BY COMPARATIVE GENOMIC HYBRIDIZATION IN FETAL BRAIN TUMORS

Tomoru Miwa 1,2, Shizuo Oi 2, Yuichiro Nonaka 2, Hikaru Sasaki 1, Kazunari Yoshida 1

Abstract

It is extremely rare that brain tumors are discovered at a fetal stage. Their pathological diagnosis is various and the treatment is not yet established. As for the malignant fetal brain tumors, they show rapid progress and the prognosis is very bad. We experienced six cases of brain tumors that were discovered by ultrasonic echo or MRI during 30-35 weeks at a fetal stage and we also used MRI for following them. Operation (biopsy: 3 cases, resection: 0 case) or autopsy was performed after birth and their pathological diagnoses were 2 cases of immature teratoma, and a case of primitive neuroectodermal tumor (PNET), congenital neuroectodermal tumor, desmoplastic infantile astrocytoma (with glioblastoma like components), hamartoma, each. All cases except hamartoma showed rapid growing and died within several weeks or months after birth. Because of their high intracranial pressure, 2 cases were forced to drain cerebrospinal fluid in emergency just after birth. Chemotherapy was done for 2 cases but they showed chemoresistance. We also analyzed genetic aberrations of these tumors by comparative genomic hybridization (metaphase CGH). Interestingly, they present none or little number of chromosomal aberrations within few weeks after birth, regardless of their malignancy. This result might suggest a difference between children and fetus about tumorigenic pathway with genetics. Fetal malignant brain tumors showed rapid growing and aggressiveness just after birth irrespective of pathological diagnosis. It seemed that there are some growing factors clinically and genetically at perinatal stage compared with general pediatric brain tumors, and their treatment and management are still difficult.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii173–iii177.

PL-011. ASSOCIATION OF THE PRESENCE OF NEUROSPHERES IN TUMOR CULTURES WITH TREATMENT RESPONSE AND SURVIVAL OF CHILDREN WITH ASTROCYTOMA

Enrique Lopez 1, Andrea Ponce de León 1, Carolina Sepulveda 1, Luis Zarate 1, Jaime Diego-Pérez 1

Abstract

INTRODUCTION: Tumors in the nervous central system are the second most common type of cancer in children, and astrocytomas account for 50% of these tumors. The poor prognostic factors used today are age, tumor location and histology. Until recently, it has been observed that only a fraction of the tumor cells have carcinogenic potential according to in-vitro models. In addition, cells with carcinogenic potential may form neurospheres that have the ability to proliferate in repeated passages, which is one of the characteristics of stems cells and could be associated with the survival and response to the treatment. PATIENTS AND METHODS: This study was carried out at the Hospital de Pediatría, Centro Médico Nacional Siglo XXI in Mexico City with patients younger than 17 years of age, and diagnosed with astrocytoma from May 1, 2010 to May 31, 2012. A primary culture was done with each tumor sample. The presence or absence of neurospheres was documented in the basal culture and each of the passages. OBJECTIVE: To associate the formation of neurospheres in the basal culture and each of the first four passages with the clinical outcome and response to treatment in patients with astrocytoma. RESULTS: 24 patients were included. 11 patients (45.8%) presented neurospheres in basal cultures, seven patients (29.1%) presented neurospheres in the first passage, six patients (25%) in the second passage and only five patiens (20.8%) presented neurosphere formation in the third passage. Patients with positive neurosphere formation in the original tissue culture had a 39.51% survival rate at 24 months, compared with 81.48% survival rate in patients who did not present neurospheres in the basal culture. CONCLUSION: We found an association between the development of neurospheres and patient survival; however, it was not statistically significant due to the sample size for this study.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii173–iii177.

PL-012. WHOLE GENOME SEQUENCING REVEALS MICROGLIA AS KEY MICROENVIRONMENTAL DRIVERS OF NEUROFIBROMATOSIS-1 GLIOMA FORMATION AND GROWTH

Winnie Pong 1, Li Ding 1, Michael McLellan 1, Ibrahim Hussain 1, Ryan Emnett 1, Scott Gianino 1, Samantha Higer 1, Jeffrey Leonard 1, Abhijit Guha 2, Elaine Mardis 1, David Gutmann 1

Abstract

Current treatments for brain tumors largely target growth control pathways de-regulated in neoplastic cells. To identify new therapeutic opportunities, we performed whole genome sequencing of three low-grade gliomas (pilocytic astrocytoma; NF1-PA) from children with the neurofibromatosis-1 (NF1) brain tumor predisposition syndrome. While bi-allelic NF1 gene inactivation and loss of total NF1 protein expression characterized these NF1-PAs, tumor purity analyses revealed that 30–50% of the cells in these tumors were stromal cells, specifically Iba1+ microglia. To define the contribution of these tumor-associated microglia to glioma formation and maintenance, we leveraged several novel Nf1 genetically-engineered mouse (GEM) strains. First, pharmacologic inhibition of microglia function attenuated Nf1 mouse glioma cell proliferation. Second, genetic elimination of microglia using CD11b-TK transgenic mice reduced Nf1 mouse glioma cell proliferation. Third, Nf1 GEM glioma-associated microglia are resident brain macrophages, which express the CX3CR1 chemokine receptor, such that reduced Cx3cr1 expression delayed Nf1 GEM glioma formation. Collectively, these findings establish microglia as essential non-neoplastic stromal elements critical for Nf1 mouse glioma formation and maintenance. Importantly, ongoing glioma-associated microglia RNA sequencing efforts have begun to identify new targets for future stroma-directed brain tumor therapy.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii173–iii177.

PL-013. PEDIATRIC GBMs: H3F3A MUTATIONS AND CORRELATION WITH METHYLATION PROFILING AND HISTONE CODE

Chitra Sarkar 1, Pankaj Pathak 1, Prerana Jha 1, Suvendu Purkait 1, Vikas Sharma 1, MC Sharma 1, Vaishali Suri 1, Mohammed Faruq 2, Mitali Mukherjee 2

Abstract

Mutations in the H3F3A-ATRX-DAXX chromatin remodelling pathway have been recently reported in pediatric GBMs. However, correlation of these mutations with methylation profiling and global histone methylation has not been studied. Hence, this study was undertaken to gain insight into this correlation in pediatric GBMs. Twenty seven pediatric and 23 adult supratentorial GBMs were studied for H3F3A, ATRX, DAXX and IDH-1 mutations. Expression of H3K27me3, H3K4me3, H3K36me3 and H3K9me3 were evaluated by immunohistochemistry. Genome wide methylation profiling by Illumina Infinium assay was done in 21 pediatric GBMs. H3F3A mutation was identified only in pediatric GBMs (52%). Of these, K27M and G34R accounted for 37% and 15% respectively. ATRX mutation was noted in 46% of pediatric GBMs and was significantly associated with H3F3A mutations. No pediatric GBM showed DAXX or IDH1 mutation. Methylation profiling revealed 110 hypermethylated and 62 hypomethylated genes in H3F3 mutants versus wild type. Hypermethylated genes in the H3F3 mutants had significantly more enrichment for genes related to neuronal differentiation and neuron fate commitment as compared to wild type. Histone expression showed distinct differences between pediatric and adult GBMs. Pediatric GBMs showed significantly higher frequency of immunonegativity as compared to adults for H3K27me3 (72% vs. 42%) and H3K4me3 (45% vs. 11%). Interestingly, distinct bivalent pattern of histone methylation was found in pediatric vs. adult GBMs in that K4−/K27− (double immunonegative) was enriched in pediatric (36%) versus adults (6%) and double immunopositive (K4 + /K27+) was enriched in adults (59%) versus pediatric GBMs (18%). Further, enrichment of K4-/K9+ was also significantly more in pediatric GBMs vs. adults. This is the first study demonstrating differences in methylation profiling between H3H3 mutants versus wild type and also highlighting distinct global histone modifications in pediatric versus adult GBMs. These indicate different epigenetic alterations in pathogenesis of pediatric GBMs, which may have prognostic and therapeutic relevance.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii173–iii177.

PL-014. IDENTIFYING DIFFERENTIAL ROLES FOR COLLAPSIN RESPONSE MEDIATOR PROTEIN4 (CRMP4) VARIANTS IN MEDULLOBLASTOMA GROWTH AND METASTASIS

Balasubramanian Sivasankaran 1, Radhi Praba Velayutham 1, Isabella R Fraschilla 1, Kevin J Morris 1, Tobey J MacDonald 2, Tracy-Ann Read 1

Abstract

Medulloblastoma (MB) is an aggressive tumor type of neuronal origin, which occurs predominantly in the cerebellum of children. Although current advancements in MB treatment have led to improved survival rates, metastasis remains the primary reason for treatment failure and represents a significant clinical challenge. Therefore, understanding the mechanism of MB cell dispersal is critical for developing more effective therapies. The recent identification of CRMP4 as a candidate oncogene in human MBs through genetic and expression profiling screens, and the demonstration of other CRMP family variants having opposing biological effects on tumor cells, has prompted us to investigate whether CRMP4 variants would exhibit differential malignant potential in medulloblastoma. CRMP4 is a developmentally regulated gene that exists in two isoforms. It is also part of a family of cytoplasmic phosphoproteins, implicated in axon guidance and outgrowth. To characterize their role in medulloblastoma, we have cloned the corresponding human cDNAs of two CRMP4 variants and stably expressed them in the human D556 MB cell line. After validating the transgene RNA and protein expression, the functional effects of the two variants were analyzed in-vitro and in-vivo. Interestingly, variant1 exhibited enhanced migratory potential while variant2 induced proliferation in D556 cells. Further, when transplanted into the cerebellum of immunocompromised mice, variant2-expressing cells generated highly aggressive, fast growing tumors compared to controls. Moreover, using a metastatic mouse model of MB, we have found that CRMP4 is upregulated specifically in the CD15+ cancer stem cell fraction of these tumors compared to non-metastatic mouse MB. Together, these findings implicate CRMP4 as an oncogene, which promotes the proliferative and migratory potential of medulloblastoma, and suggest that upstream targeting of this signaling pathway may be exploited to inhibit both MB growth and metastasis.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii173–iii177.

PL-015. MOLECULAR PROFILING OF CNS-PNETS IDENTIFIES RECURRENT HALLMARK ABERRATIONS OF VARIOUS WELL-DEFINED ENTITIES

Dominik Sturm 1, Paul Northcott 1, David Jones 1, Andrey Korshunov 1,2, Daniel Picard 3, Peter Lichter 1, Annie Huang 3, Stefan Pfister 1,2, Marcel Kool 1

Abstract

Childhood CNS primitive neuro-ectodermal tumors (CNS-PNETs; WHO °IV) are classified as poorly differentiated embryonal tumors, and the absence of a clear histomorphological pattern and divergent differentiation render histologic diagnosis contentious. We recently identified subgroups of CNS-PNETs with biological characteristics similar to other childhood brain tumors. This study investigates the heterogeneity of CNS-PNETs in relation to other CNS tumors. We profiled a cohort of 185 tumor samples with an institutional diagnosis "CNS-PNET" for genome-wide DNA methylation patterns and copy-number alterations, complemented by transcriptomic profiling of a smaller subset (n = 59). Selected cases were screened for recurrent mutations and expression of established protein markers. Molecular features of CNS-PNET were compared to the (epi-)genetic make-up of >800 other childhood brain tumors including embryonal, astrocytic, and ependymal entities, and their molecular subgroups. The histology of 48 CNS-PNETs was revisited by five experienced neuropathologists. Unsupervised clustering methods of DNA methylation and gene expression profiles were able to clearly segregate pediatric brain tumor samples by histological entity and molecular subgroup. Exceptionally, CNS-PNETs were scattered across the entire spectrum of entities. Genome-wide profiles and molecular aberrations of CNS-PNETs frequently matched or closely resembled those of their adjacent tumor subgroups, displaying hallmark features of GBM (e.g. mutations of IDH1/H3F3A), ETMR (amplification of 19q13.42), or AT/RT (deletions/mutations of SMARCB1). Identified clusters were associated with distinct protein expression patterns of previously established subgroup markers INI-1, LIN28A, and OLIG2. After blinded histopathological evaluation, a high proportion of tumors were re-classified in line with proposed molecular affiliations. The correct classification of CNS-PNET remains difficult. We could reliably re-classify many cases based on recurrent genetic aberrations, suggesting that a significant proportion of CNS-PNETs may comprise a variety of other tumor subtypes. These findings indicate that the establishment of useful markers is needed in order to assist the histopathological evaluation of this group of tumors.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii173–iii177.

PL-016. BRAF-InsT VARIANT IN PEDIATRIC GLIOBLASTOMA: CASE REPORT AND IN VITRO RESPONSE TO BRAF INHIBITION

Tsun-Wen Yao 1, Jie Zhang 1, Berry Anna 1, Tilman Brummer 2, Nalin Gupta 1, Theodore Nicolaides 1

Abstract

INTRODUCTION: A 3-bp insertion in BRAF (BRAF-insT) resulting in the duplication of threonine 599 has been reported in pilocytic astrocytoma. This conserved residue is located within the kinase domain of BRAF and, upon phosphorylation by Ras, plays a key role in Raf activation. BRAF-insT displays an in vitro kinase activity and cellular MEK/ERK activation potential comparable to those of BRAFV600E, a common activated BRAF mutant in glioma. Here, we describe the first case of pediatric glioblastoma containing the BRAF-InsT mutation and explore the potential of using the selective oncogenic BRAF inhibitor PLX4720 against BRAF-InsT in vitro. METHODS: Human glioma samples were screened for BRAF-InsT mutation by melt-curve analysis, followed by sequencing of atypical results, in a CLIA-certified laboratory. Vector, wild type BRAF, BRAFV600E, and BRAF-InsT overexpressing NIH3T3 fibroblast cells were investigated for their response to PLX4720. Molecular signaling was measured by Western blotting and cell proliferation was quantified by WST-1 assay. RESULTS: We identified a BRAF-InsT mutation in a 17 year old male with tectal glioblastoma. BRAF-insT and BRAFV600E overexpressing NIH3T3 cells treated with PLX4720 showed a significantly reduced MEK and ERK activation levels compared to DMSO treated control cells. This is concordant with a decrease in cell proliferation upon exposure to PLX4720. PLX4720 did not suppress MEK/ERK signaling in wild type BRAF overexpressing 3T3 cells. Instead, PLX4720 treatment results in a slight increase in cell proliferation on wild type BRAF transfected 3T3 cells. CONCLUSIONS: We report the first case of pediatric glioblastoma containing a BRAF-insT mutation, an activated BRAF allele predominantly described in pilocytic astrocytomas. We demonstrated that PLX4720 efficiently suppresses MAPK pathway signaling and cell growth in cells transformed with BRAF-insT. Our data suggests that treatment with clinically approved BRAFV600E inhibitors, such as vemurafenib, may be effective in glioma patients carrying BRAF-insT mutation.

Neuro Oncol. 2013 Nov;15(Suppl 3):iii173–iii177.

PL-017. THE HISTONE H3.3K27M MUTATION IN PEDIATRIC GLIOMA REPROGRAMS H3K27 METHYLATION AND GENE EXPRESSION

Kui-Ming Chan 1, Dong Fang 1, Haiyun Gan 1, Rintaro Hashizume 2, Chuane Yu 1, Mark Schroeder 1, Nalin Gupta 2, Sabine Mueller 2, David James 2, Robert Jenkins 1, Jann Sarkaria 1, Zhiguo Zhang 1

Abstract

INTRODUCTION: Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor. Recent studies have identified somatic mutation of the H3F3A gene that encodes the histone H3 variant, H3.3, and results in lysine 27 to methionine change in encoded protein (H3.3K27M) in 60% of pediatric diffuse intrinsic pontine glioma (DIPG). The median survival of this group of patients after diagnosis is approximately one year. In human cells, unlike canonical histone H3 proteins (H3.1/H3.2) that are encoded by 13 distinct genes, there are two genes encoding histone H3.3. H3K27 is conserved among all histone H3 proteins (canonical and variant H3.3). Thus, the mechanism by which the K27M mutation drives tumorigenesis is largely unknown. METHOD: We analyzed several modifications on histones in two DIPG lines as well as three cell lines expressing the H3.3K27M transgene using Western blot, immunofluorescence and ChIP-seq. In addition, we also determined gene expression in two DIPG lines using RNA-seq. RESULTS: We found that the levels of H3K27 di- and tri-methylation (me2 and me3) are reduced globally in two H3.3K27M patient samples and also in three different cell lines exogenously expressing the H3.3K27M transgene. Remarkably, in addition to global reduction of H3K27 me2 and me3, a dramatic increase in H3K27me3 and Ezh2 (the catalytic subunit of H3K27 methyltransferase) at hundreds of gene loci is observed in H3.3K27M cancer cells. The loss and gain of H3K27me3 peaks at gene promoters, is associated with increased and reduced expression of the associated genes, respectively, and genes with increased H3K27me3 are linked with cancer pathways. CONCLUSION: The H3.3K27M mutation is a dominant mutation that reprograms the H3K27 methylation landscape and gene expression, which in turn, may contribute to tumorigenesis. These findings may have implication for the discovery of future treatment of DIPG.

Articles from Neuro-Oncology are provided here courtesy of Society for Neuro-Oncology and Oxford University Press

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